Population genetic consequences of hemizygosity and the transition
to homosyly on S-locus genes
One of the most notable, recent discoveries on the S-locus is that it is
hemizygous and present only in thrums in all systems where its genetic
architecture has been investigated, including in Primula (Huuet al., 2016; Li et al., 2016), Turnera (Shoreet al. , 2019), possibly Fagopyrum (Matsui & Yasui,
2020), Linum (Gutiérrez-Valencia et al., 2022), andGelsenium (Zhao et al. , 2023), representing different
families and orders of flowering plants. The hemizygosity of the S-locus
should affect patterns of molecular diversity. Specifically, tight
genetic linkage provided by recombination suppression and thrum-specific
occurrence of S-locus genes are expected to cause a reduction of genetic
diversity inside the S-locus compared to other genomic regions
(Gutiérrez-Valencia et al. , 2021). Our results demonstrate that
the mean πS of S-locus genes
(CCMT , CYPT ,GLOT , and KFBT ;
πS: 0.0012 ± 0.0006) is lower than that of their
paralogs (CCM1 , CYP734A51 , GLO1 , and KFB1 ;
πS: 0.0034 ± 0.0007) located elsewhere in the genome
(Table 3). This result corroborates previous studies that found an
overall decrease in genetic diversity between the S-locus and its
flanking regions in Primula (Potente et al., 2022) andLinum (Gutiérrez-Valencia et al., 2022). Thus, our work
confirms predictions that S-locus genomic architecture influences
patterns of molecular evolution in S-locus genes.
The non-recombining nature of the S-locus also affects its response to
natural selection. Specifically, absence of recombination is expected to
reduce S-locus Ne , decreasing the efficacy of purifying selection
(Gossmann et al. , 2011) on S-locus genes compared to genes
outside the S-locus. Thus, increased degeneration due to accumulation of
deleterious mutations is expected in these genes (Charlesworth &
Charlesworth, 2000; Huu et al. , 2016). Conversely, if selection
to maintain function were strong, purifying selection should be more
efficient on S-locus genes than on their paralogs due to the dominant
nature of the hemizygous S-locus (Gutiérrez-Valencia et al. ,
2021; Potente et al., 2022). Regarding the former hypothesis, a
greater accumulation of transposable elements in S-locus non-coding
regions compared to the rest of the genome was detected, supporting the
conclusion that purifying selection on the S-locus might be relaxed
(Potente et al ., 2022). However, whether the efficacy of
purifying selection differs between coding regions of S-locus genes and
their paralogs remains poorly understood (Potente et al ., 2022).
Our results indicate that, on average, S-locus genes exhibit higher
accumulation of non-synonymous mutations than their paralogs, implying
purifying selection is less effective on the former
(πN/πS = 1.01 ± 0.37 and 0.53 ± 0.25,
respectively; Table 3), conformant with predicted effects of reduced
S-locus Ne . However, patterns of selective constraints within and
outside the S-locus vary among gene duplicates. For example, the
strength of purifying selection is similar betweenCYPT and CYP734A51, albeit slightly
stronger in the former (πN/πS = 0.28 and
0.38, respectively). Conversely, purifying selection is less efficient
in the S locus for KFB (πN/πS =
1.83 [KFBT ] and 0.23 [KFB1 ]),
whereas CCM shows the opposite pattern
(πN/πS = 0.91
[CCMT ] and 1.50 [CCM1 ]; Table
3). Taken together, the results imply that the effects of hemizygosity
on purifying selection vary among P. vulgaris S-locus genes,
corroborating previous results in P. veris (Potente et
al ., 2022).
A key question for the genetics of distyly concerns whether the strength
and nature of selection differ between S-locus genes with and without a
demonstrated function in distyly. Among the three, nine, and five
protein-coding genes identified in the S-locus of Gelsemium ,Linum , and Primula, respectively, (Li et al., 2016;
Gutiérrez-Valencia et al. , 2022; Potente et al., 2022;
Zhao et al., 2023) only two, namely CYPTand GLOT of Primula , have been
functionally characterized, showing that CYPTdetermines short styles and female self-incompatibility (Huu et
al., 2016, 2022), while GLOT determines high
anthers in thrums (Huu et al., 2020). However, it remains
unclear whether CCMT, PUMT ,
and KFBT play a role in Primula distyly.
The markedly reduced and non-floral specific expression ofCCMT, PUMT , andKFBT compared to CYPTand GLOT in both P. vulgaris andP. veris (Cocker et al., 2018; Potente et al.,2022) cast doubt on whether the former three genes are essential for
distyly. In the distylous Gelsemium elegans (Gentianales), the
homolog of Primula CCMT was absent from
the genome, while homologs of PUMT andKFBT were present but did not localize to the
putative S-locus and were expressed in both pin and thrum flowers (Zhaoet al., 2023). Taken together, previous evidence suggests thatCCMT, PUMT , andKFBT may not be essential for the core traits
of distyly (i.e., reciprocal placement of sexual organs and
self-incompatibility), hence they might be under relaxed purifying
selection. If this is true, one might expect thrums to exhibit higher
accumulation of non-synonymous mutations inCCMT , KFBT , andPUMT than in CYPT andGLOT . Indeed, our results support this
prediction, for we found weaker purifying selection onCCMT , KFBT , andPUMT (πN/πS =
0.91, 1.83, and 10.36, respectively) compared toCYPT (πN/πS =
0.28; Table 3A). It is unlikely that the results are explained by
positive directional selection on advantageous non-synonymous mutations
of the three genes above in thrums, because positive selection should
cause rapid fixation of advantageous mutations, hence absence of
polymorphism at non-synonymous sites (Hahn, 2020), which is not what we
found (Table 3A). To summarize, in P. vulgaris purifying
selection seems stronger on the only two S-locus genes for which a key
function in distyly has been demonstrated (namely,CYPT and GLOT ) than onCCMT , KFBT , andPUMT , which were not found in the S-locus of
other species and for which no differential expression between pin and
thrum flowers was detected. Discovering whether the three genes above
may play a role in controlling ancillary traits of distyly (e.g., pollen
size and number, male incompatibility) requires additional functional
studies in Primula and other distylous taxa.
