Microbotryozyma lacustris sp. nov. (Basidiomycota, Ustilentylomataceae) and Cyberlindnera basumtsoensis sp. nov. (Ascomycota, Phaffomycetaceae), two novel yeasts isolated from freshwater Lake Basom Tso, China

135 Microbotryozyma lacustris sp. nov. (Basidiomycota, Ustilentylomataceae) and Cyberlindnera basumtsoensis sp. nov. (Ascomycota, Phaffomycetaceae), two novel yeasts isolated from freshwater Lake Basom Tso, China Lin Tian1, Dorji Phurbu1, Yan-Yan Zheng1 1 Key Laboratory of Plateau Fungi, Institute of Plateau Biology of Xizang Autonomous Region, Lhasa 850000, China Corresponding author: Yan-Yan Zheng ([email protected].cn) Copyright: © Lin Tian et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract High-altitude lakes in Tibet represent unique and underexplored reservoirs of microbial diversity. An investigation of yeast diversity in Basom Lake, a high-altitude lake in Tibet, China, led to the discovery of two novel species: Microbotryozyma lacustris sp. nov. and Cyberlindnera basumtsoensis sp. nov. Phylogenetic analyses of the ITS region and D1/D2 LSU rRNA gene sequences, complemented by phenotypic characterization, confirmed their distinct taxonomic status. Microbotryozyma lacustris represents the third species described in its genus and the first documented occurrence of Microbotryozyma in a freshwater habitat. Cyberlindnera basumtsoensis further expands the ecological diversity of the genus Cyberlindnera. This study significantly enriches the taxonomic framework of both genera and underscores the value of high-altitude lakes as reservoirs of novel yeast diversity. Key words: Basom Lake, Cyberlindnera, Microbotryozyma, novel species, phylogeny Introduction High-altitude lakes in Tibet represent unique and relatively pristine ecosystems that serve as promising reservoirs for microbial diversity (Yang et al. 2022). Basom Lake (approx. 3,490 m a.s.l.), the largest glacial-dammed lake in southeastern Tibet, is characterized by its oligotrophic conditions and is surrounded by snow-capped mountains and dense forests (Wang and Dou 1998; Fang et al. 2018; Li et al. 2020a; Luo et al. 2021). Previous studies have confirmed that Tibetan aquatic systems host a high diversity of fungi, including numerous novel yeast species (Han et al. 2017; Tsuji et al. 2018; Zhou et al. 2019). A recent survey further revealed highly diverse fungal communities in Basom Lake, dominated by Ascomycota, Chytridiomycota, and Basidiomycota (Zhou et al. 2025). The genus Microbotryozyma (Microbotryales) represents a relatively understudied yeast lineage. Established by Suh et al. (2012) to accommodate strains isolated from the plant bug Collaria oleosa, the genus initially contained only the type species M. collariae. The subsequent description of M. swertiae from the leaf surface of Swertia yunnanensis expanded the genus to its current two recognized species (Li et al. 2020b). While both known species were originally Academic editor: Peng Zhao Received: 2 October 2025 Accepted: 26 November 2025 Published: 15 December 2025 Citation: Tian L, Phurbu D, Zheng Y-Y (2025) Microbotryozyma lacustris sp. nov. (Basidiomycota, Ustilentylomataceae) and Cyberlindnera basumtsoensis sp. nov. (Ascomycota, Phaffomycetaceae), two novel yeasts isolated from freshwater Lake Basom Tso, China. MycoKeys 126: 135–150. https://doi. org/10.3897/mycokeys.126.173807 MycoKeys 126: 135–150 (2025) DOI: 10.3897/mycokeys.126.173807 136 MycoKeys 126: 135–150 (2025), DOI: 10.3897/mycokeys.126.173807 Lin Tian et al.: Microbotryozyma lacustris sp. nov. and Cyberlindnera basumtsoensis sp. nov. isolated from terrestrial sources, recent evidence suggests a broader ecological distribution. Environmental