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Exophiala yunnanensis and Exophiala yuxiensis (Chaetothyriales, Herpotrichiellaceae), two new species of soil-inhabiting Exophiala from Yunnan Province, China
expand article infoRuili Lv‡, Xiaoqian Yang‡, Min Qiao‡, Linlin Fang‡, Jianying Li§, Zefen Yu‡
‡ Yunnan University, Kunming, China
§ Kunming Edible Fungi Institute of All China Federation of Supply and Marketing Cooperatives, Kunming, China

Corresponding author: Min Qiao ( qiaomingy@sina.com )

Academic editor: Cecile Gueidan
© 2022 Ruili Lv, Xiaoqian Yang, Min Qiao, Linlin Fang, Jianying Li, Zefen Yu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Lv R, Yang X, Qiao M, Fang L, Li J, Yu Z (2022) Exophiala yunnanensis and Exophiala yuxiensis (Chaetothyriales, Herpotrichiellaceae), two new species of soil-inhabiting Exophiala from Yunnan Province, China. MycoKeys 94: 109-124. https://doi.org/10.3897/mycokeys.94.96782
Open Access

Abstract

During a survey of soil fungi collected from Yunnan Province, China, two new species of Exophiala, E. yunnanensis and E. yuxiensis, were isolated from the soil of karst rocky desertification (KRD). The DNA sequences of these respective strains, including internal transcribed spacers (ITS), large subunit nuclear ribosomal RNA (LSU rRNA), partial small subunit (SSU) and β-tubulin (tub2) were sequenced and compared with those from species closely-related to Exophiala. Exophiala yunnanensis differs from the phylogenetically closely related E. nagquensis and E. brunnea by its smaller aseptate conidia. Exophiala yuxiensis is phylogenetically related to E. lecanii-corni, E. lavatrina and E. mali, but can be distinguished from them by its larger conidia. Full descriptions, illustrations and phylogenetic positions of E. yunnanensis and E. yuxiensis were provided.

Keywords

Exophiala, multi-locus phylogeny, morphology, new species, taxonomy

Introduction

Exophiala J.W. Carmich. (Chaetothyriales, Herpotrichiellaceae) was established with E. salmonis J.W. Carmich. as type species (Carmichael 1966) in Alberta, Canada. Due to their yeast-like melanised colonies, these fungi are often also referred to as “black yeasts” (Matsumoto et al. 1987). The genus is characterised by annellidic conidiogenous cells producing slimy heads of conidia, conidiophores upright or bent, not or irregularly branched, smooth, light olive to brown. However, there are several synanamorphs recorded in this genus (Thitla et al. 2022). Nearly all species are recognisable within the order by the way they produce cells by budding (De Hoog et al. 2011).

Exophiala spp. are widely distributed and can be isolated from bulk soil, biological crusts, rock surfaces, air, natural water masses, rhizosphere, plant tissues, and infected animals and human tissue (Addy et al. 2005; Bates et al. 2006; Neubert et al. 2006; Bukovská et al. 2010; Julou et al. 2010; De Hoog et al. 2011). Most studies on Exophiala species focused on their importance as etiologic agents of disease in animals and humans (Zeng and De Hoog 2008; Najafzadeh et al. 2013; Wen et al. 2016). Several Exophiala species are opportunistic pathogens of immunocompetent humans (Woo et al. 2013; Yong et al. 2015), in rare occasions causing nervous system phaeohyphomycosis (Chang et al. 2000) or causing cutaneous and subcutaneous skin infections, including E. spinifera (H.S. Nielsen & Conant) Mcginnis, which has the strongest pathogenicity to human skin (Vitale and De Hoog 2002). Furthermore, some Exophiala species, such as E. salmonis, E. aquamarina de Hoog et al. and E. equina (Pollacci) de Hoog et al. may cause cutaneous or disseminated infections of cold-blooded animals (De Hoog et al. 2011). Therefore, the classification and identification of this genus are significantly important for clinical diagnosis, treatment and prevention.

