Random Recipes

    \f  \b   \b  Œ§\b\b\r\t   \r \t \r    \b\b \b  ‚   \r  — \r ‹ \b  \r  \t \b   ‚       \b\r \t    \b\b \b   ¨¥     —  \f\b          ‹       \b\r\b \b \r  ‹    \b\r  ‹         \b ©  \r­­­\t\r€  Š‹ \t\b\b \b \b  \r  ¢  \r  \b     \b ƒ \r      Š  ŒŠŽ  ”  \b\r  ”ˆ\n ¢\r   \b\r\b   \b\r\b    \r\r \t Š   \r  \t    \n \t —   Œ \b\r\b ‰  \n  ƒ\r ª‹   \f\b \b\r\b \r 
\rŽ \t \r\r   \b \r¥    \r\r  \b\b \t  \b    —\r\r    «    \r \t  \b\b  \r  \b\r   \t  \r\b   \b   \t   Œ‰ \n  \b \t \r \b\r    • \b Ÿ\r   \n  \r  \t    \b  \b \r   \t ©‹\r  \r Œ\f–‰Ž \t   \b‹\b   \b \r\b    \r  ƒ \b   \b    Š \f Œ‰  • \r   \f   \r    Š  \b \b  \b  • \b\r   \b  \b \b \r  \f–‰ \r    ¦   \b    \r \t  \b \r  \b\r \t  \r\r  \r \r     Œ
 \b \t \t    \b\b\r \r   \b    \b\r  \r   \r\b \b\r   \b\r \r  ¨\n\b\r  •\r\b – ¥ Œ\n•–Ž —  \b  \t\b\b \b \b         \t   ‹    \b\b   \b   \r\r \b   •  \b       \r ¨•\r¥   ©    — \tŒ \r\b\r \r€ \n \t      \b   \b   \b‹  —      \b\b\t  ¦    \r \t \b\r \t   \t\b\b \b   \b\b \t \b‹   \b    \r      \b \r\r °   \b     \b    \b \b \b 
¢\b\b \b  \r \t     ¢ \r   \b ¢\r —  ‹\r      \r\f\r\f\n\t\f\n\b\r\t\t\b
 ƒ„…€… ±\r \f\r\n\t\b\t\t\n\r\n  \n\b\n\b\b\b \b… Š \r\n\b\b\t … Š \b \t
\n … \t\n  ¦\b ¥   Š‡ \f\b  … §\b \b\n\b\n … –  \r \b   \t \b \n\b\t    \r  ¡ ª ‰      \b\b\t         Š\b\r \b ” \t  ˆ\r ³   •      \r  \b \b\r   \t \f\b ‰  •\r\b  Œ \n   Ž    •\r –µ   \n\b\b  \b \r  \fˆ ‰ ‰\b  \r \t ƒ\b\b   \b  \b  \b    \b\r \t 
 \r  \b\b ‰\r  ¡\b   \t\r  \r  …\r\b  ‰\r   ¡\b ª\r \t    \r \b   \t\b\b  \r   Š  ¡  ‚  \b   \b\t \r   \b\r\b        ¡    \b   \b  ƒ \b\r \b\r \b  \r†  \f \t    \b\r     \b   \r \r  ‹   \r  …\b\t† \b  …\b†  °  \r   \r  \r\b  \b     \b ƒ  \n ˆ\r \b\r     \r   \t\r     \r    \r \t\r  …\t\b  ‰\r   \b ‹\r \t    \r  \b   \t\b\b \r     £ ¡   \r   \b ‹\b   \b\r   \r   \r   \b\r  \r Œ\b\r \b\r Ž†  ²‚  \t\r \r‡  \b   \r   \r   \b  \f \b \r‡  \b  \r \b   \t  \r  \f ™˜˜š   \b  \b\b \r  \b\r \r   \r  ƒ   \b        \t     \b         \r  Š ƒ\b\b \r‡\r \r  °  \b  \t\r     \b\b  \r    \t \r\r         \r   \b \r ‹ \r  \t \b      \b \b    \b \b£   \b      \t\b¥ \b £    \b      \t\b¥ £   \b   \t  \b  ¨\r¥£  \r  \b \b   \b\b  •      ­€ †\r\f Œ ¤ \rŽ  \r\b  \t\b\b ¢ \b  °      ¢  \t   ¢   \t\b\b \b \b£ \r \t\b\b  \b    \f  \r \b  ƒ\b\b    ƒ    \r\b ƒ  \r\r\r    \r  \b \b\b \b   \b  \b \b\b    \b  \r    \b   \t \r\b   \b\b \b \r\b   ¦   \b    \b \t  \b\b \b  \b  \b\f\f Œš¤ \b  \r£  ‹  ¤\r \r Ž 
\n\r\f\f\n\b\f\t\n
   \t \b\r ”ˆ\n   \r  \t\n\n\r §\b\b\r\t ¡ •  ‰\r ³ §—  \r ‰  ”ˆ\n   \b \b  \n\b\r\n\t\n\b\n\b \b­ ‰ ª\n ¡\r \t ”ˆ\n \r \r  \n\b€\b\n\n\r\n\b ‰ ª\n ‰ \n ”\t ³ §‹ ³ ¢ \n  ¡  \r  \r ”ˆ\n  \t \b     \n\b\r\n\t\n\b\n\b\b­ \r‡\r  \r  \b    \t  \r        \b‹ \b\b\r \n\n  \b  \b   \fˆ  \b  ¡ ”      ‰    \r\r °   \b \b \r  ‰ Š  \b  \b\b        \b\r \b °   ‹\r\r£     \b\b\b   \t\b\b      \f    ‹  \b\b  \b\b \r\b\b   \b\b     \f 
 Š Œ
 ”\r  \f\b ˆœ ‰\nŽ \t \b    ˆ\b ‰ • Œˆ‰•Ž   \b\r \t   \r\b\b \t \b\b  ‹ \t  \b    \b \t \b \b \t   \b‹ \b        \t °  \r \b\b \b \t \b\r ¢  \t \b    \b ·    \b  °      \r\r  ‹\r\r  \b      Œ\b·Ž    \b   \t \b ‹ \b  ‡  \t \b \t\b \b \b\r  \t  \b  \b  \b     \b   \t \b\b  \b  ‚  \b\b ƒ\r   \b  ° \b\b\b \b\b \b    \r  \t \r\b\b \t  \b\b     \r \b‡  \t    \b   •      ƒ\r  \b  \t     \b  \b       ­\b‹­  ¸¸¸¸¸ \t¸\b \b€   ˆ‰•    \r ­\t \t\t  ¹\n\tˆº›˜žž¯š˜€ \f   \r   \b  ƒ  \bƒ  \r  \t\b   \b  §\t   • ˆ‡\b – Š ¥\n\b  \b µ    \t \n\b   ¡   \f\b \t     ˆ \r\b  \r  \n\b ¦ –µ \t \b  §\r ª\r    Œ‰        \r\b µ \t  ‹\r \t \b    \t  ‰  ¢\r  \r\t\n\r\f\f \n\b­ \b\b  \r   \f\b œ Š    \r …•† \t    • •\b \nˆ” •\r  ‚ \t  \b\b\r \b\b  \b\b \b\r \b   \f   ”   \r  \b  \f\b ‰  •\r\b  \r \f˜  \n\b  ‰¥ \r     \t \r\b\b   ©  \b \r  ŠŠ     ŠŠ¥ …‚ ¦\b –\r† \r         ¡ \r  ˆ\r  \n\b\r  \r\r\r  \t\b\b ž¯ \r   \b   …  ©†  \b\b   \t   ”  \b\r \r\b\b\b   \r  ŠŠ  ”  \b  \b \b\b  • ²  \t \f\b ‰  •\r\b   \n \b \r \t    ŠŠ \t     \n\r \n\b Š‡\b   

\r •   \f \f\b \b ƒ ˆ\t ±\b ¡\b ¡\b ¡   Œˆ‹ \b ‰ ¡\r \b –\b \b£       
Ž ‰ ‰ \n ‰ ‰\t ‹ ª  \n
 ‰\n  ±\t  \f     \r\b\b \b\b   \b\r¹ ‚       \b \t £ \t   ŠŠ  \n\b Š  \t  ŠŠ ¥ \b\r    ‹­\b\r­­\b¸­€ \b\r    \r …‚ ˆ¥    \r\r \r ƒ \t‹    \b \b \b  \r          ³    \b \t¥ \b      \r   \t \b   \r \t \r \t   \b\r \t \b    ƒ\b † \t …\r† \t \b‹   ƒ\b\b \b\r   \b  \r \b  ­¹¢º­¬‘š€ \t\f ŒŠ¡¤\b  ‡Ž \b\n \n\b‚\n\r\b\b\f    ” §\r  \t‹ \b  \b \b   \b\b\n\n\r  \b\n\n Œˆ ŒžŽ š››˜  \b\t  \b\n\n \r\r ¥ ³¡ Š  Ž \r    ¦‹   \r  \r  ‚  \b   \r  \t  \t\b\b   \b  \b  \r\b\b \b  \n\b‹ \b\b      \f\r  \r\r   \r  ³   \f‹ \b‡ \t\n\n\r    \b\b\b   ‚ ƒ   \r  \b\b   Œ\b\r  š››¯ ‹  š›™›Ž    \b  \bƒ      \r ° \b   \b\r\b   \b\n\n\n   \t \b \t \t\n\n\r         \b  \b \b  ‚   \r \r \b  ƒ \b\b  §\b •½  ¢ \b \n\n \b\n \b   \b\b \b  \b  \b   \b ‹    Œ•½ Š\b\r   \r   \r   \t \b\b ¢     Š  \t\b\b     \b  ” •½  \t \n\n\b\n \b   \b    \t    \t   ‹\r  \b  \b‹  Œ¡Š\b¾  \b \b‹   \b \b   \t   \t \b  Œ±  \b \b‹  °  \r   \t \b\r  Œ‰\t  \t ƒ\b        \t Œ±\b  Š\b‹  \b\r\r    ‚\n\t  \n  \b ƒ\b \r    \b   \b   ‚ \b \r \b  ‹ \t      \t \b   ‹     \n Œ‰\b    –Ÿ ‚ ¦ \r   ƒ\b  ‹ \r \b \b      ” \b\r     \b\r   \b\t  °  \b‹ \r 
\b ¦    \r  \r \t  \b \n\r‹ ¦  —  ƒ\b  ƒ„\n… ƒ\f\t„…\b\b† ¡\r \b\n\n \b   ±”• –Ÿ   \n ¦  \r  ¥  \t‹  Š\b‹ \b ‹    \t \t \b\r  ¿   \b\n\n\r ‰‹   \t §\n
£  \r\b \b  \b Œ\tŽ  Š Á  ¿   \b\n\n\r     ³ \r \b‡   \b\b  \b  \b\r     \b\b  \b\b \b\r \b‹  \n   \t ƒ\b   \b  \b\n\n\r ¨Š\b‹ •\r\b
ƒ¥ ‰ §\r  Ž \t\b \b\b \r  \t    \b\b\n \n\r ¨‚ ”‹ ¦\b Š\b‹ •\r¥ Ÿ\b  Ž  \r†\n\n\r \t\b\b \b  \b   \b‹  \b \b ¨‚
\r\r ¡\b Š\b‹ œ¥ Œ  Ž  Š\b‹ \r  ‹\b    \b\r \t \t \b\b  \b\r \t  \r \t \r  \f ¨\n ¦ ¥  \nƒ\b Š‹\r ƒ\b    \r\r ¢  \r \t  ‡  ƒ   \r   \r \t  \t  \b\b \r ŒŠ  š››®Ž  ‰\b     ƒ \t \b‹          \b‡  \t   Œ‰   š››‘Ž  ‚ °\b\b   \r\b \r  \r °     \b  \r  \b     
   Œš›™™Ž \b   ¨\b\r\b \r¥    \b\r     \b « \b\b \b\r       ƒ\b \t \b   \b   ¥        ‚  \b‹\b        \b‹\b   ƒ \t \b \r \b\b    ƒ    
ƒ Œ \r¤ ‹\t \bŽ Š ³ ˆ\r ¡ §\r  ‡ • Š\b\t‹  
\b \b \b   \r \t  \r \r \r\r  \b ‡\n\t\n\r •½ § ˆ…\n\n\r\t‰\n\n\b\n€\r\b\tŠ … ‰‹ŒŽ \n\n\n‘\r ‚  –  à Š\b  \b\r •  ³ Ä ³  \b\b \b\r  \b\t\n  \bƒ \b \t \t   ’\n\n\b\b\n \n\n\r  ³ ª\r\b \n‰ §Ä  \r‡–\b ³ 
\b\b   \b\n\n\n \r\r \b\b      \b\b\n\n\r ‹    
 \n§ §\r ‰ ª\b Š\r    \b\t  \b\n\n \b\b\n \n\r     ª – ‰ ‡ § ” §Ä   ‰\b  \b\b \b\t\n    \b­  š\b \b « \t\r   \t“”\n ¡Š\b¾ •Å ‰\b\b  §\r ‰ •\r \r\t\r    \b\r¥ ƒ  \t ‡¹ € \b\n\n\n\r” ‰\t  \n\b\n\r\b ” §\r ‰ \n\r\b ”• Ÿ Ÿ\n ‰\b  \b  \b © \b \b‹      \b\n\n\r ‰\b  §\r ‰ ‡‡\r\b \n •
\f  •\r   \r \b \b \b‹ \r  \n   \b\n\n\r ‰  ¡‹ ‰ ‰ ‰   ”    \b‹  €‘\n\t  \b \r   \b    \b\n\n\r ± ³ ±\r ³ §\r ‰ \n\b\n\r\b ” ¡Š\b¾ •Å \f\b   \b‹         \b\n€\b\n\n\r
ƒ      \f\b \b\r\b \n Œš››’´š›™›Ž  ‡  \f˜
\r \b \b  \r   \t \b  ‹    ‡   – \b\r \f˜ 
\r     \r \t   \b\b 
\b\b \b\b\r\t  
\r  Š     \b Œ  Ž \f\b \b\r\b \r Œ\fŽ     ª \t  
ƒ  \t\b¥ \b‹  \r‡  ƒ ¹   \t \b\b  \r \t\b\b   ƒ\b Ž  \t       \r‡\r  \f \t\b       \b \b   ƒ    §\t  \r   \t    Š \b\r\b ‰ ŒŠ‰Ž \r   \r  Œ 
\r \f\b  
\fŽ  \r\r   \b \r \r\b \t   ‹\r    —  \b    ƒ\r \b\b\r \t \b\b \b  \r   \r\b\b \b\r 
\b  
Š Š‰  \b  \r      \f\n  \b \f˜ 
\r \t  Š‰ \r\r      ƒ\b \r    Š‰ \b \t \r\b \r      \r  ‹   \r‡ \f˜ \t \b  \t \t    \t\b \b\r      \b\b  \r   \b \t      —    \r \b     \r \t    \r \b \t†\n\n\r\n \r \t \b \r \b   \r  \t‹\r  \r£  \r    \b\b  \r \t \r   \t  ƒ  — \t\b     \t  \r   \t\b\b     \t \b    \r  ‹ \b  \r  \b \b\r \b‹£   \r \b \b   \b\b \r\b  \b \b  \t \b        \t\b  £ \t\b  \b      \r  Œ\r ­ Ž \r\r \b‹£    \t\b  \r \r \b    \b   \b\r   \t  \r\r   \r  Œ \r   \bŽ£ \b\r \b  \b  ƒ  \b\r  
\r  \b\b        \t\b¥  \b\b \r   \b  \t    \r  \r \b    \r\r \n\r —  
\r •\b \t \b    ƒ£    \b       \t \b \b \b \bƒ    \t  \b \b   \b   …    \b\b  †£   \b   \b   \r‡  \t \b     ‹ \f˜   \f Æ •    \t  \t   \t \b     \f˜ \t \b\r\b \b\b \b\b\b   §\t  \b    \b     \r \b   \r \t       \f‘   \r \b  \r \b ¡\b  \r‡ Œ¡Ž \t  \b    ˆ‹ \b   ‰\r   – \r‡ \r   \f Œ  \r \t \t  \t \t\b  \t  \b\r 
\rŽ \t \b\b \b    ƒ\b \r ¡  \t‹ \t  \n\r  \r  ¡ \b       ‹    \r‡ \b\r \r  \t \b\r \r \t   \r‡\r  ƒ \r  ‹\r      \n Ç   \b \r‡    \b \t ¡ \t ¢‹\b ‹‹     \b \t   ‹ \r‡\r      ¦ \b  ¡  \b\b ‹  
\b \t  \t\b\b \r \b  \r ¡ ”  – \t \b ‹  
\b¥     ¡ \t \t  ¦ \t  \b\b \r   Š‰ \t 
\b  \t  \b ¡  \r  ‹   \b \b ‹   \r \t ¨\b\b ¥ Œ \r    \b     Ž  ¦ \t    ¨ ¥ \b \b\b \b  ‹\r  Š‰  –  \r‡\r \f˜  \b\bÆ \n  \r\b 
\b ˆ…\r\f  \f\t\t\f\f\b\n ”\b\r \r \b    §\b\b  
\f \b\b\t\r  \r   ‰   ‡  ¥  \t   \b  —\b   Š‰  \f˜   ƒ\r \b\r   ¦ \b \b 
\b \t  ƒ  \b\r \t\b  \b  ‹  \r \t\b\b  \r  \r \t \b  \b   ¦  \t \f˜   \r \b\b \b  \t \b    – ¢  \t   \r   
\b  \b   \b ˆ \r \t \r  —   \t‹ \t   \b       \t    \r \b   \t\b  \b\r   \b \t \t  \r\b  \r  \f \b \t  ‰\r    —    \t \b      —  \r   ¢\b \t\r\r  –   \t \b\b Š\b\r   \b\r
 \b\r  
\b    ¡\r   \b  \r  \b\b \b\r \n \b\b    \b   — Œ \r  \r\b \b\rŽ      \r  \r \r‡   \b\b   \r     • \t  ‰\r   \b       \b  \t  \r \r‡   ‰ \t  ‰ ¡\r  \r          \t \b   \n  \b\b \t       \r    \r     ¦ ƒ   \b\r\b        \r  ¦    \t\b  \b  \b  \b\r \t  ‹ \r    \r Œ´•Ž   – ¢       §\t \t       \t   \n‡\r\b       \b\r\b  \t    \b  –        \t‹  ‡    \b \b \t    ‰ ¡\r    \r  \t  ƒ\b \b ‹  \t   \r \b      Š \t   ƒ\b\b \r\r  ¥ ‹     \t    \b    ‰\b \f  \r     ‰   \r   •\b\b \t \t \b \b  \b  ˆ\b ‰ \r  \r   \t \r\r      \t \b\b — \r  \r\b \b   \n \t    \r   \r‡\r  ƒ \r    \t ³ § \t ƒ    —  \b   \b Š 
\r ŒŠ
Ž  \t    \r ª  Š
  ”   \t \b \t\b   Š
\b \b  ƒ   \t‹     \r \t \t\b \b \b  \f˜ \b\r  \b   \r\b \b  – \t        ƒ  \b \t \b     \t¥      \t  \t ƒ    \t \b  \b\r  \r\b ƒ  – Š
  \r   ¥ \b‹  \t‹ ƒ  \t      ƒ  Š
       \b\rÆ \n   \r  Š‰ \b   \r‡   ƒ     ƒ  \r    \b\r  Š‰    \b   – ƒ \t \b\b \n\n\t•\r\n–\t‰\r —\n\n\r  \t   \b ‹    \r\b \b  \t\b\b      Š  ƒ \b  – ³ § \t ƒ  \r \t  ƒ \b     \t   \r \f˜  \t \t    Š‰ \b\r\b ‰ \n   Š ‰  ¡\b ¡\b\r  ¦ \t‹   ‹  \r    \r  \b\t  \b  \b   \t \b\t   ¢\b \r \r   
\f  ¦ \b \b‡ \t    \t\b \b\b   \r   — \b \b\r  \b\t\b        \f  \t  \b\b      ¦ \b\b \b‡  \t \t \r\r    \r\r     ‹   \r  \t \t\b    \r \b  \t \t \r       ‹   \f  \b \t \t \b    \t  ‹  ‰ \r‡  ¦ \b  ”\b\r    
\f  ‡      \t \t \b      \b\r    \b     \r  –   \t \b \r \r  \b     \r\b\b   \t \r\r   \b  
\f    \b  \b  ƒ \t \t    \b  §\t \t    \r \t      \t\t  \b\r  ƒ   \b \t   \b\r      \t   §\b\b •\b  \t‹ \t\b\b   \t  \t \b  \t\b‹   \b\r \t \r \t   \r  \r    – \b — \b \t      \t    §\t \t    \b  \b\b \r \b \t     \b\r \b\r   \r \t \r \b \r     \r\r  
\r  \b\bÆ   \r‡\r   ‹\t\r \t  \b \t\b \b\b \r        \b  \b\t  \b   ¨¥ \r  \b\r \b\b \r\r     \f     \f˜  \b\b \b\r     ª    \b\b  \b  \r   \f\b  \n      \r     \b\b     \b \t  \b\r  \t   \b \b         \b\r    \b\b \r  \f˜  – ¨\f¥  \f˜ \t  \b\b   \n \b\r \r \b   ‰  ‰\b \f       ˆ\b ‰  \f   \t     \r   ƒ\b   \b\b  \t   \r        \r  \n  \b \r  \r   \r   \t \f \r\b  \t      \r\b Œ\b\r \nŽ ³ –\b Œ \b  Š‹\bŽ ‹‹   \r \t  \r \b‹   …–  \b\r\b \b  \b\b\r† \t\b ‹   \b\b ‰ \r\b  ¦ \b¥      Š  \r\r \b\r \t  ³¥ — ¨‹ ¥ \r \t  \b\r  ¨‹ \r¥ 
     \r \t ¡\b   \b\r\b  ƒ   \b  \t \t \t   \t    – \b\r\b \b\r \b\r \t \f
ƒ Œ
ƒ  ªŽ \r\b\b   \r   \b\b¹ 

\f\f Œ\b‹  ‰\nŽ ª\t\r  \r\t\r  \r\b  \b ­\f Œ”‹  ‰\nŽ \b \r   \r\b ‹\r  
\f Œ
ƒ  ªŽ ”\b\r —\r   ¡   \b\b 
\f  ‡  – \f–• \b \r  \n\r \r \f˜  – \f–• • \f‰    \f\n  \b\b   \r    \f–• \t \r\b ƒ \r­\r­\r¸\b  ¹\r¸º™¬Â\r¸º™š€  \b      ‰\b ƒ ‰ Œ‰Ž  ¦‹\r \r Œ¦Ž   \t  \f–•  \b Œ ” Ž  – \r \b \t‹     \f\b ‰ ¡\b ¡\b\r     \b \r    \f\b \r ¡\b ¡\b\r Œ\f¡¡Ž  –
•™\b ”ˆ\n ¢    ”  \t    ª ¥”\b\b Œ‰”\nŽ \t \r \t  ¡Šš   \b       \b‹   \t   \t \t \b  \b  \r­ ƒ € – \b       \b\b   ‹\t \b­­­  ƒ€    \r   Š‹  ‹  \r­€       –  ‰ \r    \f \b  ¦‹ Œ\n\b\r \f\b \n\r   – \f\b \f¡¡  ” \t   \r‡ \b \t‹   ƒ  \b\b \b\r       \b\r    \r    \r  – ƒ \r \b   \f¡¡      \r‡   \t‹ \b \t  \b – Œ–\b  ”‹Ž    \f‰¡¡  ‰—    \r\f \b”•– Œ \f ”‡Ž  –     –ªš        \t  ­€ – \t \b  ƒ  \b  \b   \b\t\n   ƒ\b  \n\b – \r     \b\r  \b\r \b\b \b —    \b   \b\n\n\r –  \b  \n\f\t\n\n\r\f‰ ¦\b      \r\r  \b\b \r \b     \b \r €\f\f\f Œ œ‹ ªŽ \r \b¹   Œ ¦   ‰\nŽ \b‹\r  \r\b  ƒ
   \b‹ \t \b \b  ƒ\b         ƒ  \r  – ¡\b    \r \t \b\b\t   \b\b\b   \t \b\r ‹   \b    \b  \t\r    \t \b        \t\b  \b‹  \b\b     \t \r ¢\b  – ‹    \r   \t ƒ\b\b  –  \t \r   \b    • \t  \b\r   ”\r    \f\n \b \n\b  \r \t     ¡   \f\n ¡  \t \t \r\b    ƒ\b —       \f\n   \b     ³ –\b \t    \r \f\n ¡   ƒ     – \f\n  \t \b  ” Š \b   \r    •‡  \t‹\b  \n\t \b   \r   \b\r  ¨”‹¥ ª 
 ‰  \f \t \b   \t   \b  \r \b \r          \f\n \r\b  \t \r \b    \t \f\n —\b ­˜­ \t \r \b  –\t ‡  \r   \r\b  
\b \t \b   – \b  \b \t    ‹  \r \b\r  \f™› \t    \b  Š\r‹‹ –\b  š›™ž   §\t \f˜        \b\r  \t  \t   ‹\b\b     \r\b \r  \r       ¡     \b   \r‡\r      ƒ  \t\b  \r\b —     \n ®›› \b\r    \t \t \b \b  
\f \t \t \b\b          – \r \t     \b \r ‹‹ \b \r \t‹ \r    \b\b   \t\b  \t    \b  
\f   –        \t   \t    ‡\r \t‹    Œ\rŽ  –   ¢   \r‡   \f˜ ‹ \b   \t‹        \b\b — \b\b    §\t  \t\b    \t ƒ \t‹         \b \t   \b      \r‡   ‹\r   ¢ \b    \r\r  ™¯› \b \r Æ ¥ ‹ \b\b   \b \r \b‹ \r  \f \t \b  \b\b   \r  \r \b\b   \r     \r\b \b\r   \t  ‹ \r  \r       \t \r  \b‹\r   
 \t  \b \r\r      \b\b   \t    ¦ \b\b ‹\t       \t‹\r  \b  \b  \b   \r    \b \b\r \b\r  \f˜     \b\r \b\r \b\r   \r \t  \r\b \b\r  š›™›    \b        ƒ\r    \r  —   … Œ‹ ¤  ‹Ž ‡    \r‡\r  ‹\r   \b \r   \b \t‹  \b\t\n  \r\b  § \f\b   \n\t\t\b\b \b\b\b ”•\b\t€– Œ  ‰Ž  –   \b   \f‰    \b    \f–•  \f \r‡   \b \t‹  \r    \r †  \n\b  \b  \r   \b\n\n\r –       \f‰ \r  \r‡   …\n  \b\b \b\r­ \b  \r †   \f\b \r  \b    ‰ \r  – \t   \r\b\b \b\b \r­€ \t\t
\f\t \b Œ ª ‰ \b ¦\r \bŽ    \r    \b \t \r‡  \f‘  – \r  \r  \r  \b \b    \b \r \b  \r    \f\b\b \b Œ ¡ Ž  – \b    Š‰   \b\b      \b\n\n \b\n \t   \n ¡\b\r\b ‰­ ¡\b ¡\b\r\b ‰­\b\r\b ‰ \n \r  Ê   \n\r – \t\b\b \b  \b \t‹  \b\b  \n\b  \n\b ƒƒ†”– –     \t     Š   ŒŠ ‰ \f ¦\r ”£ \r  ­€ Ž  \r\b\b \r‡  ª ‰ ¡   ³ –\b Œ  \b Š‹\bŽ     Š   \t    ‰ ŒŠ‹Ž         \b \r \b‡   \r  ƒ     \r  \b \r  ”ˆ\n  ‹  \r \b\r   \b  \r  \r —  \b\r \b\r \b \r\b \r —  €\f\b ” \r  \b\r   \t \t  \r \b ‰  \t \n\n\b\t Œ¡ ³Ž  \b\t\n Œª ‰Ž  \b \r     \f \t \r ‡     \t‹   \r \t \r     \t \b      
\t\f\t\t \f         \f–•  \b  \b   \b\b\t\r  \n\f
ƒ‚   \r     \b  \r\b  \t \t  ª ‰  \b           \b    \f\n •\r  –     \r   \b ¢  \r     ƒ \b\r \t       \t \r  •—\r\bƒ•\f
‘‰‰‡ – \f–• \r‡   …–ƒ \b  \b\b  \r\b \r†   \f‰ \r  \f\b   ‰ \t  \b \r‡ \t   \f ”‡  \t\t\t\t ˆ\t ƒ   \f–•    \b  \t \b  \n        \b  \b      \b \t‹    \t‹\r \r  ‰  Œ\n\r\b  \n\r• Ž \r\r   \f–• \b    \b\r   \b \b  \b   ƒ \r£ \r   …ˆ      – † —   \f¡\n   \r \b     \f \b Š\b \r  –‹  Œ  ¡\b   –‹ Ž  \b ¦\r\b \b  \b \t  Š\b  \t \r   \b\r     ˆ \b ‰  \r \f˜    \f–• \b  \b   \b  \b   ‹\r \b  \r     \b\r\b \b \b         ‰    \f–• \b   \b  \b\r  \r   \r      \t \t\b \b  ‹ \r\b ƒ \b  \b  \r   \r  \b   \b   \f–•  ” § \t    \b  ƒ    \f–•  \f   \r  ” §\t‹ \t \t    \r   \f–• \r   … †    \t‹\r \r \r  ‰ ¡  ³Ä  ” ” § ¡ ³ \b ¦\r\b ª ‰  ‹  ‰   – … † \b  \n\n \b    ­˜­ \r\r   ‰  ¡   Š‰   \t \f–•  \n\b    … \r  † \t  \t‹\r \r¥ \b \b \b   \t \t\b   –
\r \r \b \r  \b\r   \n\b   \t  \b  \t  \t \t\b  \b  \t   \n\r  ‹\r ª ‰   ‹ \r       ‰ \b  \b    \t ƒ  \f\f Œ‹ ¤\r \r £ ‹¤—  ‹ —Ž ‡  \f˜          ‡   \f˜ \f\b  \bƒ  \t‹ \r     ‡   – \r    \f\b \b\r\b \n  \t   \b\r   \t \r\b \r \b \b  † \n\n\r\b\b\t \b\n\n\r   \n\t\n ‘\n”\b\n\n\b\n\n\r – \b   \t \t  \t  \t  Š \t \t   ³  Œ \b   \b\r\b  \b\b   \b\r    \t\r Ž  – \f\r\b Œ \b   \b\r\b  \b\b     ‹\t\b\r \r\b  \b  \b   \r ¢ \r  \b\r\b \bŽ  – \n\t \t \t   ³ ¦ Œ ƒ   \b \b\r \r  \f\nŽ  \f \b \r  
ƒ   \r  \t \t   \b  \b\b \b\b \t    \f\n \t  \b  \b     \t   \f˜ 
 \r  \n \f\n \n\t  \t \b  \b  \b  –     Œ‹ ¦\r\b ‰ –‹ ³ –\b ¦\b  ª §   Ž        Œª ‰ ” §\t‹\t  ”‹ Š\r\tŽ  \t    ¡ Œ¡ Ž  \f    \b ƒ\b\b \b \t      \b      –  \t    \t \b  –    \b \t‹  \b\b  \t     \r  \t  \t \b     \b  \t    \t    \t  ‰\b   \b  \b\b     \r  \b\b  \t        \t \t     \r  Œ  ƒŽ ‹ \t \t  \b   \b\r Œ\n\tŽ       \b\r Œ ŠŽ  \f
\t
\f
\nˆ \f\t\f
…\tŒ\f\t†\f\t
ƒ    \b   \b ¦Ë   Š   \b\b\t\r …•‡ \t‹\b      \b ¯›    \b  \r\b   Š  \t\b\r     \r      \b   \b\b \b\r  \t\b\b  •‡  \t‹\b  —    \r       \f     \b \b\r       \r    \b  \t‹ \b    \t   § \r  \b \b\r  \b \b\b \b  \r\r  \b\r  \r     \b       \t  †\n\b\n  \r\b \b   \t‹  § \t   \b\b \f\b \b\r\b \r  § \t ¡   \f\n  ™˜˜ž´™˜˜‘  \b    \b\r\b  \t\b\t  – \t \t\b \b‹   ¡   •‡ \t‹\b   ” Š \b   \f\b \b\r\b \n  š›™›†  • \b\b  \t \t  \r\b\r \f˜  \f \t \b \b\r    \r \t  \b\r\b \r  \r\b •‡ \t‹\b     \b ª Œ \r    \b ‹  ³ –\bÆŽ   
¥ \r \t …‰  \b\b 
  ¡  \t      Š\r‹‹   \fÅ † ‰ Æ \t\n\r\f
…\b\r\t
‡Ž\t
ƒ    \b   ”\b ¡   \b\b\t\r …ª      ƒ    \b        \b          ƒ  \r  § ‹  \t\r– ”\b   \b  ‹   \b     \b\b  \r \r   \b  \r  \b   ˆ \b    ƒ    \b\b   § \r \r     \b \r   \r  \b‡    \b   \f   \n\b\n \t \t    \b  \b   \b    \b     \f \t   \b \b  \b    \t  \b •‡ \t‹\b  \r  ” Š \b  \f\n ¡ ¡    ‡   
ƒ    \b   
  ¡\b   \b\b \t\r …\f \t \r \b  \t  \b   ”
 ‰   \f\n \n\t \b \r‡\r  \b ƒ   \t\b \b\r  –\r    \b  \b \b\b   \b\b \b\b  \b\b  \r   \r   \b \b\r  \f  \b \t\b\b \r\b\r  \r \b \b\r\b     \b  \b\b  \b    \b    \b\r\b  \b\b  \b \t\b\b   \r \b   \b      \t \t  \b  \f\b \b\r\b \n Œ\f\nŽ  •
 \t  ”  ‰ \n Ê \b \b\b •\r  – \b\b \t  \b   ‰       \r\b \t     \r  \r 
 \b \t    ‰   
ƒ Š  ¦\b •  \b \b\b  \t \t \b  §    § \b •\b\b\t  Š \b \r\b ‰  §         \n – \b \b\b ¡   ‰  •  \b \b\b      \t \b \b  \b \b\b    \b       \b\b  \b   \b\r\b   ” ‰ \b — \b   \r  \b  \f\n  § \b   \t\b \t ¡  \b\r\b \b\r\b —\b \b\n‘\n –‹\r  \b  —\b       \b  —\b  \t    \b \r   \b \r  \f  \b \b  \b\b  \b \t‹  \b        \t   \r       \b        \b  §      ‰  ˆ\t œ‹  \b\b¥ \n\t 
ƒ\b\b  –\r \r  ”\r –\r \n\t   ‰\r ”\b   ¦\b\b ¦ ”\r \r \t  –\r   \b        §         \r  \b\b    \b   ¦  \b\r ‹\t         \b\r
\b •  §   \b\r\b ‰ \n   \t  \b          §  \b \b\b  \f\b \b\r\b \r    \r\b\b   \f\b Š\b \r  \n  \t     \b‹   \b  § \b\r  \r  \r\b\r  \r\r    ƒ\b\b  \r     \b \b\r\b    – \n\t \b \t\b  \r \r  \b\r\b \r†  \t\n\r\f
…\tƒ  ª  Ì
\b •Ì  ª §  \f\b ” ¡ \r  \n\t  \b  ª 
 ‰ \r  \n\t \b  \f\n ¡ ¡    ‡   \t
\f\t
… –        ¥   \b  \t   \r‡ \b  \r         ¦\b ‰\b ¨‰¥ §\r \t \r        š›™›  ‰  \b\b   \b\r \t‹\r \t \r \t\b\t  §\r   \b\r\b    \t\b \b\r\b \f  ™˜ž¯´™˜¯š \t     \b 
  ‰   \t  \t \n\r\b  \n\r•   \t  ™˜¯š ¨\r¥  ™˜‘¯  §   ‹\t   \b  \r ‹\r   \r\r   \r  ƒ\b \r \b  ™˜¯¬  \b      \b  \r    \b \r  ™˜¯‘   \b \t   \b   ƒ\b \r  ™˜®’  \f    \b   \r \b\b  \r   ˆ\t ±\b \b \t ‹     ‰       \t    \b\r \b  •\b\b\t  \b ‰      \b   \r \b\r \b \b  \n\b\r \b\b    ‰   \b \b   ˆ\b Š  ¦\b           \n\b \n\b\b \b\r \t\b\b \t  \b \r\b ‰   \b   \b  \t\b\b   — \r \b  \b  \r   \b ¨¥  \n\n\f Œ \t‹\t¤  ‹Ž \n\t…\f\t\f\b\f
Š“ˆ”•–”“”‹ \f  \t  \r  \t     \b – \f\r\b  ¬™  š›™›   \r  \b ™›¯   –  ‹\t \r \b\r   \r \t‹   \r  \b  \r      ƒ‹ \t†\n\n\r\n \r Œ™˜’™´‘žŽ  \t      \b\r\b       \r \b     ¢   \t  ¬››   \t ‹\t  \n      \r  \t  \t      ™˜žž \b   ™˜®š  \b   Š‹‹ \b\b\r      \t  \b    \t‹   \t     Š‹‹  \b  \r            \b\r \b\r  Š\b §\b  ‰  ” ¡\r\b Š ¡\b‹   ¦\b\b\r  \f  ¨¥ –        Š ŒƒŽ   \r   \r \b ™˜˜‘ \b \b  \t\b\n\n\r     \b \r‡  ³ ˆ‹\b \t  \t \b  \b\r\b \t‹  Š‹‹  § \b    \b \r   Š \b\r\b ‰ \t  \b  \f \b \b    ¦\b Œˆ\bŽ \t  \b \t    \r  §    \t \b        ‰ \b  ‰ \r ŒŽ  ™˜®›  \t‹  \r  \b\b  \n  ³  \b   ª \t  \t \b   \b\r   Š\b    \b \b   †\n\b’\n ‰ \n      \t \n\t \t \b   \f\n       \t ” ‰   \f\n \t \b   § ¡  \f\n  \b  \f \b    \b \t \r  \b     \n \f \b†    ††\n”  \t  \b       –‹  \r   ” 
 ‰   \n\t \b  – \t\b   \b     \t \b \n\t ‹   \b     \f\n†  \t†…\b    ¤ ‹\t  \b ‰\b §\r  – \f\r\b   ‡    – \f\n  \b \f\n œ\r \b\r \n\t  ‹ \r  \b  \r \b\r   \f\n \r\b \b\r\b \r‡  \f\rƒ\r \n \r\b \b\r\b  \r‡ 
\r \n \r\b \b\r\b  \r‡ \r€‚\r \n\b \r\b \b\r\b  \r‡ \rƒ\r
 \r\b \b\r\b  \r‡ \b‚ƒ\r  \n \r\b \b\r\b  \r‡ €\fƒ\r ˆ \n \r\b \b\r\b  \r‡ —\b\n\t
 \b\b\t –  \b\r\b      \f\n œ\r \b\r \n\t    \f\n       ƒ \f \b \b\r\b \r  \t       ¡” \r  \r   \f™›  š›™ž        ¡” \r  \b  š››ž  \t\b\f\t\b \t\t – ¡ \f\n \r\b  \b\r\b  \r‡ ‹  \b  \f\n œ\r \b\r \n\t \r\b  \r  \b \t  Œ\b\r         \b\r   \r \rŽ   \t \t\b\b    \f\b \b\r\b \r   ƒ  –   \b       ƒ \f\b \b\r\b \r   \t\b\b     \f\n \r\b \b\r\b  \r‡  \t \b     \b   –   ƒ   \f\n œ\r \b\r \n\t \r\b        \b   \b\r \t \r   \b    \b\r\b  \r‡  ‰\r \b\r\b  \r‡   \r  –  \t\b\b  \b           ˆ     ƒ \f \b \b\r\b \r       \b  \r   \f\n \r\b \b\r\b  \r‡ \b\b    \b\b     \b \f\n œ\r \b\r \n\t \r  \f\n \t   \f\n \r\b \b\r\b  \r‡ \t  \r \b   \b\r   \r \t   \r \f\b \b\r\b \r  ˆ   \f\n œ\r \b\r \n\t \b\b  \b  \t \b   \b\r \b \t     \b\b ”      ƒ    \b  \b\r   \b   \b –   \b  \t     \b  \f\n
ƒ    \f\n œ\r \b\r \n\t  \n\t    \f\n œ\r \b\r \n\t       \b  \b\r  \b\r   \r \b\r  – \b  \t \b   \r   \b   – \r    \f\n  ‹          ˆ   \t\b\b  \t  \f\n œ\r  \b\r \n\t     \f˜     \r \f™›  ˆ \b   \t \b   ­†˜  Œ—\b¤‹\b Ž \f\t
 \n\t‡\n\b \t \b    –  ™˜‘¯   \b \b\b \b   ™®ž \b   £  ™››      – \f\n  \b\b   –  \r\r     ¡  \f™ 
ƒ  ™˜®™   \t    \t     \f\n¥  Š \b  ™˜˜’  •     ‹\b        \b \b\t  \n\n\f Œ \t‹\t¤  ‹Ž ¡\b‹ Š
¡ \b – \f\r\b     ” ‰  •  ‰  §  ”   Œ\fŽ §  ”‰  Œ
ƒŽ §  ”  \f  ŒªŽ  †\n\b’\n\n\t‡\n\b ´ƒ  ¦  \b\r – \f\r\b  \b\n\n\r\b\b\t ¦  ¡ – \f\r\b  \t‡  ‡   \r   \b  – ¢\b  \r      \b  \t  \r       \b \b  ŒŽ ‰   —\b      ŒŽ   \b  \b \b   ŒŽ \f     \b  \b \r  –  \t\b\b \t    \b     ¥    \t   \f\n œ\r \b\r \n\t     \f\n ¡      \b\b\t\r  \b    \b       ƒ \f   – \f\n œ\r \b\r \n\t  \r \t \r      \f\n œ\r  \b\r \n\t \r\b   –   \b   \f\n œ\r \b\r \n\t       \b\b   \b\b       \f  \b      \f\n œ\r \b\r \n\t \r\b  \b\b      \f     \f\n œ\r \b\r \n\t \r\b ¥    \f\n œ\r \b\r \n\t  \b\b       \f  \f  \t      \t  \b      \f  \r     \f  – \f\n œ\r \b\r \n\t  \f˜  \f™› \t\b\b     ¡  \f\n   \b\r   \f\b \b\r\b \r  – \b\b      – \f\n
ƒ     ‹ \t   \f˜   \r \f™›  \t \t\b\b  \t  \f™›  –\b  –  \b\r\b   \f˜ \t         ¡ ”  \r \b     \f™› \t \b  •  \b   \f\n œ\r \b\r \n\t  \f˜ \f\n œ\r \b\r \n\t \r\b    \r‡     \f\n
ƒ    –  \r\b \b\r\b  \r‡        ³ ˆ     \f\n œ\r \b\r \n\t \r\b   ‰ – \f\n œ\r \b\r \n\t \r\b      \b      \f\n œ\r \b\r \n\t    – \f\n œ\r \b\r \n\t  \b\b   \t  ˆ – \t \t\b\b    \f™›  –\b  \b\r \r  \r\b \r  \t   \f˜    §\b\b
\r   ‡   \f\b    –ƒ •\r Œ\f–•Ž \t \b   \f‰ ” \b\r   Å\f\f \f\b \b\r \r  Š Œ\n ‰\nŽ  \n\r \n \f¯  Ÿ   \f–•   \t        \t \f‰   \f\b  Š\b\r\b ‰  Œ\fŠ‰Ž    \t  \f\n  £ \f–• \t       \fŠ‰   \fŠ‰ \b \n\b  ¡  ‰   \b     \r   \f \b  Š\b ˆ\b \b  \r \b\b\t\r  ‰  \r Œ§\t‹\t \b \r    ƒ \t‹ Œ§\t‹\t \n\n\b\b\n\b \b\n\n\r Œ‰\r\b §\t‹\t    \t\r   Œ ƒ\b\r   Ž  \r  \r \b\r\b \b  \b  Œ  \b \r  \t  \b \r \b     \f‰ ” \b \r  \b \b  \n\n \b\r\b    ­˜­ Œ‰  \f–•  \b  \b   \r‡  \r \f\n  \f‰ \r  \b   Í\b  \f–•   \b\b \r‡   \b \b\b  ° \r \r \b\b \r   \b\b \b \t   \t‹\r   \b \r        \t \r  \b Œ \b  \f‰    \t \r   ¢\b  \b     £ ¡ ‰  Œ\t  \f\b ‰  ¡\b ¡\b \rŽ  \b\t\n Œ       \t \n\b ™\t\n\n  \n\n \b\n\t ‰\r\r      \n\n\b\t  \t \b  \r  §\t  \t   •\r\b Š \r Œ  •ŠŽ   \r       \b    ­˜­ Œ‰ ‹     \f–• \b \r  \b  \t \b   \b \b   \r             \t \b\r\b       \f˜          \f–• \b    \b  \r ƒ\r  \r        \t  \r \b \r   \b \t \r \r     ¡\r \b   ƒ\b      \r  \r \b\b \r \t  \r\b\b \b\r\b \b \b  \b   ­˜­  \b   \f\n  \f‰ \b\r ” \t  ”\b\r  \r \t \r\b  \r     \b\r   \b \n\n \b\r ”\b\r      \t \r    \b \r     \b\b \b\r\b —\b  \r‡\r    ˆ\b ‰ \r \b\b  \f       \b  \b  \b  \b \b \b   \b   \r‡\r  \b    \r \b\b  \f  \f‰ ” \b\r \r     ƒ\r   \b   ¡\r  \t  \b \b\b      •\r \t ¢   \b  \t\n\r\f\n\t\f\n\r\f ” §\t‹\t \r  \b\b \r  \t\n  œ ˆ\b ¡‹ \b ˆ  ‡  ¡\r \r   \f \b Š\b \r ˆ \b ‰ \r   \b \b  \b\r\b   ƒ\b  \r  \r       \b\b   \t ƒ   \b \r  ”\b\r  \b\r\b    \b  †\n\n   \b\b \r \b\r  \b \r \t « Œ      ¢  \fŠ‰ \b \n\b   –\t      \b  ³ ¡\r    \t \t   \r \r  \b    \r \t \b \b\b     \b \b   \t \r\b    \r\b   \b\b   ™™  ¡\r     \b\b   \f\n
ƒ  Œ \b  ­˜­ Ž    \f \fŠ‰ \b \n\b  \f‰ \b\r ” \r  ‹\r \b  \b   \t     \n\r  \f\n
ƒ    \b\b   \t  ƒ \r    ¢ 
ƒ    \b 
ƒ\r   \f–•         \t  \r\b \r  \t  \b\r   \b    \b   ‚ \b \b \r   \t —   \b   \r \f–•         \b\t  \b‹   \f     \b   \f•– \r  ‹ \r   \b    \b\b  \r\b    \t      •\r  – \b\b    \t\b\b ‹      \b  \r      \f     \b    \f    \b     \f–•     \b  \b‡  \r\b      „        \b\r \b  \t  \b  \b   \f ¢   \b   \b   \f–•     ¢  \b\b \b \r\b    \b    \r    \f–•     \f–•   \b \t   „  \b  ‹ \b \r  ™\r–™   \n  \n\b ”  ‹\t   \r \t  ƒ  \t \t      \t \b  \b ‹     ‹ \t \r\r   \b  —  \t‹  —  Œ   \b    ‰ Ž   \b       Œª ‰£ ‹  ¤\r \r Ž  ‰ Œ ‹£ ‹¤— ‹ —Ž  ‘‡
‘‰„„      \t‹ \r \r \b  \f˜  \b       \f–•   \n\b \r  \n\n\f \tš›œžŸžŽŽŽ Œ \t‹\t¤  ‹Ž  ¡• \f\b    –ƒ •\r Œ\f–•Ž ˆ \r  \r \b\r\b \b  \b   ¡ \b\n\n\n\r\b\b\b \b\n\t\n\n\r §\t‹\t ”  \r   \b \b °\r  \b \r  \b\n\n\n\r\b\b\b §\t‹\t ” •\r\b \r  \r  ƒ \t‹  \b\n\n\n\r\b\b\b ¡ ³\f ‰ \n ‰       \b\t\n\b\n\b \r €\n \r¡\r ‰ ª\n ‹ \f\b    –ƒ •\r Œ\f–•Ž  \r ‰ ª\n  \nœ §\t   \t \r\b   \r ‰\r\b  §\t‹\t ” \f\b    ƒ •\r Œ\f–•Ž     \b\r  \b\n\n\n\r\b\b\b \b\n\t\n\n\r ƒ\b\n\n\r \b\b\n\n\r\b\b  ’       Î Î  \t\b\f \r\f\t\f\f‰ \f
\b\f\t\n‚ \t\f\n\f
\f\b\f\n\r\f \b\n\b\f\n  ™’›™’´™’›š™  ¢\b\b” š¬¯›´š¯ž¯   ™› ™›š˜­š›™››ž¬¬ž¯€ €…   \r\b \b Œ–\b ‰ ªŽ  ‰
  \r  \r\b \b   œ\r ” \b Š¡ Œ‰  \f\b \n ‰\nŽ  \b… ‰
  \r  \n\b\n\b\b Œ¡  £  §Ž  … ‰
  \r  \b  \r \b\n   Î  ¯  Š ´” Á   ’   ‡\r\b
\f\t\f\b\f\f\n\f\f\f\n˜

\t˜ \r –     ¡\b\r\b\n        ‡\b\b Œ±   ¦ \b\n\n\r\b\b\t \t   ” –§  \b
\n ‰‹ ³ ”—‹ ¦      \b   \b    €
\n\b\n\n\n\r ¬’’´¬’˜  \f\b\r\t\f…\n\r\f\b\f\n \n\r\f\b\n  •   \r\r ŒŽ  \n‘\n ŒŽ   \r \b   ˆ  \r      \t\b  \n‘\n   \t  \b  Š Á   ’    \b\b\f\n\f\r\f
\f\t\f\n\t   ¡Ä\r \b‹ \b \r  \b \r      ™›¬¬´™›¬‘  §\t    \r   \b \b\r  \b\r\b   \t\b  ‰\n\b\n \t   \b\r     \b \r \t\r –  …†     \b Š  \b\b \r   \nŠ •‹ \t  \b £ §\t‹\t   ‰ €”\n\n\n\b\n–\t\n\n\n •‹ \t      …‡†  \b ³³  – \r \b\t\n\b\n\b\b     \r ƒ  ¡   ³   \r \b \b \r     ¬’®´¬®¬  ¡\b\r \b\r     \n
\r š®¯´¬›¬   ’   \f\b\t\b\b†
\f\n\t\t\n\f  \b   °   \r   §‹  ‰    \b\b \r\r \b  \b    \b „\b       ƒ\b  \t ” \n\n‘   ˜›› \b ¡‡  •   ‰\b   \b\b  \b‹    ‰\t¡‹  Œ™˜‘¯Ž \b  \t   ƒ  \b \b\b \r   \r    ‹  Š –\b \r‡    \r       \b\b \b‹ \b \t\b\b    \r\b \r  –    ƒ  ¦\r  \t      \r \r   \b\r \t‰\n\b\b\t\n\b          \t \b\n\b\n  †\n\b\n  \r  \t ¢  Š‹  \b\b ƒ \t  \t    \r \r  ‰\r  \r \t  \b  \r  \r  \b\n\b\n ¦ \b\n\b\n   \b   \t  \n\n\b – \b   \r \b\b    \b       \b \b \b\r\b   \r   \b £   •\b  – \r   ƒ  \t  \b \t  \b ‹ \t  \b    \r\b\b\b \b\b   ‰\n   \b \b  ‡\n\n\b    \b  \b\r\b\b     \t \n\n\b   \t\b\b  \t\r – \r   …\r‡  \b  ¿À     \r‡ \b\b † Œ  ™’Ž    \b       \r \b\b   \b\b\r \b\b    ‹Æ \n\r  \r  \t\b \b\b   \r  \b\t\b\n\t\n Œ  \n\b \t\b\n\t\n Ž \t     ‚\n\r   ‹  \b\r\b \b\b   \b \t\b\b Œ”Ñ\b ™˜®‘ §\t‹\t ™˜‘‘Ž   \r    \b  \b\r\b \r          …     \b †    ¹ \f   \r \b           \r \b  \b \r    –  \b\r   \t    \b   \b\r\b \b\b \r \t \b   \b  \b   ”ˆ\n     \b  ‰\n\b\n  \b\r \b \b   \t\r ¡ – ‹‹  Š Á  ”Ñ\b \tÑ \n   \r \b \n  \r\n \b\t\r£\b\t ™´¬’›  §\t‹\t ” –  \r\b\b\r\b   \b \r    \b  †\n\b’\n\n \t‡\n\b ¬´š›  ‰ ‰ ‹‹ – § ‰ ”Ñ\b ¡ \b\b  ‰—à §Ñ \b\b \r  \t    \r\r  \r \b\r  \b š‘™´¬››   ’    \n \n ‰\t ” \n ‹\b §\r‹\r \b  \n\b†\n\n\r ¯®´™›™  ‹ – ¢    šš´šž  ” œ ±\r ³œ ˆ \r\b   ‹\t    \b\n ’š¯´’š‘  §\t‹\t ” – \r\b   \r \r    \b\n\n\r\b\b\t ’ž™´’¯¯  §\t‹\t ” –     \b\r\b \b      †\n\b’\n\n\t‡\n\b ¯ž¬´¯’®   ³ ‡\b\t\t\b…\n –\r ³   ¦ ³ €\n\n‡\b\t\t Š—\r \f\b \n ¡\b  \f\t\r    \r   ƒ  \b     \b  ‹\t   \r   ¦\b  \f  \b  \t \r­€  \t   \b Œ§\t‹\t \t\b \r\r  \b \r \b       – \t\b \b  \b   \r    \r‡  \b\r  –    š‘ž˜  \t    ¯š’› \t   \b‡ \r \t     ™˜®‘´š›™›       \b \t ‘˜  ™’ž    \b  §\t  \r\r   \b  \t ƒ     \b  \r   ™›  \b   ®›  \t\b     ™š ’         \b  ™¯ \r  \r\b \r Œ§\t‹\t ™˜˜šŽ  ‰        ™š ’    \t\b   ™®¯¬ \t \b \r\b \b  \t        \b\b\t   \b\r  \b\r      \b      \b  §\t œ ”    Ž       ™› \r  …   \bƒ       \r\b †  –    \b\r   \r\b      ™˜‘› \b\r\b \b   \r ‰ \b\r  \b\r    \f  \b\r  \n ‰  Š—\r  ˆ\b     \r \b       \r \r      \b\b  ‹\t\b\r  \b\b  \r  
   ¦\b   \t\b  ‹\b  \t             \b   \r    \t \r \b  \b\b ¢\r    \r  Š\f \r   Š—\r \r € \b  ™˜˜˜      \t\b \b Œ‹ š›™›Ž  ” ±\r¥      \r ‹\t       \b  \t    \t           \b     œ\r ”  ³\r ¦   \b   ‹\b \b \t\b   \t \r      \b \b \r\r  \r  \b \r   – \r \r  ¢ \t \t\b     \b \b\b\r \t  \b\r  \b\b   • ƒ\b      \b \r\b \r\b \t   \r   Œª\b\r‹ ³ \t  \b   \b\r Š  –\b ³¦ ”\r  \b\b \b‹  \r \t  \t¹ \r†\n\n\r” ™´™’  §‹ ”‰  ”
\b\b Š ‰  ª ˆ §\r ‰Š \b\b    \b \b‡ \b  \r  \b  \b\b ™™š˜´™™¬¬  ª\b\r‹  ³ \n\n\n\r\n\b\b\b\n ¿\n\n‰¡  ‰ ˆ  ”\b\b –ŏ \n \n ‰\r ¡\b\b\r  ‰\t¡‹ \n  ‰\b \r\b  \b   \b \b\n\b ‡\t ™´š›  ¦\r §œ  ‰œ §\r   §– ¦ ³ \b\n\b\n   \b   \r    †\n\b\n \b\b\t ‡\b\b ™™’¬´™™’˜  —     \n \b \r  \n\r š›™›   \n†\n\b˜ €\r\t \b\r     \b  \n \n\b\n\r\n”\n •     \t      \b \r  \n\b \b\b  \t ‰      \t   \t\b ƒ\b\b  ƒ\b\b   \r \r  –       \t  ‰      \t      •    \t  —  ‰ \b   \t \r\b  \r€ Ž  ­\t\f\f – \b\b\r  \r\b   \r  \n Ò \b \r  \b‹ \b  \b\b \b  ‹ \t    ¦ Ò   \b  \r  ‹  – \t\b\b\r  \r    \b \b   \b  ‰  ‹\t   ™››   \b‹ \b  \b \r         ‹  –    \b\b  \b \r \r   \b\b \t\b\b  ƒ\b  \n\b\r   \b\b        \r        \r   \b \b\b   \r  \r   \b  \b \r  –       \r  ¡\b \t      \t  \b\t  \b    \b\b  \t\b   \b  \b\b \t\b   Ì\b  Ì  Ì\b  \bÌ  –  \b‡  \b\r   –  \b \b \r  — \b\r\b   \b  \t \b   ‹\t   –  ‹\r \b  \b \t \r‡ \r  ‹\r   \t   \r\b\b      \b ™˜®›  –      \b  \b\r\b \t      \r  ¡         \b \b\t       \b  \b\t\r \b   –      \r\b           \r \b   ‹ \r    •\r Œ ƒ\b   ‹\t \b    \bŽ        \b Œ    \bŽ   \r‡   \b \r      \f      ƒ\b \b\r\b   \r  – \b  ‹ \r   \b\b    \n \n
\n\n†\n\n\r\b‚” ¿Š”À  \b  \b   \b\b   \b   ƒ    Ì\b \b  \rÌ    \t ƒ ‹\r   \r  ‡  •\r \b\r    \b  \b ‹\r    \b     \b\r \r\b \b\r \b ‹\t    \r\r   \b \t\b     \r \t   \b    \r  – \b       Š” \r  \n \b  \b   \b   \b    \b   \r  \b\b  – \t \t     Ì\b\t \bÌ       \n ” §\t‹\t    \t\bÒ \b\r \b\r  \b¢\b   \r  Ì   Ì  \r\f\t\t\t\f\b \t€\t •\r\b       – €\n\n\b\n\n”\n \n\r Œ\t  \r\b  \r   €\n\b\n\n\r\b \n\b\n \t \b  \b  ™˜‘¯    ‹      \r\b    – \b \r  Ì\bÌ  Ì\rÌ \t     \b””\n \n   \n­\n\n \n”\n Œ\fˆŽ  \b\n\n\r\b\n\b  \b\b\b \r\b  \r   \r‹\r \b \r\n”\n– \n\n Œ     \r  ¡ š››™Ž \t \t\b \t      \t\b\t –  \b\r    \r\r    \b \b  Š \r\b      —    \f \b  \b\b          ŒÌ\r\b \b  \b\rÌŽ  \b\b  ŒÌ\r      ÌŽ   \b \r \t \t\b\b \b     \r  – \b\b  ‹    ‹    \n\b‘ \b\b  \r\b\b\b  \b\b \r  \fˆ ‰  ” ‰ \b \r­\b­ ƒ € ¡ ” §  \n\n\b \b\b  \r\b\b\b  \b\b  \fˆ ‰  ” \n\r \b \r­\b­  € ¡ ”  —        \b  –        \b \b  \t        \b\r  –      \r\b       \b\r    \b   –  \b   \t  \t  \t‹   \b\b   \f     ƒ\r \b    \r\b     \t   ˆ š››¯  \r \t‹ \t \r‡   €\n\n\b\n\n”\n \n\r  Ô ‰  – \t    \b  Œ  Ž  \r   \fˆ   \b    ‰ š››®  –   \b  \t \b    \r ¢\b    \fˆÒ \b \r    š \n¤‘\n\b \b \r ‹­\b€ Ž  ž \n \b\t \n\r\b\n\b \b \r ‹­€ Ž  ¥ ¤ \b \r  ‹­€ Ž  ˆ š››®  – \n\b †\b\n\n\r \b \b \r\b  \r     ‰ \n  ” š››®  \n   \b \r  ‰    \b   \r  ‚\b\n\n¦\n \b \r ‹­\t ­ € \t \b   ™¯› \b\r  ¬¯     \b \r\b\b \b  \r\b   \n\r š››‘  – \b\n\n\r\b \n\b\n\b \b \b\b\b \r\b   \r  ˆ \n  ˆ š››‘  – \n\b\b¦ ‡\n \b\b\n\n\r§ \b \t‹\r     \b\b\b \r\b  \r  ‰ \n  ³ š››˜  – \b\b\n \n\r\b\n\b\n \b \b\b\b \r\b   \r  \n  • š››˜  – \b ””\n\n   \fˆ \b\b \r‡ \r  \r \b\b    \t  Ò   –  \b       ‰\b  \r \r    \r   \b     \r  \b Ì•\r\b ‰\b Ì       \b\r    š››˜  \n  \b \r   ª \b  \r ‹­\t€ Ž      \fˆ \b ””\n\n \r\b ‰\b  \r \t     \b \b\b \r\b  \r  \b\r   š›   ‰\r    \b    \r\b   \r  ˆ š››˜  – \b\n\n\r\b \n\n    \b\n\n\r\b\n\r \b \b\b\b \r\b   \r  \n  \b\b\b    \r \r  \r\b   \f \r\r\b  \t  \b\b\b \b\r\b \b    \t  \r  ƒ   \b       \b   \b   \r\b    \r  \b     \b \b\r\b \b       \t \b  \b\b \b\b  \f   ƒ \r    \b\r   \fˆ \b””\n\n \r\b \b \r     \t \b  \b  \r   \b\b \r\b \r  Š    š›™›   \r  \t \r\b  \b\b   \r\b  \t \b \r\r    \r\b \b \t \b\b   \b \r  ‹ \b   \b\b \b\r \t  \b\t \b  \r\f\t\t\b\f \f˜  \n\r š›™› \r  \b\r  \b\b   \t\b  –   ƒ\b\b   \r   \b  \b  \r \r  – \f\n \b\b  \r\b\b\b  \b\b \r  \fˆ ‰  ” • \b \r­\b­‡\b € ¡ ”   —     \r   \b    š››˜  \b  \t   \r\b   \b \r \t     \b  – \n†\n\b˜
\r \r\b     ˆ\b ’› \b\r  š™    \t \r         ‹\r  \b         \f   \t \r    \r  \b  ˆ  \b    Š” †\n\n\b\b\b\t\b \b\t\n\n\r\b”\b Œ  \t   \n\n
\n Ž  \n    \t \t\b\r      \n\n\b\n\r\n ”\n –     \b  \r \b\b      \t   \b  \r \t \r       ‰    —      \b
\r\b   ‰Ò \t‹\r \b\r\r  \t \r  \t       \r \t‹   \r  ‰   \b   –     ‰\r    \b\b\t ¡ Š Œˆ\t ±\bŽ ‰ \b\b Œ¡ Ž ‹ ‡ Œ‰ \nŽ \r \b\b Œ‰\nŽ ”  Œª Ž  – ‰ ŒŽ  – ‰\r   \t   \r         \b  •     \b\b \b   \n   \b       ‰   \t\b\b     \t\b\b \r 
   \b \r \b\b   \b \r \t    \t\b  ‚\b\t\t\t\f\t \b        \r\r  ‰         \b \b \t  ‰  – \b\b \t\r    \b \b     \r\b   –    ƒ    \b \b   ‰ \t\b\b   \t‹   \b  \r\b      \b  •\f\f – ‰ \t\b\b    \r  \b    \t   – ‰       \b \t‹     \r \t‹\r   \b\b Œ\b \b  \b\bŽ  \n  \r\b    \b  \b \b\r  ƒ  – ‰    \t \r  \b \r  ƒ\b ƒ\b  \b\r   \t‹  \b         \b     – ‰ \b \b ‹   \t \r   \r\b \b \b\b    \b     ‘ \r\b   – ‰ \t\b\b \b ‹  \b \b‹ \t  \r‡ \r   \b‹  \b \r  \r   ƒ     \b  \f – ‰ \t\b\b \t‹ \t \b \b\r\b    \r\b \b \t     \r    \r  \b\r  \b\b \b\b     \b \b \t        —   – ‰ \t\b\b \t‹   \b  \b   \r       \r Œ\r\b   \bŽ  \r\b  \r      \t    \b  \r\b \b \b  ƒ  – ‰ \t\b\b    \r  \t ‹\r \r          \b     \b  – \t\b\b \b \r \t   \r  \b      \t\f – ‰ \t\b\b \b \b  \t\b\b \b \b\b ƒ  \t \b  \b\r   ƒ      – ‰ \t\b\b ‹   \t  \r \r  Š” ˆ\b • ¡  \t\b\b \b ‹  \r\r \r \t   \r   Š” ‹\r  \t     \r\b   – ‰ \t\b\b ‹    \b  \r   \r \r  \r \b \r\b   \b      \b\r\r    \b \b \b \b     \r  – \t\b\b  ƒ\b \b  \r\r \b\r\r \t \b     \r \t \b ŒÌÌ   \b Ì\b\rÌ \r   Ì\b\t \bÌ      Ì\bÌ  Ž    ÌÌ    Ì\b  \bÌ \t\b\b \b  \r  – ‰ \t\b\b \b \t‹     \b\r    \t     \f \r   Ì  Ì  \b\r \b‹   — \b\b   \b \t\b Œ\b\r  \b\b  \t     \b  \r\r  \b\b   Ž  \b\r  \b \b  \b    \b\r\b   Š  \b     \b \b\r  ¢\b \b     \n   \r\b  Ò  \b \n \b    \b \b \b    \t      ƒ\b\b   \r \r  •\r\b        \b    – \b\b\r  \r 
\b\r  \t ‰ \t         \t\b  – ‰     \b\b  \t   \r     Ì  Ì  †…\r\f €\n \b\n\n\r\b\n\b\n Œ¦ ª£  ¤ \rŽ —      – \b\r\b ‰ \n   \b \r \r  \r\b \b\b\r \b\b   Š\b\r \r    ‰ ƒ\r ªœ  … \b  \t\b † – \t — \r \t  \f\b ‰  •\r\b
  Œ\f‰•
Ž   \t  \t     Š       \t  ƒ\b\b    \t \r    –   \r \t …‰ † – ‰\n \r  ‹ \b\r   Š \n   •  ¦ ª‹  –  \b \b        ª‹¥   \b\r  \t\b  \r\b  \t  \r \t     \t  \f \t \t\b   \t   \b\b\r \r    – \b ª\b\r  \t ‰\b\b ‰  ƒ\b\b ‹  \t\b    ‡\b    \r \r \t  \t‹     \b ‡  – ‹ ‹ \f‰•
 \t \b ¦  Š \t \r \t   \b\r \r \r \r\b   ‰ \b …\f    \b \b\r  \b\r† …– \b       \r  \r† …\n  ”ˆ\n \r  \r†  …\b\b \r   \r \b  † \n \b \r \t  \b\r     \t  \r \r\b     ˆ \n  ‰\b  \b\r  \t   \r  – \b  ‰\b Œ‰”\n\n‰ ‰ \b\r  \b\r  Š\b\b\b ”Ž ”\r \b\r£ ³ ‰ Œ\r ‰ Ž ¦ –\r \n\t£ \n ¡\r\b Œ§ Ž \n\bƒ\b ¡‡  \r
\b \b\r£ ‰ ‰ Œ \n‹Ž ‰\n •\b\b\t£  ³Ä  ” Œ Š\bŽ §   –\t   \r \b  \t  \b   ƒ\b\b 
\r   \f\b –\b \n\t  \b  ƒ     \f˜  – \b ¢ \t    ª‹ § ¡‹ ‹‹ €  \b \b\b      – \b   \t  \b  \b\r\b    \b \t \r \tÆ ¡  \b \t  ƒ ¥ \b \r    \n\b‹ •‹  –   \r \t\b\b  …§\r \b \r  \r\r \b †      \r    \r  š    ­­€ ‰\n  \b  \n\b‹  \b   ƒ\b\b   ƒ\b \b \r   …  \r ‰ † ­€… Œ™¤ Ž \b\f\n\f\t\b ‰\n \r    Š \n   •  Š\t ªœ  ¡ – ³  Ÿ\b‹  ‰\b   ‰\n \b \r  ¡ – ³  Ÿ\b‹  —     \f \r  \r \b     \f \b\r\b ‰  \t \b  – \b\b   \b       ™¯  \r   —  \t  \t  \r   ™‘  \f ¡\b ¡ \r  § Œ\fŽ š››‘ \t  ®› \b\r         \r  \r      £   \t ¢\b \b\b \r   \r       \f   ‰   –\b\r  ¦      \b\r      \t      ¢\b   \r\b \r \t   ‹ \b  ™¯ ‰ š›™›     ƒ  \t \b      \r\b \r    \t \b  \b\b \b\b\t    ‹   \f \b\r\b ‰  \t      Œ   €Ž \t     \f  \f  ¡\b ¡ –  – \b\b\t\r \b\r \t \b   ¬  š›™›š›™¬       ƒ    \f \b\r\b ‰  ”—
 Œ
 ¡ \f   \f  ¡\b ¡Ž ¡\b   ³ˆ‹‹ Œ\n ¡  –Ž \b \b ± Œ¡ \f  \f ¡\b ¡Ž   \n Œ\n ¡ \f  \f ¡\b ¡Ž \n \n‹ ª Œ\n ¡  \bŽ š Œ\b\b\b¤ Ž \t\b\b \b\r      \r\b \r  \f \b\r\b ‰  ‡\n\r\t ” 
 ‡ \n  ª \n‡  ³  ‰\b \n  \n\r   §— \n ³ˆ‹‹ Ÿ   ±  Š   Š\b\b ‰    ±  Ž    ¦ \b  ‹Æ ‰\b   ¢ —     \r   \r   \b   \t   \r\b      – \b\r  ‹\r \b  \r  \b\b  \r Œ    \t\b \rŽ     \f \b\b \b    \b \b\r     …‡\r \t\b  \r†  \n   \r\b \b       \b\r š™  Œ\b\b    ªŽ   ®š \b  \r  – \f  ˆ‹    Š \r    \r  \b\b\t   ¨  ¥ \t   ¦\b\r Œ ™Ž  \f  \r   \r  \b\b   \b  \r \r   ƒ  ”\t‹ Š £ \b\r \r    ™˜˜¬      \b  \b\b \t  \r    š››˜ Œ  ¬›Ž  –  \r     \b\r\b  ƒ\b  ¨\b\r  ¦\b¥ Œ šŽ ¨¡\b ¡  ¡ ¡¥ Œ ¬Ž \t     ¨\n\b ‰\b ‰  ‰\b¥ Œ žŽ \t  \r   \r  ¨\n‡\r ¥ Œ ¯Ž  – ƒ  \t  \b   \r \t\b\b  \b‡ \t   £  ƒ\b \t \t\b\b    \b    \r  ¨ \b¥  \b \b    \r   \t\b Œ  ®®Ž¹ \b\b —    ¨•\r  §¥ Œ ’Ž  ¨•\r\b   ‰ •¥ Œ ®Ž  §\t    \r       \n\b §ƒ\b¥ \b \b  Œ™˜’žŽ \t      \r    \b\b\r   \b    
\b ¥  \b\b  \b\r\b \t\n\n ‹  Œ™˜¯ž’®Ž£   \t \t —\n\r\t\n\t\t\n\n  Œ™˜¯žŽ \t \t  \b  ŠÆ ¨\t\r
\b •\r¥ Œ ‘Ž \b \b  \b    \t  ƒ\r ‡\n \b\r    \b\r  \b  \r    ƒ  Æ ¡ \r \n \b\n \t ‹  \r\t  \b\t  \t\b   \b  ¨– •\r\b •\r¥ Œ ˜Ž  \b\b\r     \t\b \r\b \b \r\b\r     ƒ \r   ª \b\r\b  \r  \b \b\b\r     ¨‰\r\r  •¥ Œ ™›Ž     \b\b\b \r          \b\r \t   ‹\b\b£   \b  \b \r    \r \r   \b   \r     – \b\b\t  \t ƒ  \r  \t\b £   \b‹ \r   \t     \b\r  \r \r\n\t\n ‰\n    \b‹\b       ‰ \r        \b Æ ¦   \r         \t     \b\b  \r \t       \t\b      \b\r \t \b   •\r\b  ”  Š  \f\b Œ•”Š\fŽ   \n   Š •\r ¥ \n–
   \t   \b   \b      \b\b     \b     …\r† Œ  ™’ ¯š ™’˜Ž     \b   ƒ …\r  \b† Œ  ‘žŽ£  \b    š® \t    \t … \r    \r†Æ ‰   \b \b  ƒ\b \r \b  \b\b\r    Œ  ™›¬Ž          \r …   \t† Œ ™’™®Ž \r  \b\b \b  \b  \b     –  \b \b\b \b  ƒ\b    \n\b \b \b \t  \t \r‡  \b \r Œ ˜®Ž     \b  \b  \t \r    \n \t\b  \b  \r\b\b   \t\b   \r    ˜\n\b \n \b  \n\n\b\t\n Œ  ˜›Ž    \b\b    \b\b — \t  \b  \b\r  \b£   \t\b\b \b   \t \t\b  ƒ     \n\n\b\t\n \r Œ  ž˜Ž  \t Œ  ˜™Ž¹   ‹   \tÆ – \r Š\b\r  \b  \r     \b  \t  \r\b ƒ  \r\b \b\r        ¡
\b\b¥ Œ™˜˜®Ž \b\n\r \n‡ – \t \t  \b \b\b   ƒ     \t   \b\n\n\r \t\n\r\f\tŽ
…\n\r\fŒ\t
™
\n\f
\f


‰\f™–”“”™ ˆš›˜“˜ˆ”œ“–ˆ˜œš˜•™ ‡ ™“šœ‚\n\t™
\t\r…\n\bƒ\t
\f™‡\t\b\f…ž–”™ Ž   \t\n   \t   \b   \b    \b \b\r   \t  \b   ‡\r Æ • \r       \r  ™ž     \t  \r\b£     \b \b    \b  \b  \b\r    ƒ  — \r  —\r   ¨\b¥  ¨¥  – ‹ \t \b     ƒ\r‡   ƒ \t    \t   ¬™ ³\b  š™ ˆ š›™›   \b Š  
\r \t    \b\r  \f˜ \t \b  —  –   \t  \b  \r     ¦\b\b –  ‹  \t‹ \b \b   \b  \b\r  §\t   ‹ \t      \b      ‡     \b\r  ‰  \t\b   — \r     \b  \b\r    \b   ƒ \r  \r\b \b\r  \r\fŽ
…\n\r\f\r\t‚
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\b\r\f™ \n\f \r\f™–”“”™ ˆš›˜”˜››“ˆ–˜›ˆ“˜Ÿ™‡ ™ –š“‚\b™ ™“–Ÿ™‡\t\n
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\bŠ ³ Š\b  š›™š \r­€ \n\f\t\n\t\f‡\n‡\n\r \n\r Š—\r  š›™¬ \r­\b­€ ¯„\n\f\t\n\t\f\b ¿\t  \r Š\b\r  \n\b \b\r  \b Ÿ\b\rÀ ³\b´™ \n\r \b  \n\b \b ¬›™ž¤ \r  \bš›™ž \r­€ \n\f\t\n\b\b\t\fŠ“”‹ \n\r Š\r‹‹   Š\r‹‹ ‚\b \n\b ‹ £ \b\r\b‹¤‹      © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. the enigma of calonectria species occurring on leaves of Ilex aquifolium in europe christian lechat , Pedro w. crous and Johannes z. groenewald ascoFrance, 64 route de chizé, F-79360, villiers en Bois, France; corresponding author e-mail: lechat@ascofrance.fr cBs-Knaw Fungal Biodiversity centre, uppsalalaan 8, 3584 ct utrecht, the netherlands abstract: species of calonectria are common saprobes and plant pathogens on a wide range of hosts occurring in subtropical to tropical regions of the world. the aim of the present study was to resolve the status of new calonectria collections obtained on Ilex leaves from France. Based on dna sequence data of their -tubulin and histone gene regions, as well as morphology, the new collections matched the ex-type strain of cylindrocladium ilicicola . on the host and in culture, yellow to brownish-yellow perithecia were observed that did not strain red in 3 % Koh. Based on these results, c. ilicicola and its purported teleomorph, ca. pyrochroa , were shown to represent two distinct species, as the latter has bright red perithecia that strain purple in Koh. a new combination, ca. lauri , based on tetracytum lauri, is subsequently proposed for c. ilicicola . calonectria lauri is distinct from ca. ilicicola , a pathogen commonly associated with cylindrocladium black rot of peanut. Finally, ca. canadiana is proposed as new name for cy. canadiense , which is a nursery pathogen involved with root rot of several tree genera in quebec, canada. article info: submitted: 1 september 2010; accepted: 29 september 2010; Published: 2 november 2010. nt roduc tI species of calonectria are members of nectriaceae hypocreales ascomycetes ) (lombard 2010a–c). nectriaceae are characterised by having uniloculate, orange to purple, super�cial ascomata (rossman et al. 1999). calonectria is easily distinguished from other members of the family based on its cylindrocladium anamorphs. Formerly cylindrocladium also included members of cylindrocladiella , a genus that accommodates cylindrocladium -like species with small conidia (Boesewinkel 1982, victor et al . 1998) and nectricladiella teleomorphs (schoch et al . 2000). other morphologically similar genera that have also since been separated from this complex include Xenocylindrocladium (decock et al . 1997), curvicladiella (crous et al . 2006a) and dematiocladium (crous et al . 2005). Following the approach of crous et al. (2006b, 2008, 2009a, b) with other fungal groups, lombard et al. (2009, 2010a–d) chose to use the older calonectria name for the genus, irrespective whether the teleomorph or cylindrocladium anamorph, unnamed microconidial, megaconidial, or chlamydospore-like synanamorph was observed. all taxa are since accommodated in calonectria , which is a monophyletic genus (lombard et al . 2010a–c). most species of calonectria occur commonly in soil, especially in subtropical to tropical regions of the world. although the genus was originally regarded as saprobic (graves 1915), taxa have since been proven to be important plant pathogens, associated with a wide host range of plants, causing disease symptoms ranging from leaf spots to stem cankers, damping off, cutting rot, root and fruit rot (crous et al 2004b, 2006a, lombard et al . 2009, 2010a, d). major diseases attributed to calonectria infections include cylindrocladium black rot of arachis hypogea (peanut), and red crown rot of glycine max (soybean) (crous et al . 1993, wright et al. 2010), as well as root rot and leaf diseases of numerous diverse hosts (crous et al 2004b, 2006a). over the past few years, a species of calonectria was collected from leaves of Ilex aquifolium in France. Presently four species of calonectria have been described from Ilex ( aquifoliaceae ), namely calonectria morganii on Ilex paraguayensis in argentina, and Ilex vomitoria in Florida (usa); calonectria avesiculata on Ilex spp. in georgia and Florida (usa), cylindrocladium ilicicola (as calonectria pyrochroa ) on Ilex aquifolium on clare Island (Ireland), and calonectria spathulata on Ilex paraguariensis in Key words: hypocreales calonectria cylindrocladium Ilex aquifolium tuB hIs systematics Ima Fu gus volu me 1 o 2: 101–108   echat et al  Brazil (crous 2002). hawksworth & sivanesan (1976) also reported a calonectria species on Ilex aquifolium from slapton, south devon, england, which appears to be undescribed, with ascospores 3-septate, 14–22 ×3–4 µm. the collection obtained from France and treated in this study, is morphologically distinct from taxa presently reported from Ilex In recent years there have been several revisions focused on either calonectria or its anamorph genus, cylindrocladium (rossman 1979, Peerally 1991, crous & wing�eld 1994, crous 2002). the �rst attempt to provide a molecular phylogeny of the genus was that of schoch et al . (2001) based on -tubulin dna sequences. this gene region, however, proved insuf�ciently variable to reliably distinguish all species complexes in the genus (Kang et al . 2001a, b, henricot & culham 2002, crous et al . 2004b, 2006a). since then, a concerted effort has been made to generate a multi-gene phylogeny for taxa in the genus, and identify the best suited gene for species delimitation (lombard et al . 2009, 2010a–d). Based on these �ndings, a combination of -tubulin dna sequence data, supplemented with either calmodulin or elongation factor 1- , proved the most effective in distinguishing all known taxa. the aim of the present study was to compare the new collections on Ilex from France to all species known in the genus, using morphology and dna sequence analysis of their -tubulin and histone gene regions in order to determine if it represented a novel taxon. ate Ia ls an d met hods solates single ascospore isolates were obtained from leaves of Ilex aquifolium as explained in crous & wing�eld (1994). Isolates were incubated on plates of 2 % malt extract agar (mea), 2 % potato-dextrose agar (Pda) and oatmeal agar (oa) (crous et al. 2009c) for 7 d at 25 c under continuous near-uv light, to promote sporulation. reference strains are maintained in the cBs-Knaw Fungal Biodiversity centre (cBs) utrecht, the netherlands. na isolation, ampli�cation and analyses genomic dna was isolated from fungal mycelium grown on mea, using the ultracleantm microbial dna Isolation Kit (moBio laboratories, Inc., solana Beach, ca, usa) according to the manufacturer’s protocol. two loci were ampli�ed and sequenced as explained in crous et al . (2004b) and lombard et al . (2010c), namely, part of the β-tubulin gene (tuB), ampli�ed with primers t1 (o’donnell & cigelnik 1997) and cyltuB1r (crous et al . 2004b); and part of the histone h3 gene (hIs) using primers cylh3F and cylh3r (crous et al 2004b). Part of the nuclear rdna operon spanning the 3’ end of the 18s nrrna gene (ssu), the �rst internal transcribed spacer (Its1), the 5.8s nrrna gene, the second Its region (Its2) and the 5’ end of the 28s nrrna gene (lsu) was ampli�ed for some isolates as explained in lombard et al . (2010c). the generated sequences were compared with other fungal dna sequences from ncBI’s genBank sequence database using a blastn search; tuB sequences with high similarity were added to the alignment and the result of sequences of the other loci were used as con�rmation (not shown). the additional genBank sequences were manually aligned using sequence alignment editor v. 2.0a11 (rambaut 2002). Phylogenetic analyses of the aligned sequence data were performed using PauP (Phylogenetic analysis using Parsimony) v. 4.0b10 (swofford 2003) and consisted of neighbour- joining analyses with the uncorrected (“p”), the Kimura 2-parameter and the hKy85 substitution models. alignment gaps were treated as missing data and all characters were unordered and of equal weight. any ties were broken randomly when encountered. For parsimony analyses, alignment gaps were treated as a �fth character state and all characters were unordered and of equal weight. maximum parsimony analysis was performed using the heuristic search option with 100 random (Its) or simple (lsu) taxa additions and tree bisection and reconstruction (tBr) as the branch- swapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. the robustness of the trees obtained was evaluated by 1 000 bootstrap replications (hillis & Bull 1993). tree length (tl), consistency index (cI), retention index (rI) and rescaled consistency index (rc) were calculated. sequences derived in this study were lodged at genBank ( ), the alignment in treeBase ( ), and taxonomic novelties in mycoBank ; crous et al . 