Three new Scheffersomyces species associated with insects and rotting wood in China

Ran-Ran Jia; Shi-Long Lv; Chun-Yue Chai; Feng-Li Hui

doi:10.3897/mycokeys.71.56168

Abstract

Three species of Scheffersomyces were identified during a diversity study of yeasts. All three are associated with insects and rotting wood in China. Phylogenetic analyses of a genomic dataset combining ITS and nrLSU revealed that these new collections are distinct from known species, thus three new species are introduced i.e. S. jinghongensis, S. paraergatensis, and S. anoplophorae. In our phylogenetic analyses, Scheffersomyces jinghongensis possesses a strong independent lineage and is closely related to S. titanus. S. paraergatensis is closely related to S. ergatensis, while S. anoplophorae is related to S. stambukii. Several differences in physiological traits and molecular data indicate that S. jinghongensis, S. paraergatensis, and S. anoplophorae are three newly identified species.

Keywords

Debaryomycetaceae, Saccharomycetales, taxonomy, d-xylose-fermenting yeast

Introduction

Kurtzman and Suzuki (2010) introduced the genus Scheffersomyces Kurtzman. ex M. Suzuki. (2010) to include d-xylose-fermenting species in the Pichia stipitis clade, viz. P. segobiensis, P. spartinae, and P. stipitis. The genus Scheffersomyces was later expanded by including seven related Candida species as new combinations and by three novel species, S. illinoinensis, S. quercinus, and S. virginianus, which were discovered in rotting wood (Urbina and Blackwell 2012). More recently, several new species and combinations from the genus Scheffersomyces, (S. cryptocerci, S. parashehatae, S. titanus, and S. xylosifermentans) have been identified in wood-ingesting insects (Suh et al. 2013; Urbina et al. 2013; Liu et al. 2016b), and in rotting wood (S. amazonensis, S. ergatensis, S. henanensis, and S. stambukii) (Ren et al. 2013; Urbina and Blackwell 2013; Lopes et al. 2018). This genus comprises 21 species, though the status of S. gosingicus and S. lignicola is currently unclear due to nomenclatural issues regarding the Melbourne Code (Index Fungorum, accessed 17.07.2020). Of the 19 valid species, five of them are known to form ascospores (Kurtzman 2011; Ren et al. 2013, Liu et al. 2016b) and 14 of them display asexual morphs (Urbina and Blackwell 2012; Suh et al. 2013; Urbina and Blackwell 2013; Urbina et al. 2013; Lopes et al. 2018).

Species in the genus Scheffersomyces are characterized by pseudohyphae formation, an inability to utilize nitrates, and the possession of the co-enzyme Q-9 (Kurtzman and Suzuki 2010; Kurtzman 2011). Asexual reproduction occurs via multilateral budding on a narrow base; sexual reproduction occurs via the formation of 1–2 hat-shaped ascospores released soon after formation (Kurtzman 2011). Several Scheffersomyces species, such as S. henanensis, S. shehatae, and S. stipitis, strongly ferment d-xylose, which is important for producing bioethanol from the residue of plant waste (du Preez and van der Walt 1983; Agbogbo and Wenger 2006; Ren et al. 2013; Suh et al. 2013; Lopes et al. 2018). Despite the existence of these microbes, obtaining high ethanol yields from pentose sugars on a large scale remains a challenge (Chandel et al. 2011) because scientists have yet to identify microbes that convert pentose sugars into ethanol at high yields, while withstanding fermentation inhibitors (Agbogbo and Wenger 2006; Long et al. 2012). Therefore, there is a need to identify new yeasts capable of efficient xylose fermentation in order to produce bioethanol.

Several d-xylose-fermenting yeasts were collected from different regions in China during a study on fungal diversity in insects and rotting wood. Two Scheffersomyces species, S. henanensis and S. titanus, have already been described in published studies (Ren et al. 2013; Liu et al. 2016b). In this study, the other three new species are described and characterized based on their morphology and phylogenetics, increasing the species diversity of Scheffersomyces in China.

