Yeast Diversity in the Qaidam Basin Desert in China with the Description of Five New Yeast Species

The Qaidam Basin is the highest and one of the largest and driest deserts on Earth. It is considered a mars analog area in China. In contrast to numerous studies concerning its geology, geophysical, and chemistry, relatively few studies have reported microbial diversity and distribution in this area. Here, we investigated culturable yeast diversity in the northeast Qaidam Basin. A total of 194 yeast strains were isolated, and 12 genera and 21 species were identified, among which 19 were basidiomycetous yeasts. Naganishia albida, N. adeliensis, and Filobasidium magnum were the three most dominant species and were distributed in thirteen samples from eight locations. Five new species (Filobasidium chaidanensis, Kondoa globosum, Symmetrospora salmoneus, Teunia nitrariae, and Vishniacozyma pseudodimennae) were found and described based on ITS and D1D2 gene loci together with phenotypic characteristics and physiochemical analysis. Representative strains from each species were chosen for the salt-tolerant test, in which species showed different responses to different levels of NaCl concentrations. Further, the strain from soil can adapt well to the higher salt stress compared to those from plants or lichens. Our study represents the first report of the yeast diversity in the Qaidam Basin, including five new species, and also provides further information on the halotolerance of yeasts from the saline environment in mars analog.


Introduction
Mars analogs are places on Earth that possess one or more geological, geochemical, and geomorphic features similar to those found on Mars, either current or past [1,2]. The possibility of the emergence of life on the early Mars stage has been shown due to the presence of liquid water, carbon, electron donors, and acceptors that can drive microbial metabolism [3][4][5][6][7][8]. Due to the expensive and long-term dependent organization of space exploration, terrestrial analogs can serve as models for studies on the habitability of Mars, which has been a priority of Mars science investigations [5]. Generally, the terrestrial analogs on Earth are characterized by extreme environmental conditions, such as hyper-arid desert, saline or hypersaline habitat, thermal acidic spring, and cryogenic and dry Antarctica [9][10][11][12][13][14][15] Microbial composition and ecology in different Mars analogs have drawn attention due to their ecological and astrobiological importance. Microbiological studies in Mars analogs performed in the past decades have contributed to our better understanding of the strategy and spatiotemporal limit of life on Earth [16][17][18][19][20][21][22][23]. However, these studies mostly focused on bacteria [18,20,24,25], and the diversity and ecology of fungi in Mars analogs remain largely unknown.
Yeasts are unicellular or dimorphic fungi with a stable free-living unicellular state during their life cycles [26]. Yeasts are considered ubiquitous and have been found in highly diversified substrates and environments, including extremely dry or cold habitats [27]. An increasing number of studies have shown that yeasts can inhabit the Antarctic, glacier, acidic, hypersaline, hyper-arid, and desert areas [28][29][30][31][32][33][34]. Nevertheless, the occurrence and diversity of yeasts in extreme environments have not been fully investigated.
The Qaidam Basin is located in the North Tibetan Plateau, China, and is the highest desert in terms of altitude and one of the largest and driest deserts on Earth. It has an average elevation of~2800 m and is considered a terrestrial analog site of Mars [12]. This area harbors Mars-like extreme environments, e.g., hyper-arid, salt deposits, and high-UV, and similar mineral compositions and geomorphological structures to terrains on Mars [12,35]. The geology, geophysiology, and chemistry of the Qaidam Basin have been well characterized [36][37][38], and microbial diversities focusing on bacteria and archaea in different parts of this area using culture-independent methods have been performed in recent years [39][40][41]. However, the fungal diversity in the Qaidam Basin remains to be revealed.
In this study, we collected soil, soil crust, plant, and lichen samples in the northeast Qaidam Basin in Qinghai province and investigated the occurrence and diversity of yeasts in these samples using a culture-based approach. A total of 21 species, including five new species, were identified from the isolated yeast strains. In addition, the salt-tolerant ability of the representative yeast strains isolated was tested to evaluate their biotechnological potential. To our knowledge, this is the first report of the diversity of culturable yeasts within the Qaidam Basin. The aim of this study is to characterize the yeast community inhabiting this specific environment and identify potential salt-tolerant yeasts that could be of importance for astrobiology and biotechnology.

Sampling Sites
A total of 27 samples, including 19 surface soil and soil crust samples and eight whole plant tissue and lichen samples, were selected from Delingha city in the Qaidam Basin located in Qinghai province in April 2021 ( Figure 1 and Table 1). Samples were sealed in sterilized plastic bags and transported to the laboratory at ambient temperatures.

Soil Chemical Analyses
The soil was suspended 1:5 (w/v) in sterile water and shaken at 180 rpm at 20 • C for 30 min. Soil pH and salinity were determined using the HQ40d Portable Meter (Hach Company, Loveland, CO, USA) ( Table 1).

