1 INTRODUCTION
The repeated shift from outcrossing to selfing is a central topic in
plant evolution
(Stebbins, 1957;
Cutter, 2019). Previous studies used phenotypic traits typically
associated with selfing to estimate, for example, the number and tempo
of transitions to selfing in phylogenies of ancestrally outcrossing taxa
(Goldberg & Igic, 2012; de Vos et al ., 2014). However, missing
knowledge of the genes that control mating systems has hindered the
study of molecular processes associated with transitions to selfing
until recently, especially in non-model organisms. Current advances in
genomics now facilitate the identification of the genes and mutations
associated with mating-system shifts.
A prime model to investigate the transition from outcrossing to selfing
has been the shift from distyly to homostyly in Primula (Barrett,
2019). Distyly is characterized by the co-occurrence in populations of
two types of self-incompatible individuals, called pins and thrums,
distinguished by the reciprocal arrangement of male (anthers) and female
(stigma) sexual organs in their flowers (Figure 1A; Ganders, 1979; Lloyd
& Webb, 1992; Keller et al. , 2014). This floral heteromorphism
represents an adaptation for outcrossing reported in at least 26
angiosperm families (Naiki, 2012). Conversely, homostyly is a floral
homomorphism that enables selfing. It is characterized by
self-compatible individuals bearing flowers with both stigma and anthers
at the same level in the corolla tube (Figure 1A; Barrett, 2019).
Evidence supporting higher selfing in homostylous than distylous plants
has been reported in diverse taxa (Belaoussoff & Shore, 1995; Schoenet al. , 1997; Mora-Carrera et al. , 2021). Independent
shifts from distyly to homostyly have been documented both within and
among species (Zhou et al. , 2012; Kissling & Barrett, 2013; de
Vos et al. , 2014; Ruiz-Martín et al. , 2018; Costa et
al. , 2019).
It has long been known that the S-locus supergene controls distyly and
the shift to homostyly (Lewis & Jones, 1992). However, the molecular
and functional characterization of the S-locus has been performed only
recently. The breakthrough occurred in the Primula system, where
the S-locus comprises five genes (CCMT ,CYPT , GLOT ,KFBT , and PUMT ) and is
hemizygous in thrums (S/0) but absent in pins (0/0; Figure 1A; Liet al. , 2016; Potente et al. , 2022). Two S-locus genes
were recently shown to control key traits in thrum flowers:GLOT determines high anthers, whileCYPT determines short stigma and
self-incompatibility (Huu et al ., 2016; 2020; 2022).
Specifically, experimental silencing of GLOT inPrimula forbesii thrums lowered anther position, producing
flowers with both anthers and stigma in the middle of the corolla tube
(i.e., short-homostyly). However, self-incompatibility was retained,
preventing self-fertilization in short-homostyles (Huu et al. ,
2020). Conversely, silencing of CYPT inPrimula veris was associated with both style elongation and loss
of self-incompatibility, thus turning self-incompatible thrum flowers
into self-compatible, homostylous flowers with both stigma and anthers
at the mouth of the corolla tube (i.e., long-homostyly; Huu et
al., 2016; 2022). Although both short- and long-homostyly have been
reported, the latter type is most common in Primula (Charlesworth
& Charlesworth, 1979; Lewis & Jones, 1992), likely because
self-compatibility in long-homostylous flowers enables
self-fertilization and reproductive assurance (Mora-Carrera et
al., 2021). Therefore, we hereafter refer to long-homostyly simply as
homostyly (Figure 1A).
The shift from distyly to homostyly has been intensely studied in
populations of P. vulgaris from Somerset, England, that display
variation of thrums, pins, and homostyles (Crosby 1940; 1949). Targeted
Sanger sequencing of the five individual CYPTexons of homostyles from the mentioned populations revealed that all
tested thrums shared the same functional CYPT allele (CYPT -1; Figure 1B). Contrariwise, 21
homostyles harbored six different CYPT alleles,
each with a unique, potentially disruptive mutation
(CYPT -2 to CYPT -7;
Figure 1B; Li et al., 2016; Mora-Carrera et al., 2021).