In the past, taxonomic and diagnostic schemes for Exophiala were morphological characteristics, but the anamorphic states of some species are highly pleomorphic (De Hoog et al. 1995; Haase et al. 1995; Thitla et al. 2022), which make them difficult to be recognised and circumscribed (Naveau 1999; Zeng and De Hoog 2008), so only a small number of Exophiala species are, in fact, recognisable using morphology. With the development of molecular systematics, more and more species were redefined, re-designated or described mainly depending on genetic, morphological, physiological and ecological features (Haase et al. 1999; De Hoog et al. 2003; Vitale et al. 2003; De Hoog et al. 2006). At present, 80 names in Exophiala were recorded in Index Fungorum, amongst them E. nigra (Issatsch.) Haase & de Hoog, E. placitae Crous & Summerell, E. prototropha (Bulanov & Malama) Haase et al. and E. werneckii (Horta) Arx, have been moved to Nadsoniella Issatsch., Neophaeococcomyces Crous & M.J. Wingf., Pullularia Berkhout and Hortaea Nishim. & Miyaji, respectively. Currently, 68 species have been accepted into this genus after a brief review of Thitla et al. (2022) and Crous et al. (2022), who described new species from Thailand and Australia.

During a survey of fungi from rocky desertification area, two unknown fungi were found. Based on morphology and phylogenetic analysis combined ITS, SSU, LSU and tub2, we proposed two new species, E. yunnanensis and E. yuxiensis.

Materials and methods

Isolation and morphological characterisation of strains

Soil samples were collected from Yiliang and Yuxi in Yunnan Province, southwest China. Samples were placed in plastic bags, labelled and transported to the laboratory. All the samples were stored at 4 °C before further processing. Fungal strains were obtained by serial dilutions (1,000 to 1,000,000 fold) and spread on to the surface of Rose Bengal agar with antibiotics (40 mg streptomycin, 30 mg ampicillin per litre) added in a 9 cm diam. Petri dish, followed by incubation at 25 °C for 5 days (Zheng et al. 2021a). Representative colonies were picked up with a sterilised needle and transferred to potato dextrose agar (PDA, 200 g potato, 20 g dextrose, 18 g agar, 1000 ml distilled water). After 7 days, colonies were transferred to cornmeal agar (CMA, 20 g cornmeal, 18 g agar, 1000 ml distilled water). Characteristics of colonies, growth rate and other morphological aspects from PDA were observed after 10 days. Microscopic characteristics including mycelium, 10 conidiophores and 30 conidia were examined and measured after 7 days on CMA using an Olympus BX51 microscope. Pure cultures were deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming, Yunnan, P.R. China (YMF, formerly Key Laboratory of Industrial Microbiology and Fermentation Technology of Yunnan), China General Microbiological Culture Collection Center (CGMCC), the Guangdong Microbial Culture Collection Center (GDMCC) and Japan Collection of Microorganisms (JCM).

DNA extraction, PCR amplification and sequencing

Total DNA was extracted following the protocol of Zheng et al. (2021b). The internal transcribed spacer (ITS), the large subunit nuclear ribosomal RNA (LSU rRNA), the partial small subunit (SSU) and the β-tubulin (tub2) were amplified using the primer pairs ITS1/ITS4 (White 1990), LR0R/LR5 (Vilgalys and Hester 1990), NSSU131/NS24 (Kauff and Lutzoni 2002) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The PCR amplifications were conducted in 25 µl final volumes which consisted of 1.0 µl DNA template, 1.0 µl of each forward and reverse primers, 12.5 µl 2 × Master Mix and 9.5 µl ddH2O. The PCR reaction cycles were as follows: initial denaturation at 94 °C for 5 min; followed by 35 cycles of denaturation at 94 °C for 40 s; the annealing extension dependent on the amplified loci (48 °C for LSU, 54 °C for ITS, 51 °C for SSU and 58 °C for tub2) for 1 min and extension at 72 °C for 2 min; a final extension at 72 °C for 10 min. PCR products were sequenced by TSINGKE Biological Technology in Kunming, China.