Materials and methods

Sample collection, morphological studies, and isolation

Samples of insects and rotting wood were collected from Henan Province and Yunnan Province from 2015–2017. Strains of yeast were isolated from the insect guts according to the methods described by Liu et al. (2016a, b). Prior to dissection, the insects were placed in Petri dishes for 1–3 days without food, which eliminates some of the contaminating organisms found in the gut. Surface disinfection was performed by submersion in 95% ethanol for 1–2 min, followed by rinsing in a 0.7% saline solution. The insect gut was removed aseptically under a dissecting microscope. The gut segments were streaked on acidified yeast extract–malt extract (YM) agar plates (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, 2% plain agar; pH adjusted to 3.5 with HCl) and incubated at 25 °C for 3–4 days. Strains were isolated from the samples of rotting wood via the enrichment technique with YM broth, supplemented by 0.025% sodium propionate and 0.01% chloramphenicol (Ren et al. 2013). All yeast isolates were purified by repeated streak-inoculation on YM agar plates and preserved in 15% glycerol at -80 °C.

The morphological, physiological, and biochemical properties were determined according to those used by Kurtzman et al. (2011). The beginning of the sexual stage was determined by incubating single or mixed cultures of each of the two strains on cornmeal (CM) agar, YM agar, or 5% malt extract (ME) agar at 25 °C for 6 weeks (Ren et al. 2013, Liu et al. 2016b). The assimilation of carbon and nitrogen compounds and related growth requirements were tested at 25 °C. The effects of temperature from 25–40 °C were examined in a liquid culture and on agar plates.

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from the yeast using an Ezup Column Yeast Genomic DNA Purification Kit, according to the manufacturer’s instructions (Sangon Biotech, Shanghai, China). The nuc rDNA ITS1-5.8S-ITS2 (ITS) region was amplified using primer pairs ITS1/ITS4 (White et al. 1990). The D1/D2 domain of nrLSU rDNA (nrLSU) was amplified using the primer pair NL1/NL4 (Kurtzman and Robnett 1998). The PCR protocols used for the ITS and nrLSU were those outlined by Liu et al. (2016a). PCR products were directly purified and sequenced by Sangon Biotech Inc. (Shanghai, China). We confirmed the identity and accuracy of the resulting sequences by assembling them using BioEdit and comparing them to sequences in GenBank (Hall 1999). The sequences were then submitted to GenBank (https://www. ncbi.nlm.nih.gov/genbank/; Table 1).

Table 1.

Download as

CSV

XLSX

Sequences used in molecular phylogenetic analysis. Entries in bold are newly generated for this study.

Species	Strain	ITS	D1/D2
Candida broadrunensis	CBS 11838^T	HQ263349	NG_064320
Candida thasaenensis	CBS 12529^T	NR_111028	NG_055174
*Scheffersomyces anoplophorae*	NYNU 15730^T	KU128714	KU128724
*Scheffersomyces anoplophorae*	NYNU 15733	MT133542	MT133540
Scheffersomyces coipomoensis	NRRL Y-17651^T	HQ652070	HQ651966
Scheffersomyces cryptocerci	CBS 12658^T	NR_120091	NG_055704
Scheffersomyces ergatensis	NRRL Y-17652^T	EU343826	U45746
Scheffersomyces gosingicus	CBS 11433 ^T	HQ999978	HQ999955
Scheffersomyces henanensis	CBS 12475 ^T	HQ127627	HQ127626
Scheffersomyces illinoinensis	NRRL Y-48827^T	JN943261	JN703959
Scheffersomyces insectosa	NRRL Y-12854^T	NR_111587	NG_055695
*Scheffersomyces jinghongensis*	NYNU 17926^T	MG255722	MG255714
*Scheffersomyces jinghongensis*	NYNU 17977	MT133547	MT133543
Scheffersomyces lignicola	CBS 10610 ^T	HQ652074	AY845350
Scheffersomyces lignosus	NRRL Y-12856^T	R_120020	NG_055694
*Scheffersomyces paraergatensis*	NYNU 16782^T	KY213803	KY213826
*Scheffersomyces paraergatensis*	NYNU 16969	MT133541	MT133546
Scheffersomyces parashehatae .	CBS 12535^T	NR_138230	NG_055697
Scheffersomyces queiroziae	NRRL Y-48722^T	HM566445	HM566445
Scheffersomyces quercinus	NRRL Y-48825^T	JN943260	JN703957
Scheffersomyces segobiensis	NRRL Y-11571^T	NR_111217	NG_055696
Scheffersomyces shehatae	NRRL Y-12858^T	JN943264	JQ025409
Scheffersomyces spartinae	NRRL Y-7322^T	HQ876044	U45764
Scheffersomyces stambukii	CBS 14217^T	KT033721	KT033720
Scheffersomyces stipitis	NRRL Y-7124^T	JN943257	U45741
Scheffersomyces titanus	CBS 13926^T	KP054263	KP054262
Scheffersomyces virginianus	NRRL Y-48822^T	NR_120018	NG_055702
Scheffersomyces xylosifermentans	CBS 12540^T	KY105362	KY109586
Saccharomyces cerevisiae	CBS 1171^T	NR_111007	NG_042623

