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Genetic differentiation at chromosomal inversion regions. Heatmap showing in red inversion regions that have significantly higher mean genetic differentiation (F ST ) compared to the 99% CI of the collinear genomic background. Nonsignificant (NS) tests are indicated in gray. F ST was measured per locality according to their direction along Crab-Wave and Low-High axes as indicated on the y axis (see main text and Fig. 1B). The three groups on the y axis indicate localities that are expected to show a consistent signal depending if they are implicated in the Crab-Wave, Low-High, or both axes of divergence. Groups in the x axis indicate chromosomal inversion regions that have a signal of differentiation consistent with the expectations for A = Crab-Wave divergence, B = Low-High divergence, or C = variable across the species range.
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The study of parallel ecological divergence provides important clues to the operation of natural selection. Parallel divergence often occurs in heterogeneous environments with different kinds of environmental gradients in different locations, but the genomic basis underlying this process is unknown. We investigated the genomics of rapid parallel ad...
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... varied in their Crab-Wave genome-wide genetic differentiation [ranges of mean F ST in 500-base pair (bp) nonoverlapping windows: Spain = 0.09 to 0.12; France = 0.03; United Kingdom = 0.01 to 0.03; Sweden = 0.05 to 0.07; fig. S5]. We summarized genome-wide genetic variation with a principal components analysis (PCA) of 10,263,736 biallelic SNPs (minor allele frequency > 5% in at least one pool). The first four PCA axes, containing most of the variation (75.7%), depicted a genetic structure consistent with geography (Fig. 1C). This genetic structure agrees with ...
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... wanted to test whether candidate chromosomal inversion regions are likely implicated in parallel ecological divergence to both Crab-Wave and Low-High divergence. We first tested whether genetic differentiation within inversions was significantly higher than a random expectation drawn from the collinear genomic background in each locality ( Fig. 5; see details in the Supplementary Materials). While in Spain, United Kingdom, and France genetic differentiation is expected to reflect selection along both environmental axes, in Sweden the axes' effects are separate (y axis in Fig. 5). Our findings indicated that some chromosomal inversion regions are likely repeatedly involved in ...
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... significantly higher than a random expectation drawn from the collinear genomic background in each locality ( Fig. 5; see details in the Supplementary Materials). While in Spain, United Kingdom, and France genetic differentiation is expected to reflect selection along both environmental axes, in Sweden the axes' effects are separate (y axis in Fig. 5). Our findings indicated that some chromosomal inversion regions are likely repeatedly involved in the Crab-Wave divergence (e.g., in LG6 and LG14) and others in the High-Low divergence (e.g., LG9 and LG12). The patterns observed for other inversions are more complex, as some are predominantly differentiated in Spain and Sweden (group ...
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... Fig. 5). Our findings indicated that some chromosomal inversion regions are likely repeatedly involved in the Crab-Wave divergence (e.g., in LG6 and LG14) and others in the High-Low divergence (e.g., LG9 and LG12). The patterns observed for other inversions are more complex, as some are predominantly differentiated in Spain and Sweden (group C in Fig. 5), while others show no clear pattern (group D in Fig. 5). Next, following findings from Faria et al. (22) that most inversions are polymorphic in one or both of the Crab and Wave habitats, we tested whether inversion regions have more, the same, or less nucleotide diversity than collinear regions. Despite some variation across ...
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... regions are likely repeatedly involved in the Crab-Wave divergence (e.g., in LG6 and LG14) and others in the High-Low divergence (e.g., LG9 and LG12). The patterns observed for other inversions are more complex, as some are predominantly differentiated in Spain and Sweden (group C in Fig. 5), while others show no clear pattern (group D in Fig. 5). Next, following findings from Faria et al. (22) that most inversions are polymorphic in one or both of the Crab and Wave habitats, we tested whether inversion regions have more, the same, or less nucleotide diversity than collinear regions. Despite some variation across localities, we found overall patterns that are highly consistent ...
