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et al. Key words: Auxarthron Knuckle-joints Molecular phylogeny Multiseptate appendages Onygenaceae that forms Onygenales that forms
Renispora et al. 1999, Sole et al. 2002c, Sugiyama IMA FUNGUS · VOLUME 4 · NO 1: 89–102 NO 1: 4 · · VOLUME FUNGUS IMA . However, . However, Auxarthron and Auxarthronopsis albicans,
contains a few genera that have The order Onygenales contains a few genera that have analysis of one or more rDNA regions (ITS, LSU and SSU). regions (ITS, LSU analysis of one or more rDNA locus the rDNA Unlike several other groups of fungi where onygenalean fungal fails to resolve the phylogeny of taxa, gene with rDNA phylogeny was mostly well resolved comparisons (Gräser evolved An exception lay in some recently et al. 2002). little variation is asexual dermatophyte lineages, where microsatellites or observed even in the ITS region, and where species delimitation other highly labile loci are necessary for by molecular methods. mesh-like peridia, the reticuloperidium (sensu Currah 1988), bearing elongate appendages; these include Auxarthron, Pectinotrichum, and Uncinocarpus (Currah 1988). Castan- edomyces is another genus of in having a membranous elongate appendages, but differs The generic name peridium similar to that in Aphanoascus. Auxarthron was established by Orr et al. (1963) for species with “enlargements at the septa in the peridial hyphae; lightly coloured, elongate appendages which are non-septate except for one, two or three basal septa with characteristic The generic name means ‘swollen swellings at such septa”. joints’, and the genus now comprises 13 species (Sole The monophyly of Auxarthon 2002a, b, Sigler et al. 2002).
and the new generic name isolated from the vicinity of from the isolated Onygenales 1 hair – and Auxarthron, A. volatilis-patellis, Nannizziopsis , . Further, the pale cream ascomata, punctate ascospores, the pale Onygenaceae. Further, , and Sanjay K. Singh , and Sanjay 2
, Yvonne Gräser , Yvonne 1 is an assemblage of fungi which has evolved is an assemblage of fungi which has An interesting onygenalean ascomycete was isolated from soil collected from a hollow tree near ascomycete was isolated from soil collected An interesting onygenalean
, but differs morphologically. , but differs The most recent monographic treatment of Onygenales Institute of Microbiology and Hygiene (Charité), Humboldt University, Dorotheenstr 96, Berlin 10117 Germany Berlin 10117 Dorotheenstr 96, and Hygiene (Charité), Humboldt University, Institute of Microbiology National Facility for Culture Collection of Fungi, MACS’ Agharkar Research Institute, G. G. Agarkar Road, Pune - 411 004, India; corresponding Road, Pune - 411 Agarkar Agharkar Research Institute, G. G. Collection of Fungi, MACS’ National Facility for Culture phylogenetic study of LSU, SSU and ITS sequences, and presence of more than three swollen septa on the of more than three swollen septa on the LSU, SSU and ITS sequences, and presence phylogenetic study of within peridial appendages, do not support a placement and swollen septa in the peridial hyphae suggested that this was a new species of Auxarthron the peridial hyphae suggested that this was and swollen septa in Abstract: with a malbranchea- The keratinophilic nature associated Park situated in central India. Bandhavgarh National to oblate, punctate ascospores, mesh-like reticuloperidia, and subglobose like asexual morph, appendaged this isolate in support the inclusion of author e-mail: [email protected] © 2013 International Mycological Association © 2013 International Mycological conditions: distribute and transmit the work, under the following are free to share - to copy, You Attribution: Bandhavgarh National Park, India National Bandhavgarh 2 1 Rahul Sharma , a new genus of , a new Auxarthronopsis Non-commercial: No derivative works: Any of the above conditions can be waived if you get 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. Nothing in this license impairs or restricts the author’s moral rights. permission from the copyright holder. Submitted: 19 February 2012; Accepted: 17 April 2013; Published: 10 June 2013. 17 Accepted: Article info: Submitted: 19 February 2012; is introduced to accommodate this new fungus. The distinguishing features of this new taxon are the multiple The is introduced to accommodate this new fungus. 