DNA barcoding analysis of more than 9 000 yeast isolates contributes to quantitative thresholds for yeast species and genera delimitation

Abstract

DNA barcoding is a global initiative for species identification through sequencing of short DNA sequence markers. Sequences of two loci, ITS and LSU, were generated as barcode data for all (ca. 9k) yeast strains included in the CBS collection, originally assigned to ca. 2 000 species. Taxonomic sequence validation turned out to be the most severe bottleneck due to the large volume of generated trace files and lack of reference sequences. We have analysed and validated CBS strains and barcode sequences automatically. Our analysis shows that there were 6 and 9.5 % of CBS yeast species that could not be distinguished by ITS and LSU, respectively. Among them, ∼3 % were indistinguishable by both loci. Except for those species, both loci were successfully resolving yeast species as the grouping of yeast DNA barcodes with the predicted taxonomic thresholds was more than 90 % similar to the grouping with respect to the expected taxon names. The taxonomic thresholds predicted to discriminate yeast species were 98.41 % for ITS and 99.51 % for LSU. To discriminate current yeast genera, thresholds were 96.31 % for ITS and 97.11 % for LSU. Using ITS and LSU barcodes, we were also able to show that the recent reclassifications of basidiomycetous yeasts in 2015 have made a significant improvement for the generic taxonomy of those organisms. The barcodes of 4 730 (51 %) CBS yeast strains of 1 351 (80 %) accepted yeast species that were manually validated have been released to GenBank and the CBS-KNAW website as reference sequences for yeast identification.

Yamadazyma cocois f.a., sp. nov., an ascomycetous yeast isolated from coconuts

“…But we do not consider this strain as the same species, since the raw sequence read had even more differences: matching 483 of 486 nucleotides and a single gap (Dr. Boundy-Mills, personal communication). Thus, the similarity between the strains is less than the average level 99.51 % for D1/D2 domains of LSU which allows us to formally consider the strains as different species [26]. However, due to the impossibility of receiving the additional data we cannot exclude that the related strain is the same species from another habitat.…”

Selection of yeasts for their anti‐mold activity and prospective use in table olive fermentation

“…According to ITS sequences, fungal isolates obtained from olive drupes and brine were identified as P. crustosum (Visagie et al, ), Aspergillus sect. Nidulantes (Samson et al, ), Malassezia restricta (Castellá, Dall'Acqua Coutinho, & Cabañes, ), and Galactomyces candidum (Vu et al, ). The list of fungal taxa and relative GenBank accession numbers are reported in Table .…”

Grease Waste as a Reservoir of Lipase-Producing Yeast and Description of Limtongella siamensis gen. nov., sp. nov

“…After incubation, cell morphology was studied by using a phase-contrast microscope. A study of pseudohyphae formation was performed via slide culture on potato dextrose agar (PDA) at 25 • C. Growth at various temperatures was determined by cultivation yeast on YM agar incubated at 15,25,30,35,37,40 and 45 • C. Ascospore formation was examined for individual strains, and the culture was mixed at weekly intervals for three months on YM agar, YPD agar, PDA agar, corn meal agar (CMA), 5% malt extract agar, Gorodkowa agar, and Fowell's acetate agar [31] at 25 • C. The assimilation of carbon and nitrogen compounds was investigated in a liquid medium.…”

“…According to the criterion proposed by [39], the yeast strain showed greater than 1% nucleotide substitutions of the D1/D2 domain of the LSU rRNA gene, compared with the type strains of species representing a distinguished species (the multiple alignment of the D1/D2 domain of the large subunit (LSU) rRNA gene, SSU, RPB I, RPB II and EF-1α, and genes of the strains DMKU-JMGT1-45 T , DMKU-JMGT4-14, C. incommunis and Deakozyma indianesis are shown in Figures S1-S5, respectively). Meanwhile, according to the guidelines proposed by [40], the discrimination of current yeast genera possessing similarity thresholds of lower than 96.31% for ITS and 97.11% for LSU are taken to be different taxa. Based on the analysis of sequence similarity of the D1/D2 domain of the LSU rRNA gene and the ITS region, the strains DMKU-JMGT1-45 T and DMKU-JMGT4-14 differ from their closest species, Candida incommunis CBS 5604 T , by 10.41% nucleotide substitutions (53 nucleotide substitutions and 25 gaps of 509 nucleotides) and 20.96% nucleotide substitutions (87 nucleotide substitutions and 46 gaps of 415 nucleotides) of the ITS region.…”