Citations
... Such contact zones are ideal contexts for assessing barriers and processes involved in reproductive isolation and speciation [97,98]. In the snails, these contact zones are replicated at various levels of population independence, from zones in the same geographic area where populations share the same gene pool [25], to replicate zones between similar ecotypes in different geographic regions [41]. ...
... These snails live in high densities in areas that are easy for us to access (shores) and are characterised by steep environmental gradients in temperature, desiccation risk, predation, and wave action. The heterogeneous environment promotes small-scale adaptation, resulting in ecological [39], physiological [40], and genetic differentiation [20], replicated both at a local geographic scale and over latitudinal gradients [25,27,41]. Furthermore, these stress-tolerant snails are tractable organisms for field manipulations, laboratory experiments and crossbreeding, and the first assembled genome and a genetic linkage map for Littorina saxatilis [29] have boosted studies of the genomic architecture of divergence (Box 1). ...
... Pair 1. High-and low-shore L. saxatilis Closely related populations (within either the Crab or Wave ecotype, see below) present in high and low parts of rocky Atlantic shores of Europe and with a continuum of intermediate phenotypes through the mid-shore level [41,42]. Shell shapes are very similar but high-shore snails tend to be larger and have higher spires [43]. ...
... Variable selection pressures may decrease the degree of gene reuse [51], resulting in an even stronger dependence of gene reuse on divergence time [52]. Although decreasing phenotypic and ecological similarity were not the focus of this review, they remain important factors in the relationship between gene reuse and divergence time, as seen in recent studies [3,53]. ...
... Recent timescales: the effect of divergence through reduction in allele sharing Our review of case studies on the genomic basis of repeated adaptation provides empirical support for the expectation that there should be a decline in gene reuse as the divergence time increases, when considering recently diverging (< one million generations) lineages (Box 4 and the supplemental information online) [4,5,10,[12][13][14]53,[56][57][58][59][60][61][62][63][64]. Two of these case studies explicitly support our predictions that this decrease in gene reuse is associated with a reduction in allele sharing between lineages (Box 3) [12,14]. ...
... See the supplemental information online for details about the case studies and how they were selected. See [2][3][4][5]9,10,[12][13][14]53,[56][57][58][59][60][61][62][63][64][69][70][71][72][73][74][75][76][77][78][79][80][81]. [10,[62][63][64]. ...
... In Spain, the tidal range is much greater and the two ecotypes are distributed on a perpendicular up-down shore axis (vertical zonation) with the Crab ecotype primarily in the barnacle belt in the high shore, where predation is most intense, and the Wave ecotype among blue mussels in the low shore, where wave action is strongest. The Spanish ecotypes overlap in a wide contact zone in the mid shore, characterized by a mosaic distribution of barnacle and mussel patches (Johannesson et al., 1993(Johannesson et al., , 1995Morales et al., 2019;Rolán-Alvarez et al., 1997. There are some indications that the barrier to gene flow between ecotypes is stronger in Spain than in Sweden: background F ST is higher, around 0.1 (Butlin et al., 2014;Morales et al., 2019;Westram et al., 2021), and only a few intermediate genotypes were observed in putatively hybrid samples using reduced representation data (RADseq, Kess et al., 2018). ...
... The Spanish ecotypes overlap in a wide contact zone in the mid shore, characterized by a mosaic distribution of barnacle and mussel patches (Johannesson et al., 1993(Johannesson et al., , 1995Morales et al., 2019;Rolán-Alvarez et al., 1997. There are some indications that the barrier to gene flow between ecotypes is stronger in Spain than in Sweden: background F ST is higher, around 0.1 (Butlin et al., 2014;Morales et al., 2019;Westram et al., 2021), and only a few intermediate genotypes were observed in putatively hybrid samples using reduced representation data (RADseq, Kess et al., 2018). Yet, an investigation of the pattern of differentiation in Spain with a combination of genome-wide data and detailed spatial coverage of the contact zone has been lacking. ...