1>? + of arthroconidial and aleurioconidial asexual forms. Sequence regularly punctate ascospores, and the production clearly separate this fungus from monophyletic analysis of ITS1-5.8S-ITS2, SSU and LSU regions Auxarthronopsis bandhavgarhensis Phylogenetically, similarity. and other taxa bearing some morphological to Amauroascus purpureus is closest gen. sp. nov. was devoted to evolutionary and molecular phylogenetic studies (Guarro et al. 2002). It included four new genera, 14 new species, and six new combinations based on sequence INTRODUCTION Onygenales integument of birds, to utilize keratin that forms part of the This substrate, like cellulose, is reptiles, and mammals. by large number of abundant in soil as it is regularly shed integumental elements or as vertebrates either as effete Burrows actively ingested materials mixed into excreta. habitats for inhabited by small mammals are excellent these fungi since they contain ample substrate, and even in hot dry summers. moisture – at all times of the year, The unusual form of the ascomata in most onygenalean members, a mesh-like reticuloperidium with various types of appendages, is suggested to be an adaptation to active dispersal via attachment to arthropods, birds, and mammals (Currah 1985). During our studies on keratinophilic fungi geographical regions of India, an interesting from different ascomycete was isolated from a burrow-like habitat, in this case a hollow tree, near Bandhavgarh National Park which has one of the highest densities of wild tigers in the world. doi:10.5598/imafungus.2013.04.01.09 VOLUME 4 · NO. 1 90 ARTICLE
134524). Fungal BiodiversityCentre,Utrecht, The Netherlands(CBS Agharkar ResearchInstitute,Pune,India,andatCBS-KNAW 932), Maharashtra Association forCultivationofSciences’ National FungalCultureCollectionofIndia(NFCCI–WDCM Herbarium (AMH),withpureculturesdepositedinthe lactophenol mounts. dextrose 4g,agar20gL of ourtaxon. regions ofrDNA toestablishthephylogeneticrelationships data, weconductedmolecularstudiesusingthreedifferent To determineifsuchachangewassupportedbysequence species withmorethanthreeseptateperidialappendages. if theexistingcircumscriptionwasextendedtoinclude area suggestedthatitmightbeaccommodatedin That genuswasfoundtobepolyphyletic(Soleetal.2002a). L Agar (PDA;200gpotato(extract),dextrose20g,agar Carrot Agar (PCA)(HiMedia,Mumbai)andPotatoDextrose Mumbai), Cornmeal Agar (CMA)(HiMedia,Mumbai),Potato 1 hyphae, and,unlikeAuxarthron,itformsincompositoperidia Amauroascus, hasascomatacomposedoflooselywoven sequence data. Another morphologicallysimilargenus, was demonstratedbySoleetal.(2002a)basedonITS \ ; >
voltages. voltages. " ; visualized underJEOL 610andJEOL JSM6360A (JEOL, min), thencoatedwithgold-paladium/platinummixtureand μL Taq polymerase(5UμL (1 UμL each); 0.5μL primer(50pmolμL mixture contained28μL H et al.1990).FortheITSregion,50μL ofthePCRreaction (Rehner &Samuels1994,Vilgalys &Hester1990,White LR7, LR7R,LR12(LSU),andNS1,NS4,NS3,NS8(SSU) pairs Mass266/V9D,ITS1/ITS4(ITS),5.8SR,LROR, using universalprimers.PCRwasperformedwithprimer "%>+< |%+"%E >|% E|% /* glucose agar(DifcoLaboratories)for21dat28ºC. The 1999) afterthefungushadbeengrownonSabouraud N-trimethylammonium bromide)method(Gräser DNA wasextractedusingtheCTAB (N-cetyl-N,N, %'* { | < (Submission ID12372). and sequencealignmentshave beensubmittedto TreeBase (NFCCI 2185)havebeendeposited inGenBank(Table 1) replicates. The ITS,LSU,andSSUsequencesofour isolate of brancheswasassessedby bootstrapanalysiswith1000 with the‘pairwisedeletionofgapsoption’. The robustness the Kimuratwo-parameterdistancemodel(Kimura1980) et al.2011). The phylogenetic treewasconstructedusing performed withtheMEGA v. 5computerprogram(Tamura Neighbour-Joining (NJ)method(Saitou&Nei1987)were format fromGenBank.Phylogeneticanalysesusingthe previous studiesandex-typestrainswereretrievedinFASTA For phylogeneticanalysis,sequencesofmaterialused in 3100 Avant, Applied Biosystems,FosterCity, California). sequencer (Beckman-Coulter, Fullerton,Californiaor ABI using thesameprimers(Whiteetal.1990)onanautomated * % * * / À / À* " Sequencing andphylogeneticanalysis 16 μL H 28S rRNA,25μL ofthePCRreactionmixturecontained > &U? VE >? >|% °C for5min,(9515572 Branchburg, NY)withthefollowingreactionconditions:95 thermocycler (PerkinElmer, RocheMolecularSystems, Steinheim). PCRwasperformedinaPerkinElmer9600 was overlaidwithonedropoflightmineraloil(Sigma, dNTPs (250mMeach),1μL primer(50pmoleμL photographed underUV. on 1.2%agarosegelstainedwithethidiumbromideand VE >? " < > VE > &U? the followingconditions:95°Cfor5min,(95151 in anEppendorfMastercycler(Eppendorf,Hamburg)with oil (Genei,Bangalore). The PCRreactionwasperformed ng μL NJ), and5μL genomicDNA (10ngμL -1 ); andwasoverlaidwithonedropoflightmineral 2 -1 O; 2.5μL PCRbuffer (10x);1μL dNTPs(250mM ) (Genei,Bangalore);2.5μL genomicDNA (10 2 -1 O, 5μL PCRbuffer (10x),4μL ) (AppliedBiosystemsRoche, -1 ); 1μL Taq polymerase -1 ). The mixture). The IMA FUNGUS -1 et al. ), 0.4 Auxarthronopsis gen. sp. nov.