sequencing has detected Microbotryozyma in Lake Yamdrok (Yamzho Yumco) in Tibet, and strains of M. collariae have been recovered from freshwater environments in Japan (Urano et al. 2019; Hao et al. 2021). These findings indicate that Microbotryozyma species may inhabit both terrestrial and aquatic ecosystems (Wurzbacher et al. 2010). The genus Cyberlindnera (Saccharomycetales) represents a metabolically versatile and ecologically widespread lineage (Kurtzman et al. 2008a, 2011). The genus, originally proposed as Lindnera before being renamed due to nomenclatural priority (Kurtzman et al. 2008b), exhibits diverse reproductive strategies and physiological capabilities (Lachance et al. 2011). Species of Cyberlindnera are cosmopolitan in distribution, having been isolated from diverse habitats including plant substrates, insect frass, soil, and aquatic systems (Wang et al. 2015b). This broad ecological distribution suggests important roles in carbon cycling and ecosystem functioning (Soto-Robles et al. 2019). Beyond their ecological significance, several Cyberlindnera species possess considerable biotechnological potential, with applications in single-cell protein production, synthesis of valuable compounds, and biofuel production from lignocellulosic biomass (Sousa-Silva et al. 2021; Bonthong et al. 2025). Recent advances in molecular systematics and genomics have further clarified the taxonomic framework of this genus (Barros et al. 2021). Despite these advances, the yeast diversity in Basom Lake remains insufficiently explored, particularly regarding the representation of these two genera in high-altitude freshwater ecosystems. During a fungal diversity survey of this habitat, we isolated five yeast strains that could not be assigned to any known species based on preliminary sequence analysis. Phylogenetic and phenotypic characterizations confirmed that these isolates represent two novel species. Here, we formally describe these species, designated as Microbotryozyma lacustris sp. nov. and Cyberlindnera basumtsoensis sp. nov., thereby expanding the known diversity and ecological ranges of their respective genera while contributing to our understanding of yeast diversity in high-altitude aquatic environments. Materials and methods Isolation A total of 500 mL of lake water was vacuum-filtered through a membrane using a sintered glass filter holder to capture fungal cells. Following filtration, the membrane was transferred into a sterile centrifuge tube and immediately transported to the laboratory for further processing. Serial dilutions of the fungal suspension were prepared from the membrane. From each dilution, 100 µL was spread onto yeast extract–malt extract (YM) agar plates containing 1.0% (w/v) yeast extract, 2.0% (w/v) malt extract, 0.4% (w/v) glucose, and 2.0% (w/v) agar. Chloramphenicol was added at a final concentration of 50 mg L−1 to inhibit bacterial growth. For each dilution, three replicate plates were prepared and incubated at 17 °C for seven days. Yeast-like colonies were selected and repeatedly streaked onto YM agar to obtain pure cultures. For long-term preservation, the purified strains were stored in glycerol suspensions at –80 °C. All type strains are maintained in a metabolically inactive state at the China General Microbiological Culture Collection Center (CGMCC) and the Japan Collection of Microorganisms (JCM). 