ARTICLE 89

et al. et

1: 89–102 1: · no Key words: Knuckle-joints Molecular phylogeny Multiseptate appendages

Renispora e 4 e 4 lum s · vo gu Fun IMA . However, . However, Auxarthron and albicans,

contains a few genera that have The order contains a few genera that have analysis of one or more rDNA regions LSU (ITS, and SSU). analysis of one or more rDNA locus Unlike several other groups of fungi where the rDNA onygenalean resolve the phylogeny taxa, fungal fails to of gene phylogeny well was mostly resolved with rDNA comparisons et al. 1999, Sole et al. 2002c, Sugiyama (Gräser evolved An exception lay in some recently et al. 2002). little variation is asexual lineages,where region,and where microsatellites or ITS the observed even in other highly labile loci are necessary for species delimitation by molecular methods. 1988), Currah (sensu reticuloperidium the peridia, mesh-like bearing elongate appendages; these include Auxarthron, Pectinotrichum, and (Currah 1988). Castan- of Onygenales that forms is another edomyces in having a membranous elongate appendages, but differs The generic name peridium similar to that in . species for Auxarthron was established by Orr et al. (1963) lightly hyphae; peridial the in septa the at “enlargements with non-septate are which appendages elongate coloured, characteristic with septa basal three or two one, for except The generic name means ‘swollen swellings at such septa”. 13 species (Sole joints’, and the genus now comprises . 2002). The monophyly of Auxarthon 2002a, b, Sigler et al. 2002).

and the new generic name Auxarthronopsis isolated from the vicinity of vicinity the from isolated Onygenales 1 Auxarthron, . Further, the pale cream ascomata, punctate , the pale cream ascomata, punctate ascospores, Further, . A. volatilis-patellis, volatilis-patellis, A. , , and Sanjay K. Singh , and Sanjay 2 purpureus

, Yvonne Gräser , Yvonne 1 is an assemblage of fungi which has evolved An interesting onygenalean ascomycete was isolated from soil collected from a hollow tree near

