Reproductive isolation in a three-way contact zone.

Contact zones between divergent forms within a species provide insight into the role of gene flow in adaptation and speciation. Previous work has focused on contact zones involving only two divergent forms, but in nature, many more than two populations may overlap simultaneously and experience gene flow. Patterns of introgression in wild populations are, therefore, likely much more complicated than is often assumed. We begin to address this gap in current knowledge by investigating patterns of divergence and introgression across a complex natural contact zone. We use phenotypic and genomic data to confirm the existence of a three-way contact zone among divergent freshwater resident, saltwater resident and saltwater migratory three-spined stickleback (Gasterosteus aculeatus) on the island of North Uist, Scottish Western Isles. We find evidence for hybridization, mostly between saltwater resident and saltwater migratory forms. Despite hybridization, genomic analyses reveal pairwise islands of divergence between all forms that are maintained across the contact zone. Genomic cline analyses also provide evidence for selection and/or hybrid incompatibilities in divergent regions. Divergent genomic regions occur across multiple chromosomes and involve many known adaptive loci and several chromosomal inversions. We also identify distinct immune gene expression profiles between forms, but no evidence for transgressive expression in hybrids. Our results suggest that reproductive isolation is maintained in this three-way contact zone, despite some hybridization, and that reduced recombination in chromosomal inversions may play an important role in maintaining this isolation.

The WZA: A window-based method for characterizing genotype-environment associations.

Genotype-environment association (GEA) studies have the potential to identify the genetic basis of local adaptation in natural populations. Specifically, GEA approaches look for a correlation between allele frequencies and putatively selective features of the environment. Genetic markers with extreme evidence of correlation with the environment are presumed to be tagging the location of alleles that contribute to local adaptation. In this study, we propose a new method for GEA studies called the Weighted-Z Analysis (WZA) that combines information from closely linked sites into analysis windows in a way that was inspired by methods for calculating FST . Performing GEA methods in analysis windows has the advantage that it takes advantage of the increased linkage disequilibrium expected surrounding sites subject to local adaptation. We analyse simulations modelling local adaptation to heterogeneous environments to compare the WZA with existing methods. In the majority of cases we tested, the WZA either outperformed single-SNP (single nucleotide polymorphism)-based approaches or performed similarly. In particular, the WZA outperformed individual SNP approaches when a small number of individuals or demes were sampled. Particularly troubling, we found that some GEA methods exhibit very high false positive rates. We applied the WZA to previously published data from lodgepole pine and identified candidate loci that were identified in the original study alongside numerous loci that were not found in the original study.

SNP discovery and genetic structure in blue mussel species using low coverage sequencing and a medium density 60 K SNP-array.

Blue mussels from the genus Mytilus are an abundant component of the benthic community, found in the high latitude habitats. These foundation species are relevant to the aquaculture industry, with over 2 million tonnes produced globally each year. Mussels withstand a wide range of environmental conditions and species from the Mytilus edulis complex readily hybridize in regions where their distributions overlap. Significant effort has been made to investigate the consequences of environmental stress on mussel physiology, reproductive isolation, and local adaptation. Yet our understanding on the genomic mechanisms underlying such processes remains limited. In this study, we developed a multi species medium-density 60 K SNP-array including four species of the Mytilus genus. SNPs included in the platform were called from 138 mussels from 23 globally distributed mussel populations, sequenced using a whole-genome low coverage approach. The array contains polymorphic SNPs which capture the genetic diversity present in mussel populations thriving across a gradient of environmental conditions (~59 K SNPs) and a set of published and validated SNPs informative for species identification and for diagnosis of transmissible cancer (610 SNPs). The array will allow the consistent genotyping of individuals, facilitating the investigation of ecological and evolutionary processes in these taxa. The applications of this array extend to shellfish aquaculture, contributing to the optimization of this industry via genomic selection of blue mussels, parentage assignment, inbreeding assessment and traceability. Further applications such as genome wide association studies (GWAS) for key production traits and those related to environmental resilience are especially relevant to safeguard aquaculture production under climate change.

Species delimitation using genomic data to resolve taxonomic uncertainties in a speciation continuum of pelagic seabirds.

