Divergent selection and drift shape the genomes of two avian sister species spanning a saline–freshwater ecotone

The role of species divergence due to ecologically based divergent selection—or ecological speciation—in generating and maintaining biodiversity is a central question in evolutionary biology. Comparison of the genomes of phylogenetically related taxa spanning a selective habitat gradient enables discovery of divergent signatures of selection and thereby provides valuable insight into the role of divergent ecological selection in speciation. Tidal marsh ecosystems provide tractable opportunities for studying organisms' adaptations to selective pressures that underlie ecological divergence. Sharp environmental gradients across the saline–freshwater ecotone within tidal marshes present extreme adaptive challenges to terrestrial vertebrates. Here, we sequence 20 whole genomes of two avian sister species endemic to tidal marshes—the saltmarsh sparrow (Ammospiza caudacutus) and Nelson's sparrow (A. nelsoni)—to evaluate the influence of selective and demographic processes in shaping genome‐wide patterns of divergence. Genome‐wide divergence between these two recently diverged sister species was notably high (genome‐wide F_ST = 0.32). Against a background of high genome‐wide divergence, regions of elevated divergence were widespread throughout the genome, as opposed to focused within islands of differentiation. These patterns may be the result of genetic drift resulting from past tidal march colonization events in conjunction with divergent selection to different environments. We identified several candidate genes that exhibited elevated divergence between saltmarsh and Nelson's sparrows, including genes linked to osmotic regulation, circadian rhythm, and plumage melanism—all putative candidates linked to adaptation to tidal marsh environments. These findings provide new insights into the roles of divergent selection and genetic drift in generating and maintaining biodiversity.     

Model‐based demographic inference of introgression history in European whitefish species pairs'

Parallel phenotypic differentiation is generally attributed to parallel adaptive divergence as an evolutionary response to similar environmental contrasts. Such parallelism may actually originate from several evolutionary scenarios ranging from repeated parallel divergence caused by divergent selection to a unique divergence event followed by gene flow. Reconstructing the evolutionary history underlying parallel phenotypic differentiation is thus fundamental to understand the relative contribution of demography and selection on genomic divergence during speciation. In this study, we investigate the divergence history of replicate European whitefish (Coregonus lavaretus), limnetic and benthic species pairs from two lakes in Norway and two lakes in Switzerland. Demographic models accounting for semi‐permeability and linked selection were fitted to the unfolded joint allele frequency spectrum built from genome‐wide SNPs and compared to each other in each species pair. We found strong support for a model of asymmetrical post‐glacial secondary contact between glacial lineages in all four lakes. Moreover, our results suggest that heterogeneous genomic differentiation has been shaped by the joint action of linked selection accelerating lineage sorting during allopatry, and heterogeneous migration eroding divergence at different rates along the genome following secondary contact. Our analyses reveal how the interplay between demography, selection and historical contingency has influenced the levels of diversity observed in previous whitefish phylogeographic studies. This study thus provides new insights into the historical demographic and selective processes that shaped the divergence associated with ecological speciation in European whitefish.     

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.     

The accumulation of reproductive isolation in early stages of divergence supports a role for sexual selection

Models of speciation by sexual selection propose that male–female coevolution leads to the rapid evolution of behavioural reproductive isolation. Here, we compare the strength of behavioural isolation to ecological isolation, gametic incompatibility and hybrid inviability in a group of dichromatic stream fishes. In addition, we examine whether any of these individual barriers, or a combined measure of total isolation, is predicted by body shape differences, male colour differences, environmental differences or genetic distance. Behavioural isolation reaches the highest values of any barrier and is significantly greater than ecological isolation. No individual reproductive barrier is associated with any of the predictor variables. However, marginally significant relationships between male colour and body shape differences with ecological and behavioural isolation are discussed. Differences in male colour and body shape predict total reproductive isolation between species; hierarchical partitioning of these two variables' effects suggests a stronger role for male colour differences. Together, these results suggest an important role for divergent sexual selection in darter speciation but raise new questions about the mechanisms of sexual selection at play and the role of male nuptial ornaments.     