Sequence alignment and phylogenetic analysis

Preliminary BLAST searches with ITS, LSU, SSU and tub2 gene sequences of the new isolates against NCBI databases had identified species closely related to our two isolates. Based on this information, ITS, LSU, SSU and tub2 sequences of 62 strains were downloaded and used in the phylogenetic analysis with Cyphellophora oxyspora (CBS 698.73) as outgroup. The GenBank accession numbers of sequences used in the phylogenetic analysis are shown in Table 1. DNA sequence data were aligned using ClustalX 1.83 (Thompson et al. 1997) with default parameters. Aligned sequences of multiple loci were concatenated and manually adjusted through BioEdit version v. 7.0.4.1 (Hall 1999) and ambiguously aligned regions were excluded. The combined sequence was converted to a NEXUS file using MEGA6 (Tamura et al. 2013) and it was uploaded to TreeBASE (www.treebase.org; accession number: S29757).

Table 1.
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Species, strains and their corresponding GenBank accession numbers of sequences used for phylogenetic analyses. Exophiala strains of the present study were marked in bold. Tex-type cultures; “-” The gene fragment representing this strain was not attainable.

Species Strain no. GenBank accession no.
ITS LSU SSU tub2
Exophiala abietophila CBS 145038T MK442581 NG066323 – –
Exophiala alcalophila CBS 520.82T JF747041 AF361051 JN856010 JN112423
Exophiala angulospora CBS 482.92T JF747046 KF155190 JN856011 JN112426
Exophiala aquamarina CBS 119918T JF747054 – JN856012 JN112434
Exophiala asiatica CBS 122847T EU910265 – – –
Exophiala attenuata F10685 KT013095 KT013094 – –
Exophiala bergeri CBS 353.52T EF551462 FJ358240 FJ358308 EF551497
Exophiala bonariae CBS 139957T JX681046 KR781083 – –
Exophiala brunnea CBS 587.66T JF747062 KX712342 JN856013 JN112442
Exophiala campbellii NCPF 2274 LT594703 LT594760 – –
Exophiala cancerae CBS 120420T JF747064 – – JN112444
Exophiala capensis CBS 128771T JF499841 MH876538 – –
Exophiala castellanii CBS 158.58T JF747070 KF928522 JN856014 KF928586
Exophiala cinerea CGMCC 3.18778T MG012695 MG197820 MG012724 MG012745
Exophiala clavispora CGMCC 3.17512 KP347940 MG197829 MG012733 KP347931
Exophiala crusticola CBS 119970T AM048755 KF155180 KF155199 –
Exophiala dermatitidis CBS 207.35T AF050269 KJ930160 – KF928572
Exophiala ellipsoidea CGMCC 3.17348T KP347955 KP347956 KP347965 KP347921
Exophiala embothrii CBS 146560 MW045819 MW045823 – –
Exophiala equina CBS 119.23T JF747094 – JN856017 JN112462
Exophiala eucalypti CBS 142069 KY173411 KY173502 – –
Exophiala eucalyptorum CBS 121638T NR132882 KC455258 KC455302 KC455228
Exophiala exophialae CBS 668.76T AY156973 KX822326 KX822287 EF551499
Exophiala frigidotolerans CBS 146539T LR699566 LR699567 – –
Exophiala halophila CBS 121512T JF747108 – JN856015 JN112473
Exophiala heteromorpha CBS 232.33T MH855419 MH866871 – –
Exophiala hongkongensis CBS 131511 JN625231 – – JN625236
Exophiala italica MFLUCC 16-0245T KY496744 KY496723 KY501114 –
Exophiala jeanselmei CBS 507.90T AY156963 FJ358242 FJ358310 EF551501
Exophiala lacus FMR 3995 KU705830 KU705847 – –
Exophiala lavatrina NCPF 7893 LT594696 LT594755 – –
Exophiala lecanii-corni CBS 123.33T AY857528 FJ358243 FJ358311 –
Exophiala lignicola CBS 144622T MK442582 MK442524 – –
Exophiala macquariensis CBS 144232T MF619956 – – MH297438
Exophiala mali CBS 146791T MW175341 MW175381 – –
Exophiala mansonii CBS 101.67T AF050247 AY004338 X79318 –
Exophiala mesophila CBS 402.95T JF747111 KX712349 JN856016 JN112476
Exophiala moniliae CBS 520.76T KF881967 KJ930162 – –
Exophiala nagquensis CGMCC 3.17284 KP347947 MG197838 MG012742 KP347922
Exophiala nidicola FMR 3889 MG701055 MG701056 – –
Exophiala nigra CBS 535.94T KY115191 KX712353 – –
Exophiala nishimurae CBS 101538T AY163560 KX822327 KX822288 JX482552
Exophiala oligosperma CBS 725.88T AY163551 KF928486 FJ358313 EF551508
Exophiala opportunistica CBS 109811T JF747123 KF928501 – JN112486
Exophiala palmae CMRP 1196T KY680434 KY570929 – KY689829
Exophiala phaeomuriformis CBS 131.88T AJ244259 – – –
Exophiala pisciphila CBS 537.73T NR121269 AF361052 JN856018 JN112493
Exophiala placitae CBS 121716T MH863143 MH874694 – –
Exophiala prostantherae CBS 146794T MW175344 MW175384 – –
Exophiala polymorpha CBS 138920T KP070763 KP070764 – –
Exophiala pseudooligosperma YMF 1.6741 MW616557 MW616559 MW616558 MZ127830
Exophiala psychrophila CBS 191.87T JF747135 – JN856019 JN112497
Exophiala quercina CPC 33408 MT223797 MT223892 – –
Exophiala radicis P2772 KT099203 KT723447 KT723452 KT723462
Exophiala salmonis CBS 157.67T AF050274 AY213702 JN856020 JN112499
Exophiala sideris CBS 121818T HQ452311 – HQ441174 HQ535833
Exophiala spinifera CBS 899.68T AY156976 – – EF551516
Exophiala tremulae CBS 129355T FJ665274 – KT894147 KT894148
Exophiala xenobiotica CBS 128104 MH864829 MH876272 – –
Exophiala yunnanensis YMF1.06739 MZ779226 MZ779229 MZ781222 OM095379
Exephiala yuxiensis YMF1.07354 OL863155 OL863154 OM149370 OL944581
Cyphellophora oxyspora CBS 698.73T KC455249 KC455262 KC455305 KC455232

