Multi-gene phylogeny and taxonomy of the genus Bannoa with the addition of three new species from central China

The genus Bannoa is a small group of ballistoconidium-forming yeasts in the family Erythrobasidiaceae (Cystobasidiomycetes). Prior to this study, seven species belonging to this genus have been described and published. In this study, phylogenetic analyzes of Bannoa based on the combined sequences of the small ribosomal subunit (SSU) rRNA gene, the internal transcribed spacer (ITS) regions, the D1/D2 domains of the large subunit rRNA gene (LSU) and the translation elongation factor 1-α gene (TEF1-α) were conducted. Three new species, namely B. ellipsoidea, B. foliicola, and B. pseudofoliicola, were delimited and proposed based on morphological and molecular evidence. B. ellipsoidea was found to be closely related to the type strains of B. guamensis, B. hahajimensis, and B. tropicalis, but with 0.7–0.9% divergence (4–5 substitutions) in the LSU D1/D2 domains and 3.7–4.1% divergence (19–23 substitutions and one−two gaps) in the ITS regions. B. foliicola was found to belong to the same clade as B. pseudofoliicola from which it differed by 0.4% divergence (two substitutions) in the LSU D1/D2 domains and 2.3% divergence (13 substitutions) in the ITS regions. The distinguishing morphological characteristics of the three new species, with respect to closely related taxa, are discussed. The identification of these new taxa significantly increases the number of Bannoa that have been described on the surface of plant leaves. Additionally, a key for the identification of Bannoa species is provided.


Introduction
The genus Bannoa, belonging to Erythrobasidiales, is a kind of ballistoconidium-forming Basidiomycota. It was established by Hamamoto et al. (2002) based on a single species, Bannoa hahajimensis, which was isolated from dead leaves of plants collected in the Ogasawara Islands, Japan. Additionally, the anamorphic species obtained in this study were described as three new species of Sporobolomyces, namely S. bischofiae, S. ogasawarensis, and S. syzygii. However, these three species formed a monophyletic cluster with Bannoa hahajimensis in the Erythobasidium clade based on sequence analyzes of the SSU rRNA gene , the D1/D2 domains of the LSU rRNA gene (Hamamoto, 2011), and a combined gene dataset of SSU rRNA, ITS regions, LSU rRNA, and first and second codon of TEF1-α (Nagahama et al., 2006). This monophyly was confirmed by Wang et al. et al. based on a seven-gene dataset consisting of SSU rRNA, D1/D2 LSU rRNA domains, ITS regions, RPB1, RPB2, TEF1-α and CYTB (Wang et al., 2015a). Recently, the genus Bannoa was emended by Wang et al. based on the phylogenetic analysis of a seven-gene dataset (Wang et al., 2015b). In this seven-gene phylogeny, the genus represented a well-supported clade containing a teleomorphic species B. hahajimensis and three Sporobolomyces species, including S. bischofiae, S. ogasawarensis, and S. syzygii, which were formerly recognized as the Bannoa clade Nagahama et al., 2006;Hamamoto, 2011;Wang et al., 2015a). Recent incorporations into the genus are B. guamensis, B. rosea, and B. tropicalis isolated from the surfaces of diseased and healthy leaves of plants in the Euphorbiaceae, Asteraceae, and Poaceae plant families (Parra and Aime, 2019). The genus Bannoa was located in the family Erythrobasidiaceae but was phylogenetically distinguished from other recognized species in the genus Erythrobasidium (Wang et al., 2015b).
Yeasts in the genus Bannoa are best known for their orange to salmon-red appearance in culture (Hamamoto, 2011;Wang et al., 2015b). The sexual morph of Bannoa is characterized by the production of unicellular basidia on a clamp connection formed after mating, while the asexual morph is characterized by budding cells, some of which are ballistoconidia that can be ovoidal or ellipsoidal Hamamoto, 2011;Parra and Aime, 2019).
The worldwide species diversity of Bannoa has not been extensively studied. Until now, only seven species have been accepted in the genus Bannoa (Wang et al., 2015b;Parra and Aime, 2019). Among them, B. hahajimensis, B. ogasawarensis, and B. syzygii are reported to occur in China and Japan (Zang et al., 1998;Chiang et al., 2001;Hamamoto et al., 2002;Li et al., 2020), while B. bischofiae has only been identified in Japan , and B. guamensis, B. rosea, and B. tropicalis have been isolated in South America (Parra and Aime, 2019). In addition, several studies suggest the existence of other potentially new species that could belong to this genus (Matheny et al., 2006;Vaïtilingom et al., 2012;Wang et al., 2015a,b;Parra and Aime, 2019;Li et al., 2020).
During investigations of basidiomycetous yeasts from the Henan Province, central China, seven ballistoconidium-forming yeasts were collected. Morphological characteristics and phylogenetic analyzes based on SSU, ITS, LSU D1/D2, and TEF1-α sequences indicated that these yeasts represented three undescribed species of the Bannoa genus. This study confirms the taxonomic affinities of these new species, explores the species diversity of Bannoa in central China, and infers evolutionary relationships between representative species of Bannoa.

