DISCUSSION
We implemented a seascape genomics approach to test for differences in
neutral and adaptive genetic connectivity between two intertidal marine
gastropods, the direct developer Nucella lapillus , and the
broadcast spawning Steromphala umbilicali s, within similarly
distributed ranges in the UK and Ireland. Neutral genetic structure
conformed to expectations of lower connectivity in the direct developing
species, with pairwise FST being on average 11.4x higher
across N. lapillus populations than equivalent S.
umbilicalis populations. These findings agree with other studies
comparing direct and indirect development as reproductive strategies
across marine species (e.g., Sherman et al. 2008; Hoffman et al. 2011b),
although reports of contrasting findings also exist (Ayre & Hughes
2000; Richards 2007). Putative
outlier locus datasets identified more extensive genetic structure in
both species than was identified by neutral genetic loci, suggesting a
role for adaptive divergence despite ongoing gene flow. These results
contribute to a growing body of literature showing that high gene flow
and adaptive divergence can co-occur in marine organisms (e.g., Gleason
& Burton 2016; Hoey & Pinsky 2018; Sandoval-Castillo et al. 2018;
Xuereb et al. 2018; Selmoni et al. 2020). Seascape genomic modelling
indicated a greater role of environment on genetic structure in N.
lapillus than S. umbilicalis , with the latter being more
attributable to spatial geographic patterns that coincide with the
seasonal occurrence of oceanic fronts in the Irish Sea.
As hypothesized, patterns of genetic structure indicated greater
connectivity across our study area in the broadcast spawning S.
umbilicalis than the direct developer N. lapillus , regardless of
the size of habitat gaps between sites. Although there were significant
but low levels of differentiation (FST) between many
sites and a marginally significant effect of IBD, there was a lack of
distinct neutral genetic structure across the sampled area for S.
umbilicalis . Larval connectivity (AEM vectors), geographic structure
(dbMEM vectors), and environmental variables (PCs representing summer AT
and SST, and wave exposure) best explained the variation in this
dataset, with the largest proportion of explainable variation attributed
to geographic structure. However, when partitioned, no partial RDA
models were significant, supporting a general lack of explainable
structure in the dataset. Taken together, the genetic homogeneity ofS. umbilicalis populations within our study area can at least
partially be attributed to a combination of predicted larval dispersal
over small and medium distances and multi-generational stepping-stone
dispersal over larger distances. Surprisingly, predicted larval
connectivity accounted for only a small proportion of variation in the
neutral genetic dataset, perhaps because we did not account for
interannual variability or climate change in larval connectivity
matrices. Although Coscia et al. (2020) show interannual variability to
be low over short time-scales (6 years), incorporating this variability
may become more important over longer time-scales like those considered
here (100 years). Further, what we considered to be large habitat gaps
in our sampling design seemingly only constitute medium-scale dispersal
distances for S. umbilicalis , given that genetic connectivity
across these gaps was high.
Although we found much more genetic structure in N. lapillus thanS. umbilicalis , we found greater connectivity between populations
separated by large habitat gaps than would be expected for a direct
developing species (e.g., sites 2/3 and 10; Fig. 3). This finding
supports the contribution of drifting/rafting (e.g., Colson & Hughes
2004), and stepping-stone dispersal (e.g., Crandall et al. 2012) to
population connectivity in this species. Neutral genetic structure inN. lapillus indicated 5-11 genetic clusters of geographically
proximate sites, and while there was a significant relationship between
shortest marine coastal distance and FST, no significant
effect of IBD was detected. Neutral genetic structure was best explained
by two environmental PCs representing winter AT and summer AT and SST.
The effects of environmental gradients (e.g., SST, AT) on putatively
neutral SNP loci may be explained by either isolation by adaptation
(IBA; Nosil et al. 2009), whereby strong ecological selection against
immigrants results in adaptive population divergence that restricts gene
flow and allows neutral loci to diverge via genetic drift
(Thibert-Plante & Hendry 2010), or loose linkage to loci under
selection (Gagnaire et al. 2015). Additionally, total explainable
variation was low for this dataset, supporting the contribution of
additional factors to describing neutral genetic structure in this
species. Future efforts would benefit from incorporating simulations of
putative long-distance dispersal of N. lapillus (e.g., rafting).
