ARTICLE 177 > et al. 2009) and Key words: N'" #'" echinulate ascospores Gymnoascaceae ITS Onygenales phylogeny IMA FUNGUS · VOLUME 4 · NO 2: 177–186 2: · NO 4 · VOLUME FUNGUS IMA . The generic concept and reveal its phylogenetic and reveal its phylogenetic Microscopy For microscopic observation and photomicrography, lactophenol mounts were prepared and observed under a Zeiss ;6 ; N 4 ;6 / | prism. For scanning electron microscopy, NJJ a Nikon-Y-IDT 6 and sputter coated with platinum. Photomicrographs were ¤+?¤"4 *J; #J D extraction, PCR and sequencing DNA ` |; 6 N? ? by homogenization in a FastPrep 24 tissue grown on PDA when collected and was stored at room temperature when collected and was stored at 1952) was until processed. Hair baiting (Vanbreuseghem > at room temperature after 1–2 month incubation in the dark N' / et al. 2002) in the microdilution drop trail method (Sharma in an Eppendorf needle and crushed in sterile distilled water of the ascospore suspension (in small tube. Fifty to 100 mL which was plate, drops) was placed on one side of a PDA To trail of the drop. tilted to spread ascospores with the on oatmeal agar initiate sporulation, the fungus was grown ; ~/; 6 ~; " 6 ";> / N' / and G. boliviensis , ) was ., isolated from soil from Kalyan railway station, ., isolated from soil from G. petalosporus et al. 2002). sp. nov as the type . In the present ) into Gymnoascus G. reessii Arachniotus, Gymnascella Onygenales due to the recent with verruculose ascospores with verruculose Gymnoascus Gymnoascus, especially et al. 2002) and has now become (Gymnoascaceae, Onygenales
, and Narasimhella is consequently now broadened to include species with verruculose ascospores. A key to the A ascospores. to include species with verruculose is consequently now broadened Gymnoascus Gymnoascus verrucosus new species, Gymnoascus verrucosus A Gymnoascus forms yellowish to brownish spherical
These species formed a monophyletic clade in previous Gymnoascus 4 @ and its deer antler-shaped short peridial (ornamentation visible only under SEM) verruculose ascospores 6 made up open mesh-like gymnothecial ascomata small peridial appendages originate from The appendages. the fungus is not formed in culture The characteristic morphology of peridial hyphae. of thick-walled, smooth gene rDNA analysis of different growth and forms arthroconidia. Phylogenetic where it has very restricted Gymnoascaceae and SSU) demonstrates its placement in sequences (ITS, LSU, Article info: " I NN ; NJ#N> ; I #K NJ#*> ~ I NK NJ#* accepted 19 species is also provided. relatedness to other species of relatedness to other species of Rahul Sharma, and Sanjay Kumar Singh and Sanjay Kumar Rahul Sharma, | < / / < 4;/"R ; ` ` ; ~ ? P## JJP Abstract: A new species of new A ? I based on morphological and molecular data. With this new based on morphological and molecular primarily on the basis of ascospore morphology. #' ITS sequence-based phylogenetic studies (Solé 6 |genus has or lumpy. ascospores are smooth, irregular, treatments (Orr et been the subject of three monographic al #$* ; 6 #$' " species. one of the largest genera of genera. Solé et al. transfer of species from several other (2002) merged four genera (viz. I#J KK$'L NJ#* JP JN J* Collection and isolation soil sample was collected from Kalyan railway station, A 4 #$ #PR| The soil sample was dry a sterile sealed polythene bag. * #JR MATERIAL AND METHODS MATERIAL INTRODUCTION The genus © 2013 International Mycological Association © 2013 International Mycological conditions: distribute and transmit the work, under the following are free to share - to copy, You Attribution: 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. VOLUME 4 · NO. 2 Sharma & Singh
G 4~ x ` boathook-shaped appendages. The asci were globose to 6 ; et al. (1997). The PCR '? @ " N'" |; @"PL@"K @" K '" K '"L+ +L+ |
