ARTICLE 27
l t . ) ) o d d d ) a s n e ae tri a aea z rate n you get and f k ) { nate ru es lib a m tw V ig C . 2014 rr o on, an
i Dr e , , Pandey al corrhiza !*# es akaei aea, individual t y r f alia tat d k r fr i 2012 n t e to ca
. D r e l a or liverworts d ra among which m ) li 2012 a ( e li th 2006 D
, a of the species een t e l. o ib g tr b e d n us 8(1): 27–47 (2017) 27–47 8(1): , tr V
· Key words: orchid m species delimitation < econd, application a one with e use b us a , 2015 hil
S S 2011 b . Aust it et a es s l j d i . . ave U l a ) su n h , roc G t m A n
a h lotti i a sequence divergence e o \
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the above conditions can be waived i e 1 a s f < X !*! t ella fr j hiloglottis A spec ll , using eight loci analysed
a n ideae d ) V l C a hilo h r es t k t rima, i
C MA FUN MA so for the populations or species p , a I l ase y ea Diu i b Tulas us escence can P so e 2014, 2015 l
e chids . rthrochilus including phylogenies o . 2014 l n rthrochilus, Chiloglotti i l d ( 2014, O ree w hil legalcode. Any o r / A ib . a o A l o h t 3.0 o a associate with / t tr coa R , Jacquemyn et a roc
ella , e
i t d
m h h n e t d #M Tulasnella Tulasnella e Rannala 2010, Fu i X+z escent o
l rt & an ial by-nc-nd A
w / an 2010 , r enetic histor g d the species. Formal namin \ 1 . l Tulas g g methods h Linde et a te wn fr f !!# ; i ( 2000) and the similarity within and between lo licenses o
/ Yan eana, l. y an coa m l ( al. i est of T. sphagneti T. X+z li rchidacea associates with y e t kn i O rr d e ( namin et a eana !*# / l
and . 4, Jacquemyn y as ayes mith et a l E araca e e genera te es B a S stud ecies associated with i
P h t w p A @ , 201 i e d e ecies: one associated with ec s . araca rchids y a variety o n % y two methods. First, application o l a e.g. Kottk creativecommons.org oc s preferred and essential to prevent confusion of taxonomic s preferred and essential to prevent confusion n t ith // an z < V % % % % o P b l sp o formall M concerned ' i b a sp i ( ( ' model of ph T. warcupii T. , Leon M. Smit , Leon M. T. prima T. 1 l a w : ib and u . t . associated with the orchid genera associated with the orchid c y !*_{ ] | *! @ !*_ ound at http: z Tulasnell R i f o a a #** z n / ca i e et al v z Caleana, on J a ospores M Tulasnell idi h ollowing conditions , www.index f and 1 ( taxonom a l
soil surface. s ll as Suare microscope. !!# a ( g p % e b . g for taxonomic enus. Asexual ll l ) n this work, which can be g ona e a f associated ª< i
n J t V as l e Philli u Drakaea as . recent studies on l a or T i rom some Australian rom some , f u D species are currently only known from orchid hosts, with each fungal species are currently erred to in Epulorhiz ) f ella s associated with orchids operat n a d re yan ymen ores y sp. nov. V < V h R p
i ,
2 in Australia. Here we formally describe three of those species, Australia. Here we formally describe in d gnate ' i T. calospora T. . (2008) indicating that there are l ormerl E _ ` % f @ uce es Tulasnell antharellale Tulas associates with J d spp. nov., as well as an additional spp. nov.,
d from orchids i C
f et a n , J lla y e l T. sphagnet T. were , a and Drakaea N % asne 1 l accepted species in the species epithets in T 1 2011). For example, Ma 2011). ates pro u 0 T. secunda T. . l . T. warcupii T. l *$`_ J *$_* J T ª ; z% examination under a dissectin 90 O enerate basidiospore formation a 5 y so = g s: , subsequent studies on i t y y ave most k ecies o ecies y basidia and basidios :
e phagnum and h s a cosmopolitan saprotrophic fungal genus that s a cosmopolitan saprotrophic fungal genus l f
spores were visible above the casin i Tulasnell a while S p i , ia
z y , (Tulasnellacea f A ' c . a r s a e ll ve wor : i rom the copyright holder. Nothing in this license impairs or restricts the author’s Nothing in moral rights. rom the copyright holder. f ree to share - to copy, distribute and transmit the work, under the ree to share - to copy, , onl Cru f ME 8 · N vat mm , ) i U co zocton er asne L l d n- e group *$`_ J *$_* *$"* O 006 u o 2017 International Mycological Associatio 2017 International Mycological such as the size and shape of basidiospores A ; U U% A V ; z + \ % / z/ `!* z { ormation o h Article info: V | # + % !*`{ z | ` H oreophilu * # % Tulasnell species showing a strong relationship with an orchid genus. In this study, relationship with an orchid genus. In this species showing a strong T. secunda T. email: [email protected] Chiloglottis Abstract: % % % ' % Attribution You are You Celeste C. Linde Celeste C. New s © 2 1 growing in Caleana, Chiloglottis, For any reuse or distribution, you must make clear to others the license terms o No N permission on [various media] after two months”. Although Warcup and Although Warcup on [various media] after two months”. J 1980) utilized morphological characters of the sporophores been able to 1967 < been designated amongst \ % often slow to form and diffuse. Indeed, sporophores could onl Indeed, sporophores often slow to form and diffuse. O ' treatments of |*! $" !*_ !" !* !# approximatel In some cases, such as in ' % t \ X+ morphs o be detected b < INTRODUCTIO ( 2 Rhi f T approximatel Unfortunatel fungorum.org) with Kirk V 28 ARTICLE ' ¯ isolates of ' ¯ ¯ C H H < Z % + mith etal ATERIAL a Li !* . v. v. P ( es Many oftheorchidspeciesassociatedwith k 2014 va sub n araca i . f es aea , Lind g (2010).We used twotypesofisolationmedia togrow A Chiloglottis r d tri , o lid C rt ) e al collections th a m alreadycharacterisedonsequencedatabyLinde a tri h l . et a a . ) l ( roc , alongwitha be i l inclusive of C r o valida e e T eana y so an T r . , wetreata T u hilo T et a 2010 an u c l hil Chiloglotti u l . (2014). u Dr hi l d as asne l l S l asne d at soreop us asne ds T g s l ) C a n . AND METH AND , , u lottis, T. secund Drakaea k assoc 201 e i . tur . or in l ) C Chiloglotti o ae asne ll andelsewhere y Li lla a hiloglotti lla n lla lo g C in A terature on 4, Phillip z # enera f g . turfosa _ osa Pa rthrochilus, Caleana ae s h enetic linea i lla ate hil s ere: f T urther phylogeneticspeciesisolated , r from Australia, todetermineif aca u us. p ( f sp. g d l ollows Miller Roche etal ecies involvedareeithe as s s row T w A T orc l s ' rthr ( ea n . i ” eta inclusive o ( t However, basedonprevious e Peakall h O i p T n a n ll r oc hid u Sphagnum g a s D a i ma l g l ( i ) 21) were . 