Yeast Isolation
All samples were processed immediately after returning to the laboratory. Approximately 2 g of soil, soil crust, and fragmented plant and lichen tissue samples were suspended in 10 mL of sterile water and shaken at 180 rpm for 24 h. Aliquots of 0.2 mL of each suspension were spread on potato dextrose agar (PDA, w/v, 20% potato infusion, 2% glucose, 2% agar), 1/2 PDA, and 1/10 PDA plates supplemented with 500 mg L −1 of ampicillin and 500 mg L −1 of streptomycin sulfate. The plates were incubated at 17 • C and 25 • C, respectively, for 3-7 d. All the yeast colony morphotypes were isolated, purified, and preserved in 20% glycerol at −80 • C for further study.

Molecular Phylogenetic Analysis
Genomic DNA was extracted from yeast cells that were actively growing on a YPD medium (w/v, 1% yeast extract, 2% peptone, 2% glucose, 2% agar) following the protocol described by Makimura et al. [42]. The ITS region (including the 5.8 S rRNA gene) and D1/D2 domain of the LSU rRNA gene were amplified and sequenced using the primers ITS1 and NL4 as described previously [43].
Sequences were inspected and assembled using the SeqMan program in the Lasergene 7 software package (DNASTAR Inc., Madison, WI, USA). They were then aligned using MAFFT version 7 using the G-INS-I algorithm [44]. The alignment was manually edited and trimmed, and minor gaps in all alignments were deleted. Maximum likelihood (ML) and Bayesian analyses were performed for separate and combined gene sequences using RAxML-HPC V.8 [45] and MrBayes 3.2.1 [46] on the CIPRES Science Gateway (http://www. phylo.org (accessed on 23 January 2022)), respectively. ML analysis was implemented with the rapid bootstrap algorithm with 1000 replicates and a subsequent search for the bestscoring tree using the GTRGAMMA model. The best-fit evolution model of separate and combined nucleotide sequences was determined using the Bayesian Information Criterion (BIC) jModeltest [47]. Five million generations were run with four Markov chains, sampling every 5000 generations.
Branches that received bootstrap values (BP) for Maximum likelihood and Bayesian posterior probabilities (BPP) greater than or equal to 70% and 0.95, respectively, were considered as significantly supported. The GenBank accession numbers for the sequences of the ITS region, LSU rRNA gene D1/D2 domain used in this study are listed in Supplementary Table S1. The alignments and trees were deposited in TreeBase (www.treebase.org (accessed on 1 March 2022), No. 29479).

Phenotypical Characterization
The phenotypic and physiological characters were examined according to standard methods used in yeast taxonomy [48]. The potential sexual cycles of all new species were investigated using CMA (w/v, 2.5% corn meal infusion, 2% agar), PDA, V8 (w/v, 10% V8 juice, 2% agar), and YCB (w/v, 1.17% yeast carbon base, 2% agar). A loopful of cells of each test strain was incubated singly or mixed on an agar plate and incubated at 17 • C for one month. The ballistoconidium-forming activity of all new species was observed by the inverted-plate method [49] on CMA at 17 • C. After 3 to 14 d, the glass slide containing the discharged spores was removed for examination under the microscope.

Salinity Tolerance Analysis
Salinity tolerance tests were performed on the representative strains of each species using spot plates. All the growth experiments were conducted at 20 • C in YPD agar plates supplemented with 0%, 3%, 6%, 9%, 12%, and 15% (w/v) NaCl, respectively. Briefly, strains were precultured in 3 mL YPD broth for 48 h at 20 • C with shaking at 180 rpm. Optical density (OD) at 600 was measured, and the cell suspension was adjusted to OD 0.8 before a ten-fold dilution series was carried out. An aliquot of 4 µL of each dilution of cell suspension was then spotted onto YPD agar plates supplemented with different concentrations of NaCl and incubated for five days at 20 • C.

Statistical Analysis
Standard statistical analyses were performed in the R project (v3.3.1) [50]. The statistical significance was calculated using the ANOVA or Kruskal-Wallis test executed by the aov function and kruskal.test function of R package multcomp and FSA, respectively.

Yeast Diversity
Yeast strains were isolated from 10 of the 19 soil samples and six of the eight plant and lichen samples collected from Delingha city in the Qaidam Basin. The soil samples with more than 34.32‰ salinity and the soil crust samples were yeast negative (Table 1). A total of 194 yeast strains were isolated, and the abundance of yeasts varied depending on the substrate, incubation temperature, and medium ( Figure 2). A total of 153 and 41 yeast strains were isolated from the plant and lichen tissues and soil samples, respectively. The PDA, 1/2 PDA, and 1/10 PDA agar produced 95, 68, and 31 yeast strains, respectively. A total of 91 and 103 yeast strains were isolated from the plates incubated at 17 • C and 25 • C, respectively (Supplementary Tables S2-S4). Comparative analysis indicated that both the numbers of yeast strains and species from plant and lichen tissue samples were significantly higher than those from soil samples, whereas no significant difference in the abundance and species number was observed regarding the temperature and medium used for yeast isolation ( Figure 2). In addition to the shared species, different substrates, temperatures, and media harbored their own yeast species (Figure 2). cell suspension was then spotted onto YPD agar plates supplemented with different concentrations of NaCl and incubated for five days at 20 °C.