ARTICLE Hester 1990, White et al. 1990, Rehner & Samuels 1994). identity of 93 % with G. petalosporus ;@// *P*K#> ; ~/ 6 I # 3+ ~/ ; #P' # $J G. reessii /x" NK$ #> <¤*K'N'P # " N K 3+ ~/ #J > # 3+ |@~ NKJ 4 '$ G. aurantiacus ;@// NN*$P> ; #P # > J K 3+ KJ 3+-1 > # 3+ # _ 3 -1) G. littoralis /x" PKP *> <¤*K'NN # @ ` x N K 3+ #J Onygenaceae (Onygenales) members. For some of the NCBI ng μl-1) of template DNA and was overlaid with one drop of accessions, current names (Solé et al. 2002) are used here ` ~/ ` x @ 4 / @ nrLSU of nine Gymnoascaceae and four Arthrodermataceae $K / K 6 " et al. 2002) were retrieved *J $K / # ` x | ; #N$K # G. hyalinosporus /x" KP' N> instructions on an ABI 3100 Avant Prism automated DNA AB015775.1), G. petalosporus _;4 #K> _N$*$N # G. sequencer (Applied Biosystems). littoralis /x" PKP *> <¤*K'*PJ # G. reessii /x" NK$ #> <¤*K'*P$ # G. ruber ; #N$ # $ Phylogenetic analysis other species of Gymnoascaceae, Onygenaceae, and DNA sequences obtained from the sequencer were manually Arachnomycetaceae @ ""_ 6 Gymnoascaceae, / + N J# IL Onygenaceae, and three Arthrodermataceae 6 www.technelysium.com.au). The sequences of the ITS, | ;<#N$'K* #> $K G. ` x " et al. (2002), Sugiyama et al. petalosporus |+ JJ#> 4$$#NJ #> (2002) and Doveri et al. (2011). Alignment was performed $' 'K G. reessii <4 PP#$> ;x*#P* #> /+_"@;+ ILL L L $' 'P Gymnoascus 4`; K @ et al. 2011). species. The ITS sequence of G. verrucosus differs from @ @ x;" ILL its closest neighbour G. petalosporus $' K' L L L L L @xNI"#*K | ? PJ KP' |¤ " ! | #$' differs from G. boliviensis #J# K aligned sequences using Kimura-2 parameter distance model PK KKK @ 6 #$'J neighbour, G. hyalinosporus, $* N The robustness of the branches was assessed by bootstrap *# KK @ < #$'K #JJJ x |¤ remaining Gymnoascus species were even more distant 4 6 + 4+ 4 6 ~ 4~ from G. verrucosus. analyses was also conducted with aligned sequences.
TAXONOMY RESULTS Gymnoascus verrucosus Rahul Sharma & S.K. The fungus studied formed minute gymnothecia in soil near Singh, sp.nov. ? @ 4 x 4x'JJJJ# of Gymnoascus reessii in having thick-walled, branched, < PM and septate peridial hyphae but without the characteristic
178 IMA FUNGUS A new species of Gymnoascus with verruculose ascospores ARTICLE
Fig. 1. Neighbour-joining tree of the LSU sequence of Gymnoascus verrucosus and 17 other species belonging to the families Gymnoascaceae, Arthrodermataceae, and Trichocomaceae @ 4`; K J Bootstrap values above 50 % are shown.
Etymology: The species epithet recalls the warted ascospores. Peridial appendages deer antler-shaped, smooth, yellow- 6 #JM Diagnosis: Deer antler shaped smooth, tapered peridial 15 μm long, 1.0–1.5 μm wide. Asci formed at the centre of appendages and warty globose ascospores differentiate the the ascoma, hyaline to pale yellow, globose to subglobose, new species from the rest in the genus. KM 3 '? Ascospores pale yellowish, globose and 3 μm diam to subglobose and 3.0 × 2.5 μm, walls smooth Type: India: MaharashtraI 6 under LM but minutely tuberculate under SEM. 9 Mar. 2009, Rahul Sharma ;4 $PKP ?? > | Distribution: Currently known only from the type locality, rounded ends. When the hyphae transform into arthroconidia, Kalyan, Maharashtra, India. the culture does not remain so rigid or hard and the inocula can be easily lifted with an inoculating needle. Ascomata formed only in soil near hair baits, not known in culture, DISCUSSION spherical, 50–90 μm diam (including appendages), yellowish to orange-brown, reticulum type open. Peridial hyphae deep The yellow to orange-brown gymnothecium (Fig. 4C–D and 5a) yellow to brown, smooth, septate, thick-walled, 1.5–2.0 μm is made up of a reticulum of thick-walled hyphae, which shows wide, originating radially from the centre of the ascoma. Gymnoascus verrucosus to be a typical member of Onygenales
VOLUME 4 · NO. 2 179 Sharma & Singh ARTICLE
Fig. 2. Neighbour-joining tree of the SSU sequence of Gymnoascus verrucosus and 17 other species belonging to families Gymnoascaceae, Arthrodermataceae, and Onygenaceae @ 4`; K J Bootstrap values above 50 % are shown.
