Auxarthronopsis, a new genus of Onygenales isolated from the vicinity of Bandhavgarh National Park, India.

An interesting onygenalean ascomycete was isolated from soil collected from a hollow tree near Bandhavgarh National Park situated in central India. The keratinophilic nature associated with a malbranchea-like asexual morph, appendaged mesh-like reticuloperidia, and subglobose to oblate, punctate ascospores, support the inclusion of this isolate in Onygenaceae. Further, the pale cream ascomata, punctate ascospores, and swollen septa in the peridial hyphae suggested that this was a new species of Auxarthron. However, phylogenetic study of LSU, SSU and ITS sequences, and presence of more than three swollen septa on the peridial appendages, do not support a placement within Auxarthron, and the new generic name Auxarthronopsis is introduced to accommodate this new fungus. The distinguishing features of this new taxon are the multiple (≥10) swollen septa on the appendages attached to its reticulate, loosely mesh-like peridium, the finely and regularly punctate ascospores, and the production of arthroconidial and aleurioconidial asexual forms. Sequence analysis of ITS1-5.8S-ITS2, SSU and LSU regions clearly separate this fungus from monophyletic Auxarthron and other taxa bearing some morphological similarity. Phylogenetically, Auxarthronopsis bandhavgarhensis gen. sp. nov. is closest to Amauroascus purpureus, A. volatilis-patellis, Nannizziopsis albicans, and Renispora flavissima, but differs morphologically.

INTRODUCTION

Onygenales is an assemblage of fungi which has evolved to utilize keratin that forms part of the integument of birds, reptiles, and mammals. This substrate, like cellulose, is abundant in soil as it is regularly shed by large number of vertebrates either as effete integumental elements or as ingested materials mixed into excreta. Burrows actively inhabited by small mammals are excellent habitats for these fungi since they contain ample substrate, hair – and moisture – at all times of the year, and even in hot dry summers. 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 from different geographical regions of India, an interesting 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.

The most recent monographic treatment of Onygenales was devoted to evolutionary and molecular phylogenetic studies (Guarro ). It included four new genera, 14 new species, and six new combinations based on sequence analysis of one or more rDNA regions (ITS, LSU and SSU). Unlike several other groups of fungi where the rDNA locus fails to resolve the phylogeny of taxa, onygenalean fungal phylogeny was mostly well resolved with rDNA gene comparisons (Gräser , Sole , Sugiyama ). An exception lay in some recently evolved asexual dermatophyte lineages, where little variation is observed even in the ITS region, and where microsatellites or other highly labile loci are necessary for species delimitation by molecular methods.

The order Onygenales contains a few genera that have 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 Onygenales that forms elongate appendages, but differs in having a membranous peridium similar to that in Aphanoascus. The generic name Auxarthron was established by Orr 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 swellings at such septa”. The generic name means ‘swollen joints’, and the genus now comprises 13 species (Sole , b, Sigler ). The monophyly of Auxarthron was demonstrated by Sole based on ITS sequence data. Another morphologically similar genus, Amauroascus, has ascomata composed of loosely woven hyphae, and, unlike Auxarthron, it forms incompositoperidia. That genus was found to be polyphyletic (Sole ).

Morphological study of the material from the Bandhavgarh area suggested that it might be accommodated in Auxarthron, if the existing circumscription was extended to include species with more than three septate peridial appendages. To determine if such a change was supported by sequence data, we conducted molecular studies using three different regions of rDNA to establish the phylogenetic relationships of our taxon.