Sample collection and yeast isolation
Plant leaves were collected from the Baotianman Nature Reserve (33°30′44″N, 111°55′47″E) in the Henan Province of central China. The protected area measures 4,285 ha and is classified as a world biosphere reserve by the United Nations Educational, Scientific and Cultural Organization (UNESCO). The local climate is a typical transitional climate spanning a northern subtropical zone to a warm temperate zone, with cold and dry winters and fresh and rainy summers, and with annual mean temperatures around 15°C (Hu et al., 2022). Plant leaf samples were stored in sterile flasks and transported under refrigerated conditions within 24 h of collection. Yeast strains were isolated from plant leaves by the ballistospore-fall method as previously described (Nakase and Takashima, 1993;Hu et al., 2022). The semi-withered leaf samples were stuck with vaseline to the inside of the lid of petri plates containing yeast malt (YM) agar (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, and 2% agar) and incubated at 25°C for 7 days, and the media was refreshed daily until colonies gradually formed. Different yeast morphotypes were picked and purified by streaking onto fresh YM agar. All purified yeast strains were suspended in YM broth supplemented with 20% (v/v) glycerol and stored at −80°C ( Figure 1). Cultures of isolates obtained for this study were preserved in the Microbiology Lab, at Nanyang Normal University, Henan, China. All isolates used in this study and their origins are described in Table 1.

Morphological and physiological characterization
Morphological and physiological traits of the yeast strains were characterized according to methods established by Kurtzman et al. (2011). Colony traits were observed on YM agar after 2 weeks of incubation at 25°C. Mycelium formation was investigated by cultivation on corn meal (CM) agar (2% cornmeal infusion and 2% agar) in slide culture at 25°C for 2 weeks. The sexual processes of all strains were examined for individual strains and strain pairs on potato dextrose agar (PDA) (20% potato infusion, 2% glucose, and 1.5% agar), CM agar, and yeast carbon base plus 0.01% ammonium sulfate (YCBS) agar at 25°C for 2 months Parra and Aime, 2019). Glucose fermentation was carried out in a liquid medium using Durham fermentation tubes. Carbon and nitrogen source assimilation tests were conducted in liquid medium and starved inocula were used for the nitrogen assimilation tests (Kurtzman et al., 2011). Cycloheximide resistance tests were also performed in a liquid medium, while urea hydrolysis assays were conducted on agar slants. Acid production and reaction with diazonium blue B (DBB) were investigated on a solid medium in petri dishes. Growth at various temperatures (15, 20, 25, 30, 35, and 37°C) was determined by cultivation on YM agar. Cell morphology was examined using a Leica DM 2500 microscope (Leica Microsystems GmbH, Wetzlar, Germany) with a Leica DFC295 digital microscope color camera using bright field, phase contrast, and differential interference contrast (DIC) optics. Novel taxonomic descriptions and proposed names were deposited in MycoBank (http://www.mycobank. org; 8 January 2023).