Table 1. Fungal species and LSU, SSU and ITS nrDNA GenBank accession numbers used in the study. ARTICLE Taxon ITS SSU LSU Arthrodermataceae Arthroderma cajetani AY176736.1 A. ciferri EF413624.1 EF413625.1 A. curreyi AJ315165.1 A. otae AY176735.1 Ctenomyces serratus AJ877222.1 U29391.1 AY176733.1 Gymnoascaceae Gymnoascus aurantiacus AB015772.1 AY176747.1 G. littoralis FJ358272.1 G. marginosporus AJ315168.1 G. petalosporus AY176748.1 G. reesii AY176749.1 G. ruber AY177296.1 AY176746.1 Onygenaceae Amauroascopsis reticulatus AJ271418 Amauroascus aureus AJ271433 AY176705.1 Am. echinulatus AJ271562 Am. mutatus AJ271567 AB075321.1 Am. niger AJ271563 AY176706.1 Am. oblatus AJ271421 Am. purpureus AJ271564 AY176707.1 Am. queenslandicus AB361646.1 AJ315175.1 Am. volatilis-patellis AJ133435 AB075324.1 Aphanoascus fulvescens AJ315172.1 A. mephitalis AB015779.1 AJ176725.1 Apinisia racovitzae AJ271429 Auxarthron alboluteum AB361630.1 AY124494.1 AB359411.1 A. californiense AF038352.1 AY176711.1 A. chlamydosporum AJ271425 A. concentricum AJ271428 A. conjugatum AJ271573 AB075325.1 A. compactum AJ271574 AB015767.1 } [ AY177298.1 AY124501.1 AB359417.1 A. kuehnii AB040691 AB015766.1 AB040691.1 A. pseudoauxarthron AJ271572 A. pseudoreticulatus AJ271420 A. reticulatum AJ271568 AB359430.1 A. umbrinum (A. thaxteri) AJ271571 AY124498.1 } / ;[ AJ271569 AY124492.1 AY176712.1 Auxarthronopsis bandhavgarhensis HQ164436 JQ048939 JQ048938 Byssoonygena ceratinophila AJ315176.1 Chlamydosauromyces punctatus AY177297.1 Nannizziopsis albicans AJ271432 Neogymnomyces demonbreunii AJ315842.1 AY176716.1 Onygena corvina AB075364.1 O. equina U45442.1 Pectinotrichum llanense AJ390391.1 AJ315178.1 AF299348.1 AB015784.1 AY176719.1 Shanorella spirotricha AJ271430 Uncinocarpus reesii AJ271419 L27991.1 AY176724.1 Trichocomaceae Byssochlamys nivea GU733368.1 AY176750.1 Eurotium herbariorum GU733351.1 AY176751.1 Petromyces alliaceus AB002071.1 AY176752.1
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(5), andarthrodermataceous (4)fromprevious studies sequences ofonygenaceous (16taxa),gymnoascaceous several otheronygenaleanspecies. Inaddition,thenrLSU demonbreunii (ATCC 18394, AY176716.1), and92%with 219.58, AY176712.1), 93% with Neogymnomyces 32622, AY176707.1)and homology of94%with search with689bpof5’endnrLSUshowedamaximum 301–A, 293–C,395–Gand258–T. A nucleotideBLAST of the5’endwithfollowingnucleotidecomposition: of NFCCI2185resultedina1247bpnucleotidesequence JQ048939 (SSU).Sequencingofthepartial28SrDNA gene with accessionnosHQ164436(ITS),JQ048938(LSU), NFCCI 2185andthesequencesaredepositedinGenBank partial 18Sand28SrRNA genes,weresequencedfor The completeITS1,ITS2and5.8Sregionsalongwith Phylogeny survival insoil. (Fig. 1a,b). This showed thespecies’ capacityforlong-term with horsehair, abundantascomatadevelopedonthehair preparation. However, after>6yrwhenthesoilwasre-baited could bemadeasthematerialwasdestroyedinslide was noted(Sharma2002). At thattime,however, noisolation ascoma formedinahair-baitingplateanditsunusualform E??