Speciation is a continuous and complex process shaped by the interaction of numerous evolutionary forces. Despite the continuous nature of the speciation process, the implementation of conservation policies relies on the delimitation of species and evolutionary significant units (ESUs). Puffinus shearwaters are globally distributed and threatened pelagic seabirds. Due to remarkable morphological status the group has been under intense taxonomic debate for the past three decades. Here, we use double digest Restriction-Site Associated DNA sequencing (ddRAD-Seq) to genotype species and subspecies of North Atlantic and Mediterranean Puffinus shearwaters across their entire geographical range. We assess the phylogenetic relationships and population structure among and within the group, evaluate species boundaries, and characterise the genomic landscape of divergence. We find that current taxonomies are not supported by genomic data and propose a more accurate taxonomy by integrating genomic information with other sources of evidence. Our results show that several taxon pairs are at different stages of a speciation continuum. Our study emphasises the potential of genomic data to resolve taxonomic uncertainties, which can help to focus management actions on relevant taxa, even if they do not necessarily coincide with the taxonomic rank of species.

Rapid genomic convergent evolution in experimental populations of Trinidadian guppies (Poecilia reticulata).

Although rapid phenotypic evolution has been documented often, the genomic basis of rapid adaptation to natural environments is largely unknown in multicellular organisms. Population genomic studies of experimental populations of Trinidadian guppies (Poecilia reticulata) provide a unique opportunity to study this phenomenon. Guppy populations that were transplanted from high-predation (HP) to low-predation (LP) environments have been shown to evolve toward the phenotypes of naturally colonized LP populations in as few as eight generations. These changes persist in common garden experiments, indicating that they have a genetic basis. Here, we report results of whole genome variation in four experimental populations colonizing LP sites along with the corresponding HP source population. We examined genome-wide patterns of genetic variation to estimate past demography and used a combination of genome scans, forward simulations, and a novel analysis of allele frequency change vectors to uncover the signature of selection. We detected clear signals of population growth and bottlenecks at the genome-wide level that matched the known history of population numbers. We found a region on chromosome 15 under strong selection in three of the four populations and with our multivariate approach revealing subtle parallel changes in allele frequency in all four populations across this region. Investigating patterns of genome-wide selection in this uniquely replicated experiment offers remarkable insight into the mechanisms underlying rapid adaptation, providing a basis for comparison with other species and populations experiencing rapidly changing environments.

On the genetic architecture of rapidly adapting and convergent life history traits in guppies.

The genetic basis of traits shapes and constrains how adaptation proceeds in nature; rapid adaptation can proceed using stores of polygenic standing genetic variation or hard selective sweeps, and increasing polygenicity fuels genetic redundancy, reducing gene re-use (genetic convergence). Guppy life history traits evolve rapidly and convergently among natural high- and low-predation environments in northern Trinidad. This system has been studied extensively at the phenotypic level, but little is known about the underlying genetic architecture. Here, we use four independent F2 QTL crosses to examine the genetic basis of seven (five female, two male) guppy life history phenotypes and discuss how these genetic architectures may facilitate or constrain rapid adaptation and convergence. We use RAD-sequencing data (16,539 SNPs) from 370 male and 267 female F2 individuals. We perform linkage mapping, estimates of genome-wide and per-chromosome heritability (multi-locus associations), and QTL mapping (single-locus associations). Our results are consistent with architectures of many loci of small-effect for male age and size at maturity and female interbrood period. Male trait associations are clustered on specific chromosomes, but female interbrood period exhibits a weak genome-wide signal suggesting a potentially highly polygenic component. Offspring weight and female size at maturity are also associated with a single significant QTL each. These results suggest rapid, repeatable phenotypic evolution of guppies may be facilitated by polygenic trait architectures, but subsequent genetic redundancy may limit gene re-use across populations, in agreement with an absence of strong signatures of genetic convergence from recent analyses of wild guppies.

A large and diverse autosomal haplotype is associated with sex-linked colour polymorphism in the guppy.

Male colour patterns of the Trinidadian guppy (Poecilia reticulata) are typified by extreme variation governed by both natural and sexual selection. Since guppy colour patterns are often inherited faithfully from fathers to sons, it has been hypothesised that many of the colour trait genes must be physically linked to sex determining loci as a 'supergene' on the sex chromosome. Here, we phenotype and genotype four guppy 'Iso-Y lines', where colour was inherited along the patriline for 40 generations. Using an unbiased phenotyping method, we confirm the breeding design was successful in creating four distinct colour patterns. We find that genetic differentiation among the Iso-Y lines is repeatedly associated with a diverse haplotype on an autosome (LG1), not the sex chromosome (LG12). Moreover, the LG1 haplotype exhibits elevated linkage disequilibrium and evidence of sex-specific diversity in the natural source population. We hypothesise that colour pattern polymorphism is driven by Y-autosome epistasis.

Drainage-structuring of ancestral variation and a common functional pathway shape limited genomic convergence in natural high- and low-predation guppies.

