Phylogeny and taxonomy of three new Ctenomyces (Arthrodermataceae, Onygenales) species from China

Abstract Twelve Ctenomyces (Arthrodermataceae, Onygenales) strains were obtained and identified during a survey of keratinophilic fungi in soils from China. We used molecular identification combined with morphological evidence to delimit species, circumscribing five species in the genus. Three new species are herein described: C.albussp. nov., C.obovatussp. nov. and C.peltricolorsp. nov. We also described, illustrated and compared the novel species with related species in the morphology.

The main diagnostic criteria of Ctenomyces (sensu Oorschot 1980) are that conidia are verrucose, thick-walled, lightly pigmented, commonly with ampulliform swellings and mostly longer than 8 μm.Oorschot (1980) regarded Ctenomyces as a sexual morph of Myceliophthora.Furthermore, he transferred Chrysosporium asperatum to Myceliophthora as M. vellerea in a taxonomic revision of Chrysosporium and allied genera.Myceliophthora vellerea was regarded as a synonym of the asexual morph of Ctenomyces serratus (Guarro et al. 1985, Chabasse 1988).Phylogenetic analyses, based on ITS rDNA, demonstrated Myceliophthora vellerea to be a synonym of Ctenomyces serratus (van den Brink et al. 2012). De Hoog et al. (2017) expanded the breadth and understanding of dermatophytes in Arthrodermatacea based on multi-locus molecular data and Ctenomyces serratus is used as the only previously validated species of the genus Ctenomyces.
Investigation of keratinophilic fungi has been given more attention in some countries (Anbu et al. 2004, Zarrin and Haghgoo 2011, Shadzi et al. 2002).Many researchers have shown that keratinophilic fungi distribution is closely related to human and animal activity (Sharma and Sharma 2010).Therefore, we conducted a survey of keratinophilic fungi in places with high human activity in Guizhou, Shanxi and Gansu provinces in China and isolated 12 strains.By combining the ITS sequence and a multi-gene phylogeny and the morphological characteristics, we identify and describe three new species and one new record of Ctenomyces from China.

Isolates
Twelve Ctenomyces strains were obtained from soil samples collected in Guizhou, Shanxi and Gansu province of China using a baiting technique (Vanbreuseghem 1952).Sterile chicken feather and human hair were combined with the soil samples and the samples were placed in sterile Petri dishes, which were moistened with sterile distilled water.The baited soil sample Petri dishes were incubated at 25 °C for 1 month and remoistened as necessary.When fungal growth was observed, those feathers with fungal growth were mixed with 9 ml of sterile water in an Erlenmeyer flask and 1 ml of suspensions were evenly spread on plates containing Sabouraud's dextrose agar (SDA) with chloramphenicol and cycloheximide medium.The plates were incubated at 25 °C.The pure culture were then transferred to potato dextrose agar (PDA) plates for purification, the isolates were inoculated to test-tube slants and stored at 4 °C.
All holotypes and isotypes were deposited in the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS).Type strains and ex-type living cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC) and the Institute of Fungus Resources, Guizhou University (GZUIFR).Taxonomic information of the new taxa was deposited in MycoBank (www.MycoBank.Org).

Morphology
Isolates were transferred to potato dextrose plates, incubated at 25 °C for 14 days and subjected to macroscopic examination.Fungal microscopic features were examined with a Nikon Ti-U microscope (Nikon, Japan) and photographed.Diagnostic features were then illustrated on the basis of these observations.Finally, the fungi were morphologically identified according to colony characteristics, conidiogenous structures and conidia (sensu Oorschot 1980).

DNA extraction, PCR amplification, sequencing
Total genomic DNA was extracted from fresh sporulating cultures after 14 days at 25 °C using a Fungal DNA Mini Kit (Omega Biotech, Doraville, GA, USA) according to the manufacturer's protocol and then stored at -20 °C.Three regions were amplified and sequenced, including the internal transcribed spacer (ITS) region using primers ITS1 and ITS4 (White et al. 1990); partial fragments of the RNA polymerase II largest subunit 2 (RPB2) gene region using primers 5F-Eur and 7cR-Eur (van den Brink et al. 2012); partial fragments of the translation elongation factor 1-alpha (EF1A) gene region using primers EF1-983F andEF1-2218R (van den Brink et al. 2012).The PCR mixture was prepared using a commercial kit (TSINGKE Biological Technology, Kunming, China) and contained 5 μl 10 × reaction buffer, 0.4 μl dNTPs (25μM), 0.2 μl T6 DNA polymerase (5 U/μl), 1 μl of each primer and 2 μl DNA template in a final volume of 25 μl.Reaction mixtures were pre-heated at 98 °C for 2 min and PCR was performed as follows: 30 cycles of 10 s at 98 °C, 10 s at 55 °C and 10 s 72 °C, with a final extension at 72 °C for 5 min and cooling at 4 °C.The PCR conditions were the same for all three markers.The resulting PCR products were sequenced by TSINGKE Biological Technology (Kunming, China) using the corresponding primers.