... The marine snail Littorina saxatilis is a renowned study system of repeated differentiation between Crab and Wave ecotypes, yet differences in the strength of reproductive isolation across its distribution has been largely overlooked. Few studies have directly compared ecotype divergence among different regions using the same methods (Butlin et al., 2014;Morales et al., 2019). Using low coverage whole genome sequencing and analyses of shell features and behavioural traits in two continuous transects in Spain and Sweden spanning the whole Crab-Wave axis, we confirmed parallel phenotypic and genetic divergence in these shores and the role of chromosomal inversions. ...
Speciation typically occurs in a time frame too long to be observed directly. This issue can be over-come by studying pairs of populations at different points in the speciation continuum, ideally within clades so that patterns are not confounded by differences among taxa. Such comparisons are possible in the marine snail Littorina saxatilis because it shows repeated occurrence of ecotypes adapted to either crab predation or wave action that differ in age and environmental context. Here, we explored transects spanning hybrid zones between the crab and wave ecotypes to contrast barriers to gene flow in Spain and Sweden, using low coverage whole genome sequencing, shell features, and behavioural traits. The two countries showed parallel divergence but distinct patterns of differentiation between the ecotypes: a continuous cline in Sweden but two highly genetically and phenotypically divergent, and partly spatially-overlapping clusters in Spain. Spanish early-generation hybrids were not observed but a low level of gene flow still seems to occur. In both countries, highly differentiated loci are clustered in genomic regions covered by chromosomal inversions but also occur in collinear regions. Despite being the same species and showing similar levels of phenotypic divergence, the Spanish ecotypes are closer to full reproductive isolation than the Swedish ecotypes. We discuss potential mechanisms contributing to the evolution of these different levels of reproductive isolation, particularly the age of the population, the strength of selection, the spatial context, and the role of assortative mating.
... The ecotypes evolve repeatedly across similar types of meter-scale environmental gradients 25 . In L. saxatilis, the parallel evolution of 'wave' and 'crab' ecotypes involves more than 10 chromosomal inversions 21,26 . Many of these show signatures of divergent selection, and contribute to phenotypic differences between the ecotypes 24,[27][28][29] . ...
... LG2 ( Figure 1). The genomic signatures were different from those of the putative inversions ( Figure S2 to S5), and this may be a region of low recombination associated with the centromere 37,26 . ...
... The WGS data from L. fabalis were analysed using the same reference genome as used in previous studies of L. saxatilis 26 , and this allowed us to search for similarities in the architecture of RI. We found a weak but significant correlation in the mean FST per contig between ecotypes across the two species (r²=0.211, ...
Chromosomal rearrangements lead to the coupling of reproductive barriers, but whether and how they contribute to completion of speciation remains unclear. Marine snails of the genus Littorina repeatedly form hybrid zones between taxa segregating for inversion arrangements, providing opportunities to study this question. Here, we analysed two adjacent transects across hybrid zones between large and dwarf ecotypes of Littorina fabalis covering wave exposure gradients in a Swedish island. Applying whole-genome sequences we found 12 putative inversions reaching near differential fixation between the opposite ends of each transect, and being in strong linkage disequilibrium. These inversions cover 20% of the genome and carry 93% of divergent SNPs. Bimodal hybrid zones in both transects indicate that the two ecotypes of Littorina fabalis maintain their genetic and phenotypic integrity following contact due to strong coupling between inversion clines that strengthened the reproductive barrier. The bimodality resulting from the linked inversions extends into collinear parts of the genome, suggesting a genome-wide coupling. Demographic inference suggests that the coupling built up during a period of allopatry, and has been maintained for more than 1K generations after secondary contact. Overall, this study shows that the coupling of multiple chromosomal inversions contributes to strong reproductive isolation. Importantly, two of the inversions overlap with inverted genomic regions associated with ecotype differences in a closely-related species (L. saxatilis), suggesting the same regions, with similar structural variants, repeatedly contribute to ecotype evolution in distinct species.