2014), asne Mc p i hil S , and es, wedescribefournew n s grow so . nov. us S C an l etal. a . phagnum lla ormick 2010,Philli , t & d ed f T. warcupii C from Australia was T. sphagneti f S ( i C r fr aleana ng as o . After assessing . After impliglotti 2010 lements m o P m i & n D i a nvest Jacquemyn r r Chilo Sphagnum h , ' a aca Peakall& Tulasnell < V ummoc ( k i inclusive ps sp.nov. r aea ( s l i C ea 2014 gate etal ) g i , and . lottis p spp n a Linde rio a ' a n k d e et ff. ) ) a d s r . . , , . t de tal et al / H<@ #@+ colonies consistedofasinglegenotype.Cultureswerestored were platedontoagarplatescontainingantibiotics " of theDepartment Agriculture, Victoria (VPRI). z #M "z'[ #M*! H<@ #@+ mg/mL). Germinatingpelotonsweretransferredtoeithe macerate times withsteriledistilledwaterafterwhichthetissuewas m + ª + ' ª a DNA a A o ( S homo \ ' w ] ' *! # @+ % / % *!! z +VJ ; (Centrum “BalancedFormula”,Wyeth ConsumerHealthcare, 15g/L agarandhumanvitaminmineralsupplements 96 PlantKitaccordingtothemanu with thehomogenisedagarblocksandincubatedatroom se H]* ! % ' / most similarsequencesavailable fromGenBank(http://www base readambiguities.Oursequences werealignedwiththe v. 8(http://www.geneious.co ncbi.nlm.nih.gov). Alignments wereperformedinGeneious Sequencher v. 4.7(GeneCodes, Ann Arbor, MIto correct fo z;<'#*#! sequencer. Sequenceswereedited usingtheprogram /z / z H ; # * % ; X ]A D s prev y g extract E' ra enizer sis k d aea. s ] "z'[ H<@ #@+ i < V < V n ster , i T Calean ous u E ' / f l or 5sat5.5m as i @@+ + ' @ '+ #@+"z'[ ly on, sequenc ° il n note e e ll di a #@+"z'[ st isolatesfromorchidsinthe X+ a "z'[ ill d , g ' e ( J al IsolationMedia d as i n acompre watertore # / # m % ; K P i / s. Petridishescontainin ng an araca @ X+ @ ] e ea t ' Tulasnell @ a r V et al.(2010)forthePCR se l X+ f . ' l acturer’s instructions acturer’s !*! l ease pe eana h d e e y) ! " ens r J p t e . w a ) ( @ a h , FIM; Clements l. 2012 i ve eva isolatesfrom as y l et al I otons, w T l etal MA FUNGUS Chilo h ogenet u ed l asne @z . (2014) ) se . g l using g 1990 uat enera lottis, v ( lla hi FIM e z;< i on r c i a c z h g a r ) r . l New orchid associated Tulasnella species ) ) ) ) ) ) ) ) ) ARTICL 2010 2010 2010) 2010 2010 2010 2010 2010) 2010 2010 ...... l. l l l l al. al. a a a t t t t t al t t al. 2010 t a ce e et a e e e et al et al e e e e n e r E efe Roche Roche Roche Roche Roche Roche Roche Roche Roche R ( ( ( ( ( ( (Roche ( (Roche ( ( o. n 2 7 6 5 8 9 6 7 1 # 92 91 98 o 2 2 2 2 i nk 7 7 7 55 55 a 59 59 59 5924 $ # 59 6 6550 6 ` 196800 196 196 196 196805 *$`"!# nB M M @ Ge access KY09511 KY44 KY44 KY44592 KY44 KY44 KY44 KY44592 KF47 HM KF47 KF47 H_ HM196797 HM HM s* r ecto ll P P PH PH P Co RP RP RP R R R R RP RP d d d d d d d d d n n n n an an a a a an l l l dl dl dl num num num num num num num num oodla ood ood ood g g g g g g g g woo woo w w w woodlan woodlan w woo woodland R woodland RP s s s s s s s s s k k k k k k k k ks pha pha pha pha pha phagnum Spha S S S S Spha Spha S S ptu oc oc oc oc oc oc oc oc ly tat i ne ne ine ine ine ine ine mm mm mm mm mm mm mm mm p p p p p ab l lpine l lpine l l l uca a a a a a a ammoc H A h h A h A A h A ha A ha h A Alpi h Alpi h Eucalyptus E Eucalyptus Eucalyptus Eucalyptus Eucalyptus Eucalyptus Eucalyptus Eucalyptus Eucalyptus Eucalyptus J J J J J J VJ VJ VJ VJ +V + +V + + +V +V +V J J +VJ J J VJ angaroo +V +VJ +V K V V +V n i +V g J ri pper pper Kangaroo VJ +] +] +] +] +] +V +] ; +] +VJ +VJ O +] +] +] ; @ + UE U ª U ª + H H UE UE + @ ; n U U U E E E U E E U U U U E U V V "U " V 6 2E * V _U know 47 47 #$ 1 #$ ! f 6 55 # #$ _ 886 i #` `U # # "U # # # # 26 * 6016 60 0 # * * # .2 . . . . * .4 S, " " " " " " " 8 8 ! 0 0 $ $ ! 0 P V 4 ` $ V 5 ` #_V 5 ! `V ! `V ` $ V # * V 5 G * # #"V # #`V * * # # * # * * # #`V * # #`V 14 # ##` 1 # ##`V # *#" V 149.9722 # * # ` #_V 1 # 150 1 ## $!$V # * # * # ` $ * # * ! ## **V * ! # 1 da li a a a a os v f . . . . . ff ff ff p. p. p. p p a s s s s s a a s s s s i i lotti lottis tur lotti lotti lottis lottis turfos lottis turfos lotti lottis formicifera g g g g g g g g g ost hilo hilo hilo hiloglottis trilabra hilo hiloglottis hiloglottis seminuda hiloglottis seminuda hiloglottis diphylla eanesi eanesi Chilo C Chilo C C Chilo Chilo Chilo Chiloglottis C Chiloglottis seminuda Chiloglottis formicifera Chiloglottis formicifera C C j Chiloglottis j C C C y. e e p y otyp l pe H o Ty Holot H . ) o * 9 ! ` n ¯* ¯ ' << * # #!` # # ate #$ l * * !## LM541 @ @ @#*! @#*` * @#*` @ @#`* #! " # # _ C Iso 12033 ( * !#! /@ "# * *#!` *#!` *#*! ¯* *#*! ¯ * * CLM15 ! / /@#!" /@#!$ / / / / / / isolates examined in this stud a ll e a n m i as l u i T a a pr ll ll y e 1. net n g e l a as asne l l h ab u u T Identit T sp T V O LUME 8 · NO . 1 29 30 ARTICLE Table 1. (Continued). Identity Isolate no. Type Host GPS, if known Origin Habitat Collectors* GenBank Reference accession no. Chiloglottis # _$V /@#`` ; @ z/ Eucalyptus woodland CCLHM196794 trapeziformis 149.0976E (Roche et al. 2010) Chiloglottis # _$V /@#_* ; @ z/ Eucalyptus woodland CCL HM196799 trapeziformis 149.0976E (Roche et al. 2010) Chiloglottis # _$V /@#_ ; @ z/ Eucalyptus woodland CCL HM196789 trapeziformis 149.0976E (Roche et al. 2010) Chiloglottis aff. ## $!$V ; +] /@#__ Eucalyptus woodland RP HM196782 jeanesii 150.0552E +VJ (Roche et al. 2010) Chiloglottis aff. ## $!$V ; +] /@#"! * Eucalyptus woodland RP HM196779 jeanesii 150.0552E +VJ (Roche et al. 2010) Chiloglottis aff. ## $!$V ; +] /@#"! Eucalyptus woodland RP HM196788 jeanesii 150.0552E +VJ (Roche et al. 2010) Chiloglottis aff. ## $!$V ; +] /@#"* * Eucalyptus woodland RP @*$`_"# jeanesii 150.0552E +VJ (Roche et al. 2010) Chiloglottis aff. ## $!$V ; +] /@#"* Eucalyptus woodland RP HM196784 jeanesii 150.0552E +VJ (Roche et al. 2010) Linde Chiloglottis aff. ## $!$V ; +] /@#"" Eucalyptus woodland RP HM196787 de jeanesii 150.0552E +VJ (Roche et al. 2010) Chiloglottis aff. ## $!$V ; +] al et al /@#"$ Eucalyptus woodland RP HM196795 jeanesii 150.0552E +VJ (Roche et al. 2010) . Chiloglottis aff. ## $!$V ; +] /@#$! Eucalyptus woodland RP HM196785 jeanesii 150.0552E +VJ (Roche et al. 2010) Chiloglottis aff. ## $!$V ; +] /@#$* Eucalyptus woodland RP HM196786 jeanesii 150.0552E +VJ (Roche et al. 2010) # ! `V V /@#$# Chiloglottis valida Eucalyptus woodland RP HM196804 *$ # *U H +VJ (Roche et al. 2010) ## $!$V ; +] /@#$ Chiloglottis valida Eucalyptus woodland RP HM196801 150.0552E +VJ (Roche et al. 2010) Chiloglottis # _ *V CLM405 z+;= z/ Eucalyptus woodland SR HM196796 trapeziformis 149.1097E (Roche et al. 2010) Chiloglottis # _ *V VA;=!* << # z+;= z/ Eucalyptus woodland SR HM196790 trapeziformis 149.1097E (Roche et al. 2010) Chiloglottis # _ *V CLM407 z+;= z/ Eucalyptus woodland SR @*$`_$# trapeziformis 149.1097E (Roche et al. 2010) Chiloglottis # _$V CLM416 ; @ z/ Eucalyptus woodland SR HM196809 I MA FUNGUS trapeziformis 149.0976E (Roche et al. 2010) Chiloglottis # _ *V VA;=!# < " z+;= z/ Eucalyptus woodland SR HM196807 trapeziformis 149.1097E (Roche et al. 2010) New orchid associated Tulasnella species ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ARTICL . l 2010 2010 2010 . . a l l . l l. l. . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 . 