137 MycoKeys 126: 135–150 (2025), DOI: 10.3897/mycokeys.126.173807 Lin Tian et al.: Microbotryozyma lacustris sp. nov. and Cyberlindnera basumtsoensis sp. nov. Phenotypic characterization The morphological, physiological, and biochemical characteristics of the strains were assessed following standard methods (Kurtzman et al. 2011). Assimilation of carbon and nitrogen sources was examined in liquid media after subjecting the cells to a starvation period prior to inoculation. Fermentation tests were conducted using inverted Durham tubes (Chai et al. 2023). Cell morphology was observed after three days of incubation in YM broth at 17 °C by both light microscopy and scanning electron microscopy (Leica DM2500). Pseudohypha formation was evaluated on cornmeal agar (CMA; containing 2.5% cornmeal and 2% agar, w/v) with a coverslip placed over the colony to induce a semi-anaerobic environment favorable for pseudohyphal development. The sexual stage was inspected on V8 juice agar (10% V8 juice, 2% agar) and 5% malt extract agar (MEA; 5% malt extract, 1.5% agar). Growth was assessed under several conditions, covering a temperature range (17, 20, 25, 30, 35, and 37 °C), high glucose concentration (50% w/w), and vitamin-free medium. Starch production was also tested. Each strain was inoculated alone or in combination onto agar plates using a loopful of cells and incubated at 17 °C for up to two months, with periodic monitoring (Wei et al. 2024). Molecular phylogenetic analysis Genomic DNA was extracted from yeast cells according to the protocol described by Kurtzman (2000). The D1/D2 domains of the large subunit (LSU) rRNA gene were amplified with primers NL1 and NL4 (Kurtzman and Robnett 1998), and the internal transcribed spacer (ITS) region was amplified using primers ITS1 and ITS4 (Schoch et al. 2012). Each PCR reaction mixture consisted of 1.0 μL of each primer (10 pM/μL), 3.0 μL of genomic DNA (10 ng/μL), and 20 μL of 1× PCR master mix (T3 Super PCR Mix, Tsingke Biotechnology Co., Ltd.). Amplification was carried out in an AB 2720 thermal cycler (Applied Biosystems, Foster City, CA, USA). PCR products were confirmed by agarose gel electrophoresis and subsequently sent to Sinogenomax (Beijing, China) for sequencing. Preliminary identification of yeast strains was conducted by BLAST searches against the GenBank database using the D1/D2 and ITS sequences as queries (Altschul et al. 1997). Multiple sequence alignments of the ITS region and D1/D2 LSU rRNA gene domains were generated with the MAFFT program (White 1990), incorporating reference sequences obtained from GenBank (Tables 1, 2). Phylogenetic trees were reconstructed using MEGA v7.0 under the Maximum Likelihood (ML) criterion, with the best-fit substitution model selected through model testing (Wang et al. 2015a). BI analyses were conducted using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003). Two MCMC chains were run from random trees for 1,000,000 generations, resulting in a total of 10,000 trees. The first 25% of trees sampled were discarded as the burn-in phase of each analysis. The posterior probabilities (BPP) were calculated from the remaining trees (Rannala and Yang 1996). Branch support was assessed with 1,000 bootstrap replicates (Kumar et al. 2016). Colacogloea peniophorae CBS 684T and Trigonopsis californica CBS 10351T were designated as outgroup taxa. 138 MycoKeys 126: 135–150 (2025), DOI: 10.3897/mycokeys.126.173807 Lin Tian et al.: Microbotryozyma lacustris sp. nov. and Cyberlindnera basumtsoensis sp. nov. Table 1. Taxa used in the study of Microbotryozyma lacustris sp. nov. and their GenBank accession numbers. Taxon Strain GenBank accessions ITS D1/D2 Kalmanago commelinae SOMF 30249 TMT636665 MT636655 Liroa emodensis FO 17516 TDQ238743 AY512858 Microbotryum anomalum GLM 59392 EF621921 EF621960 Microbotryum bistortarum TUB 015861 EF621932 EF621975 Microbotryum bosniacum M-0066097 DQ238740 EF621977 Microbotryum cordae B 700006023 DQ238726 EF621978 