One possible explanation for the lack of sharedCYPT mutations among the homostyles is that
homostyly evolved independently multiple times. However, the same study
also found that six homostyles from two different populations had the
same CYPT allele as that of thrums (i.e.,CYPT -1). This result raised the possibility
that homostyly initially arose via CYPT silencing caused by either a structural rearrangement (such as an
inversion or a translocation) involving any of theCYPT exons or an inactivating mutation in theCYPT promoter, followed by multiple, unique
mutations in CYPT exons, as those found inCYPT-2 to CYPT-7(Mora-Carrera et al. , 2021; Charlesworth, 2022). Both types of
mutations (structural rearrangements in CYPT or
silencing of CYPT promoter) cannot be detected
using Sanger sequencing of individual CYPTexons. Determining whether homostyly in P. vulgaris arose
multiple times via independent mutations inCYPT exons or once through a shared structural
rearrangement involving CYPT exons or a
mutation in the CYPT promoter requires the
mapping against a genomic reference of extensive genomic sequences
covering both the S-locus and its upstream region. Both types of
resources are now available from whole genome resequencing data (WGR)
and published genomes for P. vulgaris (Cocker et al .,
2018) and its close relative P. veris (Potente et al .,
2022)
Furthermore, the availability of WGR data and reference genomes in the
selected study group facilitates the testing of population genetic
predictions concerning the evolution of the entire S-locus and S-locus
gene-paralogs in thrums, pins and homostyles. First, the thrum-specific
segregation of the hemizygous S-locus should cause a 3/4th reduction of
effective population size (Ne ) (Huu et al., 2016), hence a
decrease of genetic diversity in S-locus genes compared to the rest of
the genome (Gutiérrez-Valencia et al. , 2021). Secondly,
hemizygosity could have contrasting effects on the efficacy of purifying
selection on S-locus genes. On the one hand, the reduction of Nein the S-locus should make purifying selection less efficient (Huuet al., 2016). On the other hand, similarly to what happens in
the Y sex-chromosome (Gossmann et al ., 2011), selection to
maintain function of S-locus genes and the exposure of recessive
deleterious mutations under hemizygosity should enhance the efficacy of
purifying selection. However, the extent to which the efficacy of
purifying selection differs between genes within and outside the S-locus
remains poorly understood (Potente et al ., 2022). Finally, the
transition to homostyly could also reduce genetic diversity in S-locus
genes due to increased homozygosity in homostyles (Mora-Carrera et
al., 2021). The high-quality annotation of the five S-locus genes and
their four paralogs (CCM1 , CYP734A51 , GLO1 , andKFB1 ) in Primula (Li et al., 2016; Potente et
al., 2022), combined with sequences of these nine genes extracted from
WGR data, provides an ideal opportunity to test the above predictions
for the molecular evolution of the S-locus.
Additionally, the ability to assess the ploidy level of specific genomic
regions extracted from WGR data now enables the testing of predictions
about the changing frequencies of haploid and diploid S-locus genotypes
during the transition from distyly to homostyly. In a pioneering study,
Crosby (1949) proposed a model for how the frequencies of thrum, pin,
and homostylous phenotypes change over time (Figure 1C and D). This
model rested on then accepted genetic model for the S-locus, which
assumed that thrums were typically heterozygous dominant at the S-locus,
pins homozygous recessive, and homostyles stemmed from thrums via
recombination at the S-locus (Bateson & Gregory, 1905). Crosby assumed
that the viability of homozygous homostyles was either 35% lower than
or equal to the viability of pins, thrums, and heterozygous homostyles.
The assumption of lower viability for homozygous homostyles rested on
previous studies by Mather and Winton (1941) proposing that homozygous
dominant thrums (S/S) had lower viability than heterozygous thrums.
Crosby’s model is applicable also under the recently demonstrated
hemizygosity of the S-locus in Primula , by assuming that
homostyles with a diploid S-locus (S*/S*-genotypes; where S* indicates
an S locus with disrupted CYPT ) can have either
lower or equal viability as that of homostyles with a haploid S-locus
(S*/0-genotype), thrums (S/0-genotype), and pins (0/0-genotype; see
Figures 1C and D).
In P. vulgaris , repeated phenotypic surveys conducted in
Somerset, England, have shown that, when homostyles are present at high
frequency, thrums tend to be less frequent and, in some cases, absent,
compared to pins (Crosby, 1949; Curtis & Curtis, 1985; Mora-Carreraet al. , 2021). These findings align with the predictions of
Crosby’s model under lower viability of S*/S*-homostyles (Figure 1C).
However, one Somerset population consisted exclusively of homostyles
(Curtis & Curtis, 1985; Mora-Carrera et al., 2021), suggesting
that the fixation of homostyly is possible, as expected under the model
with equal viability for S*/S*- and S*/0-homostyles. However, previous
phenotypic surveys could not discriminate between S*/S*- and
S*/0- genotypes for homostyles. Recently developed sequencing
technologies enable the estimation of sequencing depth at the S-locus
(Gutiérrez-Valencia et al. , 2022), allowing us to determine
whether the S-locus is haploid or diploid in both homostylous and thrum
individuals. Therefore, it is now possible to estimate whether the
observed frequencies of S*/S*- and S*/0- homostyles in
natural populations support the model assuming lower or equal viability
for S*/S*- genotypes in relation to the other genotypes.
Here, we analyze WGR data from nine populations of P. vulgariswith varying frequencies of pins, thrums, and homostyles, to answer the
following questions: 1) Do all homostyles carrying different disruptedCYPT alleles share either a mutation in the
promoter region and/or a structural rearrangement involvingCYPT exons that might disruptCYPT function, allowing for the possibility of
a single origin of homostyly? 2) Do S-locus genes have lower genetic
diversity and efficacy of purifying selection than their paralogs? 3) Do
homostyles have lower genetic diversity in S-locus genes than thrums? 4)
Do observed frequencies of S* /0- andS*/S*- homostyles in natural populations better match genotypic
frequencies predicted under the assumptions of lower or equal viability
for S*/S*- homostyles? Our study illustrates how knowledge of the
genes controlling mating systems combined with high-quality genomic
resources generates novel insights into the genotypic changes and
evolutionary consequences associated with phenotypic transitions from
outcrossing to selfing.