2004a). morphology characteristics in culture were determined after 7 d on mea, Pda and oa (crous et al. 2009c). morphological descriptions were based on sporulating cultures on synthetic nutrient-poor agar (sna) (nirenburg 1981, lombard et al . 2009) and carnation leaf agar (cla) (crous et al . 2009c). slide preparations were made from sporulating cultures (sna for anamorph, cla for teleomorph) in clear lactic acid, with 30 measurements determined per structure, and observations made with a nikon smz1500 dissecting microscope, and with a zeiss axioscope 2 microscope using differential interference contrast (dIc) illumination. colony characters and pigment production were noted after 7 d of growth on mea, Pda and oa (crous et al 2009c) incubated at 25 ºc. colony colours (surface and reverse) were rated according to the colour charts of rayner (1970). calonectria lauri sp. nov.   re sul Phylogeny approximately 600, 480 and 680 bases were determined for the isolates indicated in table 1 for tuB, hIs and Its, respectively. of the β-tubulin gene, 522 bases were used for phylogenetic analyses in the manually adjusted alignment containing 32 isolates (including the outgroup sequence). of these 522 characters (including alignment gaps), 180 were parsimony- informative, 47 were variable and parsimony- uninformative, and 295 were constant. neighbour- joining analysis using the three substitution models, as well as the parsimony analysis, yielded trees with exactly the same topologies. Parsimony analysis of the alignment yielded a single most parsimonious tree (tl 381 steps; cI 0.816; rI 0.953; rc 0.778), which is shown in Fig. 1. taxonomy alonectria lauri (vanderw.) lechat & crous, comb. nov. mycoBank mB517423 (Fig. 2) Basionym : tetracytum lauri vanderw., Parasitica 1: 145. 1945. (as “ laurii ”). candelospora ilicicola hawley, Proc. roy. Irish acad. 31: 11. 1912. [non calonectria ilicicola Boedijn & reitsma, 1950] cylindrocladium ilicicola (hawley) Boedijn & reitsma, reinwardtia 1: 57. 1950. typus : reland , clare Island, Ilex aquifolium , hawley , K (m) 61269!, holotype of cy. ilicicola , ImI 76542 isotype. etherlands south-east limburg, vijlenerbos, vijlen, Ilex aquifolium , aug. 1970, h.a van der aa , epitype cBs h-15110, ex-epitype culture cBs 749.70. ascomata perithecial, solitary, scattered, subglobose to ovoid, 450–550 µm high × 380–420 µm diam, super�cial, not obviously stromatic but dif�cult to remove from the subtratum because basal cells of ascomata remain immersed in the substratum, yellow to brownish-yellow, dark-red at base, not changing colour in 3 % Koh or lactic acid, warted except at ostiolar region, ostiole papillate, composed of palisade-like, cylindrical to narrowly ellipsoidal cells. ascomatal wall 50–65 µm thick of two regions; outer region comprising warts 50–55 µm thick, composed of globose to nearly angular, thick-walled cells, 10–30 × 5–16 µm, yellow, wall 1.5–2 µm thick; inner region 5–10 µm thick, composed of �attened, ellipsoidal cells, 12–18 × 3–5 µm, hyaline; warts globose to subglobose 25–40 × 15– 30 µm, yellow. asci clavate, long stipitate, 110–130 × 17–22 µm, 8-spored, multiseriate. ascospores narrowly fusiform with rounded ends, lightly curved, guttulate, hyaline, smooth, (53–)60–86(–89) × 6.5–8(–9) µm, 3-septate, not conctricted at the septa or constricted when overmature. conidiophores consisting of a stipe bearing a penicillate arrangement of fertile branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, 40–150 × 3–5 µm; stipe extensions septate, straight to �exuous, 120–200 µm long, 2.5–3 µm wide at the apical septum, terminating in an obpyriform to ellipsoid vesicle, (5–) 7–8(–10) µm diam. conidiogenous apparatus with primary branches aseptate or 1-septate, 15–20 × 4–5 µm; secondary branches aseptate, 8–15 × 4–5 µm; tertiary branches aseptate, 10–15 × 4–5 µm, each terminal branch producing 2–4 phialides; phialides doliiform to reniform, hyaline, aseptate, 6–12 × 2.5–4 µm; apex with minute periclinal thickening and inconspicuous collarette. conidia cylindrical, rounded at both ends, straight, (45–) 55–68(–73) × (4–)5–6(–7) µm (av. 60 × 5.5 µm), (1–)3-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. megaconidia and microconidia unknown. culture characteristics : colonies on mea sienna to brick on the surface, and sienna in reverse; sienna on oa (surface); sienna to umber on Pda (surface), and umber in reverse; chlamydospores on mea moderate, occurring throughout the medium, with sparse to moderate sporulation on aerial mycelium. additional specimens examined : etherlands , hilversum, on leaves of Ilex aquifolium , 11 nov. 2008, w. gams , cPc 15683 cBs 128031, cPc 15684, cPc 15685. rance Pressigny (52), on leaves of Ilex aquifolium , 05 dec. 2009, a. gardiennet , ag09308, cBs h-20476 , c ulture cPc 17978 cBs 126269; Forêt de chizé, villiers en Bois (79) on leaves table 1. collection details and genBank accession numbers of isolates of calonectria lauri included in this study. train no. ubstrate ountry ollector(s) enBank accession no. (t B, , It cPc 15683 leaves of Ilex aquifolium netherlands w. gams Fr694682, Fr694676, Fr694679 cBs 126269 cPc 17978 leaves of I. aquifolium France a. gardiennet Fr694683, Fr694677, Fr694680 cBs 553.69 ImI 299390 root of Buxus sempervirens Belgium Fr694684, Fr694678, — cBs 749.70 I. aquifolium netherlands h.a. van der aa Fr694685, gq267250, gq280584 cBs: cBs Fungal Biodiversity centre, utrecht, the netherlands; cPc: culture collection of P.w. crous, housed at cBs; ImI: International mycological Institute, caBI-Bioscience, egham, Bakeham lane, uK. tuB: partial beta-tubulin gene; hIs: partial histone h3 gene; Its: Internal transcribed spacers 1 and 2 together with 5.8s nrdna.   echat et al  Fig. 1. single most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the β-tubulin sequence alignment. the scale bar shows 10 changes and bootstrap support values from 1000 replicates are shown at the nodes. the tree was rooted to cylindrocladiella lageniformis (genBank ay725652). calonectria lauri sp. nov.   Fig. 2. calonectria lauri and its cylindrocladium anamorph. a, B. yellowish perithecia in vivo (a), and in vitro (B). cylindrocladium anamorph. vertical section through perithecium, showing wall anatomy. e. ascospores. F– conidiophores. I– conidiogenous apparatus with phialides. m. ellipsoid to obpyriform vesicles. n. three-septate conidia. scale bars: a, B 200 µm, c, e–h, m 8 µm, d, J–l, n 10 µm, I 5.5 µm.   echat et al  of Ilex aquifolium , 19 sept. 2006, c. lechat , cll696. elg um gent, on roots of Buxus sempervirens , July 1969, a. roos ImI 299390 cBs 553.69. notes : the name calonectria ilicicola is already occupied, and thus the next available epithet for this species in calonectria is that of tetracytum lauri calonectria lauri is phylogenetically closely related to ca. citri (known on citrus from Florida). morphologically the two species can be separated in that ca. citri has ellipsoid to pyriform or obovoid vesicles, and 3-septate conidia that are slightly shorter and narrower, (25–)53– 60(–65) 3–4(–5) µm (crous 2002). dI scuss the genus calonectria is based upon calonectria pyrochroa (on Platanus leaf litter, France, lectotype BPI), which rossman (1979) found to be indistinguishable from ca. daldiniana (on magnolia grandi�ora leaf litter, Italy, holotype ro). a separate collection from decaying leaves of Pittosporum undulatum collected in madeira (cuP-mm 2407) produced a cylindrocladium anamorph with clavate vesicles, which later led rossman (1983) to conclude that the oldest anamorph epithet that could be linked to ca. pyrochroa was c. ilicicola Brayford & chapman (1987) reported a wilting disease of laurus nobilis in nurseries on the Isles of scilly, and later on arbutus andrachnoides and gaultheria shallon in west devon, uK. the causal organism was identi�ed as c. ilicicola , but incorrectly linked to the teleomorph name, ca. ilicicola . Based on a molecular comparison of ex-type strains, crous et al . (1993) showed ca. ilicicola was the teleomorph of c. parasiticum , a major pathogen associated with cylindrocladium black rot of peanut. In a later study, crous & wing�eld (1994) accepted the relationship between ca. pyrochroa and c. ilicicola , as there were no cultures available at the time to refute this proposed link (crous 2002). Following a revision of cylindrocladium strains in the cBs culture collection, crous et al. (2006a) discovered a strain linked to a specimen that closely matched the type of c. ilicicola and subsequently designated cBs 749.70 (on Ilex aquifolium , the netherlands) as ex-epitype strain for c. ilicicola . sequence data derived from the ex-epitype strain, and morphology, proved to be identical to that of the new collection obtained from France (Figs 1–2), con�rming it to be c. ilicicola however, isolate cBs 126269 produced a calonectria teleomorph in culture, which is clearly distinct from ca. pyrochroa . the latter species (and its synonyms) have scarlet-red perithecia, which turn purple in 2 % Koh (rossman 1979). the present collection (on the host and on cla in culture), forms yellow to brownish yellow perithecia that do not discolour in Koh (except at the perithecial base). the teleomorph of c. ilicicola could therefore not be ca. pyrochroa as currently accepted (lombard et al. 2010c). Because the name ca. ilicicola is already occupied by the pathogen causing cylindrocladium black rot of peanut (crous et al . 1993), a new name, ca. lauri , is proposed for this species, which appears to occur commonly on laurus , Ilex, as well as several other hosts in europe (Brayford & chapman 1987). Presently no cultures are available of ca. pyrochroa and further collections will have to be made from Platanus leaf litter in France to help clarify the morphology of its cylindrocladium anamorph. aPPen IX In the recent treatment of the genus calonectria lombard et al . (2010c) allocated the name cylindrocladium canadense to calonectria as ca. canadensis (J.c. Kang, crous & c.l. schoch) l. lombard, m.J. wingf. & crous, but overlooked the older existing name, ca. canadensis (ellis & everh.) Berl. & voglino. a new combination is required to resolve this homonym as follows: alonectria canadiana l. lombard, m.J. wingf. & crous, nom. nov. mycoBank mB517424 Basionym : cylindrocladium canadense J.c. Kang, crous & c.l. schoch, syst. appl. microbiol. 24: 210. 2001. calonectria canadensis (J.c. Kang, crous & c.l. schoch) l. lombard, m.J. wingf. & crous, stud. mycol. 66: 56. 2010, non calonectria canadensis (ellis & everh.) Berl. & voglino, addendum to syll. Fung. 4: 212. 1886. ac Kn owl dg ement the authors thank the technical staff, arien van Iperen (cultures), marjan vermaas (photo plates), and mieke starink- willemse (dna isolation, ampli�cation and sequencing) for their invaluable assistance. drew minnis (usda, Beltsville, usa) is also thanked for bringing the homonym associated with epithet “ canadensis ” to our attention. Finally, we thank alain gardiennet for the supply of specimens. reFe en Boesewinkel hJ (1982) cylindrocladiella , a new genus to accommodate cylindrocladium parvum and other small- spored species of cylindrocladium . canadian Journal of Botany 60 : 2288–2294. calonectria lauri sp. nov.   Brayford d, chapman au (1987) cylindrocladium ilicicola on cuttings of evergreen ornamental shrubs in the uK. Plant Pathology 36 : 413–414. crous Pw (2002) taxonomy and pathology of cylindrocladium (calonectria) and allied genera . aPs Press, st. Paul. crous Pw, allegrucci n, arambarri am, cazau mc, groenewald Jz, wing�eld mJ (2005) dematiocladium celtidis gen. sp. nov. ( nectriaceae , hypocreales ), a new genus from celtis leaf litter in argentina. mycological research 109 : 833–840. crous Pw, gams w, stalpers Ja, robert v, stegehuis g (2004a) mycoBank: an online initiative to launch mycology into the 21st century. studies in mycology 50 : 19–22. crous Pw, groenewald Jz, risède J-m, simoneau P, hyde Kd (2006a) calonectria species and their cylindrocladium anamorphs: species with clavate vesicles. studies in mycology 55 : 213–226. crous Pw, groenewald Jz, risède J-m, simoneau P, hywel-Jones nl (2004b) calonectria species and their cylindrocladium anamorphs: species with sphaeropedunculate vesicles. studies in mycology 50 : 415–430. crous Pw, slippers B, wing�eld mJ, rheeder J, marasas wFo, Phillips aJl, alves a, Burgess t, Barber P, groenewald Jz (2006b) Phylogenetic lineages in the Botryosphaeriaceae . studies in mycology 55 : 235–253. crous Pw, summerell Ba, carnegie aJ, wing�eld mJ, groenewald Jz (2009a) novel species of mycosphaerellaceae and teratosphaeriaceae . Persoonia 23 : 119–146. crous Pw, summerell Ba, carnegie aJ, wing�eld mJ, hunter gc, Burgess tI, andjic v, Barber Pa, groenewald Jz (2009b) unravelling mycosphaerella : do you believe in genera? Persoonia 23 : 99–118. crous Pw, verkley gJm, groenewald Jz, samson ra (eds) (2009c) Fungal Biodiversity. [ cBs laboratory manual series 1 ]. centraalbureau voor schimmelcultures, utrecht. crous Pw, wing�eld mJ (1994) a monograph of cylindrocladium , including anamorphs of calonectria mycotaxon 51 : 341–435. crous Pw, wing�eld mJ, alfenas a (1993) cylindrocladium parasiticum sp. nov., a new name for c. crotalariae . mycological research 97 : 889–896. crous Pw, wood ar, okada g, groenewald Jz (2008) Foliicolous microfungi occurring on encephalartos Persoonia 21 : 135–146. decock c, hennebert gl, crous Pw (1997) nectria serpens sp. nov. and its hyphomycetous anamorph Xenocylindrocladium gen. nov. mycological research 101 : 786–790. graves ah (1915) root rot of coniferous seedlings. Phytopathology : 213–217. hawksworth dl, sivanesan a (1976) new and Interesting microfungi from slapton, south devonshire: ascomycotina II. transactions of the British mycological society 67 : 39–49. henricot B, culham a (2002) cylindrocladium buxicola , a new species affecting Buxus spp., and its phylogenetic status. mycologia 94 : 980–997. hillis dm, Bull JJ (1993) an empirical test of bootstrapping as a method for assessing con�dence in phylogenetic analysis . systematic Biology 42 : 182–192. Kang Jc, crous Pw, old Km, dubzinski mJ (2001a) non- conspeci�city of cylindrocladium quinqueseptatum and calonectria quinqueseptata based on a β-tubulin gene phylogeny and morphology. canadian Journal of Botany 79 : 1241–1247. Kang Jc, crous Pw, schoch cl (2001b) species concepts in the cylindrocladium �oridanum and cy. spathiphylli complexes ( hypocreaceae ) based on multi-allelic sequence data, sexual compatibility and morphology. systematic and applied microbiology 24 : 206–217. lombard l, crous Pw, wing�eld Bd, wing�eld mJ (2010a) species concepts in calonectria ( cylindrocladium ). studies in mycology 66 : 1–14. lombard l, crous Pw, wing�eld Bd, wing�eld mJ (2010b) multigene phylogeny and mating tests reveal three cryptic species related to calonectria pauciramosa . studies in mycology 66 : 15–30. lombard l, crous Pw, wing�eld Bd, wing�eld mJ (2010c) Phylogeny and systematics of the genus calonectria studies in mycology 66 : 31–69. lombard l, rodas ca, crous Pw, wing�eld Bd, wing�eld mJ (2009) calonectria ( cylindrocladium ) species associated with dying Pinus cuttings. Persoonia 23 : 41–47. lombard l, zhou Xd, crous Pw, wing�eld Bd, wing�eld mJ (2010d) calonectria species associated with cutting rot of eucalyptus . Persoonia 24 : 1–11. nirenburg hI (1981) a simpli�ed method for identifying Fusarium spp. occurring on wheat. canadian Journal of Botany 59 : 1599–1609. o’donnell K, cigelnik e (1997) two divergent intragenomic rdna Its2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. molecular Phylogenetics and evolution : 103–116. Peerally a (1991) the classi�cation and phytopathology of cylindrocladium species. mycotaxon 40 : 367–366. rambaut a (2002) sequence alignment editor. version 2.0. department of zoology, university of oxford, oxford, uK. software distributed by author ( ). rayner rw (1970) a mycological colour chart . commonwealth mycological Institute, Kew. rossman ay (1979) calonectria and its type species, daldiniana , a later synonym of . pyrochroa . mycotaxon : 321–328. rossman ay (1983) the phragmosporous species of nectria and related genera. mycological Papers 150 : 1–164. rossman ay, samuels gJ, rogerson ct, lowen r (1999) genera of Bionectriaceae , hypocreaceae and nectriaceae hypocreales, ascomycetes ). studies in mycology 42 1–248. schoch cl, crous Pw, wing�eld Bd, wing�eld mJ (2001) Phylogeny of calonectria based on comparisons of β-tubulin dna sequences. mycological research 105 1045–1052.    echat et al schoch cl, crous Pw, wing�eld mJ, wing�eld Bd (2000) Phylogeny of calonectria and selected hypocrealean genera with cylindrical macroconidia. studies in mycology 45 : 45–62. swofford dl (2003) PauP*. Phylogenetic analysis using Parsimony (* and their methods). version 4.0b.10 sinauer associates, sunderland, massachusetts. victor d, crous Pw, Janse BJh, zyl wh van, wing�eld mJ, alfenas ac (1998) systematic appraisal of species complexes within the hyphomycete genus cylindrocladiella . mycological research 102 : 273–279. wright lP, davis aJ, wing�eld Bd, crous Pw, Brenneman t, wing�eld mJ (2010) Population structure of cylindrocladium parasiticum infecting peanuts ( arachis hypogaea ) in georgia, usa. european Journal of Plant Pathology 127 : 199–206.   © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. ow to describe a new fungal species Keith a. seifert and amy y. rossman Biodiversity (mycology), eastern cereal and oilseed research centre, agriculture & agri-Food canada, ottawa, ontario K1a 0c6 canada; corresponding author e-mail: Keith.seifert@agr.gc.ca systematic mycology & microbiology laboratory, usda-ars, rm. 246, B0101a, 10300 Baltimore ave., Beltsville, md 20705, usa for the International commission on the taxonomy of Fungi, www.fungaltaxonomy.org abstract: the formal requirements and best practices for the publication of descriptions of new fungal species are discussed. expectations for dna sequences and cultures are considered. a model manuscript offers one possible approach to writing such a paper article info: submitted: 9 october 2010; accepted: 15 october 2010; Published: 2 november 2010. nt roduc tI every fungal species is unique. therefore, every description of a fungal species is also unique. the morphological, physiological, ecological, and molecular diversity in fungi means that descriptions and illustrations differ from one taxonomic group to another. there are no formal standards for the description and illustration of species, but there are some formal (or ‘legal’) requirements for proposing names that are imposed by the International code of Botanical nomenclature (IcBn; mcneill et al. 2006). Furthermore, community standards of scienti�c rigor are enforced by editors and reviewers. For the beginner, it is useful to have models to assist with the preparation of descriptions and illustrations. In this paper, formal requirements and best practices that should be considered for any description are outlined, and a model manuscript for describing a new species is provided. several ‘tricks of the trade’ and cautionary notes are also included. additional hints can found in the now somewhat dated code of Practice developed by the IctF (sigler & hawksworth 1987), and the guidebook for mycologists by hawksworth (1974). although not exclusively concerned with fungi, the book by winston (1999) also provides a valuable perspective. For ma l r qu ement the International code of Botanical nomenclature (IcBn) governs the naming of fungi. this is a complex document, but you should read the relevant articles of the code for exact wording of the regulations. the IcBn is updated every six years, after each International Botanical congress, and is available on the world wide web (see references). the most recently published code must be followed, and previous codes are considered obsolete. although there have been discussions about a possible independent mycological code, or a Biocode covering all organisms, these are still in the dialogue stage. the Phylocode (cantino & de queiroz 2010) promotes phylogenetically based non-linnaean nomenclature and is not relevant for the description of new species as presented here. In taxonomic language, species must be ‘effectively’, ‘legitimately’ and ‘validly’ published. these three words have special meanings in taxonomic terminology (as do ‘illegitimate’ and ‘invalid’), and they should not be used in other ways in taxonomic manuscripts. to be effectively published (arts. 29–31), i.e. to be made available, a description of a new species must be published in a journal that can be read by the scienti�c community. species published in newspaper articles, or mentioned in oral presentations at scienti�c meetings, for example, are not considered effectively published. at present, Key words: culture collections herbaria International code of Botanical nomenclature latin diagnoses molecular phylogenetics Ima Fu gus volu me 1 o 2: 109–116 eifert & ossman    descriptions of new species cannot be published exclusively on electronic media such as cd-roms, dvds or on the Internet. effective publication is only accepted by the IcBn when at least two paper copies are archived in a scienti�c library or other despositary; however, we feel that many more printed copies should be deposited, preferably on each continent. several mycological journals now publish articles including new species online and deposit printed copies in permanent libraries to meet effective publication requirements; the date of publication of the paper copy remains the of�cial date, not the often earlier date of publication on- line. academic Phd or other theses presented to universities as part of degree requirements are not considered effective publications, even if copies are distributed to other universities, unless they have an IsBn number or clearly state that they are to be intended effective publications (art. 30.5). to be legitimately published (art. 6), i.e. legally acceptable, a new species must have a unique binomial, i.e. it cannot have the same species epithet as another species validly published in the same genus. to be validly published (arts 32–45), a new species must be clearly designated as a new species, have a latin diagnosis, and a single, clearly designated and permanently preserved ‘type’, which �xes the application of the name. usually, the type is preserved in a public herbarium (holmgren et al. 1990) that will make the material available to interested scientists, has an on-line database of holdings, and that will assign a unique accession number, which you will then quote to clearly identify the type specimen. If there duplicates of the type specimen, or cultures derived from it, these must be explicitly distinguished from the holotype; the others are referred to as ‘isotypes’ or as ‘ex-type cultures’. to safeguard against loss and to facilitate access by other mycologists, isotypes should be deposited in several herbaria, on different continents if possible. there are many nuances to the concept of a type (arts 7–10). For a new species, you will normally propose a ‘holotype’. the holotype is usually a dried, physiologically inert specimen (or a dried culture) that includes all diagnostic morphological characters of the species. For microscopic fungi, several separate individuals can be present as long as they are part of one sample collection, i.e. made at one time in a precise locality. living cultures are now allowed as holotypes (art. 8.4), but only if they are preserved in a metabolically inactive state (i.e. by lyophilization or in liquid nitrogen), ideally in an internationally recognized culture collection (see world Federation for culture collections, s.d.). this practice is not widely used in mycology except for yeasts. cultures can be dried for use as type specimens (constantinescu 1983); take care to dry an uncontaminated, optimally developed culture, not an old one that has started to degenerate. If you wish to designate a microscope slide as a type, or to include one with the type, it is worth the effort to make a permanent preparation using the method described by Kohlmeyer & Kohlmeyer (1972). nF or ma l r qu ement to successfully describe a new species, the author must convince readers (especially reviewers and editors) that: the species is really undescribed. the species is being described in the most appropriate genus, and if molecular data are available, the genus including the new species remains monophyletic. the species is described, illustrated or otherwise characterized adequately so that it can be recognized again by subsequent workers. a suf�cient number of cultures or specimens were examined. Ideally, new species should be described based on more than one specimen or culture, and some journals demand this. with limited material but clear taxonomic novelty, the author may be able to write a convincing argument for the proposal of a new species that is acceptable to editors and reviewers. manuscripts that do not satisfy these criteria should not be published until they can be met. normally, peer reviewers and editors assess whether these criteria are satis�ed. In recent decades, partly as a result of the spirit of the un convention on Biological diversity (cBd), taxonomists are encouraged, and sometimes legally required by national laws, to deposit type specimens in public reference collections in the country where the specimens were originally collected. If cultures were isolated, there may be a similar requirement or expectation from the originating country. cultures of new species should be deposited in two or three internationally recognized public culture collections, which agree to make them available to other researchers. this latter practice is a condition for valid publication of new bacterial species (lapage et al. 1992), and is enforced as an editorial policy by some journals that publish new fungal species. It is critical that type specimens and cultures are available to other taxonomists who want to study and compare them with other material. the IcBn ow to describe a new fungal species   recommends (rec. 7a), but cannot enforce, that type specimens be deposited in public institutions with a policy to allow scienti�c researchers to examine material. a frequent problem is the unavailability of type or other specimens from under-resourced collections, or collections not curated by a mycologist. some historical collections may never be sent on loan because of their fragility and extreme importance. some nations forbid specimens or cultures from being sent abroad, under their interpretations of the cBd. a parallel situation is the reluctance of some industrial researchers (e.g. pharmaceutical companies) to allow access to cultures that they own. Balancing the question of open access to specimens or cultures against the legal or proprietary interests attached to that material is complicated, but must be considered when depositing type specimens. the scienti�c process demands reproducibility, and if this cannot be assured, responsible journals will not allow publication. the risk for the authors of species that cannot be re-examined or studied by other taxonomists is that the species will not be accepted by future scientists, and that the efforts and work of the authors of such species will be wasted and ignored. almost all mycological journals now require that names and certain nomenclatural information for all newly proposed fungal taxa, including new combinations, be deposited in mycoBank (crous et al. 2004), and that the mycoBank accession number be included as part of the description. while the minimum requirements are the deposit of the latin diagnosis and information on the type specimen, it is good practice to include as much information as possible, including illustrations, the english description, and links to molecular data, because this critical information will then be freely available to all scientists. re qu ement s or cul ur s an ol cul r d ata at present, there are no formal requirements that you must have cultures or dna sequences of a fungus before you can describe a new species. nevertheless, dna sequences and cultures signi�cantly enhance the value of a species description and you should make every effort to generate these resources. mycologists describing new species should indicate whether they have tried to obtain cultures and what methods were attempted. not all species can be cultured using currently available methods, but for most groups, culturing should be relatively straightforward after consulting the literature on related species. cultures are essential for some groups where the modern morphological taxonomy is based entirely on in vitro characters, especially hyphomycetes such as alternaria , aspergillus, cladosporium, Fusarium, Penicillium and trichoderma ; new species in these genera should not be described in the absence of cultures or sequences. For other ascomycetes, single- spore cultures may yield unexpected anamorphs that will allow the description of a more complete life cycle. dna sequences can be usually obtained from all but the most recalcitrant materials (such as fossils). there is a growing expectation that descriptions of all new species should be accompanied by molecular data, driven in part by the need for dna sequence data to integrate new species into molecular phylogenies. the growth of molecular ecology, which relies on databases of reference sequences for identi�cation of environmental sequences, has also highlighted the importance of sequencing all newly described species. therefore, an increasing number of journals, editors or reviewers insist on cultures or dna sequences before a manuscript is accepted for publication. If you do not have cultures or dna sequences, your new species can only be published in a journal with different policies. we encourage mycologists who lack resources for culturing or dna sequencing to collaborate with colleagues who can assist with this, often in return for co-authorship. eifert & ossman    nt roduc tI the paragraphs of the introduction should be presented in a logical order, i.e. how they tell the best story. remember, most people reading a scienti�c paper will only read the Introduction and discussion, so the account of your discovery should be complete and complementary between these two sections. normally, you will tell the reader about the larger projects (if any) that led to the discovery of the new species, provide information on its ecological niche and associated organisms, and give references to complementary publications where appropriate. Information should be provided about the taxonomy of genus in which the species is being described, such as the number of species already known, a brief review of recent revisions or monographs, perhaps discussion of controversies about the generic concept, and something about the biology of the species. Be diligent about citing all relevant literature. If you have dna sequence data, usually one paragraph will provide a brief review of the existing state of molecular knowledge for the group your species belongs to, and explain to the reader what experiments you have done with your own species to �t it into the existing context. another paragraph gives information about why the new species is suspected to be undescribed. this should be basic information that leads into the formal part of the paper. some of this information may be repeated and presented in more detail in the discussion. some papers may require a longer introduction. manuscripts including molecular or physiological data are often longer. situations where the new fungus could be described in one of several different genera also may require a longer introduction. sometimes this will include a more extensive review of historical literature, or discussions of taxonomic characters of particular signi�cance. you must judge whether this information is most suitable in the introduction, which the reader will read before the description itself, or if it is more logical to place it in the discussion. usually the Introduction concludes with a statement like, “therefore, we decided that our fungus represents an undescribed species, which is described and illustrated here as genus species sp. nov.” ate Ia ls an d met hods this section is often omitted from taxonomic papers that include only morphological data, but it is preferable to include as many details as possible. some of the following subheadings and paragraphs might be appropriate. ollecting and �eld sites how the specimens were collected and transported to the laboratory and preserved or incubated prior to examination may be relevant. Information about speci�c �eld sites is usually given in the ‘material examined’ section, but it might be appropriate to provide more details here if they are relevant to understanding the species. ultures and media give recipes for the isolation media employed, or cite a reference for the media. give brand names for extracts used, such as malt extract, yeast extract, and the agar used for the media. describe the inoculation methods, incubation conditions such as temperature and lighting mod l m an uscr IPt title: genus species sp. nov., an undescribed fungus ( taxonomic group ) from habit in country with interesting biological properties abstract: If your title is suf�ciently engaging, a prospective reader will probably look next at the abstract. the abstract should include all details necessary for the reader who does not have access to the whole article (i.e. someone looking at the abstract only on-line or in an abstract journal) so that they will know whether it is worth their time or money to obtain the full article. when describing a new species, you should include a summary of the diagnostic characters of the new species, especially the spore characters and dimensions. make sure to include information about where your fungus was found and what it was growing on. If you have molecular data, it is useful to mention what genes you have sequenced, and what this information tells us about the fungus, such as what family or order it belongs to, and what are the most closely related species. mention if a key to related species or comparative synoptic table is included, a feature that will increase potential readership. article info: submitted: dd month yyyy; accepted: dd month yyyy; Published: dd month yyyy. Key words: these should not reproduce words in the title. It is useful to list special techniques used in the description, e.g. electron microscopy, dna sequencing, or chemo- taxonomic methods. ow to describe a new fungal species   regime, and length of incubation before examination. determine the cardinal growth temperatures (minimum, optimum and maximum) if possible. list the culture collections where the cultures are maintained, with accession numbers, either here or in a table. Isolation methods explain all isolation methods used, such as explants from sporophores or infected host tissues, removal of spores directly from sporulating structures, transfer of actively discharged spores from Petri dish lids or spore prints, etc. If the substrate was treated before isolation, e.g. by some form of surface sterilization, these methods should be explained. give the recipes for the isolation media employed including any antibacterial compound added, or cite a reference for those media. describe the incubation conditions such as temperature and lighting conditions. If single- spore cultures were prepared, a good practice to be undertaken when possible (choi 1999, crous 2002), describe how this was done. microscopy give details of the kind of microscope used, including the illumination systems (e.g. phase contrast, differential interference contrast), the mounting media and stains employed for routine examination and for making measurements, and how many structures of each microscopic character were measured. a similar section, with details on dehydration protocols, �xation, staining, etc. should be given for electron microscopy methods, if these were used. Permanent microscopic preparations should be deposited with the type if possible, which will make your observations reproducible to later taxonomists, and limit the amount of material they might use on specimens in future studies. techniques used for illustrations are often given here, but may also be brie�y mentioned in the �gure legends. For example, whether drawings were made with a drawing tube, a camera lucida, or by freehand, and the type of camera used for photography, may be relevant. It is essential to describe techniques used for image enhancement of digital photographs, such as the sharpening �lters of Photoshop or other imaging software, whether separate photographs were combined into one image, or whether colours have been altered (microscopy society of america, 2003). modi�cation of contrast has always been standard practice in photography and need not be mentioned. Physiological tests or chemotaxonomic methods For yeasts, substrate utilization and other physiological tests are standard parts of taxonomic descriptions. For some lichen groups, spot-tests with a standard set of chemical reagents are essential. these methods must be described carefully. If chemotaxonomic methods, such as isozyme analysis or secondary metabolite pro�ling, were employed, complete methods should be given to allow the resulting data to be reproduced. na extraction, P cr ampli�cation, na sequencing, and phylogenetic analysis note what kind of material was used for dna extraction, such as cultures, single spores, or naturally occurring tissues, and give the details of the kits and methods used for dna extraction. For dna isolations from natural tissues, note whether procedures were repeated to reduce the chance of sequencing a contaminant or associated organism. give the details of Pcr ampli�cation pro�le used, the concentrations of reagents used in the reactions, and information on the brand and model of thermocycler used. Provide details of any methods used to clean or otherwise process the Pcr products before sequencing. If Pcr products or other dna fragments were cloned for sequencing, provide the relevant information for this procedure. For the dna sequencing, provide details of the cycle sequencing pro�le used, the concentration of reagents used, the brand and model of the thermocycler, the relevant information about the sequencing chemistry used, and the brand and model of dna sequencer employed. If you used a dna sequencing service, list it here. cite the literature where Pcr and sequencing primers were �rst published. If you designed the primers yourself, give details of how you did this. Include details about how you did your phylogenetic analyses, including literature citations for sequences originating from published or unpublished work of colleagues, the software used for analysis, and the details of the parameters used for the analysis. diverse methods of phylogenetic analysis are available. while the choice of methods is largely a matter of preference, there is a general agreement that it is critical to employ measures of con�dence, such as the bootstrap, Bayesian posterior probabilities, the ‘decay index’ or congruence among independent data sets. If you have used several sequences from a previously published study, you should cite that study so that it is properly credited (seifert et al. 2008). re sul many descriptions of new species will not have a results section because all of the data are included in the taxonomy section. however, if some experiments were done with the fungus, such as physiological tests or tests of antibiotic resistance, then these data should be presented in the results section, in the same order as the methods are described in the materials and methods section. often, the results of physiological tests are given in a table. eifert & ossman    general results of dna sequencing analyses are usually given in this section. these can include details of the length and composition of the dna fragments, and the results of comparisons with other sequences (e.g. Blast searches). If phylogenetic trees are presented, the tree statistics may be given in this section, or otherwise in the �gure legends. describe what the data shows, e.g. that the sequence is similar to those in a particular genus, family or order, or that the sequence is apparently unique, but leave the conclusions derived from this for the discussion section. mention support values for the critical nodes in your tree based on bootstrap frequencies or other measures of con�dence. If you have done analyses using different phylogenetic methods, or have analyzed several genes, then comparison of the results is appropriate here, but leave the conclusions for the discussion section. taX my genus species authors , sp. nov. mycoBank: mBxxxxxxx. Figs xxx–xxx the latin diagnosis comes �rst and is essential for valid publication. It should list the diagnostic characters only, i.e. those that separate it from similar ones, and not be a complete translation of the description. many journals now restrict the latin to a few lines. use published latin diagnoses for models, then if possible have yours checked by a mycologist or botanist competent in scienti�c latin. If there is no such expert in your own department, consult with colleagues in other institutions. stearn’s Botanical latin (1992) is a valuable resource for preparing latin diagnoses. holotypus: collection acronym, accession number. Immediately after the latin diagnosis, clearly and explicitly indicate the details of the single accession that will serve as holotype. If you wish to list isotypes or extype cultures here, be certain that they are clearly distinguished from the holotype or you may have problems with the validity of your new name (art. 37.7). a full description follows. think of the descriptions and illustrations together as providing a blue-print for your new species. If someone wanted to build an exact scale- model of the fungus, they should be able to do so using your paper. where both asexual and sexual states (i.e. anamorph and teleomorph) occur, the description of the sexual state is traditionally given �rst. In general, a taxonomic description begins at the broadest scale and moves towards the �nest. macroscopic characters are described next. use a colour standard, such as ridgway (1912), rayner (1970), Kornerup & wancsher (1984), or munsell (1905 and many subsequent editions) to accurately describe colours. most details will be visual, but sometimes texture and odour are useful additions. when describing microscopic characters, be as complete as possible about shape, colour, texture and size for every component of the fungus. there are standard terminologies for shape; check ainsworth & Bisby’s dictionary of the Fungi (Kirk et al. 2008, and earlier editions, see under ‘shapes’) as a starting point. Be aware that the terminology for describing three dimensional shapes sometimes differs from the terminology used to describe two dimensional shapes. the full range of observed dimensions should be given for all structures. means should be calculated for all dimensions in the description (at least the spores), along with a statistical measure of variation in these measurements, such as standard error, standard deviation, con�dence intervals or percentile ranges. If you have isolated a culture, its features are usually included in a separate paragraph. For some fungi, colony characters are described �rst; for others, this information follows the morphological description. at a minimum, give the growth rates on a speci�ed medium and explain the temperature and light regime, and give a general impression of the colour and texture of the colonies. If the fungus sporulates in culture, it can be very helpful to compare the sizes and shapes of the microscopic structures to what occurred on the natural specimen. the detail employed in culture descriptions varies considerably from one taxonomic group to another, and you should consult published descriptions for the group you are working with. If you have done any physiological tests, or determined cardinal temperatures, this information is normally put in a separate paragraph in the description. It can also be put into a table or in a graph, which may be easier for a reader to follow. substrate or host : Provide a summary of the known hosts or substrates as a separate paragraph, especially if you have more than one specimen. distribution : summarize the known disttibution, by continent, country (and by province or state for larger countries), along with relevant information on the biome, climactic or geological conditions. etymology : explain the meaning or derivation of the species epithet, and note the language of origin of the word(s) used for constructing the epithet. avoid species epithets with more than �ve syllables and those similar to other epithets in the same genus. epithets that are descriptive are most helpful, but names can be derived from any source, including acronyms or the name of a names of author or authors, and their abbreviations, should follow the standards of Kirk & ansell (1992). ow to describe a new fungal species   person, usually someone involved in the discovery of the fungus or a mycologist who has made a signi�cant contribution to the subject. additional material examined : most journals have a speci�c format for this part of the paper. For all specimens and cultures, including isotypes and ex- type cultures, list country: Province/state/territory/ county/township, city/town/Park, speci�c location details (gPs coordinates, including altitude), substrate or host, date of collection and/or isolation, collector’s name, collector’s number (if any), herbarium or culture collection abbreviations and accession numbers where the material is preserved. often, some of this information is instead provided in a table including genBank accession numbers for dna sequences. dI scuss the discussion completes the story that began in the Introduction. there are many ways to write this section, but one rule is not to introduce new data that should have been introduced in the ‘results’ or ‘taxonomy’ sections. mixing of the results and discussion in one section is generally frustrating for the reader, unless the section is very short. It is often useful to start the discussion by summarizing the diagnostic features of the fungus you have described. In a separate paragraph, you should compare your fungus to other similar species of the same genus, stating clearly how they differ. many papers will include either a diagnostic key or synoptic table (or both), either including all species of a smaller genus, or only the most similar species of a larger genus, to assist the reader in understanding why the new species is distinct. If you have dna sequence data, there are often several paragraphs of discussion relating to what they show or do not show. discuss how the data support the classi�cation of your fungus and its recognition as a distinct species. If you have done analyses using different phylogenetic methods, or have analyzed several genes, compare the results and explain your conclusions carefully, especially if contradictory evidence occurs in the different data sets or analyses. It is often useful to conclude the paper with discussion of the biology of the new species, either demonstrated by the experiments done in the paper or as an extension of �eld observations. a limited amount of speculation on this topic is usually tolerated by reviewers and editors. Illus atI the IcBn does not require that new species descriptions have illustrations (Fig. 1), but few journals allow the description of a new species without them, with the exception of yeasts. usually, at least some of the illustrations will be of the holotype specimen or culture. the package of illustrations should present the complete concept of the species to the reader so that they can con�dently identify your fungus. Provide visual information at several different size scales, from general habitat to the most detailed microscopy. expectations vary among different taxonomic groups, but often a mixture of photographs and line drawings are included. Individual photographs are visual data that are proof of observations. electron micrographs are generally unhelpful to facilitate identi�cation, but scanning electron micrographs may be necessary for documenting spore ornamentation or tissue types, and transmission electron micrographs may be necessary to prove ultrastructural observations Fig. 1. genus species (specimen or culture number, noting whether it is type). a series of photographs of the fungus, showing the �eld habit, the appearance under the dissecting microscope, and microscopic photographs showing taxonomically relevant structures and preferably some developmental sequence. all illustrations should include scale bars. In this particular example: Fig. 1. sarcinella questierii (daom 235813). Black growth on living leaves of sp. Black conidia on leaf surface. development of dictyoconidia from conidiogenous hyphae, with hyphopodia (h) arising from the same hyphae (differential interference contrast). Bars: a 1 cm, B 25 µm, c 10 µm. B–c, composite images created with combinez (hadley 2006).    