Five new additions to the genus Spathaspora (Saccharomycetales, Debaryomycetaceae) from southwest China

Abstract Spathaspora is an important genus of d-xylose-fermenting yeasts that are poorly studied in China. During recent yeast collections in Yunnan Province in China, 13 isolates of Spathaspora were obtained from rotting wood and all represent undescribed taxa. Based on morphological and phylogenetic analyses (ITS and nuc 28S), five new species are proposed: Spathaspora elongata, Sp. mengyangensis, Sp. jiuxiensis, Sp. parajiuxiensis and Sp. rosae. Our results indicate a high species diversity of Spathaspora waiting to be discovered in rotting wood from tropical and subtropical southwest China. In addition, the two Candida species, C. jeffriesii and C. materiae, which are members of the Spathaspora clade based on phylogeny, are transferred to Spathaspora as new combinations.

Most species of Spathaspora, including Sp. arborariae, Sp. brasiliensis, Sp. passalidarum, Sp. roraimanensis, Sp. suhii and Sp. xylofermentans, are economically important because of their ability to ferment d-xylose, the second most abundant sugar in lignocellulosic feedstocks (Nguyen et al. 2011;Cadete et al. 2013;Lopes et al. 2016;Morais et al. 2017). These xylose-fermenting species can be used directly for ethanol production or may provide a source of genes, enzymes and/or sugar transporters to engineer industrial strains for the efficient production of bioethanol from renewable biomass (Wohlbach et al. 2011;Cadete et al. 2013).
Spathaspora species are associated with rotting-wood substrates and the insects that occupy this ecological niche (Cadete et al. , 2013Nguyen et al. 2011;Lopes et al. 2016;Wang et al. 2016;Morais et al. 2017;Varize et al. 2018). They can be found in tropical, subtropical and temperate regions on different continents, but most species are presently known from Brazilian regions (Cadete et al. , 2013Lopes et al. 2016;Morais et al. 2017;Varize et al. 2018). In China, the genus is underexplored with only three published reports of the species Sp. allomyrinae, Sp. gorwiae and Sp. passalidarum (Ren et al. 2013;Wang et al. 2016). Here, we describe five new species of Spathaspora discovered in tropical and subtropical areas of southwest China, based on their morphological characters and molecular phylogenetic analyses.

Sample collection and isolation
Rotting wood samples were collected in two areas of Yunnan Province, China. The areas are located in the Xishuangbanna Primeval Forest Park of Jinghong (21°98'N, 100°88'E) and Jiuxi Mountain Forest Park of Honghe (24°40'N, 103°68'E). The predominant vegetation is characterised as a tropical and subtropical forest biome. The climate is hot and humid, with annual precipitation between 1,100 to 1,600 mm and an average temperature that ranges from 17.2 to 26.4 °C. Sixty decayed wood samples were collected, thirty from each area, during July to August in 2016-2018. The samples were stored in sterile plastic bags and transported under refrigeration to the laboratory over a period of no more than 24 h. The yeast strains were isolated from rotting wood samples in accordance with the methods described by Lopes et al. (2016). Each sample (1 g) was added to 20 ml sterile d-xylose medium (yeast nitrogen base 0.67%, d-xylose 0.5%, chloramphenicol 0.02%, pH 5.0 ± 0.2) in a 150 ml Erlenmeyer flask and then cultured for 3-10 days on a rotary shaker. Subsequently, 0.1 ml aliquots of the enrichment culture and appropriate decimal dilutions were spread on d-xylose agar plates and then incubated at 25 °C for 3-4 days. Different yeast colony morphotypes were then isolated by repeated plating on yeast extract-malt extract (YM) agar (1% glucose, 0.5% peptone, 0.3% yeast extract and 0.3% malt extract, pH 5.0 ± 0.2) and stored on YM agar slants at 4 °C or in 15% glycerol at -80 °C.

