Introduction

The genus Sporobolomyces was proposed by Kluyver and van Niel (1924) for red pigmented ballistoconidia-forming yeasts. While the later inclusion of cream and yellow–brown colored species (Lodder and Kreger-van Rij 1952) made this genus more heterogeneous (Hamamoto et al. 2011). The single ribosomal RNA (rRNA) gene sequence analyses, e.g., the small subunit of ribosomal RNA (SSU rRNA), the D1/D2 domains of the large subunit of rRNA (LSU rRNA) and internal transcribed spacer (ITS) region, showed that Sporobolomyces were highly polyphyletic (Boekhout et al. 2011; Fell et al. 2000; Hamamoto and Nakase 2000; Hamamoto et al. 2011; Scorzetti et al. 2002). The species of Sporobolomyces were dispersed in four classes, namely Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes and Mixiomycetes, in the subphylum Pucciniomycotina (Bauer et al. 2006; Boekhout et al. 2011; Hamamoto et al. 2011; Hibbett et al. 2007). Recently a reclassification of the basidiomycetous yeasts, including the genus Sporobolomyces, has been done by Liu et al. (2015a, b) and Wang et al. (2014, 2015a, b, c). The revised Sporobolomyces by Wang et al. (2015b) includes only 15 species; the other species previously of this genus were placed into 15 genera, Ballistosporomyces, Bannoa, Buckleyzyma, Chrysozyma, Colacogloea, Cystobasidiopsis, Erythrobasidium, Microsporomyces, Fellozyma, Phyllozyma, Ruinenia, Rhodotorula, Rhodosporidiobolus, Slooffia and Symmetrospora (Wang et al. 2015b). The genus Ballistosporomyces was established by Nakase et al. (1989) including Ballistosporomyces xanthus and Ballistosporomyces ruber. However, this genus was relegated as a synonym of Sporobolomyces by Boekhout (1991). Wang et al. (2015b) emended and reintroduced this genus to include three previous Sporobolomyces species, namely Sporobolomyces sasicola, Sporobolomyces taupoensis and Sporobolomyces xanthus (B. xanthus). The species of B. ruber (Sporobolomyces ruber) was proposed as a new combination in the genus Ruinenia (Wang et al. 2015b).

In a survey of ballistoconidium-forming yeast diversity in the phyllosphere of China, 33 and 65 leaf samples including herb and woody plants were collected from Changbai Mountain Nature Reserve that is located in Jilin Province of northeastern China in October 1998 and Tibet that is located in southwest China in July 2004, respectively. More than 200 yeast strains were isolated from those samples. Among those isolates, four strains forming ballistoconidia and yellowish-brown colored colonies isolated from three shrub plants (the family Ericaceae) leaves were classified into one group. Three rRNA fragments, including the SSU rRNA, the D1/D2 domains of LSU rRNA and ITS + 5.8S rRNA region, were used to address the phylogenetic positions of these four strains in this study. The molecular result indicated that these strains belong to the genus Ballistosporomyces, and represent two novel species in this genus.

Materials and methods

Four strains listed in Table 1 were isolated from plant leaves using the improved ballistoconidia-fall method as described by Nakase and Takashima (1993). CB 266T and CB 272 were isolated from Rhododendron chrysanthum and Ledum palustre var. dilatatum, respectively. They were collected from Changbai Mountain Nature Reserve (41°41′–42°25′N, 127°42′–128°16′E; annual precipitation 600 mm; annual average temperature 3.3–7.3 °C). XZ 44D1T was isolated from Rhododendron phaeochrysum collected from Bomi county, Tibet (29°42′N, 95°49′E; annual precipitation 876.9 mm; annual average temperature 8.5 °C). XZ 49D2 were isolated from Rhododendron sp., collected from Milang county, Tibet (29°59′N, 94°52′E; annual precipitation 654 mm; annual average temperature 8.7 °C). The phenotypic and biochemotaxonomic characterizations were examined according to the standard methods (Kurtzman et al. 2011).