Colonisation of toxic environments drives predictable life‐history evolution in livebearing fishes (Poeciliidae)

New World livebearing fishes (family Poeciliidae) have repeatedly colonised toxic, hydrogen sulphide‐rich waters across their natural distribution. Physiological considerations and life‐history theory predict that these adverse conditions should favour the evolution of larger offspring. Here, we examined nine poeciliid species that independently colonised toxic environments, and show that these fishes have indeed repeatedly evolved much larger offspring size at birth in sulphidic waters, thus uncovering a widespread pattern of predictable evolution. However, a second pattern, only indirectly predicted by theory, proved additionally common: a reduction in the number of offspring carried per clutch (i.e. lower fecundity). Our analyses reveal that this secondary pattern represents a mere consequence of a classic life‐history trade‐off combined with strong selection on offspring size alone. With such strong natural selection in extreme environments, extremophile organisms may commonly exhibit multivariate phenotypic shifts even though not all diverging traits necessarily represent adaptations to the extreme conditions.     

Genome divergence and diversification within a geographic mosaic of coevolution

Despite substantial interest in coevolution's role in diversification, examples of coevolution contributing to speciation have been elusive. Here, we build upon past studies that have shown both coevolution between South Hills crossbills and lodgepole pine (Pinus contorta), and high levels of reproductive isolation between South Hills crossbills and other ecotypes in the North American red crossbill (Loxia curvirostra) complex. We used genotyping by sequencing to generate population genomic data and applied phylogenetic and population genetic analyses to characterize the genetic structure within and among nine of the ecotypes. Although genome‐wide divergence was slight between ecotypes (F_ST = 0.011–0.035), we found evidence of relative genetic differentiation (as measured by F_ST) between and genetic cohesiveness within many of them. As expected for nomadic and opportunistic breeders, we detected no evidence of isolation by distance. The one sedentary ecotype, the South Hills crossbill, was genetically most distinct because of elevated divergence at a small number of loci rather than pronounced overall genome‐wide divergence. These findings suggest that mechanisms related to recent local coevolution between South Hills crossbills and lodgepole pine (e.g. strong resource‐based density dependence limiting gene flow) have been associated with genome divergence in the face of gene flow. Our results further characterize a striking example of coevolution driving speciation within perhaps as little as 6000 years.     

Multilocus patterns of genetic variation across Cryptosporidium species suggest balancing selection at the gp60 locus

Cryptosporidium is an apicomplexan protozoan that lives in most vertebrates, including humans. Its gp60 gene is functionally involved in its attachment to host cells, and its high level of genetic variation has made it the reference marker for sample typing in epidemiological studies. To understand the origin of such high diversity and to determine the extent to which this classification applies to the rest of the genome, we analysed the patterns of variation at gp60 and nine other nuclear loci in isolates of three Cryptosporidium species. Most loci showed low genetic polymorphism (π_S <1%) and similar levels of between‐species divergence. Contrastingly, gp60 exhibited very different characteristics: (i) it was nearly ten times more variable than the other loci; (ii) it displayed a significant excess of polymorphisms relative to between‐species differences in a maximum‐likelihood Hudson–Kreitman–Aguadé test; (iii) gp60 subtypes turned out to be much older than the species they were found in; and (iv) showed a significant excess of polymorphic variants shared across species from random expectations. These observations suggest that this locus evolves under balancing selection and specifically under negative frequency‐dependent selection (FDS). Interestingly, genetic variation at the other loci clusters very well within the groups of isolates defined by gp60 subtypes, which may provide new tools to understand the genome‐wide patterns of genetic variation of the parasite in the wild. These results suggest that gp60 plays an active and essential role in the life cycle of the parasite and that genetic variation at this locus might be essential for the parasite's long‐term success.     

Antitropicality and convergent evolution: a case study of Permian neospiriferine brachiopods

Antitropical distribution is a biogeographical pattern characterized by natural occurrences of the same species or members of the same clade in the middle‐ or middle‐to‐high‐latitudinal habitats of both hemispheres, either on land or in marine environments, without appearing in the intervening tropical environments. For most of the noted examples of Permian antitropical distribution, particularly in marine invertebrates, the causes of disjunctions have been mainly linked to either dispersal or vicariance models. Little attention has been paid to other possible mechanisms. This study investigated the antitropicality of some Permian neospiriferine brachiopods through detailed taxonomic revision, comparison of palaeobiogeographical distribution, and a phylogenetic analysis. Several species, previously assigned to Kaninospirifer, are here reassigned to other genera, especially to Fasciculatia in the northern hemisphere and to Quadrospira in the southern hemisphere during the Permian. Both Kaninospirifer and Fasciculatia appear to have been restricted to north‐western Pangea and north‐eastern Asia during the Permian, but there is no robust evidence to suggest their presence in the southern hemisphere to which Imperiospira and Quadrospira were confined. In spite of the distributional separation between the two pairs of neospiriferine genera in the Permian palaeobiogeographical regime, they share considerable numbers of morphological characters, such as a large shell, subdued fasciculation, and reduction of ventral adminicula. Notwithstanding these morphological similarities, our phylogenetic reconstruction of the neospiriferines does not support a close relationship between these genera. This therefore must indicate that these similar morphological features were independently acquired, probably with these taxa living in spatially separate but ecologically compatible environmental conditions in the mid‐latitudinal area of each hemisphere during the Permian. We regard this as an example of convergent evolution.     

