2.4 Genetic relationships of homostyles and heterostyles
Data generation - We genotyped all 613 individuals collected from the 22 natural populations using 12 microsatellites previously developed for P. vulgaris (Triest, Sierens, & Van Rossum, 2015)⁠. Briefly, the loci were amplified in two multiplex reactions with a QIAGEN Multiplex PCR kit. Detailed conditions for the amplification of microsatellites are included in Supplementary Material. Fragment size estimations were performed using an ABI Prism 3130xl genetic analyzer (Applied Biosystems) with GeneScan 500 LIZ (Applied Biosystems) as an internal standard. Alleles were scored using GeneMapper v.4.1 (Applied Biosystems). Presence of null alleles was estimated with Micro-checker v.2.2.3 (Van Oosterhout, Hutchinson, Wills, & Shipley, 2004)⁠⁠.
Analyses – For the analysis of genetic relationships, populations were divided in groups as follows: each dimorphic, heterostylous population (i.e., only SS- and LS-morphs) was defined as a single HE- group, whereas each trimorphic population was split into into a HE- (SS- and LS-morphs together) and a HO- (homostyles) group. Genetic distances between HO- and HE-groups were then estimated from the proportion of shared alleles among groups using the program MSA v.4.05 (Dieringer & Schlötterer, 2003)⁠⁠ with 1000 bootstraps. To infer the genetic relationships among HO- and HE-groups, a Neighbor-Joining tree (NJ) was inferred with PHYLIP v.3.697 (Felsenstein, 2005)⁠ using the majority rule consensus from 1000 replicated bootstraps.