> ; fungus wasisolatedsixtoeightyearsafterthesoil ascomycete withcharacteristicsfoundin Hair-baiting ofsoilsampleS214resultedinisolationan RESULTS nmrhTerminal, intercalaryorrarely Anamorph eiilapnae Branchesrare,tipsround,sub- Peridial appendages eiilhpa Reddish,rustytan,enlargedat Peridial hyphae sopr oorHaie oylo rylo-rw Hyaline Hyaline,toyelloworyellow-brown Colour Ascospore siSubglobose,globoseorovoid, Asci soatp eiuoeiimRtclprdu Incompositoperidium Reticuloperidium Reticuloperidium Ascoma type soaaClu elwbont rw ht opl ra White, yelloworbrown Whitetopalecream Yellow-brown tobrown Colour Ascomata Character Table 2. A comparisonofmorphologicalcharactersthegenus ie< m1.75–2.5×2.75–3.2μm <5μm Size hp sphericaltooblate,minutely Shape ie<0 m5010 mUpto2.5mm 500–1000μm <700μm Size arthroconidia, keratinolytic cylindrical, aleurioconidia, lateral, pyriform,oblongor uncinate which arebentorslightlycoiled (1, 2or3septa)withsimpleapices acute, largerappendagesseptate tuberculate minutely tocoarselyasperulateor septa, thickcuticularizedsmoothto punctuate topunctate-reticulate 7.2–8 ×10.6μm 8-spored, evanescent, uatrnAxrhoossAmauroascus Auxarthron Auxarthronopsis Amauroascus purpureus(IFO / ;[ } 1 Onygenales Sharma, Gräser&Singh (CBS . The Auxarthron, Auxarthronopsis aleurioconidia, slightlykeratinolytic dehiscing arthroconidiaand Terminal orintercalaryrhexolytically vegetative hyphae,upto400μm apex indistinguishablefrom septa, taperingtoacutehyaline Multiseptate withdistinctswollen sparsely asperulate network, withswollensepta, anastomosed formreticulate Pale brown,branchedand view butoblateinequatorial regularly punctate,globoseinpolar 5 ×5.5μm evanescent, Globose, 8-spored,hyaline included (Fig. 5). The newfungusformedaseparateclade that the analysedstrainsintodistinct familiesofOnygenales the analysis. The neighbour-joiningSSUtreeresolved of sequencesfrombothendswereagainexcluded also alignedwiththatofNFCCI2185.Incompleteportions gymnoascaceous, andthreearthrodermataceous,were previous studies,including17onygenaceous,three 2846, AY124494.1). The nrSSUsequencesfrom (UAMH 4098, AY124492.1) andA.alboluteum(UAMH onygenalean species,including (CBS 453.75, AB015779.1); and97%withseveralother and 708.79, AB015784.1), (UAMH 9990, AY177297.1), homology of:98%withChlamydosauromyces with 1339bpofthe5’endnrSSUshowedamaximum 275–C, 360–G,and347–T. A nucleotideBLAST search 5’end withthefollowingnucleotidecomposition:357–A, 2185 resultedina1339bpnucleotidesequenceofthe Sequencing ofthepartial18SrDNA geneofNFCCI clade alongwithAm.purpureusandvolatilis-patellis. 4). The newisolatewas placed withinthe for membersofthethreefamiliesOnygenales(Fig. nucleotide substitutionmodel.Itshoweddistinctclades (NJ) treewasconstructedusingtheKimura–2parameter were excludedfromtheanalysis. A neighbour-joining 2185. Incompletepartsofthesequencesfrombothends were retrievedfromGenBankandalignedwithNFCCI and purpureus, Am. volatilis-patellis, Nannizziopsisalbicans, punctatus. Someotherrelatedspecies, suchasAm. Neogymnomyces demonbreuniicouldnotbeincluded and , andAmauroascus keratinolytic rhexolytically dehiscingconidia, Terminal and/orintercalary Appendages lacking thick walled occasionally palebrown,smooth& Usually undifferentiated inculture, 3.5–7 μm or regularpunctaandridges spherical withpronouncedirregular dark brown Hyaline, yelloworreddishbrownto Ovoid, 8-spored,upto14μm Renispora Aphanoascus . / ;[ } 1 Chlamydosauromyces (CBS Onygenaceae IMA FUNGUS mephitalis punctatus
Auxarthronopsis gen. sp. nov.