2014 t a l l a e t t a al al al al al al al al al al al al al al al al t e e r ce t t t t t t t t t t t t t t t t e et a et a n e et a et a e e e e e e e e e e e e e e e e ) r ) ) E omme efe 011 011 012 Roche Roche Linde Linde Linde Linde Linde Linde Linde Linde Linde Linde Linde Linde Linde Linde Linde Linde Linde Linde Phillips Phillips R ( ( (Roche ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( 2 ( 2 (S 2 o. n 2 5 1 9 4 # 6 8 _ o 7 88 92 $# 77 7 _ 7 i nk 5 5 574 57 a 655 65 6 6 6590 ` "# 6586 65 ` 659 65 658 6 ` 65 6580 6 196810 196806 #"`_# nB M Ge access H HM KF47 KF47 KF476568 KF47 KF47 KF47 H_ KF47 KF47 H_ KF47 KF47 KF47 KF47 H_ KF47 KF47 KF476585 KF47 °#"`__" ° Z^*#" ` s* r ecto P ll P DP DP R Co SR S R RDP CCL RDP RDP RDP RDP RDP RDP RDP RDP RDP RDP RD R R RDP RDP RDP RDP RDP d d d d d d d n n n n n a an a a l d l l d d d d dl n d n n n h h n an a l ood ood oodla oodla ood a l dl t t t t w woodlan woodland woo w w w w oodla s oodla oodla ood ood w woo w w ores orest w ores f f f w lain heath a p ea ea ea ee ksia ll n nz nz nz ucalyptu unze andplain heat and arrah arrah fores arrah forest arrah Habitat Eucalyptus E Eucalyptus K Eucalyptus S Jarrah J Ba Eucalyptus Ku Ku Eucalyptus J Eucalyptus Sandplain heat S J Ku Ma Eucalyptus Jarrah fores J J A , A / y A , W z A A p WA A Jz Jz A A A , W A WA y W r W , , WA ver, ver, / WA WA e or e , , W l , nartaminny, z Ri g e y J rk a l p G on n aret River, W aret River, d o ole, W regory, W regory, i = b ba g p lp g G n est A a t oore ri ua +; +;= z/ @ +V arnu @ z urard outhern W ake Gnartaminn O z z ; K ; M Lake W W M R + W L WA Mar Mt Gre E Canning Mills, W J J Lake Chinocu Westdale Wal De S n E E E 7 V 7 know f 886 i 109 109 `$!V . . .4 S, 9 9 P z z z 4 4 # * V 49.0920E G 1 # _ *V 1 # _ * # 150 +z # 1 +z +z +z +z +z +z +z +z +z +z + +z + +z + +z +z +z +z i r a s ll r on on n on y no o i l d d e d s onsi ca a i ica y t ili t tri a pto ptodon pto l ptodon pto s s da da a a ida ast y y l ly ly rac racilis ly v n n s so li elas g g s livi i livi eana m m ea li l l lotti u a r g aea e aea conco aea g aea g aea aea aea aea aea g o eziformi eziformi k k k k k k k k k acalea aca r r p p rti akaea akaea a a a rmin hiloglottis ra ra r ra ra r ra r rakaea r rakaea gl ra araca aracaleana l a o ra ra Host Chilo t C t Chiloglottis diph D P D D D D D D Drakaea gl Pa te Paracaleana h P D P Dr D D D D e p y ot l pe o Ty H . 1 o 9 2 # 7 1 7 # n ` !# <<< * Den 55 5 58 59 6 66 6 68 068 = G D X< LM LM00 LM22 LM251 @ LM2 LM2 LM2 LM2 LM2 LM2 LM2 LM2 LM272 @ _ LM274 LM276 LM277 A; 9 S4 Isolate V VA;=!# <ª C C C C / C C C C C C CLM260 C C C / C C C <# B J . ) da Continued ( a secun ll y 1. t i e l asne l ent ab u Id T T V O LUME 8 · NO . 1 31 32 ARTICLE Table 1. (Continued). Identity Isolate no. Type Host GPS, if known Origin Habitat Collectors* GenBank Reference accession no. Arthrochilus 17.2697S, z Tulasnella warcupii CLM027 Holotype Eucalyptus woodland DG KF476596 oreophilus * # U Qld (Linde et al. 2014) Arthrochilus *_ #! V z CLM007 Eucalyptus woodland DG KF476600 oreophilus 145.4210E Qld (Linde et al. 2014) Arthrochilus *_ #! V z CLM022 Eucalyptus woodland DG KF476601 oreophilus 145.4210E Qld (Linde et al. 2014) Arthrochilus *_ #! V z CLM028 Eucalyptus woodland DG KF476599 oreophilus 145.4210E Qld (Linde et al. 2014) Arthrochilus 17.2697S, z CLM091 Eucalyptus woodland DG KF476598 oreophilus * # U Qld (Linde et al. 2014) Arthrochilus *_ #! V z CLM092 Eucalyptus woodland DG KF476597 oreophilus 145.4210E Qld (Linde et al. 2014) (Linde et al. 2014) Arthrochilus *_ ##**U z Unassigned CLM084 Eucalyptus woodland DG KF476594 oreophilus * *#*V Qld (Linde et al. 2014) Arthrochilus *_ ##**U z Linde Unassigned CLM085 Eucalyptus woodland DG KF476595 oreophilus * *#*V Qld (Linde et al. 2014) de Arthrochilus *_ ##**U z Unassigned /@!#* Eucalyptus woodland DG KF476602 al et al oreophilus * *#*V Qld (Linde et al. 2014) Howard,C.G. . Unassigned BB0002_2_A Australia and Z+!* *$ Unpublished Clements,M.A. z+;= z + ; = { +] + ] // / { { A] A ] { AX] A ] { X= X = I MA FUNGUS New orchid associated Tulasnella species Table 2. Isolates of Tulasnella (some as Tulasnellaceae or Epulorhiza) from Australian orchids, additional to those analysed from orchid hosts ARTICL in Drakaeinae by Roche et al. (2010) and Linde et al. (2014). All isolates collected by Warcup currently present in culture collections are shown, along with all isolates from which sequences have been obtained. Orchid hosts in Drakaeinae + z `#"!# % ]+* Pterosylis nutans is given in GenBank as “asexual morph: Epulorhiza repens”, but Bougoure et al. (2005) considered the isolate was most likely a Thanatephorus (and it is therefore omitted below). Tulasnella Host Original isolate Reference Culture Sequence Sequence Reference for E species for original collection strain ITS* LSU sequence isolate T. asymmetrica Thelymitra JH Warcup 0267 J *$_# @zHH #! "!_ DQ520101 Garnica nuda zH /* ##$M (Cruz et al. [clones c001– 2014) c006] T. asymmetrica Thelymitra Z J !#! J *$_# X°#""!_ (Suarez et al. epipactoides @zHH#! "!" 2006) /* #_M (Cruz et al. 49,51,52,56 2014) [clones c001– c005, c009] T. asymmetrica Thelymitra JH Warcup 0591 J *$_# + T. calospora JH Warcup 07 (Warcup & @zHH#! "!* no sequences Acianthus *$`_ exsertus T. calospora Diuris Z J !#"" J *$_# @zHH#! "! no sequences maculata T. calospora Thelymitra JH Warcup 0584 J *$_# @zHH#! "!# no sequences aristata T. calospora Thelymitra sp. Z J !`#" J *$_# @zHH#! "! no sequences T. calospora host not JH Warcup 0689 J *$_# @zHH#! "! no sequences T. calospora Caladenia JH Warcup 062 /;V _# "# z # * et al. reticulata !!# T. irregularis Dendrobium Z J !`# /;V _ "# Æ E zH# `! (Kristiansen et dicuphum type] = JCM 9996 al. 2001) z # *$ et al. !!# T. calospora Microtis @* (Perkins et al. z `#"! (Bougoure et 1995) al. 2005) Tulasnella sp. [presume from Z #!` DQ061110 Otero (unpub.) orchid] Tulasnella sp. [presume from Z #!_ DQ061111 Otero (unpub.) orchid] Epulorhiza Acianthus AP2 z `#"!` (Bougoure et ‘possibly’ [in pusillus al. 2005) GenBank as ‘Fungi’] V O LUME 8 · NO . 1 33 34 ARTICLE Tulasnell Epulorhiz Epulorhiz Epulorhiz Epulorhiz fungus’ T A]' Tulasnellacea T. calos Disabracteata T. calospora Diuris T. calospora spec T mycorrhizal mycorr f f [ T T T Tulas T Tulasnell Tulasnell sp. 1 Tulasnellacea sp. Tulasnellacea Tulasnellacea s s Tulasnellacea s Tulasnellacea s sp. sp. Tulasnell [as mycorrhizal [as ‘Uncultured ungus’ ungus’ as p p p p ab u u u u u * . 1 . 1 . . 8– *M# l l l l l asne asne asne asne asne ‘ ‘ 14 10 6 4 l Uncultured Uncultured e 1 6 # 2 – – i n e 9 7 5 2 ellaceae hi s ] ] ] !** . p ll ll ll ll lla za a a a a acea acea acea acea ( r Mi ora a a a a Continued s sp. sp. sp l sp sp sp. sp. p. . . . e e e e e e e e e e Pyrorchi giganteum Prasophyllum Disa bracteat Diuri corymbos nigricans m n macrop D. livida t f se S g Di P Disa Th g H Microtis s elastica, D. Drakaea orchid ‘terrestrial var. daltoni Diuris punctat Diuris punctat Diuris Microtis medi capu Microtis Lyperanthu Drakaea ciliata ragrantissim h i l l piculaea yrorc os gr ). y y i ynn ur c rr e crant ptodo ptodon, r i a ly i o t cans s s l t b ar ti m us i ’ r hi s p ac i i hyll m tra s hila h s s , D. n p t a, a ea ed . a i s D t i a a a a a . a a Kings_Park 1 Dm0 Kings_Park Pg0 7 isolates DR DR JP ZV JS64 Z 50 O 2 #! 