Interestingly, sites within habitat gaps were more likely to be
colonized by N. lapillus , which we often found in small patches
or sub-optimal habitat that did not support populations of S.
umbilicalis . This may suggest that N. lapillus is more of a
habitat generalist than S. umbilicalis , or, alternatively, that
direct developers will have a greater likelihood of establishing new
populations since founders can be a fertilized female or a drifting egg
mass, allowing multiple offspring to hatch within the same area
(Johannesson 1988). This tactic facilitates rapid population increase as
encounter rates between individuals will be high in species with low
mobility and high rates of self-recruitment. In contrast, while S.
umbilicalis was found at two sites within the north Wales–Scotland
gap, we found only one individual at one of these sites (St. Bees Head),
and a very small population in the other (Selker Bay). This suggests
that while larval dispersing species may reach distant sites more
quickly and frequently than direct developers, they are likely to be
spread over a much broader area during their planktonic larval feeding
stage (Johannesson 1988). This dispersal tactic can lead to a low
density of individuals at sites beyond a critical distance, and thus low
population sizes and reduced encounter rates for subsequent generations
of reproduction.
In contrast to a lack of neutral genetic structure in S.
umbilicalis , outlier loci distinguished four genetic clusters
indicating that selection can still be a major driver of spatial genomic
structure, even in the face of extensive gene flow. These results are
not surprising for a broadcast spawning species, for which large
populations sizes are expected to reduce the effects of genetic drift
and thus increase the probability that population differentiation
results from localized natural selection (Nielsen et al. 2009; Gagnaire
et al. 2015). The distribution of adaptive genetic structure in S.
umbilicalis corresponds to clusters separated by the habitat gaps we
investigated. This structure was significantly explained by both
geographic and larval dispersal variables, but none of the environmental
variables investigated. However, larval dispersal vectors were not
significant in partial RDA models. Rather, most of the variation was
attributed to geographic vectors representing large-scale spatial
patterns differentiating sites in the northern Irish Sea/North Channel
from all others (Fig. 6, dbMEM_2), and Irish from British (particularly
south Wales) sites (Fig. 6, dbMEM_3). Interestingly, patterns in the
spatial predictor dbMEM_2 closely resemble the “Forbes Line,” which
demarcates the general northern limit of southern species within the UK
and Ireland (Forbes 1858), and in the summer coincides with tidal fronts
in the Irish Sea (Simpson & Hunter 1974). Where they occur, fronts have
been shown to affect the dispersal (e.g., Ayata et al. 2010; Firth et
al. 2021), and survival (e.g., Gaylord & Gaines 2000) of species by
establishing spatially stratified environmental conditions in
temperature and/or salinity (Pingree et al. 1974; Pineda 1994). The
extent to which these fronts may drive adaptation in S.
umbilicalis at its northern range edge is unknown, but seasonal fronts
that form in the Irish Sea during summer (Simpson et al. 2009) may be
especially influential, as they coincide with the timing of spawning and
dispersal. Alternatively, these results may suggest that our models did
not include other important environmental variables driving selection inS. umbilicalis or that selective pressures may vary
spatio-temporally, resulting in patterns of chaotic genetic patchiness
that do not easily correlate with environmental features.
The outlier locus dataset for N. lapillus suggested the potential
for 7-8 adaptive genetic clusters that do not correspond completely with
the structure observed in the neutral dataset. This putative adaptive
structure was best described by the same environmental variables as for
the neutral dataset (winter AT and summer AT and SST) but with the
addition of wave exposure and a minor, non-significant contribution of
one geographic vector. The significance of temperature to the adaptive
structure of N. lapillus populations in our study area
corroborates previous evidence supporting thermal-mediated selection in
this species. Specifically, Chu et al. (2014) identified several fixed
single nucleotide polymorphisms (SNPs) within heat stress-mediated genes
between clades of N. lapillus in the northwestern Atlantic.
Additionally, the significance of wave exposure corroborates many
previous studies establishing relationships between exposure and
adaptation of shell morphology in N. lapillus populations from
Europe (Hughes & Taylor 1997; Guerra-Varela et al. 2009; Pascoal et al.