MATERIALS AND METHODS

Sample origin and isolation of fungus

A soil sample (S214) was collected from around Bandhavgarh National Park (23.5°N, 80.25°E), Umariya, Madhya Pradesh, India during 2001. Collections were made from the subsurface using sterile spatulas in sterile sealed polythene bags. On return to the laboratory, the samples along with the polythene bags were directly stored in plastic boxes at room temperature (range 10 °C in winter to >40 °C in summer). The present isolate was obtained from the samples in February 2007 by the hair-baiting technique (Vanbreuseghem 1952) and the microdilution drop-trail method (Sharma ). The growth rate was determined at 28 °C on Sabouraud Dextrose Agar (SDA; peptone 10 g, 40 g dextrose, agar 20 g L−1), dilute Sabouraud Dextrose Agar (dSDA; peptone 1 g, dextrose 4 g, agar 20 g L−1), Oatmeal Agar (OA) (HiMedia, Mumbai), Cornmeal Agar (CMA) (HiMedia, Mumbai), Potato Carrot Agar (PCA) (HiMedia, Mumbai) and Potato Dextrose Agar (PDA; 200 g potato (extract), dextrose 20 g, agar 20 g L−1). The morphological structures were measured in warmed lactophenol mounts.

The specimen is deposited in the Ajrekar Mycological Herbarium (AMH), with pure cultures deposited in the National Fungal Culture Collection of India (NFCCI–WDCM 932), Maharashtra Association for Cultivation of Sciences’ Agharkar Research Institute, Pune, India, and at CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands (CBS 134524).

Light and Scanning Electron Microscopy (SEM)

Light microscopy was performed using lactophenol mounts. Photographs were taken either with a Nikon Eclipse E800 research microscope (Nikon, Tokyo) mounted with a Nikon HIII camera or a Zeiss Axio Imager A-2 (Carl Zeiss MicroImaging, Gottingen) research microscope. Stereomicroscopic study of the ascomata was undertaken using a Nikon SMZ1500 microscope with an attached Nikon 8400 camera. For SEM analysis, naturally dried ascomata on baited hair were directly mounted onto stubs and briefly dried under vacuum (15–20 min), then coated with gold-paladium/platinum mixture and visualized under JEOL 610 and JEOL JSM 6360A (JEOL, Tokyo) microscopes at varying magnification and accelerating voltages.

DNA extraction and PCR amplification

DNA was extracted using the CTAB (N-cetyl-N, N, N-trimethylammonium bromide) method (Gräser ) after the fungus had been grown on Sabouraud glucose agar (Difco Laboratories) for 21 d at 28 °C. The ITS1-5.8S-ITS2, 18S, and 28S rRNA genes were amplified using universal primers. PCR was performed with primer pairs Mass 266/V9D, ITS1/ITS4 (ITS), 5.8SR, LROR, LR7, LR7R, LR12 (LSU), and NS1, NS4, NS3, NS8 (SSU) (Rehner & Samuels 1994, Vilgalys & Hester 1990, White ). For the ITS region, 50 μL of the PCR reaction mixture contained 28 μL H2O, 5 μL PCR buffer (10x), 4 μL dNTPs (250 mM each), 1 μL primer (50 pmole μL−1), 0.4 μL Taq polymerase (5 U μL−1) (Applied Biosystems Roche, NJ), and 5 μL genomic DNA (10 ng μL−1). The mixture was overlaid with one drop of light mineral oil (Sigma, Steinheim). PCR was performed in a Perkin Elmer 9600 thermocycler (PerkinElmer, Roche Molecular Systems, Branchburg, NY) with the following reaction conditions: 95 °C for 5 min, (95 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min) ×30; final extension 72 °C for 10 min. For 18S and 28S rRNA, 25 μL of the PCR reaction mixture contained 16 μL H2O; 2.5 μL PCR buffer (10x); 1 μL dNTPs (250 mM each); 0.5 μL primer (50 pmol μL−1); 1 μL Taq polymerase (1 U μL−1) (Genei, Bangalore); 2.5 μL genomic DNA (10 ng μL−1); and was overlaid with one drop of light mineral oil (Genei, Bangalore). The PCR reaction was performed in an Eppendorf Mastercycler (Eppendorf, Hamburg) with the following conditions: 95 °C for 5 min, (95 °C 1 min, 51 °C or 65 °C for 1 min, 72 °C 1 min) ×30; final extension 72 °C for 10 min. The amplification product was checked on 1.2 % agarose gel stained with ethidium bromide and photographed under UV.

