Genetic differences between genotypes and species
Following the identification and characterization of clonal variation,
subsequent analyses focused on a reduced dataset composed of one
high-coverage accession per genotype. Removal of the lower coverage
samples in each clonal group allowed more sites to pass the missing data
filter, resulting in an increase to 177,954 variant sites for this
highest-quality dataset. A PCA on the set of 40 unique genotypes
revealed the same groupings as in the total dataset (Fig. 3, Fig. S3).
For both PCAs, most accessions fell into three distinct groups of
tightly clustered accessions, corresponding to P. distichum and
the two ecotypes of P. vaginatum. For the reduced-sample dataset,
PC 1 explained 31.6% of the variance and separated P. distichumfrom P. vaginatum ; PC 2 explained 20.2% of the variance and
primarily separated the coarse- and fine-textured ecotypes of P.
vaginatum .
In addition to the three main PCA clusters, two coarse-textured P.
vaginatum genotypes were placed as intermediate between the fine- and
coarse-textured accessions along PC2 (Fig. 3, Fig. S3). These two
genotypes corresponded to GRIN accession PI 612771 and two of the
wild-collected accessions which were clones of one genotype (coarse_05,
Table S1). The intermediate placement of these genotypes suggests
possible admixture between the coarse-textured and fine-textured
ecotypes. To test this hypothesis, we created a reduced SNP dataset
containing only unique diploid P. vaginatum genotypes (33
accessions and 117,218 polymorphic sites). ADMIXTURE analysis shows that
the genetically intermediate coarse-textured individuals indeed have
genetic compositions consistent with admixture between coarse- and
fine-textured ecotypes (Fig. S4). This result suggests that there is
occasional gene flow between the two P. vaginatum ecotypes.
SNP variation in the rpl16 intron of the chloroplast genome
confirmed the three major groups identified by the GBS data. In the 1.15
kb aligned sequence, P. distichum differed from both ecotypes ofP. vaginatum by a total of 6 SNPs and one 10-bp indel, with one
additional SNP segregating within P. distichum (Table S2). WithinP. vaginatum , accessions of the fine-textured ecotype carried two
unique SNPs that distinguished them from the other two groups. These
results are consistent with the current taxonomy recognizing P.
vaginatum and P. distichum as two distinct species (Brummitt,
1983). In addition, the pattern of SNP variation within P.
vaginatum indicates that the fine-textured ecotype is characterized by
unique, derived variants.