Sanger sequencing and phenotypic characterization
Part of the small subunit (SSU) rRNA gene, the internal transcribed spacer (ITS) region and the D1/D2 domains of the large subunit (LSU) rRNA genes were amplified and sequenced using primers NS1 and NS4 (Whiteet al. , 1990, Kurtzman & Robnett, 1998, Lachance et al. , 1999), ITS1 and ITS4 (White et al. , 1990) and LR0R and LR16 (Vilgalys & Hester, 1990, Moncalvo et al. , 2000), respectively. The PCR products were purified using QIAquick PCR columns (Qiagen) following the manufacturer’s instructions. After purification, the products were sent to Macrogen (Korea) for sequencing, employing the respective PCR primers. To identify the yeast species, the sequences of the SSU, ITS region, and the D1/D2 domains of the LSU rRNA gene were compared with those available in GenBank. This comparative analysis was conducted using the ‘blastn’ search utility (McGinnis & Madden, 2004). To identify novel species, we used a cut-off of 97% sequence identity (STACKEBRANDT & GOEBEL, 1994, Vu et al. , 2016, Lachance, 2018). All sequences generated during the study were deposited in NCBI GenBank. The GenBank accession numbers of the ITS, SSU, and LSU rDNA sequences are OP293325, OP293328, and OP293331 for ATA-11A-B (=CBS 18375T), OP293326, OP293329, and OP293332 for isolated ATA-12C-B (=CBS 18376) and OP293327, OP293330, and OP293333 for the ATA-13E-S (=CBS 18374) strain, respectively.
For phylogenetic analysis, only Nakazawaea species that exhibited sequences of the SSU rRNA gene, the ITS region, and the D1/D2 domains of the LSU rRNA gene were included. Sequences were edited, assembled, concatenated, and aligned using the MUSCLE multiple alignment program in MEGA software version 11 (Kumar et al. , 2018). The phylogenetic relationship of the novel species was determined through Neighbor-Joining analysis, based on the D1/D2 domains of the LSU rRNA gene, and using Pachysolen tannophilus as the outgroup species. For this analysis, the number of substitutions between the sequences was used as the distance metric. Confidence values were estimated from bootstrap analyses of 1,000 replicates (Felsenstein, 1985). Nodes were considered supported if the bootstrap percentage was ≥50 % (Hillis & Bull, 1993). The identity matrix between Nakazawaea species was generated with Bioedit (Hall et al. , 2011).
The yeasts were subjected to morphological, physiological, and biochemical characterization under solid media conditions using the standardized methods outlined by Kurtzman et al. (2011). To assess their fermentative capacity, the ability to metabolize glucose, fructose, and sucrose was examined in Durham tubes containing fermentation base media, with a final sugar concentration of 2% (w/v), as described by Yarrow (1998). The tubes were incubated at 25°C for a period of 14 days. For cell morphology analysis, observations were made using a Nikon Eclipse Ti2-E microscope equipped with differential interference contrast (DIC) optics after 3 days of growth in YPD broth, incubated at 25°C.