Phylogenetic analyses were conducted using both the Bayesian Inference (BI) and Maximum Likelihood (ML) methods. Bayesian Inference analysis was conducted using MrBayes v.3.2 (Ronquist et al. 2012) with the NEXUS file. The following parameters were used: ngen = 1,000,000; samplefr = 1,000; printfr = 1,000. The Akaike Information Criterion (AIC) implemented in jModelTest version 2.0 (Posada 2008) was used to select the best fit models after likelihood score calculations were done. TPM1uf + I + G was estimated as the best-fit model under the output strategy of AIC. Two independent analyses with four chains each (one cold and three heated) were run until stationary distribution was achieved. The initial 25% of the generations of MCMC sampling were excluded as burn-in. The refinement of the phylogenetic tree was used for estimating Bayesian Inference posterior probability (BIPP) values. The ML trees, based on four gene loci, were constructed with the GTR+GAMMA model using RAxML version 7.2.6 (Stamatakis 2006) and the robustness of branches was assessed by bootstrap analysis with 1000 replicates. The tree was viewed in TreeView 1.6.6 (Page 1996) with Maximum Likelihood bootstrap proportions (MLBP) greater than 50% and Bayesian Inference posterior probabilities (BIPP) greater than 70%, as shown at the nodes.

Results

Molecular phylogeny

The Bayesian tree, based on ITS sequence data, confirmed that two strains were distinct from known species of Exophiala (Fig. 1), Exophiala yunnanensis is phylogenetically close to E. nagquensis CGMCC 3.17284 and ITS similarity between E. yunnanensis and E. nagquensis is 92.21%. Exophiala yuxiensis is phylogenetically related to E. lecanii-corni CBS 123.33, E. mali CBS 146791 and E. lavatrina NCPF 7893 and the similarities between the holotype of E. yuxiensis and the representative strains of three species are 90.27%, 89.86% and 85.08%, respectively.

Figure 1. 

Phylogenetic tree generated by Bayesian Inference, based on sequences of the ITS. Cyphellophora oxyspora CBS 698.73 serves as outgroup. Bayesian posterior probability over 75 is shown at the nodes. Two new species were shown in bold.