Statistical Analysis
Standard statistical analyses were performed in the R project (v3.3.1) [50]. The statistical significance was calculated using the ANOVA or Kruskal-Wallis test executed by the aov function and kruskal.test function of R package multcomp and FSA, respectively.

Yeast Diversity
Yeast strains were isolated from 10 of the 19 soil samples and six of the eight plant and lichen samples collected from Delingha city in the Qaidam Basin. The soil samples with more than 34.32‰ salinity and the soil crust samples were yeast negative (Table 1). A total of 194 yeast strains were isolated, and the abundance of yeasts varied depending on the substrate, incubation temperature, and medium ( Figure 2). A total of 153 and 41 yeast strains were isolated from the plant and lichen tissues and soil samples, respectively. The PDA, 1/2 PDA, and 1/10 PDA agar produced 95, 68, and 31 yeast strains, respectively. A total of 91 and 103 yeast strains were isolated from the plates incubated at 17 °C and 25 °C, respectively (Supplementary Tables S2-S4). Comparative analysis indicated that both the numbers of yeast strains and species from plant and lichen tissue samples were significantly higher than those from soil samples, whereas no significant difference in the abundance and species number was observed regarding the temperature and medium used for yeast isolation ( Figure 2). In addition to the shared species, different substrates, temperatures, and media harbored their own yeast species ( Figure 2).  The sequences of the D1/D2 domain of the 194 isolated strains were determined. The closest relative of each strain was recognized based on a blast search against the GenBank database using the BLASTn tool. The strains were classified to the species level based on the threshold of >99% sequence identity with the type strain of a described species in the ITS region or D1/D2 domain [51][52][53]. A total of 21 species belonging to 12 genera were identified from the isolated strains. Among the species identified, 19 species were basidiomycetous yeasts, including Dioszegia hungarica, Filobasidium magnum, Kondoa sorbi, Naganishia adeliensis, N. albida, Papiliotrema laurentii, Teunia korlaensis, Udeniomyces puniceus, Vishniacozyma dimennae, V. tephrensis, V. victoriae, and eight undescribed species, i.e., Fibulobasidium sp., Filobasidium sp., Kondoa sp., Symmetrospora sp., Teunia sp., and Vishniacozyma sp. (Supplementary Table S2), and two were ascomycetous yeast species, namely Diutina catenulata and Pichia kudriavzevii. The most abundant yeast genus was Naganishia, followed by Filobasidium and Teunia. The Naganishia species, viz. N. albida and N. adeliensis, accounted for 39.2% of the total number of isolates. To a lesser extent was the genus Filobasidium, of which an undescribed species, Filobasidium sp. formed the majority of the isolates. The strains belonging to the genus Teunia accounted for 9.8% of the total yeast strains isolated, and half belonged to an undescribed species of the genus. On the whole, 24.7% of the isolated strains belonged to undescribed species, and most of these were isolated from plant samples (Supplementary Table S2). Among the 21 species identified, three species, i.e., Filobasidium magnum, Naganishia adeliensis, and N. albida, were the most dominant species, which occurred in seven to eight samples collected from different locations, while thirteen species, i.e., Dioszegia hungarica, Fibulobasidium sp., Kondoa sorbi, Kondoa globosum sp. nov., Kondoa sp. Teunia nitrariae sp. nov., Vishniacozyma dimennae, Vishniacozyma pseudodimennae sp. nov., and Vishniacozyma tephrensis, occurred only in one sample or location (Supplementary Table S2).

Salt-Tolerant Ability
Representative strains from lichen, plant, and soil samples were subjected to salttolerant ability tests using spot plate assay. Different species had different responses to concentrations of NaCl. In detail, all tested strains of 21 species identified in this study could grow well with NaCl concentrations up to 6% (Supplementary Figure S2), while no strains could grow on the plate with NaCl concentrations of 15% (data not shown). It suggested that their growth was apparently prevented when NaCl concentration increased to 15%. Four species, such as Dioszegia hugarica, Fibulobasidium sp., Kondoa globosum, and Teunia korlaensis, were particularly sensitive to 9% NaCl concentration, while the other species, especially Diutina catenulata, Filobasidium magnum, Kondoa sorbi, Naganishia adeliensis, Papiliotrema laurenti, Pichia kudriavzevii, Symmetrospora salmoneus, and Vishniacozyma victoriae, showed to be unaffected by this level of salt exposure (Supplementary Figure S2). Several strains, such as F. chaidanensis, Kondoa sp. N. albida, Teunia nitrariae, Teunia sp., Udeniomyces puniceus, Vishniacozyma dimennae, V. pseudodimennae, and V. tephrensis, showed some ability to grow at 9% NaCl concentration. In contrast, most strains appeared to be sensitive to exposure to NaCl concentration of 12%. The strains of F. magnum, P. laurenti, and V. victoriae seemed to be relatively unaffected by this level of NaCl exposure (Supplementary Figure S2). These three species exhibited a similar tendency, that is, no difference, in all tested strains in growth ability regardless of their origin from plant and soil samples with different NaCl concentrations, except for the strain of F. magnum from lichen ( Figure 3). The strains of N. adeliensis and S. salmoneus from the soil sample showed better growth than those from plant or lichen thallus at 12% NaCl concentration, indicating that the strain from soil samples can adapt well to the higher salt stress. . Spot plate assay for growth of representative strains of selected species on YPD medium supplemented with 0%, 3%, 6%, 9%, and 12% (w/v) NaCl for 5 days at 20 °C.