, but differs morphologically. , but differs specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). must attribute the work in the manner You 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 004, India; corresponding Agarkar Road, Pune - 411 Agharkar Research Institute, G. G. National Facility for Culture Collection of Fungi, MACS’ and swollen septa in the peridial suggested hyphae a new species of Auxarthron that this was on the more than three swollen septa sequences, and presence of ITS SSU and LSU, phylogenetic of study peridial appendages, do not support a placement within Abstract: author e-mail: [email protected] The keratinophilic associated nature with a malbranchea- Bandhavgarh National in central India. Park situated like asexual morph, appendaged mesh-like reticuloperidia, oblate, punctate ascospores, and subglobose to support the inclusion of this isolate in Onygenaceae © 2013 International Mycological Association © 2013 International Mycological conditions: distribute and transmit the work, under the following are free to share - to copy, You Attribution: 2 1 Rahul Sharma , a new genus of genus new a , Auxarthronopsis Bandhavgarh National Park, India National Bandhavgarh 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. transform, or build upon this work. may not alter, You may not use this work for commercial purposes. You 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 . The distinguishing features of this new taxon are the multiple new taxon are the The distinguishing this features of accommodate this new fungus. is introduced to (≥10) swollen septa on the appendages attached to its reticulate, loosely mesh-like peridium, the finely and arthroconidial production of and aleurioconidial Sequence asexual forms. regularly punctate ascospores, and the analysis of ITS1-5.8S-ITS2, SSU and LSU regions clearly separate this fungus from monophyletic Auxarthronopsis bandhavgarhensis Phylogenetically, and other taxa bearing some morphological similarity. is closest to nov. gen. sp. flavissima was devoted to evolutionary and molecular phylogenetic was devoted to studies (Guarro et al. 2002). It included four new genera, 14 new species, and six new combinations based on sequence INTRODUCTION Onygenales to utilize forms part of the integument of birds, that This substrate, like cellulose, is reptiles, and mammals. large number of regularly shed by is it abundant in soil as integumental elements or as vertebrates either as effete Burrows actively ingested materials mixed into excreta. inhabited mammals are excellent by small habitats for and – hair 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 near Bandhavgarhwhich case a hollow tree, National Park 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). Centre,Utrecht, Biodiversity Fungal (CBS The Netherlands Agharkar ResearchInstitute, Pune, India,andatCBS-KNAW 932), Maharashtra Association for Cultivation ofSciences’ of India(NFCCI–WDCM Collection Culture Fungal National in the (AMH), withpureculturesdeposited Herbarium lactophenol mounts. dextrose 4g, agar 20gL of ourtaxon. of rDNAregions relationships the phylogenetic toestablish differentthree using studies molecular conducted data, we To determineifsuchachangewassupportedbysequence appendages. species withmorethanthreeseptateperidial to include was extended if theexistingcircumscription in Auxarthron, area suggestedthatitmightbeaccommodated That genuswasfoundtobepolyphyletic(Soleetal.2002a). L Agar (PDA; 200 gpotato(extract), dextrose 20g, agar 20g Carrot Mumbai) andPotatoDextrose Agar (PCA)(HiMedia, Cornmeal Mumbai), Potato Mumbai), Agar (CMA)(HiMedia, 1 mounted onto stubs and briefly dried under vacuum (15–20 (15–20 vacuum under dried briefly and stubs onto mounted analysis, naturally dried ascomata on baited hair were directly microscope withanattachedNikon8400camera.ForSEM of theascomatawasundertakenusingaNikonSMZ1500 Gottingen) research microscope. Stereomicroscopic study camera oraZeiss Axio Imager A-2 (CarlZeissMicroImaging, research microscope(Nikon, Tokyo) mountedwithaNikonHIII Photographs were taken either with a Nikon Eclipse E800 Light microscopywasperformedusinglactophenolmounts. (SEM) Light and ScanningElectronMicroscopy g L Dextrose 10 g,40gdextrose,agar20 Agar (SDA;peptone The growthrateat was determined 28°ConSabouraud and thedrop-trailmethod(Sharmaet microdilution al. 2002). (Vanbreuseghemtechnique 2007 bythehair-baiting 1952) present isolatewasobtainedfromthesamplesinFebruary temperature (range10°Cinwinterto>40summer). The polythene bagsweredirectlystoredinplasticboxesat room bags. Onreturnto the laboratory, the samplesalongwith polythene sealed sterile in spatulas sterile using subsurface were madefromthe 2001. Collections Pradesh, Indiaduring Park (23.5°N,80.25°E),Umariya,Madhya National ABandhavgarh fromaround soilsample(S214)wascollected Sample originandisolationoffungus MATMETHODS ERIALS AND hyphae, and,unlikeAuxarthron,itformsincompositoperidia Amauroascus, has ascomata composed of loosely woven sequence data. Another morphologicallysimilargenus, was demonstratedbySoleetal.(2002a)basedonITS and incomplete networkofhyphae. Aa loose ascomata having termusedbyCurrah (1985)forglobose -1 ). The structuresweremeasuredinwarmed morphological -1 The specimenisdepositedinthe Ajrekar Mycological Morphological study ofthematerialfromBandhavgarh Morphological ), dilute Sabouraud Dextrose ), diluteSabouraud 1 g, Agar (dSDA;peptone -1 ), Oatmeal Agar (OA) (HiMedia, Sharma, Gräser&Singh 1 . voltages. voltages. accelerating Tokyo) and magnification varying at microscopes visualized underJEOL 610andJEOL JSM6360A (JEOL, min), thencoatedwithgold-paladium/platinummixtureand µL Taq polymerase(5UµL (1 U µL each); 0.5µL primer(50pmolµL mixture contained28µL H et al. 1990). For the ITS region, 50 µL of the PCR reaction (Rehner &Samuels1994,Vilgalys &Hester1990,White LR7, LR7R,LR12(LSU),andNS1,NS4,NS3,NS8(SSU) pairs Mass266/V9D,ITS1/ITS4(ITS),5.8SR,LROR, using universal primers. PCR was performed with primer ITS1-5.8S-ITS2, 18S, and 28S rRNA genes were amplified glucose agar(DifcoLaboratories)for21dat28ºC. The 1999) after the fungus had been grown on Sabouraud N-trimethylammonium bromide) method (Gräser DNA wasextractedusingtheCTAB (N-cetyl-N,N, DNA extractionandPCRamplification (Submission ID12372). to submitted been have alignments sequence and TreeBase in GenBank(Table(NFCCI 2185)havebeendeposited 1) replicates. The ITS, LSU, and SSU sequences of our isolate of branches wasassessedby bootstrap analysiswith1000 with the ‘pairwise deletionof gaps option’. The robustness the Kimuratwo-parameterdistancemodel(Kimura1980) et al. 2011). The phylogenetic tree was constructedusing with theMEGAperformed v.(Tamura 5computerprogram (NJ) method(Saitou& Nei 1987)were Neighbour-Joining using the analyses format fromGenBank.Phylogenetic studies andex-typestrainswereretrievedinFASTAprevious used in of material sequences analysis, For phylogenetic 3100 Avant, Applied Biosystems,FosterCity, California). sequencer (Beckman-Coulter, or Fullerton, California ABI sequenced and using thesameprimers(Whiteet al. 1990) onanautomated CA) Scientific, (Axygen kit orAxygen Cleanup with PCR Hilden) cleaned (Qiagen, kit were purification PCR products QIAquick amplification resulting The Sequencing andphylogeneticanalysis 16 µL H 28S rRNA, 25 µL of the PCR reaction mixture contained and 18S For min. 10 for °C 72 extension final ×30; min) 1 °C for5min,(9515572 Branchburg, NY)withthefollowingreactionconditions:95 thermocycler (PerkinElmer, Roche Molecular Systems, Steinheim). PCRwasperformedinaPerkinElmer9600 was overlaidwithonedropoflightmineraloil(Sigma, dNTPs (250mMeach),1µL primer(50pmoleµL ng µL photographed underUV. on 1.2 % agarosegelstainedwith ethidium bromide and checked was product amplification The min. 10 for °C extension 72 final ×30; min) 1 °C 72 min, 1 for °C 65 or °C the followingconditions:95°Cfor5min,(95151 in anEppendorfMastercycler(Eppendorf,Hamburg)with oil (Genei, Bangalore). The PCR reaction was performed NJ), and5µL genomicDNA (10ngµL -1 ); andwasoverlaidwithonedropoflightmineral 2 -1 O; 2.5µL PCRbuffer (10x);1µL dNTPs(250mM ) (Genei, Bangalore); 2.5 µL genomic DNA (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 Classification Taxon ITS SSU LSU Arthroderma cajetani AY176736.1 A. ciferri EF413624.1 EF413625.1 A. curreyi AJ315165.1 A. otae AY176735.1 AJ877222.1 U29391.1 AY176733.1 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 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 A. filamentosum 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 A. zuffianum AJ271569 AY124492.1 AY176712.1 Auxarthronopsis bandhavgarhensis HQ164436 JQ048939 JQ048938 ceratinophila AJ315176.1 Chlamydosauromyces punctatus AY177297.1 Nannizziopsis albicans AJ271432 Neogymnomyces demonbreunii AJ315842.1 AY176716.1 corvina AB075364.1 O. equina U45442.1 Pectinotrichum llanense AJ390391.1 AJ315178.1 flavissima AF299348.1 AB015784.1 AY176719.1 Shanorella spirotricha AJ271430 AJ271419 L27991.1 AY176724.1 Byssochlamys nivea GU733368.1 AY176750.1 herbariorum GU733351.1 AY176751.1 Petromyces alliaceus AB002071.1 AY176752.1