Phylogenetic analysis
Sequence data from the nine genera of Arthrodermataceae and Myceliophthora lutea sequences were used in the phylogenetic analysis.Details of newly generated and refer-ence sequences retrieved from GenBank are listed in Table 1.Multiple sequence alignments for ITS, EF1A and RPB2 were achieved with MAFFT v.7.037b (Katoh and Standley 2013) and manually edited in the MEGA 6.06 (Tamura et al. 2013).
A total of 50 ITS sequences of 23 species and including Myceliophthora lutea (CBS 145.77 and MUCL 10070) as the outgroup taxon were used in the analysis.The data were analysed phylogenetically using Bayesian Markov chain Monte Carlo (MCMC) and maximum likelihood (ML).For the Bayesian analysis, two simultaneous Bayesian Inference (BI) Markov chain Monte Carlo runs were also executed for 10,000,000 generations, saving trees every 500 generations.Modeltest v3.7 suggested the GTR+I+G as the best-fit evolutionary model for dataset (Posada and Crandall 1988).After the BI analysis, each run was examined using the programme Tracer v1.5 (Drummond and Rambaut 2007) to determine whether the burn-in period was sufficient and to confirm that both runs had converged.ML analyses were performed using RAXML (Stamatakis and Alachiotis 2010) with the graphical user interface (GUI) (Silvestro and Michalak 2012) implementation and the GTRGAMMA model.The BI and ML analysis trees are available in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S23736) and a consensus tree is presented in Figure 1.
A concatenated dataset (ITS+EF1A+RPB2) of five Ctenomyces species and Myceliophthora lutea (CBS 145.77 and MUCL 10070) was assembled using SequenceMatrix v. 1.7.8 (Vaidya 2011).Concordance between genes was assessed with the 'hompart' command of PAUP4.0b10(Swofford 2002).Maximum likelihood (ML) phylogenetic analyses of the datasets were performed using RAxML (Stamatakis and Alachiotis 2010) with the graphical user interface (GUI) (Silvestro and Michalak 2012) implementation and the General Time Reversible (GTR) model.Bootstrap analysis with 1,000 replicates was used to estimate nodal support.Two simultaneous Bayesian Inference Markov chain Monte Carlo runs were also executed for 10,000,000 generations, saving trees every 500 generations.Modeltest v3.7 suggested the GTR+I+G as the best-fit evolutionary model for the dataset (Posada and Crandall 1988).After the BI analyses, each run was examined using the programme Tracer v1.5 (Drummond and Rambaut 2007) to determine whether the burn-in period was sufficient and to confirm that both runs had converged.The BI and ML analyses trees are available in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S23736) and a consensus tree is presented in Figure 2.
Culture characteristics.Colonies on PDA growing in the dark reaching 32 mm diam. in 14 d at 25 °C, white, short fluffy to powdery, appearing some annulations, rounded, margin regular and defined.Reverse yellowish.
Notes.Ctenomyces albus is distinct from other species as it is the only species with intercalary conidia in the genus.In addition, our ITS and polygenic phylogeny showed that three isolates of C. albus were in a clade sister to C. serratus (Figures 1, 2) and clearly separate from other species.Following Jeewon and Hyde's (2016) guidelines on new species delimitation, there were 37 bp (base pair) differences amongst 508 nucleotides ITS sequences between the isolate CGMCC 3.19232 and C. serratus CBS 187.61 (only ITS sequence data are available, EF1A and RPB2 are lacking), which also supports them as distinct different species.Therefore, we introduce C. albus sp.nov. in this study.Holotype.CHINA, Shanxi Province, on soil, Nov. 2017, Z.Y. Zhang (HMAS 255446, holotype, ex-type culture CGMCC 3.19225).
Culture characteristics.Colonies on PDA growing in the dark reaching 14-15 mm diam. in 14 d at 25 °C, yellowish, white in the margin; fluffy; rounded, margin regular.Reverse brown.
Culture characteristics.Colonies on PDA growing in the dark reaching 12 mm diam. in 14 d at 25 °C, pewter at the centre, white in the margin; powdery to floccose at the centre, fluffy in the margin; appearing a circle of annulation; rounded, margin regular.Reverse brown at the centre, yellowish in the margin.
Notes.Ctenomyces peltricolor is distinct from other species in its single conidia borne on ampulliform swellings and colony colour.Phylogenetically, the ITS-based phylogenetic analysis (Figure 1) showed that three isolates of C. peltricolor cluster in a single clade (BPP 1, BS 100%), consistent with the results of multigene phylogenetic analysis (BPP 1, BS 100%) (Figure 2).Therefore, based on both morphological and phylogenetic evidence, C. peltricolor was identified as a new species of Ctenomyces.
Culture characteristics.Colonies on PDA growing in the dark reaching 30 mm diam. in 14 d at 25 °C, brown, white in the margin, floccose at the centre, short fluffy in other part, appearing obvious annulation; rounded, margin regular and defined.Reverse yellowish.
Known distribution.Currently known from Australia, England, India, Argentina, Germany (sensu Oorschot 1980) and China (this study).