Sequencing and phylogenetic analysis

The resulting amplification products were cleaned with QIAquick PCR purification kit (Qiagen, Hilden) or Axygen PCR Cleanup kit (Axygen Scientific, CA) and sequenced using the same primers (White ) on an automated sequencer (Beckman-Coulter, Fullerton, California or ABI 3100 Avant, Applied Biosystems, Foster City, California). For phylogenetic analysis, sequences of material used in previous studies and ex-type strains were retrieved in FASTA format from GenBank. Phylogenetic analyses using the Neighbour-Joining (NJ) method (Saitou & Nei 1987) were performed with the MEGA v. 5 computer program (Tamura ). The phylogenetic tree was constructed using the Kimura two-parameter distance model (Kimura 1980) with the ‘pairwise deletion of gaps option’. The robustness of branches was assessed by bootstrap analysis with 1000 replicates. The ITS, LSU, and SSU sequences of our isolate (NFCCI 2185) have been deposited in GenBank (Table 1) and sequence alignments have been submitted to TreeBase (Submission ID 12372).

Table 1.Fungal species and LSU, SSU and ITS nrDNA GenBank accession numbers used in the study.

Classification	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
	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
	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
	Renispora flavissima	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

RESULTS

Hair-baiting of soil sample S214 resulted in isolation of an ascomycete with characteristics found in Onygenales. The fungus was isolated six to eight years after the soil was collected, but it was first observed in 2001 when a single ascoma formed in a hair-baiting plate and its unusual form was noted (Sharma 2002). At that time, however, no isolation could be made as the material was destroyed in slide preparation. However, after >6 yr when the soil was re-baited with horse hair, abundant ascomata developed on the hair (Fig. 1a, b). This showed the species’ capacity for long-term survival in soil.

Fig. 1.

Auxarthronopsis bandhavgarhensis (AMH 9405). A–B. Stereomicroscopic-view of mature ascoma growing on horse hair. C. Light microscopic view of unmounted ascomata with elongate appendages picked up from hair bait. D. Mesh-like reticuloperidium with central ascospore mass. E. Base of elongate appendage showing inverted Y-shaped arch with swollen septa (arrows). F. Phase contrast image of ascoma showing multiseptate peridial appendages. G. Bifurcate branching of perdial appendages (arrows). H. Dichotomously branched perdial hyphae showing knuckle joints (arrows). Bars: A = 600 μm; B = 200 μm; C = 100 μm; D = 80 μm; E = 6 μm; F = 80 μm; G = 80 μm; H = 10 μm.

Phylogeny

The complete ITS1, ITS2 and 5.8S regions along with partial 18S and 28S rRNA genes, were sequenced for NFCCI 2185 and the sequences are deposited in GenBank with accession nos HQ164436 (ITS), JQ048938 (LSU), JQ048939 (SSU). Sequencing of the partial 28S rDNA gene of NFCCI 2185 resulted in a 1247 bp nucleotide sequence of the 5’end with the following nucleotide composition: 301–A, 293–C, 395–G and 258–T. A nucleotide BLAST search with 689 bp of 5’end of nrLSU showed a maximum homology of 94 % with Amauroascus purpureus (IFO 32622, AY176707.1) and Auxarthron zuffianum (CBS 219.58, AY176712.1), 93 % with Neogymnomyces demonbreunii (ATCC 18394, AY176716.1), and 92 % with several other onygenalean species. In addition, the nrLSU sequences of onygenaceous (16 taxa), gymnoascaceous (5), and arthrodermataceous (4) from previous studies were retrieved from GenBank and aligned with NFCCI 2185. Incomplete parts of the sequences from both ends were excluded from the analysis. A neighbour-joining (NJ) tree was constructed using the Kimura–2 parameter nucleotide substitution model. It showed distinct clades for members of the three families of Onygenales (Fig. 4). The new isolate was placed within the Onygenaceae clade along with Am. purpureus and Am. volatilis-patellis. Sequencing of the partial 18S rDNA gene of NFCCI 2185 resulted in a 1339 bp nucleotide sequence of the 5’end with the following nucleotide composition: 357–A, 275–C, 360–G, and 347–T. A nucleotide BLAST search with 1339 bp of the 5’end of nrSSU showed a maximum homology of: 98 % with Chlamydosauromyces punctatus (UAMH 9990, AY177297.1), Renispora flavissima (CBS 708.79, AB015784.1), and Aphanoascus mephitalis (CBS 453.75, AB015779.1); and 97 % with several other onygenalean species, including Auxarthron zuffianum (UAMH 4098, AY124492.1) and A. alboluteum (UAMH 2846, AY124494.1). The nrSSU sequences from previous studies, including 17 onygenaceous, three gymnoascaceous, and three arthrodermataceous, were also aligned with that of NFCCI 2185. Incomplete portions of sequences from both ends were again excluded from the analysis. The neighbour-joining SSU tree resolved the analysed strains into distinct families of Onygenales (Fig. 5). The new fungus formed a separate clade that included Renispora flavissima and Chlamydosauromyces punctatus. Some other related species, such as Am. purpureus, Am. volatilis-patellis, Nannizziopsis albicans, and Neogymnomyces demonbreunii could not be included in the analysis because SSU sequences were not available in GenBank. The strong LSU and SSU sequence similarity of NFCCI 2185 to other members of Onygenales suggests membership in this order.