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(5), andarthrodermataceous (4)fromprevious studies sequences of onygenaceous (16 taxa), gymnoascaceous several other onygenalean species. In addition, the nrLSU demonbreunii (ATCC 18394, AY176716.1), and92%with 219.58, AY176712.1), 93% with Neogymnomyces 32622, AY176707.1)and homology of 94 % with search with 689 bp of 5’endnrLSUshoweda maximum 301–A, 293–C,395–Gand258–T. A nucleotideBLAST of the5’endwithfollowingnucleotidecomposition: of NFCCI 2185 resultedin a 1247 bpnucleotidesequence JQ048939 (SSU).Sequencingofthepartial28SrDNA gene with accessionnosHQ164436(ITS),JQ048938(LSU), NFCCI 2185 and the sequences are deposited in GenBank partial 18Sand28SrRNA genes,weresequencedfor The complete ITS1, ITS2 and 5.8S regions along with Phylogeny survival insoil. (Fig. 1a,b). the species’This showed for long-term capacity with horsehair,on thehair ascomata developed abundant However,preparation. the soilwasre-baited after>6yrwhen in slide be madeasthematerialwasdestroyed could was noted(Sharma2002). At thattime,however, noisolation asingle when form plateanditsunusual ascoma formedinahair-baiting in 2001 observed first was it but collected, fungus wasisolatedsixto eight yearsafter the soil was ascomycete with characteristics found inOnygenales of an of soilsampleS214resultedinisolation Hair-baiting RESUL Anamorph Peridial appendages Peridial hyphae Asci Ascoma type Ascomata Character Table 2. A comparisonofmorphologicalcharactersthegenus TS Size Shape Colour Size Colour arthroconidia, keratinolytic cylindrical, aleurioconidia, lateral, pyriform,oblongor Terminal, intercalaryorrarely uncinate which arebentorslightlycoiled (1, 2or3septa)withsimpleapices acute, largerappendagesseptate Branches rare,tipsround,sub- tuberculate minutely tocoarselyasperulateor septa, thickcuticularizedsmoothto Reddish, rustytan,enlargedat <5 µm punctuate topunctate-reticulate spherical tooblate,minutely Hyaline, toyelloworyellow-brown 7.2–8 ×10.6µm 8-spored, evanescent, Subglobose, globoseorovoid, Reticuloperidium <700 µm Yellow-brown tobrown Auxarthron Amauroascus purpureus (IFO uatrn zuffianum Auxarthron 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 1.75–2.5 ×2.75–3.2µm view butoblateinequatorial regularly punctate,globoseinpolar globose tosubglobose,finelyand Hyaline 5 ×5.5µm evanescent, Globose, 8-spored,hyaline Reticuloperidium 500–1000 µm White topalecream Auxarthronopsis included (Fig. 5). The new fungus formed a separate clade that the analysedstrainsintodistinct familiesofOnygenales the analysis. The neighbour-joiningSSUtreeresolved of sequencesfrombothendswereagainexcluded also alignedwiththatofNFCCI2185.Incompleteportions gymnoascaceous, and three arthrodermataceous, 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); and 97 % with several other and 708.79, AB015784.1), 9990, AY177297.1),(UAMH homology of:98%withChlamydosauromyces with 1339bpofthe5’endnrSSUshowedamaximum 275–C, 360–G, and 347–T. A nucleotide BLAST search 5’end withthefollowingnucleotidecomposition:357–A, 2185 resultedina1339bpnucleotidesequenceofthe Sequencing ofthepartial18SrDNA geneofNFCCI clade alongwithAm.purpureusandvolatilis-patellis. 4). The new isolate was placed within the for membersofthethreefamiliesOnygenales(Fig. nucleotide substitutionmodel.Itshoweddistinctclades (NJ) treewasconstructedusingtheKimura–2parameter were excluded fromtheanalysis. A neighbour-joining 2185. Incompletepartsofthesequencesfrombothends were retrievedfromGenBankandalignedwithNFCCI and purpureus, Am. volatilis-patellis, Nannizziopsisalbicans, punctatus. Someotherrelatedspecies, suchasAm. Neogymnomyces demonbreuniicouldnotbeincluded Renispora flavissima and Renispora , 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 Incompositoperidium Up to2.5mm White, yelloworbrown Amauroascus Renispora Aphanoascus . uatrn zuffianum Auxarthron Chlamydosauromyces flavissima (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 bifurcated, multiseptate (≥10) with distinct swollen septa 2–2.5 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 light microscopy, finely and regularly punctate under SEM, 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 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 Phenotypically, the new taxon superficially resembles 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 , 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 the apex, sometimes branched, with multiple (≥10) swollen DISCUSSION septa. Asci globose to subglobose, hyaline, evanescent, 8– spored. Ascospores unicellular, hyaline, oblate, with finely 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 new taxon are pale, narrow, and flexible. Also, the ascospores the soil collection was made, Bandhavgarh National Park, of the new species are finely and regularly punctate (with India. circular punctae), a morphology that differs from the minutely volume 4 · no. 1 93 94 ARTICLE

