3.3 Molecular basis of the transition to homostyly
We sequenced ~97% of the 1,587 bp comprising the five exons of CYPᵀfrom 17 SS- and 27 HO-individuals of P. vulgaris from seven and ten different populations, respectively. Seven different CYPᵀalleles were found in our sampling (CYPᵀ-1 to -7 ; Figure 3A) that differed from two previously reported alleles from long-homostyles here labelled as CYPᵀ-8 and -9(corresponding to CYPᵀ SLH1 andSLH2, respectively, of Li et al., 2016). All 17 SS-individuals shared the same allele (CYPᵀ-1 ), which was identical to a functional copy of CYPᵀ previously reported inP. vulgaris (GenBank: KT257665.1; Li et al., 2016). Unexpectedly, this putatively functional CYPᵀ allele also occurred in six HO-individuals. Furthermore, six CYPᵀ alleles (CYPᵀ-2 to-7 ) found in the remaining 21 HO-individuals differed fromCYPᵀ-1 by different kinds of mutations: four nonsynonymous mutations and two deletions. Specifically, the nonsynonymous mutation inCYPᵀ-2 introduced a premature stop codon in exon 2, whereas a 31 bp deletion in exon 5 (CYPᵀ-5 ) as well as an 8 bp deletion in exon 1 (CYPᵀ-6 ) introduced a frameshift leading to an early stop codon in the translation of CYPᵀ. Moreover, a mutation in exon 5 of CYPᵀ-3 changed a non-polar (phenylalanine) to a polar amino acid (serine), whereas another mutation in exon 3 of CYPᵀ-4changed an amidic (asparagine) to hydroxylic amino acid(serine). Finally, the arginine to histidine mutation in CYPᵀ-7 caused no change in amino acid side-chain polarity.
In the haplotype network, CYPᵀ-1 occupied a central node and was connected by a single mutational step to all other alleles except forCYPᵀ-5, which differed by two mutational steps (Figure 3B). Additionally, phylogenetic analysis recovered well-supported hierarchical relationships among the different CYPᵀ alleles in SS- and HO-individuals (Figure 4). Specifically, CYPᵀ-2 was shared among homostyles from different populations (T03, T04, T07, T10 and D*11; Figure 4);CYPᵀ-3 was unique to population T05 and CYPᵀ-5 was unique to population T09. Finally, two CYPᵀ alleles were detected in each of three populations: CYPᵀ-4 and CYPᵀ-6 were found in population T06, CYPᵀ-2 and CYPᵀ-4 in population T10, andCYPᵀ-2 and CYPᵀ-7 in population D*11 (Figure 4).