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.

Fig. 5.

Neighbour-joining tree based on nucleotide sequences of 18S rDNA gene of the 22 stains of Arthrodermataceae, Gymnoascaceae and Onygenaceae listed in Table 1 along with Auxarthronopsis bandhavgarhensis NFCCI 2185T. Three strains belonging to Trichocomaceae Byssochlamys nivea (GU733368.1), Eurotium herbariorum (GU733351.1), and Petromyces alliaceus (AB002071.1) 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.

Sequencing of the ITS region of NFCCI 2185 yielded a 613 bp long nucleotide sequence that included 242 bp of ITS1, 150 bp of 5.8S and 166 bp of ITS2. In contrast to the LSU and SSU, the ITS sequence of the undescribed fungus was distant from other species it was compared with. It showed maximal sequence similarities of only 84 % with two quite distinct species: Amauroascus purpureus (IFO 32622, AJ271564) and Nannizziopsis albicans (IMI 155645, AJ271432), with 90 % and 88 % query coverage, respectively. The next most closely associated sequences in the BLAST results had similar similarities of 85 %, but show very low query coverage of less than 75 %. These sequences included a mixture of Eurotiales and Onygenales. The ITS sequence of NFCCI 2185 was also compared with sequences of 29 species from ten genera of related Onygenaceae (Table 1) and a phylogenetic tree (NJ) was constructed using Kimura-2 parameter model (Fig. 6).

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.

Phenotypically, the new taxon superficially resembles Auxarthron in having peridial hyphae with swollen septa, but it is distinct genetically. This is evident in the LSU, SSU, and ITS trees where Auxarthron forms a monophyletic clade (Figs 4–6). A morphological comparison of the newly designated Auxarthronopsis with Auxarthron and Amauroascus is shown in Table 2.

Table 2.A comparison of morphological characters of the genus Auxarthron, Auxarthronopsis, and Amauroascus.