7 isolate J JP24 JP49 DR12 BB0002 JP40 Z] J Z 1 isolate S V S4 ] isolate riginal isolat isolate `# ## 1 * 66, J 28 88 `# # 5 1 1 , , , Z] 6 JP6 JP2 JP44 S _ s s #_ 6 2 s s 8 0 6 _A _ _ , e isolat for original Re f erence e Linde de tal et al collectio C ulture . n st r ain 69 S _ UH*_`_#M__ EF160067 EF IT EF Z # Z Z Z Z °#"`_#M"# DQ790804 D DQ790719– Z^*#" _!M_ Z^ Z^ Z^*#" `M` Z^ Z^ + ^* ^* ^ ^* $ " "`'$ equence Q S M –7 *#" *#" *#" `` *#" *#" ! 1 160068 `! `!* `!* 978X 790798 * "# #" #" `$ #" `! * 7 2 6 * _M * 4 `# " `" `_ ` _ $ 6 ` `* 4– M M , `# `* 0 66 * $ 8 , S L X°_$!_`$ 77 72 `! "'" DQ790751– SU equence ° , , 79 _$!_`# ` , 82 , 98 _ # I e e ( e e ( s Re (Bonnardeaux (Bonnardeaux a a a a a a a 2 ( ( ( C 2 2 (S 2 (S al (S (S (S al ( ( al ( D (unpub Davis etal (unpub Davis etal (Smith (Smith (Sommer (Sommer (Sommer Howard & MA FUNGUS Bonnardeaux Bonnardeaux Phillips unpub unpub. Sommer Sommer Sommer t a t a t a t equenc l l l l l l l 011 010 010) 010 a lements . 2012 . 2012 . 2012 . 2012 . 2012 . 2012 . 2012 . 2012 . 2012 . 2012 mith ommer ommer ommer ommer al f vi erence l l l . 2007 . 2007 . 2007 . 2007 s ) ) ) e ) ) ) e e e t ) ) ) ) ) ) ) ) ) ) ) t a t a t a et a a e et et e et et et et e e e l . . . ) ) ) ) l l l t t t t . . . f o l . r New orchid associated Tulasnella species Table 2. (Continued). ARTICL Tulasnella Host Original isolate Reference Culture Sequence Sequence Reference for species for original collection strain ITS* LSU sequence isolate E Tulasnella sp. Diuris 1 isolate DQ790802 DQ790767 (Smith et al. [as ‘Uncultured dendrobioides 2010)) mycorrhizal fungus’] Tulasnella sp. Diuris # DQ790796,99 DQ790761,64, (Smith et al. [as ‘Uncultured chryeopsis 80 2010) mycorrhizal DQ790815 fungus’] < V V\ Table 3. Tulasnella z Z J ]; V from sporophores (i.e. the “perfect state”) initiated from cultures. Tulasnella J genera in Drakaeinae are given in bold. References do not include publications where original isolates of Warcup were later sequenced (as cited \ % % = ; Tulasnella Orchid genera !_`"+,[#*+,$"&`+]$_ Comments References species T. allantospora Chiloglottis, Corybas sporophore morphology J *$_* Warcup 1981) T. asymmetrica Chiloglottis, Cryptostylis, sporophore morphology also as Tulasnella sp., isolate J *$`_ Dendrobium, Thelymitra !"` J *$`_ { *$_* J *$_# *$"* J *$_* T. calospora Acianthus, Caladenia, sporophore morphology J *$`_ Corybas, Cymbidium, J *$_* *$_# *$"* Dendrobium, Diuris, 1990) Eriochilus, Lyperanthus, Microtis, Orthoceras, Thelymitra Disa, Diuris, Microtis sequence (Bougoure et al. 2005) T. cruciata Acianthus, Chiloglottis, sporophore morphology J *$_* Thelymitra J *$_# *$"* *$$! T. deliquescens Acianthus, Microtis culture morphology (Perkins et al. 1995) [as Epulorhiza repens] T. irregularis Dendrobium sporophore morphology also as Tulasnella sp. 1, isolate J *$"! !`# J *$_# { J Warcup 1981) *$"! T. violea Drakaea culture morphology (Warcup 1981) Thelymitra sporophore morphology J *$_* J *$_# *$$! Tulasnella Arthrochilus, Caladenia, culture morphology “culturally seem Tulasnella, perfect J *$_# *$"* *$$! sp. (some as Caleana, Calochilus, states not seen” Perkins & McGee 1995, Epulorhiza sp. or Chiloglottis, Corybas, Perkins et al. 1995) Cryptostylis, Cymbidium, sp.) Dendrobium, Dipodium, Drakaea, Microtis, Thelymitra Acianthus, Caleana sequence (Bougoure et al. 2005, (as Paracaleana), Bonnardeaux et al. 2007, Smith et al. 2010, Phillips Disa, Diuris, Drakaea, et al. 2011, Sommerr et al. Lyperanthus, Microtis, 2012) Prasophyllum, Pyrorchis, Spiculaea, Thelymitra V O LUME 8 · NO . 1 35 36 ARTICLE A o li deemed tobeT. deli morpholo Furthermore, Roberts(1999) noted thatthe Australian re T. vi T. T. calos Tulasnella allantospor from Australia, includingreports ofafurtherfourspecies: !!# an fr previous workonthe Acia ( Tulas R T In placingformalnamesonphylogeneticspeciesof z P S loca 1000 pseudoreplicatesofnonparametricbootstrappin !!# associated with Tulasnell subclades ofphylogeneticgroupIVthephylogeny ' % % ClustalW followedb ; sequences inGenBank.Phylogenieswereestimatedusing from Australian orchidsfromouranalysistorecoverrelated \ % % material existsandifsequencedataareavailableforthat morphological charactersforrecentlyisolatedstrains,a framework. However, giventhelackofdiagnostic conta * * z sequences wereaddedaselectionofpreviouslysequenced et a t H * # % H ME h ' ' support wasassessed.Pairwisesequencediver < V h that anchornames ste u owever t f o robabilities upport forthenodeswasassessedwithBayesianPosterio ustralian roug ESULT = l d m A T as G V . a A" d t o l. nt io T A n l i ea . asymmetr b ns h hus n ll (2014) alongwithnewsequencesfrom 5 e ) n y androotedto t antospora J s ll p co us = . Accordin ella a . a R T or g h h constructedbyCruzetal , itisnecessar u ll tr a o S icall Tulasnella e e a a l n b a asne J id t d % # * % @ ; erts (1999)asasynonymofthelatterspecies. li species ed ( RA a BPP ent y De n indistin r Sphagnum x lla e i i n ML ca originallyfrom ca eco t g ) o d s ) inMrBa andmaximumlikelihood a toRoberts y needtobeplacedintotheph r r l sequenceswereremove l. 2011 c q obiu p manualad n r hi a Bl . ECU6and uescens. Inmakin ds ames, Roberts g , g ds ac enus. Essentiall f T. calospora Tulas T. uishable fromT. m rom Australian orchids rom Australian : o y kb ; Tulasnell ) toconsideran f h . * ;zV T x(Stamatakiseta ox T. T. ttp://tree.bio.ed.ac.uk/software y . T n es andforML treesusin i u ella rregu ( 1994 l j as c ustments tooptimiseindel r uc n \ \ T eichle a . e l Th , ar . e i ) havebeendescribed a (2011) ll T. rubropallens , T. deliquescens,and ( a t ( i e 1999) notedthat he T. as T. s a i 1971) wasposs (1967)w fr c l y g , ori ym f hl , t r p r redispositionsof ia o o inicol er y m i n m y y tra. { pe specimens i mmetrica w a namesfrom d an ( g De ML w ( g inall Ronquist a seq a a ence ofthe Chiloglotti M ll % h y n , ) andwas en no lo analysis l. d ay sou y 2008). u r g obiu from ences enetic ; et a and r p Linde d ces ibl ort m as as es g & g l y s ' r . ; . . / de tal et al T phylogenetic speciesdelimitedbyLinde w described from Australia and two species w t E' zX XzA JCM 9996),butapparentlynoneofT. cruciat co A genera ( o ( i a However, although thetypeofT. asymmetric $* *" $* *_ $* *$ H H *$$! *$"* *$_# *$_* J *$"! *$_* #! "!