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Research
Cite this article: Hollenbeck CM, Portnoy DS,
Garcia de la serrana D, Magnesen T,
Matejusova I, Johnston IA. 2022 Temperature-
associated selection linked to putative
chromosomal inversions in king scallop (Pecten
maximus). Proc. R. Soc. B 289: 20221573.
https://doi.org/10.1098/rspb.2022.1573
Received: 12 August 2022
Accepted: 8 September 2022
Subject Category:
Evolution
Subject Areas:
genomics, evolution, ecology
Keywords:
local adaptation, chromosomal inversion,
population genomics, molluscs
Author for correspondence:
Christopher M. Hollenbeck
e-mail: christopher.hollenbeck@tamucc.edu
Electronic supplementary material is available
online at https://doi.org/10.6084/m9.figshare.
c.6198521.
Temperature-associated selection linked to
putative chromosomal inversions in king
scallop (Pecten maximus)
Christopher M. Hollenbeck
1,2
, David S. Portnoy
1
, Daniel Garcia de la serrana
3
,
Thorolf Magnesen
4
, Iveta Matejusova
5
and Ian A. Johnston
6,7
1
Department of Life Sciences, Texas A&M University Corpus Christi, 6300 Ocean Drive, Corpus Christi, TX 78412,
USA
2
Texas A&M AgriLife Research, College Station, TX, USA
3
Department of Cell Biology, Physiology and Immunology, Faculty of Biology, University of Barcelona, Barcelona, Spain
4
Department of Biological Sciences, University of Bergen, Thormøhlensgt 53B, Bergen, Norway
5
Marine Science Scotland, Marine Laboratory, 375 Victoria Road, Aberdeen AB11 9DB, UK
6
Scottish Oceans Institute, School of Biology, University of St Andrews, St Andrews, Fife KY16 8LB, UK
7
Xelect Ltd, Horizon House, Abbey Walk, St Andrews KY16 9LB, UK
CMH, 0000-0003-0227-7225; DSP, 0000-0002-8178-1018
The genomic landscape of divergence—the distribution of differences among
populations or species across the genome—is increasingly characterized to
understand the role that microevolutionary forces such as natural selection
and recombination play in causing and maintaining genetic divergence.
This line of inquiry has also revealed chromosome structure variation to be
an important factor shaping the landscape of adaptive genetic variation.
Owing to a high prevalence of chromosome structure variation and the
strong pressure for local adaptation necessitated by their sessile nature,
bivalve molluscs are an ideal taxon for exploring the relationship between
chromosome structure variation and local adaptation. Here, we report a
population genomic survey of king scallop (Pecten maximus) across its natural
range in the northeastern Atlantic Ocean, using a recent chromosome-level
genome assembly. We report the presence of at least three large (12–22 Mb),
putative chromosomal inversions associated with sea surface temperature
and whose frequencies are in contrast to neutral population structure.
These results highlight a potentially large role for recombination-suppressing
chromosomal inversions in local adaptation and suggest a hypothesis to
explain the maintenance of differences in reproductive timing found at
relatively small spatial scales across king scallop populations.
1. Introduction
The field of evolutionary genetics, driven by population genomic techniques, is
increasingly concerned with the genomic landscape of divergence, which can
be defined as the distribution of diversity across the genome within and
among populations [1]. A common observation is the presence of ‘genomic
islands of divergence’among populations or species, which refers to genomic
regions of high genetic differentiation flanked by regions of low differentiation
[2,3]. Explanations for genomic islands of divergence initially focused on the
interplay of selection and gene flow, hypothesizing that these regions contained
variation important to local adaptation, thereby slowing the rate at which immi-
grant alleles move among populations, while gene flow homogenized allele
frequencies in adjacent, selectively neutral regions [3,4]. However, recent
research has demonstrated that genomic islands of divergence can arise
under a variety of conditions, including scenarios without selection or gene
flow [1,5,6].
© 2022 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution
License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original
author and source are credited.
Increasingly, chromosomal architecture is being implicated
in the process of adaptation and formation of genomic islands
of divergence [7,8], in large part because elements of chromo-
somal architecture including inversions, rearrangements and
centromere location can reduce or prevent local recombination
[9]. The effect is that alleles in regions of reduced recombination
are frequently inherited as large units, amplifying the signals of
forces that produce genomic islands across a larger genomic
region. Chromosomal inversions, which often completely sup-
press recombination in inversion heterozygotes (however, see
Navarro et al. [10]), may promote local adaptation through
the maintenance of sets of co-adapted alleles at two or more
loci (so-called ‘supergenes’; [11]). Chromosomal inversions
have been implicated in driving differences in mating systems
and local adaptation in a variety of taxa, including plants [12],
birds [13,14], insects [15,16] and fishes [17–20].
