3.2. Novel species delineation and Identification
Delineation of a new species and phylogenetic placement of Nakazawaea atacamensis sp. nov.
Three Nakazawaea isolates from different tree samples, representing a novel anamorphic and sucrose-fermenting yeast species, were isolated from N. chilensis samples in the Atacama Desert of Chile. Two isolates of N. atacamensis were obtained from bark samples from two different trees (ATA-11A-BT and ATA-12C-B), while the third isolate was obtained from bark-exuded gum from another tree (ATA-13E-S). To assess the sequence divergence among the three N. atacamensis isolates, we compared the ITS region, the small subunit (SSU) rRNA gene sequence, and the D1/D2 domains of the LSU rRNA gene. We found that the sequences of the internal transcribed spacer (ITS) and the LSU rRNA domains were 100% identical among the three strains (Figure S2 ). However, in the SSU rRNA region, the ATA-11A-BT and ATA-12C-B isolates, both derived from bark samples, exhibited a 99.8% sequence identity compared to ATA-13E-S isolate (Figure S3 ). Strain CBS 5808 (GenBank accession number AY366526) and the novel species have identical D1/D2 sequences. This strain was deposited in 1970 by J. Grinbergs in the CBS Yeast Collection as Candida conglobata , and it was isolated from tree bark in Chile. The ITS sequences of strain CBS 5808 and the novel species were also identical, showing that they represent the same yeast species.
To determine the presence of the new Nakazawaea species in a phylogenetic context, we conducted a comparative analysis using the D1/D2 domains of the LSU rRNA gene from the currently acceptedNakazawaea species. The obtained sequences were utilized to construct a Neighbor-Joining tree. This tree highlighted the clustering of N. atacamensis with N. pomicola , and also indicated that N. atacamensis represents a novel species (Figure 1 ). The purpose of the Neighbor-Joining tree shown in Figure 1 is to demonstrate that our Nakazawaea isolates are indicative of a new species rather than to establish a comprehensive phylogeny of the genus. By comparing the DNA sequences of the investigated N. atacamensisstrains with those of the genus, notable differences were observed. Specifically, N. atacamensis strains exhibited sequence divergences to the other species in the genus ranging from 1.3% to 5.2% in the D1/D2 domains. These findings provide strong evidence that the investigated strains represent a distinct anamorphic species within the Nakazawaea clade. Therefore, we propose the nameNakazawaea atacamensis f. a., sp. nov. for these three isolates. The mention forma asexualis (f. a.) is added as a reminder that the sexual state is not known (Lachance, 2012).