Morphological, physiological and biochemical studies
Morphological and physiological properties were determined according to Kurtzman et al. (2011). Induction of the sexual stage was tested by incubating single or mixed cultures of each of the two strains on cornmeal (CM) agar, 5% malt extract (ME) agar, dilute (1:9 and 1:19) V8 agar or yeast carbon base plus 0.01% ammonium sulphate (YCBAS) agar at 25 °C for 2 months (Cadete et al. 2013;Lopes et al. 2016). Assimilation of carbon and nitrogen compounds and growth requirements were tested at 25 °C. The effects of temperature from 25-40 °C were examined in liquid culture and on agar plates. Ethanol was determined with alcohol oxidase (Sangon Biotech, China) and peroxidase (Sangon Biotech, China), as described previously (Alves et al. 2007).

DNA extraction, PCR amplification and nucleotide sequencing
Genomic DNA was extracted from the yeasts using the Ezup Column Yeast Genomic DNA Purification Kit, according to the manufacturer's protocol (Sangon Biotech, China). The nuc rDNA ITS1-5.8S-ITS2 (ITS) region was amplified using the primer pair ITS1/ITS4 (White et al. 1990). The D1/D2 domain of the nuc 28S rDNA was amplified using the primer pair NL1/NL4 (Kurtzman and Robnett 1998). The following thermal profile was used to amplify the ITS and nuc 28S regions: an initial denaturation step of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 51 °C and 40 s at 72 °C, with a final extension of 10 min at 72 °C (Liu et al. 2016). PCR products were directly purified and sequenced by Sangon Biotech Inc. (Shanghai, China). We determined the identity and accuracy of the newly-obtained sequences by comparing them to sequences in GenBank and assembled them using BioEdit (Hall 1999). Newly-obtained sequences were then submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/; Table 1).

Phylogenetic analyses
The sequences obtained from this study and the reference sequences downloaded from GenBank (Table 1) were aligned using MAFFT v. 6 (Katoh and Toh 2010) and manually edited using MEGA v. 7 (Kumar et al. 2016). The best-fit nucleotide substitution models for each gene were selected using jModelTest v2.1.7 (Darriba et al. 2012), according to the Akaike Information Criterion. Phylogenetic analyses of the combined gene regions (ITS and nuc 28S) were performed using the Maximum Likelihood (ML) and Bayesian Inference (BI) methods. Candida argentea CBS 12358 was chosen as the outgroup. ML analysis was performed using MEGA v7 with the GTR+I+G model (Nei and Kumar 2000) and 1,000 rapid bootstrap replicates to estimate branch confidence. BI analysis was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0b4 (Ronquist and Huelsenbeck 2003). Two MCMC chains, started from random trees for 1,000,000 generations and trees, were sampled every 100 th generation, resulting in a total of 10,000 trees. The first 25% of the trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP) were estimated in the remaining 7,500 trees. The phylogenetic trees from the ML and BI analyses were displayed using Mega 7 and FigTree v1.4.3 (Rambaut 2016), respectively.