Bivalve molluscs are a useful model system for investi-
gating the relationship between genomic architecture and
adaptation, as there is ample evidence of local adaptation
across heterogeneous environments [21–23],aswellasagrow-
ing body of evidence documenting an exceptional degree of
genomic structural variation [24–26]. King scallop (Pecten
maximus), also known as great scallop, is a high-value mollusc
that supports a large fishery in the eastern North Atlantic
ocean, and for which attempts to describe genetic population
structure span decades [27–29]. The consensus among recent
microsatellite and single nucleotide polymorphism (SNP)-
based studies involving samples largely spanning the natural
range of the species (Spain to Northern Norway) is the exist-
ence of an ‘Atlantic’population (following the nomenclature
of [30]) in the south (Spain to the UK) and a ‘Norwegian’popu-
lation in the north, with comparatively small differences
observed among localities within these larger groups at neutral
loci [30,31]. A recent study using restriction site-associated
DNA sequencing (RADseq) was able to place the genetic dis-
continuity separating the two stocks in proximity to the
Norwegian Trench, located between the Shetland Islands
(UK) and Norway, and also reported the association of a
subset of loci with environmental parameters, notably sea sur-
face temperature, which tended to group individuals by
latitude in contrast to the neutral structure [31].
Using a recent chromosome-level genome assembly [32], a
population genomic survey of king scallop in the northeastern
Atlantic Ocean was conducted to describe the genomic land-
scape of divergence in king scallops sampled from Galicia,
Spain to north-central Norway, and a variety of genome scan
and environmental association approaches were employed to
assess population structure and genetic diversity at both
neutral and putatively adaptive loci across the genome.
2. Methods
King scallops were sampled from eight localities in European
waters of the eastern North Atlantic Ocean (figure 1). Individual
king scallops from Scotland were sub-sampled from a larger set
of individuals obtained from Marine Science Scotland survey
–20
–10
0
10
axis 2: 0.96 %
–20 –10 0 10
axis 1: 2.69 %
(a)
–5
0
5
axis 2: 6.73 %
–15 –10 –5 0 5
axis 1: 19.71 %
(b)
ESP
SW
NW
SE
NE
SLD
SNO
NNO
Norwegian
Atlantic
40
50
60
–10 –5 0 5 10
(c)
ESP
SW
SE
NW
NE
SLD
SNO
NNO
Figure 1. Study sampling distribution and neutral and outlier population structure. (a) Principal components analysis using 1852 neutral SNPs. (b) Principal com-
ponents analysis using 68 SNPs identified as selection outliers by at least one test. (c) Map of samples collected in the current study: NNO, north Norway; SNO, south
Norway; SLD, Shetland Islands; NE, northeast Scotland; SE, southeast Scotland; NW, northwest Scotland; SW, southwest Scotland; ESP, Spain. The red dashed line
represents the approximate location of the Norwegian Trench. ‘Atlantic’and ‘Norwegian’refer to populations identified by previous population genetic analyses [30].
(Online version in colour.)
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 289: 20221573
2
trawls in 2015 and 2016 and included individuals from southwest
(SW, n=15), northwest (NW, n= 32), northeast (NE, n= 31) and
southeast (SE, n= 29) Scotland, and the Shetland Islands (SLD,
n= 35). Individuals from Norway were obtained from fish markets
in the Hordaland (southern Norway; SNO, n= 20) and Trøndelag
(north-central Norway; NNO, n= 20) regions and individuals
from Galicia, Spain (ESP, n= 19) were obtained by diving. Further
information, including details of sampling location, is presented in
the electronic supplementary material, table S1.
Double-digest restriction-site-associated DNA libraries were
prepared following Peterson et al. [33] for 225 unique individuals
across the eight sample localities and were sequenced using
150 bp paired-end reads on two lanes of an Illumina HiSeq
4000 DNA sequencer. Raw sequence reads were demultiplexed
with the program process_radtags from the STACKS (v. 1.47) soft-
ware package [34]. Demultiplexed reads were processed with
the dDocent (v. 2.6) pipeline [35], which performs quality trim-
ming, read mapping and variant calling from the RAD data,
and reads were mapped to a draft of the king scallop genome
[32]. The resulting VCF file of genotypes was filtered stringently
following O’Leary et al. [36] using the programs VCFTOOLS
v. 0.1.16 [37], VCFLIB v. 1.0.0-rc1 (https://github.com/vcflib/
vcflib) and the R package vcfR v. 1.8.0 [38]. R scripts document-
ing the complete SNP filtering process can be found at https://
www.github.com/chollenbeck/king_scallop_popgen_2022.
To facilitate linkage disequilibrium (LD) pruning and later
haplotype-based tests for selection, the SNP genotypes were
phased using the program BEAGLE [39,40]. SNPs in the resulting
phased VCF file were then pruned for LD using the function
snp_autoSVD in the R package bigsnpR [41]. This pruning step
resulted in a ‘quasi-independent’set of SNPs used for parameter-
izing the selection outlier tests, which is intended to eliminate or
reduce bias caused by regions of low recombination [42].