Phylogenetic analysis

The sequences obtained from this study and the reference sequences obtained 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 combined gene regions (ITS and nrLSU) were performed using MEGA v7 for maximum parsimony (MP) analysis (Kumar et al. 2016) and PhyML v3.0 for Maximum Likelihood (ML) analysis (Guindon et al. 2010). Saccharomyces cerevisiae CBS 1171^T was chosen as the outgroup after consulting Liu et al. (2016b) and Suh et al. (2013).

MP analysis was performed using a heuristic search option with tree-bisection reconnection (TBR) branch swapping (Nei and Kumar 2000) and 1,000 random sequence additions. ML analysis was performed using GTR+I+G models for each partition (Nei and Kumar 2000) and a proportion of invariant sites with 1000 rapid bootstrap replicates. The phylogenies from MP and ML analyses were displayed using Mega 7 and FigTree v1.4.3 (Rambaut 2016), respectively. Bootstrap support values ≥ 50% are shown at the nodes.

Results

The alignment was based on the combined sequence dataset (ITS and nrLSU) and included 26 in-group taxa and one out-group taxon (Saccharomyces cerevisiae CBS 1171^T), comprised of 1085 characters in the aligned matrix. Of these, 541 characters were constant, 356 variable characters were parsimony-uninformative, and 188 characters were parsimony-informative. The MP analysis resulted in three equally parsimonious trees; the first tree (TL = 589, CI = 0.640, RI = 0.833, RC = 0.533) is shown in Fig. 1. The three MP trees were identical, except for the species order within different clades. ML analyses revealed that tree topologies of the best tree were identical to those of the MP tree (not shown). The sequences of each three new species formed a well-supported monophyletic group (MP = 98–100%, ML = 100%). S. paraergatensis and S. anoplophorae were related to S. ergatensis and S. stambukii, respectively, while S. jinghongensis possessed a strongly independent lineage that is distinct from other species (Fig. 1).

Figure 1.

Maximum parsimony phylogenetic tree generated from analysis of combined ITS and nrLSU sequences dataset for 24 taxa of Scheffersomyces and related Candida species. Saccharomyces cerevisiae CBS 1171^T is the out-group taxon. Bootstrap values ≥ 50% for MP/ML analyses are presented at the branches. Scale bar = 20 nucleotide substitutions. The species from this study are indicated in bold letters.

Taxonomy

Scheffersomyces jinghongensis C.Y. Chai & F.L. Hui, sp. nov.

MycoBank No: 835004

Figure 2

Etymology

The species name jinghongensis (N.L. fem. adj.) refers to the geographical origin of the type strain of this species.

Holotype

NYNU 17926^T.