in the analysis because SSU sequences were not available Description: Ascomata discrete, globose, white to ARTICLE in GenBank. The strong LSU and SSU sequence similarity pale cream, reticuloperidium 500–1000 μm diam, with of NFCCI 2185 to other members of Onygenales suggests appendages. Peridial hyphae branched and anastomosed to membership in this order. form reticulate network with knuckle-joints 2.0–2.5 μm wide. Sequencing of the ITS region of NFCCI 2185 yielded Peridial appendages pale, numerous, elongated, straight or a 613 bp long nucleotide sequence that included 242 bp 1>? EME < of ITS1, 150 bp of 5.8S and 166 bp of ITS2. In contrast to μm wide, tapering to acute hyaline apex indistinguishable the LSU and SSU, the ITS sequence of the undescribed from vegetative hyphae, up to 400 μm long. Asci globose, fungus was distant from other species it was compared with. eight-spored, hyaline, evanescent 5 × 5.5 μm. Ascospores It showed maximal sequence similarities of only 84 % with unicellular, hyaline, globose to subglobose, smooth under two quite distinct species: Amauroascus purpureus (IFO %# 32622, AJ271564) and Nannizziopsis albicans (IMI 155645, globose in polar view but oblate in equatorial view, 1.5–2.5 AJ271432), with 90 % and 88 % query coverage, respectively. × 2.5–3.0 μm. The next most closely associated sequences in the BLAST results had similar similarities of 85 %, but show very low Asexual morph: Conidia malbranchea-like, arthric, intercalary query coverage of less than 75 %. These sequences included or terminal on short branches, abundant, hyaline, solitary, a mixture of Eurotiales and Onygenales. The ITS sequence aseptate, 2.0–4.5 × 4.5–11.5 μm, separated by autolytic of NFCCI 2185 was also compared with sequences of 29 connective cells. species from ten genera of related Onygenaceae (Table 1) and a phylogenetic tree (NJ) was constructed using Kimura-2 Cultures: Colonies after 3 wk at 28 °C (Figs 2a, b) on SDA, parameter model (Fig. 6). white, slow growing (3 cm), cottony, slightly raised at the centre; reverse uncoloured to pale brown; on CMA, OA, and Auxarthron in having peridial hyphae with swollen septa, but dilute SDA, slow growing (3 cm diam), mostly submerged it is distinct genetically. This is evident in the LSU, SSU, and with sparse mycelium, pale brown, reverse uncoloured to ITS trees where Auxarthron forms a monophyletic clade (Figs pale brown; on PCA and PDA slow growing (2 cm diam), 4–6). A morphological comparison of the newly designated pale coloured at the centre and white in the peripheral region, Auxarthronopsis with Auxarthron and Amauroascus is shown slightly cottony, and the reverse pale brown. Ascomata in Table 2. produced sparsely on PCA and abundantly on PDA (Figs 2c, d), but without elongate appendages.
TAXONOMY Type: India: Madhya Pradesh: Umariya, buffer zone of Bandhavgarh National Park, ascomata growing on horse hair Auxarthronopsis Rahul Sharma, Y. Gräser & S.K. Singh, (keratin bait) in soil S214 collected from inside a big hollow gen. nov. tree, 16 June 2001, R. Sharma (AMH 9405 – holotype; MycoBank MB563744 NFCCI 2185 = CBS 134524 – cultures ex-type).