Four genome scan methods were applied to identify loci
potentially under the influence of natural selection: (i) a Bayesian
differentiation outlier method implemented in the program
BAYESCAN [43], (ii) a principal components analysis (PCA)-based
differentiation outlier method implemented in the R package pca-
dapt [44], (iii) an environmental association method (latent factor
mixed models; LFMM) implemented in the R package LEA [45],
and (iv) an environmental association method (redundancy
analysis; RDA) implemented in the R package vegan [46].
Environmental association analyses used sea surface tempera-
ture, extracted from a geographical grid of global monthly sea
surface temperature data from January 1990 to December 2015
obtained from Ifremer’s CORA dataset (available at http://
www.ifremer.fr/erddap/griddap/CORA.html). Temperature
values for each grid point were averaged across the entire time
period to obtain a single estimate for each point on the grid,
and the temperature estimate at the grid point nearest to the
approximate geographical location of each sampling locality
was used in the association analyses. In addition, phased geno-
types were used to calculate two haplotype-based selection
statistics: iES, a single-population measure of haplotype homo-
zygosity (in this form the average length of shared haplotypes
in a particular genomic region) indicative of positive selection
[47,48], and Rsb, the log ratio of normalized iES between popu-
lation pairs [49]. These methods were implemented in the R
package rehh [50].
Results of the selection tests were used to separate the geno-
type data into two datasets: one containing loci that were
identified as being putatively under directional selection by at
least one of the genome scan methods and one containing the
remainder of the putatively neutral loci. Population genetic struc-
ture was evaluated for both datasets separately using PCA,
implemented in the R package adegenet [51,52]. Estimates of gen-
etic diversity (expected and observed heterozygosity) for each
sample locality and pairwise F
ST
were calculated using adegenet
and the R package hierfstat [53]. Pairwise F
ST
was also estimated
for sample localities grouped by region (Norway, Scotland and
Spain), based on the results of the outlier PCA.
To further test for an association between sea surface temp-
erature and genotype, allele frequencies for outlier loci in each
locality were decomposed into a set of composite synthetic
variables with correspondence analysis (CA), as implemented
in adegenet. The first CA axis (corresponding to outlier PCA
axis 1) was used as the dependent variable in a multiple linear
regression with sea surface temperature as the independent vari-
able and neutral genetic group (Norway versus Scotland/Spain)
and latitude as covariates.
In order to test for the presence of putative chromosomal
inversions or other regions of low recombination, pairwise LD
for all loci within each locality and region (Norway, Scotland
and Spain) was calculated using the R package gaston [54]. LD
network analysis (LDna), as implemented in the R package
LDna [55], was used to further explore the existence of chromoso-
mal inversions on chromosomes 2, 8 and 12. First, a pairwise
matrix of LD values was calculated with gaston, as above, with
individuals at all localities grouped together. Single outlier clus-
ters (SOCs) of loci linked together by LD were then identified,
and the resulting LD network was visualized with LDna and
the R package ggnetwork [56]. To explore the frequency of puta-
tive inversion genotypes, PCA was conducted, as above, but
separately for SNPs contained within the boundaries, defined
by LD blocks, of each putative inversion (local PCA; [57]). To
identify putative inversion homozygotes and heterozygotes, the
find.clusters function in adegenet was used to assign individuals
to one of three clusters (presumably non-inverted homozygotes,
inversion heterozygotes and inversion homozygotes). For each
putative inversion, frequency of each inversion genotype, hetero-
zygosity and conformance to Hardy–Weinberg equilibrium
within individual localities were then calculated using adegenet.
Using the reference genome annotation, genes in the vicinity
of each outlier region were extracted by selecting genes falling
within 50 kb (25 kb upstream and downstream) of each outlier
locus. In the case of large outlier clusters, all genes located
within the bounds of the region were selected as candidate
genes, whether or not they were within 50 kb of an outlier
locus. Candidate genes were further refined by assigning outlier
loci to two separate groups based on contribution to the principal
components in the outlier PCA. Further details regarding
methods used, including specific parameters, can be found in
the electronic supplementary material, methods and in R scripts
provided at https://www.github.com/chollenbeck/king_scal-
lop_popgen_2022.
3. Results
The raw sequencing data contained 562.9 million read pairs,
with a total of 514.5 million read pairs retained after demul-
tiplexing. Following read mapping and variant calling, a total
of 747 758 putative raw variants were discovered. Stringent
filtering produced a set of 1920 SNPs that were used in all
subsequent analyses.
