Additive transcriptomic variation associated with reproductive traits suggest local adaptation in a recently settled population of the Pacific oyster, Crassostrea gigas

So far, colonization of C. gigas on European coasts has been investigated by ecological or marker-based population
genetic approaches, but the variability of phenotypic traits potentially associated
with this range expansion has only very recently been investigated 7]. Despite strong difference in expression levels between sexes, as previously reported
53], we nonetheless detected a clear difference in expression levels between progenies
of oysters originated from France and Denmark, and their hybrids. From the RDA approach
on microarrays we found that 4.3 % of the explained variance is ascribed by the progeny
in a mainly orthogonal way to sex differences, which means that despite the huge transcriptomic
differences between sexes, the more modest differences between progenies proved to
be mainly independent of the sex. Only characters being heritable could allow adaptation
because significant genetic bases of trait variation (rather than only phenotypic
plasticity) are necessary to allow response to eventual natural selection. In our
study, the use of hybrid progeny allowed to identify transcripts presumed to show
additive genetic variation. In the RDA the hybrid progeny appeared intermediate between
French and Danish, and 50 % of the transcripts contributing the most to RDA2 axis
proved to behave mainly additively. Our analyses allowed us to obtain a list of candidate
genes, the expression of which might have reflected adaptation during invasion or
were already genetically differentiated in the founding populations. By the way overall
microarray features, only a very limited proportion of the total genes showed intermediate
values in hybrids. The extensive non additivity of the transcriptome has been observed
in Drosophila (Gibson et al. 2 %, 54]) and maize (Auger et al., ~30 % 55]) in contrast with the classical assumption in quantitative genetics of predominately
additive genetics effects. Furthermore, a low proportion (2 %) of additive patterns
of gene expression was previously observed by transcriptomic analysis in larvae of
partially inbred Pacific oyster populations 50]. Finally, another study on oyster showed the non-additive nature of genetic variance
for fitness-related traits and that the non-additive genetic component of yield is
often the largest 56]. However, by focusing on a population likely to have adapted to a new environment
during invasion (Denmark), we have access to the adaptation filter on phenotypic evolution
and uncover additively behaving traits (intermediate expression in hybrids) in the
subset of transcripts that are the most differentially expressed. Interestingly, Wendling
and Wegner 7] recently investigated the adaptive potential of North Sea C. gigas populations to local Vibrio spp., proposing that dominantly inherited resistance
could facilitate fast adaptation.

In our study, ANOVA and eP
_ST
analyses gave complementary results on phenotypic traits differentiation between the
Danish and the French progenies. The majority of the mRNA differentially expressed
in the ANOVA analysis was not in common with eP
_ST
analysis. This could be explained by the fact that eP
_ST
estimates were only performed on transcripts showing additive patterns and that most
of the eP
_ST
outliers did not have a high p-value in the ANOVA analysis. Roberge et al. 37], who find similar discordant results between ANOVA and Q
_ST
estimates on salmon, suggested evolution as consequence of selection but without an
additive genetic basis for these traits. eP
_ST
estimates performed separately on males and females, demonstrates that considering
sexes separately, when sex effect is so strong on gene expression patterns like in
in gonads, could help in highlighting new candidates. Furthermore, this suggests that
sex-dependent adaptation might be involved in the observed genetically-based phenotypic
and transcriptomic variations of reproductive traits, supported by an increasing evidence
of a role for sex differentiated effects in the architecture of complex traits 57]. Sex interaction effects are common in model organisms for a wide range of traits,
and can often explain a substantial part of the genetic basis of phenotypic variation
58].