Studies of convergence in wild populations have been instrumental in understanding adaptation by providing strong evidence for natural selection. At the genetic level, we are beginning to appreciate that the re-use of the same genes in adaptation occurs through different mechanisms and can be constrained by underlying trait architectures and demographic characteristics of natural populations. Here, we explore these processes in naturally adapted high- (HP) and low-predation (LP) populations of the Trinidadian guppy, Poecilia reticulata. As a model for phenotypic change this system provided some of the earliest evidence of rapid and repeatable evolution in vertebrates; the genetic basis of which has yet to be studied at the whole-genome level. We collected whole-genome sequencing data from ten populations (176 individuals) representing five independent HP-LP river pairs across the three main drainages in Northern Trinidad. We evaluate population structure, uncovering several LP bottlenecks and variable between-river introgression that can lead to constraints on the sharing of adaptive variation between populations. Consequently, we found limited selection on common genes or loci across all drainages. Using a pathway type analysis, however, we find evidence of repeated selection on different genes involved in cadherin signaling. Finally, we found a large repeatedly selected haplotype on chromosome 20 in three rivers from the same drainage. Taken together, despite limited sharing of adaptive variation among rivers, we found evidence of convergent evolution associated with HP-LP environments in pathways across divergent drainages and at a previously unreported candidate haplotype within a drainage.

Intercontinental genomic parallelism in multiple three-spined stickleback adaptive radiations.

Parallelism, the evolution of similar traits in populations diversifying in similar conditions, provides strong evidence of adaptation by natural selection. Many studies of parallelism focus on comparisons of different ecotypes or contrasting environments, defined a priori, which could upwardly bias the apparent prevalence of parallelism. Here, we estimated genomic parallelism associated with components of environmental and phenotypic variation at an intercontinental scale across four freshwater adaptive radiations (Alaska, British Columbia, Iceland and Scotland) of the three-spined stickleback (Gasterosteus aculeatus). We combined large-scale biological sampling and phenotyping with restriction site associated DNA sequencing (RAD-Seq) data from 73 freshwater lake populations and four marine ones (1,380 fish) to associate genome-wide allele frequencies with continuous distributions of environmental and phenotypic variation. Our three main findings demonstrate that (1) quantitative variation in phenotypes and environments can predict genomic parallelism; (2) genomic parallelism at the early stages of adaptive radiations, even at large geographic scales, is founded on standing variation; and (3) similar environments are a better predictor of genome-wide parallelism than similar phenotypes. Overall, this study validates the importance and predictive power of major phenotypic and environmental factors likely to influence the emergence of common patterns of genomic divergence, providing a clearer picture than analyses of dichotomous phenotypes and environments.

Improved Reference Genome Uncovers Novel Sex-Linked Regions in the Guppy (Poecilia reticulata).

Theory predicts that the sexes can achieve greater fitness if loci with sexually antagonistic polymorphisms become linked to the sex determining loci, and this can favor the spread of reduced recombination around sex determining regions. Given that sex-linked regions are frequently repetitive and highly heterozygous, few complete Y chromosome assemblies are available to test these ideas. The guppy system (Poecilia reticulata) has long been invoked as an example of sex chromosome formation resulting from sexual conflict. Early genetics studies revealed that male color patterning genes are mostly but not entirely Y-linked, and that X-linkage may be most common in low-predation populations. More recent population genomic studies of guppies have reached varying conclusions about the size and placement of the Y-linked region. However, this previous work used a reference genome assembled from short-read sequences from a female guppy. Here, we present a new guppy reference genome assembly from a male, using long-read PacBio single-molecule real-time sequencing and chromosome contact information. Our new assembly sequences across repeat- and GC-rich regions and thus closes gaps and corrects mis-assemblies found in the short-read female-derived guppy genome. Using this improved reference genome, we then employed broad population sampling to detect sex differences across the genome. We identified two small regions that showed consistent male-specific signals. Moreover, our results help reconcile the contradictory conclusions put forth by past population genomic studies of the guppy sex chromosome. Our results are consistent with a small Y-specific region and rare recombination in male guppies.

Prior exposure to long-day photoperiods alters immune responses and increases susceptibility to parasitic infection in stickleback.

