Torulaspora

Detection of novel Torulaspora View in CoL View at ENA species with taxogenomics

The strains upon which the novel species descriptions are based ( Table S1 View Table 1 ) were obtained from various isolation programs and surveys whose details are given in Supplementary Data S 1. They were preliminarily identified using the sequences of the D1/D2 or ITS regions of the rDNA. After genome sequencing of strains suspected to represent novel species, single-copy orthogroups (SCOs) were identified from a predicted proteome dataset that included representatives of all known Torulaspora species. The resulting maximum likelihood phylogeny is shown in Fig. 1 View Fig . The putative novel species were distributed across the genus and generally have as closest relatives already known Torulaspora species. To further assess discontinuities at the species level, we calculated average nucleotide identity (ANI) values within and between representatives of the main clades of Fig. 1 View Fig . We took into consideration earlier studies suggesting 95 % or lower identity as a guideline for the separation of yeast (with et al. in italics: ( Lachance et al. 2020, Libkind et al. 2020), microsporidian ( de Albuquerque & Haag 2023), and prokaryotic ( Jain et al. 2018) species, and expanded our earlier calculations for species in this genus ( Silva et al. 2022). In the present study, all proposed novel species had ANI values that were equal or lower than 91 % when compared with their closest relatives, whereas intraspecific values were equal or higher than 96.5 % ( Fig. 1 View Fig and Fig. S1 View Fig ). Taken together, our analyses suggested the existence of an additional twelve novel species, which would add to the ten species currently recognized in the genus Torulaspora , more than doubling its size.

Recognition of the novel species using DNA barcode sequences

To investigate if the new species could be recognized using DNA barcode sequences and to determine if additional representatives could be found among sequences deposited in the NCBI archive, we prepared extensive phylogenies based on the two DNA barcode regions most frequently used for the delineation of yeast species, the D1/D2 region of the LSU rDNA and the complete ITS region. The corresponding phylogeny for the D1/D2 region is shown in Fig. S2 View Fig , the phylogeny based on the ITS region is shown in Fig. S3 View Fig , and the phylogeny combining both regions is shown in Fig. S4 View Fig . We included sequences from representative genomes used in Fig. 1 View Fig , as well as sequences retrieved from the GenBank database, whose accession numbers are listed in Table S 2. Although, we observed that the species delimitation obtained with whole-genome data ( Fig. 1 View Fig ) is not completely supported by the phylogenies based on those regions, all novel species could be recognized based on the DNA barcode sequences. The D1/D2 region ( Fig. S2 View Fig ) appeared to have less resolution than the ITS region ( Fig. S3 View Fig ) and thus we recommend the later for species identifications in the absence of whole genome sequences. In Fig. 3 View Fig , a simplified ITS phylogeny including a single representative from each species summarizes these analyses. It also depicts the number of nucleotide substitutions observed between the more closely related species. Among these, species pairs showing the lowest number of nucleotide substitutions were T. delbrueckii - T. mapucheana sp. nov. and T. pretoriensis – T. cukson sp. nov. that differed from their closest relative by five and four substitutions each, respectively ( Fig. 3 View Fig ). These substitutions were consistently found when multiple representatives were tested and are seen as apomorphies. i.e. unique for each species.

Our extensive analyses using DNA barcode sequences shown in Figs S2 View Fig , S 3 View Fig and S 4 View Fig allowed us to identify additional representatives of the novel species. For example, a considerable number of isolates of T. ventriculi sp. nov. was identified among strains isolated from soil in Cameroon and previously identified as T. globosa ( Aljohani et al. 2018) ( Fig. S3 View Fig and Table S 2). In the case of T. incommunis sp. nov. for which a single isolate was initially available, we detected one D1/D2 ( Fig. S2 View Fig ) and one ITS ( Fig. S3 View Fig ) sequence in the pool of GenBank sequences analysed that appear to belong to two additional strains of this species (Table S 2). In fact, our analyses allowed to enlarge the number of known representatives of most novel species and of all the already known species (Table S 2).

We also used these analyses to ascertain if any of the currently 15 recognized synonyms of T. delbrueckii coincides with any of the proposed novel species. These synonyms and their molecular identification are shown in Fig. S 5. All synonyms were confirmed to belong to T. delbrueckii . Moreover, for eight of these synonyms, whole-genome sequences also validated their identification as T. delbrueckii .