/ #$'K Gymnoascaceae. However, due of G. reessii in having a thick-walled reticulum, albeit an to the minute echinulations or verruculose structures on the open one. The thick-walled, septate, peridial hyphae seen Gymnoascus, in G. verrucosus are also found in G. petalosporus, but the whose species have smooth, irregular, or lumpy ascospores. latter species has a loose mesh of such hyphae (not a true The ascospores of G. verrucosus reticuloperidium), which are branched at right angles, while of Mallochia whose species possess globose, echinulate those of the former do not. Also, G. verrucosus possesses ascospores. Mallochia echinulata forms ascospores with blunt small, pointed appendages along with a completely different spines apart from coarse echinulations. The other species of type of ascospores, unlike G. petalosporus. Two other genera the genus, M. endodonta, possess two-walled ascospores of Onygenales, Auxarthon and Acitheca (Onygenaceae) unlike G. verrucosus. Also, Mallochia forms a telaperidium possess pointed appendages, but unlike those of G. (ascomata made of an envelope of thin-walled hyphae not verrucosus, they are elongate with or without septation. Also, differentiated from vegetative hyphae) while G. verrucosus Auxarthron forms an inverted Y-shaped arch at the base of forms a reticuloperidium (ascomata mesh-like made of the elongate appendage(s), unlike G. verrucosus whose richly branched and more or less anastomosed, thick walled appendages are short and branched like deer antlers. Both hyphae, mostly with thick-walled appendages). However, the genera Acitheca and Auxarthron, however, have a different molecular studies using various rDNA regions suggest that type of ascospore morphology and never have the verruculose our fungus belongs in the monophyletic genus Gymnoascus. type of sculpturing seen in G. verrucosus. The other species The inclusion of this species in the genus now means that of the clade, G. boliviensis ` et al. 1992), differs from the generic circumscription needs to be amended to include G. verrucosus in having smooth, discoid ascospores with species with verruculose ascospores. Phylogenetic analysis of an equatorial rim, which are arranged within the ascus in a the ITS region of the rDNA places this new species in subclade petaloid manner. A depression is seen on one side of some Ia of Gymnoascus, comprising G. petalosporus, G. boliviensis, of the ascospores of G. verrucosus "4 < K/ > G. reessii and G. uncinatus (Fig. 3). Morphologically, the this may be an artefact due to collapse of the ascospore wall gymnothecium of G. verrucosus "4 >
180 IMA FUNGUS A new species of Gymnoascus with verruculose ascospores ARTICLE
Fig. 3. Neighbour-joining tree of ITS sequences showing the phylogenetic position of Gymnoascus verrucosus within the genus Gymnoascus.