Isolation data

China, Yunnan Province, Jinghong, in rotting wood, under a tropical rainforest, July 2017, K.F. Liu & Z.W. Xi (ex-holotype: CICC 33270; CBS 15230).

Description

The cells are ovoid to elongate (3–4 ×3–7.5 μm) and occur singly or in pairs after being placed in YM broth for 3 days at 25 °C (Fig. 2A). Budding is multilateral. After 3 days of growth on YM agar at 25 °C, the colonies are white to cream-colored, buttery, and smooth, with entire margins. After 7 days at 25 °C on a Dalmau plate culture with CM agar, pseudohyphae were observed but true hyphae were not (Fig. 2B). Asci or signs of conjugation were not observed on sporulation media. Glucose, galactose, trehalose, cellobiose, and d-xylose (weak) are fermented, but maltose, sucrose, melibiose, lactose, melezitose, raffinose, and inulin are not. Glucose, galactose, l-sorbose, d-glucosamine, d-ribose, d-xylose, sucrose, maltose, trehalose, cellobiose, arbutin, melezitose, inulin, glycerol, erythritol, ribitol, xylitol, d-glucitol, d-mannitol, d-glucono-1, 5-lactone, 2-keto-d-gluconate, d-glucuronate, succinate, and ethanol are assimilated. No growth was observed in l-arabinose, d-arabinose, l-rhamnose, methyl α-d-glucoside, salicin, melibiose, lactose, raffinose, l-arabinitol, galactitol, myo-inositol, 5-keto-d-gluconate, d-gluconate, dl-lactate, citrate, or methanol. For the assimilation of nitrogen compounds, growth on l-lysine, glucosamine, or d-tryptophan is positive, while growth on nitrate, nitrite, ethylamine, cadaverine, creatine, creatinine, or imidazole is negative. Growth is observed at 37 °C but not at 40 °C. Growth in the presence of 0.1% cycloheximide is positive, but growth in the presence of 10% NaCl with 5% glucose and 1% acetic acid is negative. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are also negative.

Additional isolate examined

China, Yunnan Province, Jinghong, in rotting wood, under a tropical rainforest, July 2017, K.F. Liu & Z.W. Xi, NYNU 17977.

GenBank accession numbers

holotype NYNU 17926^T (ITS:MG255722; nrLSU D1/D2: MG255714) ; additional isolate NYNU 17977 (ITS: MT133547; nrLSU D1/D2: MT133543).

Notes

Two strains representing S. jinghongensis were grouped in an independent lineage and are related to S. titanus and other Scheffersomyces species. The nucleotide differences between the new species and the close relative S. titanus (Liu et al. 2016b) are 1.6% substitutions in the D1/D2 domain and 4.9% substitutions in the ITS region, respectively. Physiologically, S. jinghongensis can be differentiated from S. titanus based on growth in l-arabinose, l-rhamnose, methyl α-d-glucoside, salicin, lactose, myo-inositol, and 5-keto-d-gluconate, all of which were positive for S. titanus and negative for the new species. Additionally, S. jinghongensis ferments cellobiose and grows at 37 °C, but not for S. titanus.

Figure 2.

Morphology of S. jinghongensis. A budding cells B pseudohyphae. Scale bars: 10 μm.

Scheffersomyces paraergatensis C.Y. Chai & F.L. Hui, sp. nov.

MycoBank No: 835005

Figure 3

Etymology

The species name paraergatensis (Gr. prep.) refers to its phylogenetic similarity to S. ergatensis.

Holotype

NYNU 16782^T.

Isolation data

China, Henan Province, Nanyang, in rotting wood, under a mixed forest, July 2016, K.F. Liu & Z.W. Xi (ex-holotype: CICC 33165; CBS 14694).