Character		Auxarthron	Auxarthronopsis	Amauroascus
Ascomata	Colour	Yellow-brown to brown	White to pale cream	White, yellow or brown
	Size	<700 μm	500–1000 μm	Up to 2.5 mm
Ascoma type		Reticuloperidium	Reticuloperidium	Incompositoperidium
Asci		Subglobose, globose or ovoid, 8-spored, evanescent,	Globose, 8-spored, hyaline evanescent,	Ovoid, 8-spored, up to 14 μm
		7.2–8 × 10.6 μm	5 × 5.5 μm
Ascospore	Colour	Hyaline, to yellow or yellow-brown	Hyaline	Hyaline, yellow or reddish brown to dark brown
	Shape	spherical to oblate, minutely punctuate to punctate-reticulate	globose to subglobose, finely and regularly punctate, globose in polar view but oblate in equatorial view	spherical with pronounced irregular or regular puncta and ridges
	Size	<5 μm	1.75–2.5 × 2.75–3.2 μm	3.5–7 μm
Peridial hyphae		Reddish, rusty tan, enlarged at septa, thick cuticularized smooth to minutely to coarsely asperulate or tuberculate	Pale brown, branched and anastomosed form reticulate network, with swollen septa, sparsely asperulate	Usually undifferentiated in culture, occasionally pale brown, smooth & thick walled
Peridial appendages		Branches rare, tips round, sub-acute, larger appendages septate (1, 2 or 3 septa) with simple apices which are bent or slightly coiled or uncinate	Multiseptate with distinct swollen septa, tapering to acute hyaline apex indistinguishable from vegetative hyphae, upto 400 μm	Appendages lacking
Anamorph		Terminal, intercalary or rarely lateral, pyriform, oblong or cylindrical, aleurioconidia, arthroconidia, keratinolytic	Terminal or intercalary rhexolytically dehiscing arthroconidia and aleurioconidia, slightly keratinolytic	Terminal and/or intercalary rhexolytically dehiscing conidia, keratinolytic

TAXONOMY

Auxarthronopsis Rahul Sharma, Y. Gräser & S.K. Singh, gen. nov.

MycoBank MB563744

Etymology: Named after its phenotypic similarity to the genus Auxarthron.

Description: Ascomata solitary, globose to subglobose, 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 septa. Asci globose to subglobose, hyaline, evanescent, 8–spored. Ascospores unicellular, hyaline, oblate, with finely and regularly punctate walls. Asexual morph terminal and intercalary hyaline arthroconidia and aleurioconidia.

Type: Auxarthronopsis bandhavgarhensis Rahul Sharma et al. (2013).

Auxarthronopsis bandhavgarhensis Rahul Sharma, Y. Gräser & S. K. Singh, sp. nov.

MycoBank MB563745

GenBank HQ164436, JQ048938, JQ048939

(Figs 1–3)

Fig. 3.

Auxarthronopsis bandhavgarhensis (AMH 9405). Peridial appendages, ascospores and asexual morph. A. Multiple septa on peridial appendages (arrows). B. Elongate appendages radiating from reticuloperidia on horse hair. C. Sparsely asperulate basal portion of perdial appendage with two swollen septa. D. Enlarged portion of septa with ring of tubercles. E. Asci and ascospores. F. Finely and regularly punctate ascospores showing circular punctae. G, H. Slide culture preparation showing sessile and stalked aleurioconidia and arthroconidia (NFCCI 2185). Bars: A = 40 μm; B = 100 μm; C = 10 μm; D = 1 μm; E = 2 μm; F = 1 μm; G–H = 20 μm.

Etymology: bandhavgarhensis- referring to the locality where the soil collection was made, Bandhavgarh National Park, India.

Description: Ascomata discrete, globose, white to pale cream, reticuloperidium 500–1000 μm diam, with appendages. Peridial hyphae branched and anastomosed to form reticulate network with knuckle-joints 2.0–2.5 μm wide. Peridial appendages pale, numerous, elongated, straight or bifurcated, multiseptate (≥10) with distinct swollen septa 2–2.5 μm wide, tapering to acute hyaline apex indistinguishable from vegetative hyphae, up to 400 μm long. Asci globose, eight-spored, hyaline, evanescent 5 × 5.5 μm. Ascospores unicellular, hyaline, globose to subglobose, smooth under light microscopy, finely and regularly punctate under SEM, globose in polar view but oblate in equatorial view, 1.5–2.5 × 2.5–3.0 μm.

Asexual morph: Conidia malbranchea-like, arthric, intercalary or terminal on short branches, abundant, hyaline, solitary, aseptate, 2.0–4.5 × 4.5–11.5 μm, separated by autolytic connective cells.