Microbotryum dianthorum TUB 011802 AY588080 DQ366871 Microbotryum holostei B 700006032 DQ238722 EF621981 Microbotryum intermedium M 0066090 DQ238723 EF621982 Microbotryum lychnidis-dioicae TUB 011796 AY588097 DQ366865 Microbotryum marginale TUB 015881 EF621940 EF621989 Microbotryum onopordi M-0066075 DQ238735 EF621990 Microbotryum parlatorei B 700007574 DQ238736 EF621991 Microbotryum pustulatum TUB015872 EF621947 EF621998 Microbotryum reticulatum M-0066067 DQ238730 EF621999 Microbotryum salviae GLM 50395 EF621923 EF621963 Microbotryum saponariae TUB 011809 AY588089 DQ366887 Microbotryum betonicae TUB 015851 EF621924 EF621964 Microbotryum major B 700006042TAY877419 DQ366858 Microbotryum lychnidis-dioicae TUB 015865 EF621936 EF621984 Microbotryum scabiosae TUB 011789 AY588083 DQ366861 Microbotryum scorzonerae TUB 015878 EF621953 EF622007 Microbotryum stygium M-0066047 DQ238737 EF622009 Microbotryum tragopogonis-pratensis TUB 015879 EF621954 EF622014 Microbotryum tuberculiforme M-0066035 DQ238744 EF622015 Microbotryum violaceum TUB 011818 TAY588099 DQ366880 Sphacelotheca cf.koordersiana JAG-55AFTOL-ID1979 DQ832221 DQ832219 Sphacelotheca polygoni-persicariae KM1 MT557670 MT566306 Sphacelotheca polygoni-serrulati PYCC 4293 AF444593 AF189974 Aurantiosporium scleriae SOMF 30248 MT636671 MT636661 Fulvisporium restifaciens DTME 306 MT636672 MT636663 Bauerago abstrusa HUV 18526 DQ238719 EF621955 Bauerago vuyckii MP 2380 TDQ238720 DQ363321 Bauerozyma artemisiae YN 25-3 TOP470312 OP470216 Microbotryozyma lacustris CGMCC 2.8854 TPX048001 PX048003 Microbotryozyma lacustris BSC-W-3-4 PX499076 PX499117 Microbotryozyma lacustris BSC-W-7-4 PX499077 PX499118 Microbotryozyma collariae ATCCMYA-4666 TJN849458 JN849460 Microbotryozyma swertiae CGMCC 2.3533 TMK050424 MK050424 Ustilentyloma fluitans RB900 AY212990 AF009882 Ustilentyloma brefeldii TUB012510 DQ238745 EF622016 Mastigobasidium intermedium CBS 7226 AF444564 AF189889 Leucosporidium fellii CBS 7287 AF444508 AF189907 Leucosporidiella fragaria CBS 6254 AF444530 AF070428 Rhodotorula creatinovora CBS 8620 AF444629 AF189925 Leucosporidium scottii CBS 5930 TAF444495 AY213000 Rhodotorula mucilaginosa CBS 316 AF444541 AF070432 Rhodosporidium sphaerocarpum CBS 5939 AF444499 AF070425 Rhodotorula qlutinis CBS 20 TAF444539 AF070430 Colacogloea peniophorae CBS 684 TDQ202270 AY629313 Note: Newly generated sequences are in bold. The superscript “T” indicates ex-type strains. 139 MycoKeys 126: 135–150 (2025), DOI: 10.3897/mycokeys.126.173807 Lin Tian et al.: Microbotryozyma lacustris sp. nov. and Cyberlindnera basumtsoensis sp. nov. Table 2. Taxa used in the study of Cyberlindnera basumtsoensis sp. nov. and their GenBank accession numbers. Taxon Strain GenBank accessions ITS D1/D2 Candida adriatica ZIM 2334 HE_574654 NG_060386 Cyberlindnera basumtsoensis CGMCC 2.8853 TPX048002 PX048004 Cyberlindnera basumtsoensis Y-18-1-13-6 PX578846 PX495968 Cyberlindnera japonica NRRL YB-2750 TKY103061 EF550323 Cyberlindnera xylosilytica NRRL YB-2097 TKP232976 EF550324 Candida maesa ATCC MYA-4698 THM461661 JQ812697 Cyberlindnera veronae NRRL Y-7818 TAF335966 EF550322 Cyberlindnera fabianii NRRL Y-1871 TAF335967 EF550321 Candida mycetangii NRRL Y-6843 TKY102221 EF550330 Candida maritima NRRL Y-17775 TKY102197 EF550332 Candida nakhonratchasimensis JCM 12474 TKY102223 AY634567 Cyberlindnera mississippiensis NRRL YB-1294 TKY103068 EF550320 Cyberlindnera amylophila NRRL YB-1287 TKY103039 EF550319 Cyberlindnera xishuangbannaensis NYNU 16752 TKY213821 KY213813 Candida stauntonica ATCC MYA-4699 THM461658 JQ812698 Candida taoyuanica ATCC MYA-4700 TFJ873419 JQ812699 Candida hungchunana ATCC MYA-4701 THQ623543 JQ812700 Cyberlindnera meyerae