Description

The cells are ovoid to elongate (2.5–5×3.5–6 μm) and occur singly or in pairs after grown in a YM broth for 3 days at 25 °C (Fig. 3A). Budding is multilateral. After 3 days of growth on YM agar at 25 °C, the colonies are white to cream-colored, buttery, and smooth with entire margins. After 7 days at 25 °C, on a Dalmau plate culture with CM agar, pseudohyphae were observed but true hyphae were not. Conjugated asci formed after 6 days at 25 °C on CM agar and 5% ME agar, with each ascus containing one or two hat-shaped ascospores (Fig. 3B). Glucose, galactose, and d-xylose are weakly fermented, but maltose, sucrose, trehalose, melibiose, lactose, cellobiose, melezitose, raffinose, and inulin are not. Glucose, galactose, d-ribose, d-xylose, l-arabinose, d-arabinose, sucrose, maltose, trehalose, methyl α-d-glucoside, cellobiose, salicin, arbutin, lactose, raffinose, inulin, glycerol, ribitol, xylitol, d-glucitol, d-mannitol, d-glucono-1, 5-lactone, d-gluconate, succinate, citrate, and ethanol are assimilated. No growth was observed in l-sorbose, d-glucosamine, l-rhamnose, melibiose, melezitose, erythritol, galactitol, myo-inositol, 2-keto-d-gluconate, 5-keto-d-gluconate, d-glucuronate, dl-lactate, or methanol. For the assimilation of nitrogen compounds, growth on l-lysine, glucosamine, and d-tryptophan is positive, while growth on nitrate, nitrite, ethylamine, cadaverine, creatine, creatinine, and imidazole is negative. Growth was observed at 30 °C, but not at 35 °C. Growth in the presence of 0.1% cycloheximide is positive, but growth in the presence of 10% NaCl with 5% glucose and 1% acetic acid is negative. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are also negative.

Additional isolate examined

China, Henan Province, Nanyang, in rotting wood, under a oak forest, August 2016, K.F. Liu & Z.W. Xi, NYNU 16969.

GenBank accession numbers

holotype NYNU 16782^T (ITS: KY213803; nrLSU D1/D2: KY213826) ; additional isolate NYNU 16969 (ITS: MT133541; nrLSU D1/D2: MT133546).

Notes

Two strains formed a group related to S. ergatensis and Candida broadrunensis, which represent a new species, S. paraergatensis. The nucleotide differences between the new species and its closest relative, S. ergatensis, were 1.1% substitutions in the D1/D2 domain and 0.8% substitutions in ITS region, respectively. Similarly, S. paraergatensis and C. broadrunensis displayed 0.9% substitutions in the D1/D2 domain and 2.4% substitutions in the ITS region, respectively. Physiologically, S. paraergatensis can be differentiated from its closest relative, S. ergatensis (Lachance et al. 2011), by its ability to ferment d-xylose and assimilate l-arabinose, raffinose, inulin, and d-gluconate and its inability to assimilate l-sorbose. Additionally, S. paraergatensis can grow in 0.1% cycloheximide and at 30 °C, but not for S. ergatensis.

Figure 3.

Morphology of S. paraergatensis. A budding cells B ascus and ascospores. Scale bars: 10 μm.

Scheffersomyces anoplophorae C.Y. Chai & F.L. Hui, sp. nov.

MycoBank No: 835006

Figure 4

Etymology

The species name anoplophorae (N.L. fem. Gen. n.) refers to the genus of the host beetle, Anoplophora leechi.

Holotype

NYNU 15730^T.

Isolation data

China, Henan Province, Nanyang, in the gut of Anoplophora leechi, in the People’s Park, July 2015, R.C. Ren & K.F. Liu (ex-holotype: CICC 33086; CBS 14170).