The genomic Torulaspora and phenotypic landscape of Our updated perspective of the genus Torulaspora , as revealed through whole genome sequences of 77 strains from 22 species, allowed us to take a snapshot of genome composition across all currently known species. Our first observation was that the genomic landscape of Torulaspora was highly variable, with a marked gene content oscillation both between and within species. Genomes of Torulaspora spp. contain, on average, 4978 (± 87) genes with a disparity of 409 genes between the most gene-rich, T. microellipsoides NRRL-Y-1549 (5219 genes), and the least gene-rich, T. asahi sp. nov. NBRC11086 (4810 genes). Although isolates of the same species exhibit some variability in gene-richness, this value was roughly similar within species. For example, the three isolates of T. mapucheana sp. nov. have an average of 4863 (± 43) genes, while the three isolates of T. microellipsoides contain an average of 5165 (± 46) genes.

This variability was reflected in the presence or absence of genes involved in the metabolism of key carbohydrate sources and propagates to the phenotypic level ( Fig. 2 View Fig ). For example, in T. delbrueckii and its closest relative, T. mapucheana sp. nov., the inability to grow on galactose was observed frequently and, in those cases, two of the three genes required for canonical galactose metabolism, GAL 1, GAL 7, and GAL 10 ( Hittinger et al. 2004), were absent or inactivated pseudogenes ( Fig. 2 View Fig ). Conversely, galactose growth in T. delbrueckii , and in all other species in the genus, was linked to the presence of these three genes. Overall, variation across the genus was seen for maltose and melibiose metabolism, as well as for the presence or absence of the associated α-glucosidase-encoding genes ( Fig. 2 View Fig ). Three classes of α-glucosidase-encoding genes were defined by analysing signature amino acids that correlate to substrate specificity (Table S 4). Following Viigand et al. (2018), we tentatively grouped the α-glucosidase genes in three categories corresponding to the type of enzymes they encode: maltases, isomaltases, and mixed maltase– isomaltase activity. Our results are generally consistent with previous observations in known Torulaspora species ( Silva et al. 2022) and suggest that maltose utilization is based on a widespread prevalence of α-glucosidase-encoding genes. Moreover, these genes appear to have been lost multiple times ( Fig. 2 View Fig , Table S 4). Thus, we observed that consumption of the substrate predicted the presence of the gene coding for the canonically associated enzyme, but not vice versa. Those latter cases (i.e. gene presence but lack of the corresponding phenotype) might be explained by gene inactivation or gene expression impairments. Such variations occurred both between and within species. For example, we found that all five isolates of T. jiuxiensis contained the three GAL genes, while they were completely absent in all five isolates of T. kanakia sp. nov. Moreover, the intraspecific variation that we previously observed for T. delbrueckii ( Silva et al. 2022) was observed here for other species, especially with respect to maltose and melezitose utilization ( Fig. 2 View Fig ).

However, the observed gene content variation at the species level was not universal. For traits, such as sucrose and trehalose consumption, the genes most commonly associated with these phenotypes were consistently found to be present in all Torulaspora genomes. As above, this consistent gene presence fails to act as a perfect predictor of a positive phenotype for sucrose, raffinose, and trehalose assimilation ( Fig. 2 View Fig ). Taken together, these results paint the picture of a genomic landscape that is highly diverse, with frequent events of gene loss that appear to be recent in terms of the ancestry of the genus Torulaspora (i.e. encompass a single species or a group of closely related species). This variation is not solely associated with the phylogeny. While some genes and traits are found consistently in one species but not another, others vary substantially among isolates of the same species, and the genus Torulaspora as a whole displays substantial variation in phenotype both within and between species (Fig. S 6).

The correlation between assimilation and fermentation abilities varied between substrates and species, but as expected, assimilation was a prerequisite for fermentation. For example, all isolates that could assimilate sucrose could also ferment it; with just two exceptions, all isolates tested for galactose assimilation were also positive for galactose fermentation. A similarly strong correlation was found for maltose assimilation and fermentation. However, raffinose and trehalose did not exhibit this correlation; most isolates that exhibited assimilation of these carbohydrates did not also ferment them. These correlations between assimilation and fermentation sometimes persisted within species. For example, all three tested isolates of T. jiuxiensis both assimilated and fermented raffinose, whereas all four tested isolates of T. ventriculi sp. nov. only exhibited assimilation. Likewise, all three tested isolates of T. jiuxiensis both assimilated and fermented melibiose, whereas the two tested isolates of T. obscura sp. nov. only assimilated this carbohydrate. However, variation within species was common; for example, one T. asahi sp. nov. isolate could ferment raffinose but not trehalose, while the other two isolates could ferment trehalose but not raffinose.