2012; Carro et al. 2019) and North America (Etter 1988, 1996).
The most supported driver of environmental adaptation in marine species
is temperature (Liggins et al. 2019), and in the present study we
provide further evidence to substantiate its important role. Temperature
is as a strong stressor in the intertidal, where it underlies many
important ecological (e.g., latitudinal distributions [Helmuth et al.
2006] and vertical zonation [Somero 2002]) and physiological
(Tomanek & Helmuth 2002) processes. Intertidal species are influenced
by both sea and air temperatures during high and low tides,
respectively. Unfortunately, detailed and consistent meteorological
records are generally unavailable for intertidal regions, where sea
temperatures are likely influenced by air and ground temperature,
substrate, aspect, and tidal range, among other factors. For this study
we used average estimates of SST and AT over large, buffered areas (10
km), and thus acknowledge that we have likely not been able to
characterize fine-scale environmental features of the local seascape
that may substantially influence adaptation and fitness in our species.
This may partially explain the lack of environmental associations
detected in our S. umbilicalis outlier dataset, despite the known
impacts of temperature on its dispersal and physiology.
S. umbilicalis spawns multiple times throughout the year in its
range centre where sea temperatures are warmer, with shorter breeding
periods towards its northern range edge (the current study area) where
recruitment failure is associated with cooler temperatures (Bode et al.
1986; Kendall & Lewis 1986). Given the importance of temperature on
larval dispersal and settlement in S. umbilicalis , fine-scale
temperature data may aid in identifying environmental associations with
outlier loci that are involved in reproductive processes.
Our inability to identify sequence matches for most of our outlier loci
is not surprising, given the distant relationship of N. lapillusand S. umbilicalis to most model species, and the
underrepresentation of genomic resources and sequencing efforts for
marine invertebrates (Lopez et al. 2019). Of loci that we were able to
match to NCBI sequences, few were within coding regions, an increasingly
common result in SNP-mapping studies. Indeed, SNPs can be as far as two
Mbp away from, and are not necessarily closest to the genes they affect
(Brodie et al. 2016). Of the few loci that were located within coding
regions, both species showed matches to the estrogen receptor (ER) gene.
Although the function of the ER gene in molluscs is still largely
uncharacterized, it has been shown to overexpress in N. lapillusin the presence of estrogenic chemicals in raw urban/industrial effluent
pollutants, with concomitant increases in reproductive maturation
(Castro et al. 2007). The other two outlier loci that mapped to coding
regions in S. umbilicalis are less well characterized but may
also be involved in reproduction (He et al. 2015; Robay et al. 2018).
Our results support the hypothesis that substantial heterogeneity of the
seascape can support connectivity of the otherwise low mobility direct
developing species, Nucella lapillus , and create adaptive
divergence in an otherwise highly connected meta-population of the
broadcast spawning species, Steromphala umbilicalis . Ultimately,
characterizing spatial patterns in connectivity, estimating genetic
diversity, and identifying locally adapted populations will lead to a
better understanding of the resilience of marine species to changing
environments and allow for improved management of fisheries and marine
protected areas (MPAs; e.g., Miller & Ayre 2008; Sinclair-Waters et al.
2018). Sea temperatures around the UK and Ireland are predicted to catch
up with global trends within the next decade, and associations between
species abundance and sea temperatures observed over less than a decade
indicate the sensitivity of many species to these changes (Mieszkowska
et al. 2020). Thus, understanding the impacts of ocean warming on
intertidal marine species, and their potential to adapt to these changes
is an important goal.
Acknowledgements: This research was funded by the Ecostructure
project (part-funded by the European Regional Development Fund (ERDF)
through the Ireland-Wales Cooperation Programme 2014-2020). The authors
would like to acknowledge all members of the Ecostructure project
(http://www.ecostructureproject.eu/) who helped in the sample collection
and processing for this work, and Data2Bio LLC (Ames, Iowa) who provided
the sequencing services for this project. We are grateful to Niall
McKeown, Peter Lawrence and Ally Evans for helpful discussions of the
data and results. Data analysis was supported by the Supercomputing
Wales programme, which is part-funded by the European Regional
Development Fund (ERDF) via the Welsh government.