Fig 1.
Overview of the experimental design.
A) The rDNA locus in chromosome XII of the model yeast Saccharomyces cerevisiae, and a schematic representation of its rDNA unit. Boxes and lines represent the rDNA repeat structure and are not to scale. B) A three-step strategy is used to identify intra-genomic variants within a pool of 43 Z. rouxii related strains. Abbreviations: ETS, external transcribed spacer; ITS, internal transcribed spacer; IGS, intergenic spacer.
Table 1.
ITS restriction fingerprinting and direct sequencing of ITS regions and 26S rDNA D1/D2 domains.
Clusters G-1 to G-9 and singleton ITS profiles (-) are determined based on restriction digestion patterns of ITS amplicons with the endonucleases HaeIII, HhaI and HinfI. Restriction fragments lower than 70 bp are omitted from the analysis. Strains in bold are chosen as representatives of each cluster and/or unique ITS genotypes and are submitted to direct ITS and D1/D2 sequencing. Overlapping peaks in chromatograms due to ambiguous sites and/or indels are indicated as unreadable sequences. Closest-sequence matches are reported together with identity (%). Abbreviations: na, not applicable; nd, not determined; cp, copy.
Table 2.
Pairwise comparisons between intra-genomic ITS1 and ITS2 variants cloned from each strain.
Numbers of transition (si) and transversion (sv), as well as the number of indels are computed in pairwise comparisons between copies 1 and 2 of ITS1 and ITS2 segments with MEGA6. The total number of nucleotides in indels is reported in brackets. Identity (%) indicates percentage of identical nucleotides between copies 1 and 2 of full-length ITS region or ITS1/ITS2 segments scored within each strain. Abbreviation: cp, copy.
Fig 2.
Phylogenetic relationships within the Z. rouxii complex inferred from ITS sequences.
This phylogeny was inferred through the Neighbor-Joining method using the ITS sequence of Z. bailii CBS 680T (GenBank accession number AY046191) as outgroup. The percentage values of replicate trees in which the associated taxa clustered together in the bootstrap test (10,000 replicates) are shown next to the branches (only values higher than 60% are reported). The evolutionary distances were computed using the Tamura-Nei method [47]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. All positions containing gaps and missing data were eliminated. Z. rouxii and Z. rouxii-like clades are colored in red, Z. mellis clade in green, and Z. sapae and Z. sapae-like clades in blue. Black triangles represent strains with homogeneous 26S rDNA D1/D2 domains and heterogeneous ITS regions. Black circles indicate strains without any rDNA heterogeneity. White triangles indicate strains with heterogeneous 26S rDNA D1/D2 domains and homogeneous ITS rDNA regions.
Fig 3.
Neighbor-net network of ITS sequences.
Both intra- and inter-strain variants are considered. For display purposes, bootstrap scores are not shown. The scale bar represents the split support for the edges. Blue and red edges mark splits separating the three major clusters. Clusters described in the text are denoted by Roman numerals.
Table 3.
Pairwise comparison of D1/D2 sequences cloned from strains with intra-genomically variable D1/D2 sequences.
Intra-genomic D1/D2 variants cloned from individual strains are indicated as D1/D2 copies. The number of transitional (si) and transversional (sv) mutations, as well as number of indels and the involved nucleotides, were computed with MEGA6. The number of nucleotides in pairwise-aligned sequences is indicated as nt (tot), whereas the number of nucleotides in indels (N° nt) is reported in brackets. Identity (%) indicates the percentage of identical nucleotides between D1/D2 variants within individual genomes.
Fig 4.
Phylogenetic relationships within the Z. rouxii complex based on 26S rDNA D1/D2 sequences.
This phylogeny was inferred using the Neighbor-Joining method using D1/D2 sequence of Z. bailii CBS 680T (GenBank accession number U72161) as outgroup. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (10,000 replicates) are shown next to the branches (only values higher than 60% are reported). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tajima-Nei method [48]. All positions containing gaps and missing data were eliminated. Z. rouxii, Z. sapae, and Z. mellis clades are colored in red, blue, and green, respectively. Black triangles represent strains with homogeneous 26S rDNA D1/D2 domains and heterogeneous ITS regions. Black circles indicate strains without rDNA heterogeneity. White triangles indicate strains with heterogeneous 26S rDNA D1/D2 domains and homogeneous ITS rDNA regions.
Fig 5.
Neighbor-net network of D1/D2 sequences.
A dataset of 30 intra-genomic and inter-strain variants is considered. The scale bar represents the split support for the edges. For display purposes, bootstrap scores are not shown. Strains belonging to Z. sapae and Z. rouxii clusters are denoted by red and blue labels, respectively.
Fig 6.
Patterns of ribosomal homogenization detected in Z. rouxii complex strains.
Four evolutionary outcomes (or categories) result from homogenization of divergent intra-genomic ribosomal variants. Red, blue and green rectangles mark Z. sapae, Z. rouxii and Z. mellis ribosomal sequences, respectively; asterisks mark ITS sequences partially divergent from Z. sapae and Z. rouxii (minor clusters Z. sapae-like and Z. rouxii-like, according to Fig 2). r, s, and m indicate Z. rouxii, Z. sapae, and Z. mellis D1/D2 copy variants, respectively. Abbreviation: cp, copy.
Fig 7.
Model for the rDNA evolution in the Zygosaccharomyces rouxii complex.
A schematic course of the evolution in rDNA arrays is shown. Small rectangles represent ITS regions, whereas the big ones 26S rRNA genes; 18S and 5.8S rRNAs are omitted for simplicity. Black circles represent unviable spores, while the blue/red circles rare viable spore. Outcrossing followed by nuclear fusion (1) or incomplete lineage sorting (2) give rise to divergent repeats in individual genomes, and set evolutionary processes in motion leading to different patterns of intra-genomic variation. A polymorphic/hybrid ancestor can rarely produce viable meiotic spores with chimeric rDNA arrays (category I). Over time some lineages partially homogenize rDNA arrays (categories II and III), whereas other descendants can retain both ribosomal variants due to low levels of homogenisation by recombination (category IV). Abbreviation: ILS: incomplete lineage sorting. Alternatively, outcrossing between two divergent haploid cells results in a transient heterokaryon (without nuclear fusion) (3) which undergoes introgression, i.e. the transfer of genetic materials from a donor to a recipient cell. The blue rDNA array and the introgressed red array donated by the pink parental cell recombine, leading to chimeric rDNAs (category I).