Sixty-eight loci putatively under the influence of selection
were identified by at least one of the four methods. Eleven
loci were identified with all four methods. Fifty-three loci
were significantly associated with sea surface temperature,
based on at least one environmental-association test, and 30
loci were significantly associated with both environmental-
association methods. The genomic distribution of loci
putatively under directional selection was non-random,
with most loci grouped into one of three large regions (ran-
ging from 12 to 22 Mb) on chromosomes 2, 8 and 12
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 289: 20221573
3
(figure 2). The regions on chromosomes 2 and 12 exhibited
high estimates of global F
ST,
reduced heterozygosity, and
increased levels of haplotype homozygosity (iES) in Spain
(figure 3a–d; electronic supplementary material, figure S3A-
D). The approximately 17 Mb region on chromosome 8 exhib-
ited generally high estimates of pairwise F
ST
and showed
allele frequency differences similar to the other two regions
but did not exhibit a reduction in heterozygosity or an
increase in iES across the entire 17 Mb region in Spain (elec-
tronic supplementary material, figure S4A-D). For
chromosome 8, the distribution of Rsb (indicative of direc-
tional or divergent selection) contained several smaller
peaks rather than a single large peak.
PCA revealed that individuals grouped into three distinct
‘regional’groupings: Norway, Scotland and Spain. The PCA
involving only neutral loci revealed a primary component
of variation (explaining 2.69% of the total variation) that dif-
ferentiated Norway from Scotland and Spain, and a
secondary component of variation (0.96% of the total) that
differentiated Spain from Scotland and Norway (figure 1a).
Estimates of pairwise F
ST
based on the neutral dataset were
at least three times larger for comparisons between localities
in Norway and localities in Scotland or Spain (ranging from
0.036 to 0.045) than comparisons between localities in
Scotland/Spain (ranging from 0.008 to 0.010). Fine-scale sub-
structure was also detected between Scottish localities, with
SW Scotland differing significantly at neutral loci (F
ST
=
0.0043–0.0049) from NE Scotland and the Shetland Islands,
and there was a small, but significant difference (F
ST
=
0.005) between the two Norwegian localities (electronic
supplementary material, table S2).
The PCA conducted using outlier loci revealed a contrast-
ing pattern. The primary component of variation (19.5% of the
total) differentiated Spain from Norwayand Scotland, and the
secondary component of variation (6.71% of the total) differ-
entiated Norway from Scotland and Spain (figure 1b). SNPs
contributing most to each outlier PC tended to group together
in the genome, with loci contributing a larger effect to PC1 (the
temperature/latitude-associated pattern; figure 1b, PC1;
figure 2) tending to be located in the large regions identified
on chromosomes 2, 8 and 12. The majority of loci that were sig-
nificantly associated with sea temperature by the LFMM or
RDA methods (41 of 53) fell into these regions. The SNPs
contributing most to outlier PC2 (figure 1b, PC2) were located
on chromosomes 3, 10, 13 and 19. A comparison of regional
pairwise F
ST
confirmed this pattern, showing that SNPs
which strongly differentiated Spain from the other localities
(high pairwise F
ST
) tended to be located in the same regions,
while SNPs that differentiated Norway from the other
localities also tended to group together in the genome (elec-
tronic supplementary material, figure S1).
The regression-based test for genotype-environment
association with outlier loci was significant, both with sea
surface temperature as the sole independent variable (adj.
R
2
= 0.877; p< 0.001) and after correcting for the effects of
neutral genetic group and latitude (adj. R
2
= 0.991; p<
0.001). Visualization of the CA and allele frequencies from
the SNPs contributing most to CA axis 1 showed a north/
south gradient in allele frequencies, with alleles in SW
Scotland often intermediate to Spain and other Scottish
localities (electronic supplementary material, figure S2).
Visualization of pairwise LD revealed that the three
clusters of outlier loci on chromosomes 2, 8 and 12 fell into
well-defined blocks of extended LD (figure 3e–g; electronic
supplementary material, figures S3 and S4E-G), suggesting
a reduction in recombination over a large segment of each
of the chromosomes. For chromosome 12, LD was strongest
in Spain, with a block of LD (r
2
> 0.99) spanning at least
10 Mb (figure 3g). The same block of LD was apparent in
chromosome 12 in Scotland and Norway, but at reduced
levels of LD, as measured by r
2
(figure 3e,f). For chromosome
2, the LD block was more apparent in Scotland and Norway,
but largely because it was not possible to measure LD in
Spain owing to fixation of many of the SNPs in the LD
block. The LD block on chromosome 8 was largest (greater
than 17 Mb) and most clearly defined in Norway (electronic
supplementary material, figure S4E), although elevated LD
could still be seen in the same chromosomal region in Scot-
land (electronic supplementary material, figure S4F), and
LD was not able to be estimated at all loci owing to fixation
of several loci in Spain. LDna identified five SOCs
containing more than three loci on chromosomes 2 (19 loci;
chr2: 43819604–55023173, 8 (six loci; chr8: 8584060–
22490951) and 12 (10 loci; chr12: 1468734–12766088)
(electronic supplementary material, figure S5 and table S3).
Three of these SOCs (one on each chromosome) corre-
sponded to regions containing LD blocks identified with
previous analyses. In addition, two overlapping SOCs with
relatively lower median r
2
(containing four and six loci)
were identified adjacent to the major SOC on chromosome 2.