Phylogenetic analyses
The combined nuclear dataset (ITS and nuc 28S) was analysed to infer the interspecific relationships within the larger Spathaspora/Candida albicans/Lodderomyces clade of Debaryomycetaceae. The dataset consisted of 72 sequences including the outgroup, Candida argentea (culture CBS 12358). A total of 944 characters including gaps (391 for ITS and 553 for nuc 28S) were included in the phylogenetic analysis. The best nucleotide substitution model for ITS and nuc 28S was GTR+I+G. ML and BI analyses of the combined dataset resulted in phylogenetic reconstructions with similar topologies and the average standard deviation of split frequencies was 0.011210 (BI). In the ML phylogenetic tree (Figure 1), thirteen strains formed five single clades with high to full support (100% in ML and 0.99 or 1.00 in BI) and clustered in the clade that comprised most species of Spathaspora. Phylogenetically, S. elongata and S. mengyangensis clustered together with high support (84% in ML and 0.91 in BI), while S. jiuxiensis and S. parajiuxiensis clustered together with strong support (100% in ML and 1.00 in BI). Two strains of S. rosae formed a unique lineage with S. allomyrinae, but with low support.  Etymology. Elongata refers to the elongate ascospores of this yeast. Description. After 3 days of culture in YM broth at 25 °C, the cells are ovoid (3-4 × 3-7 μm) and occur singly or in pairs (Fig. 2a). Budding is multilateral. Sediment is formed after a month, but a pellicle is not observed. After 3 days of growth on YM agar at 25 °C, colonies are white to cream-coloured, butyrous and smooth with entire margins. After 14 days at 25 °C, on Dalmau plate culture on CM agar, pseudohyphae are present, but true hyphae are not formed (Fig. 2b). Sporulation occurs on dilute (1:19) V8 agar after 14 days at 25 °C. Unconjugated asci are formed from single cells with one elongated ascospore which are tapered and curved at the ends (Fig. 2c). Glucose, galactose, maltose and sucrose are weakly fermented. Xylose fermentation is absent using Durham tubes, but ethanol is produced from xylose when determined with alcohol oxidase and peroxidase tests. Glucose, d-ribose, d-xylose, d-arabinose, sucrose, maltose, trehalose, methyl α-d-glucoside, cellobiose, salicin, arbutin, inulin, ribitol, d-glucitol, d-mannitol, 2-keto-d-gluconate, succinate, citrate and ethanol are assimilated. No growth occurs with galactose, l-sorbose, d-glucosamine, l-arabinose, l-rhamnose, melibiose, lactose, raffinose, melezitose, glycerol, erythritol, xylitol, galactitol, myo-inositol, d-glucono-1, 5-lactone, 5-keto-d-gluconate, d-gluconate, d-glucuronate, dl-lactate or methanol. For the assimilation of nitrogen compounds, growth on ethylamine, l-lysine, glucosamine or d-tryptophan is present, whereas growth on nitrate, nitrite, cadaverine, creatine, creatinine or imidazole is absent. Growth is observed at 37 °C but not at 40 °C. Growth in the presence of 1% acetic acid is present,  Notes. Four strains, representing Sp. elongata, clustered in a well-supported phylogenetic clade that is closely related to Sp. mengyangensis, another new species proposed in this paper and C. subhashii. The nucleotide differences between Sp. elongata and Sp. mengyangensis were 2.5% substitutions in the D1/D2 domain and 5.2% substitutions in the ITS region (Groenewald et al. 2016). Similarly, Sp. elongata and C. subhashii showed differences of 3.9% substitutions in the D1/D2 domain and 5.9% substitutions in the ITS region (Groenewald et al. 2016). Physiologically, Sp. elongata can be differentiated from its close relative, Sp. mengyangensis, based on its growth in citrate and the presence of 1% acetic acid, which are present for Sp. elongata and absent for Sp. mengyangensis. Moreover, Sp. elongata weakly ferments glucose, galactose, maltose and sucrose and grows at 37 °C, but Sp. mengyangensis does not.  Etymology. Mengyangensis refers to the geographical origin of the type strain of this species.
Description. In YM broth after 3 days at 25 °C, cells are ovoid (3-7 × 5-7.5 μm) and occur singly or in pairs (Fig. 3a). Budding is multilateral. Sediment is formed after a month, but a pellicle is not observed. After 3 days of growth on YM agar at 25 °C, colonies are white to cream-coloured, butyrous and smooth with entire margins. After 14 days at 25 °C on Dalmau plate culture on CM agar, pseudohyphae are present, but true hyphae are not formed (Fig. 3b). Sporulation occurs on CM agar after 14 days at 25 °C. Unconjugated asci are formed from single cells with one elongated ascospore which are tapered and curved at the ends (Fig. 3c). Xylose fermentation is negative using Durham tubes, but ethanol is produced from xylose when determined with alcohol oxidase and peroxidase tests. Glucose, d-ribose, d-xylose, sucrose, maltose, trehalose, methyl α-d-glucoside, cellobiose, salicin, arbutin, inulin, ribitol, d-glucitol, d-mannitol, 2-keto-d-gluconate, succinate and ethanol are assimilated. No growth occurs with galactose, l-sorbose, d-glucosamine, l-arabinose, d-arabinose, l-rhamnose, melibiose, lactose, raffinose, melezitose, glycerol, erythritol, xylitol, galactitol, myo-inositol, dglucono-1, 5-lactone, 5-keto-d-gluconate, d-gluconate, d-glucuronate, dl-lactate, citrate or methanol. For the assimilation of nitrogen compounds, growth on ethylamine, l-lysine, glucosamine or d-tryptophan is present, whereas growth on nitrate, nitrite, cadaverine, creatine, creatinine or imidazole is absent. Growth is observed at 30 °C, but not at 35 °C. Growth in the presence of 10% NaCl plus 5% glucose, 0.01% cycloheximide and 1% acetic acid is absent. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are negative. Notes. Phylogenetic analyses show that Sp. mengyangensis is closely related to Sp. elongata and C. subhashii; however, the independent phylogenetic position and different physiological characters can distinguish Sp. mengyangensis from its sister species Sp. elongata (as mentioned above). Similarly, Sp. mengyangensis differed from C. subhashii by 2.8% substitutions in the D1/D2 domain and 7.8% substitutions in the ITS region (Groenewald et al. 2016). Physiologically, Sp. mengyangensis can be differentiated from C. subhashii by the ability to assimilate d-ribose, trehalose, d-glucitol and d-mannitol and the inability to assimilate galactose, l-arabinose and melezitose. In addition, C. subhashii can grow at 40 °C, but Sp. mengyangensis cannot.  Etymology. Jiuxiensis refers to Jiuxi Mountain, the mountain from which it was collected.