eifert & ossman of spore production (especially types of conidiogenesis). line drawings are interpretations; they are not proof, but can present a complex concept in one image and be extremely helpful for someone trying to identify your species. with colour photographs now published with increasing frequency, and the relative economy and ease of digital photography and computerized imaging, the preparation of informative illustrations is one of the most exciting aspects of describing a new species. Find the best model illustrations for the group of organisms where your species �ts, and then do better! ac Kn owl dg ement In addition to the usual acknowledgements for a scienti�c paper (e.g. mentors, sources of funding), it is traditional to acknowledge any taxonomic specialists that you have consulted during your decision to describe a new species. similarly, it is customary to acknowledge the curators of any collections who have provided specimens or cultures that was used in your study. For this paper, we wish to thank the other members of the International commission on the taxonomy of Fungi, especially david hibbett and david hawksworth, and the peer reviewers, for valuable comments on earlier drafts of this paper. the �nal sentence usually identi�es funders of the research. reFe en do your best to cite the relevant historical and modern literature, including revisions, monographs, identi�cation keys, and molecular studies that you have consulted. reviewers often see submitted papers in some �elds of taxonomy that only cite literature more than 25 years old. study the guidelines of the journal carefully for citation formats. cantino Pd, queiroz K de (2010) International code of Phylogenetic nomenclature. version 4c. choi y-w, hyde Kd, ho wh (1999) single spore isolation of fungi. Fungal diversity : 29–38. constantinescu o (1983) dried reference fungal cultures. a review and a simpler technique. Bulletin of the British mycological society 17 : 139–143. crous Pw (2002) adhering to good cultural practice (gcP). mycological research 106 : 1377–1378. crous Pw, gams w, stalpers Ja, robert v, stegehuis g (2004) mycoBank: an online initiative to launch mycology into the 21st century. studies in mycology 50 : 19–22. hadley a (2006) combinez. version 5. Published by the author. hawksworth dl (1974) mycologist’s handbook: an introduction to the principles of taxonomy and nomenclature in the fungi and lichens. Kew: commonwealth mycological Institute. holmgren, PK, holmgren nh, Barnett lc (1990) Index herbariorum. Part I. the herbaria of the world. th edn. [regnum vegetabile vol. 120.] utrecht: Bohn, scheltema & holkema. Kirk Pm, ansell ae (1992) authors of Fungal names: a list of authors of scienti�c names of fungi, with recommended standard forms of their names, including abbreviations. [Index of Fungi supplement.] wallingford, uK: caB International. Kirk Pm, cannon PF, minter dw, stalpers Ja, eds (2008) ainsworth and Bisby’s dictionary of the Fungi . 10 th edn. wallingford, uK: caB International. Kohlmeyer J, Kohlmeyer e (1972) Permanent microscopic mounts. mycologia 64 : 666–669. Kornerup a, wanscher Jh (1984) methuen handbook of color. rd edn. london, uK: methuen. lapage sP, sneath Pha, lessel eF, skerman vdB, seeliger hPr, clark wa (eds) (1992) International code of nomenclature of Bacteria and satutes of the International committee on systematic Bacteriology and statutes of the Bacteriology and applied microbiology section of the International union of microbiological societies. Bacteriological code (1990 revision). washington, dc, usa: american society for microbiology. mcneill J, Barrie Fr, Burdet hm, demoulin v, hawksworth dl, marhold K, nicolson dh, Prado J, silva Pc, skog Je, wiersema Jh, turland nJ, eds (2006) International code of Botanical nomenclature (vienna code) . [regnum vegetabile vol. 146.] liechtenstein: gantner verlag, ruggell. microscopy society of america (2003) msa policy on digital imaging. www.microscopy.org/resources/digital_imaging. cfm munsell ah (1905) a color notation . Boston: g. h. ellis. (for a pop-up computer version). rayner rw (1970) a mycological colour chart . Kew: commonwealth mycological Institute. ridgway r (1912) color standards and color nomenclature. washington, dc, usa: published by the author. seifert Ka, crous Pw, Frisvad Jc (2008) act: appropriate citation of taxonomy. Inoculum 59 (3): 4; Persoonia 20 105. sigler l, hawksworth dl (1987) International commission on the taxonomy of Fungi (IctF): code of practice for systematic mycologists. mycologist : 101–105; mycopathologia 99 3–7; microbiological sciences : 83–86. stearn wt (1992) Botanical latin: history, grammar, syntax, terminology and vocabulary . 4 th edn. newton abbot: david & charles. winston Je (1999) describing species: practical taxonomic procedure for biologists. new york: columbia university Press. world Federation for culture collections. s.d.   © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. hat is Johansonia Pedro w. crous , robert w. Barreto , acelino c. alfenas , rafael F. alfenas and Johannes z. groenewald cBs-Knaw Fungal Biodiversity centre, uppsalalaan 8, 3584 ct utrecht, the netherlands; corresponding author e-mail: p.crous@cbs.knaw.nl departemento de Fitopatologia, universidade Federal de viçosa, 36.570 viçosa, mg, Brazil abstract: the bitunicate ascomycete genus Johansonia is presently treated as a member of saccardiaceae , a family regarded as incertae sedis within the ascomycota . recent collections on leaves of a leguminous host, dimorphandra mollis, in mato grosso, Brazil, led to the discovery of a new species of Johansonia , described here as J. chapadiensis. Based on dna sequence data of the nuclear ribosomal dna (lsu), Johansonia is revealed to represent a member of dothideomycetes capnodiales . although its family could not be resolved, it clustered basal to schizothyriaceae and mycosphaerellaceae , and could well represent a species of saccardiaceae . dna sequence data of other members of saccardiaceae would be required, however, to con�rm this classi�cation. article info: submitted: 19 october 2010; accepted: 24 october 2010; Published: 2 november 2010. nt roduc tI the genus Johansonia is based on J. setosa (saccardo 1889), a species known from leaves of sapindaceae collected in south america (müller & von arx 1962). due on its super�cial discoid ascomata, bitunicate asci and hyaline, 1-septate ascospores, müller & von arx (1962) were of the opinion that the genus belonged to schizothyriaceae . In a later study, however, von arx & müller (1975) again placed it in saccardiaceae suborder dothideaceae in dothideales , based on the ascomata having an epithecium of branched hyphal elements. Barr (1993) again placed it in Phillipsiellaceae in loculoascomycetes , while lumbsch & huhndorf (2007) concluded that it was a member of saccardiaceae , a family they regarded as incertae sedis in ascomycota . In recent studies on dothideomycetes (schoch et al . 2006, 2009), no mention is made of Johansonia . as there are presently no dna sequence data represented for any species of Johansonia in genBank, its taxonomic position remains obscure. during a recent visit to Brazil, we collected fresh material of a species of Johansonia on leaves of a legume. the aims of the present study were, therefore, to identify the species of Johansonia , and at the same time to see if the taxonomic position of the genus could not be resolved. ate Ia ls an d met hods Isolates leaves bearing ascomata were soaked in water for approximately 2 h, after which they were placed in the bottom of Petri dish lids, with the top half of the dish containing 2 % malt extract agar (mea; crous et al . 2009c). ascospore germination patterns were examined after 24 h, and single ascospore and conidial cultures established as described earlier (crous et al. 1991, crous 1998). colonies were subcultured onto potato-dextrose agar (Pda), oatmeal agar (oa), mea (crous et al. 2009c), and incubated at 25 under continuous near-ultraviolet light to promote sporulation. reference strains are maintained in the cBs-Knaw Fungal Biodiversity centre (cBs) utrecht, the netherlands. na isolation, ampli�cation and analyses genomic dna was isolated from fungal mycelium grown on mea, using the ultracleantm microbial dna Isolation Kit (moBio laboratories, Inc., solana Beach, ca, usa) according to the manufacturer’s protocols. the primers v9g (de hoog & gerrits van den ende 1998) and lr5 (vilgalys & hester 1990) were used to amplify part of the nuclear rdna operon spanning the 3’ end of the 18s rrna gene (ssu), the internal transcribed spacer 1, the 5.8s rrna gene, the internal transcribed spacer 2 (Its) and the �rst 900 bases at the 5’ end of the 28s rrna gene (lsu). the primers Its4 (white et Key words: dothideomycetes Johansoniella Its lsu systematics Ima Fu gus volu me 1 o 2: 117–122 rous et al    teratosphaeriaceae Fig. 1. the �rst of 1000 equally most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the lsu sequence alignment. the scale bar shows 10 changes, and bootstrap support values from 1000 replicates are shown at the nodes. the novel sequence generated for this study is shown in . Branches present in the strict consensus tree are thickened and important lineages are colour-coded. Johansonia   al . 1990) and lsu1Fd (crous et al . 2009b) were used as internal sequence primers to ensure good quality sequences over the entire length of the amplicon. the Pcr conditions, sequence alignment and subsequent phylogenetic analysis followed the methods of crous et al . (2006, 2009a). sequences were compared with the sequences available in ncBI’s genBank nucleotide (nr) database using a megablast search and results are discussed in the relevant species notes where applicable. Based on the Blast results, the novel sequence was added to the alignment of Frank et al 2010 (treeBase study s10547). alignment gaps were treated as new character states. sequences derived in this study were lodged at genBank, the alignment in treeBase ( ), and taxonomic novelties in mycoBank ( ; crous et al . 2004). the morphological description is based on preparations made from host material in clear lactic acid, with 30 measurements determined per structure, and observations made with a nikon smz1500 dissecting microscope, and with a zeiss axioscope 2 microscope using differential interference contrast (dIc) illumination. colony characters and pigment production were noted after 2 wk of growth on mea, Pda and oa (crous . 2009c) incubated at 25 ºc. colony colours (surface and reverse) were rated according to the colour charts of rayner (1970). growth characteristics were studied on mea plates approximately 1700 bases, spanning the Its and lsu regions, were obtained from the sequenced culture. the lsu region was used in the phylogenetic analysis for the generic placement (Fig. 1) and Its to determine species- level relationships (see notes under species descriptions). the manually adjusted lsu alignment contained 77 taxa (including the Phaeobotryosphaeria visci outgroup sequence) and, of the 731 characters used in the phylogenetic analysis, 171 were parsimony-informative, 96 were variable and parsimony-uninformative and 464 were constant. only the �rst 1000 equally most parsimonious trees were retained from the heuristic search, the �rst of which is shown in Fig. 1 (tl 776, cI 0.485, rI 0.839, rc 0.407). the phylogenetic tree of the lsu region (Fig. 1) show that the taxonomy crous, r.w. Barreto, alfenas & r.F. alfenas, sp. nov. : named after the location where the holotype was Johansoniae brasiliensis morphologice similis, sed ascosporis m, discernitur. mato grosso, chapada dos guimarães, on leaves dimorphandra ( leguminosae ; False Barbatimao), 18 aug. P.w. crous, a.c. alfenas & r. alfenas , (cBs h-20484 – holotypus, cultures ex-holotype cPc 18475, 18474 cBs 128068). leaves with brown spots, but ascomata also occurring on dead and green leaf areas. mycelium super�cial, consisting of septate, branched, medium brown, verruculose to warty, 2–5 µm wide hyphae. ascomata on lower leaf surface, super�cial, situated on a hyphal stroma (occurring loosely on surface), discoid, dark brown, up to 300 µm diam, 200 µm high. exciple 15–20 µm diam, consisting of 3–6 layers of brown textura angularis to textura globulosa . asci in parallel layer, bitunicate with ocular chamber, sessile, narrowly ellipsoid to subcylindrical or clavate, 8-spored, 32–45 × 11–19 µm. Paraphyses intermingled among asci, hyaline, branched, septate, 1.5–2.5 µm wide, becoming somewhat darkened and branched towards the apical region, forming an epithecium. ascospores hyaline, thick-walled, medianly 1-septate, thick-walled, constricted at the septum, prominently guttulate, (13–)15–19(–24) × (5–)6–7 µm. ascospores after 24 h on mea germinating from both ends, with germ tubes parallel to the long axis of the spore, developing lateral branches; ascospores remaining hyaline, prominently constricted, not distorting, 5–7 µm wide. setae brown, erect, straight to curved, separate and surrounding ascomata, thick- walled, brown, smooth, with basal t-cell devoid of rhizoids, with slight taper towards apical cell, which is thin-walled, pale brown, and acutely to obtusely rounded, 5–10-septate, 130–260 × 4–5 µm; 2.5–3 µm wide at apical septum. culture characteristics : colonies spreading, erumpent, with sparse aerial mycelium and diffuse, submerged margins. on Pda surface pale mouse-grey (centre), olivaceous-grey (middle) with smoke-grey to cream outer region; reverse olivaceous-grey; colonies reaching 5 mm diam. on oa smooth, somewhat slimy, surface umber to dark mouse- grey; margin diffuse, reaching 8 mm diam. on mea, surface smoke-grey; reverse greyish-sepia, reaching 10 mm diam additional specimen examined Pernambuco: Poço do macaco, on sp., 18 sept. 1960, osvaldo soares de silva : the generic name Johansonia is based on J. setosa a species described from living leaves of collected in south america. the genus is characterised by having loose, super�cial, discoid ascomata situated on a hyphal stroma, and an exciple covering the bitunicate asci. , which are intermingled among asci, are hyaline, rous et al    branched, septate, and become somewhat darkened and branched towards the apical epithecium. ascospores are hyaline and 1-septate. ascomata are surrounded by brown, erect, straight to curved, septate setae (müller & von arx 1962). Based on these features, J. chapadiensis is a typical morphologically, J. chapadiensis closely resembles J. brasiliensis (Fig. 3). the two species can be distinguished in that ascospores of J. chapadiensis are smaller, (13–) 15–19(– 24) × (5–)6–7 µm, than those of J. brasiliensis, (18–24 × 6–7 µm). Furthermore, asci of J. chapadiensis are narrowly ellipsoid to subcylindrical or clavate, 32–45 × 11–19 µm, while those of J. brasiliensis are broadly ellipsoid, obovoid to subcylindrical, never clavate, and larger, 40–58 × 15–23 µm. Finally, setae in J. chapadiensis are more acutely rounded, 2.5–3 µm diam at the apical septum, while those of J. brasiliensis are bluntly rounded, and wider at the apical septum, 4–6 µm diam. although there are only 12 species of listed in Index Fungorum, von arx & müller (1975) were of the opinion Johansoniella maranhensis represented a further species . Batista et al. (1966) introduced the monotypic generic name ( ), based J. maranhensis , which they regarded as closely related . morphologically, the description appears Fig. 2. (cBs h-20484 – holotype). leaves colonised with ascomata on leaf surface from above (B, c), below (d), and a vertical section though an ascoma (e). F, germinating ascospores. vertical section through ascoma. ascospores. Bars: B, c 300 µm; d, e 150 µm; g, h 20 µm; F, I–l 10 µm. Johansonia   somewhat different, as the ascomata are described as having an upper wall layer (though this may be an epithecium), and setae around the ascomata, as well as on top of the ascomata. regardless of these supposed differences, von arx & müller (1975) treated in synonym with . a re-examination of the holotype specimen (urm 47621) found it to be depauperate, and hence the status of could not be resolved in the present study. an attempt to make a key to the species described to date based on published descriptions has not proven feasible, as too many species either have similar ascospore dimensions, or are insuf�ciently known. Based on published descriptions, most taxa only seem distinct if aspects such as dimenions of the ascospores, asci and setae are combined with host and distribution. however, as most taxa have been recorded once only, the value of these characters seems unreliable, and hence a key would only be feasible once the specimens of all described taxa have been re-examined to help resolve recent studies focused on elucidating the higher order phylogeny of dothideomycetes (schoch et al . 2009) and (crous et al . 2009b) did not treat , as the present collection represents the �rst known cultures of this genus. von arx & müller (1975) were of the opinion that belonged to , a treatment accepted by lumbsch & huhndorf (2007), though they regarded it as a family incertae sedis . Based on the dna phylogeny generated in the present study (Fig. 1), we can reveal that belongs to the ), and is closely related to and . however, whether it is a member of the (von arx & müller 1975, lumbsch & huhndorf 2007), could not be con�rmed, as presently there are no known cultures of this family available for comparison. Parts saccardiaceae have been transferred to von arx & müller (1975), and thus its close relationship to the suggests a likely family for this genus, pending further collections and study. we thank the technical staff, arien van Iperen (cultures), marjan vermaas (photo plates), and mieke starink-willemse (dna isolation, ampli�cation and sequencing) for their invaluable assistance. the curator of urm (recife, Brazil), dr leonor m. maia, is acknowledged available for study. arx Ja von, müller e (1975) a re-evaluation of the bitunicate ascomycetes with keys to families and genera. studies in Barr me (1993) redisposition of some taxa described by J.B. ellis. Batista ac, Bezerra Jl, cavalcanti aaas da silva (1966) , um novo gênero de atas do Instituto de micologia universidade Federal de Pernambuco, crous Pw (1998) spp. and their anamorphs associated with leaf spot diseases of crous Pw, gams w, stalpers Ja, robert v, stegehuis g (2004) mycoBank: an online initiative to launch mycology into the 21st century. crous Pw, groenewald Jz, risède J-m, simoneau P, hyde Kd (2006) species and their anamorphs: crous Pw, groenewald Jz, summerell Ba, wing�eld Bd, wing�eld mJ (2009a) co-occurring species of teratosphaeria on ascoma on leaf. asci and ascospores. Bars: a 300 µm; B–d 10 µm.    rous et al crous Pw, schoch cl, hyde Kd, wood ar, gueidan c, hoog gs de, groenewald Jz (2009b) Phylogenetic lineages in the crous Pw, verkley gJm, groenewald Jz, samson ra (eds) Fungal Biodiversity . [cBs laboratory manual series 1 crous Pw, wing�eld mJ, Park rF (1991) mycosphaerella nubilosa a Frank J, crous Pw, groenewald Jz, oertel B, hyde Kd, Phengsintham P, schroers h-J (2010) microcyclospora and novel genera accommodating epiphytic fungi causing sooty hoog gs de, gerrits van den ende ahg (1998) molecular diagnostics of clinical strains of �lamentous basidiomycetes. lumbsch ht, huhndorf sm (eds) (2007) outline of ascomycota - müller e, arx Ja von (1962) die gattungen der didymosporen Beitrage zur Kryptogamen�ora der schweiz 11 rayner rw (1970) a mycological colour chart . commonwealth mycological Institute, Kew. saccardo Pa (1889) Johansonia setosa sylloge Fungorum (abellini) schoch cl, shoemaker ra, seifert Ka, hambleton s, spatafora Jw, crous Pw (2006) a multigene phylogeny of the schoch cl, crous Pw, groenewald Jz, Boehm ewa, Burgess et al. (2009). a class-wide phylogenetic assessment of vilgalys r, hester m (1990) rapid genetic identi�cation and mapping of enzymatically ampli�ed ribosomal dna from several white tJ, Bruns t, lee J, taylor J (1990) ampli�cation and direct sequencing of fungal ribosomal rna genes for phylogenetics. : Innis ma, gelfand dh, sninsky JJ, white tJ (eds), Protocols: a guide to methods and applications : 315–322.   © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. Int roduc tI Biological diversity (biodiversity) encompasses the variety of life forms occurring in nature, from the ecosystem to the genetic level, as a result of evolutionary history (wilson 1992). the idea that fungi form a kingdom distinct from plants and animals gradually became accepted only after whittaker (1969). Presently, the “fungi” as a mega-diverse group span three kingdoms, most belonging to the ( while others are classi�ed in the and ) (cavalier-smith 1998, James et al. 2006b). the word “fungi”, lower case and not in italics, is commonly used as a collective term for organisms traditionally studied by mycologists from all three kingdoms (hawksworth 1991). the myxomycetes have also been traditionally studied by mycologists (everhart & Keller 2008, rojas & stephenson 2008), and are included here. estimates for the number of fungi in the world range up to ca. 13.5 m species (mcneely et al. 1990, hawksworth 1991, 2001, hawksworth & Kalin-arroyo 1995, hyde 1996, hyde 1997, tangley 1997, groombridge & Jenkins 2002, Brusca & Brusca 2003, rossman 2003, crous et al. 2006, adl et al. 2007, Kirk et al. 2008). It might be expected that the predicted numbers of fungi on earth would have been considerably greater than the 1.5 m suggested by hawksworth (1991), which is currently accepted as a working �gure although the 10 edition of ainsworth & Bisby’s dictionary of the et al. 2008) provided a total of 98 998 for the number of fungal species accepted to date (excluding taxa treated under and ). Kirk et al. (2008) reported 1 039 species chromistan fungal analogues 1 165 as protozoan in which 1 038 are regarded as protozoan fungal analogues: ( dictyostelia, myxogastria, Protostelia , cercozoa ) which were previously treated as egypt’s geographical position at the junction between two large continents (africa and asia), and its inclusion as part of the mediterranean basin, has indelibly in�uenced both the people and the biota of the country socially, economically and biologically. egypt is part of the sahara of north africa, it has an area of about 1 m km , divided by the river nile into a western part including the libyan desert (681 000 ) and an eastern part comprising the eastern desert (223 000 km ), and the sinai Peninsula (61 000 km ). the nile basin, comprising the valley in the south (upper egypt) and nile delta in the north (lower egypt), forms a riparian oasis (40 000 km ) that constitutes the densely inhabited farmlands the history, fungal biodiversity, conservation, and future perspectives for mycology in egypt ahmed m. abdel-azeem Botany department, Faculty of science, university of suez canal, Ismailia 41522, egypt; e-mail: zemo3000@yahoo.com abstract: records of egyptian fungi, including lichenized fungi, are scattered through a wide array of journals, books, and dissertations, but preliminary annotated checklists and compilations are not all readily available. this review documents the known available sources and compiles data for more than 197 years of egyptian mycology. species richness is analysed numerically with respect to the systematic position and ecology. values of relative species richness of different systematic and ecological groups in egypt compared to values of the same groups worldwide, show that our knowledge of egyptian fungi is fragmentary, especially for certain systematic and ecological groups such as agaricales , glomeromycota , and lichenized, nematode-trapping, entomopathogenic, marine, aquatic and coprophilous fungi, and also yeasts. certain groups have never been studied in egypt, such as trichomycetes and black yeasts. By screening available sources of information, it was possible to delineate 2281 taxa belonging to 755 genera of fungi, including 57 myxomycete species as known from egypt. only 105 taxa new to science have been described from egypt, one belonging to chytridiomycota 47 to ascomycota , 55 to anamorphic fungi and one to Basidiomycota article info: submitted: 10 august 2010; accepted: 30 october 2010; Published: 10 november 2010. Key words: checklist distribution fungal diversity lichens mycobiota species numbers Ima Fu gus volu me 1 o 2: 123–142 ahmed m. abdel-azeem    Kassas (2002) mentioned four gaps related to biodiversity knowledge: the number of species on earth; the diversity of the less conspicuous organisms such as fungi, bacteria, algae, and protozoa; the role played by each species among biotic elements of ecosystems; and the human ability to documentation of the egyptian fungi may be dated back to 4500 B.c., when ancient egyptians produced a number of hieroglyphic depictions of plants (many of which are psychedelic) on walls and within texts throughout egypt. temples with countless pillars are shaped like huge mushrooms with tall stems, umbrella caps, and mushroom engravings distributed all over the country (Fig. 1). these are shaped like sporophores, and some like . others look like bracket fungi and are decorated with pictures of an incredible variety of plants (arthur 2000). In the egyptian Book of the dead, the Papyrus of ani (Budge 1967), mushrooms are called “the food of the gods,” studies on fungi in egypt started at the beginning of the century on lichens (e.g. delile 1813a, b, nylander 1864, 1876, müller 1880a–c, 1884, stizenberger 1890, 1891). In the early 20 century, sickenberger (1901) and steiner (1893, 1916) provided information for collections of lichens from egypt in the 19 and early 20 century. In the , delile (1813a) presented a scienti�c study of egyptian fungi into the early19 century (mouchacca 2008), in which he described the gastromycete now known as Phallus roseus ; Fig. 1) which he had collected in damietta and assiut in 1798 and 1799, respectively. It should be noted that some early works repeat previous records, sometimes ambiguously as a result of the misinterpretation of synonyms and erratic use of infraspeci�c ranks; further, in the case of sickenberger, misspellings of scienti�c names By the beginning of the 20 century, special attention was being given to phytopathogenic fungi on wild and domesticated plants of economic importance (e.g 1902, reichert 1921, Fahmy 1923, shearer 1924, Briton- Jones 1922, 1923, 1925, Bishara 1928, melchers 1931, sirag Both reichert and melchers are considered the pioneer scientists in the documentation of egyptian fungi. Israel reichert (1891–1975) went to study in germany. here he obtained his doctorate on die Pilz�ora Ägypten in which 237 species were recognized, of which 42 were new to science. unfortunately, none of his specimens were retained in egypt, or if they were, there is no record of their whereabouts today. however, earlier material collected before 1914 was present in the Botanisches museum in Berlin-dahlem, which reichert used when compiling his list of 1921, but it is In 1927 leo e. melchers went to egypt at the invitation of the egyptian minister of agriculture as chief mycologist for 18 months. he met a series of dif�culties such as there being no records available on the occurrence, distribution, or dates of any mycological observations conducted previously by any investigator in egypt, and no mycological reference collection existing in the country. his checklist, however, included 345 species of fungi, especially those causing plant diseases no studies were carried out on the soil fungi until the 1930s, yet it was to be expected that, in such a country with rich agricultural traditions, knowledge of these fungi should have attracted considerable interest. research on egyptian soil fungi was probably commenced by younis salem sabet (1898–1977). sabet graduated in 1921 from the high school of agriculture (now the Faculty of agriculture of cairo university), and soon after was sent to england to study botany at the university of london, where he obtained a Bsc (hons) in 1925. after his return, he joined the ministry of agriculture in the Plant Breeding section. In 1927 he was appointed lecturer in Botany in the faculty of science of the newly established egyptian university, and in 1935 published his pioneering study, which was followed by many other publications (sabet 1936, 1938, 1939a). his exploration led to the discovery of three taxa which were described later as new to science. sabet took the initiative in the establishment of some scienti�c organisations, and served as a member and president for several years in some others. Particularly of note were the egyptian academy of sciences, egyptian Botanical society, egyptian science union, egyptian association for scienti�c culture, society of applied microbiology, egyptian Phytopathological society, society for the history of science, near the end of the 1930s, new aspects of mycological research were introduced into egypt by several investigators such as mycorrhizal fungi (mostafa 1938, sabet 1939b, 1940, 1945, yousef 1946); biocontrol (mostafa & gayed 1953), rhizosphere (montasir et al. 1956, naim et al. 1957), air (saad 1958, zaki 1960), and stored seeds and grains In 1956 late magdy a. ragab (department of Botany, Faculty of agriculture, university of cairo) isolated 16 new species for the �rst time from soil, water and some plant however, the credit for initiating real research concerned with egyptian fungi must be given to abdel-al h. moubasher Fig. 1. a. mushroom-like pillars (upper part), which are common in egyptian temples. B. description of Phallus roseus by mycology in egypt   (Botany department, Faculty of science, assiut university; Fig. 2). In the early 1960s, with colleagues and students, he broadened the scope of mycological research in egypt by conducting many studies on fungi. these included aspects such as: cellulose-decomposition, thermophily, osmophily, seed and grain mycobiota, phylloplane fungi, mycotoxins, and aquatic fungi. moubasher, with his colleagues and students, have published more than 150 scienti�c papers to date, and in 1993 he published his major contribution to mycology in the arabic world the lavishly illustrated soil fungi of qatar and other arab countries (moubasher 1993). he also invited outside specialists to run courses from the 1980s and trained many Phds students. specialists included colin Booth and el-abyad & abu-taleb (1993) summarized the habitat diversity of egyptian fungi, and in 1997 the late samy m. el-abyad (Botany department, Faculty of science, cairo university; Fig. 2) presented his pioneering attempt to update the checklist of egyptian fungi: 1 246 species were recorded of which 173 were referred to , 41 to , 222 , 143 to and 667 to . different ecological and taxonomic groups were not separated cited in the checklist, such as protozoan fungal analogues ( ), lichens, yeasts, aquatic and marine fungi, entomopathogenic fungi, nematophagous fungi, and mycorrhizal fungi. a large numbers of taxa, either reported in routine isolations or as novel taxa, are completely absent from this list. this may be due to his inability to trace the majority of references, which is actually the main reason why updated information documenting the fungi of egypt was needed today. amongst records lacking in the el-abyad (1997) checklist are seven species (melchers 1931), chaetomium gelasinosporum and c. uniporum (aue & müller c. mareoticum (Besada & yusef 1969), (lundqvist 1969), Podospora aegyptiaca (lundqvist 1970), thermoascus aegyptiacus (udagawa & ueda 1983), and gelasinospora hippopotama (Krug et al. In addition to the previous efforts of reichert (1921), melchers (1931), el-abyad & abu-taleb (1993), and el- abyad (1997), several other studies have added to the documentation of egyptian fungi: moubasher (1993), lado (1994), mouchacca (1995, 1999, 2001a, b, 2003a, b, 2004, 2005, 2008, 2009a, b; Fig. 2), moustafa & abdel-azeem (2005a, b, 2006, 2010), moustafa (2006), and seaward & the late abdel razak abo-sedah organized the second african regional mycological congress, in cairo in 1992, under the auspices of the Ima committee for the Fig. 2. a selection of prominent mycologists who have contributed greatly to our knowledge of mycology in egypt. a. abdel-al h. moubasher. samy m. el-abyad. Jean mouchacca. abdul-wahid F. moustafa. Farida t. el-hissy. F. younis s. sabet. Israel reichert. youssef a. youssef. ahmed m. abdel-azeem    development of mycology in africa. then in 1993 he founded the regional center for mycology and Biotechnology (rcmB) in al-azhar university, cairo. the major tasks of this centre were the establishment of a fungal culture collection, the application of fungi in public health, agriculture, environment and industry, and supporting researchers as well as research projects. the centre actively participated in organizing further african regional and international conferences and meetings in cairo in 1994, vancouver in 1994, zimbabwe in 1995, cairo in 1996 on “regulations of fungal activities”, and again in cairo in 1999 on “Fungi and the environment”. the center had collaborative agreements with the former International mycological Institute (ImI) in the uK, and collaborative activities with egyptian universities as well as with others in the uK, south africa, mauritius, zimbabwe and austria. the centre also initiated and published the african Journal of mycology and Biotechnology from 1993 to 2001, which contained numerous contributions by egyptian authors, and From the beginning of 2005 to the end of 2007, the Biodiversity monitoring and assessment Project (Biomap) had as its primary objective to develop and strengthen biodiversity research, monitoring and assessment across egypt. In this project an extensive e-database was established to map the distribution of species across egypt, and document up to as mentioned above, the information concerning the fungi of egypt is still incomplete and cannot be fully documented without an updated checklist of all taxa reported for the country. the p resent contribution assesses the diversity of fungi in egypt. In addition, major groups of fungi are discussed brie�y to highlight the extent of their diversity, followed by examples of habitats that are unique and deserve greater attention. t hese data show that the present contribution is a preliminary one concerning the diversity of egyptian fungi, and therefore this summation is intended to enhance our knowledge of, and stimulate research into, the fungi of egypt. mate an the present contribution is based on an exhaustive revision of the available literature and sources of the egyptian fungi reported from the 19 th century to the present, including dissertations, published papers, compilations and checklists. name corrections, authorities, and taxonomic assignments of all taxa reported in this article were checked against the Index Fungorum database ). In addition, websites of international mycological centres such as the atcc (usa) ( ), caBI (uK) <194.203.77.76/grc/index.htm> , cBs (the netherlands) ( ), mucl (Belgium) cabri.org/ htdig/index-ebrcn.html> ) and the catalogue of the culture collection of the assiut university mycological center (aumc 2010) were also consulted. the systematic arrangement in the present article follows Kirk et al. (2008). this study extended to more than eight years in documenting and updating the information on egyptian fungi. a ll results of the present study can be checked against the number of fungi recorded in egypt is 2 281 species, out of which 105 taxa have been described from egypt as new to science: one in chytridiomycota , 47 in 56 in anamorphic fungi, and one in . reichert introduced 24 of the new taxa, representing 24.7 % of the novel taxa, followed by Jean mouchacca and his colleagues (laboratoire de cryptogamie, muséum national d’histoire naturelle, Paris), who described 18 new species (17.1 % of the total), and abdul-wahid F. moustafa (Fig. 2) and his colleagues and students at the suez canal university who contributed 11 new taxa. the kingdom contains 115 000 known species. they are extremely diverse in their cell structure, patterns of nutrition, metabolic needs, reproduction, and habitat. this kingdom contains a grab-bag of organisms that do not �t into the other kingdoms. are extremely dif�cult to classify so for the purpose of this survey, they are grouped by their nutritional patterns. Protozoan fungal analogues are heterotrophic and most are decomposers that feed on dead plants and animals by endocytosis (Kendrick 2000). according to Kirk et al. (2008) there are about 1 165 fungal protozoan analogues described. slime moulds are a small and relatively homogenous group of eukaryotic organisms, and these are referred to as ( ). In egypt the slime moulds have never been the target of any widescale study (lado 1994, stephenson & stempen 1994), except for the pioneer study of abdel-raheem (2002) on abdel-raheem (2002) reported 20 species belonging to 17 genera in his �rst inventory of the protozoan fungal analogues ( myxomycota ) of upper egypt from wood, bark of living and dead trees and leaf litter. exhaustive examination of all available literature concerning protozoan fungal analogues in egypt led to the discovery of reports of Protostelium irregulare (as “ irregularis” ; olive & stoianovitch 1969) and eidamella spinosa (Kowalik & sadurska 1973). the protozoan fungal analogues occurring on decaying wood, bark, leaf litter and papyrus papers presently amount to 57 species belonging to 25 genera. For more details refer to the PBI: global Biodiversity of eumycetozoans ) and Farghaly (2008). In addition, three species representing three genera of cercozoa (previously Plasmodiophoromycota have been recorded: Plasmodiophora , spongospora, and woronina . no dictyostelid cellular slime moulds are so far known from egypt (cavender et al. 2010). mycology in egypt   the kingdom ) is a collection of eukaryotic, walled microorganisms that produce heterokont, wall-less cells in their life-cycles, including some fungal- like groups that are not considered to be ancestors of any members of the (lutzoni et al. 2004). Kirk et al. (2008) estimated the chromistan fungal analogues as 1 039 known species and included the phyla , and along with some taxa of the late Farida t. el-hissy (Botany department, Faculty of science, assiut university; Fig. 2) was the founder of aquatic mycology research in egypt. el-hissy and her students published more than 60 papers on this topic. however, the plant pathogenic have been the target of many research investigations since 1921, and in the present study, 186 taxa of chromistan fungal analogues were recorded, of which 172 belong to 40 genera of . Four species and two genera of labyrinthista were recorded, while are represented by six species within three genera. For more details refer to el-helaly et al . (1963, 1966), ali hassanein et al . (1972), Khallil et al . (1995), and this phylum was once considered part of the chytrids. however, most of the true chytrids ( produce a limited mycelium while usually make extensive mycelia. thus, they super�cially resemble the water moulds to which they were thought to have been af�liated. like the chytrids, neocallimastigomycota are the only members of the fungi in which motility has been retained. In overall growth habit, the blastocladiomycetes tend to be eucarpic, in which there is an extensive vegetative growth habit in which some part of the organism participates in reproduction (asexual and sexual). members of this phylum do exhibit a complete alternation of generation between a haploid gametophyte and a diploid sporophyte (Barr 1990, James et al. 2006a). et al. (2008) give a world total for of 179 species. In egypt, 27 species and one variety belonging to seven genera of were found in this study. For more details see ragab (1956), yusef (1964), gad et al. (1967), gad & sadek (1968), el-hissy (1974), el-hissy et al. (1997), el-abyad (1997), shoulkamy et al. (2001), and are a phylum of fungi that reproduce through the production of motile spores (zoospores), typically propelled by a single, posteriorly directed �agellum. these organisms, often referred to as chytrid fungi or chytrids, have a global total of approximately 1 000 described species et al. 2006a). Based on biochemical characteristics, including chitin in cell walls, the α-aminoadipic acid lysine synthetic pathway, and storage carbohydrates (i.e. glycogen), Bartnicki-garcia (1970) classi�ed as true fungi and this is supported by current molecular studies et al. 2007). In the past some authors considered the chytrids as a transitional group between protists and fungi because of their production of motile zoospores (Barr 1990). et al. (2008) give the number known as the study of gaertner (1954) on of africa is considered one of the pioneer mycological studies in egypt. however, the real start of research on chytrids in egypt must be credited to samy Kamel mohamed hassan (minia university) who obtained his Phd from the university of warsaw for work on chytrids and aquatic fungi in 1982. later, hassan and mohamed abdel-wahab el-naghy (minia Intensive revision of the nomenclature showed that 84 species belonging to 32 genera of were recorded in egypt. For more details see el-naghy et al. (1985, 1987), hassan (1991a-d, 1993), hassan & Fadl-allah (1991), are a particularly ecologically diverse group of fungi, occurring as saprobes ( ), harmless inhabitants of arthropod guts ( ), plant mutualists forming ectomycorrhizas ( endogonales ), and pathogens of animals, plants, amoebae, and especially other fungi (all and some are mycoparasites) (James & o’donnell 2007). conversely, some have a negative economic impact as they cause storage rots or plant diseases, while others can cause life-threatening opportunistic infections in diabetic, immuno-suppressed, and immuno-compromised patients. In addition, several species cause serious human infections (de hoog according to Kirk et al. (2008) the total world number of is 1 065 species. data collected from previous studies show the in egypt to be fragmentary because members