In general, SNP loci within the three LD blocks showed
similar patterns of allele frequency differences among
localities (electronic supplementary material, figure S2C),
but the frequency of putative inversion genotypes across
localities differed among the three LD blocks. For chromo-
somes 2 and 12, local PCA grouped individuals into three
genotype clusters (figure 4; electronic supplementary
material, figure S6). For chromosome 2, putative inver-
sion genotypes did not deviate from the expectations of
Hardy–Weinberg equilibrium in all localities and allele fre-
quencies were similar in Scotland and Norway, with one
inversion allele being completely fixed in Spain (electronic
Chr1 Chr2 Chr3 Chr4 Chr5 Chr6 Chr7 Chr8 Chr9 Chr10 Chr11 Chr12 Chr13 Chr14 Chr15 Chr16 Chr17 Chr18 Chr19
0.25
0.50
0.75
1.00
chromosomal position
global FST
selection outlier
true
false
temperature-associated
true
false
Figure 2. Global F
ST
plotted against genomic position for all 19 Pecten maximus chromosomes. Blue points represent loci identified as being under the influence of
natural selection by at least one test. Triangular points indicate loci significantly associated with sea surface temperature. Grey boxes highlight chromosomal regions
spanning several megabases containing selection outliers. (Online version in colour.)
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 289: 20221573
4
supplementary material, figure S6). For chromosome 12, one
putative inversion allele that was nearly fixed in Spain was
found in intermediate frequencies in Norway and at lower
frequencies in Scotland. In addition, an excess of heterozy-
gotes (α= 0.1) was found in northern Norway (NNO,
figure 4; p= 0.016) and also in the SW Scotland locality
(SW, figure 4; p= 0.070). The LD block on chromosome 8
revealed a more complex pattern of divergence than the puta-
tive inversions on chromosomes 2 and 12, with a component
of variation differentiating Spain from the other localities and
a component where Norway was differentiated from all other
localities (electronic supplementary material, figure S7).
0
0.25
0.50
0.75
FST
outlier
false
true
(a)
0.0
0.1
0.2
0.3
0.4
heterozygosity
(b)
106
2 × 106
3 × 106
4 × 106
5 × 106
iES
locality
ESP
SW
SE
NW
NE
SLD
SNO
NNO
(c)
0
1
2
3
010203040
position (Mb)
log(P)Rsb
comparison
Spain :: Norway Spain :: Scotland
Scotland :: Norway
(d)
chromosome 12
Norway
(e)
chromosome 12
Scotland
(f)
chromosome 12
Spain
(g)
r2
0
0.25
0.50
0.75
1.00
Figure 3. Signatures of selection at 97 SNPs on Pecten maximus chromosome 12. (a) Pairwise F
ST
(Scotland/Spain) plotted against genomic position for chromosome
12; (b) smoothed expected heterozygosity plotted against genomic position for each locality; (c) iES, a statistic that measures the average length in base pairs of
shared haplotypes (where larger values indicate larger regions of extended homozygosity, an indicator of a selective sweep) plotted against genomic position for
chromosome 12; (d) log of the p-value for test of statistical significance of Rsb, the log-ratio of iES for pairs of populations, plotted against genomic position for
chromosome 12; (e,f,g) heatmap of pairwise linkage disequilibrium (r
2
) for all loci on chromosome 12 for (e) Norwegian localities ( f) Scottish localities and (g)
Spain. Locality abbreviations: NNO, north Norway; SNO, south Norway; SLD, Shetland Islands; NE, northeast Scotland; SE, southeast Scotland; NW, northwest Scot-
land; SW, southwest Scotland; ESP, Spain. (Online version in colour.)
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 289: 20221573
5
A total of 2840 genes were identified to be in proximity to
an outlier SNP or contained within one of the three LD
blocks. Of these, 2774 were in proximity to SNPs contributing
a larger effect to outlier PC1 (latitudinal effect), while 66
genes were in close proximity to genes contributing more to
outlier PC2 (differentiating Norway from Atlantic localities).
4. Discussion
A chromosome-level reference genome and a genotyping-by-
sequencing approach was used to explore the genomic
landscape of divergence in king scallop in the NE Atlantic.
Neutral population genetic structure was in concordance
with the results of previous studies [30,31], supporting the
existence of distinct Atlantic (Spain to the UK) and Norwegian
populations, with a genetic discontinuity occurring in the
proximity of the Norwegian Trench separating the Shetland
Islands, Scotland, from Norway. Smaller scale neutral genetic
differences were also observed within the two larger popu-
lations, but a more complete sampling is needed to resolve
whether these differences represent distinct local subpopu-
lations [58] or whether an isolation by distance effect is
present. Putatively adaptive genetic variation revealed two
patterns of structure, with each pattern being driven by loci
localized to separate regions of the genome. The first, minor
pattern was spatially congruent with the neutral pattern of
variation (distinguishing Atlantic and Norwegian groups)
but driven by outlier loci with large differences between Nor-
wegian and Atlantic groups and may reflect regions of the
genome involved in local adaptation related to larger-scale
regional (Atlantic versus Norwegian) conditions. The second,
more pronounced pattern of adaptive genetic variation
observed, as summarized by outlier PC1, was characterized
by a latitude-associated pattern in which the southernmost
locality, Spain, showed a high degree of divergence in allele
frequencies from other localities. This component of variation
was significantly associated with sea temperature and was
almost entirely driven by loci localized to three large LD
blocks on chromosomes 2, 8 and 12.