ascospores are seen in Fig. 5D (without a depression). verruculose ascospore surfaces, and G. ruber with bivalvate The generic name Kraurogymnocarpa was introduced ascospores. The SSU and LSU results, therefore, do not by Udagawa & Uchiyama (1999) with the new species K. support the recognition of K. trochleospora as distinct from lenticulospora as type. The new genus was distinguished Gymnoascus. from Gymnoascus on the basis of distinct bivalvate Species delimitation in the broad generic concept of 6 Gymnoascus (sensu Solé et al. 2002) is primarily based on lack of enlargements or swellings at the septa on peridial shape, size, and sculpturing of ascospores. The verruculose appendages. A second species, K. trochleospora (syn. ascospores (with small tubercules over the entire surface) Pseudoarachniotus trochleosporus) was added to the genus of G. verrucosus make it distinct from all currently known based on the similar, but not identical, ascospores. Only one species of the genus. Only Gymnoascus udagawae forms species of Gymnoascus forms bivalvate ascospores, G. ruber, ascospores that seem to have warts, which are sporadic whose ascospores are smooth, unlike Kraurogymnocarpa. but do not resemble the prominent verruculose pattern of G. The original authors did not perform a molecular analysis, verrucosus. but stated in their paper that such studies may supply more The treatment of the new isolate as a new species of evidence on their relationships (Udagawa & Uchiyama Gymnoascus is supported by the sequence analysis of the 2001). Sigler et al. (2002) sequenced the SSU region of ITS, LSU and SSU regions of rDNA, which places it near G. 6? Kraurogymnocarpa trochleospora and petalosporus, G. boliviensis, G. reessii, and G. uncinatus, all of which form thick-walled differentiated peridial hyphae genera of Gymnoascaceae, all of which are now placed suggesting they evolved from an ancestor having a thick- in Gymnoascus (Sole et al. 2002). When we included the walled reticuloperidium. The genus now comprises 19 sequence of K. trochleospora in our SSU and LSU analysis, species, which form discoid (with or without equatorial it was also placed within the monophyletic Gymnoascus rim), bivalvate, globose, subglobose, irregular or lumpy clade (Figs 1–2). The ascospores of K. trochleospora ascospores with smooth, irregular or verruculose walls. A key have a combination of characters found in two different to the known species is given below, adapted from Solé et al. species of Gymnoascus, viz. G. verrucosus which has (2002) to accommodate the new species.
VOLUME 4 · NO. 2 181 Sharma & Singh ARTICLE
Fig. 4. Gymnoascus verrucosus 6? | £ *J 3 > < £ #J 3
182 IMA FUNGUS A new species of Gymnoascus with verruculose ascospores ARTICLE
Fig. 5. Gymnoascus verrucosus, (AMH 9454) A–D. Peridial hyphae, asci and ascospores. A. Thick-walled, septate peridial hyphae. B. Portion of peripheral region of ascoma showing bi- or trifurcating peridial appendages. C. Mature asci, note the smooth peridial hyphae. D. Tuberculate ascospores compactly arranged. Bar: A = 10 μm.
Key to accepted Gymnoascus species
Note: @ / #$'K ` et al. (2012).
# / > > ¤ ...... japonicus Above characters not combined ...... 2
2 (1) Ascospores with an equatorial depression or furrow ...... 3 Ascospores without such features ...... 4
* N > ...... ruber > / ~ USA ...... 4 (2) Ascospores 3–4 μm long ...... 5 ; PM' 3 ...... 12
5 (4) Ascospores hyaline to pale yellow ...... Ascospores yellow, yellow-orange or reddish brown ...... 7
VOLUME 4 · NO. 2 183 Sharma & Singh ARTICLE
Fig. 6. Gymnoascus verrucosus. (AMH 9454) A. Ascomata with loose central mass of asci and ascospores and thick-walled, septate peridial hyphae radiating towards periphery. Note typical bi- or trifurcating peridial appendages resembling deer antlers. B. Two asci traced from SEM x I ; £ NJ 3 > x £ N 3
K ; > 6 G ...... hyalinosporus ; "4> ...... verrucosus
K ; > ...... ' ; > ...... 9
' ; ? > > ? yard soil, Bolivia ...... boliviensis ; \ > > rat dung, horn, horse ringworm, a human lesion, pigeon or duck feathers, apparently worldwide ...... petalosporus