NRRL Y-17236 TKY103066 EF550327 Cyberlindnera euphorbiae NRRL Y-17232 TKY103041 EF550326 Cyberlindnera xylebori NBRC 11048 TKY103116 AB534167 Cyberlindnera suaveolens NRRL Y-17391 TEU307977 EU544674 Cyberlindnera saturnus NRRL Y-17396 TEU307970 EF550316 Cyberlindnera subsufficiens NRRL Y-1657 TEU307975 EF550318 Candida takata ATCC MYA-4702 TJQ906769 JQ906764 Candida vartiovaarae NRRL Y-670 TKY102489 EF550315 Candida mengyuniae CBS 10845 TEU043159 EU043158 Cyberlindnera samutprakarnensis CBS 12528 TAB695388 AB598079 Cyberlindnera jadinii NRRL Y-1542 TDQ249199 EF550309 Cyberlindnera misumaiensis NRRL Y-17389 TKY103070 U73581 Cyberlindnera lachancei NRRL Y-27008 TKY103063 EF550313 Cyberlindnera petersonii NRRL YB-3808 TKY103077 EF550311 Millerago phaffii IBUN-04084 TON311286 ON264698 Millerago galiae CBS 8842 TKY102096 NG058980 Barnettozyma californica CBS 252 TNR138212 KY106168 Barnettozyma hawaiiensis CBS 8760 TKY101728 NG058701 Barnettozyma vustinii CBS 11554 TNR137724 NG058702 Barnettozyma xylosica NBRC 111558 TNR154882 NG058714 Barnettozyma populi CBS 8094 TNR153632 NG058630 Barnettozyma menglunensis NYNU 1811121 MK682797 MK682804 Trigonopsis californica CBS 10351 TKY105760 KY109968 Note: Newly generated sequences are in bold. The superscript “T” indicates ex-type strains. 140 MycoKeys 126: 135–150 (2025), DOI: 10.3897/mycokeys.126.173807 Lin Tian et al.: Microbotryozyma lacustris sp. nov. and Cyberlindnera basumtsoensis sp. nov. Results Phylogenetic analyses During a survey of yeast diversity in Basom Lake (Basum Tso), Nyingchi, Tibet, China, a total of 40 water samples were collected from various depths. Among the isolates, five yeast strains exhibiting unusual phenotypic characteristics were recovered and could not be identified as any known species based on BLAST searches of the ITS and D1/D2 sequences. Subsequent phylogenetic analyses confirmed that these five strains represent two distinct novel species. Strains CGMCC 2.8854, BSC-W-3-4, and BSC-W-7-4 were assigned to the genus Microbotryozyma, while strains CGMCC 2.8853 and Y-18-1-13-6 were affiliated with the genus Cyberlindnera. Phylogenetic analysis showed that strains CGMCC 2.8854, BSC-W-3-4, and BSC-W-7-4 formed a distinct, well-supported clade that is sister to Microbotryozyma collariae (type strain ATCC MYA-4666). Nucleotide comparisons with strain ATCC MYA-4666 revealed the following sequence divergences: CGMCC 2.8854 differed by 44 mismatches (29 substitutions and 15 indels, 8.78%) in the ITS region and seven substitutions (1.39%) in the D1/D2 domain; BSC-W-3-4 exhibited 35 substitutions and 17 indels in ITS, and eight substitutions and two indels in D1/D2; and BSC-W-7-4 displayed 36 substitutions and 17 indels in ITS, along with eight substitutions and two indels in D1/D2 (Fig. 1, Suppl. material 1: figs S1, S2). An unpublished strain, YYB134 (GenBank accession MT408741), also isolated from Tibet, shares identical D1/D2 sequences with CGMCC 2.8854, confirming that they represent the same taxon (Suppl. material 1: fig. S1). These results suggested that the CGMCC 2.8854 clade represents a novel species in the genus Microbotryozyma. Phylogenetic analysis showed that strains CGMCC 2.8853 and Y-18-1-13-6 formed a distinct, well-supported clade. Although this clade is located at the base of the genus in the phylogenetic tree, there is insufficient morphological and phylogenetic evidence to support its description as a novel genus; therefore, both strains are retained in the genus Cyberlindnera (Fig. 2, Suppl. material 1: figs S3, S4). Nucleotide comparisons with Cyberlindnera misumaiensis NRRL Y-17389 (type strain) revealed the following sequence divergences: CGMCC 2.8853 