Discussion
Members of the family Arthrodermataceae (Onygenales) were common in nature, mostly found as saprobes in soil on keratin-rich substrates or associated with vertebrate causing dermatophytosis and other infections.The widely accepted morphology-based taxonomy of dermatophytes in the genera Trichophyton, Microsporum and Epidermophyton was established by Emmons (Emmons 1934).There are three common ecological groups, anthropophilic, zoophilic or geophilic (de Hoog et al. 2017).
Trichophyton and Epidermophyton usually belonged to anthropophiles or zoophilic taxa, Microsporum were considered to zoophilic, Arthroderma was geophilic.Some species cannot be clearly attributed to one of these groups due to insufficient data.Geophilic dermatophytes have their reservoir in the soil around burrows of specific terrestrial mammals, feeding on keratinous debris.Hence, the difference between geophilic and zoophilic dermatophytes is not always well-resolved.The genus of Ctenomyces is usually saprobic or closely related to keratin-rich substrates (Wijayawardene et al. 2017, Deshmukh et al. 2018).In addition, these strains of our study were isolated by using hair and feathers as baiting material.Therefore, we infer the genus Ctenomyces to be geophilic and/or zoophilic.
Phylogenetic studies based on the ITS (Graser et al. 2008), partial LSU, the ribosomal 60S protein, partial β-tubulin and translation elongation factor 3 for Arthrodermataceae have shown that the genus Trichophyton was polyphyletic and resulted in establishing nine genera, i.e.Arthroderma, Ctenomyces, Epidermophyton, Guarromyces, Lophophyton, Microsporum, Nannizzia, Paraphyton and Trichophyton and it suggested that ITS was the optimal barcoding marker (de Hoog et al. 2017).Therefore, we selected the ITS sequences for phylogenetic analysis of Arthrodermataceae in this study and our results are consistent with the previous studies (de Hoog et al. 2017).Ctenomyces vellereus (strains CBS 479.76 and CBS 715.84) had ITS, EF1A and RPB2 sequence data.Hence, we selected the ITS, EF1A and RPB2 genes for phylogenetic analysis of Ctenomyces.The results show that the ITS-based and multigene-based phylogenetic analyses have similar results.Although our study revealed that Ctenomyces was closely related to Arthroderma in Arthrodermataceae, Onygenales, the species of Ctenomyces nearly all produce ampulliform swellings, a feature absent in Arthroderma.Guarro et al. (1985), Chabasse (1988) and van den Brink et al. (2012) proposed that C. vellereus was a synonym of C. serratus, however, these two species have several different characters, the conidia of C. serratus are ellipsoid, 12-23 × 10.5-12 μm, while those of C. vellereus are subglobose, pyriform or ellipsoid, 4-13 × 3-9 μm (sensu Oorschot 1980).Van den Brink et al. (2012)

Table 1 .
Strains included in the phylogenetic analysis.T= type strains, strain and sequences generated in this study are shown in bold.