Etymology: Named after its phenotypic similarity to the genus Substratum: Isolated on horse hair from soil. Auxarthron. Distribution: Known only from the type locality, Bandhavgarh, Description: Ascomata solitary, globose to subglobose, India. white to pale cream. Peridium made of mesh of interwoven hyphae with swollen septa. Appendages straight, tapering at 1>? DISCUSSION septa. Asci globose to subglobose, hyaline, evanescent, 8– spored. Ascospores The typical mesh-like peridium of Auxarthronopis and regularly punctate walls. Asexual morph terminal and bandhavgarhensis and other features, including the ability intercalary hyaline arthroconidia and aleurioconidia. to grow on horse hair (keratinophilic nature), make this fungus a relatively typical member of Onygenales (Currah Type: Auxarthronopsis bandhavgarhensis Rahul Sharma et 1985). However, it could not be placed in the morphologically al. (2013). similar genus Auxarthron, which never forms multi-septate appendages. The new genus Auxarthronopsis shows Auxarthronopsis bandhavgarhensis Rahul Sharma, a resemblance to Auxarthron in its mesh-like reticulo- Y. Gräser & S. K. Singh, sp. nov. peridium and in its possession of knuckle joints; however, MycoBank MB563745 the ascomata of Auxarthron are mostly yellow to brown at GenBank HQ164436, JQ048938, JQ048939 maturity, while the ascomata of the new taxon are white to (Figs 1–3) pale cream. The peridial hyphae of Auxarthron species are dark coloured, rigid, and relatively broad, while those of the Etymology: bandhavgarhensis- referring to the locality where \ * the soil collection was made, Bandhavgarh National Park, India. circular punctae), a morphology that differs from the minutely
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Fig. 1. hyphae showing knuckle joints(arrows).Bars: A =600μm;B200 C=100μm;D80E6 μm;F=80GH 10 μm. ascoma showingmultiseptateperidial appendages.G.Bifurcatebranchingofperdialappendages (arrows). ascospore mass. microscopic viewofunmounted ascomata withelongateappendagespickedupfromhairbait. D.Mesh-likereticuloperidiumwith central Auxarthronopsis E. Baseofelongateappendageshowing inverted Y-shaped archwithswollensepta(arrows).
bandhavgarhensis (AMH 9405). Sharma, Gräser&Singh A–B. Stereomicroscopic-view ofmature ascomagrowingonhorsehair. C.Light H. Dichotomouslybranchedperdial F. Phasecontrastimageof IMA FUNGUS Auxarthronopsis gen. sp. nov. ARTICLE
Fig. 2. Auxarthronopsis bandhavgarhensis (NFCCI 2185T). Colonies at 28 °C after 3 wk of incubation. A. Colony front on different media. B. Reverse of colony on different media. C. Enlarged view of the colony on PDA with abundant ascomata near peripheral region. D. Developing ascomata on the periphery of colony on PDA. Bars: C = 1 cm; D = 300 μm.
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Fig. 3. 2185). Bars: A =40μm;B 100 μm;C=10D1E =2μm;F1G–H20μm. ascospores showingcircularpunctae. appendage withtwoswollensepta. appendages (arrows). Auxarthronopsis B. Elongateappendagesradiating from reticuloperidiaonhorsehair.
bandhavgarhensis (AMH9405) D. Enlargedportionofseptawithring oftubercles. G, H.Slideculturepreparationshowing sessileandstalkedaleurioconidiaarthroconidia(NFCCI Sharma, Gräser&Singh . Peridialappendages,ascospores andasexualmorph.A.Multipleseptaonperidial E. Asci andascospores. F. C. Sparselyasperulatebasalportion ofperdial Finelyandregularlypunctate IMA FUNGUS Auxarthronopsis gen. sp. nov. ARTICLE
Fig. 4. Neighbour-joining tree based on nucleotide sequences of 28S rDNA gene of the 25 stains of Arthrodermataceae, Gymnoascaceae and Onygenaceae listed in Table 1 along with Auxarthronopsis bandhavgarhensis (NFCCI 2185T). Three strains belonging to Trichocomaceae Byssochlamys nivea (AY176750.1), Eurotium herbariorum (AY176751.1), and Petromyces alliaceus (AY176752.1) served as outgroup. The branch lengths are proportional to distance values calculated in MEGA 5 and values at nodes represents bootstrap percentage of 1000 replicates. Bootstrap values above 50 % are shown.