(a) Evidence for chromosomal inversions
While localized reduction in recombination can be caused by
several possible aspects of chromosome architecture, including
proximity to centromeres [59] and structural variation (par-
ticularly chromosomal rearrangements such as inversions;
[9]), two pieces of evidence suggest that the three large outlier
regions and blocks of LD identified correspond to chromoso-
mal inversions. First, the LD blocks identified are all
relatively large (approx. 11–15 Mb) with well-defined bound-
aries, suggesting the presence of inversion breakpoints. As
an example, on chromosome 12, LD in Spain drops sharply
from nearly 1 to background levels (less than 0.01) moving
between adjacent SNPs that span the boundary of the LD
block between the SNPs at positions 12 083 837 and 12 091
012. Second, in certain localities increased LD can be seen
between loci flanking either side of the block of LD
(figure 3e), which would occur if these loci are adjacent in
2 052 968
2 374 464
2 445 430
3 352 856
5 932 388
6 543 028
8 041 319
9 752 326
9 929 586
10 421 552
12 083 837
individual
SNP position
genotype
0/0
0/1
1/1
–7.5
–5.0
–2.5
0.0
2.5
–5 0 5
PC1
PC2
inversion genotype
A/A
A/B
B/B
0
0.25
0.50
0.75
1.00
ESP SW SE NW NE SLD SNO NNO
locality
genotype frequency
inversion genotype
A/A
A/B
B/B
ESP
SW
NW
SE
NE
SLD SNO
NNO
(a)
(b)
(c)
(d)
Figure 4. Population frequencies of inversion genotypes on chromosome 12. (a) Genotype heatmap of all individuals (x-axis) at SNPs contained within the putative
inversion on chromosome 12 (y-axis). Colours represent SNP genotypes (blue, homozygote; green, heterozygote; red, alternate homozygote; grey, missing genotype)
and yellow boxes indicate genotype clusters in (b). (b) Local PCA of putative inversion on chromosome 12 showing clusters of inversion genotypes. (c) Population
frequencies of inversion genotypes. (d) Sample map with inversion genotype frequencies. (Online version in colour.)
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 289: 20221573
6
particular inversion genotypes or if there are multiple small
inversions present. The hypothesis of the existence of
inversions can be tested in future studies using alternative
methods to those presented here, including genetic mapping
[60], whole-genome resequencing [20] or polymerase chain
reaction-based methods [61].
Chromosomal inversions linked to adaptation have
recently been described in several marine systems, notably
in Atlantic cod [17,62] and threespine sticklebacks [63], as
well as in the rough periwinkle, an intertidal marine snail
[60]. The presence of chromosomal inversions is concordant
with recent findings that genomic structural variation may
be extremely prevalent in bivalve molluscs. Calcino et al. [24]
recently showed using highly contiguous reference genome
assemblies from several molluscan species that individual
bivalves tended to be hemizygous at approximately 4–7 per
cent of the genome, with the king scallop genome showing
6.14% hemizygosity. In addition, a high-quality assembly of
the Mediterranean mussel genome and resequencing of 14
individuals [25] revealed that approximately 25% of genes
were found to be missing owing to the presence–absence
variation in at least one of the resequenced individuals. The
authors of these studies have suggested that widespread geno-
mic structural variation in molluscs may support an ability to
rapidly adapt to heterogeneous environmental conditions
despite a high degree of connectivity, a hypothesis further
supported by the results of this study.
(b) Adaptive genetic variation
One key result of the present study that highlights the benefits
of establishing a genomic position for loci in a population
genomics context is the finding that patterns of divergence
revealed by each of the two primary outlier PCs tended to
be driven by loci grouped in separate regions of the genome.
The loci contributing to the weaker, secondary pattern of adap-
tive genetic variation (spatially congruent with neutral
structure) were found as singletons or pairs of outlier SNPs
on chromosomes 3, 10, 13 and 19, and potentially represent
loci that promote local adaptation across larger regional popu-
lations (Norwegian and Atlantic). However, the fact that allele
frequency patterns within these loci are congruent with the
overall neutral signal make it difficult to rule out the effects
of purely demographic processes [64]. One observation that
supports the effects of selection rather than drift alone is the
presence of related genes in multiple areas of the genome exhi-
biting the same signal: five of the 66 genes identified within
50 kb of these outlier loci (a tandem array of three genes on
chromosome 19 and an array of two genes on chromosome
10) were serine/threonine kinases, a family of proteins that
have been observed to be highly upregulated in scallop
gonads [65,66]. Phenotypic differences between Norwegian
and Atlantic king scallops have been documented involving
growth rates [67] and in proteomic comparisons [68], but
further work is needed to determine the underlying
mechanisms behind these between-region differences.
