Targeted resequencing reveals geographical patterns of differentiation for loci implicated in parallel evolution

Parallel divergence and speciation provide evidence for the role of divergent selection in generating biological diversity. Recent studies indicate that parallel phenotypic divergence may not have the same genetic basis in different geographical locations – ‘outlier loci’ (loci potentially affected by divergent selection) are often not shared among parallel instances of phenotypic divergence. However, limited sharing may be due, in part, to technical issues if false‐positive outliers occur. Here, we test this idea in the marine snail Littorina saxatilis, which has evolved two partly isolated ecotypes (adapted to crab predation vs. wave action) in multiple locations independently. We argue that if the low extent of sharing observed in earlier studies in this system is due to sampling effects, we expect outliers not to show elevated F_ST when sequenced in new samples from the original locations and also not to follow predictable geographical patterns of elevated F_ST. Following a hierarchical sampling design (within vs. between country), we applied capture sequencing, targeting outliers from earlier studies and control loci. We found that outliers again showed elevated levels of F_ST in their original location, suggesting they were not generated by sampling effects. Outliers were also likely to show increased F_ST in geographically close locations, which may be explained by higher levels of gene flow or shared ancestral genetic variation compared with more distant locations. However, in contrast to earlier findings, we also found some outlier types to show elevated F_ST in geographically distant locations. We discuss possible explanations for this unexpected result.     

Putative chromosomal rearrangements are associated primarily with ecotype divergence rather than geographic separation in an intertidal,poorly dispersing snail

Littorina saxatilis is becoming a model system for understanding the genomic basis of ecological speciation. The parallel formation of crab‐adapted ecotypes that exhibit partial reproductive isolation from wave‐adapted ecotypes has enabled genomic investigation of conspicuous shell traits. Recent genomic studies suggest that chromosomal rearrangements may enable ecotype divergence by reducing gene flow. However, the genomic architecture of traits that are divergent between ecotypes remains poorly understood. Here, we use 11,504 single nucleotide polymorphism (SNP) markers called using the recently released L. saxatilis genome to genotype 462 crab ecotype, wave ecotype and phenotypically intermediate Spanish L. saxatilis individuals with scored phenotypes. We used redundancy analysis to study the genetic architecture of loci associated with shell shape, shape corrected for size, shell size and shell ornamentation, and to compare levels of co‐association among different traits. We discovered 341 SNPs associated with shell traits. Loci associated with trait divergence between ecotypes were often located inside putative chromosomal rearrangements recently characterized in Swedish L. saxatilis. In contrast, we found that shell shape corrected for size varied primarily by geographic site rather than by ecotype and showed little association with these putative rearrangements. We conclude that genomic regions of elevated divergence inside putative rearrangements were associated with divergence of L. saxatilis ecotypes along steep environmental axes—consistent with models of adaptation with gene flow—but were not associated with divergence among the three geographical sites. Our findings support predictions from models indicating the importance of genomic regions of reduced recombination allowing co‐association of loci during ecological speciation with ongoing gene flow.     

Genetic evidence for ecological divergence in kokanee salmon

The evolution of locally adapted phenotypes among populations that experience divergent selective pressures is a central mechanism for generating and maintaining biodiversity. Recently, the advent of high‐throughput DNA sequencing technology has provided tools for investigating the genetic basis of this process in natural populations of nonmodel organisms. Kokanee, the freshwater form of sockeye salmon (Oncorhynchus nerka), occurs as two reproductive ecotypes, which differ in spawning habitat (tributaries vs. shorelines); however, outside of the spawning season the two ecotypes co‐occur in many lakes and lack diagnostic morphological characteristics. We used restriction site‐associated DNA (RAD) sequencing to identify 6145 SNPs and genotype kokanee from multiple spawning sites in Okanagan Lake (British Columbia, Canada). Outlier tests revealed 18 loci putatively under divergent selection between ecotypes, all of which exhibited temporally stable allele frequencies within ecotypes. Six outliers were annotated to sequences in the NCBI database, two of which matched genes associated with early development. There was no evidence for neutral genetic differentiation; however, outlier loci demonstrated significant structure with respect to ecotype and had high assignment accuracy in mixed composition simulations. The absence of neutral structure combined with a small number of highly divergent outlier loci is consistent with theoretical predictions for the early stages of ecological divergence. These outlier loci were then applied to a realistic fisheries scenario in which additional RAD sequencing was used to genotype kokanee collected by trawl in Okanagan Lake, providing preliminary evidence that this approach may be an effective tool for conservation and management.     