... However, some of these arguments were questioned (Butlin, Galindo, & Grahame, 2008) and it has been found that genomic parallelism is limited (Koch et al., 2022;Ravinet et al., 2016). Furthermore, the Iberian populations share a demographic history that is clearly different from the remaining European populations (Blakeslee et al., 2021;Morales et al., 2019;Panova et al., 2011), while the Swedish and British populations have probably shared a post-glacial colonization history. A single study has examined and compared different models of the demographic history of the ecotypes across a wide geographical range. ...
... Recently, the significance of chromosomal inversions in the adaptive divergence of L. saxatilis has been highlighted Koch et al., 2021;Morales et al., 2019). Each inversion is likely to have its own unique evolutionary history, which can be influenced by a range of demographic and selective processes, including divergent and balancing selection. ...
Parallel evolution of phenotypic divergence offers compelling evidence supporting the influence of natural selection on the divergence process. However, the existence of ecotypes adapted to different habitats in separate locations is not a definitive proof of parallel evolution. Here, we leverage a large pool-seq dataset of the rocky-shore gastropod, Littorina saxatilis to compare and contrast two explicit scenarios of ecotype formation. Populations of this species inhabit contrasting habitats, where the main selective pressure is either wave action or crab predation. Using approximate Bayesian computation to jointly infer demographic parameters and account for pool-seq specific sources of uncertainty, our analysis reveals that ecotype formation at a large geographical scale has occurred in parallel. Parameter estimates provide strong support for a demographic history marked by the spatial separation of the ancestral populations that later gave rise to parallel evolution of ecotypes in Spain and Sweden. Additionally, ecotype formation occurred in the face of continuous gene flow between the diverging ecotypes. These results shed new light on a important model of speciation driven by ecological factors and emphasize the effectiveness of combining pool-seq with ABC for studying parallel evolution across diverse geographical regions.
... These polymorphic inversions and their associations with ecotypes are repeated across multiple nearby sites in Sweden . Strong genetic differentiation between ecotypes occurs at genomic regions corresponding to some Swedish inversions suggests that similar associations exist in the United Kingdom, France and Spain (Kess & Boulding, 2019;Morales et al., 2019). However, while a signal of crab-wave divergence has been inferred across Europe, there is currently no direct evidence that the inversions detected in Sweden are polymorphic across the species range. ...
... L. saxatilis and L. arcana are considered sister species, with near complete reproductive isolation (Stankowski et al., 2020). There is also increasing evidence of a strong phylogeographic break in L. saxatilis around the Bay of Biscay, separating populations in the Iberian Peninsula from those in the North (Doellman, Trussell, Grahame, & Vollmer, 2011;Morales et al., 2019;Tirado, Saura, Rolán-Alvarez, & Quesada, 2016, & Panova et al. 2011. ...
... Samples were split into three genetic groups for analysis: Northern saxatilis, Iberian saxatilis and L. arcana (Figure 1). These were chosen based on known genetic differences to reduce the impact of geographic isolation (Northern vs Iberian; Doellman et al., 2011;Morales et al., 2019;Panova et al., 2014;Stankowski, Zagrodzka, Galindo, et al., 2023;Tirado et al., 2016) and reproductive isolation (L. saxatilis vs L. arcana; Stankowski et al., 2020) while still maintaining a sufficient sample size to identify polymorphic inversions. ...
... The second dataset, referred to as 'inversion-and-outlier-free' dataset, is a subset of the sites in the inversion-free data set, but filtered to reduce the impact of parallel selection on inferences of population history still further. Because the L. saxatilis genome consists of hundreds of thousands of very short contigs, we simply removed all assembly contigs that contained outlier loci according to analyses performed in Morales et al. (2019) who compared crab and wave samples from many locations across Europe. The outlier loci included SNPs with an F ST in the top 1% of the crab-wave empirical distribution or a BayPass (Gautier 2015) Bayes factor greater than 20. ...