Fig. 1. hyphae showing knuckle joints(arrows).Bars: A =600µm;B200 C=100µm;D80E6 µm;F=80GH 10µm. appendages. multiseptate peridial ascoma showing inverted showing appendage mass. E.Baseofelongate ascospore Y-shapedsepta (arrows).F. arch withswollen with central reticuloperidium up fromhairbait.D.Mesh-like picked appendages ascomata withelongate view ofunmounted microscopic Auxarthronopsis bandhavgarhensis bandhavgarhensis (AMH 9405) Dichotomously branched perdial branched (arrows). H.Dichotomously appendages ofperdial G. Bifurcatebranching Sharma, Gräser&Singh . A–B. Stereomicroscopic-view of mature ascoma growing on horsehair.of matureascomagrowing Stereomicroscopic-view Phase contrast image of Phasecontrastimage ima fUNGUS C. Light 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. Auxarthronopsis 2185). Bars: A =40µm;B 100µm;C=10D1E =2µm;F1G–H20µm. (NFCCI and arthroconidia sessile andstalkedaleurioconidia punctae. G,showing circularH.Slideculturepreparation showing ascospores portion ofseptawithringtubercles.E. septa. D.Enlarged with twoswollen appendage Asci andascospores. F. (arrows). B.radiating from onhorsehair.appendages Elongate reticuloperidia bandhavgarhensis (AMH9405) Sharma, Gräser&Singh Multiple septa onperidial morph. A.Multiple and asexual ascospores appendages, . Peridial C. Sparsely asperulate basalportionof perdial Finely and regularly punctate Finelyandregularly 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 volume 4 · no. 1 97 98 ARTICLE