Genome‐wide association analyses reveal polygenic genomic architecture underlying divergent shell morphology in Spanish Littorina saxatilis ecotypes

Gene flow between diverging populations experiencing dissimilar ecological conditions can theoretically constrain adaptive evolution. To minimize the effect of gene flow, alleles underlying traits essential for local adaptation are predicted to be located in linked genome regions with reduced recombination. Local reduction in gene flow caused by selection is expected to produce elevated divergence in these regions. The highly divergent crab‐adapted and wave‐adapted ecotypes of the marine snail Littorina saxatilis present a model system to test these predictions. We used genome‐wide association (GWA) analysis of geometric morphometric shell traits associated with microgeographic divergence between the two L. saxatilis ecotypes within three separate sampling sites. A total of 477 snails that had individual geometric morphometric data and individual genotypes at 4,066 single nucleotide polymorphisms (SNPs) were analyzed using GWA methods that corrected for population structure among the three sites. This approach allowed dissection of the genomic architecture of shell shape divergence between ecotypes across a wide geographic range, spanning two glacial lineages. GWA revealed 216 quantitative trait loci (QTL) with shell size or shape differences between ecotypes, with most loci explaining a small proportion of phenotypic variation. We found that QTL were evenly distributed across 17 linkage groups, and exhibited elevated interchromosomal linkage, suggesting a genome‐wide response to divergent selection on shell shape between the two ecotypes. Shell shape trait‐associated loci showed partial overlap with previously identified outlier loci under divergent selection between the two ecotypes, supporting the hypothesis of diversifying selection on these genomic regions. These results suggest that divergence in shell shape between the crab‐adapted and wave‐adapted ecotypes is produced predominantly by a polygenic genomic architecture with positive linkage disequilibrium among loci of small effect.     

The role of local ecology during hybridization at the initial stages of ecological speciation in a marine snail

Hybrid zones of ecologically divergent populations are ideal systems to study the interaction between natural selection and gene flow during the initial stages of speciation. Here, we perform an amplified fragment length polymorphism (AFLP) genome scan in parallel hybrid zones between divergent ecotypes of the marine snail Littorina saxatilis, which is considered a model case for the study of ecological speciation. Ridged‐Banded (RB) and Smooth‐Unbanded (SU) ecotypes are adapted to different shore levels and microhabitats, although they present a sympatric distribution at the mid‐shore where they meet and mate (partially assortatively). We used shell morphology, outlier and nonoutlier AFLP loci from RB, SU and hybrid specimens captured in sympatry to determine the level of phenotypic and genetic introgression. We found different levels of introgression at parallel hybrid zones and nonoutlier loci showed more gene flow with greater phenotypic introgression. These results were independent from the phylogeography of the studied populations, but not from the local ecological conditions. Genetic variation at outlier loci was highly correlated with phenotypic variation. In addition, we used the relationship between genetic and phenotypic variation to estimate the heritability of morphological traits and to identify potential Quantitative Trait Loci to be confirmed in future crosses. These results suggest that ecology (exogenous selection) plays an important role in this hybrid zone. Thus, ecologically based divergent natural selection is responsible, simultaneously, for both ecotype divergence and hybridization. On the other hand, genetic introgression occurs only at neutral loci (nonoutliers). In the future, genome‐wide studies and controlled crosses would give more valuable information about this process of speciation in the face of gene flow.     

Selection on outlier loci and their association with adaptive phenotypes in Littorina saxatilis contact zones

A fundamental issue in speciation research is to evaluate phenotypic variation and the genomics driving the evolution of reproductive isolation between sister taxa. Above all, hybrid zones are excellent study systems for researchers to examine the association of genetic differentiation, phenotypic variation and the strength of selection. We investigated two contact zones in the marine gastropod Littorina saxatilis and utilized landmark‐based geometric morphometric analysis together with amplified fragment length polymorphism (AFLP) markers to assess phenotypic and genomic divergence between ecotypes under divergent selection. From genetic markers, we calculated the cline width, linkage disequilibrium and the average effective selection on a locus. Additionally, we conducted an association analysis linking the outlier loci and phenotypic variation between ecotypes and show that a proportion of outlier loci are associated with key adaptive phenotypic traits.     