Understanding the factors that have shaped the current distributions and diversity of species is a central and longstanding aim of evolutionary biology. The recent inclusion of genomic data into phylogeographic studies has dramatically improved our understanding in organisms where evolutionary relationships have been challenging to infer. We used whole-genome sequences to study the phylogeography of the intertidal snail Littorina saxatilis, which has successfully colonized and diversified across a broad range of coastal environments in the Northern Hemisphere amid repeated cycles of glaciation. Building on past studies based on short DNA sequences, we used genome-wide data to provide a clearer picture of the relationships among samples spanning most of the species natural range. Our results confirm the trans-Atlantic colonization of North America from Europe, and have allowed us to identify rough locations of glacial refugia and to infer likely routes of colonization within Europe. We also investigated the signals in different datasets to account for the effects of genomic architecture and non-neutral evolution, which provides new insights about diversification of four ecotypes of L. saxatilis (the crab, wave, barnacle, and brackish ecotypes) at different spatial scales. Overall, we provide a much clearer picture of the biogeography of L. saxatilis, providing a foundation for more detailed phylogenomic and demographic studies.
... In situations where obtaining DNA from each individual sample is impractical, an alternative approach is to group several individuals together prior to DNA extraction. For instance, muscle tissue from multiple individuals can be combined, extracting DNA from the entire group of individuals (Morales et al., 2019;Ross et al., 2019). ...
Next‐generation sequencing of pooled samples (Pool‐seq) is an important tool in population genomics and molecular ecology. In Pool‐seq, the relative number of reads with an allele reflects the allele frequencies in the sample. However, unequal individual contributions to the pool and sequencing errors can lead to inaccurate allele frequency estimates, influencing downstream analysis. When designing Pool‐seq studies, researchers need to decide the pool size (number of individuals) and average depth of coverage (sequencing effort). An efficient sampling design should maximise the accuracy of allele frequency estimates while minimising the sequencing effort. We describe a novel tool to simulate single nucleotide polymorphism (SNP) data using coalescent theory and account for sources of uncertainty in Pool‐seq.
We introduce an R package, poolHelper , enabling users to simulate Pool‐seq data under different combinations of average depth of coverage and pool size, accounting for unequal individual contributions and sequencing errors, modelled by adjustable parameters. The mean absolute error is computed by comparing the sample allele frequencies obtained based on individual genotypes with the frequency estimates obtained with Pool‐seq.
poolHelper enables users to simulate multiple combinations of pooling errors, average depth of coverage, pool sizes and number of pools to assess how they influence the error of sample allele frequencies and expected heterozygosity. Using simulations under a single population model, we illustrate that increasing the depth of coverage does not necessarily lead to more accurate estimates, reinforcing that finding the best Pool‐seq study design is not straightforward. Moreover, we show that simulations can be used to identify different combinations of parameters with similarly low mean absolute errors. This can help users to define an effective sampling design by using those combinations of parameters that minimise the sequencing effort.
The poolHelper package provides tools for performing simulations with different combinations of parameters (e.g. pool size, depth of coverage, unequal individual contribution) before sampling and generating data, allowing users to define sampling schemes based on simulations. This allows researchers to focus on the best sampling scheme to answer their research questions. poolHelper is comprehensively documented with examples to guide effective use.
... two minor-allele reads observed across all populations. Recent studies have uncovered an important role of chromosomal inversions in the adaptive divergence of L. saxatilis ecotypesKoch et al., 2021;Morales et al., 2019). Each inversion likely has its unique evolutionary history that may be influenced by various demographic and selective processes, such as divergent and balancing selection, and may differ from the population history. ...
... The poolABC package is available on the CRAN repository. Genomic data from Littorina saxatilis populations was previously processed inMorales et al. (2019). All the custom scripts used by the authors can be found in the GitHub repository: https://github.com/hmora ...