Description

The cells are spherical or ovoid (2.5–6 × 2.5–7.5 μm) and occur singly or in pairs (Fig. 4A) when placed in YM broth after 3 days at 25 °C. Budding is multilateral. After 3 days of growth on YM agar at 25 °C, the colonies are white to cream-colored, buttery, and smooth with entire margins. After 12 days at 25 °C on a Dalmau plate culture with CM agar, pseudohyphae were observed but true hyphae were not (Fig. 4B). Asci or signs of conjugation were not observed on sporulation media. Glucose, galactose, trehalose, cellobiose (weak), and d-xylose (weak) are fermented, but maltose, sucrose, melibiose, lactose, melezitose, raffinose, and inulin are not. Glucose, galactose, d-glucosamine, d-xylose, maltose, trehalose, cellobiose, salicin, glycerol, ribitol, d-glucitol, d-mannitol, d-glucono-1, 5-lactone, 2-keto-d-gluconate, 5-keto-d-gluconate, succinate, citrate, and ethanol are all assimilated. No growth was observed in l-sorbose, d-ribose, l-arabinose, d-arabinose, l-rhamnose, sucrose, methyl α-d-glucoside, arbutin, melibiose, lactose, raffinose, melezitose, inulin, erythritol, xylitol, l-arabinitol, galactitol, myo-inositol, d-gluconate, d-glucuronate, dl-lactate, or methanol. For the assimilation of nitrogen compounds, growth on l-lysine, glucosamine, or d-tryptophan is positive, while growth on nitrate, nitrite, ethylamine, cadaverine, creatine, creatinine, and imidazole is negative. Growth is observed at 37 °C, but not at 40 °C. Growth in the presence of 0.01% cycloheximide is positive, but growth in the presence of 0.1% cycloheximide, 10% NaCl with 5% glucose, and 1% acetic acid is negative. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are also negative.

Additional isolate examined

China, Henan Province, Nanyang, in the gut of Anoplophora leechi, in the People’s Park, July 2015, R.C. Ren & K.F. Liu, NYNU 15733.

GenBank accession numbers

holotype NYNU 15730^T (ITS: KU128714; nrLSU D1/D2: KU128724) ; additional isolate NYNU 15733 (ITS: MT133542; nrLSU D1/D2: MT133540).

Notes

Two strains, representing S. anoplophorae, were clustered in a well-supported clade and were phylogenetically related to S. stambukii (Lopes et al. 2018). The nucleotide differences between the new species and its closest relative, S. stambukii, were 2.3% substitutions in the D1/D2 domain and 6.6% substitutions in the ITS region, respectively. Physiologically, the ability to assimilate d-glucosamine and the inability to assimilate d-gluconate are the primary differences between S. anoplophorae and its closest relative, S. stambukii. Additionally, S. stambukii can grow in 5% glucose medium with 10% NaCl, while S. anoplophorae cannot.

Figure 4.

Morphology of S. anoplophorae. A budding cells B pseudohyphae. Scale bars: 10 μm.

Discussion

This study introduced and characterized three new d-xylose-fermenting species based on their morphology and phylogeny: S. jinghongensis, S. paraergatensis, and S. anoplophorae. While these new species share high morphological similarities with their closest relatives, S. titanus, S. ergatensis, and S. stambukii, they are different species due to their physiological traits and nucleotide differences in the D1/D2 domain and ITS region.

The genus Scheffersomyces accommodates a monophyletic group based on phylogenetic analyses of two sequence datasets (nrSSU and nrLSU) (Kurtzman and Suzuki 2010). Since it was first proposed, 18 new species and combinations have been categorized in this genus. Kurtzman and Robnett (2013) compared the species type of 70 recognized genera by analyzing sequence divergence in five-genes (nrSSU, nrLSU, EF-1a, RPB1, and RPB2), and determined that the genus Scheffersomyces is polyphyletic. S. spartinae was placed in a clade with Spathaspora passalidarum, distinct from the type species S. stipitis, although this placement was weakly supported by statistical analyses. However, Urbina and Blackwell (2012) suggested that the genus can be divided into three groups based on phylogenetic analysis of a multilocus dataset (nrLSU, ITS, COXII, and MtSm) with 14 described species. These results were later supported by Ren et al. (2013) and Suh et al. (2013). The results of our phylogenetic analyses of combined gene sequences (ITS and nr LSU) with all currently known species indicated that the genus is not monophyletic, but that it consists of three phylogenetically distinct groups (Fig. 1): (i) S. stipitis (the type species), S. coipomoensis, S. shehatae, and their related species, (ii) S. stambukii and S. anoplophorae (described in this paper) and (iii) S. gosingicus, S. spartinae, and C. thasaenensis. These results suggest that the genus Scheffersomyces should be limited to species of the group comprising the type species S. stipitis. The remaining two groups, which have previously been considered members of Scheffersomyces, could become two novel genera, although their phylogenetic relationships with other genera were not fully examined by this study (Fig. 1). As such, a careful phylogenetic analysis of Scheffersomyces species is required to clarify the possible heterogeneity of the genus.