Ecotypes of an ecologically dominant prairie grass (Andropogon gerardii) exhibit genetic divergence across the U.S. Midwest grasslands' environmental gradient

Big bluestem (Andropogon gerardii) is an ecologically dominant grass with wide distribution across the environmental gradient of U.S. Midwest grasslands. This system offers an ideal natural laboratory to study population divergence and adaptation in spatially varying climates. Objectives were to: (i) characterize neutral genetic diversity and structure within and among three regional ecotypes derived from 11 prairies across the U.S. Midwest environmental gradient, (ii) distinguish between the relative roles of isolation by distance (IBD) vs. isolation by environment (IBE) on ecotype divergence, (iii) identify outlier loci under selection and (iv) assess the association between outlier loci and climate. Using two primer sets, we genotyped 378 plants at 384 polymorphic AFLP loci across regional ecotypes from central and eastern Kansas and Illinois. Neighbour‐joining tree and PCoA revealed strong genetic differentiation between Kansas and Illinois ecotypes, which was better explained by IBE than IBD. We found high genetic variability within prairies (80%) and even fragmented Illinois prairies, surprisingly, contained high within‐prairie genetic diversity (92%). Using Bayenv 2, 14 top‐ranked outlier loci among ecotypes were associated with temperature and precipitation variables. Six of seven BayeScan F_ST outliers were in common with Bayenv 2 outliers. High genetic diversity may enable big bluestem populations to better withstand changing climates; however, population divergence supports the use of local ecotypes in grassland restoration. Knowledge of genetic variation in this ecological dominant and other grassland species will be critical to understanding grassland response and restoration challenges in the face of a changing climate.     

Development and preliminary evaluation of a genomewide single nucleotide polymorphisms resource generated by RAD‐seq for the small yellow croaker (Larimichthys polyactis)

Recent advances in high‐throughput sequencing technologies have offered the possibility to generate genomewide sequence data to delineate previously unidentified genetic structure, obtain more accurate estimates of demographic parameters and to evaluate potential adaptive divergence. Here, we identified 27 556 single nucleotide polymorphisms for the small yellow croaker (Larimichthys polyactis) using restriction‐site‐associated DNA (RAD) sequencing of 24 individuals from two populations. Significant sources of genetic variation were identified, with an average nucleotide diversity (π) of 0.00105 ± 0.000425 across individuals, and long‐term effective population size was thus estimated to range between 26 172 and 261 716. According to the results, no differentiation between the two populations was detected based on the SNP data set of top quality score per contig or neutral loci. However, the two analysed populations were highly differentiated based on SNP data set of both top F_ST value per contig and the outlier SNPs. Moreover, local adaptation was highlighted by an F_ST‐based outlier tests implemented in LOSITAN and a total of 538 potentially locally selected SNPs were identified. blast2go annotation of contigs containing the outlier SNPs yielded hits for 37 (66%) of 56 significant blastx matches. Candidate genes for local adaptation constituted a wide array of biological functions, including cellular response to oxidative stress, actin filament binding, ion transmembrane transport and synapse assembly. The generated SNP resources in this study provided a valuable tool for future population genetics and genomics studies of L. polyactis.     

Transcriptome-based single nucleotide polymorphism markers for genome mapping in Japanese pear (Pyrus pyrifolia Nakai)

The development of single nucleotide polymorphism (SNP) markers in Japanese pear (Pyrus pyrifolia Nakai) offers the opportunity to use DNA markers for marker-assisted selection in breeding programs because of their high abundance, codominant inheritance, and potential for automated high-throughput analysis. We developed a 1,536-SNP bead array without a reference genome sequence from more than 44,000 base changes on the basis of a large-scale expressed sequence tag (EST) analysis combined with 454 genome sequencing data of Japanese pear ‘Housui’. Among the 1,536 SNPs on the array, 756 SNPs were genotyped, and 609 SNP loci were mapped to linkage groups on a genetic linkage map of ‘Housui’, based on progeny of an interspecific cross between European pear (Pyrus communis L.) ‘Bartlett’ and ‘Housui’. The newly constructed genetic linkage map consists of 951 loci, comprising 609 new SNPs, 110 pear genomic simple sequence repeats (SSRs), 25 pear EST–SSRs, 127 apple SSRs, 61 pear SNPs identified by the “potential intron polymorphism” method, and 19 other loci. The map covers 22 linkage groups spanning 1341.9 cM with an average distance of 1.41 cM between markers and is anchored to reference genetic linkage maps of European pears and apples. A total of 514 contigs containing mapped SNP loci showed significant similarity to known proteins by functional annotation analysis.     