The second observed pattern of adaptive genetic variation
involved significant differences in frequencies of temperature-
associated alleles within putative inversions, which was in
contrast to neutral population structure. Reduction in hetero-
zygosity and elevated iES for putative inversions located on
chromosomes 2 and 12 observed in Spain are evidence for
strong positive selection (i.e. selective sweeps) in these genomic
regions [48]. Significant heterozygote excess for the putative
inversion on chromosome 12 observed in Norway and SW Scot-
land suggests that balancing selection may also have an
important role in shaping inversion allele frequencies. While
inversion heterozygotes may incur a fitness cost owing to the
inviability of gametes when recombination occurs within
inverted regions, selection for inversion heterozygotes genotypes
via overdominance, frequency-dependent selection, or selection
in spatially/temporally heterogeneous environments can
overcome this barrier [8]. A well-documented example of hetero-
geneous selection favouring chromosomal inversions is seen in
Drosophila melanogaster, where allele frequencies in some popu-
lations have been shown to fluctuate seasonally in response to
variables such as temperature, independently of neutral popu-
lation structure [16]. Balancing selection associated with
inversion polymorphisms in response to heterogeneous environ-
mental conditions has also been described in related Drosophila
subobscura [69] and Anopheles mosquitos [70]. Further evidence
for spatio-temporally heterogeneous selection in this study is
the observation that SW Scotland, which has warmer sea temp-
eratures compared to the other northern localities sampled
here, exhibited heterozygote excess, suggesting that putative
inversion heterozygotes may be favoured at intermediate lati-
tudes within the Atlantic population. The excess of putative
inversion heterozygotes in north-central Norway is also consist-
ent with this, as these individuals were sampled in an area
intermediate to groups shown to have different life-history
characteristics in the south and north of Norway [71].
However,onelimitationofthecurrentstudyisalackofspatially
continuous sampling along the European coast, which would
help to further elucidate whether the patterns detected here are
clinal in nature or spatially discrete, as well as defining the spatial
scales at which local adaptation is important.
(c) Structural variation, genomic islands and adaptation
Genomic islands of divergence are hypothesized to arise under
a variety of conditions, and these mechanisms can be broadly
classified by whether zero (selection is not involved), one, or
multiple loci within an island are targets of selection [6].
Chromosomal rearrangements, by producing regions of
low recombination, can be involved in any of these scenarios.
In the case of king scallop, it is unlikely that purely
demographic processes (e.g. allele surfing owing to range
expansion; [72]) are responsible for the observed signal,
based on the fact that the primary outlier signal contrasts
with neutral structure. Without further data, it is difficult to dis-
tinguish mechanisms that involve a single target of selection,
for example genetic hitchhiking associated with directional or
divergent selection acting upon a single locus [73] or back-
ground selection against deleterious alleles [74], from multi-
locus mechanisms, such as co-adapted gene complexes or
‘supergenes’[11]. The physical size of the putative inversions
(approx. 11–15 Mb) and the existence of multiple peaks of
reduced diversity and/or increased divergence within individ-
ual islands (figure 3; electronic supplementary material, figures
S3 and S4) observed here, are potential evidence for selection
acting on multiple loci within each large region [6].
Connecting the genomic landscape revealed here to adap-
tive mechanisms will require further work; however, one
hypothesis relates to the fact that temperature is known to
be among the most important factors influencing timing of
gametogenesis and spawning in bivalve molluscs, including
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 289: 20221573
7
P. maximus [75,76]. Previous studies have reported the major
spawning period for P. maximus to be roughly May through
to August [77], with some studies reporting additional
peaks in spawning activity in autumn or winter, notably in
Spanish populations [78]. It has also been noted that natural
populations tend to exhibit one of two different reproductive
cycles—one in which individuals rebuild gonads quickly
after spawning and another in which individuals wait until
the next year to rebuild gonads [79], and populations exhibit-
ing these different reproductive tendencies have been
identified at relatively small spatial scales within the Norwe-
gian [71] and Atlantic [80,81] populations. Further, transplant
studies have demonstrated that individuals relocated early in
life to areas with different reproductive cycles maintain the
reproductive cycle of their source population in the new
environment, suggesting heritable differences in the timing
of reproductive development [71,81,82]. However, few differ-
ences in neutral genetic variation have been identified at the
same geographical scales [27,30,31], consistent with the high
potential for dispersal owing to a relatively long pelagic
larval duration (18–42 days; [75]. The evidence for an associ-
ation between temperature and specific components of
genetic variation, originally reported by Vendrami et al.