Comprehensive population genetic analyses of variability in S-locus
genes and their paralogs had never been performed until now, due to
missing knowledge of relevant genes, unavailability of sequences from
said genes, and inadequate population sampling. Here, we expanded on
previous Sanger sequencing analyses of CYPT in
Somerset (England) populations (Mora-Carrera et al ., 2021) by
analyzing also sequences of S-locus genes and their paralogs extracted
from WGR data of Slovakian, Swiss, and Turkish populations of P.
vulgaris . First, homostyles, found exclusively in three Somerset
populations, exhibited lower genetic diversity than thrums for both
S-locus genes and their paralogs (Table 3), corroborating previous
reports of reduced genetic diversity in homostyles (Husband & Barrett,
1993; Ness et al. , 2010; Yuan et al. , 2017; Zhou et
al. , 2017; Zhong et al. , 2019). Second, both S-locus genes and
their paralogs have markedly lower genetic variation in English
populations than in other Eurasian populations of P. vulgaris(Table S2). This finding suggests a recent genetic bottleneck in English
populations. This bottleneck could be associated with colonization of
England following glacial retreat during the Last Glacial Maximum (ca.
10,000-12,000 years ago), as suggested for other plant species (Birks,
1989). Future genomic and demographic investigations will determine
whether the signatures of genetic bottlenecks detected in S-locus genes
and their paralogs apply to the entire genome, thus helping to infer the
timing and mode of P. vulgaris colonization of the British Isles.
Does lower viability of S*/S*-homostyles prevent the fixation of
homostyly in P. vulgaris?
Theoretical and experimental work suggests that, all else being equal,
once selfing originates, the selfing phenotype should increase in
frequency and eventually become fixed over time (Fisher, 1941; Lande &
Schemske, 1985; Charlesworth et al. , 1990). In the transition
from distyly to homostyly, Crosby’s model (1949) predicted that the rate
of increase and ultimate fixation of homostyles in a population depends
on whether homostyles with diploid S-locus have lower or equal viability
as the other genotypes in the population (Figure 1C and D). The
assumption of lower viability for S*/S*-homostyles of P. vulgarisexpanded upon evidence from crossing experiments in P.sinensis suggesting that homozygous dominant thrums had 30%
lower viability than heterozygous thrums (de Winston & Mather, 1941).
More recently, results of crossing experiments in a Primulahybrid (Primula x tommasinii) were interpreted as evidence
of inviability for S/S-thrums (Kurian & Richards, 1997). Furthermore,
population surveys of pin-to-thrum ratios in P. oreodoxaindicated that thrums were overrepresented at the seed
(~1:3) but not adult stage (~1:1),
implying that differences in viability could occur during the life cycle
(Yuan et al. , 2018). However, genotyping of thrums was not
carried out, thus preventing the determination of whether the decrease
of thrums from seed to adult stage was caused by lower viability ofS /S -thrums. Our observed frequencies ofS* /0- and S*/S*- homostyles from the two trimorphic,
English populations EN4-T and EN5-T of P. vulgaris are consistent
with Crosby’s prediction of a recent transition to homostyly (20-30
generations) under 30-40% lower viability ofS* /S* -homostyles (Table 4), supporting the model that
assumes lower fitness for S*/S*- homostyles than
S*/0- homostyles (Figure 1C).
Conversely, the occurrence of a monomorphic, homostylous population ofP. vulgaris in England, first reported by Curtis and Curtis
(1985) 38 years ago and recently sampled by Mora Carrera et al .
(2021 and present study) is congruent with the assumption of equal
viability for S*/S* homostyles. All 11 genotyped homostyles in this
population (here named EN6-M) carry the S* /S*- genotype
(Table 1 and Figure 3), thus EN6-M could represent a case in which
homostyly increased in frequency over time and became fixed in the
population by displacing pins and thrums, as predicted under the
assumption of equal viability for S*/S* homostyles (Figure 1D).
Alternatively, EN6-M could have been established by anS* /S*- homostyle stemming from a nearby population, thus it
might have been monomorphic homostylous from the beginning. Indeed,
Curtis and Curtis (1985) reported that this monomorphic population was
located only about 240 m away from a trimorphic population which might
have served as a source for the initial homostyle that gave origin to
EN6-M. Finally, EN6-M had a very low population size (n = 19;
Mora-Carrera et al., 2021) suggesting that stochasticity could
have played a role in the fixation of S*/S*-homostyles in this
population and that homozygosity of an S-locus with disruptedCYPT might have detrimental effects on
population growth.
To summarize, our results suggest that a diploid S-locus with
inactivated CYPT* may not per se be
incompatible with homostyle viability. However, the occurrence of two
copies of the remaining S-locus genes [i.e.,CCMT , GLOT ,KFBT , and PUMT ] in
the genome of a homostyle could have detrimental effects on viability at
different stages of the life cycle, possibly stemming from gene-dosage
effects (Rice & McLysaght, 2017; Ascencio et al ., 2021; Liet al ., 2015; Tasdighian et al ., 2017). Future research
combining S-locus genotyping and characterization of function and dosage
effects of S-locus genes at different life-cycle stages with fitness
measurements in the field and in greenhouse experiments is essential to
address whether differences in viability prevent the widespread fixation
of homostyly in P. vulgaris .