` had notexaminedt fr ' t al o assoc isolates from of E' V\ matching sequencesweresubsequently excludedfromthe fr % ' Chiloglottis Z^*#" `_ E 1, asdoallothersequencesfrom Australian orchids(data of clade from from the Australian orchid D o d T T = c P w three ph *!! * rregu y errestrial orchidthatissistertothetwocladesconsistingof $# *$"* *$_# J D 1999) ashousedat ADW, itwasnotexamined.Warcup’s . as u u rchid generaandonelithophytic/epiphyticorchidgenus reop f thesefourlinea eca n o o n exceptionisareportof hil e hi e rakaea, . pes are . (2014).Furthersequencesfrom ;z ; h endrobium ll l l d m m T. asymmetricafalloutsideofthecladesdepictedinFig. T. irregulari r r asne asne c ect y Tulasnell e e % h T D us r y y D lo desc nameCaleana hil Dr . ca i l in mmetr rakaea ate ar i r ons wereor e a g g lla lla , us A y a i l k a s branchesinEcuador. Representativesequences enetic anal lo k Caleana, Chilo d rthrochilus l aea f a f lineages couldnotbelocatedin ADW (Roberts1999),and t os r J , rom otherorchid aea o w ed ibed g i nd ( so r b f D enetic speciesof a i H p rom ca Chiloglottis t i ) Chiloglottis isolateshavebeenobtainedfrom21terrestrial z # J ra ora. Indeed,typematerialo t l a t s T. irregulari ates h C C V hat werethesourceofapparentlynovel . , n isonlydistantly relatedtoLSUsequences o ( * k D aleana. hiloglottis Phillip d T Chiloglotti aea C g ra . C T. eichleriana,T. tomaculum es wereaddedtothe72fun hiloglotti < V E' c k i , alean n t y r aea ! *!! ucia pe materialorvouchercollectionsfor i C ( g E s wasformerlyplaced), hi alean s . i y T. asymmetric na C ( , . A numberofadditionalse s stu e sis ofisolatesfrom there arereportso thatfellwithinthecladeofisolates t T U 5andE a Fo , a t and s ll . as s s g y g grow a Tulas r t werecollecteda g enera a ( enera, andtheisolateso l !`# J Tulasnella 1967, 1971 . y dy (orfrom lottis °#"`__" °#"`_# i 2011 ( n mmetr he Z c revea + ) n i tw ADW D ng ! ella A * ra ) , rt Chiloglottis a i k a i o ca * h C v a n a aea l (Warcup 085,MAFF an $ r e from is listedbyRoberts n U 6 ll ochilus iolea P z S d a Tulas < ) T d aracaleana t n phagnum c u an et al o f l d h l d f us f D V T asne ) ( ese sequences su T fr isolated T / f ( Warcup 1981 T . a d ter thespecies *$`_ a r g . a n t . . a . s ; c e a C ) , al sequences o . c ella I (exclusively r r withinth V A m MA FUNGUS k r ll Chiloglotti ucia Chiloglottis alean ( ll ll b , ucia antospor 2014 _ # _ "# rthrochilu a aea antospor sequent A D a ) formed q i so s n r r t , uences t unde f a akaea pec t d a these a ) hr l tw ) ates , , o f , the the t rom r h i o es a et at T s l ) a o e y s - r . , , . , . , New orchid associated Tulasnella species T. eichleriana AY373292 T eichleriana KC152389 ARTICL 100/1 ECU5 KC152397 72/0.98 ECU5 KC152398 100/1 EC6 KC152401 ECU6 KC152402 JN015192 Terrestrial orchid 12030 C. turfosa 13102_2 C. turfosa 13102_1 C. turfosa E 13065_2 Chiloglottis sp. 13065_1 Chiloglottis sp. Tulasnella sphagneti 12033_1 C. aff. valida 100/1 13058 Chiloglottis sp. 13139_1 Chiloglottis sp. 13143_1 Chiloglottis sp. KF476543 C. formicifera 100/1 HM196783 C. aff. jeanesii HM196784 C. aff. jeanesii HM196788 C. aff. jeanesii HM196786 C. aff. jeanesii HM196779 C. aff. jeanesii 100/1 HM196785 C. aff. jeanesii HM196796 C. trapeziformis HM196782 C. aff. jeanesii HM196795 C. aff. jeanesii HM196787 C. aff. jeanesii HM196804 C. valida CLM306 C. formicifera 100/1 CLM308 C. formicifera HM196791 C. aff. jeanesii HM196792 C. aff. jeanesii Tulasnella prima HM196801 C. valida HM196800 C. seminuda HM196799 C. trapeziformis HM196807 C. trapeziformis KF476556 C. trilabra KF476552 C. diphylla HM196794 C. trapeziformis HM196806 C. trapeziformis HM196809 C. trapeziformis 0.99 HM196793 C. trapeziformis HM196805 C. reflexa HM196803 C. diphylla HM196810 C. trapeziformis 99/1 HM196797 C. seminuda HM196789 C. trapeziformis HM196798 C. seminuda HM196790 C. trapeziformis KF476573 P. lyonsii KF476568 P. minor KF476574 P. terminalis KF476575 D. elastica 100/1 KF476578 D. gracilis KF476586 D. glyptodon JX138567 D. glyptodon KF476579 D. confluens KF476591 D. gracilis KF476588 D. concolor KF476590 D. livida Tulasnella secunda KF476583 D. glyptodon KF476584 P. hortiorum KF476585 D. isolata KF476592 D. confluens KF476593 D. elastica 0.76 HQ386778 D. livida HQ386743 D. glyptodon KF476577 D. glyptodon KF476576 D. livida KF476580 P. triens KF476602 CLM031 A. oreophilus KF476596 CLM027 A. oreophilus 0.74 KF476599 CLM028 A. oreophilus 1/100 KF476597 CLM092 A. oreophilus KF476598 CLM091 A. oreophilus Tulasnella warcupii 1/100 KF476600 CLM007 A. oreophilus 0.75 KF476601 CLM022 A. oreophilus 1/100 KF476594 CLM084 A. oreophilus KF476595 CLM085 A. oreophilus 1/100 T. tomaculum AY373296 T. tomaculum KC152380 0.04 Fig. 1. Rooted MrBayes tree for Tulasnella < V % E % ; ; % 1 _! ; 1 ! _! % % Tulasnella isolate was collected from is indicated after the isolate number or GenBank number. Sequences from the holotype of each species is indicated in bold. from Australian isolates of Tulasnella from Arthrochilus, barcode gap between all Tulasnella lineages studied here is Chiloglottis, Drakaea, and Caleana (data not shown). M` % H % lineages from Chiloglottis ` # V % Recognition of novel taxa between all other Australian Tulasnella lineages and close Support for three of the novel taxa was high across the % $ "M ! ` < V eight loci analysed by Linde et al. (2014) V O LUME 8 · NO . 1 37 38 ARTICLE divergence threshold II f a ab a not clusterwithorareclosetoanyo ' % ` ; ' clades hadlongsubtendingbasalstemsinthephylogenies < V Fig. 2. described from Australia, orindeedan contain sequencesfrommaterialof = ; < < V putative newph were represente our ph E' Tulasnella E Tulasnella E Tulasnella eichlerian Tulasnell Tulasnell Tulasnell Tulasnella sphagnet Tulasnella prim Table an nd isthereforereco ssociated with ove t + d Barcode gap;percentagesequence divergenceamongTulasnell T ' 4 y u . J ' ] lo h a a a l asne e g H t s warcupi omaculum enetic speciesarepreviousl ecunda C CU5 di * U lla a 6 ver y d lo sp. Arthrochilus III S ' ' i g g phagnum by i a f ence esta enetic species or thisdataset. g on nised asafourthph . . 14.8±1. 0.2 ±0.1 1 0 2 0 # Wi . . ` # / * 0.1 ± * / ! .5 ± .2 ± .2 ±0. " ly T t / hi . as twoan 0 n taxa 0 ' ' .5 ! . 2 6 # y mmetr bli s d h ( one T e ) u d 20 1 15.4 ±1. 1 T 1 i l ( ca f as . 5 5 8 Linde the Tulas y p . .2 ±1. . .1 ±1. 6 6 b i y othersequencesof r n so a ± ±1. i unreco etween t m e y n lo l ll a d 2 f ates respect ! eta a our phylogenetic . n g 0 6 6 6 6 7 sp. Arthrochilus T ella enetic species. . i rre l. g *" *# 1 *# 1 T 1 prev 2014 Chilo . sp . . 50±151. . 11.4 ±1. 12.7±1.5 15.0±1.5 5.1 ±1.6 *# $ / * 5.1 ±1.5 6 nized, and `!