In males, functional annotation of outlier eP
_ST
estimates pinpointed mRNA encoding proteins involved in sperm motility. The A-kinase
anchor protein 7 isoform gamma [Genbank:AM866859], identified as outlier eP
_ST
both in males and females and more expressed in Danish progeny, is a A-kinase anchoring
protein (AKAPs) promoting the selective sequestration of intracellular cAMP-dependent
protein kinase (PKA) involved in epithelial sodium channel regulation 59]–61].This protein has been suggested to have additional and perhaps unique function in
spermatozoa as a scaffolding protein for the Rho-GTPase pathway that regulates sperm
motility. Furthermore, Kington et al. 62] found high expression levels of proteins having an important role in sperm motility
of C. gigas involved in Rho signalling pathways. Sperm motility is commonly used as criteria
of male gamete quality linked with the fertilization success and therefore potentially
favouring colonization of new habitats. Furthermore, Filamin-A [Genbank:CU686267],
more expressed in Danish progeny, is an Actin-binding protein that participates in
the anchoring of membrane proteins for the actin cytoskeleton and is involved in sperm
morphogenesis in mammals 63]. Finally, as cells protection against oxidative injury in marine invertebrates 64], Alternative oxidase (AOX, [Genbank:BQ426710]), more expressed in Danish progeny,
may have a role in oxidative protection in gonads, and potentially in gamete quality.
In females, the Molluscan insulin-related peptide 5 [Genbank:CU987248] was more expressed
in Danish progeny. In mammals, the role of insulin pathway in fertility is well known
65] and in fish the insulin pathway has been positively associated to gamete quality
66]. In C. gigas, an Insulin-related peptide receptor has previously been identified in oysters by
Gricourt et al. 67] as well as several factors of the insulin signalling pathway. Jouaux et al. 68] found that insulin pathway elements can modulate germinal cells proliferation during
food deprivation in the first stages of gametogenesis with expected consequences on
fertility.

A comparison of divergence in neutral markers, F
_ST
, to divergence in phenotypic traits, Q
_ST
, has widely been used as a method to assess the relative strength of genetic drift
and selection 34]. The Q
_ST
levels typically exceed that observed in F
_ST
suggesting an important role of natural selection on quantitative traits 34], 69]. In our study, we did not have access to Q
_ST
values but we restricted P
_ST
values to additively behaving transcript with intermediate expression levels in hybrid.
We found that mean eP
_ST
(0.04) was similar to the estimated F
_ST
values 24] on the same populations, while the outliers eP
_ST
had values greater than 0.15. Our results therefore suggest that diversifying selection
has most probably acted on outlier gene expressions. Finally, gene expression can
also be controlled by epigenetic mechanisms, potentially in a transgenerational manner,
meaning in a way, by genetic mechanisms non-DNA dependents. There are yet only a few
papers studying epigenetics in oysters 70], 71] and this is clearly an emerging topic. Moreover, few studies until now focused on
epigenetic-mediated adaptation in invasive species 72].

Sex-ratio and population expansion

In our study, we observed a female biased sex-ratio in the Danish progeny. Sex-ratio
biased to female in invasive species has also been observed in an estuarine shrimp,
Palaemon macrodactylus73] and was proposed as a good descriptor to detect invading populations in signal crayfish,
Pacifastacus leniusculus74]. Although sex determinism in C. gigas, an alternative and irregular protandrous hermaphrodite, is complex, it seems to
be determined both by environmental and genetic factors 75]. In our study, environmental effects were minimized by rearing progenies in common
garden conditions, indicating that the observed differences in sex-ratio are genetically
based. However interactions with environmental conditions have been suggested to result
from regulatory pathways involved in sex determination 76]. Two genetic models have been proposed for sex determinism in C. gigas. The first suggests the presence of 2-genotypes, a dominant male M allele and a protandric
recessive F allele 77]. The second propose a 3-genotypes model, FF for true female oysters, MM for true
male oysters and FM for individuals that may mature as females or males 75]. Furthermore, an energy-mediated sex determinism was proposed 78], in which sex-ratio could be a possible way to select faster-growing populations
when more females are produced in the first year of the life. The greater condition
index observed in the Danish progeny could translate a greater reproductive effort
in this new-expanded Danish population. This index is strictly correlated to ripeness
and gonadal occupation production during gametogenesis in oysters 79]. In species like C. gigas, having an “r” demographical strategy, characterized by high fecundity, reproductive
success is greatly dependent on the quantity of gametes produced, especially oocytes,
as well as their quality. Furthermore, Cardoso et al. 80] found that in Northern European locations, oysters produce smaller eggs in larger
quantities, suggesting an increasing reproductive output. The authors proposed that,
since smaller oocytes are thought to have a longer development time, the environmental
conditions along the Northern European coasts may result in increased larval dispersal
and possibly in further population expansion. In this context, a greater reproductive
effort together with a female-biased sex-ratio in C. gigas could favour a rapid colonization of new habitats.