Yeasts of the genus Scheffersomyces have been found to occupy habitats rich in xylose, including rotting wood (Ren et al. 2013; Lopes et al. 2018), wood-feeding insects (Suh et al. 2013; Urbina et al. 2013; Liu et al. 2016b), and the resulting frass (Lachance et al. 2011). We have reported the isolation and identification of several d-xylose-fermenting yeasts from wood-feeding insects and rotting wood (Ren et al. 2013; Liu et al. 2016b), including those detailed in this study. Although the samples of insects and rotting wood were collected in a relatively small geographical area in China, the d-xylose-fermenting yeasts are diverse and several of them were identified as new species (Ren et al. 2013; Liu et al. 2016b). These yeasts demonstrated several physiological traits related to the full utilization of lignocellulosic biomass, such as assimilating and fermenting xylose and/or cellobiose (Ren et al. 2013; Suh et al. 2013). Surveying d-xylose-fermenting yeasts in insects and rotting wood from various regions in different climates will help identify valuable biological and genetic resources to aid in the production of ethanol from biomass.

Acknowledgments

We sincerely thank Dr. Kai-Fang Liu, Mr. Zhi-Wen Xi, and Mr. Rong-Cheng Ren for their help with collecting specimens. This project was supported by Grant No. 31570021 from the National Natural Science Foundation of China (NSFC), P. R. China, No. 2018001 from the State Key Laboratory of Motor Vehicle Biofuel Technology.

References

Agbogbo FK, Wenger KS (2006) Effect of pretreatment chemicals on xylose fermentation by Pichia stipitis. Biotechnology Letters 28: 2065–2069. https://doi.org/10.1007/s10529-006-9192-6

Chandel AK, Chandrasekhar G, Radhika K, Ravinder R, Ravindra P (2011) Bioconversion of pentose sugars into ethanol: A review and future directions. Biotechnology and Molecular Biology Reviews 6: 8–20.

Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9: 772. https://doi.org/10.1038/nmeth.2109

du Preez JC, van der Walt JP (1983) Fermentation of Dxylose to ethanol by a strain of Candida shehatae. Biotechnology Letters 5: 357–362. https://doi.org/10.1007/BF01141138

Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59: 307–321. https://doi.org/10.1093/sysbio/syq010

Hall TA (1999) Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic acids symposium series 41: 95–98.

Katoh K, Toh H (2010) Parallelization of the MAFFT multiple sequence alignment program. Bioinformatics 26: 1899–1900. https://doi.org/10.1093/bioinformatics/btq224

Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870–1874. https://doi.org/10.1093/molbev/msw054

Kurtzman CP (2011) Scheffersomyces Kurtzman & M. Suzuki (2010). In: Kurtzman CP, Fell JW, Boekhout T (Eds) The Yeasts – a Taxonomic Study, 5^th edn, vol. 2. Elsevier, Amsterdam, 773–777. https://doi.org/10.1016/B978-0-444-52149-1.00065-3

Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73: 331–371. https://doi.org/10.1023/A:1001761008817

Kurtzman CP, Robnett CJ (2013) Relationships among genera of the Saccharomycotina (Ascomycota) from multigene phylogenetic analysis of type species. FEMS Yeast Research 13: 23–33. https://doi.org/10.1111/1567-1364.12006

Kurtzman CP, Suzuki M (2010) Phylogenetic analysis of ascomycete yeasts that form coenzyme Q-9 and the proposal of the new genera Babjeviella, Meyerozyma, Millerozyma, Priceomyces, and Scheffersomyces. Mycoscience 51: 2–14. https://doi.org/10.1007/S10267-009-0011-5