Spathaspora jiuxiensis
Description. In YM broth after 3 days at 25 °C, cells are ovoid to elongate (3-6 × 3.5-9 μm) and occur singly or in pairs (Fig. 4a); pseudohyphae are present. Budding is multilateral. Sediment is formed after a month, but a pellicle is not observed. After 3 days of growth on YM agar at 25 °C, colonies are white to cream-coloured, butyrous and smooth with entire margins. After 12 days at 25 °C on Dalmau plate culture on CM agar, pseudohyphae and true hyphae are formed (Fig. 4b). Asci or signs of conjugation were not seen on the sporulation media used. Glucose and maltose are weakly fermented. Xylose fermentation is negative using Durham tubes, but ethanol is produced from xylose when determined with alcohol oxidase and peroxidase tests. Glucose,sucrose,maltose,trehalose,cellobiose,salicin,arbutin,melezitose,inulin,ribitol,succinate and ethanol are assimilated. No growth occurs with galactose,melibiose,lactose,raffinose,glycerol,erythritol,xylitol,galactitol,citrate, For the assimilation of nitrogen compounds, growth on l-lysine, glucosamine or d-tryptophan is present, whereas growth on nitrate, nitrite, ethylamine, cadaverine, creatine, creatinine or imidazole is absent. Growth is observed at 35 °C, but not at 37 °C. Growth in the presence of 0.01% cycloheximide is present, but growth in the presence of 0.1% cycloheximide, 10% NaCl plus 5% glucose and 1% acetic acid is Notes. The two strains, both representing Sp. jiuxiensis, cluster in a well-supported clade in the phylogenetic analysis and are closely related to Sp. parajiuxiensis. The nucleotide differences between these two new species were 1.4% substitutions in the D1/D2 domain and 4.6% substitutions in the ITS region (Groenewald et al. 2016). These two sister species can also be differentiated by a few physiological characteristics; Sp. jiuxiensis can assimilate dl-lactate and Sp. parajiuxiensis can grow at 37 °C.  Etymology. Paraluxiensis refers to its close phylogenetic relationship to Sp. luxiensis. Description. In YM broth after 3 days at 25 °C, cells are ovoid to elongate (3.5-4 × 7-15 μm) and occur singly or in pairs (Fig. 5a); pseudohyphae are present. Budding is multilateral. Sediment is formed after a month, but a pellicle is not observed. After 3 days of growth on YM agar at 25 °C, colonies are white to cream-coloured, butyrous and smooth with entire margins. After 12 days at 25 °C on Dalmau plate culture on CM agar, pseudohyphae and true hyphae are formed (Fig. 5b). Sporulation occurs on 5% ME agar after 14 days at 25 °C. Unconjugated asci are formed from single cells with one elongated ascospores which are tapered and curved at the ends (Fig. 5c) Glucose and maltose are weakly fermented. Xylose fermentation is negative using Durham tubes, but ethanol is produced from xylose when determined with alcohol oxidase and peroxidase tests. Glucose,sucrose,maltose,trehalose,cellobiose,salicin,arbutin,melezitose,inulin,ribitol,succinate and ethanol are assimilated. No growth occurs with galactose, l-sorbose, l-arabinose, d-arabinose, l-rhamnose, melibiose, lactose, raffinose, glycerol, erythritol, xylitol, galactitol, myo-inositol, dglucono-1, 5-lactone, 5-keto-d-gluconate, d-gluconate, d-glucuronate, dl-lactate, citrate, l-arabinitol or methanol. For the assimilation of nitrogen compounds, growth on l-lysine, glucosamine or d-tryptophan is present, whereas growth on nitrate, nitrite, ethylamine, cadaverine, creatine, creatinine or imidazole is absent. Growth is observed at 37 °C, but not at 40 °C. Growth in the presence of 0.01% cycloheximide is present, but growth in the presence of 0.1% cycloheximide, 10% NaCl plus 5% glucose and 1% acetic acid is absent. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are negative.