Seasonal disease and parasitic infection are common across organisms, including humans, and there is increasing evidence for intrinsic seasonal variation in immune systems. Changes are orchestrated through organisms' physiological clocks using cues such as day length. Ample research in diverse taxa has demonstrated multiple immune responses are modulated by photoperiod, but to date, there have been few experimental demonstrations that photoperiod cues alter susceptibility to infection. We investigated the interactions among photoperiod history, immunity and susceptibility in laboratory-bred three-spined stickleback (a long-day breeding fish) and its external, directly reproducing monogenean parasite Gyrodactylus gasterostei. We demonstrate that previous exposure to long-day photoperiods (PLD) increases susceptibility to infection relative to previous exposure to short days (PSD), and modifies the response to infection for the mucin gene muc2 and Treg cytokine foxp3a in skin tissues in an intermediate 12 L : 12 D photoperiod experimental trial. Expression of skin muc2 is reduced in PLD fish, and negatively associated with parasite abundance. We also observe inflammatory gene expression variation associated with natural inter-population variation in resistance, but find that photoperiod modulation of susceptibility is consistent across host populations. Thus, photoperiod modulation of the response to infection is important for host susceptibility, highlighting new mechanisms affecting seasonality of host-parasite interactions.

What can be learned by scanning the genome for molecular convergence in wild populations?

Convergent evolution, where independent lineages evolve similar phenotypes in response to similar challenges, can provide valuable insight into how selection operates and the limitations it encounters. However, it has only recently become possible to explore how convergent evolution is reflected at the genomic level. The overlapping outlier approach (OOA), where genome scans of multiple independent lineages are used to find outliers that overlap and therefore identify convergently evolving loci, is becoming popular. Here, we present a quantitative analysis of 34 studies that used this approach across many sampling designs, taxa, and sampling intensities. We found that OOA studies with increased biological sampling power within replicates have increased likelihood of finding overlapping, "convergent" signals of adaptation between them. When identifying convergent loci as overlapping outliers, it is tempting to assume that any false-positive outliers derived from individual scans will fail to overlap across replicates, but this cannot be guaranteed. We highlight how population demographics and genomic context can contribute toward both true convergence and false positives in OOA studies. We finish with an exploration of emerging methods that couple genome scans with phenotype and environmental measures, leveraging added information from genome data to more directly test hypotheses of the likelihood of convergent evolution.

Contingent Convergence: The Ability To Detect Convergent Genomic Evolution Is Dependent on Population Size and Migration.

Outlier scans, in which the genome is scanned for signatures of selection, have become a prominent tool in studies of local adaptation, and more recently studies of genetic convergence in natural populations. However, such methods have the potential to be confounded by features of demographic history, such as population size and migration, which are considerably varied across natural populations. In this study, we use forward-simulations to investigate and illustrate how several measures of genetic differentiation commonly used in outlier scans (FST, DXY and Δπ) are influenced by demographic variation across multiple sampling generations. In a factorial design with 16 treatments, we manipulate the presence/absence of founding bottlenecks (N of founding individuals), prolonged bottlenecks (proportional size of diverging population) and migration rate between two populations with ancestral and diverged phenotypic optima. Our results illustrate known constraints of individual measures associated with reduced population size and a lack of migration; but notably we demonstrate how relationships between measures are similarly dependent on these features of demography. We find that false-positive signals of convergent evolution (the same simulated outliers detected in independent treatments) are attainable as a product of similar population size and migration treatments (particularly for DXY), and that outliers across different measures (for e.g., FST and DXY) can occur with little influence of selection. Taken together, we show how underappreciated, yet quantifiable measures of demographic history can influence commonly employed methods for detecting selection.

A genetics-based approach confirms immune associations with life history across multiple populations of an aquatic vertebrate (Gasterosteus aculeatus).

Understanding how wild immune variation covaries with other traits can reveal how costs and trade-offs shape immune evolution in the wild. Divergent life history strategies may increase or alleviate immune costs, helping shape immune variation in a consistent, testable way. Contrasting hypotheses suggest that shorter life histories may alleviate costs by offsetting them against increased mortality, or increase the effect of costs if immune responses are traded off against development or reproduction. We investigated the evolutionary relationship between life history and immune responses within an island radiation of three-spined stickleback, with discrete populations of varying life histories and parasitism. We sampled two short-lived, two long-lived and an anadromous population using qPCR to quantify current immune profile and RAD-seq data to study the distribution of immune variants within our assay genes and across the genome. Short-lived populations exhibited significantly increased expression of all assay genes, which was accompanied by a strong association with population-level variation in local alleles and divergence in a gene that may be involved in complement pathways. In addition, divergence around the eda gene in anadromous fish is likely associated with increased inflammation. A wider analysis of 15 populations across the island revealed that immune genes across the genome show evidence of having diverged alongside life history strategies. Parasitism and reproductive investment were also important sources of variation for expression, highlighting the caution required when assaying immune responses in the wild. These results provide strong, gene-based support for current hypotheses linking life history and immune variation across multiple populations of a vertebrate model.