Cultures: Colonies after 3 wk at 28 °C (Figs 2a, b) on SDA, white, slow growing (3 cm), cottony, slightly raised at the centre; reverse uncoloured to pale brown; on CMA, OA, and dilute SDA, slow growing (3 cm diam), mostly submerged with sparse mycelium, pale brown, reverse uncoloured to pale brown; on PCA and PDA slow growing (2 cm diam), pale coloured at the centre and white in the peripheral region, slightly cottony, and the reverse pale brown. Ascomata produced sparsely on PCA and abundantly on PDA (Figs 2c, d), but without elongate appendages.

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.

Type: India: Madhya Pradesh: Umariya, buffer zone of Bandhavgarh National Park, ascomata growing on horse hair (keratin bait) in soil S214 collected from inside a big hollow tree, 16 June 2001, R. Sharma (AMH 9405 – holotype; NFCCI 2185 = CBS 134524 – cultures ex-type).

Substratum: Isolated on horse hair from soil.

Distribution: Known only from the type locality, Bandhavgarh, India.

DISCUSSION

The typical mesh-like peridium of Auxarthronopis bandhavgarhensis and other features, including the ability to grow on horse hair (keratinophilic nature), make this fungus a relatively typical member of Onygenales (Currah 1985). However, it could not be placed in the morphologically similar genus Auxarthron, which never forms multi-septate appendages. The new genus Auxarthronopsis shows a resemblance to Auxarthron in its mesh-like reticulo-peridium and in its possession of knuckle joints; however, the ascomata of Auxarthron are mostly yellow to brown at maturity, while the ascomata of the new taxon are white to pale cream. The peridial hyphae of Auxarthron species are dark coloured, rigid, and relatively broad, while those of the new taxon are pale, narrow, and flexible. Also, the ascospores of the new species are finely and regularly punctate (with circular punctae), a morphology that differs from the minutely asperulate to punctate-reticulate ascospores of Auxarthron. Ascospores of A. pseudoauxarthron possess similar circular pits on the ascospore walls, but in that species the spores are spherical and the ascomatal appendages lack knuckle joints. In general, species of Auxarthron do not possess more than three septa in the peridial appendages, whereas those of Auxarthronopsis have more than 10 septa. The phylogenetic interrelationships of Auxarthron and Amauroascus have been studied by Sole using ITS sequences, who found the former genus to be monophyletic and the latter polyphyletic. Our nrSSU and nrLSU trees also showed Auxarthron as monophyletic. The new taxon was excluded from the Auxarthron clade (Figs 4– 5), and the ITS sequence comparisons also showed Auxarthronopsis as distinct from Auxarthron, but closer to Amauroascus purpureus, Nannizziopsis albicans, and Amauroascus volatilis-patellis. The ITS similarity of Auxarthronopsis bandhavgarhensis to the nearest neighbour, Amauroascus purpureus, was less than 85 % (119 bp out of 620 bp; 92 substitutions and 27 indels). Morphologically, A. purpureus is also distinct in possessing purple ascomata that lack knuckle joints and peridial appendages, and forming globose, frilled ascospores (Ito & Nakagiri 1995). The other phylogenetically closest neighbour, Nannizziopsis albicans (Fig. 6), differs from the new taxon in having a peridium of undifferentiated peridial hyphae without appendages, and in lacking a well-developed reticuloperidium (Guarro ). Phylogenetically these two species differ at 128 of 627 nucleotide positions, including 74 substitutions and 54 indels. Another phylogenetically related species, Amauroascus volatilis-patellis, differs from Auxarthronopsis bandhavgarhensis in forming a peridium of undifferentiated hyphae lacking appendages, and in producing ascospores which are punctate-reticulate. It also differs at 166 of 623 nucleotide positions, 102 of which are substitutions and 64 of which are indels.