DNA extraction, PCR amplification, and sequencing
Genomic DNA was extracted from the yeasts using the Ezup Column Yeast Genomic DNA Purification Kit according to the manufacturer's protocol (Sangon Biotech, China). Briefly, yeast cells were homogenized in snailase reaction buffe, followed by using with solution Frontiers in Microbiology 03 frontiersin.org to subsequently remove protein. The resulting DNA was further purified by adsorption column and resuspended in 50 μL TE Buffer. A total of four nuclear loci were sequenced, including the small ribosomal subunit (SSU) rRNA gene, the internal transcribed spacer (ITS) regions, the D1/ D2 domains of the large subunit (LSU) rRNA gene, and the translation elongation factor 1-α gene (TEF1-α). The primer pairs NS1/NS8 (White et al., 1990), ITS1/ITS4 (White et al., 1990), NL1/NL4 (Kurtzman and Robnett, 1998), and EF1-526F/EF1-1567R (Rehner and Buckley, 2005) were used for amplifying SSU, ITS, LSU, andTEF1-α, respectively. PCR was conducted as described by Toome et al. (2013) for SSU, ITS, and LSU. For TEF1-α, a touchdown PCR protocol was used as described by the research group (Wang et al., 2014). PCR products were directly purified and sequenced by Sangon Biotech Inc. (Shanghai, China). We determined the identity and accuracy of the newly obtained sequences by comparing them to sequences in GenBank and assembled them using BioEdit (Hall, 1999). Newly obtained sequences were then submitted to GenBank (https://www. ncbi.nlm.nih.gov/genbank/; Table 2).

Phylogenetic analysis
The phylogenetic relationship of the new Bannoa species and related species was determined by analysis of concatenated sequence datasets of four loci (SSU, ITS, LSU D1/D2, and TEF1-α). Naohidea sebacea CBS 8477 was used as the outgroup, according to Wang et al. (2015a). For the dataset, each gene region was aligned using MAFFT v7.110 with default settings (Katoh and Standley, 2013), and then manually adjusted in BioEdit 7.1.3.0 (Hall, 1999). The"Concatenate Sequence" function in PhyloSuite v1.16 was used to combine the aligned sequences of the different loci (Zhang et al., 2020). Manual gap adjustments were done to improve the alignment and ambiguously aligned regions were also excluded. Multi-locus phylogenetic analyzes were performed with MEGA11 software (Tamura et al., 2021) using the neighbor-joining (NJ) and maximum likelihood (ML) methods. The Kimura-2 parameter distance correction (Saitou and Nei, 1987) and the general time reversible (GTR) models (Nei and Kumar, 2000) were, respectively, used for the NJ and ML analyzes. Confidence levels of the clades were estimated from bootstrap analysis (1,000 replicates) (Felsenstein, 1985).