and tree basedonnucleotide Fig. 5. Neighbour-joining Byssochlamys nivea Byssochlamys Bootstrap values above50%areshown. to distancevaluescalculated inMEGAbranch lengthsareproportional of 5andvaluesatnodesrepresentsbootstrap percentage1000replicates. Onygenaceae listed in Table 1 along with Auxarthronopsis (GU733368.1) , Eurotium herbariorum , Eurotium

sequences of 18SrDNAsequences geneofthe22stains ofArthrodermataceae Sharma, Gräser&Singh (GU733351.1),

bandhavgarhensis

and Petromyces alliaceus Petromyces NFCCI 2185 T . to Three strainsbelonging (AB002071.1) served as outgroup. served (AB002071.1) The , Gymnoascaceae Trichocomaceae 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. volume 4 · no. 1 99 100 ARTICLE

1 Key tothegeneraofOnygenaleswithperidialappendages Thetype ascospores. species of orbacilliform reniform pitted finely differentiatedforms and lack appendages, hyphae, which septa. with swollen reticuloperidium ascomata, withamesh-like inthedistinctnessof with thesegenera inconsistent Neogymnomyces. was placedclosetoAmauroascus, substitutions and64ofwhichareindels. differs at 166 of 623 nucleotidepositions,102of which are It ascospores whicharepunctate-reticulate.also producing of undifferentiatedand in hyphae lackingappendages, bandhavgarhensis Auxarthronopsis volatilis-patellis, related species,Amauroascus 74 substitutions and 54indels. Another phylogenetically two species differ at 128 of 627 nucleotidepositions,including (Guarro etthese reticuloperidium al.1991).Phylogenetically hyphae withoutappendages,andinlackinga well-developed ofundifferentiateda peridium taxon inhaving new peridial neighbour, (Ito & Nakagiri 1995). closest The otherphylogenetically ascospores frilled globose, forming and appendages, peridial ascomata thatlackknucklejointsand purple possessing Morphologically,indels). A.purpureus than 85%(119 bpoutof620bp;92substitutionsand27 the nearest neighbour, to bandhavgarhensis ofAuxarthronopsis The ITSsimilarity albicans, and Nannizziopsis from comparisons alsoshowedAuxarthronopsis from theAuxarthron Auxarthron showed trees also nrLSU and Our nrSSU latter polyphyletic. and the who foundtheformergenustobemonophyletic been studiedbySoleetal. (2002a) usingITS sequences, of Auxarthron interrelationships Auxarthronopsis 2(1) walled, verruculoseseptatehyphaealongwithtwokinds of globose to pulvinate ascomatawithpaleyellow, fairly thick virgineus to us.Neogymnomyces available ITS and LSUsequences(JN038187,JN038186)werenot data; however, it could not be includedinouranalysisas its Doveri , of Neogymnomyces species Recently,appendages. peridial with long,hyaline, anew thick-walled, smoothandseptateperidialhyphae,along ascomatawithhyaline, golden-yellow discrete, spherical 3(1)