belonging to this group either have long been overlooked or simply reported as rare taxa during abdel-Kader (1973) carried out a pioneering study in which he was able to isolate 11 species from a range of soils collected from various egyptian localities. the second most relevant study is probably that of al-alfy (1995) who reported 21 species from various substrates including soil, dung, stored seeds and grains, and the phyllosphere. In his recent contribution on in egypt, moustafa (2006) reported 33 species, out of which nine were considered new revision of all available data showed that zygomycota in egypt comprises 70 taxa including eight varieties and seven special forms within 35 genera. In addition, absidia aegyptiaca (sartory et al. 1939) is omitted from the list, as no living or other type of authentic material is apparently preserved; furthermore the name was not validly published as it lacked a ahmed m. abdel-azeem    latin diagnosis (mouchacca 1995). For more information on egyptian zygomycota refer to Kharboush (1969a, b), Besada & yusef (1968), abdel-rahman et al. (1990), moubasher (1993), el-abyad & abu-taleb (1993), swelim et al. (1994), mouchacca (1995), el-abyad (1997), abdel-azeem (2003), moustafa (2006), ali & Ibrahim (2008), a�fy et al. (2009), and moubasher et al. (2010). currently comprises 169 described species (Kirk et al. 2008). the phylum is not as diverse as other phyla of fungi with only three families and such a modest number of species. however, they make up for this uniformity by being among the most abundant and widespread of all fungi. as far as we know, all species of are mutualistic with plants, forming endomycorrhizas. although there are various types of mycorrhizas, involving different fungal and plant symbionts, the arbuscular mycorrhiza type is the most widespread occurring in around 80 % of plant the pioneering work of mostafa (1938) and sabet (1939b, 1940, 1945; Fig. 2) is now accepted as the starting point of research on egyptian glomeromycota (Kelley 1950, abdel- moneim & abdel-azeem 2009). these studies were followed by many other investigations concerned mainly with the ecology and physiology of endomycorrhizas in egypt, viz. Fares (1986), Ishac et al. (1986), abdel-Fattah (1991), aboulkhair & el-sokkary (1994), mankarios & abdel-Fattah (1994), abdel- Fattah & mankarios (1995), abdel-Fattah & rabie (1995), abdel-Fattah et al. (1996), abdalla & abdel-Fattah (2000), abdel-Fattah (2001), and abdel-azeem et al. (2007). however, surveys of egyptian glomeromycota are limited, and had never been the sole target of any study until Fares (1986) conducted a survey of vesicular arbuscular mycorrhizas, followed by agwa (1990) on mycorrhizas and nodulation in some egyptian plants. after 10 years, agwa (2000) studied the arbuscular mycorrhizal fungi associated with medicinal plants as glomales in egypt (I)”. agwa & abdel-Fattah (2002) followed up their work “ glomales in egypt (II)” as an ecological view of some saline affected plants in the delta of the mediterranean coast. a study of the distribution of glomales in the egyptian Protectorates was published by agwa & al-sodany (2003) as “ glomales in egypt (III)”, which surveyed the distribution and ecology in some plants in the el-omayed Biosphere reserve. later, other relevant studies were carried out by several investigators such as el- zayat et al. (2007) and abdel-moneim & abdel-azeem (2009) on the wadi allaqi and saint Katherine Protectorate, respectively. recently, mansour (2010) screened 71 soil and root samples for endomycorrhizas in north sinai and adopted some of them as biocontrol agents against fusarium-wilt of tomato. eight genera and 19 species have been recorded in egypt since 1938: acaulospora, entrophospora, gigaspora, glomus, Paraglomus, sclerocysti, scutellospora . Both Paraglomus occultum were recorded and never cited in any publication related to egyptian . For more details see sabet lichens are unique associations composed of two to three different organisms living together in a mutualistic relationship in which the fungal partner forms the external structure. the name used is that of the fungal parter, and the photosynthetic partner or partners have independent scienti�c names. estimates for the number of lichen fungi worldwide vary, but a draft global checklist has 18 882 names of lichen-forming egyptian lichens have received the attention of many researchers since the early 1800s (delile 1813a, b, nylander 1864, 1876, müller 1880a–c, 1884, stizenberger 1890, 1891, sickenberger 1901, steiner 1893, 1916, werner 1966, galun & garty 1972, temina et al. 2004, 2005, seaward & sipman 2006). egyptian investigators have participated in a few studies of lichens, namely in north sinai (Khalil 1995) and on trees (Koriem 2003), and there have also been some physiological studies on the bionts (Koriem 2006). Khalil (1995) recorded 43 species belonging to 18 genera, all of which are ascolichens without any basidiolichens at all, and only one of these had a perithecioid ascoma ( seaward & sipman (2006) reported 157 taxa of lichenized fungi (149 species and 8 infraspecific taxa) and six lichenicolous fungi (fungi obligately growing on lichens). Foliose lichens are very scarce, only being represented by the Xanthoria (7 species) and (1 species). the fruticose growth form is better represented, with members of the genera and at the family level, accommodated the most taxa (39), followed by roccellaceae (16), and (12). For more information concerning egyptian lichens please see: check-lists of lichens and lichenicolous Fungi ( galun & garty (1972), Khalil (1995), Koriem (2003, 2006), constitute by far the largest group of fungi so far known, accommodating a relatively large assemblage of taxa estimated to be 65 % of all described fungi (Kirk et al. 2008) occurring in various habitats; aquatic or terrestrial, under moderate or stress conditions (Kodsueb et al. 2008a, b, Kruys & ericson 2008, thongkantha 2008). a large number of species are economically important (e.g. spp., Kvas et al. spp., damm et al. 2009, hyde et al. spp., crous 2009), while few are edible (morels and truf�es), and some are used also in the production of food (including bread), drinks, organic acids, mycofungicides, fungal biofertilizers, cosmetics and this phylum encompasses biologically diverse forms. many are free-living saprobes including species which may be cellulose decomposers, chitinolytic, keratinolytic, or mycology in egypt   coprophilous, others are parasitic forms including species which cause very serious plant diseases like powdery-mildew, wood-canker, ergot, rot, blight, scab, leaf curl, and leaf-spots (e.g. alves et al. 2008, aveskamp et al. 2008, simonis et al. 2008, wulandari et al. 2009). others that are considered symbiotic forms contain species which live in association with characteristically, when reproducing sexually, produce non-motile spores (ascospores) in a distinctive “ascus”. however, some members of the do not reproduce sexually and do not form asci or ascospores ). these asexual members are assigned to based upon morphological and/ or physiological similarities to ascus-bearing taxa, and in particular by phylogenetic comparisons of dna sequences. In old classi�cation systems these were often placed in a separate arti�cial phylum, the deuteromycota (or “fungi imperfecti”). molecular analyses can now place these genera and species among ascus-bearing taxa, or more rarely in the �rst reports of mutualistic non-lichenized ascus- forming fungi from egypt were those of terfezia tirmania, by reichert (1921), and then melchers (1931) who recorded six species. later, sabet (1935) recorded some chaetomium species. the saprobic did not receive attention, and therefore information remained limited until the early 1970s, when some research on the group was initiated by moubasher and his co-workers during their studies on soil fungi. since then, fragmentary information has been accumulating, but these fungi had never been the main objective of any egyptian study focusing on their ecology, distribution, and substrate preferences, untill the study of three hundred and three species of teleomorphic (including ascosporic yeasts) have been recorded from all terricolous substrates of egypt (moustafa & abdel-azeem 2010, and unpubl.). In their studies, 10 species of edible were recorded from egypt within the genera morchella, terfezia, tirmania . In total, 328 taxa were recorded in this survey, of which only 32 species are ascosporic yeasts. Binyamini (1973) reported Peziza vesiculosa as a coprophilous fungus from occupied Palestine, and some samples were even collected from north sinai during the occupation in 1967, but never cited as an egyptian record in any checklist. In addition, was recorded for the �rst time in egypt by el- saadawi & shabbara (1999) as an association between a For more details see sabet (1936, 1939a), Binyamini (1973), el-saadawi & shabbara (1999), abdel-hafez et al. (1995), Ibrahim (1995), el-abyad (1997), zaki et al. (2005), moustafa & abdel-azeem (2005a, b, 2006, 2008, 2010), and records of phytopathogenic fungi in egypt were scattered through the literature until 1921, when Israel reichert (Fig. 2) carried out his pioneer study of egyptian fungi. this was followed by a comprehensive checklist of plant diseases and fungi occurring in egypt by melchers (1931). records concerning aspects of plant pathology in egypt continued to be accumulated during many decades until el-helaly et al. (1963, 1966) started to update the information, and another updated bibliography of agricultural studies conducted in egypt between the period 1900 to 1970 appeared et al. 1972). this revealed records of 82 species of teleomorphic plant pathogenic . For more details please check, natrass (1933), abou el-seood (1968), ghoniem (1985), el-desouky & el-wakil (2003), Phillips et al. anamorphic genera are gradually disappearing into the overall ascomycete system, though it will take many years a school of medical mycology in egypt was formed at the beginning of 1967, when the late youssef a. youssef (ain shams university, Faculty of science; Fig. 2) published two papers on fungus infection of the human ear. youssef and his students and colleagues became interested in medical mycology, serology and fungi affecting human health. For more details see youssef & abdou (1967a, b), hassan et al. (1980a–e, 1981), youssef & Karam el-din (1988a, b), Karam et al. (1994 a–c, 1995, 1996), and youssef et al. (1989, In 1979 Ismail abdel-razak m. el-Kady received credit as the egyptian mycologist working on mycotoxin producing fungi in egypt. el-Kady and his coworkers studied the majority of aspects related to toxinogenic fungi, e.g. factors affecting mycotoxin production, toxinogenic taxa in food and feed, and mutagenic effects of fungal toxins. For more details see el- Kady & moubasher (1982 a, b), and el-Kady et al. In 1987, mamdouh s. haridy (minia university, Faculty of science) became the pioneer egyptian mycologist in yeast identi�cation and taxonomy, having completed his Phd thesis on the taxonomy of yeasts (“taxonomie milchwirtschaftlich wichtiger hefen”, technical university, munich). he conducted a series of extensive studies on the egyptian saprobic yeasts from different ecological habitats and sources (haridy 1992a, recently, other areas of egyptian mycology have been established, such as on the identi�cation of human plant pathogens by molecular techniques. youssuf a. (Botany department, Faculty of science, south valley university) focused on the identi�cation of plant pathogens and saprobic fungi of food by means of molecular techniques (gherbawy 2004, gherbawy & abdelzaher 2002, gherbawy & Farghaly 2002, gherbawy & voigt 2010), and sherif m. (microbiology department, Faculty of shams university) extended the research of youssef a. youssef using the molecular techniques in species identi�cation of human pathogens (zaki et al. 2005, about 905 filamentous or yeast-like anamorphic fungi have been reported from egypt. these taxa colonize, survive and multiply in air, litter, soil, plant surfaces, the human body and other substrates. of these, only 28 are species ahmed m. abdel-azeem    of anamorphic ascomycetous yeasts, which belong to three genera. Furthermore, five genera of basidiomycetous yeasts For more information consult al-doory (1968), abdel- Fattah (1985), sherief (1985), Bagy & abdel-hafez (1985), Khater (1989), shalouf (1989), shindia (1990), abdel-mallek et al. (1995), abdul wahid et al. (1996), hamdi & hassanein (1996), el-tanash (1997), shalaby (1999), mahmoud (1999), Ismail & sabreen (2001), teramoto et al. (2001), abdel- wahab (2002), Farghaly et al. (2004), nofal & haggag (2006), et al. (2007), abdel-hamed (2008), and Kottb (2008). marine fungi form an ecological, and not a taxonomic group (raghukumar 2008, Jones et al. 2009, hyde et al. 2000). among these, the obligate marine fungi grow and sporulate exclusively in seawater, and their spores are capable of germinating in seawater (hyde et al. 1998). on the other hand, facultative marine fungi are those obtained from freshwater or a terrestrial , and have undergone physiological adaptations that allow them to grow and possibly also sporulate in the marine environment (Kohlmeyer & Kohlmeyer 1979). these fungi belong mostly to ascomycetes, their anamorphs, and a few basidiomycetes. among the straminipilan fungi, those belonging to labyrinthulomycetes , comprising the thraustochytrids, aplanochytrids, and labyrinthulids are obligate marine fungi (raghukumar 2002), and those belonging to the oomycetes are also fairly widespread in the about 3000 fungi (exclusive of yeasts) have been reported from aquatic habitats of which (1 527 spp.) and anamorphic taxa (785 spp.) are the most diverse groups, followed by (576 spp.) with (21 spp.) as the least diverse group (vijaykrishna et al. 2006, anwar abdel aleem (Faculty of science, university of alexandria), or Peripatetic aleem as he was known among his colleagues, is one of the most brilliant arab marine botanists and oceanographer extraordinaire. he is considered one of the pioneer marine egyptian mycologists, with studies on marine fungi dating back to 1950 (aleem 1950a–c, 1952a–c, 1953, 1962, 1974, 1975, 1978, 1980a, b, aleem & mailbari In egypt, obligate and facultative marine fungi are considered as forgotten fungi (Jones 2001) because they never featured in research topics until 1993, which is considered the starting point of marine mycology research in egypt. this provided mohamed abdel-wahab (Botany department, Faculty of science, south valley university, sohag, egypt) the possibility to publish his pioneering study on the egyptian obligate mangrove-inhabiting fungi of the red sea in 1996. three contributions of el-sharouny et al. (1998, 1999) and abd-elaah (1998) shed light on the ecology and taxonomy of mangrovicolous, algicolous and aquatic fungi of the red sea in upper egypt. abdel-wahab (2000) obtained his Phd on the biodiversity of fungi in subtropical mangroves; he recorded 25 fungi on intertidal wood of avicennia marina collected from three mangrove stands of the red sea coast of egypt. abdel-wahab et al. (2001a, b) published three new species, halosarpheia unicellularis swampomyces aegyptiacus s. clavatispora, from red sea mangroves. Pang et al. (2002) erected as a new lignicolous freshwater ascomycete order with the new Patescospora separans from egypt. abdel-raheem (2004) studied the effect of different techniques on diversity of freshwater hyphomycetes in the river nile (upper egypt). abdel-wahab (2005) examined the diversity of marine fungi on intertidal decayed wood of a. marina and on decayed prop roots of rhizophora mucronata in mangrove stands in the southern part of the egyptian red sea coast; 39 species were identified on decayed wood of a. marina, of which 19 were new records for egypt and the red sea. Freshwater fungi are those relying on freshwater for at least part of their life-cycle (wong et al. 1998, raja et al. 2009). abdel-aziz (2008) studied the diversity of aquatic fungi in lake manzala, which was the first report of aquatic fungi from the lake. sixty taxa including 26 ascomycetes and 34 anamorphic fungi were recorded, of which 19 species were new records for egypt. el-sharouny et al. (2009) studied the fungal diversity in brackish and saline lakes in egypt; 97 fungi (40 ascomycetes, 55 anamorphic fungi and 2 basidiomycetes) were identified from 764 collections, obtained from 545 samples, of which 70 the revision of all available data sources reveals that the total number of marine and aquatic fungi known in egypt is 207 taxa (87 , 117 anamorphic taxa, and 3 ). there is no checklist of aquatic egyptian fungi so far. For more details on these fungi see the website ( ), search mangrove fungi ( ), and check relevant studies (Khallil 2001, abdel-aziz 2004, abdel-wahab et al. 2009, the taxonomy of the entomopathogenic fungi has received much attention since the 1970s. more than 700 species of fungi are associated with insects, spiders, and mites (samson et al. 1988, hajek & st. leger 1994, sung et al. 2007, aung the invertebrate pathogenic fungi can be classi�ed in the , and allied anamorphic fungi; no truly entomopathogenic basidiomycetes have been documented (samson et al. 1988). entomopathogenic fungi range from commensals or mutualists, through ectoparasites which do not seriously affect their hosts, to pathogens which are lethal and include representatives of all the groups of fungi (hawksworth et al. Few records appeared reporting the occurrence of entomogenous fungi in egypt until natrass (1932) published preliminary notes on some of these fungi in egypt. he recorded �ve species: empusa grylli ( aspergillus �avus mucor racemosum, metarhizium anisopliae . In the beginning of the 1960s, mycology in egypt   egypt started to apply biocontrol methods to insects by entomopathogenic fungi, and gad et al. (1967) studied the there are several studies on this ecological group of fungi in egypt, such as Badran & aly (1995), shoulkamy et al. (1997), shoulkamy & lucarotti (1998), hafez et al. (1997), sewify (1997), abdel-Baky (2000), sewify com/journal/119022140/abstract?cretry 1&sretry 0 - fn1#fn1&> hashem (2001), abdel-sater & eraky (2002), ali (2003), <3.interscience.wiley.com/journal/119022140/ abstract?cretry 1&sretry 0 - fn1#fn1> abdel-mallek et al. (2003 a, b), el-hady (2004), mourad et al. (2005), abdel-mallek & abdel-rahman (2006), el-maraghy et al. (2006), and moubasher et al. (2010). as a result of these only 18 species belonging to 13 genera were recorded as entomopathogenic fungi of egypt. For more details please In egypt, the study of nematophagous fungi dated back to 1963 when hamdy ( department of Plant Pathology, nematology laboratory, national research centre, dokki, cairo) isolated and illustrated four species belonging to two genera. various studies on the biocontrol of nematodes by fungi have been the target of many studies in egypt; the most relevant are: ali (1994, 1995), ali & Barakat (1994), aboul-eid et al. (1997a, b, 2006), ashour & moustafa (1999), and amin & moustafa (2000). out of these various data and information only 10 species belonging to seven genera were recorded as the Basidiomycota contains about 31 503 described species, which represents 31.8 % of the known species of true Fungi (Kirk et al. 2008). this group includes mushrooms, puffballs, bracket fungi and some yeasts (Petersen et al. 2008, wannathes et al. 2009). many Basidiomycota decay dead organic matter, including wood and leaf litter symbiotic lifestyles (intimate mutually bene�cial or harmful associations with other living organisms) are well developed in the Basidiomycota . they include major plant pathogens, such as “rusts” ( uredinales ) and “smuts” ( ustilaginales ), which attack wheat and other crops, and some human and animal pathogens. not all symbiotic Basidiomycota cause harm to their partners. Indeed, some form ectomycorrhizas with the roots of plants, principally forest trees such as oaks, pines, dipterocarps, and eucalypts (smith & read 1997, rinaldi et al. 2008). other symbiotic Basidiomycota form associations with insects, including leaf-cutter ants, termites, scale insects, wood wasps, and bark beetles (wheeler & Blackwell 1984, mueller et al. 1998). the �rst information on hyphenate macro-basidiomycota (phytopathogenic or saprobic) in egypt dates back to delile (1813a), melchers (1931), and morse (1933). In her study on the genus , morse referred to some samples collected from egypt. after six decades more information about macro- basidiomycota came to light through a series of studies carried out by several investigators, such as mouchacca (1977), zakhary (1979), salem & michail (1980), zakhary (1983), malenÇon (1984), assawah (1991), chen (1999), abu el-souod et al. (2000), el-Fallal (2003), el-Fallal & Khedr (n. dat.), el-Fallal & el-diasty (2006), Kim et al. (2006), and an exhaustive revision of all the available literature and sources mentioned since 1931 shows that 108 taxa belonging to 65 genera, 104 species, and 4 varieties of egyptian macro- though many basidiomycetes are saprobes or wood-rotters, Basidiomycota contains two common and destructive groups of plant pathogens: rusts and smuts. rust fungi are the largest group of fungal plant pathogens, containing 7 000 species that possess the most complex life-cycles in the kingdom fungi (sert 2009). they are obligate biotrophs and cause disease on most crops, ornamentals, and many other plants (hawksworth et al. 1995). In addition to basidia and basidiospores, rusts produce other types of spores such as teliospores spermatia, aeciospores, and uredospores. rusts that produce all �ve types of spores are referred to as macrocyclic, while rusts that lack one or more spore type are referred to as microcyclic. unlike rusts, smuts produce only basidiospores and teliospores which can survive in the soil away from a host plant. smuts commonly infect the ovaries of grains and are easily recognized by the formation of galls the initial research and documentation of rust and smut diseases in egypt was by reichert (1921), Briton-Jones (1922), Philp & selim (1941), abdel-hak & abdel-rehim (1950), ragab & mahdi (1966), and assawah (1969). later, in-depth research was carried out by egyptian and other investigators, with different targets such as taxonomy, pathogenicity, biocontrol and serology. the most relevant studies are: sherif et al. (1991), el-shamy (1996), Baka & gjaerum (1996), mennicken et al. (2005), abd el Fattah et al. (2009), abd el-ghany (2009) and et al. Baka & gjaerum (1996) gave the �rst serious modern taxonomic treatment of local rusts, reporting 23 rust species on various monocotyledonous and dicotyledonous plants in the nile valley (see mouchacca 2003b). as a result of these studies, 112 species of plant pathogenic belonging to total recorded species after the omission of duplicate names, name correction, allowance for synonyms and taxonomic assignments of all reported taxa from egypt, the number of the egyptian fungi recorded is 2 281 taxa belonging to 755 genera (table 1). at the generic level, some genera exhibit an extraordinary high species richness such as (100 spp.) and (83). other genera show moderate richness such chaetomium (53 spp.), (49), (41), ahmed m. abdel-azeem    It is generally accepted that only about 7 % of all fungi have so far been discovered, and about 93 % still wait to be discovered. Fungi are neglected organisms and they are not well protected, but like animals and plants, they are endangered by human activities. although the 1992 convention on Biological diversity extends protection to all groups of organisms, it is worded in terms of “animals, plants and microorganisms” and fungi do not �t well into these categories. In egypt and up to now fungal biodiversity and conservation topics have been overlooked. as a result, countries which signed the convention have almost universally overlooked fungi in preparing their biodiversity conservation plans: fungi are truly the orphans of rio (minter threats to fungi throughout the globe are of concern since they are not only beautiful but also play a signi�cant role in human welfare. three steps were suggested by moore et al. (2001) for fungal conservation: (1) conservation of habitats; (2) in situ conservation of non-mycological reserves/ ecological niches; and (3) ex situ conservation especially for saprobic species growing in culture. to help collections of fungal cultures to maintain appropriate standards, the world Federation for culture collections (wFcc) has formulated guidelines which outline the necessary requirements (hawksworth 1991, smith et al. 2001, smith 2003). there are 573 microbial culture collections in 68 countries registered in the world directory of collections of microorganisms (dcm) ( ). In egypt only two centers are recorded: emcc (wdcm583) egypt microbial culture collection, cairo microbiological resources centre (cairo mIrcen), ain shams university, and nodcar wdcm822 marwa mokhtar abd rabo, national organization of drug control and research. however, moubasher and his colleagues founded the assiut university mycological centre (aumc) in 1999 where more than 6 000 fungal isolates belonging to more than 500 species are being preserved under low temperature (5 °c), deep-freezed (-80 °c), and lyophilized; this is the biggest reference culture collection in the arab countries. the centre also has a collection of dried specimens (i.e. a fungarium) which is rare in arab countries. In spite of this the aumc is not yet registered with the wFcc. the number of habitats that potentially support specialized fungi is enormous. the fungi described as new to science during 1981 to 1990 were associated with 1 982 host genera or substrata (hawksworth & rossman 1997). some unexplored substrata and habitats from which these fungi were found include the rumens of herbivorous mammals, algae, lichens, mosses, marine plants, including mangroves the egyptian fungi are presently represented by 2 281 taxa (1 035 species and 395 genera) out of the 101 202 world estimate. In comparing the fungal diversity recorded in egypt with other countries, it is important to mention that some ecological groups of fungi are completely ignored or have never been studied in a comprehensive way in egypt, such as trichomycetes (a group of enigmatic fungi occurring in the hindguts of insects and other invertebrates; lichtwardt 2002), in addition to hypersaline and black yeasts. other groups needing more exploration such as algicolous fungi, invertebrate associated fungi, mycorrhizas, endophytic fungi, lichens, wood deteriorating, and coprophilous fungi. the potential fungal resources of egypt are globally important and there are vast areas that are still unexplored. at present, egypt needs more investigators and funds to explore and develop this research �eld and, therefore, the extensive collection of fungi in unexplored areas remains a priority. this review will be followed by an updated checklist of all recorded egyptian fungi up to the present, a bibliographic study of egyptian mycological research, and a book on the fungi of egypt, supplemented with provisional keys to all table 1. numbers of recorded egyptian fungi. roups and Phyla el-abyad (1997) Present survey 25 25 40 21 32 17 35 teleomorphic genera 80 251 181 261 32 87 total no. of genera recorded in egypt 360 755 total no. of species recorded in egypt 1246 2281 mycology in egypt   all my thanks to the late samy m el-abyad (Botany department, Faculty of science, cairo university) for his courage in documenting the egyptian fungi in 1997. I express my appreciation to Paul m Kirk (caBI europe, uK) for data on species names in the Index Fungorum database; the late John c Krug (centre for Biodiversity and conservation Biology, royal ontario museum, toronto) for providing some unavailable papers. I am further indebted to Jean mouchacca (laboratoire de cryptogamie, muséum national d’histoire naturelle, Paris), yaacov Katan (department of Plant Pathology and microbiology, Faculty of agriculture, Food and environmental quality sciences, the hebrew university of Jerusalem, Israel) and hussien m rashad (ashtoum el-gamil Protectorate, Port said, egypt) for their unfailing help during this work. I also owe thanks to robert a (Forest Pathology and wood microbiology research laboratory, minnesota university) and el-sayeda m gaml el-din (Botany department, Faculty of science, suez canal university) for critical reading the manuscript. I also thank Pedro w crous (cBs- Knaw Fungal Biodiversity centre, utrecht) who worked closely with me in preparing and editing the manuscript and photoplates, and the abdalla me, abdel-Fattah gm (2000) In�uence of the endomycorrhizal glomus mosseae on the development of peanut pod rot abd-elaah ga (1998) the occurrence of fungi along the red sea coast and variability among isolates of as revealed by : 303–311. abdel-azeem am (2003) ecological and taxonomical studies on ascospore-producing fungi in egypt . Phd thesis, Faculty of science, suez canal university, egypt. abdel-azeem am (2009) operation wallacea in egypt. I- a preliminary study on diversity of fungi in the world heritage site of saint Katherine, egypt. assiut university Journal of Botany abdel-azeem am, abdel-moneim ts, Ibrahim me, hassan maa, saleh my (2007) effect of long-term heavy metal contamination on diversity of terricolous fungi and nematodes in egypt - a case study. water, air, and soil Pollution abdel-aziz Fa (2004) Biodiversity of aquatic fungi, from the river . Phd thesis, south valley university, egypt. abdel-aziz Fa (2008) diversity of aquatic fungi on abdel-Baky nF (2000) spp. an entomopathogenic fungus for controlling white�ies and aphids in egypt. abd el Fattah aI, alamri s, abou-shanab raI, hafez ee (2009) Fingerprinting of ustilago scitaminea (sydow) in egypt using differential display technique: chitinase gene the main marker. research Journal of agriculture and Biological sciences : 674–679. abdel-Fattah gm (1985) studies on thermophilic xylan-decomposing fungi in humus . msc thesis Faculty of science, mansoura university, egypt. abdel-Fattah gm (1991) some physiological and ecological studies on vesicular-arbuscular (va) mycorrhizal fungi . Phd thesis, mansoura university, egypt. abdel-Fattah gm (2001) measurement of the viability of am fungi colonized in roots using three different stains and its relation to growth and metabolic activities of soybean plants. abdel-Fattah gm, mankarios at (1995) Functional activity of glomus mosseae in the protection of soybean from infection by the pathogenic fungus chalara elegans egyptian Journal of abdel-Fattah gm, rabie gh (1995) Improved growth and tolerance of cowpea to irrigation with waste ef�uents from fertilizer’s factories using mycorrhizal fungus ( glomus fasiculatum zagazig Journal abdel-Fattah gm, abo-hamed sa, mohamed za (1996) the role of va mycorrhizal fungus (glomus mosseae) and kinetin in alleviation of salinity stress in Pisum sativum plants. 1- Plant growth, photosynthetic pigments, nodulation, proline and nutrient . 1 International conference on fungi: hopes and challenges. cairo, 2–5 september 1996, al-azhar university, abd el-ghany tm, el-taher em, el-sheikh hh (2009) ef�cacy of fungal rust disease on willow plant in egypt. australian Journal of Basic and applied sciences abdel-hafez sII, el-said ahm, maghraby ta (1995) studies on fungi isolated from skin diseases and associated fungi of students in qena and red sea governorates, egypt. assiut Bulletin of Faculty of science, assiut university abdel-hak t, abdel rehim mF (1950) studies on long smut of sorghum in egypt. laboratory research committee monthly report, ministry of agriculture, egypt. november volume : 226– 229 (in arabic). abdel-hamed na (2008) ecological, physiological and taxonomical studies on the genus Fusarium in egypt . msc thesis, Faculty of science, assiut university, egypt. abdel-Kader mI (1973) mucorales in egyptian soil . msc thesis. Faculty of science, assiut university, egypt. abdel-mallek ay, abdel-rahman maa (2006) mycopathogens of the corn leaf aphid, rhoplosiphum maidis (Fitch.) infesting wheat plants in assiut, egypt . ninth arab congress of Plant Protection, abdel-mallek ay, omar sa, Bagy m K (1995) In�uence of licid on fungi of human hair and keratin degradation. Journal of Islamic : 119–126. abdel-mallek ay, abdel-rahman maa, hamam gha (2003a) survey of entomopathogenic fungi naturally infecting cereal aphids (homoptera: aphididae) in southern egypt . Proceedings of abstracts of the 9 european meeting of the IoBc/wPrs working group “Insect Pathogens and entomopathogenic abdel-mallek ay, abdel-rahman maa, omar sa, hamam gha (2003b) survey of entomopathogenic fungi naturally infecting cereal aphids infesting wheat plants in assiut. Bulletin of Faculty of science, assiut university abdel-moneim ts (2010) occurrence of nematophagous fungi in Ismailia governorate, egypt . the �rst international conference ahmed m. abdel-azeem    on basic and applied mycology, 9–11 march 2010, assiut, egypt, abdel-moneim ts, abdel-azeem am (2009) operation wallacea in egypt. II- diversity of arbuscular mycorrhizal fungi in different elevation wadis in saint Katherine Protectorate, egypt. abdel-raheem am (2002) myxomycetes from upper egypt. abdel-raheem am (2004) study of the effect of different techniques on diversity of freshwater hyphomycetes in the river nile (upper abdel-rahman tma, salama am, ali mIa, tharwat ha (1990) Fibrinolytic activity of some fungi isolated from self-heated composted fertilizer. abdel-salam mm (1933) damping-off and other allied diseases of 11 abdel-sater ma, eraky sa (2002) Bulbs myco�ora and their relation abdel-wahab ghF (2002) studies on the myco�ora of stored food msc thesis, Faculty of science, mansoura university, egypt. abdel-wahab ma (1996) studies on mangrove-inhabiting fungi of the red sea . msc thesis, Faculty of science, south valley university, sohag, egypt. abdel-wahab ma (2000) diversity of fungi in subtropical mangroves Phd thesis, Faculty of science, south valley university, sohag, abdel-wahab ma (2005) diversity of higher marine fungi from abdel-wahab ma, el-sharouny hm, Jones eBg (2001a) two new intertidal lignicolous species from red sea abdel-wahab ma, el-sharouny hm, Jones eBg (2001b) sp. nov. (halosphaeriales, ascomycota) based on morphological and molecular evidence. : 255– abdel-wahab ma, nagahama t, abdel-aziz Fa (2009) two new corollospora species and one new anamorph based on morphological and molecular data. mycoscience 50 : 147– 155. abdel-wahab ma, Pang Kl, nagahama t, abdel-aziz Fa, Jones eBg (2010) Phylogenetic evaluation of anamorphic species of and with the description of eight new genera and four new species. mycological Progress 10.1007/s11557-010-0661-x> wahid oa, moustafa aF, moustafa am (1996) Fungal population in the atmosphere of Ismailia city. abou el-seood ms (1968) survey of some fungal diseases of weeds and their relation to some economic crops . msc thesis, Faculty of agriculture, assiut university, egypt. aboul-eid hz (1963) studies on some aspects of nematode biological control . msc thesis, Faculty of agriculture, cairo university, egypt. aboul-eid hz, abdel-Bari am, Korayem ha, noweer ea (1997a) concomitant occurrence of nematode-antagonistic fungi and bacteria associated with prevalent nematodes in mansouria egyptian Journal of agronematology aboul-eid hz, abdel-Bari am, ameen ha, noweer ea (1997b) the morphological identify of tweleve nematode-antagonistic fungi and the bacterium Pasteuria penterans isolated from el-mansouria region soils (giza, egypt). egyptian Journal of aboul-eid hz, hasabo sa, noweer ema (2006) effect of a nematode- trapping fungus dactylaria brochopaga on meloidogyne incognita infesting olives and coconut palms in egypt. International Journal aboulkhair Ks, el-sokkary Ih (1994) effect of salinity, boron and sodium on the growth and root infection by vam, and of seedlings of three tree species. Journal of agricultural abu el-souod sm, assawah s, Bedaiwy m (2000) survey of mushrooms and polypores fungi in delta region of egypt Proceeding of 1st International conference of Biological sciences (IcBs) Faculty of science, tanta university 7–8 may, adl sm, leander Bs, simpson agB, archibald Jm, et al. (2007) diversity, nomenclature, and taxonomy of Protists. a�fy as, mahmoud ma, emara ha, abdelkreem KI (2009) Phenolic compounds and cod removal from olive mill wastewater by chemical and Biological Procedures. australian Journal of Basic and applied sciences agrios gn (2005) Plant Pathology . 5 edn. elsevier academic Press, agwa he (1990) vesicular arbuscular mycorrhizae and nodulation in . Phd thesis, tanta university, egypt. agwa he (2000) arbuscular mycorrhizal fungi ( glomales ) in egypt. I. a �eld survey of arbuscular mycorrhizal fungi associated with medicinal plants and effect of inoculation on growth of some plants. african Journal of mycology and Biotechnology : 1–12. agwa he, abdel-Fattah gm (2002) arbuscular mycorrhizal fungi (glomales) in egypt II. an ecological view of some saline affected plants in the deltatic mediterranean coastal land. acta Botanica agwa he, al-sodany ym (2003) arbuscular-mycorrhizal fungi ) in egypt. III: distribution and ecology in some plants in el-omayed Biosphere reserve. egyptian Journal of Biology al-alfy sm (1995) ecological studies on mucorales fungi in Ismailia . msc thesis. Faculty of science, suez canal university, egypt. al-doory y (1968) theisolation of keratinophilic fungi from african : 113–116. aleem aa (1950a) a fungus in ectocarpus granulosus c. agardh : 119–120. aleem aa (1950b) Phycomycetes marins de diatomees et d’algues dans la region de Banyuls-sur-mer (Pyrenees-orientales). vie et aleem aa (1950c) Phycomycetes marins parasites de diatomees et d’algues. comptes rendus hebdomadaires des seances de aleem aa (1952a) marine fungi from the west coast of sweden. mycology in egypt   aleem aa (1952b) olpidiopsis feldmanni sp. nov. champignon marin parasite d’algues de la famille des Bonnemaisoniacees. omptes rendus hebdomadaires des seances de l’academie aleem aa (1952c) sur la présence de melanopsamma tregoubovii ollivier (Pyrénomycète) dans la manche occidentales. Parasite dilophus fasciola (roth) howe. Bulletin du laboratoire aleem aa (1953) marine fungi from the west-coast of sweden. aleem aa (1962) marine fungi from the white sea. zhurnal sssr : 1582–1595. akademia nauk, moscow and aleem aa (1974) marine fungi from romania. cercetari marine, aleem aa (1975) marine fungi from rumania. rapport commission International pour l’exploration scienti�que de la mer aleem aa (1978) new records of marine fungi from the red sea. Bulletin Faculty of science, King abdel-aziz university, Jeddah aleem aa (1980a) Pythium marinum sparrow (Phycomycetes) Porphyra leucosticta thuret in the mediterranean sea. aleem aa (1980b) distribution and ecology of marine fungi in sierra leone (tropical west africa). aleem aa , malibari aa (1981) studies in tropical marine fungi I. mangrove fungi from sierra leone (west africa). 1. Bulletin Faculty of science, King abdul aziz university, Jeddah ali hassanein mde, morsi aae, el-sherif mae (1972) bibliography of agricultural studies conducted in egypt . Part one ali ahh (1994) studies on some fungal nematode antagonists in relation to nematode egg hatching and reproduction. ali ahh (1995) chaetomium spirale a potential fungus as a biocontrol agent for the reniform nematode on cotton. egyptian Journal of ali ahh, Barakat mle (1994) utilization of trichoderma harzianum as a biological agent against root-knot nematode meloidogyne incognita . egyptian Journal of Biological Pest control : 67–77. ali Fu, Ibrahim zm (2008) Production and some properties of �brinolytic enzyme from rhizomucor miehei (cooney & emerson) schipper. Journal of applied sciences research ali mam (2003) Biological and ecological studies of certain pets of honey bee . Phd thesis. Faculty of agriculture, ain shams university, cairo, egypt. alves a, crous Pw, correia, a, Phillips aJl (2008) morphological and molecular data reveal cryptic speciation in amin wa, mostafa Fam (2000) management of infecting sun�ower by integration of glomus mosseae trichoderma viride, t. harzianum and egyptian Journal of agronematology arthur J (2000) mushrooms and mankind: the impact of mushrooms on human consciousness and religion . Book tree, escondido, ashour eh, mostafa Fam (1999) effect of certain heavy metals on spp. on the growth effect of the nematophagous arthrobotrys oligospora , trap formation, root-knot nematode infection and enzymes production. Pakistan Journal assawah mw (1969) Pileolaria terebinthis on pistachio in uar assawah mw, el-arosi h (I960) Fungi associated with wheat, barley assawah ws (1991) Biochemical studies on some mushrooms in . Phd thesis. Faculty of science, tanta university, tanta, aue r, müller e (1967) vergleichende untersuchungen an einigen chaetomium arten. Berichte der schweizerischen Botanischen aumc (2010) catalogue of culture collection . 2 edn. assiut university mycological center, assiut. aung om, soytong K, hyde Kd (2008) diversity of entomopathogenic fungi in rainforests of chiang mai Province, thailand. aveskamp mm, gruyter J de, crous Pw (2008) Biology and recent developments in the systematics of , a complex genus of Badran ram, aly mzy (1995) studies on the mycotic inhabitants culex pipiens collected from fresh water ponds in egypt. : 105–110. Bagy mmK, abdel-hafez aaI (1985) myco�ora of camel and goat hairs from aI-arish, egypt Baka za, gjaerum hg (1996) egyptian . I. rusts on wild plants from the nile valley. Barr dJs (1990) Phylum In: margulis l, corliss Jo, melkonian m, chapman dJ (eds), handbook of Protoctista Bartnicki-garcia s (1970) cell wall composition and other biochemical markers in fungal phylogeny. In: harborne JB (ed.) Phytochemical Phylogeny , 81-103, academic Press, london, Besada wh, yusef hm (1968) on the myco�ora of uar soil. Proceedings of the egyptian academy of science Besada wh, yusef hm (1969) chaetomium mareoticum sp. nov. transactions of the British mycological society Binyamini n (1973) coprophilous Fungi of Israel. III. Israel Journal of Bishara I (1928) cotton insects. ministry of agriculture, egypt, Briton-Jones hr (1922) the smuts of millet. ministry of agriculture, egypt. technical series Bulletin Briton-Jones hr (1923) a wound parasite of cotton bolls. ministry of agriculture, egypt.technical series Bulletin Briton-Jones hr (1925) mycological work in egypt during the period ministry of agriculture, egypt. technical series Brusca rc, Brusca gJ (2003) . 2 edn. sinauer Budge, eaw (1967) the egyptian Book of the dead (the papyrus of . dover Publications, mineola, new york, usa. ahmed m. abdel-azeem    cavalier-smith t (1998) a revised six-kingdom system of life. cavender Jc, landolt Jc, ndiritu gg, stephenson sl (2010) dictyostelid cellular slime moulds from africa. chen c (1999) genetical and molecular systematic study on the genus montagnea Fr., a desert adapted gasteromycete. msc thesis virginia Polytechnic Institute and state university, usa. crous Pw (2009) taxonomy and phylogeny of the genus crous Pw, rong Ih, wood a, lee s, glen h, Botha w, slippers B, Beer wz de, wing�eld mJ, hawksworth dl (2006) how many species of fungi are there at the tip of africa? studies in damm u, woudenberg Jhc, cannon PF, crous Pw (2009) species with curved conidia from herbaceous delile ar (1813a) delile ar (1813b) el-abyad ms (1997) Biodiversity of Fungal Biota in egypt. up-dated . [Publication of national Biodiversity unit no. 7.] egyptian environmental affairs agency. el-abyad ms, abu-taleb a (1993) II-soil Fungi. ecology of fungal Publication of national Biodiversity unit. no. 1. habitat : 237–262. egyptian environmental affairs agency, el-desouky sm, el-wakil aa (2003) occurrence of root rot and vine decline of cantaloupe and watermelon in egypt. el-Fallal aa (2003) agaricales from the countryside and grasslands of east delta region, egypt. Journal of environmental sciences el-Fallal aa, Khedr aa [undated] Podaxis pistillaris (lin. ex. Press) Fr., an edible gasteromycete, from zaranik protected area, north sinai, egypt. James ty, letcher Pm, longcore Je, mozley-standridge se, Porter d, Powell mJ, grif�th gw, vilgalys r (2006a) a molecular phylogeny of the �agellated fungi ( ) and description of a new phylum ( James ty, Kauff F, schoch cl, et al. (2006 b) reconstructing the early evolution of the fungi using a six gene phylogeny. Jones eBg (2001) the forgotten fungi: facultative marine fungi. In: International marine and Freshwater mycology symposium Jones eBg, sakayaroj J, suetrong s, somrithipol s, Pang Kl (2009) classi�cation of marine , anamorphic taxa and Kaewchai s, soytong K, hyde Kd (2009) mycofungicides and fungal Karam el-din aa, youssef ay, soliman aK, elias m (1994a) comparative serological study on cryptococcosis. Karam el-din aa, youssef ay, el-Kholy em (1994b) Prevalence of fungi in clinical specimens from patients with fever of unknown origin, leukemia and cancer. african Journal of mycology and Karam el-din aa, youssef ay, hassan y (1994c) Prevalence of pathogenic yeast fungi in clinical specimens from patients with candidiasis. african Journal of mycology and Biotechnology : 37–47. Karam el-din aa, youssef ay, taher em (1995) mycotic keratitis: seasonal, etiological and Biochemical study. african Journal of Karam el-din aa, youssef ay, zaki sh (1996) distribution of pathogenic and potentially pathogenic fungi among soil fungal �ora in egypt african Journal of mycology and Biotechnology : 23–39. Kassas m (2002) Biodiversity: gaps in knowledge. Kelley a (1950) mycotrophy in plants . chronica Botanica, waltham, Kendrick B (2000) the Fifth Kingdom. 