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.