[31], combined with the evidence that temperature-associated
genetic variation is associated with putative chromosomal
inversions reported here, supports the hypothesis that differ-
ences in the optimal timing of reproductive development and
spawning may be driven by localized selection operating on
suites of co-adapted genes contained within chromosomal
inversions that allow for the maintenance of locally adapted
variation despite the high potential for connectivity at small
spatial scales. Inversion-mediated adaptive divergence in
the face of high potential for gene flow is exemplified in the
well-characterized gastropod Littorina saxatilis, in which eco-
types characterized by differences in the frequencies of
inversion polymorphisms have been observed along interti-
dal transects on the scale of tens of metres [60,83]. While
the divergent phenotypes explored to date in the Littorina
system have been largely morphological, similar observations
have been made involving Atlantic cod and Pacific herring,
where genetic variation associated with reproductive timing
and strategy has also been associated with putative chromo-
somal inversions [17,84].
The size of the LD blocks and the large number of genes
contained within them make it difficult to identify specific
candidate genes that may be the targets of selection, particu-
larly because of the possibility that a single gene under
selection within the inversion could influence allele frequen-
cies within the entire region. However, a number of genes
previously linked to gonad-specific expression in scallops
are present in the putative inversions. These include several
serine/threonine protein kinases and phosphatases (two
tandem serine/threonine kinases on chromosome 2 and
four serine/threonine protein phosphatases on chromosome
12) and two adenosine deaminase-like genes on chromosome
8, all of which have been shown to be differentially expressed
in male and female scallop gonads [65,66]. In addition, mul-
tiple genes related to serotonin transport and signalling were
found in the putatively inverted regions (two 5-hydroxytryp-
tamine receptor-like genes on chromosome 12 and one
sodium-dependent serotonin transporter-like gene on
chromosome 2). Serotonin is known to be intimately involved
in the process of oocyte maturation in scallops and other
bivalves [85], and is known to be an effective inducer of
spawning in many bivalve molluscs [86]. Overall, the suite
of inversion-associated genes identified here will be a rich
set of candidate genes for future studies to attempt to identify
the targets of selection associated with these regions.
5. Conclusion
Observing and understanding the genomic landscape of diver-
gence, which is now possible owing to ever-improving
genome sequencing and assembly techniques, allows for a
more sophisticated view of microevolutionary processes
because it incorporates the effects of local recombination, in
addition to migration, drift and selection. The results pre-
sented here demonstrate an association between sea
temperature and genetic variation in specific regions of the
genome characterized by local reductions in recombination
and highlight the importance of establishing genomic context
in disentangling the effects of microevolutionary forces.
These results suggest a mechanism by which broadcast
spawning species with a high degree of connectivity can main-
tain genetic differences that allow for local adaptation. Further
work in king scallops and other taxa to better characterize
these systems will help to improve our understanding of
how chromosome structure variation contributes to
evolutionary change.
Ethics. Tissue samples for the study were obtained by Marine Science
Scotland survey trawls (Scotland), per Scottish Government field col-
lection protocols or through purchase/recreational collection
(Norway and Spain).
Data accessibility. Data and code (Rmd files) necessary for reproducing
the results of the study can be found at https://www.github.com/
chollenbeck/king_scallop_popgen_2022. Raw DNA sequence data
can be found at the NCBI Short Read Archive (SRA) under BioProject
Accession PRJEB20627. Additional raw data files (unfiltered SNP
data) can be found on Dryad: https://dx.doi.org/10.5061/dryad.
ttdz08m26 [87].
Data are provided in the electronic supplementary material [88].
Authors’contributions. C.M.H.: conceptualization, data curation, formal
analysis, investigation, methodology, project administration, writ-
ing—original draft, writing—review and editing; D.S.P.: formal
analysis, investigation, writing—review and editing; D.G.: conceptu-
alization, data curation, formal analysis, funding acquisition,
investigation, methodology, project administration, writing—review
and editing; T.M.: conceptualization, resources, writing—review
and editing; I.M.: conceptualization, resources, writing—review and
editing; I.A.J.: conceptualization, funding acquisition, investigation,
methodology, project administration, supervision, writing—review
and editing.
All authors gave final approval for publication and agreed to be
held accountable for the work performed therein.
Conflict of interest declaration. We declare we have no competing interests.
Funding. This study was initiated as part of the European Marine Bio-
logical Research Infrastructure Cluster (EMBRIC) project funded by
the European Union’s Horizon 2020 research and innovation pro-
gramme under grant agreement no. 654008. The sequencing service
was provided by the Norwegian Sequencing Centre (www.sequen-
cing.uio.no), a national technology platform hosted by the
University of Oslo and supported by the ‘Functional Genomics’
and ‘Infrastructure’programmes of the Research Council of
Norway and the Southeastern Regional Health Authorities.
Acknowledgements. Dr Daniel Garcia de la serrana is a Serra Húnter
Tenure Track Lecturer. We also thank Dr Jorge Hernández Urcera
from the Instituto de Ceincias Marinas for providing the scallops
from the Spanish coast.
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 289: 20221573
8
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