* g h .7 ±1. # " # u e t / / * / * h l ar agnet g i * ) ous i is lottis t h ve $ ` 6 Linde ree h do ly at ly i , de 1 14. ** T . warcu 7. tal et al a ` 6 9 % / * ± ±1. u I a R d # v a sp c o r s morp we areprov % ' ] % n time,certainmorpholo outinel ar haracters are pecies ofthe nique forparticulartaxa,butthiscanonl s tow ata, i re notformallydescribedherependingdiscoveryoffurther ur descriptionso 1 enner 2016 . '% ecies characteristics. .7 pii # 6 i Tulas Here wediagnosethenewspeciesonbasisof ous ' h f o voucherspecimensorculturesstillexist. hi y 18 $ * T l T o n usedtoidentif . secun c " u gi ella . h 6 # / * l asne ca morp ± 1 / * _ idi ) l 1 # n g da . g enus. Inaddition,itissequencedatathatare 9 lla d p h ata arecompre rovided b f o speciesfromhostsin themorpholo ot l o 1 T h gi . tomacu 0 4. r . ca 6 5 Ì# # _ ig ± ±1. y l isolatesofT orous spec c < f g 1 or com ical featuresma . h 4 6 4 aracters areactua l um V gy z p o leteness, ratherthanas 1 11.2 ±1. T h . e 7. ens i f es culturesando 0 i u c ± hl ( i l d ve across as Hibbett 1 er e D .7 li 4 n i ra an y m e turnouttobe y k ll i a tat beknownif ae a lly < I , MA FUNGUS andhence i e i on an di nae V t 1 U/\ a 4.5 ± a ' f g l h . nost k 2011, y nown d phal 1 t . h 6 i c. e New orchid associated Tulasnella species Table 5. Presence of and support for clades of six phylogenetic species of Tulasnella from Australian orchids in the genera Arthrochilus, Chiloglottis, Drakaea and Caleana in phylogenetic trees constructed separately for each of eight loci, as indicated on trees presented as Fig. 2 ARTICL V V 'V" et al. (2014). Values are bootstrap/Bayesian posterior probability. +: clade present (support ;V _! ;]] ! "! { | /@*_ { | % Fig. 1 of the present work (all other clades in this tree representing the phylogenetic species are also 100/1.00); n=1: one isolate only, falls outside of other clades, and separate to any other singletons; na: not present in analysis. E no. isolates ITS mtLSU C4102 C12424 C14436 C3304 C4722 C10499 T. prima ## 100/1.00 97/1.00 100/1.00 100/1.00 85/0.99* 100/1.00 100/1.00 99/1.00 T. sphagneti 9 100/1.00** na na nanana nana T. secunda 21 100/1.00 + 95/1.00 86/0/96 96/1.00 100/1.00 99/1.00 94/1.00 T. warcupii 6 100/1.00 94/0.88 100/1.00 100/1.00 '! $ 99/1.00 n=1 100/1.00 T. sp. Arthrochilus II 2 100/1.00 98/0.99 100/1.00 100/1.00 98/1.00 + n=1 na T. sp. Arthrochilus III 1 n=1 n=1 n=1 n=1 n=1 n=1 na n=1 TAXONOMY Substrate or host| A ' Chiloglottis orchid species. Tulasnella prima J @ sp. nov. MycoBank MB817404 Distribution| ' z H #z Eucalyptus woodlands and forests. Current known distribution coincides with that of the Chiloglottis hosts. Etymology| A Tulasnella found in the host, Chiloglottis. Notes: / ]Xz rings. Culture edges lack concentric rings, are broad and Type: Australia: New South Wales: Blue Mountains, Mt diffused. Culture appearance is quite variable, with some J A H Chiloglottis trilabra, + 22 Mar. 2007, C.C. Linde & R. Peakall CLM159 (MEL #@+ "z'[ M ) ! " M { E' ª]A< "*! diam, branched, often at right angles, septate, lacking clamp { ! ) { Diagnosis: Tulasnella prima can be diagnosed by the refractive internal bodies, and then small; sometimes uneven E (with undulate outline); sometimes with swollen elements to T. tomaculum and T. prima % | < V| $ ) ' ! ) % ITS1 *"V *" =| #' terminal or intercalary, sometimes in short chains. =/ |/ =z # '## /=|'' #" =| z * z| / =| " |/ `* '| `" '| "! =| *!* |z **_ Additional material examined| V * |/ * # =|z * _ /| *#! =| *# '*## / | / *! z| *'* z=| * /| * ` |= * " '|z= Tulasnella secunda J @ sp. nov. *`# z|= *` /|' *`" |/ *"! =|z *$! z| / MycoBank MB817406 *$ /| !#' ! / | / *' * z/|= *_' *" H #/ =|/ ' z|'' #_ /|z= 5.8S starting from < V* | ] '| ' |/ *#$ |/ ** |/ Etymology: Referring to the second Tulasnella described that * /| ITS2 "V | ] * |' associates with Drakaeinae orchids. *` /|z |/ _ z| / # '## / | / #_ /| $ |/ ' ` / | / $ /| `$ z|= _*'_ /z| = Type: Australia: Western Australia: Paganoni Swamp _ |/ __'"* / =z|/ z /= " |/ "" z|= $* Reserve, Karnup, isolated from Drakaea elastica, 2008, R.D. =|/ $# /| $`'$" = |zzz *! z|' *!_ z| Phillips [C.C. Linde CLM009Ç @U ! "*$ M { E' *!$'**! ''|/ **`'**_ z|'' * ! |/ * ` |/ *#` type culture VPRI 42808). =| *#$ /| *#'* z |=z *_'*" //| z= * ! '|/ * =|z * _ |= *`# /| *"! |= *"_ Diagnosis: Tulasnella secunda can be diagnosed by the '| / *$* =| / * |= |' #_ |' * '|= E T. z * /|= # =|z ` =| $ |z ` /| tomaculum and T. secunda respectively. < V| ITS1 " ='|z/ $!' $# //=|/ =/ $ ' $` /=| *"V #'# |'z * z|/ $"' $$ =|z #! '#!# /=|= / #! z|= #!_'#!" =|z __ /| * " =|z *#! /|z *# =| * ' z/| # " |/ ##* =|z ##" =| * # '/| * '* ` /|/ * " |/ *`# /|' *`` |' *_! '|/ *_$ =|z *"* /| *"$ z|/ ! /|z **' Clade-based diagnosis| % < V * z/|'' *$' ! /z|z/ # |/ #" |/ 5.8S no phylogeny in Fig. 1 containing HM196790 and HM196786. differences. ITS2 "V | ] # /| _ z| # /| # '#_ ==/|zz " =|z ' ` / | V O LUME 8 · NO . 1 39 4 0 ARTICLE Table 6. Basepair differences and their positions, among Tulasnella tomaculum, T. sphagneti, T. prima, T. secunda and T. warcupii. Polymorphic basepair differences in two or more isolates of a species are given by the most common basepair/alternative basepair. 1 5 6 7 8 9 15 16 17 18 19 24 27 28 30 37 41 44 51 62 63 64 74 90 92 T. tomaculum G G T G C T C G T T T A G C C G T --TC G - T T T. sphagneti . C G A C . C C C . . . C C C . ' A . C. prima . C G A . C . . . . C . . ' A . T. secunda . . C A/. . . . . A . C C . . . A . ' ' . . ' .. T. warcupii . A C . . C . C . ... . . . ' ' ... C . C 97 99 105 107 111 113 117 120 121 122 123 124 125 126 129 131 135 136 144 145 146 147 149 150 152 T. tomaculum C A C T C G C G G C - T G G C A A G C C G - T C T T. sphagneti C . . . A . ' C C . A . . . . G . . C T. prima . C C . A . ' C . ./A . . . G C A . T. secunda ...... A . A ' . . . . . . G C C T. warcupii . . . . . . . ' ...... ' . . C Linde de 153 155 160 162 163 172 174 180 182 183 184 195 196 197 209 212 214 215 218 231 232 233 234 259 376 tal et al T. tomaculum G A T C T G C T A A C C T T T - A - T C A T A T T . T. sphagneti A G C . C A .. . C C . A C . . . . . C T. prima . G C . ./C z . . C . C . ./C A C A . . . C C T. secunda . . C C A . C . . A . ..A ' ' C . . C . . . T. warcupii . . C . C . C . G . . A C A . .. G . .. 378 391 410 411 414 421 422 423 424 425 426 431 432434 435 436 438 445 447 448 449 452454455 456 T. tomaculum T C T C C C C T G A T C T G G C G C G T G C C T C T. sphagneti C . . A . . C C . C A/...... C T. prima C . . A . . C z / . C A/. . . . . C A/. C I MA FUNGUS T. secunda . . . . . . . . . . A A . . A . . . C . T. warcupii . . C G . . . A . A . . . . . A . C . . C . New orchid associated Tulasnella species 7 3 ARTICL 4 5 00 A . T 6 A . . ' . G ' 5 ' A T A 7 ./ . . G G G 508 6 1 4 . C 6 . ' CC A A . C . 698 A . . T 506 6 E 45 50 55 . C . T 6 A G A 75 A .. G . T C 5 . A 696 A C G C T C 505 4 0 5 4 5 . . . C C 6 . G T G 7 G A G A A A A G .. 69 A . G 504 9 . G . T A 623 A . G A 74 . C . ' C ' 548 549 ' - 690 . . G. C 503 7 6 5 1 C . . . T C 60 C T . 74 A . C ...... G 689 C ./ C .G. 50 7 28 9 . . T . 606 . . A . . G . . . C . 688 C . . G . 77 7 44 2 40 542 54 . G . A . 600 . . C 7 . C . . C . 5 . G T G 68 G . G . A . 49 99 26 C C C G . 5 . C T . 7 C C T . . C 686 . . . C 493 2 98 9 ' G A G - . 725 . C . C T . 527 532 .. A T . 681 . . G . 4 55 6 9 90 . . G ' C C C 724 C C A T . . C C . 