In the nrLSU and nrSSU trees (Figs 5–6), Auxarthronopsis was placed close to Amauroascus, Renispora, and Neogymnomyces. However, the new taxon is morphologically inconsistent with these genera in the distinctness of the ascomata, with a mesh-like reticuloperidium with swollen septa. Renispora flavissima has ascomata of poorly differentiated hyphae, which lack appendages, and forms finely pitted reniform or bacilliform ascospores. The type species of Neogymnomyces, N. demondreunii, forms discrete, spherical golden-yellow ascomata with hyaline, thick-walled, smooth and septate peridial hyphae, along with long, hyaline, peridial appendages. Recently, a new species of Neogymnomyces, N. virgineus, was described by Doveri based on morphological and molecular data; however, it could not be included in our analysis as its ITS and LSU sequences (JN038187, JN038186) were not available to us. Neogymnomyces virgineus forms irregularly globose to pulvinate ascomata with pale yellow, fairly thick walled, verruculose septate hyphae along with two kinds of peridial appendages: one long, tapering and smooth, and the other comparatively short, often branched, verruculose, and rounded at the tips. Neogymnomyces also differs in lacking swollen septa in the peridial hyphae and the appendages, and in possessing an arthro- and aleurioconidial asexual morph characterised by frequent swollen cells.

Another genus that possesses knuckle joints is the monotypic Pectinotrichum (Varsavsky & Orr 1971). Pectinotrichum llananse forms a reticuloperidium with long appendages and knuckle joints, but differs from Auxarthronopsis in having pectinate hyphae, large tubercules on the appendages and peridial hyphae, a Chrysosporium asexual morph, and almost smooth ascospores. Currah (1994) transferred Pectinotrichum llanense to Auxarthron, but its distance from Auxarthron was substantiated by the molecular studies of Sugiyama . Phylogenetically that species is distant from Auxarthronopsis as it separates out from the main Onygenaceae clade in SSU analysis (Fig. 5) and clusters with Ctenomyces serratus. Varsavsky & Orr (1971), who originally noted that the pectinate hyphae of P. llanense resemble the ctenoid appendages of Ctenomyces serratus, suggested that genetic studies of these genera would provide more evidence of a close relationship between them (Varsavsky & Orr 1971).

Knuckle-joints are a common feature in species with well developed reticuloperidia, viz. Gymnoascus reesii, Pectinotrichum llananse, and almost all Auxarthron species. Their purpose may be to provide strength to the mesh-like spherical peridium which protects the central ascospore mass from mite grazing in nature (Summerbell 2000). The swollen septa in Auxarthronopsis are slightly different in that they have a ring of tubercles visible under SEM (Fig. 3c, d) making knuckle joints relatively prominent in LM even though they are found on thin, smooth or sparsely asperulate, peridial hyphae.

Currah (1985) suggested that the morphological discontinuity seen in several genera of Onygenales might be bridged by as yet unrecorded forms. Perhaps Auxarthronopsis represents one such intermediate genus.

The order Onygenales now comprises 18 genera that have peridial appendages. These are usually borne on either a membranous cleistoperidium or a completely hyphal, mesh-like reticuloperidium. They may also be formed on an incomplete hyphal mesh-like incompositoperidium. A key to all appendaged genera of the order is provided, including Ctenomyces, the peridium of which includes membranous as well as hyphal elements.

1            Ascomata cleistoperidium type ....................................................................................................................................... 2

Ascomata reticuloperidium or incompositoperidium type .................................................................................................. 3

2(1)        Ascomata made of only cleistoperidium ................................................................................................ Castanedomyces

Ascomata made of inner layer cleisto-type, outer layer reticulo-type ............................................................. Ctenomyces

3(1)        Peridial appendages straight with simple or curved apices, branched or unbranched, uncinate, never coiled ................ 4

Peridial appendages coiled ............................................................................................................................................ 11

4(3)        Peridial hyphae or appendages with knuckle joints ......................................................................................................... 5

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

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 ...................................................................................................... Histoplasma (syn. Ajellomyces)

Ascospores irregularly ridged ........................................................................................................................... Kuehniella

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 ....................................................................................................................................... Spiromastix

Ascospores regularly punctate-muricate ........................................................................................................... Polytolypa