Recurrent selection explains parallel evolution of genomic regions of high relative but low absolute differentiation in a ring species

Recent technological developments allow investigation of the repeatability of evolution at the genomic level. Such investigation is particularly powerful when applied to a ring species, in which spatial variation represents changes during the evolution of two species from one. We examined genomic variation among three subspecies of the greenish warbler ring species, using genotypes at 13 013 950 nucleotide sites along a new greenish warbler consensus genome assembly. Genomic regions of low within‐group variation are remarkably consistent between the three populations. These regions show high relative differentiation but low absolute differentiation between populations. Comparisons with outgroup species show the locations of these peaks of relative differentiation are not well explained by phylogenetically conserved variation in recombination rates or selection. These patterns are consistent with a model in which selection in an ancestral form has reduced variation at some parts of the genome, and those same regions experience recurrent selection that subsequently reduces variation within each subspecies. The degree of heterogeneity in nucleotide diversity is greater than explained by models of background selection, but is consistent with selective sweeps. Given the evidence that greenish warblers have had both population differentiation for a long period of time and periods of gene flow between those populations, we propose that some genomic regions underwent selective sweeps over a broad geographic area followed by within‐population selection‐induced reductions in variation. An important implication of this ‘sweep‐before‐differentiation’ model is that genomic regions of high relative differentiation may have moved among populations more recently than other genomic regions.     

Experimental evidence for trait utility of gill raker number in adaptive radiation of a north temperate fish

North temperate fish in post‐glacial lakes are textbook examples for rapid parallel adaptive radiation into multiple trophic specialists within individual lakes. Speciation repeatedly proceeded along the benthic–limnetic habitat axis, and benthic–limnetic sister species diverge in the number of gill rakers. Yet, the utility of different numbers of gill rakers for consuming benthic vs. limnetic food has only very rarely been experimentally demonstrated. We bred and raised families of a benthic–limnetic species pair of whitefish under common garden conditions to test whether these species (i) show heritable differentiation in feeding efficiency on zooplankton, and (ii) whether variation in feeding efficiency is predicted by variation in gill raker numbers. We used zooplankton of three different size classes to investigate prey size dependency of divergence in feeding efficiency and to investigate the effect strength of variation in the number of gill rakers. Our results show strong interspecific differences in feeding efficiency. These differences are largest when fish were tested with the smallest zooplankton. Importantly, feeding efficiency is significantly positively correlated with the number of gill rakers when using small zooplankton, also when species identity is statistically controlled for. Our results support the hypothesis that a larger number of gill rakers are of adaptive significance for feeding on zooplankton and provide one of the first experimental demonstrations of trait utility of gill raker number when fish feed on zooplankton. These results are consistent with the suggested importance of divergent selection driven feeding adaptation during adaptive radiation of fish in post‐glacial lakes.     

Limited gene exchange between two sister species of leaf beetles within a hybrid zone in the Alps

Analysing genomic variation within and between sister species is a first step towards understanding species boundaries. We focused on two sister species of cold‐resistant leaf beetles, Gonioctena quinquepunctata and G. intermedia, whose ranges overlap in the Alps. A previous study of DNA sequence variation had revealed multiple instances of mitochondrial genome introgression in this region, suggesting recent hybridization between the two species. To evaluate the extent of gene exchange resulting from these hybridization events, we sampled individuals of both species inside and outside the hybrid zone and analysed genomic variation among them using RAD‐seq markers. Individual levels of introgression in the nuclear genome were estimated first by defining species‐specific SNPs (displaying a fixed difference between species) a priori and second by using model‐based methods. Both types of analyses indicated little gene exchange, if any, between species at the level of the nuclear genome. Whereas the first method suggested slightly more gene flow, we argue that it has likely overestimated introgression in the phylogeographic context of this study. We conclude that strong intrinsic barriers prevent genetic exchange at the level of the nuclear genome between the two species. The apparent discrepancy observed between introgression occurring in the nuclear and mitochondrial genomes could be explained by selection acting in favour of the latter. Also, these results have consequences for the phylogeographic study of each species, since we can assume that genetic diversity in the overlapping portion of their ranges is not the product of introgression.     