Molecular phylogenetic analysis
The combined SSU + ITS + LSU D1/D2 + TEF1-α dataset was analyzed to infer the taxonomic positions of the novel species described in this study. The dataset included sequences from 20 fungal samples representing 16 taxa including the outgroup, Naohidea sebacea CBS 8477 T . After alignment and trimming, the combined dataset contained 3,075 characters including gaps with 1,396 characters for SSU, 428 characters for ITS, 504 characters for LSU, and 747 characters for TEF1-α alignment, of which 358 characters were parsimony-informative. The best model for the SSU + ITS + LSU D1/ D2 + TEF1-α dataset estimated and applied in the ML analysis was GTR + I + G with equal frequency of nucleotides. ML and NJ analyzes produced almost identical topologies, thus only the ML generated tree is displayed with ML and NJ supported values above 50% at the nodes ( Figure 2). Our phylogenetic analyzes indicated that Bannoa formed a monophyletic group with high support (100% ML; 100% NJ; Figure 2). Ten phylogenetic species of the genus Bannoa are delineated, three of which were new: B. ellipsoidea, B. foliicola, and B. pseudofoliicola.
Bannoa ellipsoidea was grouped with the three described species, B. guamensis, B. hahajimensis, and B. tropicalis Parra and Aime, 2019), with strong support (80% ML; 80% NJ; Figure 2). The LSU D1/D2 sequences of B. ellipsoidea differed by 0.7% The yeast isolation procedures.  Fell et al. (2000), Scorzetti et al. (2002), and Vu et al. (2016), strains that differ by two or more nucleotide substitutions in the D1/D2 domains or 1-2% nucleotide differences in the ITS regions may represent different taxa. The differences in both the LSU D1/D2 and ITS sequences were significant enough for B. ellipsoidea to be considered a distinct Bannoa species. Bannoa foliicola formed a subclade with B. pseudofoliicola described in this study with full support (100% ML; 100% NJ; Figure 2). B. foliicola differed from its nearest phylogenetic neighbor, B. pseudofoliicola CBS 6936 T , by 0.4% divergence (2 substitutions) in the LSU D1/D2 domains and 2.3% divergence (13 substitutions) in the ITS regions. According to the criteria mentioned above, this data clearly supports the distinction between B. foliicola and B. pseudofoliicola at the species level.

Phenotypic characterization
All strains of the three new species formed orange-colored colonies ( Figures 3A, 4A, 5A) and ovoidal, ellipsoidal, and cylindrical vegetative cells (Figures 3B, 4B, 5B) like other Bannoa species. Ballistoconidia were formed and were ovoidal and ellipsoidal in shape ( Figures 3C, 4C, 5C). B. ellipsoidea produced more abundant ballistoconidia than strains of the other two new species. Sexual structures were not observed in the cultures of single or mixed strains on PDA, CM agar, or YCBS agar.
Description: On YM agar, after 7 d at 25°C, colonies are orange, smooth, glistening and butyrous in texture ( Figure 5A). The margin is entire. In YM broth, after 7 d at 25°C, cells are ellipsoidal and cylindrical, 3.2-3.7 × 7.7-12.6 μm and single, budding is polar ( Figure 5B). After 1 mo at 25°C, a pellicle and sediment are present.
Members of Bannoa have not been sufficiently studied, and the diversity of Bannoa is poorly understood. All known Bannoa species, including B. ellipsoidea, B. foliicola and B. pseudofoliicola described in the present study, have a relatively narrow geographic distribution range, found primarily in subtropical Asia and tropics South America. However, some strains of Bannoa have been isolated in different parts of the world; for example, Bannoa sp. MP3490 (DQ631900) has been obtained from Panama, Bannoa aff. Ogasawarensis MCA7670 (MK990652) and Bannoa aff. Ogasawarensis MCA 7643 (MK990651) from Vanuatu, Bannoa aff. Guamensis MCA7799 (MK990655) from Cameroon, and Bannoa sp. BRIP 28272 (OK001795) from Australia. In addition, several environmental sequences of Bannoa have also been reported from Mexico (James et al., 2016), from Austria, and from Australia (Raghavendra et al., 2017). This suggests this genus could be broadly distributed and further large-scale studies are needed to explore the diversity and distribution of Bannoa species worldwide. Ultimately, these findings will greatly improve our understanding of the diversity, distribution and ecology of Bannoa.
In conclusion, three new Bannoa species isolated from the surface of plant leaves in China are identified based on morphology and phylogeny; viz. B. ellipsoidea, B. foliicola and B. pseudofoliicola. This study enriches the species diversity of the genus, which will also promote its taxonomy and phylogeny.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Author contributions
C-YC and X-YC isolated the yeast strains and performed the taxonomic characterization. TL performed the phylogenetic analysis. C-YC drafted the manuscript and prepared the tables and figures. F-LH designed the study and revised the manuscript. All authors contributed to the article and approved the submitted version.