680 . C T 4 45 9 23 25 52 75 ' G G T ' 5 ' . - A 7 . . . G . 5 A . G . 6 .C. C C G G 486 3 1 4 92 7 . . C ' 5 G ' A ' 721 ... . - ' C / 6 ' ' ' - 48 0 2 90 7 8 C ' C ' T ' 5 G G - G 720 G G . A . 522 52 . C 6 . A .. ... - 4 1 88 2 81 G G G G - G G . A . 719 . . A . T . 5 C C G C 669 A G C G - 4 75 5 ' 8 17 7 . = . C C 5 - C C C G A A ' T C 668 C . . T 4 47 7 /. C . ' C ' 577 ' . - . 71 G/A A A . T . . G G G 66 G . G A 472 1 5 7 17 518 519 G . ' C G 5 G C C 713 A C G 5 . G 66 . . .. C 471 4 0 66 . . G 5 C G C 712 C ' C T ' ' - 66 . . . A C 47 11 15 516 69 G G A G T . 565 . GC T . 7 G . . 5 . G C G. . 663 G C G C A 4 4 62 10 63 A ' A G A 5 C . G A 7 C C . . . C T . 662 . A C ' T 4 8 09 60 C C C ' - C 55 C G T . 7 G . G A AGA A 513 51 A C G C 660 . C . C T 4 9 1 5 ' C - 557 . C . 708 . G . . . G A 6 . ' A C G 459 8 5 58 A A C 555 . G . 707 A A . G G G C . 6 . . C 74 54 03 09 510 51 57 ' A C 5 - . 7 . G . C C . 5 . . T A 6 . . G 45 m i i i i i um u l l i i ii a ii a net da pii d da d g n a a a a acu agnet a m m h h m i i r r rima p p to p secu . tomacu . tomaculum . . warcup . sp . secun . tomaculum . . warcu . secun . warcup . sp . secun T T T T T T T. sphagnet T. T. warcupi T. T T. secunda T. T. prim T. T T. sphagnet T. T T. tomaculum T. T. T. sphagnet T. T. warcupi T. T T. T. prim T. T T T T. V O LUME 8 · NO . 1 41 4 2 ARTICLE Fig. 3. B . T. sphagnet Tulasnell D C B A i (CLM541); a ]Xz C. T. secunda (C LM009 ) and D . T. warcupi H<@ Linde de i (CLM027). tal et al . #@+ "z'[ A. Tulasnella prima I MA FUNGUS (C LM159 ) ; New orchid associated Tulasnella species z/ " =|z `!'`* = |z/ `$ z|= _# |/ _` |/ "! Diagnosis: Tulasnella sphagnetii can be diagnosed by the ARTICL '|/ "$ z|= $ /| $` /| *!* =|z *!_ =|z E *!$'**! =|// ** =|' ** z| / **"'**$ ' |z/ between T. tomaculum and T. sphagneti respectively: Locus *## /| ** /|' *#'*_ ///|''=z *$ '| * * < V| ITS1 *"V *" =| # '|/ |/ * `'* _ =|/z *_"'*_$ '/| = * |= # ' ` /|' _ |z ## /|' # =| #$ =|/ z|/ =| E #_ / |/ = "' $ /|/ * =|z ' $ |/ ` '| `$ |/ _$ |/ "* =| *! |z *!" ='| " |/ _!' _* / |z/ _` /| " =|z /| **! z|/ * =|z * " /| *# =| *# |/ "_ / | = $* =|z $' $ =/| #!! /| #! *#` =|z ** z| * z| * /| * ` '|= *`* |'/ z|= #!" '|/ *` '|z *`` z|' *`_ |' *`" /|' *_* |/ *_ |/ *"# =|z *$# z| *$ /| !` /| !_ |/ *` Clade-based diagnosis| % < V /| *_ z|= *" /| ! |/ * =| _ | " H * H_` _# Z^*#" `_ (A:). 5.8S < V* | ] *#" |/ *! |/ * # /| ITS2 "V | ] * | *` Substrate or host| \ ' Caleana and /|z |/ ` =| _ z|/ # /| ## |/ /| Drakaea orchid species. ` |/ _ =| `! =|z ` |/ `$ z|= _ z|= __ '|/ "! |z "* =|/ " |/ "$ z|= $ /| $` /| Distribution| V ' ' z $" |= *! =| *!`'** z=z = = z|=/ //z z= extending from high rainfall areas to the margin of the arid **" |/ * |/ *# =| *#_ /| **'* 'z|== zone, occurring in open areas within eucalypt forests and * '*_ '//| z *$ |/ * # =|z * ` |z *_$ |= woodlands, Banksia woodlands and sandplain heath. Most *"_'*"" /z| / *$!'*$* z'|/= * |= * | #` ' |/ $ /|= * =|z =| _' " =|z/ `! known from yellow sands, laterite, sandy clay soil, etc. /| ` =|z "# =|z "`' "_ / |z/ $#' $ ='' Current known distribution coincides with that of Caleana | / $"' $$ = |/= #!* /| #!'#! zz|== #!_ (inclusive of Paracaleana) and Drakaea. /| # _ |/ ##! =|z ##_ =| Notes: E O A]' Clade-based diagnosis| % < V 2011” by Phillips et al. !** / % ' H * *#*# * !#! pink colour due to bacterial associates that are not affected by streptomycin in the isolation medium. Application of Substrate or host: Roots and collars of Chiloglottis valida, C. tetracyclin eliminates bacteria and cultures then assume an aff. valida, and C. turfosa growing in Sphagnum hammocks ' ]Xz in alpine areas in eastern Australia. aerial mycelium giving it a velvety look. Cultures also have concentric rings with culture edges diffused. Cultures often Distribution| V ' z show scalloped edges. Hyphae from cultures are cylindrical, habitats associated with Sphagnum hummocks. Current ' ) known distribution coincides with that of Chiloglottis hosts septate, lacking clamp connections; wall slightly thickened to within this particular habitat. ! ) { % { sometimes uneven (with undulate outline); often with swollen Notes: / ]Xz *! ) ' ! ) concentric rings. Culture edges lack concentric rings, are thick) and globose to clavate when terminal, and globose to broad and diffused. Hyphae from cultures cylindrical, 2–5.5 ellipsoid when intercalary; when terminal, subtended by one ) or two swollen, clavate elements, but not in chains. Refractive { ! ) { bodies within the hyphae are more common and obvious in rarely with refractive internal bodies, and then in narrower this and T. warcupiii than in the other two species. hyphae; sometimes uneven (with undulate outline); rarely _ ) ' Additional material examined| V * walled, subglobose and terminal. Swollen elements are less common in this species than in the other three. Tulasnella sphagneti J @ sp. nov. MycoBank MB817405 Additional material examined| V * H #; Tulasnella warcupii J @ sp. nov. Etymology: Referring to the Sphagnum habitat of the orchid MycoBank MB817407 host. H #X Type: Australia: New South Wales| +] Etymology: After J. H. Warcup who was instrumental in A Chiloglottis aff. studying mycorrhizal fungi associated with orchids in valida growing in a Sphagnum hummock, 19 Jan. 2012, C.C. Australia. Linde CLM541 & E. Triponezz @U ! " # M { E' type culture VPRI 42811). Type: Australia: Queensland| z Range State Forest, Atherton, isolated from Arthrochilus V O LUME 8 · NO . 1 43 44 ARTICLE outline N i Dis S h w Clade-based diagnosi = *!# z *'* '=z " * =/|'' # z|= #" = /z|'''' *"' * ''|=z * ' *# |z *`$ |/ *_" =| *"` |/ *"$ z|= !* /| !# =| z *#* /| * =| * " |/ *`# /|' *`` |/ |/ ## =|/ #_ /|' _ /| *!* |/ * ! /| *#! $ / * / / $_ /= _ | _` z|= _ z/|= `$'_! |/ ` |/ `* /| $ =|z " |z #$ =|z ` /| $ |/ ' ` / | / ITS2 "V | ] *# /|= /| ` ph IT g and thisistheonlyoneoffourspeciestoshowchains of described here,itistheslowest velvet **` *_$ z |=z * '*` //|z *$ '|/ * # =|z * ` |/ * M ) *M M often atri refractive internalbodies;sometimesuneven as advocatedbyRenner(Renner 2016) culture VPRI42809 A T Di Here wedescribefournew species of DI oreo species revea belon f E E n assoc ou =|/ / | / | _ ) . tomacu !"* M ! " * l yp ` = |z/ " /| `$ |/ _ '| __ /|z _" z| ! dditional materialexamined ubstrate orhost:R oodla otes o S agnos / y S b #!# n tr 1 *"V ' # = |z/ ` h lo ose e d phil C ibu " ae z| g |= g l in =/|z y : e USS ) en in ' ed n = todistinctlymonilioid,withshort,repeated,globose d "# t u _' $ i io i ( d g s not s i s T. prim y ]z ]Xz b at z|'= . *"* l totheDiurideae **$'* ! : n | , g inFi ements. g y an l assoc 1 um z z | i e. Usuall ht an / T on w I A 5.8 ON u *!_ i cea =|z' "` p l asne g #!_ r. r. i S a a n . /| i 2010 n g , i a 1 bl t d @ M M ! " ! @U { d les, septate,lackin h * | |' * ] | < V* = T. T. ti containin ) oo ept y t ll o . /=|=z "_ y a warcup z|= *" | n with A |z A secu T noaerialm t |/ hi s , s h . warcup rt | % < V C.C. Linde&D.GomezCLM027 stu nner t a "" h /= n roc *!