Next-generation sequencing of pooled samples (Pool-seq) is a popular method to assess genome-wide diversity patterns in natural and experimental populations. However, Pool-seq is associated with specific sources of noise, such as unequal individual contributions. Consequently, using Pool-seq for the reconstruction of evolutionary history has remained underexplored. Here we describe a novel Approximate Bayesian Computation (ABC) method to infer demographic history, explicitly modelling Pool-seq sources of error. By jointly modelling Pool-seq data, demographic history and the effects of selection due to barrier loci, we obtain estimates of demographic history parameters accounting for technical errors associated with Pool-seq. Our ABC approach is computationally efficient as it relies on simulating subsets of loci (rather than the whole-genome) and on using relative summary statistics and relative model parameters. Our simulation study results indicate Pool-seq data allows distinction between general scenarios of ecotype formation (single versus parallel origin) and to infer relevant demographic parameters (e.g. effective sizes and split times). We exemplify the application of our method to Pool-seq data from the rocky-shore gastropod Littorina saxatilis, sampled on a narrow geographical scale at two Swedish locations where two ecotypes (Wave and Crab) are found. Our model choice and parameter estimates show that ecotypes formed before colonization of the two locations (i.e. single origin) and are maintained despite gene flow. These results indicate that demographic modelling and inference can be successful based on pool-sequencing using ABC, contributing to the development of suitable null models that allow for a better understanding of the genetic basis of divergent adaptation.
... Rearrangements can form "supergenes" often associated with linked traits and coadapted alleles (Wellenreuther et al. 2014;Campagna 2016;Charlesworth 2016;Iijima et al. 2018; Adams and Castoe 2019; Merritt et al. 2020;Anderson and Tung 2021). They often occur in populations along ecotones or on the outer edge of the species distribution (Rieseberg 2001;Morales et al. 2019;Faria et al. 2019aFaria et al. , 2019bKess et al. 2021) and are also frequently associated with hybrid populations between closely related species (Marhold and Lihová 2006;Payseur and Rieseberg 2016;Bedoya and Leaché 2021;Potter et al. 2022;Dobry et al. 2023aDobry et al. , 2023b. Rearrangements are generally detected in four ways: 1) cytogenetically, if they capture structural features such as the centromere and alter the morphology of the chromosome, 2) through molecular cytogenetics, with fluorescent probes that hybridize to known positions on the chromosomes (inversions can be observed or putative rearrangements can be validated), 3) population genetic/ genomic inferences such as linkage disequilibrium (Faria et al. 2019a(Faria et al. , 2019b and differences in population structure using PCA from patterns of allele frequencies that reflect the differentiation of specific loci (Li and Ralph 2019; ,and 4) bioinformatically using comparative genomics (Koochekian et al. 2022). ...
Chromosome rearrangements are often implicated with genomic divergence and are proposed to be associated with species evolution. Rearrangements alter the genomic structure and interfere with homologous recombination by isolating a portion of the genome. Integration of multi-platform next generation DNA sequencing technologies has enabled putative identification of chromosome rearrangements in many taxa, however, integrating these data sets with cytogenetics is still uncommon beyond model genetic organisms. Therefore, to achieve the ultimate goal for the genomic classification of eukaryotic organisms, physical chromosome mapping remains critical. The ridge-tailed goannas (Varanus acanthurus BOULENGER) are a group of dwarf monitor lizards comprised of several species found throughout Northern Australia. These lizards exhibit extreme divergence at both the genic and chromosomal levels. The chromosome polymorphisms are widespread extending across much of their distribution, raising the question if these polymorphisms are homologous within the V. acanthurus complex. We used a combined genomic and cytogenetic approach to test for homology across divergent populations with morphologically similar chromosome rearrangements. We showed that more than one chromosome pair was involved with the widespread rearrangements. This finding provides evidence to support de novo chromosome rearrangements have occurred within populations. These chromosome rearrangements are characterised by fixed allele differences originating in the vicinity of the centromeric region. We then compared this region with several other assembled genomes of reptiles, chicken and the platypus. We demonstrated that the synteny of genes in Reptilia remains conserved despite centromere repositioning across these taxa.