3 edn. Focus Publishing, newbury. massachsetts, usa. Khalil hme (1995) the effect of microenvironmental variation on the distribution of the macrolichens in gebel dalfa, north sinai, egypt msc thesis, Faculty of science, suez canal university, egypt. Khallil am (2001) Ingoldian and other �lamentous fungi of hot Bulletin of the Faculty of science, assiut university Khallil am, el-hissy Ft, ali e (1995) seasonal �uctuations of aquatic fungi recovered from egyptian soil (delta region). Journal of mycology in egypt   Kharboush mam (1969a) on the ecology of soil fungi. I-distribution of the soil fungal �ora in the different types of soil in the uar. Bulletin of Faculty of science alexandria university Kharboush mam (1969b) on the ecology of soil fungi. II-some ecological aspects of the soil fungal �ora in the uar. Bulletin of Faculty of science alexandria university Khater hm (1989) survey of soil from different sites at cairo for yeast . msc thesis. Faculty of science, ain shams university. Kim hm, Paik s, ra Ks, Koo KB, yun Jw, choi Jw (2006) enhanced production of exopolysaccharides by fed-batch culture of ganoderma resinaceum the Journal of microbiology Kirk P, cannon PF, minter dw, stalpers Ja (2008) ainsworth & Bisby’s dictionary of the Fungi. edn. caB International, wallingford, uK. Kodsueb r, mcKenzie ehc, lumyong s, hyde Kd (2008a) diversity Kodsueb r, mcKenzie ehc, lumyong s, hyde Kd (2008b) Fungal succession on woody litter of magnolia liliifera ( Kohlmeyer J, Kohlmeyer e (1979 ) marine mycology. the higher . academic Press, new york, u.s.a. Koriem am (2003) light and electron microscopic study of the lichen Xanthoria Parietina infecting fruit trees in egypt . 8 arab congress of Plant Protection, 12–16 october 2003, el-Beida, Koriem am (2006) First isolation and cultivation of the fungus and the alga of the lichen Xanthoria parietina infecting mango trees in egypt . 9 arab congress of Plant Protection, 19–23 november Kottb mr (2008) survey and characterization of trichoderma and gliocladium species and speci�cation of their biocontrol ability msc thesis, Faculty of science, suez canal university, egypt. Kowalik r, sadurska I (1973) micro�ora of papyrus from samples of Krug Jc, Khan rs, Jeng rs (1994) a new species of Kruys Å, ericson l (2008) species richness of coprophilous ascomycetes in relation to variable food intake by herbivores. Kvas m, marasas wFo, wing�eld Bd, wing�eld mJ, steenkamp et (2009) diversity and evolution of species in the lado c (1994) a checklist of myxomycetes of mediterranean : 117–185. lichtwardt r (2002) Biogeographical implications of trichomycete . (abstract). Imc7 Book of abstracts, p. 48. lundqvist n (1969) and ( lundqvist n (1970) new ( s. lat svensk Botanisk tidskrift lutzoni FF, Kauff cJ, cox d, et al. (2004) assembling the fungal tree of life: progress, classi�cation, and evolution of subcellular traits. mahmoud yag (1999) First environmental isolation of var. and var. from the gharbia malençon g (1984) Phallus roseus a. delile 1813, alias Itajahya rosea (delile) ed. Fischer 1929. de la société mycologique mankarios at, abdel-Fattah gm (1994) ecology of va-mycorrhiza in mansour maI (2010) endomycorrhizal fungi as a biocontrol mean of tomato-fusarial –wilt in north sinai . msc thesis, Faculty of education, university of suez canal, al-arish, egypt. mcneely Ja, miller Kr, mittermeier r, werner tB (1990) conserving the world’s Biological diversity . International union for conservation of nature and natural resources, gland, melchers le (1931) a check list of plant diseases and fungi occurring in egypt. transactions of the Kansas academy of science mennicken m, maier w, oberwinkler F (2005) a contribution to the rust �ora ( uredinales ) on zygophylloideae ( ) in minter dw (2010) conservation of fungi: the orphans of the rio de Janeiro convention. In: the First International conference on Basic and applied mycology, 9–11 march 2010, assiut, egypt montasir ah, mostafa ma, elwan sh (1956) development of soil micro�ora under l. and moore d, nauta mn, evans se, rotheroe m (eds) (2001) conservation, Issues and solutions . cambridge university morse ee (1933) a study of the genus : 1–33. morton JB, redecker d (2001) two new families of glomales archaeosporaceae and Paraglomaceae , with two new genera archaeospora and Paraglomus , based on concordant molecular and morphological characters. mycologia 93 : 181– 195. mostafa ma (1938) mycorrhiza in tropaeolum majus l. and mostafa ma, gayed sK (1953) Interaction between trichoderma and Fusarium vasinfectum and its possible bearing on the biological control of the cotton-wilt disease in egypt. extrait du : 191–211. moubasher ah (1993 ) soil fungi of qatar and other arab countries the scienti�c and applied research centre, university of qatar. moubasher ah , abdel-rahman ma, abdel-mallek ay, hammam gha (2010) Biodiversity of entomopathogenic fungi infecting cereal and cabbage aphids in assiut. In: the First International conference on Basic and applied mycology, 9–11 march 2010, mouchacca J (1977) les champignons de la momie de ramsès. comptes rendus de l’academie des sciences de Paris 285 mouchacca J (1995) check-list of novel fungi from the middle east mouchacca J (1999) a list of novel fungi described from the middle east, mostly from non-soil substrata. nova hedwigia 68 : 149–174. mouchacca J (2001a) Biodiversité des récentes découvertes fongiques, dans les états arides de l’est méditerranéen (moyen- ahmed m. abdel-azeem    mouchacca J (2001b) new fungi described from north east africa and other arab countries since 1940. what conclusions could be drawn from this scienti�c activity? cairo university african mouchacca J (2003a) annotated basic references of novel fungal taxa introduced from the middle east (1940–2000). mouchacca J (2003b) a selection of bibliography on the biodiversity and phytopathology of african fungi (–1994). mouchacca J (2004) novel fungal taxa from the arid middle east (1940–2000): omissions from previous notes. mouchacca J (2005) mycobiota of the arid middle east: check-list of novel fungal taxa introduced from 1940 to 2000 and major recent biodiversity. Journal of arid environments mouchacca J (2008) novel fungal taxa from the arid middle east introduced prior to the year 1940. I - non lichenized ascomycetes. mouchacca J (2009a) novel fungal taxa from the arid middle east introduced prior to the year 1940. II – anamorphic fungi: mouchacca J (2009b) novel fungal taxa from the arid middle east introduced prior to the year 1940. III – anamorphic fungi: moustafa aF (2006) Fungi of egypt. zygomycetes . aumc descriptions, no. 1. assiut. moustafa aF, abdel-azeem am (2005a) zygopleurage zygospora (sepg.) Boedijn, a new record to the egyptian ascomycetes. moustafa aF, abdel-azeem am (2005b) the genus in moustafa aF, abdel-azeem am (2006) some new records to the egyptian ascomycetes with a provisional key to their identi�cation. assiut university Journal of Botany 35 : 87–103. moustafa aF, abdel-azeem am (2008) thielavia gigaspora , a new thermotolerant ascomycete from egypt. moustafa aF, abdel-azeem am (2010) an annotated check-list of egyptian ascomycota reported from soil and other terricolous mourad aK, zaghloul oa, Kady el, nemat Fm, morsy me (2005) a novel approach for the management of the chalkbrood disease infesting honeybee apis mellifera l. (hymenoptera: apidae) colonies in egypt. 611. mueller ug, rehner sa, schultz tr (1998) the evolution of müller J (1880a) les lichens d’egypte. revue mycologique 2 : 38–40. müller J (1880b) enumeratio lichenum aegyptiacorum hucusque müller J (1880b) enumeratio lichenum aegyptiacorum hucusque müller J (1884) enumerationis lichenum aegyptiacorum. supplement toulouse naim ms, mahmoud saz, hussein am (1957) qualitative and quantitative studies on the rhizosphere micro�ora of some egyptian cotton varieties. ain shams science Bulletin : 65–83. natrass rm (1932) Preliminary notes on some entomogenous fungi in egypt. ministry of agriculture, egypt. technical series Bulletin natrass rm (1933) a new species of ( h. toruloidea ) on deciduous trees in egypt. transactions of the British mycological ndiritu gg, winsett Ke, spiegel Fw, stephenson sl (2009) a checklist of african myxomycetes. nofal ma, haggag wm (2006) Integrated management of powdery nylander w (1864) lichenes in aegypto a cel. ehrenberg collecti. nylander w (1876) lichenes in aegypto a cel. larbalestier collecti. olive ls, stoianovitch c (1969) monograph of the genus Pang Kl, abdel-wahab ma, sivichai s, el-sharouny hm, Jones eBg Jahnulales ( ): a new order of lignicolous freshwater ascomycetes. mycological research Petersen rh, desjardin de, Krüger d (2008) three type specimens Phillips aJl, oudemans Pv, correia a, alves a (2006) characteri- sation and epitypi�cation of Botryosphaeria corticis , the cause of blueberry cane canker. Philp J, selim ag (1941) rust-resistant wheats for egypt. ragab ma (1956) a contribution to the fungi of egypt. ragab ma, mahdi mt (1966) studies on tolyposporium ehrenbergii the cause of long smut of sorghum in egypt (uar). raghukumar c (2002) Bioremediation of coloured pollutants by terrestrial versus facultative marine fungi. In: Fungi in the marine environment (ed. Kd hyde): 317–344. [Fungal diversity research series no.7.] Fungal diversity Press, hong Kong. raghukumar c (2008) marine fungal biotechnology: an ecological raja ha, Ferrer a, shearer ca (2009) Freshwater ascomycetes: a new genus, ocala scalariformis gen. et sp. nov, and two new ayria nubispora sp. nov. and rivulicola cygnea sp. nov. redecker d, raab P (2006) Phylogeny of the (arbuscular mycorrhizal fungi): recent developments and new reichert I (1921) diePilz�ora Äegypten. engler’s Botanischen rinaldi ac, comandini o, Kuyper tw (2008) ectomycorrhizal fungal diversity: separating the wheat from the chaff. Fungal diversity rojas c, stephenson sl (2008) myxomycete ecology along an elevation gradient on cocos Island, costa rica. Fungal diversity : 117–127. rossman ay (2003) a strategy for an all taxa Inventory of Fungal Biodiversity. In: Biodiversity and terrestrial ecosystems ( eds cI Peng, ch): 169–194. taipei. mycology in egypt   saad sI (1958) studies in atmospheric pollen grains and fungus spores at alexandria. annals of allergy sabet ys (1935) a preliminary study of egyptian soil fungi. Bulletin of the Faculty of science, egyptian university, cairo sabet ys (1936) Preliminary study of Penicillium egyptiacum v. sabet ys (1938) contributions to the study of Penicillium egyptiacum v. Beyma. transactions of the British mycological society sabet ys (1939a) on some fungi isolated from soil in egypt. of the Faculty of science, Fouad I university, egyptian university, : 1–112. sabet ys (1939b) cotton mycorrhiza. sabet ys (1940) mycorrhizal habit in the date palm ( sabet ys (1945) reactions of citrus mycorrhiza to manurial treatment. Proceeding of egyptian acadamy of science salem ma, michail sh (1980) Inonotus psuedohisbidus on in egypt. transactions of the British mycological society : 107–110. samson ra, evans hc, latg JP (1988) atlas of entomopathogenic . springer, Berlin heidelberg new york. sartory a, meyer J, taw�k z (1939) contribution à l’étude d’une mucoracée, absidia aegyptiacum n. sp., ferment alcoolique de la Bouza d’egypte. comptes rendus hebdomadaires des séances seaward mrd, sipman hJm (2006) an updated checklist of lichenized and lichenicolous fungi for egypt. willdenowia sert hB (2009) additions to rust and smut fungi of turkey. sewify g (1997) occurrence and pathogenicity of entomopathogenic fungi in egypt. In: national conference of pest and diseases of vegetables and fruits in egypt Ismailia, suez canal university, g, hashem ym (2001) effect of the entomopathogenic metarhizium anisopliae (metsch.) sorokin on cellular defence response and oxygen uptake of the wax moth l. (lep., Journal of applied entomology shalaby Keem (1999) Physiological studies on some chitin-degrading . msc thesis, Faculty of science, zagazig university, egypt. shalouf hms (1989) studies on fungal leaf spots of some plants in egypt . msc thesis Faculty of science, mansoura university, shearer ca, descals e, volkmann-Kohlmeyer B, Kohlmeyer J, marvanová l, Padgett d, Porter d, thorton ha, voglmayr h, raja ha, schmit JP (2007) Fungal biodiversity in aquatic shearer a (1924) cotton wilt 3 annual report, cotton research Board, ministry of agriculture. egypt sherief aa (1985) studies on xylan-decomposing fungi egypt . Phd thesis, Faculty of science, mansoura university, egypt. sherif s, ghanem eh, sha�k I, mustafa ee, abdel-aleem mm (1991) Integrated control of wheat loose smut in egypt. assiut Journal of shindia aae (1990) studies on fungal degradation of composts in egypt . Phd thesis, Faculty of science, zagazig university, egypt. shoulkamy ma, lucarotti cJ (1998) Pathology of shoulkamy ma, lucarotti cJ, el-Ktatny mst, hassan sKm (1997) Factors affecting coelomomyces stegomyiae infections in adult shoulkamy ma, abdelzaher hma, shahin aaB (2001) ultrastructural changes in the muscles, midgut, hemopoietic organ, imaginal discs and malpighian tubules of the mosquito aedes aegypti larvae infected by the fungus coelomomyces stegomyiae sickenberger e (1901) lichenes. memoires de l’Institut d sirag el-din a (1931) the citrus twig gum disease in egypt. of agriculture, egypt. technical series Bulletin simonis Jl, raja ha, shearer ca (2008) extracellular enzymes and soft rot decay: are ascomycetes important degraders in fresh smith d (2003) culture collections over the world. smith d, ryan mJ, day Jg (eds) (2001) the uK national culture collection Biological resource: properties, maintenance and smith se, read dJ (1997) mycorrhizal symbiosi. 2 ed. academic steiner J (1893) Beiträge zur lichenen�ora griechenlands und sitzungsberichte der Kaiserlichen akademie der wissenschaften steiner J (1916) aufzählung der von J. Bornmüller im oriente gesammelten Flechten. annals naturhistorische museum wien stephenson sl, stempen h (1994) myxomycetes: a hand book of . timber Press. Portland, oregon, usa. stizenberger e (1890) lichenaea africana. Berichte über die tätigkeit der st. gallischen naturwissenschaftlichen gesellschaft stizenberger e (1891) lichenaea africana. Berichte über die tätigkeit der st. gallischen naturwissenschaftlichen gesellschaft sung gh, hywel-Jones nl, sung Jm, luangsa-ard JJ, shrestha B, spatafora Jw (2007) Phylogenetic classi�cation of swelim ma, Baka zam, el-dohlob sm, hazzaa mm, el-sayed tI (1994) myco�ora of stored poultry fodder in egypt and their ability tangley l (1997) how many species are there? us news and world report aug. 18, 1997. 4 years this committee was unable to reach consensus upon changes. some mycologists have decided to ignore existing rules or to take nomenclatural risks. genetic sequence phylogenetic analyses have revealed many new relationships leading to binomial recombinations and even a Phylocode. having reached an impasse it can be asked if mycologists wish to eliminate dual nomenclature? If the answer is yes, it may be asked how to resolve conflicts, and then to create a process or body capable of dealing with such conflicts. (gams): this presentation was submitted without a formal abstract and too late to be included in the printed program. gams discussed the effects of ‘teleotypification,’ which permits — after a teleomorph discovered for a fungus previously known only as an anamorph (and for which there is no existing legitimate name for the holomorph) — designation of an epitype exhibiting the teleomorph stage for the hitherto anamorphic name, even when there is no hint of the teleomorph in the protologue of that name. several examples were forwarded to show that teleotypification is not the same as ordinary epitypification. For further information, norvell et al    From august 1–10, Imc9 delegates returned questionnaires in which they were to circle a y (yes) or n (no) to 24 questions on 4 topics. we discovered during our first tabulation that one number (#19) appeared twice, bringing the actual number of questions to 25, and have renumbered the text below accordingly. of the 174 questionnaires received, 7 were declared ‘spoiled’ as the respondents had placed an X over an option so that we could not determine whether agreement or rejection was intended. Both raw numbers and majority percentages are shown. we note that protocols followed at the 2005 International Botanical congress in vienna with respect to the preliminary mail-in ballots decreed that proposals receiving 60 % or higher support merited further discussion by the attending nomenclature section, while 75 % support virtually ensured passage for all but the most controversial proposals. In the results reported below, opinions showing one code for the future nomenclature of all organism names would be ideal .............................................................................................................................................................. Fungi should continue to be covered under the Botanical code (IcBn) ............................................................................................................................................................... Fungi should continue to be covered under the IcBn provided it is renamed the “Botanical and mycological code” .............................................................................................................................................................. Fungi should be covered by a separate mycological code (Icmn) ............................................................................................................................................................... under either IcBn or Icmn, decisions on fungal nomenclature should be voted at an International mycological congress (and not an International Botanical congress), guided by a secure advanced web publication and ............................................................................................................................................................ latin diagnoses/descriptions should continue to be required ............................................................................................................................................................... english diagnoses/descriptions rather than latin should be required ............................................................................................................................................................. either latin or english diagnoses/descriptions should be required .............................................................................................................................................................. diagnoses/descriptions in any language should be permitted ............................................................................................................................................................... deposition of key nomenclatural information in one or more approved depositories (e.g. mycoBank) should be made mandatory for the valid publication of new fungal names ............................................................................................................................................................ 11 historic names not included in t date) should no longer be treated as validly published ............................................................................................................................................................... deposited names should be automatically protected against any unlisted names after a date to be agreed .............................................................................................................................................................. an accurate and free list should be prepared of names in use or available for use ............................................................................................................................................................ names with key information deposited (e.g. in mycoBank) should be automatically available provided other code ............................................................................................................................................................ electronic on-line only publication should be accepted without restriction ............................................................................................................................................................. electronic on-line only publication should be accepted only when key nomenclatural information has been deposited y-113 n-36 ............................................................................................................................................................ Im 9 edinburgh nomenclature essions   For journals publishing online and printed copies, the dates of names should be those when the works are available in ............................................................................................................................................................ For journals publishing online and printed copies, the dates of names should be those when the works are distributed ............................................................................................................................................................... special group committees should be empowered to create lists of acceptable and rejected names in particular groups Fusarium, trichocomaceae ............................................................................................................................................................ the established system allowing dual nomenclature for anamorphs and teleomorphs should continue via art. 59 ............................................................................................................................................................... article 59 should revert back to its status prior to changes in the 2006 vienna code, i.e. keeping separate anamorph ............................................................................................................................................................... a system of progressively establishing one name for each fungus should be enacted via modification of existing articles (e.g. art. 59) ............................................................................................................................................................ the historical practice of allowing valid names for different morphs of a species should be prohibited in the future via modification of existing articles .............................................................................................................................................................. the ability to select a “teleotype” (a type of epitypification) with a sexual state for a fungus previously only known in the asexual state should be continued .............................................................................................................................................................. article 59 (that permits the dual system) should be deleted provided other changes ensure this would not retroactively .............................................................................................................................................................. we thank John mcneill (royal Botanic garden edinburgh) for his perennially wise counsel and cheerful guidance. we further thank special presenters vincent demoulin, Paul Kirk, and walter gams; José dianese (Brazil) for assisting in tabulating questionnaire results on 3 august; and all those who participated in the nomenclatural demoulin v (2010) Proposals to amend articles 15, 36, and 45. taxon gams w, Jaklitsch wm, Kirschner r, réblová m (2010) three proposals to amend article 59 of the concerning taxon greuter w, hawksworth dl, mcneill J, mayo ma, minelli a, sneath Pha, tindall BJ, trehane P, tubbs P (eds) (1998) draft Biocode (1997): the prospective international rules for the scientific taxon hawksworth dl, crous Pw, dianese Jc, gryzenhout m, norvell ll, seifert Ka (2009) Proposals to amend the to make it clear that it covers the nomenclature of fungi, and to modify the governance with respect to names of organisms treated as fungi. taxon hawksworth dl, cooper Ja, crous Pw, hyde Kd, Iturriaga t, Kirk Pm, lumbsch ht, may tw, minter dw, misra JK, norvell l, redhead sa, rossman ay, seifert Ka, stalpers Ja, taylor Jw, wingfield mJ (2010) Proposals to make the pre-publication deposit of key nomenclatural information in a recognized repository a requirement for valid publication of organisms treated as fungi taxon 111 mcneill J, Barrie Fr, Burdet hm, demoulin v, hawksworth dl, marhold K, nicolson dh, Prado J, silva Pc, skog Je, wiersema Jh, turland nJ (eds) (2006) International code of Botanical nomenclature (vienna code) adopted by the seventeenth International Botanical congress vienna, austria, July 2005 [regnum vegetabile no. 146.] ruggell: a.r.g. ganter verlag. nakada t (2010) a proposal on the designation of cultures of fungi taxon redhead sa, Kirk Pm, Keeling PJ, weiss lm (2009) Proposals to exclude the phylum from the taxon [reproduced with minor amendments from 113 : 503–511      © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. species of are some of the most important pathogens of woody plants in the world. these fungi have been known as tree pathogens since their first discovery by danish botanist martin vahl. while the taxonomy of these spp. has been controversial and widely debated over an extended period of time, application of the biological species concept (Korhonen 1978, anderson & ullrich 1979, et al. 1998, qin et al. 2007) and more recently dna sequence comparisons (coetzee et al. 2000a, 2003a, 2005, et al. 2004, Keča et al. 2006, mwenje et al. 2006, et al. 2010) have resolved many problems relating to the delineation of species. at least 40 species are now recognised in (volk & Burdsall 1995, lima et al. 2008, Pildain et al. 2010) and it is likely that other species will armillaria root rot, the disease caused by pathogenic spp. can result in serious losses to productivity in tree plantations, fruit tree orchards and in gardens (gregory et al. 1991, hood et al. 1991). In native forests, spp. cause disease but this is most typically a natural process et al. 1991). Interestingly, species of these fungi exist as clones covering huge areas of land and in these situations they are considered to be amongst the largest and oldest only a single native sp. occurs in south africa (coetzee et al. 2000a). this fungus, a. fuscipes is occasionally found on native trees (Kotzé 1935, referred to as a. mellea ). In contrast, it can be a serious pathogen in plantations of non-native spp. and on fruit trees planted in moist areas that have been cleared of native forest (lundquist 1986, 1987, coetzee et al. 2000a). a more sp. in south africa is a. mellea that was discovered in the company (dutch east India company) gardens in the centre of cape town (coetzee et al. 2001). the fungus in that situation represents a single genetic entity that was shown to be at least 358 years old. It was most likely introduced into the city when gardens were established to provide sailors travelling to the east with fresh produce some years after the discovery of the a. mellea clone in cape town, armillaria root rot was found killing plants in the historic Kirstenbosch Botanical gardens ( ) on the foothills of south africa’s iconic table mountain et al. 2003b). the fungus in that situation was never seen fruiting but isolates were identified as those of a. gallica and it was suggested that the fungus was introduced into the gardens with plants brought from asia (coetzee et al. 2003b). a few of the isolates collected on the plants were also thought to represent a. mellea , but the identification was tentative and based only on rFlP comparisons, without comparison of dna sequence data against other during may 2010, sporocarps of what appeared to be an sp. were found in large clumps in the upper corner , martin P.a. coetzee , Pedro w. crous department of genetics, Forestry and agricultural Biotechnology Institute (FaBI), university of Pretoria, Pretoria, 0002, south africa; corresponding author e-mail: mike.wing�eld@fabi.up.ac.za cBs-Knaw Fungal Biodiversity centre, uppsalalaan 8, 3584 ct utrecht, the netherlands department of ecosystem and conservation sciences, college of Forestry and conservation, university of montana, missoula, mt 59812, usa abstract: during may 2010, sporocarps of what appeared to be an armillaria sp. were found in large clumps in historic Kirstenbosch Botanical gardens on the foot of table mountain, cape town, south africa. these sporocarps could be physically linked to the roots of unidenti�ed dead trees and Protea spp. the aim of this study was to identify the armillaria sp. found fruiting in Kirstenbosch. to achieve this goal isolates were made from the mycelium under the bark of dead roots linked to sporocarps. the Its and Igs-1 regions were sequenced and compared to sequences of armillaria spp. available on genBank. cladograms were generated using Its sequences to determine the phylogenetic relationship of the isolates with other armillaria spp. sequence comparisons and phylogenetic analyses showed that the isolates represented a. mellea . they were also identical to isolates of this species previously discovered in the company gardens in south africa and introduced from europe apparently by the early dutch settlers. armillaria mellea is alien and apparently invasive in cape town, fruits profusely and has the potential to spread to sensitive native forests on the foothills of the city. article info: submitted: 30 october 2010; accepted: 4 november 2010; Published: 15 november 2010. Key words: Ima Fu gus volu me 1 o 2: 149–153 ing�eld et al    of Kirstenbosch Botanical gardens and close to rycroft’s gate. these sporocarps could be physically linked to the roots of unidentified dead trees and spp. (Fig. 1B, c). upon removal of the bark from the dead roots, sheets of white mycelium typical of armillaria root rot were found. the aim of this study was to identify the sp. found fruiting in Kirstenbosch using data that were not available at the time of the discovery of armillaria root rot in cape town mate an Isolation and purification of isolates followed the methods outlined in coetzee et al . (2003). cultures were maintained on malt extract yeast agar (mya) (15 g/l malt extract, 2 g/l yeast extract, 15 g/l agar). cultures are stored in the culture collection (cmw) of the Forestry and agricultural Biotechnology Institute (FaBI), university of Pretoria and with the centraalbureau voor schimmelcultures (cBs), utrecht, Fig. 1. armillaria root rot in Kirstenbosch Botanical gardens. native woody shrubs and forest deeper in the valleys common on the cape clusters of fruiting bodies found on a stump. robust fruiting bodies of sp. showing a yellow cap, prominent Fungal phoenix   Isolates for dna extractions were grown at 25 °c in the dark for 3 wk in conical flasks containing liquid malt extract yeast (my). mycelium was harvested using a tea strainer, freeze-dried and lyophilised. dna extractions followed the protocol of coetzee et al. (2000b). a nanodrop spectrophotometer (thermo Fisher scientific, usa) was used to quantify the dna. the Igs-1 and Its regions of the rdna operon were amplified using primer pairs P-1 / o-1 and Its-1 / Its-4, respectively. Pcr reaction mixture and conditions were the same as those published by coetzee et al. (2000b), except that Faststart tag dna polymerase was used instead of an expand high Fidelity Pcr system. the Pcr products were purified prior to sequencing using a msB® spin Pcrapace kit (Invitek, germany). dna sequences for the Igs-1 and Its-1 regions were obtained in both directions using the same primers employed for their amplification. the sequence reactions were carried out using an aBI PrIsm dye terminator cycle sequencing ready kit with amplitaq dna Polymerases Fs (applied Biosystems) following the manufacturer’s instructions. chromatographs were analysed and contigs assembled in clc main workbench v. 5.7 (clc bio, denmark). na sequence comparisons and phylogenetic dna sequences were compared against those available in the ncBI genBank database using a Blastn search. Igs-1 and Its sequences generated in this study were aligned against those of isolates cmw 3975 and cmw 3978 available on genBank and originating from the company gardens in cape town. this was done to determine nucleotide variation between the isolates from the company gardens and those Phylogenetic analysis was conducted with a sub-set of the Its-1 dataset generated by coetzee et al . (2003). the dataset was amended with dna sequences for a. fuscipes from south africa and a. mellea from europe, asia, western usa, eastern usa and the company gardens, south africa. sequences were re-aligned using maFFt v. 6 (Katoh & toh 2008). cladograms were generated using a heuristic tree search algorithm in PauP v. 4 with branch swapping set to tBr and random addition of sequences (10 replicates). trees were rooted to a. fuscipes . Bootstrap analysis (1000 replicates) was done to gain support for the grouping of taxa using the same settings as above but with addition of the macro-morphology of basidiocarps produced by the fungus was similar to that described for a. mellea (watling et 1982) (Fig. 1d–g). the cap colour of the basidiocarps was distinctly yellow and they had thick annuli and stipes tapering towards the base. the basidiocarps also had a caespitose two isolates (cmw 36264 and cmw 36265) were retrieved from infected roots and these produced rhizomorphs typical of armillaria spp. in culture (Fig. 1h). the rhizomorphs displayed a dichotomous growth habit and were produced in abundance. white aerial mycelium was observed on the surface of the rhizomorphs at areas that had grown out of na sequence comparisons and phylogenetic the Igs-1 and Its dna sequences of isolates from Kirstenbosch were most similar to sequences of a. mellea in genBank. comparisons of Igs-1 and Its sequences revealed the absence of nucleotide variation between isolates from Kirstenbosch and a. mellea from the company the Its dataset included 925 characters of which 183 characters were parsimoniously informative. a heuristic search generated 6 trees with tree lengths of 250 steps (Fig. 2). the consistency index was 0.864, and retention index 0.941. the isolates from Kirstenbosch formed a monophyletic group with a. mellea from the company gardens with strong bootstrap support and together these were placed in a clade that included sequences of a. mellea from europe (99 % sporocarps linked to infected roots from which cultures were made in this study were morphologically similar to those of previously found in the cape town city centre. dna sequence comparisons also showed that the cultures were those of a. mellea and the sequences were identical to those from the company gardens. although vegetative compatibility tests were not done to test whether these represent the same clone as those in the city centre, there was no Igs-1 or Its nucleotide variation between isolates from the two locations there are three possible means of introduction of into Kirstenbosch Botanical gardens, via air-dispersed basidiospores, on infected plant material or on infested wood armillaria mellea in the company gardens fruits profusely every year at the onset of the first rains in autumn. although the fungus clone is entirely surrounded by roads and buildings, the basidospore cloud is likely to easily spread within the city and at least up the foothills of table mountain, on which Kirstenbosch is situated. while a. mellea might have been introduced into Kirstenbosch separately to that of the clone found in the company gardens and as a. gallica must have been, it would perhaps more easily have spread to this nearby location via basidiospores. one further possible route of introduction to consider relates to the cultivation practices used in the garden. Flowerbeds and paths are frequently covered with wood and bark mulch. as armillaria spp. are common wood rotting fungi, it is possible that a. mellea was introduced into the gardens through this substrate. ing�eld et al    sporocarps have not previously been found in Kirstenbosch. this may simply be related to the fact that they are ephemeral and have not been present when mycologists or plant pathologists might have been visiting the botanical garden. when these sporocarps were discovered, they were relatively widespread and all were morphologically similar. the infected roots from which isolates were made were also from a number of locations, none of which had been associated with the infection by a. gallica . It is possible that a. gallica also fruits in the garden, but at a different time to or it is less prone to fruiting. regular observations will Peripheral surveys of the native forest on the foothills of table mountain and that extending out of the Kirstenbosch Botanical gardens have not revealed evidence of armillaria root rot. the fact that a. mellea is able to fruit profusely in the gardens suggests that it may spread to native forests in the vicinity (Fig. 1a) and more careful surveys should be undertaken to determine whether this is already occurring. certainly this invasive alien fungus has the capacity to result in serious disease problems in the native environment as has been true with the introduced invasive on leucodendron argenteum (silver trees) in Kirstenbosch (van wyk 1973, linde et al . 1997). this potential risk to Kirstenbosch and the native forest associated with it the dutchman Jan van riebeeck was the founder and first commander of cape town between 1652 and 1662. one of his tasks was to establish a vegetable and fruit garden to provide ships of the dutch east India company sailing between the netherlands and east asia with fresh produce and to offset serious problems due to vitamin c deficiency [for a fascinating account of the ship’s surgeons of the dutch east India company see Bruijn (2009)]. this is the origin of the company gardens and the historic avenue of oak ( quercus robur ) trees that line government avenue, the death of which prompted the discovery of a. mellea in cape town (coetzee et al. 2001). at the time of this discovery, popular press took an interest in the problem ( ) and referred to the tragic death of historic trees as “van riebeeck’s curse”. the appearance of a. mellea fruiting profusely in Kirstenbosch, another historic garden of great national importance, suggests that the fungal “van riebeeck’s curse” remains not only present but is growing in importance. It further illustrates the devastating impact that invasive alien pathogens can have on natural woody ecosystems many years after their introduction. Fig. 2. cladogram generated from Its dna sequence data. Bootstrap values are indicated above the tree branches. the year during which isolates from Kirstenbosch and the company gardens were reported are indicated next to the taxon name. Isolates obtained during this study Fungal phoenix   we thank the department of science and technology (dst)/ national research Foundation (nrF) centre of excellence in tree health anderson JB, ullrich rc (1979) Biological species of in north america. mycologia Bruijn I (2009) ship’s surgeons of the dutch east India company. leiden university Press, the netherlands. coetzee mPa, wingfield Bd, coutinho ta, wingfield mJ (2000a) Identification of the causal agent of armillaria root rot of species in south africa. mycologia coetzee mPa, wingfield Bd, harrington tc, dalevi d, coutinho ta, wingfield mJ (2000b) geographical diversity of armillaria mellea : 105–113. coetzee mPa, wingfield Bd, harrington tc, steimel J, coutinho ta, wingfield mJ (2001) the root rot fungus armillaria mellea introduced into south africa by early dutch settlers. coetzee mPa, wingfield Bd, Bloomer P, ridley gs, wingfield mJ (2003a) molecular identification and phylogeny of isolates from south america and Indo-malaysia. coetzee mPa, wingfield Bd, roux J, crous Pw, denman s, wingfield mJ (2003b) discovery of two northern hemisphere species on in south africa. Plant Pathology coetzee mPa, wingfield Bd, Kirisits t, chhetri dB, Bloomer P, wingfield mJ (2005) Identification of isolates from Bhutan based on dna sequence comparisons. Plant Pathology gezahgne a, coetzee mPa, wingfield Bd, wingfield mJ, roux J (2004) Identification of the armillaria root rot pathogen in gould sJ (1992) a humongous fungus among us. natural history gregory sc, rishbeth J, shaw cg (1991) Pathogenicity and virulence. In armillaria root disease . (cg shaw & ga Kile, eds): Forest service united states, department of agriculture, hasegawa e, ota y, hattori t, Kikuchi t (2010) sequence-based identification of Japanese species using the elongation hood Ia, redfern dB, Kile ga (1991) in planted hosts. In armillaria root disease . (cg shaw & ga Kile, eds): 122–149 Forest service united states, department of agriculture, usa. Katoh K, toh h (2008) recent developments in the maFFt multiple sequence alignment program. Briefings in Bioinformatics Keča n, Bodles wJa, woodward s, Karadzic d, Bojovic s (2006) molecular-based identification and phylogeny of species from serbia and montenegro. Forest Pathology Kile ga, mcdonald gI, Byler Jw (1991) ecology and disease in natural forests. In armillaria root disease . (cg shaw & ga Kile, eds): 102–121 Forest service united states, department of agriculture, usa. Korhonen K (1978) Interfertility and clonal size in the Kotzé JJ (1935) Forest fungi: the position in south africa. In Papers and statements on exotics . 4 British empire Forestry conference: 12. south africa. lima mla, asai t, capelari m (2008) armillaria paulensis : a new south american species. mycological research 112 : 1122–1128. linde c, drenth a, wingfield mJ, Broembsen sl von (1997) Phytophthora cinnamomi in south africa. lundquist Je (1986) Fungi associated with in south africa. Part I. the transvaal. south african Forestry Journal lundquist Je (1987) Fungi associated with in south africa, Part III, natal, the orange Free state and the republic of transkei. south african Forestry Journal : 11–19. mwenje e, wingfield Bd, coetzee mPa, nemato h, wingfield mJ species on tea in Kenya identified using isozyme and dna sequence comparisons. Plant Pathology ota y, matsushita n, nagasawa e, terashita t, Fukuda K, suzuki K (1998) Biological species of armillaria in Japan. Plant disease Pildain mB, coetzee mPa, wingfield Bd, wingfield mJ, rajchenberg m (2010) taxonomy of in the Patagonian forests of qin gF, zhao J, Korhonen K (2007) a study on intersterility groups of smith ml, Bruhn Jn, anderson JB (1992) the fungus is among the largest and oldest living organisms. volk tJ, Burdsall hh (1995) a nomenclatural study of armillaria and armillariella species ( Basidiomycotina, tricholomataceae Fungiflora, norway. watling r, Kile ga, gregory nm (1982) the genus - nomenclature, typification, the identity of armillaria mellea and species differentiation. transactions of the British mycological wyk Ps van (1973) root and crown rot of silver trees. south african      © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. Fungi are a central component of the biosphere, essential for the growth of over 90 % of all vascular plants (allen 1993), play an essential role in ecosystem services (Boumans 2002) and it is estimated that there may be as many as 1.5 million species of fungi globally (hawksworth 1991). Fungi can impact on the outcome of plants and their enemies et al . 2006) and are a life support network for most plants (Bardgett et al . 2006). van der heijden et al. (1995) stress the importance of understanding the structure and function of fungal communities is an important contributor to the maintenance of plant biodiversity. development of such a fungal ecology requires an understanding of the spatio- temporal growth and interaction dynamics both within fungal communities and between fungi and plant systems. we therefore propose that an essential step in understanding the ecology of fungi is to combine: (1) a physiologically-based model of fungal community dynamics, capturing colony growth and interactions, in heterogeneous environments; (2) non-destructive quanti�cation of community growth patterns through instrumentation; (3) models linking fungal communities to plant systems; and (4) next-generation computational approaches to simulate complex systems at scales consistent with that instrumentation. here, we report on a special Interest group meeting held during Imc9 that considered this agenda and the four components needed to progress our understanding of fungal ecology. additionally the group considered the notion of fungi as a biological l co In Falconer et al . (2005, 2007, 2008) we demonstrated the use of a physiologically-based model to explore the factors that in�uence the nature of fungal community diversity and the link between individual behaviour and the structure and function of fungal communities. the model is individual-based and incorporates the essential physiological processes of nutrient absorption, within colony biomass transport and recycling, inhibitor production and growth, and these occur differentially within a single mycelium as a consequence of local and non-local context. this differential behaviour permits different parts of the mycelium to expand and senesce concurrently. this framework was developed to capture the minimal set of physiological processes required to reproduce the observed range in phenotypic response in real colonies: uptake, redistribution of biomass, remobilisation of biomass, and growth which are known to be important for vegetative growth of fungi but have not collectively been incorporated , adam t. sampson and nia a. sImBIos centre, university of abertay dundee, dd1 1hg, dundee scotland uK; corresponding author e-mail: r.falconer@abertay.ac.uk Institute for arts, media and computer games, university of abertay dundee, dd1 1hg, dundee scotland uK department of chemistry, university of cambridge, lens�eld road, cambridge, cB2 1ew, uK laboratory of wood technology, department of Forest and water management, ghent university and ugct, university ghent centre for X-ray tomography, ghent university, coupure links 653, Be-9000 gent, Belgium abstract: this contribution, based on a special Interest group session held during Imc9, focuses on physiological based models of �lamentous fungal colony growth and interactions. Fungi are known to be an important component of ecosystems, in terms of colony dynamics and interactions within and between trophic levels. we outline some of the essential components necessary to develop a fungal ecology: a mechanistic model of fungal colony growth and interactions, where observed behaviour can be linked to underlying function; a model of how fungi can cooperate at larger scales; and novel techniques for both exploring quantitatively the scales at which fungi operate; and addressing the computational challenges arising from this highly detailed quanti�cation. we also propose a novel application area for fungi which may provide alternate routes for supporting scienti�c study of colony behaviour. this synthesis offers new potential to explore fungal community dynamics and the impact on ecosystem functioning. article info: submitted: 30 october 2010; accepted: 15 november 2010; Published: 18 november 2010. Key words: Ima Fu gus volu me 1 o 2: 155–159 Falconer et al    into previous modelling frameworks (Falconer et al . 