differed by 91 substitutions and 25 indels in the ITS region and 51 substitutions and seven indels in the D1/D2 domain; Y-18-1-13-6 exhibited 94 substitutions and 26 indels in ITS and 53 substitutions and seven indels in D1/D2. The result suggested that the CGMCC 2.8853 clade represented a novel species in the genus Cyberlindnera. Taxonomy Microbotryozyma lacustris L. Tian, Y. Y. Zheng, D. Phurbu & Q. M. Wang, sp. nov. Fungal Names: FN 572954 MycoBank: 860640 Fig. 3 Etymology. The species is named after the lake habitat where the type strain was isolated. Holotype. China • Xizang Autonomous Region, Nyingchi City, Gongbo’gyamda County, Basom Lake, from freshwater, GPS: 30°02'11"N, 93°78'53"E, 3440 m a.s.l., 141 MycoKeys 126: 135–150 (2025), DOI: 10.3897/mycokeys.126.173807 Lin Tian et al.: Microbotryozyma lacustris sp. nov. and Cyberlindnera basumtsoensis sp. nov. on 15 August 2023, Y. Y. Zheng (holotype CGMCC 2.8854T permanently preserved in a metabolically inactive state, ex-holotype JCM 10420 = ZYY1779). Description. Culture characteristics: After 3 days of incubation in YM broth at 17 °C, cells were ellipsoidal to ovoid, measuring 1.9–3.9 × 3.5–6.5 µm, and reproduced by monopolar budding (Fig. 3). After one month under the same conditions, prominent rings and sediment were present. On YM agar at 17 °C for three days, colonies were creamy, smooth, glossy, and exhibited surface ridges with serrated margins. Pseudohyphae were not formed on cornmeal agar. No ascospores or sexual structures were observed on YM, PDA, V8, or cornmeal agar after six weeks. Ballistoconidia were not produced. Physiological and biochemical characteristics: D-Glucose, sucrose, melibiose, D-arabinose, D-ribose, L-rhamnose, D-mannitol, and raffinose (delayed and weak) were assimilated. The following carbon sources were assimilated weakly or after a delay: D-galactose, sorbose, maltose, cellobiose, trehalose, lactose, melezitose, D-xylose, N-acetyl-D-glucosamine, ethanol, glycerol, galactitol, and hexadecane. Soluble starch, L-arabinose, methanol, erythritol, ribitol, D-glucitol, α-methyl-D-glucoside, Figure 1. Phylogenetic tree constructed from the combined sequences of the D1/D2 and ITS regions, showing the phylogenetic positions of the type strain CGMCC 2.8854T and related species. Reference strains included in the tree were either type strains of closely related species within the genus or strains widely cited in previous studies to ensure accurate phylogenetic comparison. Maximum likelihood bootstrap values (ML-BS ≥ 70%) and Bayesian posterior probabilities (BPP ≥ 0.9) are shown above the branches. Colacogloea peniophorae CBS 684T (accession numbers: DQ202270/AY629313) was used as the outgroup. The scale bar represents a patristic distance of 0.02. 142 MycoKeys 126: 135–150 (2025), DOI: 10.3897/mycokeys.126.173807 Lin Tian et al.: Microbotryozyma lacustris sp. nov. and Cyberlindnera basumtsoensis sp. nov. DL-lactic acid, succinic acid, citric acid, and inositol were not assimilated. Ammonium sulfate was utilized as a sole nitrogen source; potassium nitrate, sodium nitrite, L-lysine, ethylamine hydrochloride, and cadaverine dihydrochloride were not utilized. Starch-like compounds were not produced. Growth in vitamin-free medium was weak. No growth occurred on 50% (w/w) glucose–yeast extract agar. Materials examined. China • Xizang Autonomous Region, Nyingchi City, Gongbo’gyamda County, Basom Lake, from freshwater, GPS: 29°98'13"N, 93°86'59"E, 3390 m a.s.l., on 21 July 2025, L. Tian, Y. Y. Zheng, D. Phurbu & Q. M. Wang (living culture BSC-W-3-4, BSC-W-7-4). Notes. Strains CGMCC 2.8854T, BSC-W-3-4, and BSC-W-7-4, identified as Microbotryozyma lacustris, cluster