In the combined phylogenetic analyses (ITS, LSU, SSU and tub2), which contained 2218 characters, a similar topological structure was observed between the two phylogenetic trees constructed by BI and ML. The support values with BI analysis are relatively higher than the ML bootstrap support values (Fig. 2) In this tree, E. yunnanensis, E. nagquensis W. Sun et al., E. brunnea Papendorf and E. frigidotolerans Rodr.-Andr. et al. formed a clade with high statistical support (BIBP/MLBP = 100/97). Exophiala yuxiensis is phylogenetically close to E. lecanii-corni (Benedek & G. Specht) Haase & de Hoog and the clade formed by these species and six additional ones also has high statistical support (BIBP/MLBP = 100/89).

Figure 2. 

Phylogenetic tree generated by Bayesian analyses combined sequences of ITS, LSU, SSU and tub2. Bayesian posterior probability values > 70 (left) and Bootstrap values > 50 (right) are indicated at nodes (BIBP/MLBP). Cyphellophora oxyspora CBS 698.73 serves as outgroup.

Taxonomy

Exophiala yunnanensis Z.F. Yu & R.L. Lv, sp. nov.

MycoBank No: 842373
Fig. 3

Etymology

yunnanensis, pertaining to Yunnan, a province of southwest China from where the type was collected.

Description

Colonies on CMA medium after 7 days with hyphae olive green, smooth, septate, thin walled, branched, 1.6–3.0 µm wide. Conidiogenous cells slightly differentiated from simple or branched vegetative hyphae, terminal or intercalary, flask-shaped, ovoid to elongate, pale brown, loci at tips and lateral; annellated zones inconspicuous or occasionally finely fimbriate, often inserted on intercalary cells. Conidia aseptate, ellipsoidal, cylindrical or allantoid, 1–2 guttulate, smooth, brown, 2.9–4.8 × 1.8–3.3 µm, with a conspicuous scar of approx. 1 µm wide at the base, containing no evident or few small oil drops.

Figure 3. 

Exophiala yunnanensis (YMFT 1.06739, holotype) A colony on PDA after 14 days B colony on CMA after 14 days C–G conidiogenous cells H conidia and budding cells. Scale bars: 3.2 cm (A, B); 10 µm (C–H).

Culture characteristics

Colonies on PDA medium, at 25 °C, were slow-growing, mycelium immersed and partly superficial, irregular, umbonate, surface olivaceous-grey to black. Radial growth rates were 0.8–0.9 mm day-1on PDA. Colonies on CMA medium were restricted, mycelium immersed and partly superficial, effuse, cottony, reverse olivaceous-buff to olivaceous, reaching 12 mm diam. in 15 days at 25 °C.

Type

China. Yiliang County, Yunnan Province, isolated from soil of rocky desertification area, 24°96'N, 102°66'E, ca. 1886 m elev., Oct 2020, Z.F.Yu, preserved by lyophilisation (a metabolically-inactive state) in State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan (holotype YMFT 1.06739), ex-holotype live culture: YMF 1.06739; CGMCC 3.16095; GDMCC 3.725; JCM 39339.

Exophiala yuxiensis Z.F. Yu & R.L. Lv, sp. nov.

MycoBank No: MB842374
Fig. 4

Etymology

yuxiensis, pertaining to Yuxi, a city of Yunnan Province in China, from which the type was collected.

Description

Colonies on CMA medium after 7 days with hyphae pale olivaceous-green, smooth, irregularly septate, thin-walled, branched, 1.5–3 µm wide, with lateral branches originating close to septa. Conidiogenous cells slightly differentiated from hyphae, arising from hyphal tips or lateral, terminal or intercalary, variable in shape, flask-shaped, ovoid to elongate, clavate, obtuse at the base, tapering towards inconspicuous annellate loci, 5.5–10.5 × 3–5 μm; annellated zones inconspicuous or occasionally finely fimbriate, often inserted on intercalary cells of hyphae. Conidia aseptate, ellipsoidal to cylindrical, 1–2 (mostly 2) bi-guttulate, smooth, pale olivaceous-green, 4.5–8 × 3.5–5 µm, without conspicuous scar.

Figure 4. 

Exophiala yuxiensis (YMFT 1.07354, holotype) A colony on PDA after 30 days B colony on CMA after 30 days C–E conidiogenous cells F conidia and budding cells. Scale bars: 3.2 cm (A, B), 10 µm (C–F).