Phylogeny
Among the yeast strains isolated, 43 strains were unable to be identified as any species because of their significant difference from any described yeast species in the sequence of the D1/D2 domain. The ITS sequences of these strains were then determined to confirm their novelty and phylogenetic positions.
Strain CGMCC 2.6805 T was located in the Kondoa clade and closely related with K. sorbi CGMCC 2.2303 T in the tree obtained from the combined ITS and D1D2 sequences ( Figure 4B). The two strains differed from each other by 9 nt (~1.4%) and 55 nt (~8.4%) mismatches in the D1/D2 and ITS regions, respectively. An undescribed strain 'Bensingtonia' sp. CCFEE 5424 isolated from rock samples [54] and two unpublished strains 'Pucciniomycotina' sp. RP185 (AB727159) and RP186 (AB727160) isolated from plant materials in Japan showed close relationships with strain CGMCC 2.6805 T (Supplementary Figure  S1B), but they differed from the latter by more than 1% bases in the D1/D2 domain. The result suggests that strain CGMCC 2.6805 T represents a novel species in the genus Kondoa, for which the name K. globosum sp. nov. was proposed.
Three strains, CGMCC 2.6800, CGMCC 2.6801 T , and 17-256-4 isolated from sandy soil and plant tissues possessed similar sequences with no more than one nt difference in the . Spot plate assay for growth of representative strains of selected species on YPD medium supplemented with 0%, 3%, 6%, 9%, and 12% (w/v) NaCl for 5 days at 20 • C.