asperulate to punctate-reticulate ascospores of Auxarthron. spherical and the ascomatal appendages lack knuckle joints. Ascospores of A. pseudoauxarthron possess similar circular In general, species of Auxarthron do not possess more than pits on the ascospore walls, but in that species the spores are three septa in the peridial appendages, whereas those of
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and Fig. 5. Byssochlamys nivea Bootstrap values above50%areshown. branch lengthsareproportionalto distancevaluescalculatedinMEGA 5andvaluesatnodes representsbootstrappercentageof Onygenaceae listedin Table 1 alongwithAuxarthronopsis Neighbour-joining treebasedon nucleotide (GU733368.1) , Eurotiumherbariorum
sequences of18SrDNA geneofthe22stainsArthrodermataceae Sharma, Gräser&Singh (GU733351.1),
bandhavgarhensis
and Petromyces alliaceus NFCCI2185 T . Three strainsbelongingto (AB002071.1)servedasoutgroup. The , Gymnoascaceae Trichocomaceae 1000 replicates. IMA FUNGUS
Auxarthronopsis gen. sp. nov. ARTICLE
Fig. 6. Neighbour-joining bootstrap consensus tree based on nucleotide sequences of internal transcribed spacer (ITS) region and 5.8S rDNA gene of the 30 strains listed in Table 1 which includes reference strains of Auxarthron species and other onygenalean genera along with Auxarthronopsis bandhavgarhensis (NFCCI 2185T). The unrooted phylogenetic tree was drawn using 611 nucleotide of the ITS1, 2 and 5.8S rRNA gene using MEGA 5 software. The branch lengths are proportional to distance values calculated in MEGA 5 and values at nodes represents bootstrap percentage of 1000 replicates. Bootstrap values above 50 % are shown.
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" " species of differentiated hyphae,which lackappendages,andforms septa. ascomata, withamesh-likereticuloperidiumswollen inconsistent withthesegenerainthedistinctnessof Neogymnomyces. was placedclosetoAmauroascus, substitutions and64ofwhichareindels. differs at166of623nucleotide positions,102ofwhichare producing ascosporeswhicharepunctate-reticulate.Italso of undifferentiated hyphae lackingappendages,andin Auxarthronopsis bandhavgarhensisinformingaperidium related species,Amauroascusvolatilis-patellis, 74 substitutionsand54indels. Another phylogenetically two speciesdiffer at128of627nucleotidepositions,including reticuloperidium (Guarro hyphae withoutappendages,andinlackingawell-developed new taxoninhavingaperidiumofundifferentiated peridial neighbour, (Ito &Nakagiri1995). The otherphylogeneticallyclosest peridial appendages,andformingglobose,frilledascospores possessing purpleascomatathatlackknucklejointsand indels). Morphologically, A.purpureusisalsodistinctin than 85%(119 bpoutof620bp;92substitutionsand27 the nearestneighbour, The ITSsimilarityof Nannizziopsis albicans, from comparisons alsoshowedAuxarthronopsis from theAuxarthronclade(Figs4–5),andITSsequence Auxarthron latter polyphyletic.OurnrSSUandnrLSUtreesalsoshowed who foundtheformergenustobemonophyleticand been studiedbySoleetal.(2002a)usingITSsequences, interrelationships of Auxarthronopsis () Ascomata madeofonlycleistoperidium Ascomatareticuloperidium orincompositoperidiumtype 2(1) ...... 2 Ascomatacleistoperidiumtype ...... 1 Key tothegeneraofOnygenaleswithperidialappendages walled, verruculoseseptatehyphaealongwithtwokinds of globose topulvinateascomatawithpaleyellow, fairlythick available tous. ITS andLSUsequences(JN038187,JN038186)werenot data; however, itcouldnotbeincludedinouranalysisasits Doveri species of with long,hyaline,peridialappendages.Recently, anew thick-walled, smoothandseptateperidialhyphae,along discrete, sphericalgolden-yellowascomatawithhyaline, Peridialappendages coiled ...... 11 Peridialappendagesstraightwithsimpleorcurved apices,branchedorunbranched,uncinate,nevercoiled ...... Ascomatamadeofinner layercleisto-type,outerreticulo-type 3(1) In thenrLSUandnrSSUtrees(Figs5–6),Auxarthronopsis Auxarthron, butclosertoAmauroascuspurpureus, et al.(2011) basedonmorphologicalandmolecular Neogymnomyces, asmonophyletic. The newtaxonwasexcluded Nannizziopsis albicans(Fig.6),differs fromthe Neogymnomyces, Neogymnomyces virgineus havemorethan10septa. The phylogenetic However, thenewtaxonismorphologically Auxarthronopsis bandhavgarhensisto Auxarthron and Amauroascus purpureus, and et al.1991).Phylogeneticallythese N. virgineus Amauroascus volatilis-patellis. hasascomataofpoorly N. demondreunii Amauroascus , wasdescribedby formsirregularly Renispora, asdistinct ...... Castanedomyces ...... differs from Sharma, Gräser&Singh wasless , forms have and
