4.2 Multiple losses of heterostyly within species are associated with different molecular changes in the CYPᵀ gene
Phenotypic convergence implies independent origins of the derived character state (Rosenblum et al., 2014). Testing for convergence thus requires an explicit framework of relatedness to determine whether taxa characterized by the derived state share a single origin or not (Lee & Coop, 2019; Stone, Nee, & Felsenstein, 2011)⁠. The NJ tree inferred from microsatellites indicates that homostyles of the ancestrally heterostylous P. vulgaris(Mast et al., 2006)⁠ are genetically closer to heterostyles of the same population than to homostyles of other populations (Figure 2B), implying that homostyles of P. vulgaris do not share a single origin. This finding confirms previous results in Primula chungensis (Zhou et al., 2017), P. oreodoxa (Yuan et al., 2017), P. merrilliana (Shao et al., 2019), and Eichhornia paniculata(Barrett et al., 2009; Husband & Barrett, 1993)⁠. While analyses of relatedness among homostyles and heterostyles suggest independent origins of the former inP. vulgaris , they cannot reveal the molecular basis of such transitions. Therefore, we investigated whether independent shifts from heterostyly to homostyly were driven by the same or different molecular changes in the gene that controls stigma position (Huu et al., 2016; Li et al., 2016).
Recent advancements in genomics have enabled the identification of loci responsible for phenotypic convergence at the intraspecific level (Lee & Coop, 2019; Sackton & Clark, 2019)⁠. For instance, multiple loss-of-function mutations were detected in SCR or a SCR -like gene, suggesting independent losses of SI in Arabidopsis thaliana andLaevenworthia alabamica(Chantha, Herman, Platts, Vekemans, & Schoen, 2013; Shimizu, Shimizu-Inatsugi, Tsuchimatsu, & Purugganan, 2008)⁠. In the case of homostyly, recent studies indicated that disruption of CYPᵀ can cause a shift to homostyly (Huu et al., 2016; Kappel et al., 2017; Nowak et al., 2015)⁠. Accordingly, all short-styled individuals of P. vulgaris analyzed in the present study share the same functional CYPᵀ allele (CYPᵀ-1 ), whereas alleles with nonsynonymous mutations or indels (CYPᵀ-2 to -9 ) are found exclusively in homostyles (Figures 3A, B and 4). These mutations cause either changes in the polarity of amino acid side chain (CYPᵀ-3 and -4 ) or premature stop codons (CYPᵀ-2 , -5 and -6 ), both types of mutation presumably inducing loss or reduction of function (Zhang 2000). In one case (CYPᵀ-7 ), a nonsynonymous mutation did not change the amino acid side-chain polarity, thus this mutation may or may not affect protein function (Figure 3A). Notably, the alleles found in our study differ from the ones previously reported in two long-homostyles ofP. vulgaris (CYPᵀ-8 and -9 , originally namedCYPᵀ SLH1 and SLH2 , respectively; Li et al., 2016). Thus, a total of eight different putative loss-of-function mutations in CYPᵀ have been identified so far in homostylous individuals of P. vulgaris . Our haplotype network analysis shows that all disrupted CYPᵀ alleles are independently derived from the CYPᵀ allele of SS-morphsvia different mutations (Figure 3B). Overall, our results reveal that a diversity of changes in the same gene underlies independent origins of homostyly, providing new evidence on the genetic basis of phenotypic convergence within species.
Convergent phenotypes can be linked not only to mutations in coding regions of specific genes, but also to mutations in promoter regions and/or structural rearrangements. For instance, some individuals of the self-compatible A. thaliana were characterized by mutations incis -regulatory regions, inversions or splicing variants causing loss of function in the SCR or SRK genes that control self-incompatibility (Dwyer et al., 2013; Shimizu et al., 2008; Shimizu & Tsuchimatsu, 2015)⁠. In P. vulgaris , a surprising result is that six out of 27 HO-individuals have the same CYPᵀ allele as the SS-individuals (CYPᵀ -1; Figures 3A, 3B and 4). These six homostyles occur in two geographically and genetically close populations (M01 and T08; Figures 2A and 2B). The occurrence of a CYPᵀ allele with no mutations in HO-individuals suggests that other mechanisms might be responsible for the inactivation ofCYP , including mutations in its promoter region and/or structural rearrangements that cannot be detected via exon sequencing. In general, this result underscores that mutations outside genes controlling specific traits might represent important sources of phenotypic convergence.