Culture characteristics

Colonies on PDA medium, at 25 °C, were slow-growing, mycelium immersed and partly superficial, umbonate, dense, powdery or velvety, dry, margin irregular, surface olivaceous-grey, reverse olivaceous-black, attaining 1 cm diam. in 4 days. Colonies on CMA medium were restricted, mycelium immersed and partly superficial, cottony, surface olivaceous-green, some floccose aerial hyphae in the centre, front distinct, reverse pale olivaceous-black, reaching 3 cm diam. in 5–7 days.

Type

China. Yuxi City, Yunnan Province, isolated from soil of rocky desertification area, 24°44'N, 102°55'E, 1660 m altitude, Jul 2021, Z.F. Yu, preserved by lyophilisation (a metabolically-inactive state) in State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan (holotype YMFT 1.07354), ex-holotype live culture: YMF 1.07354; CGMCC 3.16094; GDMCC 3.726; JCM 39376).

Discussion

In this study, we propose two new species of Exophiala, based on combined morphological characteristics and phylogenetic analyses. Exophiala yunnanensis and E. yuxiensis are different from their phylogenetically closely-related species. Amongst them, E. nagquensis (Sun et al. 2020) and E. brunnea (Papendorf 1969) are distinguished from E. yunnanensis by their larger conidia (E. nagquensis: 4.8–10.4 × 2.6–5.0 µm; E. brunnea: 4.5–10 µm in length; E. yunnanensis 2.9–4.8 × 1.8–3.3 µm), while E. frigidotolerans differs from E. yunnanensis by ellipsoidal to reniform and larger conidia (4.0–7.0 × 2.0–4 .0 µm) (Crous et al. 2020). Additionally, E. yunnanensis resembles E. nagquensis and E. frigidotolerans in the shape of budding cells, but E. yunnanensis has smaller budding cells (Maciá-Vicente et al. 2016; Sun et al. 2020).

Exophiala yuxiensis is phylogenetically related to E. lecanii-corni, E. lavatrina Borman et al. and E. mali Crous. Amongst these species, E. mali is the most similar to E. yuxiensis by ellipsoidal to cylindrical conidia, but the conidia of E. mali are larger (8.0–10.0 × 3.0–5.0 µm vs. 4.5–8.0 × 3.5–5.0 µm) and the hyphae of E. mali are constricted at the septa in the terminal region, forming chains of disarticulating conidia (Crous et al. 2020). Exophiala lavatrina can be distinguished from E. yuxiensis by smaller conidia (4.5–7 × 2.5–4 µm) (Borman et al. 2017).

The species of Exophiala have a wide distribution, with isolation from diverse substrates, such as plants, fruit juices, shower rooms, seawater, sports drinks, arable soil, wood pulp, oil sludge and the decaying shell of babassu coconut (De Hoog et al. 1994; De Hoog et al. 2006; De Hoog et al. 2011; Feng et al. 2014; Madrid et al. 2016). Some species were reported as opportunistic pathogens on the superficial skin or internal organs in humans and animals. For example, the type species E. salmonis, was isolated from cerebral mycetoma of Salmo clarkii Richardson, 1836 (Carmichael 1966), while isolates of E. equina (Pollacci) de Hoog et al. and E. pisciphila McGinnis & Ajello cause disease on cold-blooded animals such as fish, turtles, crabs, sea horses and frogs (De Hoog et al. 2011). In addition, some species were frequently isolated as endophytes (Addy et al. 2005), although they seldom represent important components of endophytic communities.

The present work increased the number of Exophiala species to 70 in the world (Crous et al. 2022; Thitla et al. 2022). In China, Yunnan Province has diverse climate and vegetation, which provides natural advantages for the study of environmental microbial diversity. However, further extensive samplings and investigation of fungi are necessary to generate a complete knowledge about the biodiversity, distribution, habitats and adaptation mechanisms from Exophiala to environmental stresses.

Acknowledgements

This work was financed by the National Natural Science Foundation Program of PR China (31970013, 32170017), Yunnan University Research and Innovation Fund for Postgraduates (2021Y294).

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