Sanger sequencing and phenotypic characterization
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 (White et 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 a concatenated alignment of 1,895
positions encompassing the SSU-ITS-D1/D2 regions of the LSU 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. in 2011 (Kurtzmanet 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 (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.