Kurtzman CP, Fell JW, Boekhout T, Robert V (2011) Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman CP, Fell JW, Boekhout T (Eds) The Yeasts – a Taxonomic Study, 5^th edn, vol. 1. Elsevier, Amsterdam, 87–110. https://doi.org/10.1016/B978-0-444-52149-1.00007-0

Lachance MA, Boekhout T, Scorzetti G, Fell JW, Kurtzman CP (2011) Candida Berkhout (1923). In: Kurtzman CP, Fell JW, Boekhout T (Eds) The Yeasts – a Taxonomic Study, 5^th edn, vol. 2. Elsevier, Amsterdam, 987–1278. https://doi.org/10.1016/B978-0-444-52149-1.00090-2

Liu XJ, Yi ZH, Ren YC, Li Y, Hui FL (2016a) Five novel species in the Lodderomyces clade associated with insects. International Journal of Systematic and Evolutionary Microbiology 66: 4881–4889. https://doi.org/10.1099/ijsem.0.001446

Liu XJ, Cao WN, Ren YC, Xu LL, Yi ZH, Liu Z, Hui FL (2016b) Taxonomy and physiological characterisation of Scheffersomyces titanus sp. nov., a new D-xylose-fermenting yeast species from China. Scientific Reports 6: 32181. https://doi.org/10.1038/srep32181

Long TM, Su YK, Headman J, Higbee A, Willis LB, Jeffries TW (2012) Co-fermentation of glucose, xylose, and cellobiose by the beetle-associated yeast, Spathaspora passalidarum. Applied and Environmental Microbiology 78: 5492–5500. https://doi.org/10.1128/AEM.00374-12

Lopes MR, Batista TM, Franco GR, Ribeiro LR, Santos ARO, Furtado C, Moreira RG, Goes-Neto A, Vital MJS, Rosa LH, Lachance MA 6, Rosa CA (2018) Scheffersomyces stambukii f.a., sp. nov., a D-xylose-fermenting species isolated from rotting wood. International Journal of Systematic and Evolutionary Microbiology 68: 2306–2312. https://doi.org/10.1099/ijsem.0.002834

Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics. Oxford University Press, New York.

Rambaut A (2016) FigTree, version 1.4.3. University of Edinburgh, Edinburgh.

Ren YC, Chen L, Niu QH, Hui FL (2013) Description of Scheffersomyces henanensis sp. nov., a new D-xylose-fermenting yeast species isolated from rotten wood. PLoSOne 9: e92315. https://doi.org/10.1371/journal.pone.0092315

Suh SO, Houseknecht JL, Gujjari P, Zhou JJ (2013) Scheffersomyces parashehatae f.a., sp. nov., Scheffersomyces xylosifermentans f.a., sp. nov., Candida broadrunensis sp. nov., and Candida manassasensis sp. nov., four novel yeasts associated with wood-ingesting insects and their ecological and biofuel implications. International Journal of Systematic and Evolutionary Microbiology 63: 4330–4339. https://doi.org/10.1099/ijs.0.053009-0

Urbina H, Blackwell M (2012) Multilocus phylogenetic study of the Scheffersomyces yeast clade and molecular characterization of the N-terminal region of xylose reductase gene. PLoS ONE 7:e39128. https://doi.org/10.1371/journal.pone.0039128

Urbina H, Blackwell M (2013) New combinations, Scheffersomyces amazonensis and S. ergatensis. Mycotaxon 123: 233–234. https://doi.org/10.5248/123.233

Urbina H, Frank R, Blackwell M (2013) Scheffersomyces cryptocercus: a new xylose-fermenting yeast associated with the gut of wood roaches and new combinations in the Sugiyamaella yeast clade. Mycologia 105: 650–60. https://doi.org/10.3852/12-094

White TJ, Bruns TD, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR Protocols, a Guide to Methods and Applications. Academic, San Diego, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1