Phylogeny
Among the yeast strains isolated, 43 strains were unable to be identified as any species because of their significant difference from any described yeast species in the sequence of the D1/D2 domain. The ITS sequences of these strains were then determined to confirm their novelty and phylogenetic positions.
Strain CGMCC 2.6805 T was located in the Kondoa clade and closely related with K. sorbi CGMCC 2.2303 T in the tree obtained from the combined ITS and D1D2 sequences ( Figure 4B). The two strains differed from each other by 9 nt (~1.4%) and 55 nt (~8.4%) mismatches in the D1/D2 and ITS regions, respectively. An undescribed strain 'Bensingtonia' sp. CCFEE 5424 isolated from rock samples [54] and two unpublished strains 'Pucciniomycotina' sp. RP185 (AB727159) and RP186 (AB727160) isolated from plant materials in Japan showed close relationships with strain CGMCC 2.6805 T (Supplementary Figure S1B), but they differed from the latter by more than 1% bases in the D1/D2 domain. The result suggests that strain CGMCC 2.6805 T represents a novel species in the genus Kondoa, for which the name K. globosum sp. nov. was proposed.
Three strains, CGMCC 2.6800, CGMCC 2.6801 T , and 17-256-4 isolated from sandy soil and plant tissues possessed similar sequences with no more than one nt difference in the ITS and D1D2 regions, indicating they are conspecific. They were clustered in the Symmetrospora clade with strong support in the tree constructed from the combined ITS and D1/D2 sequences ( Figure 4C). S. foliicola CBS 8075 T is closely related to these three strains but without significant support ( Figure 4C and Supplementary Figure S1C). Ten to eleven nt (~1.6-1.7%) and eighteen nt (~3.1%) differences were observed in the ITS region and D1D2 domain, respectively, between these three strains and S. foliicola. S. rhododendri, S. coprosmae, and S. oryzicola were located as a sister group to the group represented by CGMCC 2.6801 T and S. follicola but differed from the group CGMCC 2.6801 T by 7-10 nt (~1.3-1.6%) mismatches in the D1D2 domains and more than 7.2% (44-51 bp) mismatches in the ITS regions. Thus, these three strains represent a novel Symmetrospora species, for which the name S. salmoneus sp. nov. was proposed.
Six strains, represented by strain CGMCC 2.6797 T , formed a well-supported clade ( Figure 4D). Four nt and one nt mismatches in the D1D2 and ITS regions, respectively, were found among these six strains indicating they were conspecific. The closest relative of the CGMCC 2.6797 T group is Teunia korlaensis, but differed from the type strain of the latter by eight to nine nt (~1.3-1.5%) in the D1D2 domains and 22 nt (~3.9%) in the ITS regions, respectively. They also had a close relationship with T. rosae in the D1/D2 tree (Supplementary Figure S1D). They differed from the type strain of the latter by three to five nt (~0.5-0.8%) in the D1D2 domains but 38 to 40 nt (~6.8-7.1%) mismatches in the ITS regions, respectively. Therefore, a novel species, Teunia nitrariae, was proposed to accommodate these six strains.
Strain CGMCC 2.6790 T was located in the genus Vishniacozyma. It was closely related to Vishniacozyma dimennae ( Figure 4E and Supplementary Figure S1E) but differed from the type strain of V. dimennae by ten nt (~1.6%) and 19 nt (~4.0%) mismatches in the D1D2 domains and ITS regions, respectively, suggesting that CGMCC 2.6790 T represents a new species in Vishniacozyma, for which the name V. pseudodimennae sp. nov. is proposed. ITS and D1D2 regions, indicating they are conspecific. They were clustered in the Symmetrospora clade with strong support in the tree constructed from the combined ITS and D1/D2 sequences ( Figure 4C). S. foliicola CBS 8075 T is closely related to these three strains but without significant support ( Figure 4C and Supplementary Figure S1C). Ten to eleven nt (~1.6-1.7%) and eighteen nt (~3.1%) differences were observed in the ITS region and D1D2 domain, respectively, between these three strains and S. foliicola. S. rhododendri, S. coprosmae, and S. oryzicola were located as a sister group to the group represented by CGMCC 2.6801 T and S. follicola but differed from the group CGMCC 2.6801 T by 7-10 nt (~1.3-1.6%) mismatches in the D1D2 domains and more than 7.2% (44-51 bp) mismatches in the ITS regions. Thus, these three strains represent a novel Symmetrospora species, for which the name S. salmoneus sp. nov. was proposed. Six strains, represented by strain CGMCC 2.6797 T , formed a well-supported clade ( Figure 4D). Four nt and one nt mismatches in the D1D2 and ITS regions, respectively, were found among these six strains indicating they were conspecific. The closest relative of the CGMCC 2.6797 T group is Teunia korlaensis, but differed from the type strain of the latter by eight to nine nt (~1.3-1.5%) in the D1D2 domains and 22 nt (~3.9%) in the ITS regions, respectively. They also had a close relationship with T. rosae in the D1/D2 tree (Supplementary Figure S1D). They differed from the type strain of the latter by three to five nt (~0.5-0.8%) in the D1D2 domains but 38 to 40 nt (~6.8-7.1%) mismatches in the ITS regions, respectively. Therefore, a novel species, Teunia nitrariae ,was proposed to accommodate these six strains.
Strain CGMCC 2.6790 T was located in the genus Vishniacozyma. It was closely related to Vishniacozyma dimennae ( Figure 4E and Supplementary Figure S1E) but differed from the type strain of V. dimennae by ten nt (~1.6%) and 19 nt (~4.0%) mismatches in the D1D2 domains and ITS regions, respectively, suggesting that CGMCC 2.6790 T represents a new species in Vishniacozyma, for which the name V. pseudodimennae sp. nov. is proposed.
Culture characteristics: After growth on YM agar for 7 days at 20 °C, cells were globose or subglobose, 3.3-6.6 μm × 2.8-6.7 μm, and occurred singly or in pairs. Budding was polar. After 7 days of growth on YPD agar at 20 °C, the streak culture was whitish to pale greyish-cream, smooth, and butyrous. The margin was entire. In Dalmau plated culture