Landscape genomics and a common garden trial reveal adaptive differentiation to temperature across Europe in the tree species Alnus glutinosa

The adaptive potential of tree species to cope with climate change has important ecological and economic implications. Many temperate tree species experience a wide range of environmental conditions, suggesting high adaptability to new environmental conditions. We investigated adaptation to regional climate in the drought‐sensitive tree species Alnus glutinosa (Black alder), using a complementary approach that integrates genomic, phenotypic and landscape data. A total of 24 European populations were studied in a common garden and through landscape genomic approaches. Genotyping‐by‐sequencing was used to identify SNPs across the genome, resulting in 1990 SNPs. Although a relatively low percentage of putative adaptive SNPs was detected (2.86% outlier SNPs), we observed clear associations among outlier allele frequencies, temperature and plant traits. In line with the typical drought avoiding nature of A. glutinosa, leaf size varied according to a temperature gradient and significant associations with multiple outlier loci were observed, corroborating the ecological relevance of the observed outlier SNPs. Moreover, the lack of isolation by distance, the very low genetic differentiation among populations and the high intrapopulation genetic variation all support the notion that high gene exchange combined with strong environmental selection promotes adaptation to environmental cues.     

Population genomics of local adaptation versus speciation in coral reef fishes (Hypoplectrus spp,Serranidae)

Are the population genomic patterns underlying local adaptation and the early stages of speciation similar? Addressing this question requires a system in which (i) local adaptation and the early stages of speciation can be clearly identified and distinguished, (ii) the amount of genetic divergence driven by the two processes is similar, and (iii) comparisons can be repeated both taxonomically (for local adaptation) and geographically (for speciation). Here, we report just such a situation in the hamlets (Hypoplectrus spp), brightly colored reef fishes from the wider Caribbean. Close to 100,000 SNPs genotyped in 126 individuals from three sympatric species sampled in three repeated populations provide genome‐wide levels of divergence that are comparable among allopatric populations (F_st estimate = 0.0042) and sympatric species (F_st estimate = 0.0038). Population genetic, clustering, and phylogenetic analyses reveal very similar patterns for local adaptation and speciation, with a large fraction of the genome undifferentiated (F_st estimate ≈ 0), a very small proportion of F_st outlier loci (0.05–0.07%), and remarkably few repeated outliers (1–3). Nevertheless, different loci appear to be involved in the two processes in Hypoplectrus, with only 7% of the most differentiated SNPs and outliers shared between populations and species comparisons. In particular, a tropomyosin (Tpm4) and a previously identified hox (HoxCa) locus emerge as candidate loci (repeated outliers) for local adaptation and speciation, respectively. We conclude that marine populations may be locally adapted notwithstanding shallow levels of genetic divergence, and that from a population genomic perspective, this process does not appear to differ fundamentally from the early stages of speciation.     

Identifying insecticide resistance genes in mosquito by combining AFLP genome scans and 454 pyrosequencing

AFLP‐based genome scans are widely used to study the genetics of adaptation and to identify genomic regions potentially under selection. However, this approach usually fails to detect the actual genes or mutations targeted by selection owing to the difficulties of obtaining DNA sequences from AFLP fragments. Here, we combine classical AFLP outlier detection with 454 sequencing of AFLP fragments to obtain sequences from outlier loci. We applied this approach to the study of resistance to Bacillus thuringiensis israelensis (Bti) toxins in the dengue vector Aedes aegypti. A genome scan of Bti‐resistant and Bti‐susceptible A. aegypti laboratory strains was performed based on 432 AFLP markers. Fourteen outliers were detected using two different population genetic algorithms. Out of these, 11 were successfully sequenced. Three contained transposable elements (TEs) sequences, and the 10 outliers that could be mapped at a unique location in the reference genome were located on different supercontigs. One outlier was in the vicinity of a gene coding for an aminopeptidase potentially involved in Bti toxin‐binding. Patterns of sequence variability of this gene showed significant deviation from neutrality in the resistant strain but not in the susceptible strain, even after taking into account the known demographic history of the selected strain. This gene is a promising candidate for future functional analysis.     

Insights into the role of differential gene expression on the ecological adaptation of the snail <Emphasis Type="Italic">Littorina saxatilis</Emphasis>

Background  

In the past 40 years, there has been increasing acceptance that variation in levels of gene expression represents a major source of evolutionary novelty. Gene expression divergence is therefore likely to be involved in the emergence of incipient species, namely, in a context of adaptive radiation. In this study, a genome-wide expression profiling approach (cDNA-AFLP), validated by quantitative real-time polymerase chain reaction (qPCR) were used to get insights into the role of differential gene expression on the ecological adaptation of the marine snail Littorina saxatilis. This gastropod displays two sympatric ecotypes (RB and SU) which are becoming one of the best studied systems for ecological speciation.     