within the genus Microbotryozyma but Figure 2. Phylogenetic tree constructed from the combined sequences of the D1/D2 and ITS regions, showing the phylogenetic positions of the type strain CGMCC 2.8853T and related species. Reference strains included in the tree were either type strains of closely related species within the genus or strains widely cited in previous studies. Maximum likelihood bootstrap values (ML-BS ≥ 70%) and Bayesian posterior probabilities (BPP ≥ 0.9) are shown above the branches. Trigonopsis californica CBS 10351 (KY105760/KY109968) was used as the outgroup. Bar, patristic distance of 0.02. 143 MycoKeys 126: 135–150 (2025), DOI: 10.3897/mycokeys.126.173807 Lin Tian et al.: Microbotryozyma lacustris sp. nov. and Cyberlindnera basumtsoensis sp. nov. are phylogenetically distinct from their closest relatives, M. collariae and M. swertiae. The D1/D2 domain sequence of strain CGMCC 2.8854 differs by seven substitutions (1.39%) from that of M. collariae ATCC MYA-4666T, while the ITS region shows 44 mismatches (8.78%, including 29 substitutions and 15 indels), values that exceed thresholds commonly accepted for species delineation in yeasts. Phenotypically, M. lacustris can be clearly distinguished from its congeners by its unique carbon and nitrogen assimilation profile. Specifically, it assimilates melibiose, L-rhamnose, galactitol (delayed), and hexadecane (delayed), all of which are not utilized by M. collariae or M. swertiae. Conversely, it fails to assimilate α-methyl-D-glucoside, potassium nitrate, or ethylamine hydrochloride, compounds that are utilized by both related species. These consistent phenotypic differences, summarized in Table 3, corroborate the phylogenetic data and firmly support its status as a novel species. Cyberlindnera basumtsoensis L. Tian, Y. Y. Zheng, D. Phurbu & Q. M. Wang, sp. nov. Fungal Names: FN 573011 MycoBank: 860641 Fig. 4 Etymology. The species is named after the place where the type strain was isolated. Holotype. China • Xizang Autonomous Region, Nyingchi City, Gongbo’gyamda County, Basom Lake, from freshwater, GPS: 30°02'11"N, 93°78'53"E, 3440 m a.s.l., on 15 August 2023, Y. Y. Zheng, (holotype CGMCC 2.8853T permanently preserved in a metabolically inactive state, ex-holotype JCM 10419 = ZYY005). Description. Culture characteristics: After 3 days of incubation in YM broth at 17 °C, cells were ellipsoidal to ovoid, measuring 2.6–3.7 × 3.0–5.5 µm, and reproduced by monopolar budding (Fig. 4). After one month under the same conditions, conspicuous rings and sediment were present. On YM agar at 17 °C for 3 days, colonies were creamy-white, butyrous, and emitted a characteristic aroma; the center Figure 3. Morphology of M. lacustris sp. nov. (strain CGMCC 2.8854T). A. Individual colonies by streaking onto Potato Dextrose Agar (PDA) after 3 days; B. Cylindrical arthroconidia on yeast extract–malt extract (YM) after three days of growth at 17 °C. Scale bars: 10 μm. 150 MycoKeys 126: 135–150 (2025), DOI: 10.3897/mycokeys.126.173807 Lin Tian et al.: Microbotryozyma lacustris sp. nov. and Cyberlindnera basumtsoensis sp. nov. 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Current Microbiology 76(12): 1537–1544. https://doi.org/10.1007/ s00284-019-01773-x Zhou D, Wang DX, Ge S, Qin Z, Ou MX, Guo XF, De J (2025) Fungal diversity, community structure and rrediction of ecological function in Basomtso Lake, Tibet. Biotechnology Bulletin 41(1): 298–311. https://doi.org/10.13560/j.cnki.biotech. bull.1985.2024-0445 Supplementary material 1 Phylogenetic trees Authors: Lin Tian, Dorji Phurbu, Yan-Yan Zheng Data type: doc Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.126.173807.suppl1