2005). we have also investigated the consequences of environmental heterogeneity for biomass distribution (Falconer et al . 2007), identifying which trait sets allowed individuals to persist in given environmental contexts. the model has been used to explore the effect of different soil management strategies on fungal invasion and interactions (Fig. 1; Kravchenko . 2010). the enhancement of this model to incorporate inhibitor production that impacts inter-colony interactions is described in Falconer et al . (2008). the model was used to generate mycelial distribution maps that emerge from fungal interactions among a community of intrinsically different individuals (Falconer et al. 2010). this is the �rst attempt to model (physiologically) the dynamics of a fungal community in terms of a fungal ecology. we introduced the concept of a biomass-based abundance distribution function, described the form of that curve, and made the �rst attempt to identify the traits that affect the form of that curve. ongoing developments are to apply the model to soil systems to understand the effect of physical and chemical processes on fungal diversity. It has been shown by experiments that the fungal colony exhibits a remarkably complex cooperative behaviour (ritz 1995, hughes & Boddy 1996), and a linked experimental- theoretical approach by Bown et al . (1999) demonstrated that community scale dynamics are a consequence of non- independent local interactions. In our development of fungal ecology, we must also consider such cooperation, and here l co In Perez-reche et al . (2010), we used probabilistic models to determine how cooperation at the individual scale led to epidemic spread at the community scale. to investigate this phenomenon we constructed a model of fungal invasion that is spatially explicit and considers heterogeneous, discrete resource distribution. In general, the transmission of the fungus from a colonised donor host ( ) to a healthy recipient host ( ) does not only depend on the d-r pair but it is in�uenced by the environment of the d-r pair. on the one hand, the rate of transmission of the pathogen may be enhanced (constructive synergy) if the fungus is using resources from several colonised hosts. on the other hand, the rate can be diminished (interfering synergy) because of several factors arising from the competition between different parts of the colony. we have addressed the question of whether synergistic effects occurring at the individual scale play an important role in epidemics spreading at the community-scale. we have also investigated the effect of synergy on properties such as the foraging strategy followed by the pathogen, the probability of epidemic invasion, and the ef�ciency of invasion. the approach is based on an extension of a spatial model for sIr (susceptible-infected- removed) processes (grassberger 1983) to incorporate synergistic effects in the transmission rate between pairs of hosts depending on the number of infected neighbours to the pair. analysis of the model by means of numerical simulations has shown that synergy at the host level has non-trivial consequences at the population level. the foraging strategy of the pathogen changes from being explorative for interfering synergy to being exploitative for constructive synergy. the invasion in the exploitative regime is temporally more ef�cient, i.e., it is quicker, than invasion using an explorative strategy. however, explorative epidemics are spatially more ef�cient than exploitative epidemics because they can lead to invasion by infecting fewer hosts. the modelling carried out so far is based on simple assumption such as equal intrinsic infectivity and susceptibility for all the hosts in the population. extensions of the model to account for heterogeneity in transmission of infection and perhaps other factors will be essential to provide quantitative predictions for possibly invasive epidemics in real populations. while it is possible to validate models of infection spread because the domain may be directly observed, such as in infected plants where the number of lesions can be determined via direct or indirect methods (Jeger 1987), this is much more challenging for opaque soil and wood systems. For these systems, in order to obtain the experimental data for model calibration and validation information regarding the spatial distribution and biomass amounts is required. one technique that has been used to determine the physical architecture of the soil and wood systems is X-ray computed tomography and progress is being made in quantifying and visualising fungal biomass in situ qu atI o Patte X-ray computed tomography enables a non-destructive view of the internal structure of an object and is therefore an extremely valuable technique in many research �elds. the continuously improving performance of equipment, Fig. 1. effect of soil structure on fungal invasions and interactions for a single soil management practice. Blue and red isosurfaces correspond to the two boundaries of different fungal species. modelling fungal colonies and communities: challenges and opportunities   rapidly increasing computing power, and faster algorithms for reconstruction and data processing make large volume scanning at high resolutions feasible. the state-of-the-art equipment at the ugct (centre for X-ray tomography at ghent university) is highly �exible, with in-house developed software for scanner control, sample reconstruction, analysis, and visualisation. this set-up allows scanning with a resolution of 0.2 mm for samples of 37 cm in diameter down to approximately 400 nm for objects about the size of a splinter. as such, apart from visualisation, 3d quantitative information can be retrieved from objects with a broad range of sizes. sub-micron resolution scanning should enable the visualisation of fungal hyphae and by using time-lapse tomography the growth of these tubular structures could be monitored (van den Bulcke et al. 2009). the latter procedure however has associated challenges. First, fungal growth can interfere with scanning during moderately long scan times. second, with lab-based X-ray sources, polychromatic X-rays, scattering, �uorescence and noise disturb the ideal acquisition (vidal et al . 2005). third, at sub-micron resolution phase contrast emerges especially at sharp edges, complicating thresholding and segmentation. Fourth, tube shift during long scans at sub-micron resolution can reduce image quality. Fifth, hyphal tubes are hollow thin-walled structures, as such having a very low X-ray attenuation. a drastic solution to some of the problems is the use of synchrotron radiation, having a monochromatic X-ray bundle, allowing faster scanning with less heating of the samples, but access to such facilities is a major bottleneck. especially the available beam time is limited and as such this is not an option for long-running experiments, of the order of days to weeks, and for repeated experiments. many of the aforementioned problems are handled at the ugct facility. Post-processing can contribute to the enhancement of image quality; the phase contrast phenomenon can be solved using dedicated �ltering (Boone et al. 2009, de et al. 2009); and tube shift can be counteracted with correction software. Proper scanning and processing can result in the visualization of fungal hyphae as illustrated in Fig. 2, obtained after scanning of a piece of Pinus sylvestris subjected to white-rot. In order to study pigmented species with rather large hyphal structures, such as (van den Bulcke et al . 2008), visualization is easier due to X-ray interference of the pigment. apart from individual hypha tracking, processing of X-ray volumes should enable the quanti�cation of the effects of material degradation on different spatial scales, which might be an important concept to implement a degradation monitoring system. with the existing scanners, frequent scanning and quanti�cation of degradation or hyphal biomass on a larger spatial scale will be a very valuable tool for non-destructive time-lapse analysis. advanced algorithms implementing X-ray physics during reconstruction will increase image quality, whereas more advanced image processing code will improve quantitative results. the �eld of X-ray tomography, both hard- and software, is rapidly evolving and therefore is promising for in situ fungal monitoring and quanti�cation in wood and perhaps soil systems in the near future, in addition to other modalities such as confocal laser microscopy (hickey et al atI tatI l our ability to exploit the experimental advantages described above is currently constrained by the limited scales at which existing simulation technologies are able to operate. For example, in spite of data at larger scales, in Falconer et al (2010) we use a domain size for the soil/fungal interactions of approx 1 cm with a voxel resolution of 30 microns; for predictions to be useful we need to work at, at the very Fig. 2. (a) three dimensional rendering of hyphal tubes of a white rot fungus winding around a small piece of Pinus sylvestris in contact with malt extract agar (�lling some cell lumina). (b) cross-sectional and (c) longitudinal view illustrating the high anatomical detail. Bar 200 Falconer et al    least, core scale (10 cm ). we need simulations to operate at realistic scales in order to accurately reproduce the observed, often emergent behaviours we wish to study. emergent behaviours are often scale-dependent, a simulation that includes only a restricted region of the system may demonstrate different emergent behaviours, and we may not know in advance what scale is appropriate. one approach to scaling up simulations is to simplify the underlying model, for example the homogenisation approach proposed by roose & schnepf (2008), where the environmental heterogeneity is carefully coarse-grained, and by model reduction, for example et al . (2009), where stripping out unnecessary model components reduces computational demands. Both raise considerable dif�culties. In each case, simpli�cation requires identi�cation of the important model components, which are unlikely to be obvious in a complex system. an alternative approach is to increase the computational power available to the simulation by taking advantage of multicore cPus, gPus and clusters. Parallelisation is widely regarded as an experts-only programming problem, and one that is strongly tied to the particular computational platform in use. however, complex biological systems simulation is a problem with an inherently high degree of concurrency in the natural system: a complex system fundamentally consists of a large number of independent, but interacting, agents and processes. approaches to parallel programming focus on highly regular numerical problems, and make building such a simulation dif�cult. simulation becomes much more straightforward with the use of concurrent programming techniques, in which the concurrent activities in a system and their relationships are speci�ed, and the execution of those activities in the most ef�cient manner across the available resources is managed automatically by software. with careful design, a simulation built this way can be truly scalable, meaning that its complexity can be increased in a near-linear fashion by dividing it across more computational resources. this approach has become particularly interesting with the rise of grid and cloud computing: researchers can now gain access as required for a short period of time to a very large number of nodes upon which to execute their simulation, rather than relying upon in-house resources. using cloud resources, we can potentially scale up by a few orders of magnitude. approaches to scalability and validation in complex systems simulation are currently being investigated by cosmos (www.cosmos-research.org), drawing on expertise in modelling, highly-dependable software engineering and concurrent programming to develop and document reusable techniques for complex systems modelling and simulation. cosmos techniques for parallel, distributed simulation of agent-based spatial models have been successfully applied to problems including those in the �elds of immunology (in- silico experimentation with lymphocyte migration (andrews . 2008) and granuloma formation (Flügge et al . 2009)) and mycology (scaling up of the Falconer et al 2005 model, in metaP Fungal colonies are a highly successful organism, demonstrating pervasive growth through harsh environments. they achieve this through their capacity to operate in a decentralised manner, reacting locally to changes in context while interoperating at the colony scale and with other organisms. Fungi have the capacity to recycle biomass, effecting dynamic reallocation of investment, capitalise on �uxes in available resource and self-heal. these properties make them attractive metaphors for managing large, complex, distributed arti�cial networks. other researchers have used other biological metaphors for similar problems. For example, ant colonies have been used as a metaphor for telecommunication routing algorithms. typically, stigmergic pheromone trails are used to pro�le rates of �ow of ants and other network traf�c and the local strength of the trail is coupled to the values in local routing tables (e.g. di caro & dorigo 1998). similarly, in the �eld of arti�cial immune systems, the properties of the immune system have been used to inspire solutions in the areas of anomaly detection and data mining (timmis et al . 2008). we have been exploring the potential for fungi as a metaphor for protecting society’s critical infrastructures. modern societies are heavily dependent upon a number of critical infrastructure networks that allow our societies to function, including water, power and transportation. these networks are open to failure through a range of processes including shortage of essential resources, breakdowns at key nodes and surges in demand and this means that effective management of such networks is challenging (schulman et al. 2004). Bebber et al . (2007) recognise that understanding how fungi grow may inform the design of man-made networks and through image analysis, have characterised fungal colony growth patterns in terms of nodes and edges of a graph. they show that fungal colonies are ef�cient transport networks that are robust to damage and react to local variation in resource. In order to translate the concept into a working solution, we have developed a graph-based implementation of Falconer et al . (2005). we are now exploring the capacity of our model to provide robust and resilient management solutions to resource limitations, allen m (1993) the ecology of mycorrhizae . cambridge: andrews Ps, Polack F, sampson at, timmis J, scott l, coles m (2008) simulating biology: towards understanding what the simulation shows. In: stepney s, Polack F, welch P (eds) Proceedings of the 2008 workshop on complex systems modelling and simulation, york, uK, september 2008 : 93– 123. york, uK: luniver Press. Bardgett r, smith rs, shiel rs, Peacock s, simkin Jm, quirk h (2006) Parasitic plants indirectly regulate below-ground modelling fungal colonies and communities: challenges and opportunities   Bebber dP, hynes J, darrah Pr, Boddy l, Fricker md (2007) Biological solutions to transport network design. of the royal society, B Bennett a, alers-garcia J, Bever J (2006) three-way interactions among mutualistic mycorrhizal fungi, plants, and plant enemies: hypotheses and synthesis. american naturalist Boone m, witte y de, dierick m, Bulcke J van den, vlassenbroeck J, hoorebeke l van (2009) Practical use of the modi�ed Bronnikov algorithm in micro-ct. nuclear Instruments and methods in Physics research section B: Beam Interactions with materials and atoms : 1182–1186. Boumans r (2002) modelling the dynamics of the integrated earth system and the value of global ecosystem services using the Bown Jl, sturrock cJ, samson wB, staines hJ, Palfreyman Jw, white na, ritz K, crawford Jw (1999) evidence for emergent behaviour in the community scale dynamics of a fungal microcosm. Proceedings of the royal society, B Bulcke J van den Boone m, acker J van, hoorebeke l van (2009) three-dimensional X-ray imaging and analysis of fungi on Bulcke J van den, masschaele B, dierick m, acker J van, stevens m, hoorebeke l van (2008) three-dimensional imaging and analysis of infested coated wood with X-ray submicron ct. International Biodeterioration and Biodegradation caro g di, dorigo m (1998) antnet: distributed stigmergetic control for communications networks. Journal of artifcial Falconer re, Bown Jl, white na, crawford Jw (2005) Biomass recycling and the origin of phenotype in fungal mycelia. Proceedings of the royal society, B Falconer re, Bown Jl, white na, crawford Jw (2007) Biomass 116 Falconer re, Bown Jl, white na, crawford Jw (2008) modelling Falconer re, Bown Jl, white na, crawford Jw (2010) linking fungal individuals to community scale patterns. Flügge aJ, timmis J, andrews P, moore J, Kaye P (2009) modelling and simulation of granuloma formation in visceral leishmaniasis. In: 2009 Ieee congress on evolutionary computation (cec 2009) : 3052–3059. trondheim, norway, gibbons Jm, wood ata, craigon J, ramsde sJ, crout nmJ (2009) semi-automatic reduction and upscaling of large models: a farm management example. ecological modelling grassberger P (1983) on the critical behavior of the general epidemic process and dynamical percolation. hawksworth dl (1991) the fungal dimension of biodiversity: magnitude, signi�cance, and conservation. heijden mga van der, Klironomos Jn, ursic m, moutoglis P, streitwolf-engel r, Boller t, wiemken a, sanders I (1995) mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. hickey Pc, swift sr, roca mg, read nd (2005) live-cell imaging of �lamentous fungi using vital �uorescent dyes. hughes cl, Boddy l (1996) sequential encounter of wood resources by mycelial cords of Phanerochaete velutina effect of growth patterns and phosphorus allocation, Jeger mJ (1987) the in�uence of root-growth and inoculum density on the dynamics of root disease epidemics – Kravchenko ar, Falconer re, grinev d, otten w (2010) Fungal colonization in soils of contrasting managements: modelling fungal growth in 3d pore volumes of undisturbed soil ecological applications muller u, Bammer r, teischinger a (2002) detection of incipient fungal attack in wood using magnetic resonance parameter ritz K (1995) growth responses of some soil fungi to spatially heterogeneous nutrients. Fems microbial ecol roose t, schnepf a (2008) mathematical models of plant-soil Philosophical transactions of the royal society, , 4597–4611. schulman P, roe e, eeten m van, Bruijne m de (2004) high reliability and the management of critical infrastructures. Journal of contingencies and crisis management : 14–28. timmis J, hart e, hone a, neal m, robins a, stepney s, tyrell a (2008) Immuno-engineering. In: hinchey m, Pagnoni a, rammig J, schmeck h (eds) Inspired collaborative computing : 3–17. [IFIP International Federation for Information Processing, no. 268.] Boston, springer verlag. vidal FP, letang Jm, Peix g, cloetens P (2005) Investigation of artefact sources in synchrotron microtomography via virtual X-ray imaging. nuclear Instruments and methods in Physics research, B: Beam Interactions with materials and atoms witte y de, Boone m, vlassenbroeck J, dierick m, hoorebeke l van (2009) Bronnikov-aided correction for X-ray computed tomography. Journal of the optical society of america, a      © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. a special Interest group meeting colletotrichum: species, ecology and interactions , was held on 1 august 2010 at the International mycological congress (Imc9) in edinburgh, uK. the meeting, organised by Paul cannon (uK) and ulrike damm (the netherlands), brought together 23 scientists from 12 countries working in different �elds of mycology, but with a common interest in the genus . seven presentations, covering a wide range of topics, ranged from systematics and population genetics to host-pathogen interactions and genome projects. this contribution provides the �rst four presentations dealt with systematics and identi�cation of major colletotrichum species complexes containing various important anthracnose pathogens worldwide. Four of these species are illustrated in Fig. 1. while the identity of many important species still require revision (hyde et al. 2009), molecular techniques improve the delimitation of species that are hard to distinguish based on morphology alone and reveal their phylogenetic relationships et al. 2009, crouch et al. 2009, damm et al. 2009). this will inevitably result in name changes but has implications for everyone working with this genus, especially plant pathologists, and will improve our understanding of the role ulrike damm gave an overview of her ongoing collaboration with Paul cannon about the phylogeny of three species complexes. the aim of this project is to delimitate species within these complexes, characterise known and new species and designate epitypes to provide the basis for accurate identi�cations of species. this goal has so far been achieved for species with curved conidia from herbaceous hosts (damm et al. 2009), which in the past were mostly identi�ed as c. dematium . multi-gene analyses and morphological characterisation revealed several diverse and distantly related species, including four new species. seven species were epitypi�ed, including c. dematium and the type species of the genus, c. lineola . a second study con�rmed most of the previously recognised groups (sreenivasaprasad & talhinhas 2005) within the c. acutatum species complex. most of these could be de�ned on the basis of type strains or strains suitable for epitypi�cation. literature reports (lubbe 2004, Johnston et al. 2005) and preliminary studies using Its sequence data indicated that c. boninense represents a species complex as well. a multilocus molecular phylogenetic , yasuyuki Kubo , Bevan weir , Kae yoshino and Paul F. cBs-Knaw Fungal Biodiversity centre, uppsalalaan 8, 3584 ct utrecht, the netherlands; corresponding author e-mail: u.damm@cbs. knaw.nl school of life sciences, university of warwick, wellesbourne, warwick, cv35 9eF, uK Key laboratory of systematic mycology & lichenology, Institute of microbiology, chinese academy of sciences, no. 10, north 4 west, Beijing 100190, P.r. china Kyoto Prefectural university, 1-5 hangi-cho, shimogamo, sakyo-ku, Kyoto, Japan landcare research, Private Bag 92170 auckland, new zealand graduate school of agriculture, Kyoto university, sakyo-ku, Kyoto 606-8502, Japan caBI europe-uK, Bakeham lane, egham, surrey tw20 9ty, uK; and royal Botanic gardens, Kew, surrey tw9 3aB, uK abstract: the presentations of the special Interest group meeting colletotrichum: species, ecology and , held on 1 august 2010 during Imc9 in edinburgh, uK, are outlined. seven research projects, ranged from systematics and population genetics to host-pathogen interactions and genome projects were presented. the meeting revealed that currently major species complexes in the genus are being revised and the identities of many pathogens clari�ed on the basis of molecular phylogenies, and that the genomes of four species are sequenced and decoded providing an enormous amount of data that are article info: submitted: 27 october 2010; accepted: 20 november 2010; Published: 23 november 2010. Key words: Ima Fu gus volu me 1 o 2: 161–165 amm et al    analysis of strains previously identi�ed as c. boninense resulted in clades that could be recognised as separate species with differences in host range, distribution and morphology, including c. boninense sensu stricto c. hippeastri and several presumably new species. most of the species in the c. boninense complex and some in the c. acutatum species complex form teleomorph states in culture. Publications on the c. acutatum and c. boninense species complexes will appear in a 2011 issue of studies in mycology. colletotrichum gloeosporioides sensu lato is a species complex with broad genetic and biological diversity grouped together by similar conidial morphology and Its sequences. Bevan weir and Peter Johnston (landcare research, auckland, new zealand) presented their research on this species complex and possible approaches to species delimitation through the genealogical concordance Phylogenetic species recognition (gcPsr). It was shown that that the taxa c. musae , c. kahawae , c. xanthorrhoeae c. nupharicola , c. fragariae, c. gloeosporioides sensu stricto, c. horii , c. theobromicola , c. ignotum , c. tropicale , c. asianum , c. siamense , c. fructicola and c. hymenocallidis as well as many putative undescribed species are part of the c. gloeosporioides sensu lato complex. they recently characterised and neotypi�ed one of these species, c. horii (weir & Johnston 2010). the gcPsr concept was used to delimit taxa within c. gloeosporioides sensu lato . this concept considers that phylogenetic trees of different genes show discordance within a species due to gene �ow between individuals. the common node where different gene trees show concordance is considered the speciation point. they applied the gcPsr with eight genes using recently developed Bayesian analysis tool, BucKy (ané et al. 2007). the gcPsr concept worked well for species delimitation along currently recognised lines, except for c. kahawae which was insuf�ciently distinct from several genetically similar non-coffee Berry disease causing taxa. It was suggested that this may be due to the recent emergence (1920) of c. kahawae as a pathogen and that insuf�cient time had passed for ecological niche specialisation to show as mutations in the genes used. they suggested that c. kahawae be recognised at the subspeci�c rank. a publication on their work on the c. gloeosporioides sensu lato species complex will appear in the 2011 studies in mycology issue on colletotrichum as well. colletotrichum acutatum causes economically signi�cant losses of temperate, subtropical and tropical crops. globally, c. acutatum populations display considerable genotypic and phenotypic diversity. riccardo Baroncelli (university of warwick, wellesbourne, uK) presented his research on evolutionary relationships in c. acutatum populations in collaboration with charles lane (Fera, sand hutton, york, uK) and Prasad sreenivasaprasad (university of warwick, wellesbourne, uK). the overall objective is to understand the evolutionary relationships within the species with particular reference to the pathogen populations associated with the strawberry production systems in the uK. more than 150 c. acutatum isolates related to different hosts worldwide have been assembled. Phylogenetic analysis of sequence data from the rdna block, mat1-2 and βtubulin-2 genes shows eight distinct genetic groups within c. acutatum . the subsets of isolates represented within these genetic groups corresponded to the previously identi�ed groups a1 to a8. almost all of the homothallic isolates capable of sexual reproduction comprise a single genetic group, a7. Isolates representing populations capable of heterothallic sexual Fig. 1. conidiomata and conidia of four species. a, e. c. acutatum (cBs 112996, ex-paratype strain). B, F. c. lineola (cBs 125337, ex-epitype strain). c. truncatum (cBs 151.35, ex-epitype strain). c. gloeosporioides (cBs 112999, ex-epitype strain). F, conidia on sna. conidiomata on sna. , e. conidiomata on stem. scale bars: a 10 μm, B 100 μm, e 200 μm. a applies to a, c, F, h. B applies to B, d, g. colletotrichum : species, ecology and interactions   reproduction belong to two distinct genetic groups a3 and a5. molecular characterisation of c. acutatum populations representing the introduction and spread of the pathogen in the strawberry production systems in the uK showed the presence of three genetic groups (a2, a3 and a4). their results suggest the existence of c. acutatum populations potentially undergoing speciation processes, related to their reproductive behaviour and host association patterns. Further lei cai (Key laboratory of systematic mycology & lichenology, Institute of microbiology, Beijing, china) and Kevin hyde (school of science, mae Fah luang university, chiang rai, thailand) presented their research colletotrichum species from asian fruits and leaves. Fruit rots (anthracnose) were previously often attributed to c. gloeosporioides c. acutatum . Identi�cations were, however, based on morphological characters or, if gene sequence data were used, comparisons were often made with wrongly applied names. colletotrichum gloeosporioides was recently epitypi�ed (cannon et al. 2008) so that living cultures and sequence data are, for the �rst time available for comparison with fresh collections. analysis of sequence data of 25 isolates (selected from 140 obtained strains based on diversity of host and morphology) from eight tropical fruits are compared with the c. gloeosporioides epitype. contrary to previous assumptions, none of these isolates from tropical fruits was c. gloeosporioides sensu stricto 2010). the �ve gene regions used in this study resolved c. asianum, c fructicola, c. horii, c. kahawae in the c. gloeosporioides species complex as distinct phylogenetic lineages with high statistical support. many tested strains could not be assigned to any known taxa in this analysis. they also reported amaryllidaceae, orchidaceae, cordyline and Jasminum sambac , with the latter including two new species et al. 2010), and updated the typi�cations of species provide excellent models for studying fungal-plant interactions (Perfect et al. 1999). several large- scale genome projects are in progress for species aiming to produce high-quality assemblies of the genome sequences to provide resources for comparative genomics and the molecular analysis of fungal pathogenicity, which allows the identi�cation of genes and proteins relevant is a destructive pathogen of maize, causing stalk rot and leaf blight, while attacks many cultivated forms of as well as arabidopsis thaliana , providing a model pathosystem in which both partners can be genetically manipulated (o’connell 2004). Both pathogens employ a hemibiotrophic infection strategy, but while the biotrophic phase of c. graminicola extends into many host cells, that of c. is con�ned to single epidermal cells. richard o’connell (max Planck Institute for Plant Breeding research, cologne, germany) gave an overview of the c. higginsianum and genome research, which he is conducting in collaboration with lisa vaillancourt (university of Kentucky, usa), li-Jun ma (mIt-Broad Institute, usa) and mike thon (cIale-university of salamanca, spain). comparing the genomes of two species with contrasting pathogenic lifestyles and host speci�cities will allow them to study lineage-speci�c expansions and contractions of gene families and identify genes undergoing rapid evolution (diversifying selection), which may be involved in interactions with the host plant, e.g. those encoding secreted effector proteins. the 57.4 mb genome of c. graminicola comprises 13 chromosomes and was sequenced at the Broad Institute (8X sanger, 11X paired- end 454) giving an assembly of 1,151 contigs in 653 scaffolds ), to carry out a whole genome sequence of the ex-epitype strain of (ImI 356878). the dna sequencing phase is nearing completion and they are going to start soon with the assembly. the meeting provided good evidence of the rapidity with which our understanding is improving of genomics, and nicely complemented the outputs of another workshop that was held earlier in 2010, in conjunction with the 10 european conference on Fungal genetics in leeuwenhorst, the netherlands. It was particularly exciting to witness the increasing power of genomic research tools, and their potential impact on our understanding of fungal systematics and speciation. It provided good opportunities to coordinate research programmes, exchange data and share alan Buddie (caBI europe-uK, egham, uK) is thanked for sharing ané c, larget B, Baum da, smith sd, rokas a (2007) Bayesian estimation of concordance among gene trees. molecular Biology asakura m, ninomiya s, sugimoto m, oku m, yamashita s, okuno t, sakai y, takano y (2009) atg26-mediated Pexophagy Is required for host Invasion by the Plant Pathogenic Fungus cai l, hyde Kd, taylor PwJ, weir Bs, waller J, abang mm, zhang Jz, yang yl, Phoulivong s, liu zy, Prihastuti h, shivas rg, mcKenzie ehc, Johnston Pr (2009) a polyphasic approach for cannon PF, Buddie ag, Bridge Pd (2008) the typi�cation of crouch Ja, clarke BB, white Jw, hillman BI (2009a) systematic analysis of the falcate-spored graminicolous and a description of six new species of the fungus from warm-season damm u, woudenberg Jhc, cannon PF, crous, Pw (2009) species with curved conidia from herbaceous Fujihara n, sakaguchi a, tanaka s, Fujii s, tsuji g, shiraishi t, o’connell r, Kubo y (2010) Peroxisome biogenesis factor PeX13 is required for appressorium-mediated plant infection by the anthracnose fungus colletotrichum orbiculare hyde Kd, cai l, cannon PF, crouch Ja, crous Pw, damm u, goodwin Ph, chen h, Johnston Pr, Jones eBg, liu zy, mcKenzie ehc, moriwaki J, noireung P, Pennycook sr, Pfenning lh, Prihastuti h, sato t, shivas rg, tan yP, taylor PwJ, weir Bs, yang yl, zhang Jz (2009) – names in current use. Johnston Pr, Pennycook sr, manning ma (2005) taxonomy of fruit- rotting fungal pathogens: what’s really out there? new zealand Kimura a, takano y, Furusawa I, okuno t (2001) Peroxisomal metabolic Function Is required for appressorium-mediated Plant Infection by colletotrichum lagenarium Plant cell lubbe cm, denman s, cannon, PF, groenewald Jz, lamprecht sc, crous Pw (2004) characterization of colletotrichum species associated with diseases of Proteaceae . mycologia 96 : 1268–1279. o’connell r, herbert c, sreenivasaprasad s, Khatib m, esquerré- tugayé m-t, dumas B (2004) a novel pathosystem for the molecular dissection of plant-fungal colletotrichum : species, ecology and interactions   Perfect se, hughes hB, o’connell rJ, green Jr (1999) colletotrichum a model genus for studies on pathology and fungal–plant interactions. Fungal genetics and Biology 27 :186–198. Phoulivong s, cai l, chen h, mcKenzie ehc, abdelsalam K, chukeatirote e, Kd (2010) colletotrichum gloeosporioides is not a common pathogen on tropical fruits. Fungal diversity sreenivasaprasad s, talhinhas P (2005) genotypic and phenotypic diversity in colletotrichum acutatum , a cosmopolitan pathogen causing anthracnose on a wide range of hosts. molecular Plant takano y, Kubo y, Kuroda I, Furusawa I (1997) temporal transcriptional pattern of three melanin biosynthesis genes, PKs1, scd1, and thr1, in appressorium-differentiating and nondifferentiating conidia of colletotrichum lagenarium tanaka s, Ishihama n, yoshioka h, huser a, o’connell r, tsuji g, tsuge s, Kubo y (2009) colletotrichum orbiculare ssd1 mutant enhances plant basal resistance through activation of a mitogen- activated protein kinase pathway. tsuji g, Fujii s, Fujihara n, hirose c, tsuge s, shiraishi t, Kubo y agrobacterium tumefaciens -mediated transformation for random insertional mutagenesis in colletotrichum lagenarium weir Bs, Johnston Pr (2010) characterisation and neotypi�cation gloeosporium kaki hori as colletotrichum horii nom. nov. 111 wikee s, cai l, Pairin n, mcKenzie ehc, su y-y, chukeatirote e, thi hn, Bahkali ah, moslem, ma, abdelsalam K, hyde Kd (2010) species from Jasmine ( Jasminum sambac    amm et al   © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. the presentations at the session demonstrated that current species recognition in lichen forming-fungi vastly underestimates the true number of species. Based on phylogenetic and population studies, many cases were presented showing that numerous distinct lineages are hidden under a single species name. the issues raised can be grouped under the following headings: naming cryptic species, numbers of cryptic species, recognition of cryptic species, supporting species separations, and phylogeographic correlations. collectively, these presentations provide a synopsis of the current state of the recognition and naming of cryptic species from cryptic lineages was discussed and approaches and options were suggested. hawksworth (2010) examined different groups as foraminifera, plant-pathogenic fungi, insects, and plants. the main species concepts were reviewed, and a pragmatic concept was proposed, de�ning a species as “groups of individuals separated by inheritable discontinuities and which it is useful to give a species name to” (hawksworth 1996, 2010). the term cryptic species was circumscribed as “populations which are phylogenetically distinct and able to reproduce themselves, by sexual means or otherwise, but which are distinguished by molecular or other features that are either not evident macroscopically or generally an increasing number of lichen-forming species are used as biomonitors or bioindicators of pollutants, environmental disturbance, or ecological continuity. consequently there was the issue of how to proceed when cryptic species or lineages are found in taxa used in such studies where identi�cations need to be made quickly during �eld assessments – and access to a modern molecular laboratory is impractical. an acceptable way of referring to such groups of species was commended by hawksworth (2010). the term “complex” or “aggregate” was supported as used when the populations are closely related, i.e. have a recent shared common ancestor. this practice is already familiar to and regularly used by botanists, citizen scientists, and ecologists dealing with complexes in plants, for example the rubus fruticosus aggr. taraxacum of�cinale aggr. In some situations, however, the option of recognizing subspecies was suggested as perhaps the most appropriate solution, for example in paraphyletic populations (Figs 1 and 2) such as that of Parmelina pastillifera and P. tiliacea s. str. et al. 2010) (Fig. 1). In contrast, in cases where the cryptic taxa are not closely related but a result of convergence, i.e. they do not either occupy the same clade or have a recent common ancestor, it has to be recognized that the “complex” approach could give a misleading impression of af�nity, as in Parmelina cryptotiliacea (núñez zapata et al. 2010) or lineages in Parmelia saxatilis (divakar et al. 2010b). and h. thorsten lumbsch departamento de Biología vegetal II,Facultad de Farmacia, universidad complutense de madrid, e-28040 madrid, spain; corresponding author e-mail: acrespo@farm.ucm.es department of Botany, the Field museum, 1400 south lake shore drive, chicago, Illinois 60605, usa abstract: this contribution provides a synopsis of the presentations and discussions during the sIg session on cryptic speciation in lichen-forming fungi held during Imc9. In several cases, a re-examination of morphology against the background of molecular phylogenetic evidence revealed, sometimes subtle, morphological and/or chemical characters, supporting the distinction of particular clades at species level. however, there are also examples of cryptic species in which no morphological characters could be identi�ed to distinguish between lineages. several cases were presented in which distinct lineages are correlated with biogeographical patterns. when and how to name cryptic species was debated, and the use of terms such as “complex” or “aggregate” commended where the taxa formed part article info: submitted: 5 november 2010; accepted: 15 november 2010; Published: 23 november 2010. Key words: Ima Fu gus volu me 1 o 2: 167–170 respo and umbsch    there is a growing body of evi dence that the approach to current species recognition in lichenized fungi, which is largely based on morphology and chemistry, vastly underestimates the number of phylogenetic species. Phylogenetic studies repeatedly indicate that numerous distinct lineages can be hidden under a single species name (arguello et al. 2007, Baloch & grube 2009, grube & Kroken 2000, Kroken & taylor 2001, molina et al. 2004, wirtz et al. 2008). In a number of cases, morphological or chemical differences have been preted as intraspeci �c variability. re-examination of morphology against the background of a molecular phylogeny often reveals, sometimes subtle, and previously overlooked or viewed as unimportant, morphological and/or chemical characters, supporting the dis tinction of these clades at species level (arguello et al. 2007, divakar et al. 2005a, 2005b, molina et al. 2004, wirtz et al. 2008). however, there are also cases of cryptic species in which no morpho characters have yet been identi�ed to distinguish distinct lineages. In several cases, distinct lineages are correlated with distinct bio geographical patterns (arguello et al. 2007, et al. 2010, molina et al. 2004, wirtz et al. 2008). Phylogenetic studies identi�ed distinct lineages that occur in different geo the large and increasing number of cryptic lineages detected in fungi means that the recognition of these lineages as separate taxa is a major issue of current fungal taxonomy Fig. 1. Phylogenetic tree of ( ). majority rule consensus tree based on 18000 trees from B/mcmc tree sampling procedure from a combined data set of nuIts rdna and mtlsu rdna sequences. Posterior probabilities ≥0.95 in the Bayesian analysis are the branches and mP boostrap values ≥0.75 below branches. Branches with signi�cative support in both analyses are in bold. (au austria, cI canary Islands, Fr France, ge germany, In India, It Italy, mo morocco, sP spain, sv slovenia, tK turkey, tn tunisia, usa united states of america). Figure provided by nuñez-zapata ryptic species in lichen-forming fungi   (crespo & Pérez-ortega 2009, hawksworth 2001). however, cryptic species in lichen-forming fungi may be compared to fungi with other biologies where morphological characters are almost absent, thus the pertinence of using this concept in lichens was discussed (hawksworth 2010, Pérez-ortega & Printzen 2010). unlike many microscopic fungi, some groups of lichens form distinctive macroscopic structures, frequently with a foliose or fruticose form, and with easily observable phenotypical differences. despite these structures, the plasticity of morphological and chemical characters in these fungi results in a relatively high number of lichens, species or genera, being “dif�cult” for identi�cation, often accompanied by a frequent lack of generative characters (divakar et al. 2010b) or the frequency of homoplasy and convergence of characters (grube & hawksworth 2007, muggia 2010, muggia although only relatively few lichens have yet been identi�ed as comprising cryptic species using molecular data (grube & Kroken 2000, Kroken & taylor 2001, crespo et al. 2002, Feuerer & thell 2002, Printzen et al. 2003, molina . 2004, argüello et al . 