Genomic divergence between Spanish Littorina saxatilis ecotypes unravels limited admixture and extensive parallelism associated with population history

The rough periwinkle, Littorina saxatilis, is a model system for studying parallel ecological speciation in microparapatry. Phenotypically parallel wave‐adapted and crab‐adapted ecotypes that hybridize within the middle shore are replicated along the northwestern coast of Spain and have likely arisen from two separate glacial refugia. We tested whether greater geographic separation corresponding to reduced opportunity for contemporary or historical gene flow between parallel ecotypes resulted in less parallel genomic divergence. We sequenced double‐digested restriction‐associated DNA (ddRAD) libraries from individual snails from upper, mid, and low intertidal levels of three separate sites colonized from two separate refugia. Outlier analysis of 4256 SNP markers identified 34.4% sharing of divergent loci between two geographically close sites; however, these sites each shared only 9.9%–15.1% of their divergent loci with a third more‐distant site. STRUCTURE analysis revealed that genotypes from only three of 166 phenotypically intermediate mid‐shore individuals appeared to result from recent hybridization, suggesting that hybrids cannot be reliably identified using shell traits. Hierarchical AMOVA indicated that the primary source of genomic differentiation was geographic separation, but also revealed greater similarity of the same ecotype across the two geographically close sites than previously estimated with dominant markers. These results from a model system for ecological speciation suggest that genomic parallelism is affected by the opportunity for historical or contemporary gene flow between populations.     

A population genomic scan in Chorthippus grasshoppers unveils previously unknown phenotypic divergence

Understanding the genetics of speciation and the processes that drive it is a central goal of evolutionary biology. Grasshoppers of the Chorthippus species group differ strongly in calling song (and corresponding female preferences) but are exceedingly similar in other characteristics such as morphology. Here, we performed a population genomic scan on three Chorthippus species (Chorthippus biguttulus, C. mollis and C. brunneus) to gain insight into the genes and processes involved in divergence and speciation in this group. Using an RNA‐seq approach, we examined functional variation between the species by calling SNPs for each of the three species pairs and using F_ST‐based approaches to identify outliers. We found approximately 1% of SNPs in each comparison to be outliers. Between 37% and 40% of these outliers were nonsynonymous SNPs (as opposed to a global level of 17%) indicating that we recovered loci under selection. Among the outliers were several genes that may be involved in song production and hearing as well as genes involved in other traits such as food preferences and metabolism. Differences in food preferences between species were confirmed with a behavioural experiment. This indicates that multiple phenotypic differences implicating multiple evolutionary processes (sexual selection and natural selection) are present between the species.     

Differential introgression from a sister species explains high FST outlier loci within a mussel species

Scanning genomes for loci with high levels of population differentiation has become a standard of population genetics. F_ST outlier loci are most often interpreted as signatures of local selection, but outliers might arise for many other reasons too often left unexplored. Here, we tried to identify further the history and genetic basis underlying strong differentiation at F_ST outlier loci in a marine mussel. A genome scan of genetic differentiation has been conducted between Atlantic and Mediterranean populations of Mytilus galloprovincialis. The differentiation was low overall (F_ST = 0.03), but seven loci (2%) were strong F_ST outliers. We then analysed DNA sequence polymorphism at two outlier loci. The genetic structure proved to be the consequence of differential introgression of alleles from the sister‐hybridizing species Mytilus edulis. Surprisingly, the Mediterranean population was the most introgressed at these two loci, although the contact zone between the two species is nowadays localized along the Atlantic coasts of France and the British Isles. A historical contact between M. edulis and Mediterranean M. galloprovincialis should have happened during glacial periods. It proved difficult to disentangle two hypotheses: (i) introgression was adaptive, implying edulis alleles have been favoured in Mediterranean populations, or (ii) the genetic architecture of the barrier to edulis gene flow is different between the two M. galloprovincialis backgrounds. Five of the seven outliers between M. galloprovincialis populations were also outliers between M. edulis and Atlantic M. galloprovincialis, which would support the latter hypothesis. Differential introgression across semi‐permeable barriers to gene flow is a neglected scenario to interpret outlying loci that may prove more widespread than anticipated.