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.