9 (7) Ascomata formed by thick-walled, well differentiated peridial hyphae with appendages ...... 10 Ascomata formed by peridial hyphae poorly differentiated from vegetative hyphae and without appendages ...... 11
#J $ ; > 6 ? ? > 6 Canada, Uganda, USA ...... uncinatus ; > 6 > 6 G ...... reessii
## $ ; ? > bat, rabbit, and tiger dung, and also linen and buried cables, apparently worldwide ...... marginosporus ; > ...... armeniacus
184 IMA FUNGUS A new species of Gymnoascus with verruculose ascospores
12 (4) Ascospores with equatorial thickenings ...... 13 ARTICLE Ascospores without distinct equatorial thickenings ...... 15
#* #N ; M' × P KMK K 3 > 6 G .... dankaliensis Ascospores smaller, without polar thickenings ...... 14
#P #* / > ? > > / x/ 4 6 _"; ...... littoralis / 6 > > > isolated from paddy soil, India ...... punctatus
#K #N ; ? > _"; ...... citrinus Ascospores a different colour, without polar thickenings ...... #
# #K ; ? > > dung, India and Somalia ...... devroeyi ; ? > ...... 17
# # / ~; > > sheep dung, owl pellets, and soil, apparently worldwide ...... aurantiacus / ~; ? > ...... #'
#' # / > ? > 4–5 × NMN K 3 > 6 USA ...... nodulosus / 6 > > PM K × 2.5–3.5 μm, not arranged > ¤ ...... udagawae
ACKNOWLEDGEMENTS using the bootstrap. Evolution 39I '*M$# ` ¤ _ D / #$$N ; Gymnoascus We thank Sugat. V. Shende (Pune University) for assistance with from Bolivian soil. Mycotaxon 45: 317–322. the scanning electron microscopy, the Department of Science and ` ¤ ` ¤ " ;4 < 4¤ NJ#N Atlas of @ "@ ` | Soil Ascomycetes. [CBS Biodiversity Series 10.] Utrecht: support to set up a National Facility for Culture Collection of Fungi Centraalbureau voor Schimmelcultures. | "~L"L~"?KKLNJJK 4;/"R; 4 #$'J ; Pune, India, and its Director, Maharashtra Association for Cultivation of base substitutions through comparative studies of nucleotide of Sciences’ Agharkar Research Institute for providing facilities. We sequences. Journal of Molecular Evolution 16: 111–120. \ `< ~ ; #$* @ Gymnoascus Baranetzky. Mycopathologia et Mycologia Applcata 21I #M#' "; " `" #$$P @ 6 REFERENCES Gliocladium analysed for nuclear large subunit ribosomal DNA sequences. Mycological Research 98I NKM*P ; "4 4 G #$$ _ 6 " | | 4 #$' @ ? I high quality genomic DNA for PCR based techniques. Nucleic for reconstructing phylogenetic trees. Molecular Biology and Acids Research 25I P$NMP$* Evolution 4I PJMPNK ; 6 ¤; D #$' @ Gymnoascus. Persoonia Sharma R, Rajak RC, Pandey AK (2002) Teaching techniques for 13I #*M#'* mycology: 19. A micro-dilution drop-trail method for isolating / ~ D `¤4 ` ¤} " ; NJJ$ onygenalean ascomycetes from hair baits. Mycologist 16: 153–157. Fungal Biodiversity. [CBS Laboratory Manual Series 1.] Utrecht: Sigler L, Hambleton S, Pare JA (2002) Chlamydosauromyces Centraalbureau voor Schimmelcultures. punctatus gen. & sp. nov. (Onygenaceae) from the skin of a / " #$'K @ 6 Onygenales: Arthrodermata- lizard. Studies in Mycology 47: 123–129. ceae, Gymnoascaceae, Myxotrichaceae and Onygenaceae. " 4 / ¤ ~ +x " ;4 ` ¤ NJJN 4 Mycotaxon 24I #MN# phylogeny of Gymnoascus and related genera. Studies in < ~ " D 4 " " D ` NJ## Mycology 47: 141–152. A comparative study of Neogymnomyces virgineus, a new Sugiyama M, Summerbell RC, Mikawa T (2002) Molecular phylogeny keratinolytic species from dung, and its relationships with the of onygenalean fungi based on small subunit (SSU) and large Onygenales. Fungal Diversity, 52: 13–34. subunit (LSU) ribosomal DNA sequences. Studies in Mycology < ¤ #$'K / I 47: 5–23.
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@ ~ ~ | " ` | 4 Vanbreuseghem R (1952) Techniques biologique pour l’isolement " NJ## 4`;KI 4 des dermatophytes du sol. Annales de la Société Belge de 6 6 Médecine Tropicale 32I #*M#' parsimony methods. Molecular Biology and Evolution 28: 2731– D 4 #$$J 2739. G |;
ARTICLE _ " _ " #$$$ @ 6 Cryptococcus species. Journal of Bacteriology 172I PN*'MPNP fungi of non-pathogenic Onygenales 1. Mycoscience 40: 277– @¤ x @ + " @ ¤ #$$J ; 290. sequencing of fungal ribosomal RNA genes for phylogenetics. _ " _ " NJJ# @ 6 I 4; ` " ¤¤ @¤ PCR fungi of non-pathogenic Onygenales 4. Mycoscience 42I N'#M Protocols: a guide to method and applications: 315–322. San N' Diego: Academic Press
186 IMA FUNGUS