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 ᵀ ,
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.