Spathaspora parajiuxiensis
Additional isolate examined. China, Yunnan Province, Honghe Prefecture, Luxi County, in rotting wood in Jiuxi Mountain Forest Park, July 2016, R.C. Ren & L. Zhang, NYNU 16632.  Etymology. Rosae was named in honour of Carlos A. Rosa for his contributions in yeast taxonomy.
Description. In YM broth after 3 days at 25 °C, cells are ovoid to elongate (4-7 × 5-16 μm) and occur singly or in pairs (Fig. 6a). Budding is multilateral. Sediment is formed after a month, but a pellicle is not observed. After 3 days of growth on YM agar at 25 °C, colonies are white to cream-coloured, butyrous and smooth with entire margins. After 7 days at 25 °C, on Dalmau plate culture on CM agar, pseudohyphae and true hyphae are formed (Fig. 6b). Asci or signs of conjugation are not seen on sporulation media used. Xylose fermentation is negative using Durham tubes, but ethanol is produced from xylose when determined with alcohol oxidase and peroxidase tests. Glucose,sucrose,maltose,trehalose,cellobiose,salicin,arbutin,inulin,ribitol,succinate,citrate and ethanol are assimilated. No growth occurs with galactose,melibiose,lactose,raffinose,melezitose,glycerol,erythritol,xylitol,galactitol,. For the assimilation of nitrogen compounds, growth on ethylamine, l-lysine, glucosamine or d-tryptophan is present, whereas growth on nitrate, nitrite, cadaverine, creatine, creatinine or imidazole is absent. Growth is observed at 35 °C, but not at 37 °C. Growth in the presence of 0.01% cycloheximide is present, but growth in the presence of 0.1% cycloheximide, 10% NaCl plus 5% glucose and 1% acetic acid is absent. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are negative.
Notes. Three strains, representing Sp. rosae, grouped in a well-supported clade and appear to be most closely related to Sp. allomyrinae (Wang et al. 2016). The nucleotide differences between Sp. rosae and its close relative, Sp. allomyrinae, were 10.2% substitutions in the D1/D2 domain and 11% substitutions in the ITS region (Groenewald et al. 2016). Physiologically, Sp. rosae can be differentiated from Sp. allomyrinae, based on growth in galactose, melezitose, xylitol and 5-keto-d-gluconate, which are positive for Sp. allomyrinae and negative for Sp. rosae. Moreover, Sp. allomyrinae weakly ferments glucose, galactose, maltose and cellobiose, but Sp. rosae does not.

Two new combinations
In addition to the previously-described taxa, two new combinations are proposed herein and their descriptions refer to relevant protologues.