Discussion
The Qaidam Basin has been proposed as the first Mars analog in China as it harbors a hyper-arid, cold, salt-deposited, and high UV environment similar to the surface of Mars, as well as Mars-like geomorphological structures and mineral composition [12]. It provides an ideal systematic model as a terrestrial analog with astrobiological and extremophile implications. This study reports the culturable yeast diversity in lichen, plants, and soils in the Qaidam Basin for the first time.
Generally, yeasts grow well in a slightly acidic environment, with the optimum pH between 4.5 to 5.5 [55]. Nevertheless, basidiomycetous yeasts, i.e., Naganishia albida, Papiliotrema laurentii, Rhodotorula glutinis, R. mucilaginosa, Sporobolomyces roseus, and Trichosporon asahii, were found to thrive at pH value above 10 [56,57]. In our study, the pH values among the sampling sites varied from 7.54 to 9.74, indicating the alkali nature of the sampling area. Yeasts were obtained in the sampling sites with pH values between 7.65 to 8.85 but not for the soil samples with pH values greater than 9. The salinity of soil samples in our study ranged from 0.16‰ to 58.51‰. Yeasts were detected in ten soil samples with salinity from 0.21‰ and 34.32‰. Meanwhile, no yeasts occurred in seven soil samples with salinity between 0.16‰ to 16.78‰, including two soil crust samples. Though pH and salinity limited the presence of yeast inhabitants to some extent, their presence did not directly correlate with these two abiotic variables. This is probably due to additional abiotic variables affecting yeast number and diversity that have not been captured in this study.
Approximately 85% of strains obtained in our study were tremellomycetous yeast species that are in good agreement with previous studies of other extreme environments [28][29][30]34,58]. Of 21 species identified in this study, six species, such as Diutina catenulata, Filobasidium magnum, Naganishia adeliensis, N. albida, Papiliotrema laurentii, and Vishniacozyma victoriae, had been reported to be resident in extreme environments, especially saline, Culture characteristics: After growth on YM agar for 7 days at 20 • C, cells were globose or subglobose, 3.3-6.6 µm × 2.8-6.7 µm, and occurred singly or in pairs. Budding was polar. After 7 days of growth on YPD agar at 20 • C, the streak culture was whitish to pale greyish-cream, smooth, and butyrous. The margin was entire. In Dalmau plated culture on corn meal agar, pseudohyphae and hyphae were not formed. Sexual structures were not observed on YCB, 5% MEA, PDA, V8, and CMA agar. Ballistoconidia were not produced on corn meal agar.
Physiologically, F. chaidanensis differed from its close relatives F. oeirensis, F. floriforme, F. elegans, and F. magnus in the ability to assimilate inulin, methanol, and DL-lactate and its ability to grow in a vitamin-free medium.
Culture characteristics: After growth on YM agar for 7 days at 20 • C, cells were subglobose to globose, 4.5-7.6 µm × 4.3-7.3 µm, and occurred singly or in pairs. Budding was polar. After 7 days of growth on YPD agar at 20 • C, the streak culture was creamcolored, smooth, and dull. The margin was entire. In Dalmau plated culture on corn meal agar, pseudohyphae were not formed. Hyphae were seldom formed. Sexual structures were not observed on YCB, 5% MEA, PDA, V8, and CMA agar. Ballistoconidia were not produced on corn meal agar.
Physiologically, K. globosum differed from its close relatives K. sorbi in its ability to assimilate potassium nitrate and ethylamine and inability to assimilate raffinose, D-xylose, L-arabinose, D-glucitol, and D-gluconate.
Symmetrospora salmoneus X.Z. Liu, F.Y Bai, and X.Y. Wei, sp. nov. (Figure 5C) MycoBank: MB842985 Etymology: The specific epithet refers to the salmon color of the colony of the strains in this species.
Culture characteristics: After growth on YM agar for 7 days at 20 • C, cells were globose and subglobose, 4.1-6.6 µm × 3.6-6.3 µm, and occurred singly or in pairs. Budding was polar. After 7 days of growth on YPD agar at 20 • C, the streak culture was salmon-pink or orange-colored, smooth, slightly wrinkled, butyrous, and semi-glossy. The margin was entire. In Dalmau plated culture on corn meal agar, pseudohyphae and hyphae were not formed. Hyphae were seldom formed when mixed strains were cultured ( Figure 5D). Sexual structures were not observed on YCB, 5% MEA, PDA, V8, and CMA agar. Ballistoconidia were not produced on corn meal agar.
Culture characteristics: After growth on YM agar for 7 days at 20 • C, cells were subglobose, globose to ovoid, 3.2-6.4 µm × 2.5-5.7 µm, and occurred singly or in pairs. Budding was polar. After 7 days of growth on YPD agar at 20 • C, the streak culture was yellowish-cream, smooth, and glossy. The margin was entire. In Dalmau plated culture on corn meal agar, pseudohyphae and hyphae were not formed. Sexual structures were not observed on YCB, 5% MEA, PDA, V8, and CMA agar. Ballistoconidia were not produced on corn meal agar.
Physiologically, T. nitrariae differed from its close relatives, T. korlaensis and T. rosae, in the ability to assimilate D-glucitol and L-lysine and inability to assimilate sucrose and melezitose. Thus, it should be proposed as a new species in Tuenia.
Culture characteristics: After growth on YM agar for 7 days at 20 • C, cells were subglobose to ovoid, 2.6-5.6 µm × 2.4-4.3 µm, and occurred singly or in pairs. Budding was polar. After 7 days of growth on YPD agar at 20 • C, the streak culture was whitish cream, smooth, and slightly glistening. The margin was entire. In Dalmau plated culture on corn meal agar, pseudohyphae and hyphae were not formed. Sexual structures were not observed on YCB, 5% MEA, PDA, V8, and CMA agar. Ballistoconidia were not produced on corn meal agar.