2007, wirtz et al. 2008, Fehrer et al. 2009, divakar et al. 2010a), assemblages of morphologically similar species where identi�cation remains dubious due to variability or ambiguity of key characters used to distinguish those taxa are common. thus, morphological identi�cation of a lichen-forming species, sometimes even a genus, can be dif�cult. therefore, cryptic taxa have been recognised historically in lichens, although not necessarily by that term. “the recognition and characterization of cryptic species is a burgeoning and exciting activity in current systematics, and a major challenge for mycologists of all kinds, not least lichenologists” (hawksworth 2010). suggestions for when to formally recognise species within cryptic lineages that are found in molecular studies were discussed (muggia 2010, Pérez-ortega & Printzen 2010), and a consensus of the session was to recognise species formally when the phylogeny was unequivocal and other evidence supported their separation, whether ultramicroscopic, “new” morphological, ecological (muggia 2010) or geographical (Parnmen et al. 2010) were ePa atI recent molecular phylogenies have supported some species separations that were previously based on subtle characters: for example, Parmelina carporrhizans and P. quercina (argüello et al. 2007, divakar et al. 2010b), caloplaca alociza and c. albopruinosa (muggia 2010). It is also frequently found that distantly related major lineages show a surprising degree of morphological convergence. examples of this phenomenon can be found within large families such as Parmeliaceae . For example, Parmelina and austroparmelina were recently separated as independent genera based on geography and phylogeny. however, all species of austroparmelina were previously included in concept of the genus Parmelina (crespo et al. 2010, divakar et al. 2010b). also there are examples in microlichens, as in capnodiales where the morphologically similar genera racodium and cystocoleus belong to independent lineages in recent phylogenetic studies (muggia et al. 2008, muggia 2010). atI a number of lichen-forming species were historically thought to have wide distributions, including cosmopolitan and pantropical species. however, while that may be so for some species, molecular analyses have repeatedly demonstrated that many lineages can be hidden under a similar morphology. several examples were discussed in the symposium (divakar 2010, muggia 2010, Parnmen et al. 2010). divakar et al. (2010) also found a correlation between reproductive modes and distribution patterns. In fertile species, cryptic lineages were frequently found, and geographically disjunct populations were discovered to represent different lineages (divakar et al. 2010a). several examples of this type were presented, including melanelixia glabra and Parmelina quercina, two species distributed in areas with winter rain (mediterranean climate) in north africa, europe and north america (argüello et al. 2007, divakar et al. 2010a, b). In sorediate taxa, cryptic Fig. 2. Parmelina pastillifera (maF 16473; upper) and P. tiliacea (maF 16632; lower) both showing isidia, but in P. pastillifera they are P. tiliacea    respo and umbsch lineages have also been found, but in this case the lineages can include specimens from different geographical regions; examples include Flavoparmelia caperata, Parmotrema reticulatum , and P. tinctorum (divakar et al. 2005, 2010). we acknowledge the participation of david l. hawksworth (university complutense of madrid, spain) in �nalizing this article; his revision enriched the manuscript with important suggestions improving the text. this work was supported by the spanish ministerio de ciencia e Innovación (cgl 2010-21646/Bos, cgl2007-64652/Bos) and a grant of the national science Foundation to h.t.l. (“hidden diversity argüello a, del Prado r, cubas P, crespo a (2007) Parmelia quercina ) includes four phylogenetically supported morphospecies. Biological Journal of the linnean Baloch e, grube m (2009) Pronounced genetic diversity in tropical crespo a, molina mc, Blanco o, schroeter B, sancho lg, hawksworth dl (2002) rdna Its and β-tubulin gene sequence analyses reveal two monophyletic groups within the cosmopolitan crespo a, Pérez-ortega s (2009) cryptic species and species pairs in lichens: a discussion on the relationship between molecular phylogenies and morphological characters. anales del Jardín crespo a, Ferencova z, Pérez-ortega s, elix Ja, divakar PK (2010) , a new australasian lineage in parmelioid lichens systematics and Biodiversity divakar PK, molina mc, lumbsch ht, crespo a (2005a) , a new lichen species related to Parmelia sulcata ) based on molecular and morphological data. divakar PK, Blanco o, hawksworth dl, crespo a (2005b) molecular phylogenetic studies on the Parmotrema reticulatum (syn. rimelia reticulata ) complex, including the con�rmation of P. as a distinct species. : 55–65. divakar PK, Figueras g, hladun n, crespo a (2010a) morphological versus phylogenetic species: an example from Parmeliaceae Fungal diversity : 47–55. divakar PK, cubas P, Blanco o, del-Prado r, núñez-zapata J, roca-valiente B, lumbsch ht, crespo a (2010b) an overview on hidden diversity in lichens: Fehrer J, slavíková-Bayerová Š, orange a (2008) large genetic divergence of new, morphologically similar species of sterile lichens from europe ( concordance of dna sequence data with secondary metabolites. Feuerer t, thell a (2002) Parmelia ernstiae – a new macrolichen from germany. mitteilungen aus dem Institut für allgemeine Botanik in grube m, hawksworth dl (2007) trouble with lichen: the re-evaluation and re-interpretation of thallus form and fruit body types in the 111 : 1116–1132. grube m, Kroken s (2000) molecular approaches and the concept of species and species complexes in lichenized fungi. hawksworth dl (1991) the fungal dimension of biodiversity: magnitude, signi�cance, and conservation. hawksworth dl (1996) microbial collections as a tool in biodiversity and biosystematic research. In culture collections to Improve the quality of life (samson ra, stalpers Ja, mei d van der, stouthamer ah, eds): 26–35. Baarn: centraalbureau voor hawksworth dl (2001) the magnitude of fungal diversity: the 1.5 million species estimate revisited. mycological research hawksworth dl (2010) cryptic speciation: how common is it and how should it be handled taxonomically? Kroken s, taylor Jw (2001) a gene genealogical approach to recognize phylogentic species boundaries in the lichenized molina mc, crespo a, Blanco o, lumbsch ht, hawksworth dl (2004) Phylogenetic relationships and species concepts in s. str. ( Parmeliaceae ) inferred from nuclear Its rdna and β-tubulin muggia l (2010) the Implications of morphological and Phylogenetic species concepts in lichens. muggia l, hafellner J, wirtz n, hawksworth dl, grube m (2008) the sterile micro�lamentous lichenized fungi cystocoleus ebeneus racodium rupestre are relatives of plant pathogens and clinically important dothidealean fungi. mycological research 112 núñez-zapata J, divakar P, del-Prado r, cubas P, crespo a (2010) cryptic species in the lichen forming fungus Parmelina tiliacea Parnmen s, rangsiruji a, mongkolsuk P, Boonpragob K, lumbsch ht (2010) species delimitations and evolutionary history of the cladia aggregata aggregate ( lecanorales Pérez-ortega s, Printzen c (2010) species delimitation in lichens: using morphology and molecular markers to �nd species vinuesa m, sanches-Puelles Jm, tibell l (2001) Intraspeci�c variation in mycocalicium subtile ( ) elucidated by morphology and the sequences of the Its1-5.8s-Its2 region wirtz n, Printzen c, lumbsch ht (2008) the delimitation of antarctic and bipolar species of neuropogonoid ( ): a cohesion approach of species recognition for usnea perpusilla complex. mycological research 112   © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. Penicillium species are commonly occurring worldwide, and have been isolated from various substrates including air, soil, various food and feed products and indoor environments (Pitt 1979, samson et al. 2010, houbraken et al. 2010). Penicillium roqueforti is a member of this genus is and this species has both adverse and bene�cial properties. the main bene�cial property of this species is its role in the production of blue-veined cheeses, such as roquefort, danish blue, and gorgonzola (nichol 2000). however, this species is also frequently encountered as a spoilage organism, and is able to damage a vast array of food and feed products, due to its ability to grow under harsh conditions. these conditions include growth at low oxygen and high carbon dioxide levels, in the presence of preservatives and/ or at low temperatures (samson et al . 2010). the taxonomy of series roqueforti was studied by samson & Frisvad (2004) using a polyphasic approach, combining partial β-tubulin sequences, extrolite patterns, phenotypic and physiological data. they showed that P. paneum and P. carneum are closely related to P. roqueforti , together forming the series roqueforti . this series shares certain characters, such as a fast growth rate on agar media, the ability to grow on malt extract agar supplemented with acetic acid and the production of the extrolite roquefortine c. despite the various shared characters, also various features are known to differentiate between these species (Frisvad & samson 2004, Karlshøj & larsen 2005, o’Brien et al . 2008). these include the growth rate at 30 °c, reverse colours on czapek yeast agar and yeast extract agar, extrolite patterns and ehrlich reaction (samson & Frisvad 2004, samson et al. 2010). various fungi were isolated during the course of a survey in a cold-store of apples in the netherlands. the apples were stored in wooden crates, which were covered by a white fungal growth of Fubulorhizoctonia psychrophila the latter species only grows at temperatures below 20 °c, and during the isolation of this species growth of an species was detected. this species appeared to be related to the series and a detailed study was performed on these isolates using a polyphasic approach. For the phylogenetic analysis, Its, partial β-tubulin and calmodulin sequences were used, and these data were combined with extrolite analysis and macro- and microscopical characteristics. the combination of these datasets show that this species is new and is here described ate l all examined strains belong to the series . the strains (table 1) were grown for 7 d as three point inoculations on czapek yeast agar (cya), malt extract agar, yeast extract sucrose agar (yes), creatine sucrose agar (crea) and oatmeal agar (oa). the effect of various incubation temperatures (9–36 °c with intervals of 3 °c) on the growth was studied on cya and oa. genomic dna was isolated using the ultraclean™ microbial dna Isolation Kit (moBio, solana Beach, ca, usa) according and robert a. samson cBs-Knaw Fungal Biodiversity centre, uppsalalaan 8, nl-3584 ct utrecht, the netherlands; corresponding author e-mail: j.houbraken@cbs. knaw.nl department of systems Biology, Building 221, søltofts Plads, technical university of denmark, dK-2800 Kgs. lyngby, denmark abstract: various fungi were isolated during the course of a survey in a cold-store of apples in the netherlands. one of these fungi belongs to the genus and produces cleistothecia at 9 and 15 °c. a detailed study using a combination of phenotypic characters, sequences and extrolite patterns showed that these isolates belong to a new species within the series . the formation of cleistothecia at low temperatures and the inability to produce roquefortine c, together with a unique phylogenetic placement, make these isolates a novel entity in the series. the name sp. nov. (cBs 128137 article info: submitted: 28 october 2010; accepted: 19 november 2010; Published: 23 november 2010. Key words: P. paneum Ima Fu gus volu me 1 o 2: 171–180    oubraken et al table 1. strains used in this study. IBt 21509 IBt 3473 IBt 6753 P. carneum atcc 46837 IBt 6885 P. carneum IBt 3466 P. carneum 112297 IBt 6884 P. carneum type, mouldy rye bread, denmark IBt 12392 P. paneum IBt 11839 P. paneum IBt 13929 P. paneum mouldy baker’s yeast, vangede, denmark IBt 21541 IBt 12407 P. paneum type, mouldy rye bread, denmark 112296 IBt 21729 P. paneum cassava chips, africa 112294 IBt 16402 nrrl 1168 P. paneum dto 70g9 IBt 29551 P. psychrosexualis type, wooden crate in cold-store of apples, the netherlands dto 70h7 P. psychrosexualis wooden crate in cold-store of apples, the netherlands dto 70h4 P. psychrosexualis wooden crate in cold-store of apples, the netherlands dto 70h9 P. psychrosexualis wooden crate in cold-store of apples, the netherlands IBt 19475 mucl 8491 P. roqueforti atcc 10110 atcc 1129 cect 2905 IBt 6754 IFo 5459 ImI 024313 nrrl 849 P. roqueforti IBt 19781 ImI 291202 P. roqueforti IBt 21543 P. roqueforti mouldy baker’s yeast, denmark atcc 24720 Frr 1480 IBt 19476 ImI 174718 ImI 291199 vKm F-1748 P. roqueforti the manufacturer’s instructions. the Its regions (Its), a part of the β-tubulin (Bena) or calmodulin (cmd) gene were ampli�ed and sequenced according the method described in houbraken et al. (2007). each dataset was aligned using the clustal w program in mega5 (tamura et al. 2007), and subsequently manually optimised. the evolutionary history was inferred by using the maximum likelihood (ml) method based on the tamura-nei model (tamura & nei 1993). the bootstrap consensus tree inferred from 1 000 replicates is taken to represent the evolutionary history of the taxa analysed (tamura et al. 2007). the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1 000 replicates) is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically as follows. when the number of common sites is < 100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIonJ method with mcl distance matrix was used. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. all positions containing gaps and missing data were eliminated. evolutionary analyses were conducted in mega5 (Felsenstein 1985, tamura et al. 2007). all phylograms were rooted with Penicillium egyptiacum cBs 244.32 nt . the newly obtained sequences were deposited in genBank under accession numbers hq442319–hq442359. Plugs with mycelium and agar were extracted from 7 d old cultures grown on cya and yes. extracts were prepared using the method described by smedsgaard (1997). each extract was �ltrated through a 0.45 PtFe �lter and subsequently analysed using hPlc with diode array detection (dad) detection. the uv spectrum and the rI value, and comparison with authentic chemical standards, were used to characterise the extrolites produced (Frisvad & thrane 1987). the Its regions and parts of the β-tubulin (Bena) and calmodulin (cmd) gene were sequenced and analysed. the Bena alignment included 432 positions, and 35 positions were parsimony informative. the bootstrap consensus tree based on the results of the maximum likelihood analysis of this alignment is shown in Fig. 1. the total length of the calmodulin alignment was 500 positions long, and 27 sites were parsimony informative. the bootstrap consensus tree derived from the maximum likelihood analysis is shown in Fig. 2. the phylogram in Fig. 3 is based on the Its regions (incl 5.8s rdna), and 585 bases were used in the maximum likelihood analysis. of these 585 characters, 16 were the result of the analysis of the three datasets shows that P. psychrosexualis belongs to the series roqueforti . the species is related to P. carneum and P. roqueforti in all three analysed loci, and P. paneum is basal to these three species. Penicillium carneum is the closest relative of P. psychrosexualis in the tubulin phylogram (99 %, Fig. 1), and P. roqueforti is basal to these two species. however, this relationship is not supported in the phylograms based on the calmodulin and Its sequences. In these datasets, P. carneum and P. roqueforti are ex in Penicillium series roqueforti   � N� +*-�!!ee)' N� N� N� N� �N �N N� N� N� N� ,.� N� +*-�!!ee)l� N� .� N� N� N� +*-�!!ee)&� N� � N� Fig. 1. Bootstrap consensus tree from a maximum likelihood analysis of partial β-tubulin sequences. the bootstrap values from 1 000 replicates are shown at the nodes, the branches in bold have a bootstrap support higher than 95 %. the tree was N�e!.*#1) +*-�!!ee)' .� N�e!.*#1) N�e!.*#1) N�e!.*#1) N�.+-1#l+.0' N�.+-1#l+.0' � N�.+-1#l+.0' ,.� N�.+-1#l+.0' N�,/3e&.+/#21!('/ N�,/3e&.+/#21!('/ .� N�,/3e&.+/#21!('/ .� N�,!*#1) N�,!*#1) N�,!*#1) +*-�!!ee)l� N�,!*#1) +*-�!!ee)&� N�,!*#1) ,.� N#%3,0'!e1) Fig. 2. Bootstrap consensus tree from a maximum likelihood analysis of partial calmodulin sequences. the bootstrap values from 1 000 replicates are shown at the nodes, the branches in bold have a bootstrap support higher than 95 %. the tree was    oubraken et al sister species and in both cases P. psychrosexualis is basal to these two species. two isolates (cBs 449.78 and cBs 112296) warrant further attention. Penicillium carneum cBs 449.78, an isolate from cheddar cheese, has a unique position in the tubulin and calmodulin phylograms (Figs 1, 2). In addition, this strain is morphologically slightly deviating from the majority of examined P. carneum isolates. Isolate cBs 449.78 is cream- brown in reverse on cya, more restricted colonies on creatine agar and slightly slower growth rate at 30 °c. the other isolate which warrants attention is P. paneum cBs 112296. this strain has a unique β-tubulin, calmodulin and Its sequence. however, extrolite analysis shows that this strain produces a typical array of P. paneum extrolites. more strains of these two types should be collected and examined to determine whether these strains should be raised to species level. taxonomy Penicillium psychrosexualis houbraken & samson, sp. nov. Penicillium subgenus sect. ser. coloniis in mea cum 0.5 % acore acetica crescentibus et item in agaro mea, cys et yes celeriter crescentibus, et formatione cleistotheciorum ad temperationem exiguam. roquefortino c haud producenti. typus : wooden crate in cold-store of apples covered by growth of Fubulorhizoctonia psychrophila , 3 apr. 2008, J. houbraken & F. van der geijn (cBs h-20501 holotype; cultures ex type – cBs 128137 IBt 29551 dto 70g9). diameter at 7 d (in mm): cya, 25 °c, 47–55; cya, 15 °c, 35–46; cya, 30 °c, 14–27; no growth on cya at 37 °c; mea >60; yes >60; dg18, 40–50; ratio cyas : cya 1.2– 1.4; creatine agar 15–25, good growth and no or weak acid strong sporulation on cya, velvety, slightly �occose in centre, dull green or dark dull green conidia, mycelium inconspicuous, exudates absent, soluble pigment absent, radial sulcate, reverse warm brown. good sporulation on yes, conidia dull-green, soluble pigments absent, reverse mustard-yellow, none sporulating edge 6–10 mm. good sporulation on dg18, conidia dull-green, reverse pale. colonies on mea dull-green towards pure-green, velvety, on oa at 25 °c sparsely produced and not visible due to the presence of a layer of conidia, formation of cleistothecia induced and sporulation reduced at low temperatures (9–15 °c, Fig. 5), cleistothecia white, soft and sterile when young, maturing slowly and becoming pale orange-brown after 3–4 mo of incubation, (50–)100–175 µm ellipsoidal, 4–5 × 3–4 µm, with two distinct equatorial ridges, often with additional secondary ridges, one Fig. 3. Bootstrap consensus tree from a maximum likelihood analysis of Its sequences. the bootstrap values from 1 000 replicates are shown at the nodes, the branches in bold have a bootstrap support higher than 95 %. the tree was rooted with Penicillium egyptiacum cBs 244.32 ,.� N�.+-1#l+.0' N�.+-1#l+.0' N�.+-1#l+.0' N�.+-1#l+.0' � N�.+-1#l+.0' N�e!.*#1) +*-�!!ee)' .� N�e!.*#1) N�e!.*#1) N�e!.*#1) N� N� .� N� .� N�,!*#1) � N�,!*#1) +*-�!!ee)l� N�,!*#1) N�,!*#1) +*-�!!ee)&� N�,!*#1) N�#%3,0'!e1) ex in Penicillium series roqueforti   Fig. 4. Penicillium psychrosexualis (cBs 128036 , ex wooden crate in cold-store of apples, the netherlands). (a–c) colonies grown at 25 °c for 7 d on (a) cya, (B) mea, and (c) yes; (d) cleistothecium; (e–F) ascospores; (g) conidiophores on dg18 with warted stipes; (h) conidiophore    oubraken et al on either side of the main ones, suggesting the presence of four ridges when observed with light microscopy, valves slightly roughened when viewed with sem. terverticillate, slightly reduced conidiophores with smooth walled stipes on mea and other agar media (Pda, Pca), on dg18 robust conidiophores with warted stipes, 3–4 µm. 10–15 × 3–4 µm. conidiogenous cells (phialides) ampulliform, 8–10 × 3–4 µm. globose, smooth, 3.5–4 Penicillium psychrosexualis produces the extrolites andrastin a, mycophenolic, patulin, roquefortine c and the uncharacterized extrolite tentatively named “fumu”. P. psychrosexualis produces the same odour P. roqueforti diagnostic features : the growth on mea containing 0.5 % acetic acid, the formation of cleistothecia at relatively low temperatures for the genus (9 °c) and fast growth rate on mea, cya and yes are diagnostic features of P. psychrosexualis an overview of characteristics of P. psychrosexualis in comparison with other members of the series is shown in table 2. similar species and taxonomy : Phylogenetically P. belongs to series . this species shares a fast growth rate on agar media, the ability to grow on mea supplemented with 0.5 % acetic acid and forms conidiophores with warted stipes on dg18. this species produces the extrolites andrastin a, mycophenolic, patulin and roquefortine c and is chemically close to P. carneum . however, P. carneum also produces penitrem a, isofumigaclavine a and cyclopaldic aicd, while P. psychrosexualis produces the uncharacterised extrolite “fumu”. Penicillium psychrosexualis produces the same odour as P. roqueforti , and is thus very different from the strong odour of P. carneum . another difference between P. psychrosexualis and the other members of the series is the production of cleistothecia by the former species. the growth rate on cya at 30 °c is a diagnostic tool to differentiate between P. and P. carneum on one hand and P. paneum on the other. Penicillium psychrosexualis has similar growth rates at 30 °c as P. roqueforti and P. carneum . this observation is concordant with the phylogeny, which also shows that these three species are closely related and that P. paneum is basal to these species. an overview of growth rates on cya at nomenclature : although the new species produces cleistothecia, we decided to describe the taxon in Penicillium rather than eupenicillium in accordance with the recommendations of hawksworth (2010) on best-practice in such instances in a period when the rules of nomenclature that permit the dual naming of pleomorphic fungi are under revision. Fig. 5. growth of Penicillium psychrosexualis cBs 128036 on oatmeal agar at various incubation temperatures. a–F: 9, 12, 18, 24, 27 and 33 °c. ex in Penicillium series roqueforti   Fig. 6. overview of growth rates of the members of on cya at various temperatures. row, top to bottom: 9, 12, 18, 24, 24 (reverse), 30 °c; columns, left to right: P. roqueforti dto 81d6, P. paneum dto 28g8, P. carneum dto 128a9 and P. psychrosexualis    oubraken et al distribution and ecology : this species has been isolated from wood and apples (elstar) stored in a cold-store in the netherlands. the conditions in the cold-store were 1.5–2.0 °c in combination with an oxygen level of 1.0–1.5 %, a carbon dioxide level of 2.0 % and a relative humidity of 92–95 %. these conditions strongly inhibit the growth of most fungi; however, a low temperature and microaerophilic conditions do not prevent growth of members of the series the taxonomy of Penicillium series roqueforti has been studied extensively in the past, mainly due to its role in cheese manufacture. these studies were based on phenotypic characters (thom 1906, 1910, raper & thom 1949, Pitt 1980, samson et al. 1977), extrolite patterns (Frisvad & Filtenborg 1989, Boysen et al. 1996, samson & Frisvad 2004, smedsgaard et al. 2004) and/or molecules (Boysen et al. 1996, skouboe et al. 1999, samson et al. 2004). this is the �rst study using a multigene approach to determine the relationship of species belonging to the roqueforti series. all three studied loci are suitable for species recognition. even the Its regions, normally not recommended for species identi�cation in Penicillium have enough variation in this series (skouboe et al . 1999, houbraken et al. 2010, samson et al. 2010). Incongruence was detected during the phylogenetic analysis of the calmodulin, β-tubulin and Its loci. Penicillium psychrosexualis was, with high bootstrap support, basal to P. carneum and P. roqueforti in the Its and calmodulin dataset, while P. roqueforti was basal to P. carneum and P. psychrosexualis in the β-tubulin dataset. the use of β-tubulin in taxonomy was debated by Peterson (2008) and he excluded this locus in his study due to his doubt about the homology of this locus between members of sections in aspergillus . Furthermore, hubka & Kolařík (2010) showed that the commonly used primers Bt2a and Bt2b could amplify the β-tubulin paralog tubc in aspergilli. the interpretation of paralogous genes with non-homologous function in the same phylogenetic analysis posses a great risk and might create incongruence within and between datasets (hubka & Kolařík 2010). a limited number of penicillia are able to produce cleistothecia and ascospores, and these species were referred to the genus eupenicillium in a number of studies. only a limited number of penicillia known to reproduce sexually belong in subgenus Penicillium . samson & Frisvad (2004) omitted these species in their monograph of this subgenus, and they recommended that a multigene study needs to be conducted to resolve the placement of these teleomorphic penicillia within the subgenus Penicillium Peterson (2000) included various eupenicillium species in his phylogenetic study of Penicillium, and showed that e. crustaceum , e. egyptiacum , e. baarnense , e. tularense, and hemicarpenteles paradoxus belonged to group 6. this group largely corresponds with the subgenus Penicillium as circumscribed by samson & Frisvad (2004). until now, only homothallic species are described in this subgenus; however, recent studies indicated that various species belonging to this subgenus are heterothallic. hoff et al. (2008) table 2. overview of selected characters of members of series (after Frisvad & samson 2004, sumarah et al . 2005, P. carneum violet roquefortine c, isofumigaclavine a&B, penitrem a, andrastin a, (penicillic acid in P. paneum roquefortine c, marcfortin a, patulin, andrastin a, citreoisocoumarin, P. psychrosexualis andrastin a, mycophenolic, patulin and P. roqueforti violet roquefortine c, isofumigaclavine a&B, Pr-toxin, andrastin a, citreoisocoumarin, *often turning strawberry-red with age; with colour diffusing into the medium. ex in Penicillium series roqueforti   showed that P. chrysogenum is heterothallic, and analysis of 12 P. chrysogenum isolates showed an equal mating type distribution, indicating the potential of this species to reproduce sexually. In addition, eagle (2009) detected either mat1-1-1 or mat1-2-1 gene fragments in isolates of P. camemberti , P. roqueforti and P. verrucosum , also indicating heterothallism. although various trials were undertaken to inducing mating in P. chrysogenum (hoff et al. 2008, eagle 2009, houbraken unpubl. data) none of them have been successful. In addition, mating trials with P. roqueforti under conditions known to induce sex in aspergillus fumigatus were unsuccessful and no cleistothecia were detected after 6 mo of incubation (eagle 2009). various growth factors induce formation of cleistothecia, such as temperature, light, nutrients and oxygen levels (han et al. 2003). In this study, we show that P. psychrosexualis , a species related to P. roqueforti , produces cleistothecia abundantly at 9 °c. the production of a sexual stage at low temperatures might be more widespread in Penicillium, and mating experiments with P. roqueforti at this temperature might result in a sexual stage. Furthermore, P. psychrosexualis might be a good model species for comparison purposes in sex induction experiments or expression studies of genes required for sex in P. roqueforti . there are also indications of a sexual stage in P. roqueforti . sclerotia were observed in cultures in P. roqueforti (samson et al. 1977, shimada & Ichinoe 1998) and it was postulated that similar structures have a dual function in the life-cycle in aspergillus sect. Flavi survival of adverse conditions is one of them; the other is providing genetic variation in populations through sexual reproduction as a cleistothecium (mcalpin & wicklow 2005, horn et al. 2009). the possible discovery of the sexual stage in P. roqueforti could have consequences for the stability of starter cultures and might have advantages in strain improvement programs using conventional genetical approaches. the effect of temperature on sexual reproduction in species belonging to the subgenus is poorly studied. many of these species are capable to grow at low temperatures and are therefore common spoilage organisms in refrigerators. mcculloch & cain (1928) found an effect of the temperature on the formation of sclerotia of Penicillium gladioli . this species produces blue-green conidial structures abundantly when incubated at 14–15 °c, but produced comparatively a high number of sclerotia and only a few conidial structures, when incubated at 22 °c or higher. this observation is opposite to the results reported here, if the assumption is followed that sclerotia are immature cleistothecia. on the other hand, large white sclerotia are occasionally seen in P. italicum , a species related to P. psychrosexualis and also belonging to the . these structures have been observed in cultures incubated in darkness at 0 °c for 3 mo (raper & thom 1949, samson & Frisvad 2004), also suggesting the members of series roqueforti have a worldwide distribution, mainly related to human environments, and occur on various substrates. Penicillium roqueforti P. and P. carneum occur on (preserved) food and silage, and only P. roqueforti has been frequently isolated as a saprobe in nature. reports of the occurrence of P. and P. paneum in nature are rare, and recently P. has been found in stone tombs in Japan (an et al. Penicillium psychrosexualis is the second saprobic species in this series and has also been isolated from wood. several reports are made on the occurrence of P. roqueforti on woods such as sawn wood (logs), wood stakes in soil, wood in sea, cut lumber, quercus robur , and very wet wood in indoor environments (Picci 1966, Pitt 1980, land et al. 1985, Kubátová 2000, seifert & Frisvad 2000, sumarah we thank Frank van de geijn (agrotechnology & Food Innovations Bv, wageningen, the netherlands) for collecting the wood and apples samples. dae-hoo Kim is greatly acknowledged for the preparations of the sem images of the ascospores, and we thank uwe Braun for an K-d, Kiyuna t, Kigawa r, sano c, miura s, sygiyama J (2009) the identity of sp. 1, a major contaminant of the stone chambers in the takamatsuzuka and Kitora tumuli in Japan, is Boysen m, skouboe P, Frisvad J, rossen l (1996) reclassi�cation of the Penicillium roqueforti group into three species on the basis of molecular genetic and biochemical pro�les. eagle ce (2009) mating-type genes and sexual potential in the ascomycete genera aspergillus and Penicillium . Phd thesis, Felsenstein J (1985) con�dence limits on phylogenies: an approach Frisvad Jc, Filtenborg o (1989) terverticillate penicillia: chemotaxonomy and mycotoxin production. : 837– Frisvad Jc, samson ra (2004) Polyphasic taxonomy of Penicillium : a guide to identi�cation of food and air- borne terverticillate penicillia and their mycotoxins. studies in Frisvad Jc, thrane u (1987) standardized high Performance liquid chromatography of 182 mycotoxins and other fungal metabolites based on alkylphenone indices and uv vIs spectra (diode han K-h, lee d-B, Kim J-h, Kim m-s, han K-y, Kim w-s, Park y-s, Kim h-B, han d-m (2003) environmental factors affecting development of aspergillus nidulans Journal of microbiology hawksworth dl (2010) naming species: progress towards one name for each species. medical mycology : doI:    oubraken et al hoff B, Pöggeler s, Kück u (2008) eighty years after its discovery, Fleming’s strain discloses the secret of its sex. horn Bw, ramirez-Prado Jh, carbone I (2009) the sexual state of houbraken J, due m, varga J, meijer m, Frisvad Jc, samson ra (2007) Polyphasic taxonomy of section houbraken J, Frisvad Jc, samson ra (2010) taxonomy of : 117–133. hubka v, Kolařík m (2010). Beta-tubulin paralog – risk of taxonomy? - In: the Biology of Fungi, 9 International mycological Karlshøj K, larsen to (2005) differentiation of species from the Penicillium roqueforti group by volatile metabolite pro�ling. Journal of agricultural and Food chemistry Kubátová a (2000) neglected spp. associated with declining trees. – In: samson ra, Pitt JI (eds), Integration of modern taxonomic methods for Penicillium and aspergillus : 299–307. amsterdam: harwood academic land cJ, Banhidi zg, albertson ac (1985) surface discoloring and blue stain in cold-tolerant �lamentous fungi on outdoor softwood månsson m, Phipps rK, gram l, munro mhg, larsen to, nielsen KF (2010) explorative solid-phase extraction (e-sPe) for accelerated microbial natural product discovery, dereplication, : 1126–1132. mcalpin ce, wicklow dt (2005) culture media and sources of nitrogen promoting the formation of stromata and ascocarps Petromyces alliaceus ( aspergillus section mcculloch l, thom c (1928) a corm rot of gladiolus caused by a nichol aw (2000) cheese/mould-ripened varieties. In: robinson rK encyclopedia of Food microbiology : 387–393. san diego: nielsen KF, sumarah mw, Frisvad Jc, miller Jd (2006) Production of metabolites from the Penicillium roqueforti complex . Journal of o’Brien m, egan d, o’Kiely P, Forristal Pd, doohan Fm, Fuller ht (2008) morphological and molecular characterisation of Penicillium roqueforti and P. paneum isolated from baled grass 112 o’Brien m, nielsen KF, o’Kiely P, Forristal Pd, Fuller ht, Frisvad Jc (2006) mycotoxins and other secondary metabolites produced in vitro by Penicillium paneum Frisvad and Penicillium roqueforti thom isolated from baled grass silage in Ireland. Journal of Peterson sw (2000) Phylogenetic analysis of species based on Its and lsu-rdna nucleotide sequences. In: samson ra, Pitt JI (eds), Integration of modern taxonomic methods for Penicillium and aspergillus classi�cation : 163–178. Plenum Press, new york. Peterson sw (2008) Phylogenetic analysis of species using dna sequences from four loci. Picci g (1966) sulla micro�ora presente nelle strutture in legno soggette all’azione dell’acqua de mare. la ricera scienti�ca Pitt JI (1980) [‘1979’] the genus Penicillium and its teleomorphic states eupenicillium and talaromyces . academic Press, london. raper KB, thom c (1949) manual of the Penicillia. samson ra, eckardt c, orth r (1977) the taxonomy of species from fermented cheeses. antonie van leeuwenhoek samson ra, Frisvad Jc (2004) subgenus new taxonomic schemes and mycotoxins and other extrolites. samson ra, houbraken J, thrane u, Frisvad Jc, andersen B (2010) Food and Indoor Fungi . cBs laboratory manual series 2. centraalbureau voor schimmelcultures, utrecht, the samson ra, seifert Ka, Kuijpers aFa, houbraken JamP, Frisvad Jc (2004) Phylogenetic analysis of subgenus using partial β-tubulin sequences. studies in mycology : 175– seifert Ka, Frisvad Jc (2000) on solid wood products. In: samson ra, Pitt JI (eds), Integration of modern taxonomic methods for Penicillium and aspergillus classi�cation : 285–292. amsterdam: harwood academic Publishers. shimada t, Ichinoe m (1998) Fungal species from imported and domestic mold-ripened cheese. Journal of the Food hygienic skouboe P, Frisvad Jc, taylor Jw, lauritsen d, Boysen m, rossen l (1999) Phylogenetic analysis of nucleotide sequences from the Its region of terverticillate species. smedsgaard J (1997) micro-scale extraction procedure for standardized screening of fungal metabolite production in Journal of chromatography a smedsgaard J, hansen me, Frisvad Jc (2004) classi�cation of terverticillate Penicillia by electrospray mass spectrometric sumarah mw, miller Jd, Blackwell Ba (2005) Isolation and metabolite production by Penicillium roqueforti P. paneum and P. crustosum tamura K, dudley J, nei m, Kumar s (2007) mega4: molecular evolutionary genetics analysis (mega) software version 4.0. tamura K, nei m (1993) estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees. molecular Biology and evolution thom c (1906) Fungi in cheese ripening: camembert and roquefort. usda Bureau of animal Industry Bulletin thom c (1910) cultural studies of species of usda Bureau of animal Industry Bulletin 118   © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. since their discovery by colin orpin in the 1970s, the anaerobic fungi, now members of the recently erected , have aroused the curiosity of mycologists, not only due to their distinctive physiology but also because of their biotechnological potential, for more ef�cient animal nutrition and also biomass conversion/biofuel production. this contribution, based on a special Interest group session held during Imc9, brought together mycologists interested in anaerobic fungi to share recent discoveries and to discuss how best to move forward research in this area. six speakers kindly agreed to speak at the session, with a further seven non-speakers in attendance. given the small numbers of mycologists now engaged in research on anaerobic fungi, this was a pleasing quorum, with expertise in genomic, phylogenetic, morphological, physiological and ecological aspects of the biology of these fungi being represented. this synopsis of the session amounts to a mini-review of the the session began with a presentation from scott Baker on the status of two genome sequencing projects ( e2 sr2). the at richness of these genomes (approaching 80 % in non-coding regions) has caused signi�cant technical problems for genomic sequencing and assembly. the Joint genome Initiative (JgI) is working through technical issues with assembling the genome, but does plan to release the sequence in the near future. In contrast, the expressed sequence tag (est) library sequencing project did not encounter any technical issues and will be made available concurrent with the eventual release of the genome sequence. until then, contact scott for information on accessing ests (scott.baker@pnl.gov). much of the interest in these fungi relates to the genes/enzymes important for biore�ning and biofuel production, notably xylose isomerases Kate Fliegerova, who also presented a poster (4.015) at the main Imc9 congress, further explored the biotechnological , audra liggenstoffer , Kerstin voigt IBers, aberystwyth university, aberystwyth sy23 3dd, wales; corresponding author e-mail: gwg@aber.ac.uk Battelle Boulevard, P.o. Box 999, msIn P8-60 richland, wa 99352 usa Institute of animal Physiology and genetics, academy of sciences of czech republic, videnska 1083, Prague 4 - Krc, 142 20 czech republic , stillwater, oK, usa centre for eukaryotic evolutionary microbiology, Biosciences, college of life & environmental sciences, university of exeter, stocker road, Friedrich schiller university Jena, Institute of microbiology, department of microbiology and molecular Biology, Jena microbial research school of Biology, newcastle university, newcastle upon tyne ne1 7ru, uK abstract: this contribution is based on the six oral presentations given at the special Interest group session on anaerobic fungi held during Imc9. these fungi, recently elevated to the status of a separate phylum ( neocallimastigomycota ), distinct from the chytrid fungi, possess several unique traits that make their study both fascinating yet challenging to mycologists. there are several genome sequencing programs underway in the us but these are hampered by the highly at-rich genomes. next-generation sequencing has also allowed more detailed investigation of the ecology and diversity of these fungi, and it is apparent that several new taxa beyond the six genera already named exist within the digestive tracts of mammalian herbivores, with others potentially inhabiting other anaerobic niches. By increased collaboration between the various labs studying these fungi, it is hoped to develop a stable taxonomic backbone for these fungi article info: submitted: 27 october 2010; accepted: 20 november 2010; Published: 23 november 2010. Key words: Ima Fu gus volu me 1 o 2: 181–185 rif�th et al    potential of these fungi. some of the cellulases of anaerobic fungi originated via horizontal gene transfer from bacteria, so these are the only fungi known to possess cellulosomes, cell-wall associated multienzyme complexes (garcia-vallve et al. 2000, steenbakkers et al. 2001). combined with their anaerobic metabolism and ability grow at elevated temperatures (39 ºc), they have great biotechnological potential. In Prague, Kate and her colleagues have explored the use of anaerobic fungi to improve the hydrolytic phase of biogas production. they have also investigated which fungi are present in the cow manure used to prime the biogas fermentations (Fliegerova et al . 2010), �nding members of the genus , the sixth and most recently discovered group of anaerobic fungi (ozkose et al . 2001) to be dominant, with the ‘most famous’ (and type) genus comprising only a small proportion of the population. this imbalance is also re�ected in the literature, possibly a result of the widespread use of wheat straw for the culture-based the application of culture-independent approaches to assessing the diversity of anaerobic fungi was the subject of audra liggenstoffer’s presentation. her Phd project at the oklahoma state university used barcoded 454 sequencing to determine the fungal symbionts present in the faeces of 30 species of larger herbivores, many from oklahoma city zoo. her �ndings have recently been published (liggenstoffer et al . 2010) and demonstrated not only con�rmation of the occurrence of anaerobic fungi in a non-mammal host (green iguana) but also the existence of eight novel groups of fungi, with these new taxa (likely to represent new genera) comprising almost 40 % of the >250,000 Its sequences obtained. whilst it can be dif�cult to be certain that zoo animals have not acquired new symbionts whilst in captivity, some of these novel groups (ng) did show some host speci�city, for example with ng6 comprising nearly all the fungi in kudu and with such a high rate of taxon discovery, the , the newest of the fungal phyla Fig. 1. network graph highlighting shared otus between different anaerobic fungal communities in different animal hosts. the graph is colour- coded by animal host phylogeny (family). circular nodes indicate animal data sets, whereas smaller square, grey nodes represent individual otus. data sets with a higher proportion of shared otus are pulled to the middle, whereas data sets with a high proportion of unique otus remain on the periphery. the distance between any two data sets is a function of the number of shared otus between the two. Figure supplied by audra liggenstoffer. anaerobic fungi: neocallimastigomycota   et al . 2007) clearly has more taxonomic gems awaiting discovery, notably in habitats beyond the digestive tracts of vertebrates. anaerobic fungal sequences do appear in environmental clone libraries (lockhart et al . 2006) but this attests more to the resilience of their resting spores (ozkose 2001) than to their active metabolism in these habitats. however, it is already clear that a robust taxonomic scheme based on gene sequence data rather than the meagre morphological traits is needed. Kerstin voigt specialises in the phylogenetics of the lower fungi and questions the acceptability of the phylum (ebersberger . 2010). In collaboration with Kate Fliegerova and Ingo ebersberger from vienna, Kerstin has taken a phylogenomic approach (concatenated supermatrices of data) to generate more robust phylogenies. a key element of this approach is the use of orthologous genes for comparisons, requiring �rst the identi�cation of the original member of any gene family within an organism, prior to any interspeci�c comparisons. application of this more robust approach for the anaerobic fungi requires sequence data from genes other than the rrna locus, however, Kerstin’s initial analyses, like audra’s 454 data, revealed the presence of four novel genera (Fig. 2). It was also clear that some genBank accessions are mis-labelled, highlighting the dif�culty in morphological If there is one feature of the that intrigues microbiologists more generally, it is their obligately anaerobic metabolism. Prior to their “of�cial” discovery by colin Fig. 2. Phylogenetic tree based on a maximum likelihood analysis using raxml v. 7.2.6 (stamatakis 2006) with the aligned Its1-5.8sIts2 rif�th et al    orpin (1974), these organism had been reported by several rumen microbiologists, and even named by liebetanz (1910) as the �agellate protozoan . during the 1960's, hungate and Prins had also noted these organisms but had dismissed them as contaminants. thus the dogma that there are no anaerobic fungi was not easily overturned. despite the near-absence of any useful fossil record, increasingly accurate molecular clock approaches consistently show that the fungi diverged from the more primitive metazoans (animals) some 1000 million years ago. at that time, between the two great oxygenation events (at 2400 and 600 myr), primitive eukaryotes (including the earliest fungi) were exposed to low atmospheric oxygen levels (<10 %) and many potential niches (e.g. the sea) were highly anoxic (euxinic) et al . 2010). mark van der giezen’s research has focused on the metabolism of anaerobic eukaryotes (van der et al . 