In thenrLSUandnrSSUtrees(Figs5–6),Auxarthronopsis Amauroascus purpureus, Auxarthron, butclosertoAmauroascus Ascomata cleistoperidium type...... Ascomata cleistoperidium Ascomata madeofonlycleistoperidium orincompositoperidiumtype Ascomata reticuloperidium Peridial appendages straight with simple or curved apices, branchedorunbranched, uncinate,nevercoiled withsimpleorcurvedapices, Peridial appendagesstraight layer cleisto-type,outerlayerreticulo-type Ascomata madeofinner Peridial appendages coiled . Peridial appendagescoiled et al. (2011)and molecular basedonmorphological eipr flavissima has ascomataof poorly Renispora as monophyletic. The new taxon wasexcluded Nannizziopsis albicans Nannizziopsis , Neogymnomyces have more than10septa. have The phylogenetic However, thenewtaxonismorphologically clade(Figs4–5),andtheITS sequence Amauroascus purpureus, N. virgineus Amauroascus volatilis-patellis. and N. demondreunii ...... (Fig. 6),differs fromthe in formingaperidium is also distinct in isalso Amauroascus , was described by , wasdescribed formsirregularly Renispora, ...... as distinct differs from Sharma, Gräser&Singh was less , forms have and

...... Pectinotrichum llananse, Pectinotrichum well developed reticuloperidia, them (Varsavsky &Orr1971). between relationship of aclose evidence more provide would serratus, suggestedthat genetic studiesof these genera llanense (1971), whooriginallynotedthat the pectinatehyphaeofP. 5) andclusterswithCtenomycesserratus. Varsavsky &Orr out from the mainOnygenaceae that speciesisdistantfromAuxarthronopsis studies ofSugiyama molecular from Auxarthron but itsdistance (1994) transferredPectinotrichumllanense Currah almost smoothascospores. morph, and asexual a hyphae, peridial and on theappendages Auxarthronopsis long appendages andknucklejoints,but differs from llananse Pectinotrichum monotypic morph characterisedbyfrequentswollencells. asexual and inpossessinganarthro-aleurioconidial swollen septaintheperidialhyphaeandappendages, at thetips.Neogymnomyces rounded and verruculose, short, oftenbranched, other comparatively one long,taperingandsmooth,the appendages: peridial well ashyphalelements. Ctenomyces, the peridium of which includesmembranousas including is provided, of theorder genera appendaged all hyphal mesh-likeincompositoperidium. incomplete A keyto mesh-like reticuloperidium. They may also beformedonan hyphal, oracompletely cleistoperidium a membranous appendages. peridial have on either borne These areusually represents onesuchintermediategenus. forms. by asyetunrecordedPerhapsAuxarthronopsis bridged of Onygenales genera seen inseveral discontinuity peridial hyphae. though theyarefoundonthin,smoothorsparselyasperulate, in LMeven knuckle jointsrelativelyprominent 3c, d)making that theyhavearingoftuberclesvisibleunderSEM(Fig. different areslightly septa inAuxarthronopsis swollen in 2000). in nature(Summerbell mass frommitegrazing The protects thecentralascospore which peridium spherical Their purposemay be to provide strength to the mesh-like Knuckle-joints are a common feature in species with feature inspecies are acommon Knuckle-joints is the joints knuckle that possesses genus Another The orderOnygenales that themorphological suggested (1985) Currah resemble the ctenoid appendages of Ctenomyces resemblethectenoidappendages ...... Pectinotrichum inhavingpectinatehyphae,largetubercules and almost all and forms a reticuloperidium with formsareticuloperidium now comprises18generathat (Varsavsky &Orr 1971). (1999). Phylogenetically et al.(1999). clade inSSUanalysis(Fig. was substantiated by the wassubstantiated viz. alsodiffers inlacking Gymnoascus reesii, Auxarthron Castanedomyces toAuxarthron, asitseparates Ctenomyces ima fUNGUS ...... mightbe species. 11 2 3 4

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 ...... 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 Peridial appendages ornamented with numerous short fine hairs, dichotomously branched, with blunt apices ...... Bifidocarpus

11(3) Peridial appendages coiled ...... 12 Peridial appendages uncinate ...... Uncinocarpus

12(11) Ascospores hyaline ...... 13 Ascospores yellow to orange ...... 14

13 (12) Ascospores smooth ...... (syn. ) Ascospores irregularly ridged ......

14(12) Ascospores ellipsoidal or oblate ...... 15 Ascospores globose, finely echinulate ...... 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 ...... Ascospores regularly punctate-muricate ...... Polytolypa

ACKNOWLEDGEMENTS REFERENCES

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