$'*** | n #!$ d V |' da d |/ co * * hil g y us z| h , and KF47 $#' $ ll ii us oreop *' # an = a us ii *$`'*$_ z| r i can ng e s y $ tr i n t celium isvisible.Cultures of | a % | < V| % | T. warcupi 6 li g / A 5 h ° z =|=/ a ) ! i b X+z rowin g g 96 e ot rt n t z '|' e *#" clampconnections; h h hil t sequence andKF47 = T roc =/ | / T di h u u e u /=|/=z g |// er t agnose l s rr l asne $# hil as . H E' { es / i ( i ) us oreop n n h with undulate wereinitially tri **# | e ree spec y { lla ll E phae from '|' a a #' # * '*# l uca d t 6601 s #! / l h oc o 7 b p at are r l *! (MEL y t yptu hil c ecies =| i an hi . Linde i us es, h ds '| d e s ' ' ' . de tal et al is ambi % of thetwoapproaches.However, Cruz rema investi U i T a cu spp. hasbeenunsuccess ' contemporary taxonomicmycology. Somespeciesare mater described withexcellentdetailsofmorphologicalcharacters. alpine areasineastern Australia revealedthefourthspecies, for s # % ' % here remainspoorl (Girlanda where thestrictapplicationo that “sequencin morp to befoundassociatedwith E ' divergence betweenthefournewspeciesdescribedinthis g al. bu a isolates from assoc three different methodsofspeciesdelimitation T. T. l t o s t t C si C t o A a r a grow T n t ack livin \ his subtro hi his o matchthat < e tud . sp u n erminate seedo E n reop f theTulasnell r re hosttomulti rthr nfortunatel aleana ster tot hilo . % e l lt 2014 s p l d ate d t V as hi u A Tulasnell rim T. spha g y assoc Caleana r h oc i s s genus,w p d part fromtheirassociationwithorchids,theecolo h i g , m n ng roup tobefoundinbotheasternandwestern Australia, es n t d i hil i i a ce eiiain + ecies delimitation a i o g ate w lottis s e s a . hil g l g l ated, withintheorchid t ll tri s us o e ) . T o net g a y h a uous, butthechallen Remarkabl i etal gy us r t u n h r bu cultures,onl corrhizal associationsofthetropical n e caseevenw \ h i p a l ou co d asne e rema g i an ' ical s i formaclade.However, theoverallphylogeny ti S is y appear farmorediverse. Previousl ti t a a , net h o T. sphagneti , fewspeciesarewellknownfromboth Chiloglotti associatewithasin o ld ll isrepresentativeofthecomplexity phagnum x n . ec n (Linde a 2011, Jacquemy sis d a f g b i +P X+zP hil thehosts ll p e issofaronlyfoundin mo s ti ofoldfun e sequence p o a pr E le xt t a o e ot ce (Linde ecies e f th p i n y n f e n r t memberso ecies fromDrakaeinae T known.Interestin d s l n i y ecu ng genera, eta u i th t , in ds ma an h e o l y o as . s er spec n one growingin l D o i Whe % ac e ar sequence n l. C assoc l e r thr f d i e 2014 t akaea ul probabl ( w alean r h n g ll Miller oss d d a arium specimensof ( r Chiloglotti a , w T. warcu species,thefun T i e T. sphagneti ee m g m i g f u e es th nameswithoutsequences i n enera e willbetomatchmodern a ) p l t es hi o a as , . Ouradditionof etal.2011), thesequence etal.2008 i t g f andtheseformaclade rovements nc r , a e & theorchid e c h T theonl chids le . l with n Sphagnum hummocksin h . ave u co l 2014).Becausetwoof C e u wou l T Chiloglotti y as ll di lements 2014 g u duetoinappropriate p C ntin d e a s l l i ng n y ata. t ` # as i s hiloglotti b orchids.Basedon Chilo Chiloglottis ) , allorchidspecies Tulasnell e i al. isdescribedhere. een an ld n t T e i Tulasnellas n y ll p doesnota from u % a A a nt. In orchid e ecies described l ( s asne Id he rt ll 2016 ll i g a n tec ow ) h ea g lottis s p I r s #M i areclosel g A ochilus MA FUNGUS s d D w ecies from p lly C enus the co , lla i rt are a Tulas T ) ntegrat ecies with r s ( hilo h pointout akaei Drakaea, , a h species u g Linde y orchids h ) ntr s ere r l s , three s , where enus in n asne ochilus p p inthis ll iq g pp as b ec ecies t n lottis b e ues. n a y ella t ear ot i i i on ng pe ae n gy es lla e h d o y y , t New orchid associated Tulasnella species of Tulasnella fungi associates with a large number of orchid Bidartondo MI, Duckett JG (2010) Conservative ecological and ARTICL species across a vast geographic range. % % ' Proceedings of the Royal Society, B-Biological Sciences 277: 485–492. ' Bonnardeaux Y, Brundrett M, Batty A, Dixon K, Koch J, et al. (2007) interactions outside Australia, which have consistently found Diversity of mycorrhizal fungi of terrestrial orchids: compatibility E \ webs, brief encounters, lasting relationships and alien invasions. well as allopatric orchid congeners (Jacquemyn et al 2015). Mycological Research 111: 51–61. H E * \ Tulasnellaceae were associated Bougoure JJ, Bougoure DS, Cairney JWG, Dearnaley JDW (2005) with four species of Anacamptis * \ < V'AH] Acianthus, *# % Ophrys and two Orchis Caladenia and Pterostylis (Orchidaceae) in southeastern \ Serapias Queensland. Mycological Research 109: 452–460. species (Pellegrino et al. !* % Tulasnella / @z U A *$_$ * \ ' Australian terrestrial orchids. American Orchid Society Bulletin " 48: 810–816. \ ] et al. 2014). A corresponding result was Cruz D, Suarez JP, Kottke I, Piepenbring M (2014) Cryptic species found along a single 1000 m transect with the same orchid revealed by molecular phylogenetic analysis of sequences genera where 16 Tulasnellaceae \ % obtained from basidiomata of Tulasnella. Mycologia 106: 708–722. 20 species of orchids (Jacquemyn et al. !* Cruz D, Suarez JP, Kottke I, Piepenbring M, Oberwinkler F (2011) pattern is found in Andean tropical rainforests where up to X Tulasnella by correlating morphology and six Tulasnella \ Stelis orchid species X+z < V' "V and Pleurothallis lilijae (Suarez et al. 2006, Kotte et al. 2008). Andean forest. Mycological Progress 10| $M #" / H @ zX ; H @ = Zz @ / !* multiple species of an orchid genus can share the same / ' % \ % taxonomy. Trends in Ecology and Evolution 27: 480–488. = @ V A / X A @, et al. the real mycorrhizal diversity associating with the orchids. (2011) Photosynthetic mediterranean meadow orchids feature Our description of four new species of Tulasnella, all associated with Australian orchids, extends the number of American Journal of Botany 98| **"M**`# formally described species known as mycorrhizal agents of XV z = X + @ ], et al. (2011) orchids. However, it is evident that more Tulasnella species Progress in molecular and morphological taxon discovery in X+z H E Fungii % previous studies on Tulasnella < V % sequences. Fungal Biology Reviews 25| #"M_ Diuris % % \ (Smith XV ZJ !*# H | et al. 2010), likely to represent many undescribed Tulasnella enlightenment at hand? Nature Reviews, Microbiology 11: 129– species. It is further evident that earlier morphologically *## J ' % VX ; z] !!` z R ' \ X+z Tulasnella duck orchids revised (Paracaleana: Orchidaceae). Australian species diversity associated with orchids in Australia. Systematic Botany 19: 211–244. 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