Discussion
The Qaidam Basin has been proposed as the first Mars analog in China as it harbors a hyper-arid, cold, salt-deposited, and high UV environment similar to the surface of Mars, as well as Mars-like geomorphological structures and mineral composition [12]. It provides an ideal systematic model as a terrestrial analog with astrobiological and extremophile implications. This study reports the culturable yeast diversity in lichen, plants, and soils in the Qaidam Basin for the first time.
Generally, yeasts grow well in a slightly acidic environment, with the optimum pH between 4.5 to 5.5 [55]. Nevertheless, basidiomycetous yeasts, i.e., Naganishia albida, Papiliotrema laurentii, Rhodotorula glutinis, R. mucilaginosa, Sporobolomyces roseus, and Trichosporon asahii, were found to thrive at pH value above 10 [56,57]. In our study, the pH values among the sampling sites varied from 7.54 to 9.74, indicating the alkali nature of the sampling area. Yeasts were obtained in the sampling sites with pH values between 7.65 to 8.85 but not for the soil samples with pH values greater than 9. The salinity of soil samples in our study ranged from 0.16‰ to 58.51‰. Yeasts were detected in ten soil samples with salinity from 0.21‰ and 34.32‰. Meanwhile, no yeasts occurred in seven soil samples with salinity between 0.16‰ to 16.78‰, including two soil crust samples. Though pH and salinity limited the presence of yeast inhabitants to some extent, their presence did not directly correlate with these two abiotic variables. This is probably due to additional abiotic variables affecting yeast number and diversity that have not been captured in this study.
Approximately 85% of strains obtained in our study were tremellomycetous yeast species that are in good agreement with previous studies of other extreme environments [28][29][30]34,58]. Of 21 species identified in this study, six species, such as Diutina catenulata, Filobasidium magnum, Naganishia adeliensis, N. albida, Papiliotrema laurentii, and Vishniacozyma victoriae, had been reported to be resident in extreme environments, especially saline, alkali, cold or dry conditions [59]. The most frequent yeast species were assigned to Naganishia in our study in that about 39.2% of isolates were identified as N. adeliensis and N. albida. The high abundance combining frequent occurrence in more than half of the samples of these two species suggested that they are resident species in the sampling area of our study [60]. Naganishia adeliensis and N. albida are versatile extremophilic species that are frequently found in acidic, alkaline, hypersaline, cold, and arid environments [59]. These two species are found as the dominant species in hypersaline soils surrounding Urmia Lake and the dry sandy soils of the desert [30,57,61]. They are characterized as capsulated yeasts that might contribute to water retention and enhance water availability [62]. Filobasidium magnum was also detected in our study at a higher abundance and frequency (Table S2). Filobasidium magnum, which has been reported from Arctic glaciers [34,63] and plants inhabiting the Negev Desert and the Dead Sea region [64], was observed in our sampling sites in the Qaidam Basin that is characterized by hyper-arid, alkaline, and hypersaline environments. Similarly, strains of F. magnum were also plant-related with frequent isolation from plants in our study, implying this species may help plants to survive in dry areas. This was confirmed by the study where the application of F. magnum to the seed of Brassica alboglabra enhanced its metal resistance and its ability to grow in multi-metal contaminated soil [65]. We also isolated 32 strains of a novel species, Filobasidium chaidanensis sp. nov., from plants, which may own similar ecological functions as F. magnum. Though the other three species, Diutina catenulata, Papiliotrema laurentii, and Vishniacozyma victoriae, were detected in our study with low abundance and frequency, they were most commonly reported in previous studies. P. laurentii and V. victoriae displayed polyextremophilic capability, including acidophilic, alkali-tolerant, halophilic, xerotolerant, and psychrophilic aptitudes [59]. Diutina catenulata is a human pathogen that has been one of the most commonly reported in beach sand and estuarine and sea waters that represent alkali and saline environments [66,67].
Our study also yielded yeast species, namely Dioszegia hungarica, Kondoa sorbi, Pichia kudriavzevii, Teunia korlaensis, Udeniomyces puniceus, Vishniacozyma dimennae, and V. tephrensis, that have not been reported in microbial diversity research of extreme environments. Meanwhile, black yeasts, such as Hortaea werneckii, Phaeotheca triangularis, Trimmatostroma salinum, and Wallemia ichthyophaga, which are the common representatives in the hypersaline environment [59], were not found in our study. Additionally, seven undescribed species, namely Fibulobasidium sp., Kondoa globosum sp. nov., Kondoa sp., Symmetrospora salmoneus sp. nov., Teunia nitrariae sp. nov., Teunia sp., and Vishniacozyma pseudodimennae sp. nov., were found for the first time. Considering their low abundance and rare occurrence, they could be classified as transient species [60]. More impressively, most of these transient species colonized in plants, giving us an apparent indication of their role in plant growth in dry and alkaline soils. On the contrary, plants may help these organisms to survive in harsh conditions by transporting nutrition from soils. With this close association with plants, the transient species may obtain an intermedia to stabilize their colonization in harsh environments and then may evolve as a resident species with high abundance. The yeast diversity was significantly higher in the presence of vegetation in our study than that in the absence of vegetation, which has been proven in the Mars Desert Research Station in Utah, USA [17], probably indicating the important role of plants in the microbial growth and colonization.
Based on the delimitation of halophilic species that yeast strains can survive at salt concentration > 1%, such as seawater [68], the strains from the soil with salinities of 12.88‰ and 34.32‰ in our study have the potential to be halophilic species. Because they were distributed in lichen, plant, and soils with lower salinities, it is intriguing to know the extent of their ability to adapt to salinity variations, which will unravel the phenotypic scales of yeasts under extreme conditions. Along with the increasing NaCl concentration, the number of species that can grow well decreased from 17 species at 9% NaCl concentration to six species at 12% NaCl concentration and zero at 15% NaCl concentration. The four species that can grow at 12% NaCl concentration in our study, i.e., F. magnum, N. adeliensis, P. laurenti, and V. victoriae, have also been frequently found in the salty environment, and some of them from hypersaline lakes have been proved to tolerate 10% to 15% NaCl [30,59]. The intraspecies salt tolerance showed different tendencies. On the one hand, all the tested strains from lichen, plants, and soils have the same salt tolerance ability, such as F. magnum, P. laurenti, and V. victoriae. On the other hand, the adaption to salt stress should be variable inside the species. The strains of N. adeliensis and S. salmoneus from soils have extensive flexibility in changing environments compared to the strains from plants and displayed superior adaption along with elevated salinity. Previous studies indicated that halotolerant yeasts developed a number of structural and metabolic strategies to cope with the harsh environmental condition, including producing resting spores such as ascospores, teliospores, and chlamydospores [59]; adjusting the composition of a phospholipid, unsaturated fatty acid, and sterol of the yeast plasma membrane [69,70]; secreting extracellular cold-related enzymes [71] or redox enzymes to reduce the acidity [72]; synthesizing polysaccharidic capsule to prevent from drying [73]. The dynamic expression patterns of salt-dependent genes, such as ENA P-type ATPases involved in the Na + /K + transport, Glycerol-3-phosphate dehydrogenase regulating glycerol synthesis, and BAT2, ARG4, or AGP2 involved in amino acid metabolites, has been well studied, which provide genomic-level knowledge on the mechanisms involved in the halotolerant behaviors of yeasts, especially black yeast [69,74,75]. The underlying molecular mechanisms of halotolerance in species other than black yeasts deserve further investigation. It will increase our understanding of microorganisms in the saline environment and identify potential targets of economically important yeasts. To sum up, the biodiversity in Qaidam Basin not only gives us knowledge of life limits but also reserves resources such as halotolerant yeasts that can be used in agriculture as an eco-friendly biofertilizer to elevate crop productivity under saline conditions.