Table 1 Yeast strains studied
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The sequences of the ITS + 5.8S rRNA and the LSU rRNA D1/D2 domains were determined by the method described previously (Wang and Bai 2004). SSU rRNA sequence was performed according to Wang et al. (2003). Sequences were aligned with MAFFT program (Standley 2013). The combined alignment dataset was analysed with jModeltest (Posada 2008) using the Akaike information criterion (AIC), and the GTR + I + G was suggested as the best nucleotide substitution model. Phylogenetic tree was constructed from the maximum likelihood (ML) in RAxML-HPC2 7.2.8 (Stamatakis 2006) for 1000 bootstrap replicates analysis. The GenBank/EMBL/DDBJ accession numbers for the sequences of the ITS region, the D1/D2 domain of the LSU rRNA and the SSU rRNA determined in this study are KP020103–KP020108, respectively.

Results and discussion

Phylogenetic and phenotypic analyses

The four strains, CB 266T, CB 272, XZ 44D1T and XZ 49D2, with yellowish-brown colonies were classified into two groups represented by strains CB 266T and XZ 44D1T, and belong to the genus Ballistosporomyces by sequence analyses of the SSU rRNA, the D1/D2 domains of LSU rRNA and the ITS-5.8S rRNA region (Fig. 1). CB 266T and CB 272 isolated from two kinds of plant leaves (Table 1) in Jilin province, northeast China, and possessed the same sequences of the LSU D1/D2 domains and the ITS region. XZ 44D1T and XZ 49D2 isolated from two counties of Tibet, southwest China (Table 1), had identical sequences of the LSU D1/D2 domains and the ITS region. XZ 44D1T and CB 266T groups differed from each other by six nucleotides (nt) in the D1/D2 domains of LSU rRNA and about 3 % mismatches (including 12 substitutions and 11 indels) in the ITS region. These two groups are closely related to B. taupoensis and B. xanthus (Fig. 1). XZ 44D1T group differed from B. taupoensis and B. xanthus by eight substitutions in the D1/D2 domains, and by 19 and 16 nt mismatches in the ITS region, respectively. CB 266T group differed from S. taupoensis and S. xanthus by two substitutions in the D1/D2 domains, however, they had 25 nt (~5 %) and 16nt (~4 %) mismatches in the ITS region, respectively.

Fig. 1
figure 1

Phylogenetic tree constructed from ML analysis of the combined sequences of the SSU rRNA, internal transcribed spacer (ITS) region (including 5.8S rRNA) and LSU rRNA D1/D2 domains, depicting the relationships between the two new species and the other species in the Agaricostilbomycetes. Bootstrap percentages over 50 % from 1000 bootstrap replicates are shown

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The above sequence comparisons showed that these two groups represent two novel species in the genus Ballistosporomyces. The distinction of these two groups from their closely related taxa was also supported by the physiological characteristics (Table 2). XZ 44DT and CB 266T groups and B. taupoensis and B. xanthus can be distinguished from one another by the assimilation of galactose, lactose, succinic acid, citric acid, sodium nitrite, l-lysine, ethylamine and cadaverine. Thus we propose these two groups as two new species, namely Ballistosporomyces changbaiensis sp. nov. and Ballistosporomyces bomiensis sp. nov. The description of these two new species will help us improve the understanding the diversity and ecology of the genus Ballistosporomyces.

Table 2 Physiological characteristics that differentiate the two new species from closely related species
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Description of Ballistosporomyces changbaiensis F.Y. Bai, Q.M. Wang, P. J. Han and A.H. Li sp. nov. Mycobank number MB 815700

Etymology: The specific epithet changbaiensis refers to the geographic origin of the type strain of this species.