(1971), whooriginallynotedthatthepectinatehyphaeof 5) andclusterswithCtenomycesserratus.V out fromthemain that speciesisdistantfromAuxarthronopsis molecular studiesofSugiyama but itsdistancefromAuxarthronwassubstantiatedbythe (1994) transferredPectinotrichumllanense asexual morph,andalmostsmoothascospores.Currah on theappendagesandperidialhyphae,a Auxarthronopsis long appendagesandknucklejoints,butdiffers from Pectinotrichum llananse monotypic morph characterisedbyfrequentswollencells. and inpossessinganarthro-aleurioconidialasexual swollen septaintheperidialhyphaeandappendages, rounded atthetips.Neogymnomyces other comparativelyshort,oftenbranched,verruculose,and peridial appendages:onelong,taperingandsmooth,the Pectinotrichum llananse, well developedreticuloperidia,viz. them (Varsavsky &Orr1971). would providemoreevidenceofacloserelationshipbetween serratus, suggestedthatgeneticstudiesofthesegenera llanense well ashyphalelements. Ctenomyces, theperidiumofwhichincludesmembranousas all appendagedgeneraoftheorderisprovided,including incomplete hyphalmesh-likeincompositoperidium. A keyto mesh-like reticuloperidium. They mayalsobeformedonan a membranouscleistoperidiumorcompletelyhyphal, have peridialappendages. These areusuallyborneoneither represents onesuchintermediategenus. bridged byasyetunrecordedforms.PerhapsAuxarthronopsis discontinuity seeninseveralgeneraof peridial hyphae. though theyarefoundonthin,smoothorsparselyasperulate, 3c, d)makingknucklejointsrelativelyprominentinLMeven that theyhavearingoftuberclesvisibleunderSEM(Fig. swollen septain mass frommitegrazinginnature(Summerbell2000). The spherical peridiumwhichprotectsthecentralascospore Their purposemaybetoprovidestrengththemesh-like Another genusthatpossessesknucklejointsisthe Knuckle-joints areacommonfeatureinspecieswith The orderOnygenalesnowcomprises18generathat Currah (1985)suggestedthatthemorphological resemblethectenoidappendagesofCtenomyces ...... Ctenomyces ...... Pectinotrichum inhavingpectinatehyphae,largetubercules Onygenaceae Auxarthronopsis andalmostall formsareticuloperidiumwith (Varsavsky &Orr1971). et al.(1999).Phylogenetically cladeinSSUanalysis(Fig. areslightlydifferent in alsodiffers inlacking Onygenales Gymnoascus reesii, Auxarthron ...... 3 ...... toAuxarthron, asitseparates arsavsky &Orr Chrysosporium IMA FUNGUS mightbe species. . 4 P.
Auxarthronopsis gen. sp. nov.
4(3) Peridial hyphae or appendages with knuckle joints ...... 5 ARTICLE Peridial hyphae or appendages without knuckle joints ...... 7
5(4) Peridial appendages without or up to 3 septa ...... Auxarthron Peridial appendages multi-septate ...... 6
6(5) Peridium with short pectinate appendages ...... Pectinotrichum Peridium lacking pectinate appendages, swollen septa on appendages ...... Auxarthronopsis
7(4) Peridial appendages straight, rarely branched ...... 8 Peridial appendages, if present, branched or uncinate ...... 9
8(7) Peridial appendages with acute apices, ascospores smooth ...... Acitheca Peridial appendages with blunt apices, ascospores ornamented ...... Nannizziopsis
9(7) Ascospores smooth ...... 10 Ascospores pitted, pitted-reticulate ...... Neogymnomyces
10(9) Peridial appendages if present, smooth, roughened (uncinate or boat-hook shaped apices) ...... Gymnoascus ...... [
11(3) Peridial appendages coiled ...... 12 Peridial appendages uncinate ...... Uncinocarpus
12(11) Ascospores hyaline ...... 13 Ascospores yellow to orange ...... 14
13 (12) Ascospores smooth ...... Histoplasma (syn. Ajellomyces) Ascospores irregularly ridged ...... Kuehniella
14(12) Ascospores ellipsoidal or oblate ...... 15 * ...... Apinisia
15(14) Peridial hyphae made up of ossiform cells ...... Arthroderma Peridial hyphae not made up of ossiform cells ...... 16
16(15) Peridial appendages thin walled, ascospores minutely roughened, pitted ...... Shanorella Peridial appendages thick walled ...... 17
17(16) Ascospores smooth ...... Spiromastix Ascospores regularly punctate-muricate ...... Polytolypa
ACKNOWLEDGEMENTS REFERENCES
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