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).