Discussion
Spathaspora is distributed worldwide with 12 species identified from rotting wood and insects. In China, three species of Spathaspora have been previously reported (Ren et al. 2013;Wang et al. 2016). In the present study, five additional species, Sp. elongata, Sp. jiuxiensis, Sp. mengyangensis, Sp. parajiuxiensis and Sp. rosae (Fig. 1), were recorded in addition to previously-known species. Thus, to our knowledge, eight species of Spathaspora are currently known from China. Of these eight species, only two species, Sp. gorwiae and Sp. passalidarum, were reported in China up until 2013(Ren et al. 2013). The remaining six species, namely Sp. allomyrinae, Sp. elongata, Sp. jiuxiensis, Sp. mengyangensis, Sp. parajiuxiensis and Sp. rosae, were recorded from 2016 to 2018. Given this history, it is most likely that more species will be found. Nonetheless, this number is significant when compared to the total diversity of 11 species of Spathaspora reported for South America (Cadete et al. , 2013Lopes et al. 2016;Morais et al. 2017;Varize et al. 2018). Further studies are needed to document the overall diversity of species of Spathaspora in China, especially in the southwest regions.
The phylogenetic relationship of Spathaspora has been unclear until now, mainly due to its polyphyletic nature (Daniel et al. 2014;Morais et al. 2017;Varize et al. 2018). In this article, we used more available type strains to revise this genus, based on a phylogenetic analysis of the combined ITS and nuc 28S rDNA sequences. As shown in Figure 1, three main groups were reconstructed and the results showed that Spathaspora is not a monophyletic group, but rather is polyphyletic with several Candida species included. Sp. passalidarum, the type species of the genus, C. jeffriesii, C. materiae, Sp. arborariae, Sp. brasiliensis, Sp. girioi and Sp. suhii form a core group that is well supported by phylogeny. This result is similar to the results of previous phylogenetic analyses of nuc 28S rDNA sequences (Morais et al. 2017;Varize et al. 2018). Therefore, two Candida species, C. materiae and C. jeffriesii, are transferred to Spathaspora as new combinations because of their phylogenetic placement within that genus.
The second group is composed of ten distinct species, including the four species Sp. elongata, Sp. mengyangensis, Sp. jiuxiensis and Sp. parajiuxiensis described in this study. Typical ascospores are formed by Sp. elongata, Sp. mengyangensis, Sp. parajiuxiensis and Sp roraimanensis, but other members of the group are known from their asexual cycle only.
The species Sp. allomyrinae, which shares the unique ascospore morphology of the genus, fell outside a larger Spathaspora clade, as in the nuc 28S-based phylogeny proposed by Morais et al. (2017). However, this species is joined by Sp. rosae, which is described in the current study, in a third cluster consisting of Spathaspora in our phylogenetic analysis (Fig. 1). Placement of Sp. allomyrinae and Sp. rosae is only weakly supported and continued assignment to the genus will require verification from more robust datasets, such as whole genome sequences. Morais et al. (2017) described the species Sp. boniae, based on two strains producing asci containing elongate ascospores with curved ends typical of the genus Spathaspora. Our phylogenomic analysis showed that Sp. boniae clusters with C. blackwellae and C. parablackwellae to form a distinct clade outside a larger Spathaspora clade. This result was also supported by previous phylogenetic analyses on this clade using nuc 28S rDNA sequences (Morais et al. 2017;Varize et al. 2018;Zhai et al. 2019). These results suggest that the genus Spathaspora should be limited to species in the group comprising the type species Sp. passalidarum. This clade, which has been treated previously as members of Spathaspora, may represent a separate genus, despite the morphological characteristics of the included species and isolates are similar to Spathaspora. Therefore, whole genome sequencing of all Spathaspora species and those of related genera, combined with the discovery of new species of the clade, is needed to clarify the possible heterogeneity of this genus.
Spathaspora is a cosmopolitan genus, but most known species have relatively-distinct habitats or regional locations. Currently, most Spathaspora species are known from East Asia (mainly in China) and South America. Although the taxonomy of Spathaspora has received much attention in recent years, many regions in the world are under-sampled and more under-described indigenous Spathaspora species will undoubtedly be discovered in the future as with most microfungal genera (Hyde et al. 2020). Our study indicates that there is a high species diversity of Spathaspora waiting to be discovered in rotting wood in tropical and subtropical southwest China and nearby areas as with other genera (Hyde et al. 2018).