In YM broth, after 7 days at 17 °C, cells are ellipsoidal, 2.5–4.5 × 5.0–6.0 µm (Fig. 2) and single, budding is polar, a ring and sediment are formed. After 1 month at 17 °C, a ring and sediment are present. On YM agar, after 1 month at 17 °C, the streak culture is yellowish-brown, butyrous, smooth or wrinkled. The margin is entire. In Dalmau plate culture on corn meal agar, pseudohyphyae are not formed. Ballistoconidia are allantoid or reniform, 2.5–6.5 × 6.0–12.0 µm (Fig. 2). Fermentation is negative. Glucose, sucrose, maltose, cellobiose, trehalose, raffinose, soluble starch (weak), d-xylose (weak) and d-glucitol (weak) are assimilated. Galactose, l-sorbose, lactose, melibiose, inulin, melezitose, l-rhamnose, glycerol, l-arabinose, d-arabinose, d-ribose, ribitol, methyl α-d-glucoside, d-glucosamine, methanol, ethanol, erythritol, galactitol, salicin, dl-lactic acid, critic acid, inositol and hexdecane are not assimilated. The assimilation of d-mannitol and succinic acid is variable. Ammonium sulfate, sodium nitrite, l-lysine, ethylamine hydrochloride, potassium nitrate and cadaverine dihydrochloride are assimilated. Maximum growth temperature is 26 °C. Growth in vitamin-free medium is variable. Starch-like substances are not produced. Growth on 50 % (w/w) glucose–yeast extract agar is negative. Urease activity is positive. Diazonium Blue B reaction is positive. The major ubiquinone is Q-10. Sexual reproduction was not observed after mating strains CB 266T and CB 272 on YM agar, YPD agar McClary acetate agar and corn meal agar at 17 °C for 4 weeks. The type strain, CB 266T, was isolated from a leaf of Rhododendron chrysanthum collected in Changbai Mountain, Jilin, China in October, 1998. This strain has been deposited in the China General Microbiological Culture Collection Center (CGMCC), Academia Sinica, Beijing, China, as CGMCC 2.02298T, and in the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands, as CBS 10124T.

Fig. 2
figure 2

Cells (a, c) grown in YM broth for 7 days at 17 °C and ballistoconidia (b, d) produced on corn meal agar after 7 days at 17 °C of new species. B. changbaiensis CB 266 T (a, b); B. bomiensis XZ 44D1 T; (c, d); bars 10 μm

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Description of Ballistosporomyces bomiensis Q.M. Wang, F.Y. Bai, A.H. Li and P. J. Han sp. nov. Mycobank number MB 815701

Etymology: The specific epithet bomiensis refers to the geographic origin of the type strain of this species.

In YM broth, after 7 days at 17 °C, cells are ellipsoidal, 2.0–4.0 × 5.0–8.0 µm (Fig. 2) and single, budding is polar, a ring and sediment are formed. After 1 month at 17 °C, a ring and sediment are present. On YM agar, after 1 month at 17 °C, the streak culture is yellowish-brown, butyrous, smooth. The margin is entire. In Dalmau plate culture on corn meal agar, pseudohyphyae are not formed. Ballistoconidia are allantoid or reniform, 2.0–6.0 × 6.5–12.5 µm (Fig. 2). Fermentation is negative. Glucose, sucrose, maltose, cellobiose, trehalose, raffinose, d-mannitol (weak) and salicin (weak) are assimilated. Galactose, l-sorbose, lactose, melibiose, inulin, melezitose, l-rhamnose, glycerol, d-xylose, l-arabinose, d-arabinose, d-ribose, d-glucosamine, d-glucitol, methanol, ethanol, erythritol, galactitol, dl-lactic acid, critic acid, succinic acid, inositol and hexdecane are not assimilated. The assimilation of soluble starch, methyl α-d-glucoside and ribitol is variable. Ammonium sulfate, l-lysine, ethylamine hydrochloride, potassium nitrate and cadaverine dihydrochloride are assimilated. Sodium nitrite is not assimilated. Maximum growth temperature is 26 °C. Growth in vitamin-free medium is negetive. Starch-like substances are not produced. Growth on 50 % (w/w) glucose-yeast extract agar is negative. Urease activity is positive. Diazonium Blue B reaction is positive. The major ubiquinone is Q-10. Sexual reproduction was not observed after mating strains XZ 44D1T and XZ 49D2 on YM agar, YPD agar McClary acetate agar and corn meal agar at 17 °C for 4 weeks. The type strain, XZ44 D1T, was isolated from a leaf of Rhododendron phaeochrysum, collected in Bomi county, Tibet, China in July 2004. This strain has been deposited in the China General Microbiological Culture Collection Center (CGMCC), Academia Sinica, Beijing, China, as CGMCC 2.02661T, and in the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands, as CBS 12512T.