3.2. Novel species delineation and Identification
Delineation of a new species and phylogenetic placement of Nakazawaea atacamensis sp. nov.
We selected 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-BTand ATA-12C-B), while the third isolate was obtained from bark-exuded gum from another independent 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 sequence identity of the internal transcribed spacer (ITS) and the LSU rRNA region was 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 Candidaconglobata , 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 validate the phylogenetic relationships of the newly discovered taxon within the genus Nakazawaea , we conducted a comparative analysis using concatenated sequences. The sequences used for the analysis included the SSU rRNA gene, the ITS region, and the D1/D2 domains of the LSU rRNA gene from various Nakazawaea species. The obtained sequences were used to construct a Neighbor-Joining phylogenetic tree. The resulting tree revealed that N. atacamensis occupied an early diverging cluster, closely related to N. peltata , N. siamensis and N. odontotermitis (Figure 1 ). By comparing the DNA sequences of the investigated N. atacamensis strains with those of the N. peltata , N. siamensis and N. odontotermitis type strains, notable differences were observed. Specifically, N. atacamensis strains exhibited sequence divergences of 6%, 8% and 9% in the rRNA concatenated sequence when compared to the N. peltata, N. odontotermitis and N. siamensis , respectively (Figure S4 ). These findings provide strong evidence that the investigated strains represent a distinct anamorphic species within the Nakazawaea clade. Therefore, we propose the name Nakazawaea 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).