﻿Exophialayunnanensis and Exophialayuxiensis (Chaetothyriales, Herpotrichiellaceae), two new species of soil-inhabiting Exophiala from Yunnan Province, China

﻿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. Exophialayunnanensis differs from the phylogenetically closely related E.nagquensis and E.brunnea by its smaller aseptate conidia. Exophialayuxiensis 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.


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, redesignated 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 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.

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 ddH 2 O. 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).
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.

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