Investigation of Genetic Relationships Between Hanseniaspora Species Found in Grape Musts Revealed Interspecific Hybrids With Dynamic Genome Structures

Abstract

Hanseniaspora, a predominant yeast genus of grape musts, includes sister species recently reported as fast evolving. The aim of this study was to investigate the genetic relationships between the four most closely related species, at the population level. A multi-locus sequence typing strategy based on five markers was applied on 107 strains, confirming the clear delineation of species H. uvarum, H. opuntiae, H. guilliermondii, and H. pseudoguilliermondii. Huge variations were observed in the level of intraspecific nucleotide diversity, and differences in heterozygosity between species indicate different life styles. No clear population structure was detected based on geographical or substrate origins. Instead, H. guilliermondii strains clustered into two distinct groups, which may reflect a recent step toward speciation. Interspecific hybrids were detected between H. opuntiae and H. pseudoguilliermondii. Their characterization using flow cytometry, karyotypes and genome sequencing showed different genome structures in different ploidy contexts: allodiploids, allotriploids, and allotetraploids. Subculturing of an allotriploid strain revealed chromosome loss equivalent to one chromosome set, followed by an auto-diploidization event, whereas another auto-diploidized tetraploid showed a segmental duplication. Altogether, these results suggest that Hanseniaspora genomes are not only fast evolving but also highly dynamic.

Keywords: Hanseniaspora guilliermondii; Hanseniaspora uvarum; MLST; biodiversity; evolution; yeast.

FIGURE 1

Phylogenetic tree of Hanseniaspora strains based on the five marker sequences ADP1, GLN4, RPN2, VPS13, and MET5. The tree was constructed with PhyML based on the concatenated sequence of 3220 sites. DBVPG 5828, which lost H. pseudoguilliermondii MET5 allele, was not included in the phylogenetic tree. H. valbyensis Y-1626^T was used as an outgroup. Branch lengths are proportional to the number of sites that differentiate each pair of strains. Branch support was estimated by the approximate likelihood ratio test approach (aLRT) and bootstrap of 100 replicates.

FIGURE 2

DNA content of Hanseniaspora strains as measured by flow cytometry. (A) Mean intensity of cell DNA at G1 (blue dots) and G2 (red dots) phases, normalized by the intensity of CBS 8772. Species name are colored in blue (H. opuntiae), orange (H. guilliermondii), turquoise (H. uvarum), green (hybrids), dark red (H. pseudoguilliermondii). (B,C,E) Intensity curve of H. opuntiae × H. pseudoguilliermondii hybrids (red) compared to the type strain of the parental species H. opuntiae in blue and H. pseudoguilliermondii in green. (D) Intensity curve of two H. uvarum strains, the type strain CLIB 303 and the triploid strain S382CB.

FIGURE 3

Read coverage from the six hybrid strains along the genomes of the two parental species H. opuntiae (in red) and H. pseudoguilliermondii (in blue). Reference scaffolds were reordered beforehand so that the genome relative position (x-axis) is directly comparable between both parental species. Mean coverage values were computed with SppIDer tool with a sliding-window of 1700 nucleotides (without overlap) and normalized by the mean coverage of Hanseniaspora values present in Supplementary Figure S8. Values are expressed in a log2 scale. Dashed gray lines indicate scaffold boundaries. The vertical green line indicates the position of the MAT locus in each subgenome.

FIGURE 4

Density of heterozygous SNPs along H. opuntiae and H. pseudoguilliermondii collinearized parental genomes in hybrid strains. Each dot represents the total number of heterozygous SNPs enclosed within 10-kb sliding windows. Scaffold order of reference genomes is the same as in Figure 3. Dashed gray lines indicate scaffold boundaries.

FIGURE 5

Analysis of individual-based genetic distance between hybrid strains. (A) Heatmap representations of the number of differentiating SNPs between strain pairs. The divergence between strains was assessed here using the number of IBS0 and IBS1 (Identity By State) SNPs. A SNP is defined as IBS0 when no allele is shared within the considered pair, and IBS1 when one allele is found in common. Therefore, as hybrids are haploid on their H. pseudoguilliermondii subgenome part (or auto-diploid in the case of CLIB 3263), only IBS0 SNPs were analyzed in that context. (B) Principal Component Analyses of the hybrid strain total SNP data. The principal components were constructed as linear combinations of 117,191 and 23,189 total SNPs identified following a joint variant analysis of H. opuntiae and H. pseudoguilliermondii reference genomes, respectively. Only the two first components are displayed, the total variance supported by both axes is indicated within brackets.

FIGURE 6

Scenario for hybrid formation. Each Hanseniaspora species is depicted by color coded budding cells: blue (H. opuntiae), yellow (H. pseudoguilliermondii), orange (H. uvarum), and different shades of green for the hybrids, depending on the proportion of each parental species. The number of vertical lines inside the mother cells represents ploidy: one line for haploids, two for diploids, three for triploids and four for tetraploids. Lines with multiple colors represent Mosaic chromosomes.