Conclusions
This is the first study that reports on the culturable yeast diversity in Delingha city in the northeast Qaidam Basin in Qinghai province, considered to be a mars analog area in China. It contributes to the general knowledge of yeast diversity in Mars analog. A total of 194 yeast isolates assigned to 12 genera and 21 species were found from ten soil samples and six plant and lichen samples with the dominance of basidiomycetous yeasts, which reveals the underestimated importance of basidiomycetous yeasts as inhabitants of the harsh environment of the Qaidam Basin desert. Furthermore, five novel species are described in this study. A significant difference in the number of yeast strains and species from plants (including one lichen sample) and soils was observed, indicating the close association between plants and yeasts. Microorganisms can be beneficial for the growth of the plant. Representative strains from different substrates of each species found in this study were subject to salt-tolerant ability from 0% to 15% NaCl concentrations. Seventeen and five species could grow at 9% and 12% NaCl concentration, while no strains could grow on the plate with 15% NaCl concentrations. Strains from soils can adapt well to the higher salt stress compared to those from plants and lichen. More comprehensive studies on yeast distribution across a wider range of sampling sites would be clearly desirable to better comprehend the distribution, ecology, diversity, and functions of yeast in the Qaidam Basin.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8080858/s1, Figure S1: The Maximum likelihood tree of (A) Filobasidium, (B) Kondoa, (C) Symmetrospora, (D) Teunia, (E) Vishniacozyma species based on D1D2 sequences; Figure S2: Growth of representative strains of all the species found in this study on YPD plates supplemented with 0%, 3%, 6%, 9%, and 12% (w/v) NaCl. Table S1: GenBank accession numbers of taxa used in phylogenetic analyses; Table S2: Occurrence and distribution of yeast species in different samples collected from the Qaidam Basin in Qinghai province. Table S3: The number of yeast strains isolated from different samples incubated at 17 • C and 25 • C; Table S4: The number of yeast strains isolated from different samples using PDA, 1/2 PDA, and 1/10 PDA media.