2005) and in particular how the mitochondrion, thought to have been acquired endosymbiotically to alleviate oxidative stress, evolved into the hydrogenosome, converting malate and pyruvate to hydrogen, co and acetate. mark described the several lines of evidence that strongly suggest a mitochondrial origin also for the hydrogenosomes of the some mycologists interested in lower fungi attended the concurrent sIg session on evolution and biodiversity of basal lineages of fungi organised by satoshi sekimoto and tim James. we were, however, fortunate that gordon Beakes had agreed to �it between sessions and to end our session with an overview of the neocallimastigomycota within the broader panoply of �agellate fungi. though he is foremost a microscopist, gordon has made a valuable contribution to work on anaerobic fungi by providing hitherto the only quantitative assessment of the signi�cance of anaerobic fungi to rumen metabolism. this study estimated that the fungi comprised some 20 % of the microbial biomass of sheep fed hay and pelleted lucerne diet (rezaeian et al 2004). surprisingly many rumen microbiologists still do not consider the fungi to be a signi�cant component of the rumen microbiota. this belief is fostered partly through ignorance (if you don’t look you don’t see- not dissimilar to the view of mycorrhizas by many plant physiologists) but also by the bias towards investigation of livestock being fed low �bre/ high concentrate diets. with an enlarging human population and increasing demand for animal products, it is likely that animal production will have to become more reliant on high �bre feedstocks, with fungi being of greater importance in a common theme from several speakers was the need for funding to catalyse research on anaerobic fungi. In addition to the biotechnological interest mentioned by scott and Kate, it is important to explore the possibilities of funding in the area of rumen metabolism, as noted above. In addition to exploring the role of the fungi in �bre digestion, there is also the interaction between the fungi and the methanogenic of the rumen, since emission of methane from livestock production is now recognised to be a major source discussions at the end of the meeting and for the duration of Imc9 also addressed how we might promote future collaboration, for instance by establishment of a repository of important isolates. since they are dif�cult to culture and require specialised equipment to exclude oxygen, it is unlikely that any of the major culture collections will take on this daunting task. however, it is hoped that a repository for frozen cultures may be found to ensure that representative cultures of the major taxa are made freely available. Furthermore, we agreed to collaborate by sharing of molecular data to work towards a stable taxonomy for these fungi, including the physiological, genetic and morphological classi�cation of the as organiser of this special Interest group, gwg would like to express his heartfelt thanks to all the participants, especially the speakers and hopes that our discussions both during and after the sIg will lead to a resurgence in work on these fungi. gwg is also dahl tw, hammarlund eu, anbar ad, Bond dPg, gill Bc, et al. (2010) devonian rise in atmospheric oxygen correlated to the radiations of terrestrial plants and large predatory �sh. Proceedings of the national academy of sciences, usa : 17911–17915. ebersberger I, gube m, strauss s, Kupczok a, eckart m, et al. (2010) a stable backbone for the fungi. nature Proceedings ). Fliegerova K, mrazek J, hoffmann K, zabranska J, voigt K (2010) diversity of anaerobic fungi within cow manure determined by garcia-vallve s, romeu a, Palau J (2000) horizontal gene transfer of glycosyl hydrolases of the rumen fungi. molecular Biology and giezen m van der (2009) hydrogenosomes and mitosomes: conservation and evolution of functions. Journal of eukaryotic giezen m van der, rechinger KB, svendsen I, durand r, hirt rP, et al. (1997) a mitochondrial-like targeting signal on the hydrogenosomal malic enzyme from the anaerobic fungus neocallimastix frontalis : support for the hypothesis that hydrogenosomes are modi�ed mitochondria. : 11–21. giezen m van der, tovar J, clark cg (2005) mitochondrion-derived organelles in protists and fungi. International review of cytology grif�th gw, ozkose e, theodorou mK, davies dr (2009) carbon source affects isolation ef�ciency of anaerobic rumen fungi. hibbett ds, Binder m, Bischoff JF, Blackwell m, cannon PF, et al. (2007) a higher-level phylogenetic classi�cation of the fungi. 111 anaerobic fungi: neocallimastigomycota   liebetanz e (1910) die parasitischen Protozoen des liggenstoffer as, youssef nh, couger mB, elshahed ms (2010) Phylogenetic diversity and community structure of anaerobic gut fungi (phylum ) in ruminant and non- lockhart rJ, dyke mI van, Beadle Ir, humphreys P, mccarthy aJ (2006) molecular biological detection of anaerobic gut fungi ( neocallimastigales ) from land�ll sites. applied and orpin cg (1974) rumen �agellates callimastix frontalis - zoospores of phycomycete fungi. Journal of applied ozkose e (2001) morphology and molecular ecology of anaerobic . Phd thesis, university of wales, aberyswyth. ozkose e, thomas BJ, davies dr, grif�th gw, theodorou mK cyllamyces aberensis gen.nov sp.nov., a new anaerobic gut fungus with branched sporangiophores isolated from cattle. rezaeian m, Beakes gw, Parker ds (2004) distribution and estimation of anaerobic zoosporic fungi along the digestive tracts stamatakis a (2006) raxml-vI-hPc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. steenbakkers PJm, li Xl, Ximenes ea, arts Jg, chen hz, (2001) noncatalytic docking domains of cellulosomes of      © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. aspergillus sparsus species group ( section ; gams et al. 1985) was established by raper & Fennell (1965) to accommodate four species isolated from tropical or subtropical soils. species assigned to this group have large globose conidial heads, which irregularly split with age, with colours ranging from light grey to olive-buff. samson (1979) suggested that a. gorakhpurensis should also be placed to this section. however, phylogenetic analysis of parts of the ribosomal rna gene cluster indicated that this species belongs to section (Peterson 1995, 2000). according to the recent data of Peterson et al. (2008) and Peterson (2008), the monophyletic section belongs to subgenus , and in addition to a. sparsus, a. biplanus, a. diversus and a. funiculosus, originally placed to this section by raper & Fennell (1965), it also includes and a. conjunctus previously assigned to section a. anthodesmis which was previously placed in the In this study, we examined available isolates of the species proposed to belong to section to clarify the taxonomic status of this section. the methods used include sequence analysis of the Its region (including internal transcribed spacer regions 1 and 2, and the 5.8 s rrna gene of the rrna gene cluster), and parts of the -tubulin and calmodulin genes, analysis of macro- and micromorphological characters ate the strains examined are listed in table 1. the strains were grown for 7 d as three-point inoculations on czapek agar, czapek yeast autolysate agar (cya), malt extract agar (mea), and oatmeal agar (oa) at 25 c and 37 c (medium the cultures were analysed according to the hPlc-diode array detection method of Frisvad & thrane (1987, 1993) as modi�ed by smedsgaard (1997). the isolates were analysed on cya and yes agar using three agar plugs (smedsgaard 1997). the secondary metabolite production was con�rmed by identical uv spectra with those of standards and by comparison to retention indices and retention times for pure compound standards (Frisvad & thrane 1993, rahbaek János varga and robert a. samson cBs-Knaw Fungal Biodiversity centre, uppsalalaan 8, nl-3584 ct utrecht, the netherlands department of microbiology, Faculty of science and Informatics, university of szeged, h-6726 szeged, Közép fasor 52, hungary; corresponding centre for microbial Biotechnology, department of systems Biology, Building 221, technical university of denmark, dK-2800 Kgs. lyngby, abstract: section includes species which have large globose conidial heads with colours ranging from light grey to olive-buff. In this study, we examined isolates of species tentatively assigned to section using a polyphasic approach. the characters examined include sequence analysis of partial calmodulin and Its sequences of the isolates, morphological and physiological tests, and examination of the extrolite pro�les. our data indicate that the revised section includes 10 species: a. anthodesmis, a. biplanus, a. conjunctus, a. diversus, a. funiculosus, a. implicatus, a. panamensis, a. quitensis, a. sparsus, and the new taxon a. haitiensis. the recently described a. quitensis and a. ecuadorensis are synonyms of a. amazonicus based on both molecular and physiological data. the white-spored species a. implicatus has also been found to belong to this section. aspergillus haitiensis sp. nov. is characterised by whitish colonies becoming reddish brown due to the production of conidial heads, and dark coloured smooth stipes. the taxon article info: submitted: 4 november 2010; accepted: 22 november 2010; Published: 26 november 2010. Key words: Ima Fu gus volu me 1 o 2: 187–195 arga et al    the cultures used for the molecular studies were grown on malt peptone (mP) broth using 1 % (w/v) of malt extract (oxoid) and 0.1 % (w/v) bacto peptone (difco), 2 ml of medium in 15 ml tubes. the cultures were incubated at 25 °c for 7 d. dna was extracted from the cells using the masterpure yeast dna puri�cation kit (epicentre Biotechnologies) according to the instructions of the manufacturer. Fragments containing the Its region were ampli�ed using primers Its1 and Its4 as described previously (white et al. 1990). ampli�cation of part of the -tubulin gene was performed using the primers Bt2a and Bt2b (glass & donaldson 1995). ampli�cations of the partial calmodulin gene were set up as described previously et al. 2005). sequence analysis was performed with the Big dye terminator cycle sequencing ready reaction Kit for both strands, and the sequences were aligned with the mt navigator software (applied Biosystems). all the sequencing reactions were puri�ed by gel �ltration through sephadex g-50 (amersham Pharmacia Biotech, Piscataway, nJ) equilibrated in double-distilled water and analyzed on the aBI PrIsm 310 genetic analyzer (applied Biosystems). the unique Its, -tubulin, and calmodulin sequences were deposited at the genBank nucleotide sequence database under accession numbers FJ491645–FJ491675, and the sequence data was optimised using the software package seqman from dnastar Inc. sequence alignments were performed by mega v. 4.0 (tamura et al. 2007) and improved manually. For parsimony analysis, the PauP v. 4.0 software was used (swofford 2002). alignment gaps were treated as a �fth character state and all characters were unordered and of equal weight. maximum parsimony analysis was performed for all data sets using the heuristic search option with 100 random taxa additions and tree bisection and reconstruction (tBr) as the branch-swapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. the robustness of the trees obtained was evaluated by 1000 bootstrap replications (hillis & Bull 1993). an a. ochraceoroseus isolate belonging to section of subgenus (Peterson et al. 2008) was used as outgroup in these experiments. the alignments were deposited in treeBase ( ) under accession number s11028. we examined the genetic relatedness of section isolates using sequence analysis of the Its region of the ribosomal rna gene cluster, and parts of the calmodulin -tubulin genes. the calmodulin data set included 566 characters, with 288 parsimony informative characters. one of the 56 mP trees is shown in Fig. 1 (tree length: 741, consistency index: 0.7247, retention index: 0.8903). during analysis of a part of the -tubulin gene, 494 characters were analysed, among which 196 were found to be parsimony informative. the single mP tree based on partial genes sequences is shown in Fig. 2 (length: 507 steps, consistency index: 0.7179, retention index: 0.8938). the Its data set included 559 characters with 58 parsimony informative characters. one of the 702 mP trees is presented in Fig. 3 (tree length: 180, consistency index: 0.8056, Phylogenetic analysis of -tubulin, calmodulin and Its sequence data indicated that aspergillus section sparsi includes 10 species. aspergillus biplanus and a. diversus are closely related to each other on all trees, while another table 1. isolates examined in this study. nrrl 22884 nrrl 5071 soil, tilaran, costa rica cBs 469.65 nrrl 5073 soil, tilaran, costa rica nrrl 5072 soil, tilaran, costa rica nrrl 5080 Forest soil, Palmar, Province of Punteras, costa rica nrrl 5074 cBs 116.56 nrrl 4744 nrrl 4569 cBs 468.91 nrrl 4568 nrrl 1785 nrrl 1786 nrrl 1933 nrrl 1937 soil, san antonio, texas, usa Polyphasic taxonomy of aspergillus section sparsi   ���N6�!'-) +30� e##!�-(/*� N6�!'-) +30� e##!�-(/)� N6�!'-) +30� e##!�-(/+� N6�!'-) +30� N6�!'-) +30� N6�#'4l/030� e##!�-(/,� N6�#'4l/030� N6�- + *l+0'0� e##!�)/0-� N6�- + *l+0'0� N6�e,+(3+e130� e##!�-(0(� N6�e,+(3+e130� N6�0- /030� e##!�)1++� N6�0- /030� N6� +1&,#l0*'0� N6�& '2l+0'0� e##!�,-.0� N6�& '2l+0'0� ��N6�& '2l+0'0� e##!�,-.1� N6�& '2l+0'0� N6�0- /030� e##!�)1+/� N6�'*-)'e 130� N6�%3+'e3),030� e##!�,/,,� N6�,e&/ el,/,0l30� N6� * 5,+'e30� N6�le3 #,/l+0'0� N6�.3'1l+0'0� N6�!'-) +30� e##!�-(/+�� N6�!'-) +30� ��N6�!'-) +30� e##!�-(/)� ��N6�!'-) +30� e##!�-(/*� ��N6�!'-) +30� N6�#'4l/030� e##!�-(/,� N6�#'4l/030� N6�- + *l+0'0� e##!�)/0-� N6�- + *l+0'0� N6�e,+(3+e130� N6�0- /030� e##!�)1++� N6�0- /030� N6�0- /030� e##!�)1+/� N6� +1&,#l0*'0� N6�& '2l+0'0� e##!�,-.0� N6�& '2l+0'0� �N6�& '2l+0'0� e##!�,-.1� N6�& '2l+0'0� N6�'*-)'e 130� N6�%3+'e3),030� e##!�,/,,� N6�,e&/ el,/,0l30� N6�- + *l+0'0� e##!�)/0.� N6� * 5,+'e30� N6�le3 #,/l+0'0� N6�.3'1l+0'0� Fig. 1. one of the mP trees obtained based on phylogenetic analysis of calmodulin sequence data of sparsi. numbers above branches are bootstrap values. only values above 70 % are Fig. 2. the single mP tree obtained based on phylogenetic analysis of -tubulin sequence data of sparsi. numbers above branches are bootstrap values. only values above 70 % are arga et al    clade includes a. panamensis, a. anthodesmis, a. conjunctus, and the recently described a. amazonicus , a. quitensis and a. ecuadorensis isolates on the trees based on -tubulin and Its sequence data (Figs 2, 3; mares et al. 2008). although mares et al. (2008) found that these three isolates have identical Its sequences, they were suggested to represent distinct species based on morphological data (length of talks, diameter of vesicles, morphology of conidia and number of phialides), and were placed in aspergillus section wentii however, these three isolates could not be distinguished from each other based on molecular, morphological or extrolite data in our study, and clearly belong to section sparsi (Figs 1–4). aspergillus amazonicus is chosen as the correct name for the taxon and a. quitensis and a. ecuadorensis are considered synonyms. aspergillus implicatus , a white-spored species originally assigned to aspergillus section candidi (maggi & Persiani 1994), also belongs to this section. this species was described to produce conidiophores surrounded by sterile hyphae, not yet seen in any other species of the aspergillus genus. unfortunately the ex-type culture showed only poor sporulation and only a few conidiophores with sterile outgrowth could be observed (Fig. 5). Phylogenetic analysis of sequence data indicated that the four examined a. sparsus isolates fall into two closely related clades. the three phylogenies were concordant, with no con�ict between the topologies of the gene trees, in accordance with the phylogenetic species recognition concept detailed by taylor et al. (2000). the ex-type strain a. sparsus (cBs 139.61 ) together with an isolate from texas, usa form one clade, while two isolates came from soil from haiti form another clade on all trees (Figs 1–3). Both of the latter isolates were found by raper & Fennell (1965) to differ from the ex-type strain of a. sparsus in producing more restrictedly growing colonies in shades of reddish brown on mea plates, while one of the isolates (cBs 464.91 nrrl 4569) also produced “small fragmentary sporulating structures adjacent to the agar surface that bear conidia similar to those of normal heads” (raper & Fennell 1965). regarding the value of the different loci for species delimitation in section , all species could be distinguished using either Its, -tubulin or calmodulin sequence data. however, the resolving power was much higher for the protein coding genes than for the Its region. the situation is more dif�cult in other sections of aspergilli, including for example sections (samson et al. 2007), (varga et al. 2007), and (J. varga, unpubl. observ.), where the Its region cannot be used reliably to among the species assigned to aspergillus section sparsi, a. panamensis produces cyclogregatin and gregatins (also called graminins or aspertetronins; anke et al. 1980a, b, N6�!'-) +30� #lle�4/60� N6�!'-) +30� #lle�4/61� N6�!'-) +30� #lle�4/62� N6�!'-) +30� N6�!'-) +30� N6�#'4l/030� #lle�4/63� N6�#'4l/030� N6�- + *l+0'0� ����N6�- + *l+0'0� #lle�0675� N6�e,+(3+e130� N6�0- /030� #lle�0822� N6�0- /030� N6� +1&,#l0*'0� N6� +1&,#l0*'0� #lle�11773� N6�& '2l+0'0� #lle�3457� N6�& '2l+0'0� N6�& '2l+0'0� #lle�3458� N6�& '2l+0'0� N6�0- /030� #lle�0826� N6�'*-)'e 130� N6�%3+'e3),030� #lle�3633� N6�,e&/ el,/,0l30� N6� * 5,+'e30� N6�le3 #,/l+0'0� N6�.3'1l+0'0� Fig. 3. one of the mP trees obtained based on phylogenetic analysis of Its sequence data sparsi. numbers above branches are bootstrap values. only values Polyphasic taxonomy of aspergillus section sparsi   Fig. 4. aspergillus amazonicus (cBs 124228). colonies of 7 d grown at 25 °c; a on cya, B on mea, c on crea. conidiophores and arga et al    1988), while a. funiculosus has been found to produce ethericin a (also called violaceol I or aspermutarubrol), and ethericin B (or funicin; König et al. 1978, 1980, nakamura et al. 1983) (table 2). ethericin a was �rst isolated and called aspermutarubrol from a. sydowii , causing the red colouration of the medium, as this unstable compound will turn into a red dye by oxidation (shibata et al. 1978). the ethericins (or violaceols) are also produced by a. versicolor and several emericella species (Fremlin et al. 2009). gregatins are also produced by a. anthodesmis and one of the a. haitiensis isolates (table 2). siderin is related to kotanins produced by some black aspergilli and a. clavatus (samson et al. 2007, varga et al. 2007), and is also produced by a. panamensis, a. anthodesmis, a. conjunctus and by an a. haitiensis isolate (nrrl 4569). auraglaucin production is shared by a. biplanus a. conjunctus and a. diversus , and is also produced by some eurotium species (gould & raistrick 1934, quilico et al. 1949). aspergillus implicatus (Fig. 5) has been found to produce a versicolorin derivative. the two a. haitiensis isolates produced quite distinct extrolite pro�les, but shared the production of several unknown compounds including those tentatively named tidmyco1-3. several of the other extrolites produced by species assigned to aspergillus section sparsi have also been detected in other species assigned to sections nidulantes , usti and versicolores , justifying the assignment of section sparsi to aspergillus subgenus nidulantes (Peterson et al. 2008). table 2. . the structures of the extrolites in brackets have not yet been elucidated. an aszonalenin, (dob-indol, fot, vurs1, vurs2, stan) gregatins, siderin (alk-769gl; amF1, amF2, amF3, antw, kota, met k, tidmyco1, tidmyco2, senmyco1, senmyco2, auroglaucin, (BlØdo, cur-678, KonI, oKsI-1121, raI-701, raI-843, sKot, vern-652, vern-655, vern- 661, vern-673, vers-965, vers-979, vers-1049, vers-1107) auroglaucin, siderin?, (alk-1538, alk-1756, blæam, conJ1, conJ2, conJ3, duts, InsuX,Jon1, Jon2, Jon3, Jon4, kola, kola2, svIF1, svIF2, ut, verruc1, verruc2, vers-1049, vers-1107), a falconensin (? by a. conjunctus auroglaucin, mycophenolic acid?, (alka-704, conJ1, kola2, oKsI-1, oKsI-2, vers-965, vers-979, vers-1049, vers-1107; oKsI-3, oKsI-4, oKsI-5, oKsI-6 by nrrl 5075) arugosin e, ethericin a, funicin ethericin B, terrein?, (aq-798, aq-1456, bianthron-1396, derh, drI, emon, hæms, nol, raI-921, raI-972, storå, sultI-1, sultI-2, vers-818, vers-856) nrrl 4568 (atrov, gyla, nIdu, tidmyco1, tidmyco2, tidmyco3, spar1, spar2, spar3) nrrl 4569 gregatins, siderin, (amF1, amF2, amF3, senmyco1, senmyco2, senmyco3, tidmyco1, tidmyco2, tidmyco3) gregatins, siderin, (aq-1456, otto), Polyphasic taxonomy of aspergillus section sparsi   Fig. 6. aspergillus haitiensis (cBs 464.91). colonies of 7 d grown at 25 °c; a on cya, B on mea, c on crea. conidiophores and arga et al    taxonomy aspergillus haitiensis varga, Frisvad & samson, nov. sect. similes, sed coloniis porphyreis et stipitibus fuscatis, laevibus distinguitur. typus : isolated from soil under sage and cactus, w. (as 113a) (cBs h-20503 -- holotypus, cultures ex- holotype cBs 464.91 nrrl 4569). on mea 50–60 mm, on cya 30–35 mm, after 14 d at 25 c, moderate growth on mea after 7 d at 37 conidial heads produced sparsely on cya, colony colour �rst white then reddish brown, colony texture �occose, reverse creamish to light brown. conidial heads radiate; stipes 5–9 µm, thick-walled, dark brown in colour; vesicles 10–25 µm wide, biseriate; metulae covering the whole vesicle, measuring 2.5–4 × 5–7 µm. conidiogenous cells (phialides) 2–2.5 × 7–8 µm. globose to ellipsoidal 4–5.6 × 5–6 µm, smooth. Fragmentary sporulating structures in addition additional isolate studied : Port de Paix, from desert w. scott (as 103b) (cBs 468.91 nrrl 4568). diagnostic features : thin whitish colonies turning to reddish brown colour on cya, brown-coloured smooth stipes, and production of unknown extrolites tentatively called tidmyco1-3. we are grateful to tineke van doorn who helped with the morphological data, uwe Braun with the latin diagnosis, and our anke h, casser I, schrage m, steglich w (1988) cyclogregatin, a new metabolite from aspergillus panamensis Journal of anke h, schwab h, achenbach h (1980a) tetronic acid derivatives anke h, schwab h, achenbach h (1980b) tetronic acid derivatives from aspergillus panamensis . Production, isolation, characterization and biological activity. Journal of antibiotics Fremlin lJ, Piggott am, lacey e, cappon rJ (2009) cottequinazoline a and cotteslosins a and B, metabolites from an austsralian marine-derived strain of aspergillus versicolor Journal of natural Frisvad Jc, thrane u (1987) standardized high performance liquid chromatography of 182 mycotoxins and other fungal metabolites based on alkylphenone retention indices and uv-vIs spectra (diode array detection). Journal of chromatography a 404 : 195–214. Frisvad Jc, thrane u (1993) liquid chromatography of mycotoxins. gams w, christensen m, onions ah, Pitt JI, samson ra (1985) Infrageneric taxa of . In: samson ra, Pitt JI, (eds), advances in Penicillium and aspergillus systematics : 55–62. new york: Plenum Press. glass nl, donaldson gc (1995) development of primer sets designed for use with the Pcr to amplify conserved genes from �lamentous ascomycetes. applied and environmental gould a, raistrick h (1934) the biochemistry of microrganisms. Xl. the crystalline pigments of species in the aspergillus glaucus hamasaki t, Kimura y, hatsuda y, sugawara s (1980) Isolation and structure of funicin, antimicrobial substance, produced by aspergillus funiculosus agricultural and Biological chemistry hillis dm, Bull JJ (1993) an empirical test of bootstrapping as a method for assessing con�dence in phylogenetic analysis. hong sB, cho hs, shin hd, Frisvad Jc, samson ra (2006) species isolated from soil in Korea. Journal of systematic and evolutionary microbiology : 477– Johnson gt, gould Bs (1953) Pigment production in certain of the König wa, Krause r, loef�er w, schanz d (1980) metabolic products of microorganisms. 196. the structure of ethericin B, a new Journal of antibiotics König wa, Pfaff KP, loef�er w, schanz d, zähner h (1978) stoffwechselprodukte von mikroorganismen, 171. ethericin a; Isolierung, charakterisierung und strukturaufklärung eines neuen, antibiotisch wirksamen diphenylethers. Justus liebigs maggi o, Persiani m (1994) aspergillus implicatus , a new species isolated from Ivory coast forest soil. mycological research mares d, andreotti e, maldonado me, Pedrini P, colalongo c, romagnoli c (2008) three new species of from amazonian forest soil (ecuador). current microbiology : 222– nakamura m, Fukuyama K, tsukihara t, Katsube y, hamasaki t (1983) structure of funicin, antimicrobial substance from aspergillus funiculosus , c acta crystallographica section Peterson sw (1995) Phylogenetic analysis of sections and wentii , based on ribosomal dna sequences. Peterson sw (2000) Phylogenetic relationships in based on rdna sequence analysis. In: samson ra, Pitt JI Integration of modern taxonomic methods for Penicillium and aspergillus classi�cation : 323–355. amsterdam: harwood Polyphasic taxonomy of aspergillus section sparsi   Peterson sw (2008) Phylogenetic analysis of species using dna sequences from four loci. Peterson sw, varga J, Frisvad Jc, samson ra (2008) Phylogeny and subgeneric taxonomy of . In: varga J, samson ra (eds), aspergillus in the genomic era : 33–56. wageningen: wageningen academic Publishers. quilico a, Panizzi l, mugnaini e (1949) structure of �avoglaucin and raper KB, Fennell dI (1965) the genus aspergillus. williams & samson ra (1979) a compilation of the aspergilli described since samson ra, houbraken J, thrane, u, Frisvad Jc, andersen B Food and airborne Fungi. [cBs laboratory manual series no. 2.] utrecht: cBs-Knaw Fungal Biodiversity centre. samson ra, noonim P, meijer m, houbraken J, Frisvad Jv, varga J (2007) diagnostic tools to identify black aspergilli. studies in shibata K, Kamihawa t, Kaneda n, taniguchi m (1978) new metabolite aspermutarubrol, from aspergillus sydowii smedsgaard J (1997) micro-scale extraction procedure for standardized screening of fungal metabolite production in Journal of chromatography a swofford t (2000) PauP*: phylogenetic analysis using parsimony version 4.0. sunderland, ma: sinauer associates. tamura K, dudley J, nei m, Kumar s (2007) mega4: molecular evolutionary genetics analysis (mega) software version 4.0. taylor Jw, Jacobson dJ, Kroken s, Kasuga t, geiser dm, hibbett ds, Fisher mc (2000) Phylogenetic species recognition and species concepts in fungi. Fungal genetics and Biology varga J, due m, Frisvad Jc, samson ra (2007) taxonomic revision aspergillus section based on molecular, morphological white tJ, Bruns t, lee s, taylor J (1990) ampli�cation and direct sequencing of fungal ribosomal rna genes for phylogenetics. In: Innis ma, gelfand dh, sninsky JJ, white tJ (eds), Protocols: a guide to methods and applications : 315–322. new york: academic Press. arga et al      © 2010 International mycological association you are free to share - to copy, distribute and transmit the work, under the following conditions: you must attribute the work in the manner speci�ed by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). you may not use this work for commercial purposes. you may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. any of the above conditions can be waived if you get permission from the copyright holder. nothing in this license impairs or restricts the author’s moral rights. subgenus nidulantes is one of the largest subgenera of the genus , including about 80 species (Peterson 2008, Peterson et al. 2008). several species of this subgenus have a teleomorph assigned to (Pitt et al. 2000, samson 2000, Frisvad & samson 2004). species of subgenus are important as opportunistic human pathogens (verweij et al. 2008, varga et al. 2008), as producers of various secondary metabolites which are useful for the pharmaceutical industry (e.g. penicillin, echinocandins, ophiobolins), and mycotoxins which are harmful to animals and humans (e.g. a�atoxins, sterigmatocystin; Frisvad et al. 2004, 2005, Frisvad & during surveys of isolates from soil samples from subtropical regions, two interesting isolates were recovered which did not match any known species of the genus. we used the polyphasic approach, including sequence analysis of parts of the -tubulin and calmodulin genes and the Its nrdna region, macro- and micromorphological analyses, and examination of the extrolite pro�les of the isolates to differentiate the new species aspergillus karnatakaensis sp. nov. we also analysed strains of species which appeared to be closely related to the new species for the production of extrolites and found sterigmatocystin in all species with a teleomorphic state studied and also in mate an the strains used in this study are listed in table 1. For macromorphological observations, czapek yeast autolysate (cya), malt extract autolysate (mea) agar, yeast extract sucrose agar (yes), creatine sucrose agar (crea), and oatmeal agar (oa) were used (samson et al. 2010). the isolates were inoculated at three points on each plate of each medium and incubated at 25 °c and 37 °c in the dark for 7 d. For micromorphological observations, microscopic mounts were made in lactic acid from mea and oa colonies and a drop of alcohol was added to remove air bubbles and excess the isolates were grown on cya and yes at 25 °c for 7 d. extrolites were extracted after incubation. Five 6 mm plugs sect. nov., a new section of the genus for sp. nov. and some allied fungi János varga and robert a. samson cBs-Knaw Fungal Biodiversity centre, uppsalalaan 8, nl-3584 ct utrecht, the netherlands department of microbiology, Faculty of science and Informatics, university of szeged, Közép fasor 52, h-6726 szeged, hungary; department of systems Biology, Building 221, søltofts Plads, technical university of denmark, dK-2800 Kgs. lyngby, denmark abstract: the new species sp. nov. is described and illustrated. all three isolates of this species were isolated from Indian soil; two from soil under a coconut palm in a coffee plantation in Karnataka, and one from soil in the machrar river bed in Bansa district. this species is closely related to, but clearly distinct, from a. aeneus based on -tubulin or calmodulin sequence data. sequences of the Its region of these two species are identical. aspergillus karnatakaensis produced terrein, gregatins, asteltoxin, karnatakafurans a and B and the unknown metabolite, provisionally named nIdu. aspergillus karnatakaensis belongs to a well-de�ned clade within subgenus together with eight other species including a. aeneus a. crustosus, a. eburneocremeus, a. and the teleomorph producing-species emericella bicolor e. discophora, e. spectabilis, e. foeniculicola. this clade is placed in a new section, sect. sect. nov. all article info: submitted: 8 november 2010; accepted: 22 november 2010; Published: 26 november 2010. Key words: Ima Fu gus volu me 1 o 2: 197–205 arga et al    of each agar medium were taken and pooled together into the same vial for extraction with 0.75 ml of a mixture of ethyl acetate/dichloromethane/methanol (3:2:1) (v/v/v) with 1 % (v/v) formic acid. the extracts were �ltered and analyzed by hPlc using alkylphenone retention indices and diode array uv-vIs detection as described by Frisvad & thrane (1987, 1993), with minor modi�cations as described by smedsgaard (1997). the column used was a 50 × 2 mm luna c-18 (II) reversed phase column (Phenomenex, ca, usa) �tted with the cultures used for the molecular studies were grown on malt peptone (mP) broth using 1 % (w/v) of malt extract (Brix 10) and 0.1 % (w/v) bacto peptone (difco), 2 ml of medium in 15 ml tubes. the cultures were incubated at 25 °c for 7 d. dna was extracted from the cells using the masterpure yeast dna puri�cation kit (epicentre Biotechnology.) according to the instructions of the manufacturer. the Its region and parts of the -tubulin and calmodulin genes were ampli�ed and sequenced as described previously (varga the sequence data was optimised using the software package seqman from dnastar Inc. sequence alignments were performed by mega v. 4.0 (tamura et al. 2007) and improved manually. For parsimony analysis, PauP v. 4.0b10 software was used (swofford 2003). alignment gaps were treated as a �fth character state and all characters were unordered and of equal weight. maximum parsimony analysis was performed for all data sets individually using the heuristic search option with 100 random taxa additions and tree bisection and reconstruction (tBr) as the branch-swapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. the robustness of the trees obtained was evaluated by 1000 bootstrap replications (hillis & Bull 1993). eurotium heterothallica was used as outgroup in these analyses (houbraken et al. 2007). the alignments were deposited in treeBase ( ) under accession number s11027. an of the aligned -tubulin sequences, a portion with 438 positions, including 107 parsimony informative characters, was selected for the analysis; mP analysis of the sequence data resulted in two similar, equally most parsimonious trees (tree length 289 steps, consistency index 0.7855, retention index 0.7919), one of which is shown in Fig. 1. the calmodulin data set consisted of 492 characters, including 188 table 1. spp. examined in this study. ubstratum, country, location nrrl 4769 t nrrl 4988 nrrl 4773 IBt 22153 soil under coconut palm in coffee plantation, cBs 102799 IBt 22154 soil under coconut palm in coffee plantation, nrrl 4649 soil from artemisia grassland, usa, wyoming, teton Basin eF652511 IBt 21910 ay339999 cBs 470.88 IBt 21911 ay340000 coal mine spoil material, wyoming, usa, cultures are deposited in/were obtained from the following collections: cBs, cBs-Knaw Fungal Biodiversity centre, utrecht, the netherlands; IBt, culture collection of Fungi, mycology group, Biocentrum-dtu, technical university of denmark, lyngby, denmark; nrrl, agricultural aspergillus karnatakaensis sp. nov.   -..,�l&l) �� N�+e1.e3e+e&.2*2� -..,�l'&)� -..,�l''# -..,�l)(( ���� ���� N�+e1.e3e+e&.2*2� -..,�l&l) -..,�l'&)� -..,�l)(( Fig. 1. one of the two equally mP trees obtained based on phylogenetic analysis of -tubulin sequence data of aenei. Fig. 2. the single mP tree obtained based on phylogenetic analysis of calmodulin sequence data of aenei. numbers above arga et al    parsimony informative sites; mP analysis resulted in a single most parsimonious tree (length 485, consistency index 0.7402, retention index 0.8040), which is presented in Fig. 2. the Its data set consisted of 451 characters, including 43 parsimony informative sites; mP analysis resulted in four equally most parsimonious trees (length 105, consistency index 0.8190, retention index 0.8541), one of which is the two isolates from Karnataka, India were found to be closely related to aspergillus aeneus based on phylogenetic analysis of protein coding sequences (Figs 1, 2), and had identical Its sequences to a. aeneus (Fig. 3). one additional isolate also from India, “ . aeneus ” nrrl 4649 ( ImI 086833) was found to be conspeci�c with these two isolates. this isolate was obtained from soil of the machrar river bed in the district of Bansa, madhya Pradesh (rai et al. 1964), and is morphologically similar to the other two Indian isolates. the three isolates are described here as a new taxon, a. karnatakaensis sp. nov . a typical characteristic is the formation of a crust of hülle cells. the strains were incubated on various media for ascoma production, but in none of the strains were ascomata or ascospores found. also, a mating experiment with the three strains did not induce ascoma production. aspergillus karnatakaensis formed a well-supported clade together with four species, e. foeniculicola, e. bicolor, e. spectabilis and e. discophora , and four species known to reproduce only asexually, including a. aeneus, a. eburneocremeus, a. crustosus and a. heyangensis on the trees based on calmodulin (Fig. 4), -tubulin, and Its sequence data (data not shown). Based on these observations, we describe sect. sect. nov. to accommodate these species within subgenus this group of species was originally assigned to section (raper & Fennell 1965, christensen et al. 1978, aspergillus karnatakaensis isolates were found to produce karnatakafurans a and B (manniche et al. 2004), terrein, gregatins, asteltoxin (until now only detected in cBs 102799) and the partially characterised metabolite nIdu. Both gregatins and nIdu are also produced by a. granulosus while karnatakafurans are produced in common with and a. multicolor . however, phylogenetic analysis of sequence data of a. multicolor (Peterson 2008) and (houbraken et al. 2007) indicated that they are among the other species found to belong to the same clade as a. karnatakaensis, emericella bicolor produces sterigmatocystin, versicolorins, some anthraquinones, and a polar extrolite with end-absorption; e. foeniculicola produces sterigmatocystin (and many other sterigmatocystin and versicolorin-related compounds), xanthocillin derivatives, and the partially characterized (but common) metabolite drI; e. spectabilis produces two members of the shamixanthone biosynthetic family (both more polar than shamixanthone itself) and a member of the sterigmatocystin biosynthetic family; a. heyangensis produces a decaturin in common with and a. karnatakaensis and nIdu, while e. discophora produces sterigmatocystin and versicolorins (zalar et al. -..,�l&l) � N�+e1.e3e+e&.2*2� -..,�l'&)� -..,�l''# Fig. 3. one of four equally mP trees obtained based on phylogenetic analysis of Its sequence data of aspergillus karnatakaensis sp. nov.   2008). decaturins are antiinsectan metabolites which have previously been identi�ed in species including P. and P. decaturense (zhang et al. 2003, li et al. 2005). aspergillus eburneocremeus has both sterigmatocystin and mer nF-8054X in common with e. heterothallica is different from all these species in producing only Pr-toxin and related mycotoxins, and has no extrolites in common with the other species in sect. . all species in sect. produce sterigmatocystin, while the species without a known teleomorph apparently cannot produce it, with the exception of a. eburneocremeus however, sterigmatocystin is common throughout the different sections of subgenus , and has even been found in sections and (Frisvad et al. 2005). other extrolites such as shamixanthones, mer nF-8054X and the related emesterones, and terrein have also been found in other species in section nidulantes aspergillus heyangensis is only known from ex-type cultures and re-examination of the cultures showed that the taxon has great similarities with the species mentioned above, including its inability to grow at 37 °c, and the shape of the conidial heads and vesicles, although this species does not produce hülle cells (Fig. 5). that species also produces the unknown metabolite nIdu, (table 2). �%N�4+;7+ +4+/7<3<� -..,�l&l) �%N�?/;<3-858;�� -..,�l&le -..,�ee' �%N� +,+-37><�� -..,�l')! ����%N�?/;<3-858;�� �%N�9;8 >,/;><�� +*/�!! #(e �%N�+>;/85+ ><��) ..,�%!e& �������%N�+<9/;/<-/7<�� +*/�!! N%! �����������%N�-+/<93 8<><�� -..,�!)e)� ������%N�;/->;?+ ><� -..,�l) e -..,�eel � -..,�l!'( N! l#e� N#!(l %'e&� -..,�l&)) arga et al    Fig. 5. aspergillus heyangensis (cBs 101751). colonies incubated at 25 °c for 7 d; a on cya, B on mea, c on crea. conidiophores aspergillus karnatakaensis sp. nov.   aspergillus karnatakaensis varga, Frisvad & sp. nov. emericellae similibus. conidiophoris cum stipitibus laevibus, conidiis subglobosis vel late ellipsoideis. aggregationibus insignibus cum tegumento ex cellulis globosis efferentibus. typus : Karnataka, near chickmagalur, netraconda estate, isolated from soil under coconut palm ( cocos nucifera in coffee plantation, 20 dec. 1996, J.c. Frisvad (cBs h-20502 on cya, at 25 °c: 31–37 mm diam after 7 d, reverse orange; on mea, at 25 °c: 12–19 mm, reverse yellow; on yes, at 25 °c: 33–45 mm, reverse pink to raspberry-red reverse; on oat, at 25 °c: 16–23 mm, hülle cells present; on cya, at 37 °c: no growth to micro-colony (<1 mm); on crea: weak to moderate growth, no acid production. conidial heads reddish brown, yellow exudate droplets on cya colonies. biseriate, smooth, light brown stipes, 2.5–4 µm wide; vesicles subglobose to subclavate, 5–8 µm diam. conidiogenous cells (phialides) 2–2.5 × 4–5 µm, metulae, 2–3 × 4–6 µm. globose or rarely subglobose, smooth to �nely roughened, hülle cells produced in crusts, globose to diagnostic features apart from producing gregatins, terrein, and karnatakafuran a and B, isolates of this species produce a series of �uorescing extrolites (more than 33) with characteristic uv spectra. also distinguished by producing varga & samson , sect. nov. sectionis nidulantium similis, sed taxis cum conidiophoris brunneolis, vesiculis ampulliformibus et capitulis conidiorum biserialibus; statu anamorphoso cum ascosporis laevibus, convexis, aequatorialiter typus species assigned to aspergillus sect. form a well- supported clade, basal to section Peterson (2008) based on Its, -tubulin, calmodulin, and rna polymerase 2 sequences (see Fig. 11 in Peterson 2008). the section includes four species able to reproduce both sexually and asexually ( emericella discophora, e. bicolor, e. spectabilis, e. foeniculicola) , and �ve species for which the teleomorph is unknown ( a. aeneus, a. eburneocremeus, a. crustosus, a. heyangensis, a. karnatakaensis all species are characterised by brownish conidiophores, �ask-shaped vesicles, and biseriate conidial heads. several species produce hülle cells abundantly in masses (except for a. heyangensis , which does not produce hülle cells at all). the teleomorph-producing species assigned to this section all have smooth convex ascospores with two equatorial crests. none of the species assigned to this section are able to grow at or above 40 °c. all teleomorph species, together with are able to produce sterigmatocystin. the relationship of e. spectabilis to a. crustosus has already been suggested by christensen et al. (1978), while e. discophora was found to be related to e. foeniculicola (zalar et al. 2008). table 2. cBs 101751 IBt 29634 IBt 22154 cBs 102799 asteltoxin, gregatins, karnatakafuran a and B, quinolactacin IBt 22153 cBs 102800 asteltoxin, gregatins, karnatakafuran a and B, physcion, quinolactacin, terrein, a decaturin, karnatakafuran a and B, terrein, guum* cBs 425.77 IBt 22833 cBs 469.88 IBt 21910 cBs 470.88 IBt 21911 cBs 156.80 IBt 22831 drI, sterigmatocystin, xanthocillin Fa wB 5097 IBt 22604 cBs 489.65 wB 5096 IBt 22607 wB 4981 IBt 22605 wB 4983 IBt 22606 cBs 429.77 IBt 22891 arga et al    Fig. 6. aspergillus karnatakaensis (cBs 102800). a, B. colonies incubated at 25 °c for 7 d, a on cya, B on mea. c, crusts of hülle cells,. F. aspergillus karnatakaensis sp. nov.   we are grateful to dr r naidu for permission to sample soil for mycological examinations in the coffee research station and associated estates near chickmagalur, Karnataka, India. tineke van doorn helped with the morphological data and uwe Braun kindly provided the latin diagnosis. we are also indebted to our referees. christensen m, raper KB, states Js (1978) two new group members from wyoming soils. Frisvad Jc (1985) secondary metabolites as an aid to classi�cation. In: samson ra, Pitt JI (eds), advances in Penicillium and aspergillus systematics : 437–443. new york. Frisvad Jc, samson ra (2004) emericella venezuelensis , a new species with stellate ascospores producing sterigmatocystin and a�atoxin B . systematic and applied microbiology 27 672–680. Frisvad Jc, samson ra, smedsgaard J (2004) emericella astellata a new producer of a�atoxin B , B and sterigmatocystin. in applied microbiology Frisvad Jc, skouboe P, samson ra (2005) taxonomic comparison of three different groups of a�atoxin producers and a new ef�cient producer of a�atoxin B , sterigmatocystin and aspergillus rambellii sp. nov. systematic and applied microbiology Frisvad Jc, thrane u (1993) liquid chromatography of mycotoxins. hillis dm, Bull JJ (1993) an empirical test of bootstrapping as a method for assessing con�dence in phylogenetic analysis. houbraken J, due m, varga J, meijer m, Frisvad Jc, samson ra (2007) Polyphasic taxonomy of section li c, gloer JB, wicklow dt, dowd PF (2005) antiinsectan decaturin and oxalicine analogues from Penicillium thiersii. Journal of manniche s, sprogoe K, dalsgaard Pw, christophersen c, larsen to (2004) Karnatakafurans a and B: two dibenzofurans isolated from the fungus aspergillus karnatakaensis Journal of natural : 2111–2112. Peterson sw (2008) Phylogenetic analysis of species using dna sequences from four loci. Peterson sw, varga J, Frisvad Jc, samson ra (2008) Phylogeny and subgeneric taxonomy of . In: varga J, samson ra (eds), aspergillus in the genomic era : 33–56. wageningen, wageningen academic Publishers. Pitt JI, samson ra, Frisvad Jc (2000) list of accepted species and their synonyms in the family trichocomaceae. In: samson ra, Pitt JI (eds), Integration of modern taxonomic methods for Penicillium and aspergillus classi�cation : 9–49. amsterdam: harwood academic Publishers. rai Jn, tewari JP, murekji Kg (1964) cultural and taxonomic studies on two rare species of – and a. aeneus , and an interesting strain of a. variecolor from Indian mycopathologia et mycologia applicata raper KB, Fennell dI (1965) the genus aspergillus. williams & samson ra (2000) list of names of trichocomaceae published between 1992 and 1999. In: samson ra, Pitt JI (eds). of modern taxonomic methods for Penicillium and aspergillus : 73–79. amsterdam: harwood academic samson ra, houbraken J, Frisvad Jc, thrane u, andersen B (2010) Food and Indoor fungi. [cBs laboratory manual no. 2.] utrecht: cBs-Knaw Fungal diversity centre. smedsgaard J (1997) micro-scale extraction procedure for standardized screening of fungal metabolite production in Journal of chromatography a sun zm, qi zt (1994) new taxa and a new record of and swofford t (2003) PauP*: phylogenetic analysis using parsimony (*and other methods). version 4.0. sunderland, ma: sinauer tamura K, dudley J, nei m, Kumar s (2007) mega4: molecular evolutionary genetics analysis (mega) software version 4.0. udagawa s, muroi t (1979) some interesting species of ascomycetes from imported spices. transactions of the mycological society of varga J, due m, Frisvad Jc, samson ra (2007c) taxonomic revision aspergillus section based on molecular, morphological varga J, Frisvad Jc, samson ra (2007b) Polyphasic taxonomy of section based on molecular, morphological varga J, houbraken J, lee hal van der, verweij Pe, samson ra aspergillus calidoustus sp. nov., causative agent of human infections previously assigned to aspergillus ustus varga J, Kocsubé s, tóth B, Frisvad Jc, Perrone g, susca a, meijer m, samson ra (2007a) aspergillus brasiliensis sp. nov., a biseriate black species with world-wide International Journal of systematic and evolutionary verweij Pe, varga J, houbraken J, rijs aJmm, verduynlunel Fm, Blijlevens nma, shea yr, holland sm, warris a, melchers wJg, samson ra (2008) emericella quadrilineata as cause of invasive zalar P, Frisvad Jc, gunde-cimerman n, varga J, samson ra (2008) Four new species of from the mediterranean zhang y, li c, swenson dc, gloer JB, wicklow dt, dowd PF (2003) novel antiinsectan oxalicine alkaloids from two undescribed  \r\f \n\t\b\n\f \n\f\n\r\f \n\f \b\n\n\f\t\b \f\b
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