Genomic evidence for convergent evolution of a key trait underlying divergence in island birds

Reproductive isolation can be initiated by changes in one or a few key traits that prevent random mating among individuals in a population. During the early stages of speciation, when isolation is often incomplete, there will be a heterogeneous pattern of differentiation across regions of the genome between diverging populations, with loci controlling these key traits appearing the most distinct as a result of strong diversifying selection. In this study, we used Illumina‐sequenced ddRAD tags to identify genomewide patterns of differentiation in three recently diverged island populations of the Monarcha castaneiventris flycatcher of the Solomon Islands. Populations of this species have diverged in plumage colour, and these differences in plumage colour, in turn, are used in conspecific recognition and likely important in reproductive isolation. Previous candidate gene sequencing identified point mutations in MC1R and ASIP, both known pigmentation genes, to be associated with the difference in plumage colour between islands. Here, we show that background levels of genomic differentiation based on over 70,000 SNPs are extremely low between populations of distinct plumage colour, with no loci reaching the level of differentiation found in either candidate gene. Further, we found that a phylogenetic analysis based on these SNPs produced a taxonomy wherein the two melanic populations appear to have evolved convergently, rather than from a single common ancestor, in contrast to their original classification as a single subspecies. Finally, we found evidence that the pattern of low genomic differentiation is the result of both incomplete lineage sorting and gene flow between populations.     

Positive selection on the mitochondrial ATP synthase 6 and the NADH dehydrogenase 2 genes across 22 hare species (genus Lepus)

Amino acid changes in mitochondrial (mt) oxidative phosphorylation (OXPHOS) genes have been suggested as a key adaptation to environmental variation. Here, we analyzed 416 sequences of ATPase synthase 6 (MT‐ATP6) and NADH dehydrogenase 2 (MT‐ND2) in 22 different hare (Lepus) species from across a wide range of habitats and climates. We used site‐ and branch‐based methods to test for positive selection on specific codons and lineages. We found four codons in MT‐ATP6 and five in MT‐ND2 under positive selection, affecting several species lineages. We investigated the association of protein variants at each locus with climate zone, using multinomial generalized linear models (glm), including species, regions, historical introgression events, and the co‐occurring protein variant at the other locus as additional explanatory variables. A significant climate effect as based on the “Köppen climate classification” was observed for MT‐ND2 protein variants as translated from our nucleotide sequences. Moreover, MT‐ND2 protein variants were significantly affected by the co‐occurring MT‐ATP6 protein variant in the same mtDNA molecule. Contrary to the expectation for non‐recombining mitochondrial DNA molecules, the presence of an evolutionarily relatively ancestral protein variant at one locus was associated with a relatively derived protein at the other locus in the same mitochondrial molecule, respectively. The relative evolutionary status of a protein variant was evaluated according to its positions relative to the respective out‐group protein variant in a network analysis of nucleotide sequences. All our results suggest a complex effect of various climatic parameters acting on multiple mtOXPHOS genes in a co‐adaptive way, favoring combinations of ancestral and derived variants.     

Sampling related individuals within ponds biases estimates of population structure in a pond‐breeding amphibian

Effective conservation and management of pond‐breeding amphibians depends on the accurate estimation of population structure, demographic parameters, and the influence of landscape features on breeding‐site connectivity. Population‐level studies of pond‐breeding amphibians typically sample larval life stages because they are easily captured and can be sampled nondestructively. These studies often identify high levels of relatedness between individuals from the same pond, which can be exacerbated by sampling the larval stage. Yet, the effect of these related individuals on population genetic studies using genomic data is not yet fully understood. Here, we assess the effect of within‐pond relatedness on population and landscape genetic analyses by focusing on the barred tiger salamanders (Ambystoma mavortium) from the Nebraska Sandhills. Utilizing genome‐wide SNPs generated using a double‐digest RADseq approach, we conducted standard population and landscape genetic analyses using datasets with and without siblings. We found that reduced sample sizes influenced parameter estimates more than the inclusion of siblings, but that within‐pond relatedness led to the inference of spurious population structure when analyses depended on allele frequencies. Our landscape genetic analyses also supported different models across datasets depending on the spatial resolution analyzed. We recommend that future studies not only test for relatedness among larval samples but also remove siblings before conducting population or landscape genetic analyses. We also recommend alternative sampling strategies to reduce sampling siblings before sequencing takes place. Biases introduced by unknowingly including siblings can have significant implications for population and landscape genetic analyses, and in turn, for species conservation strategies and outcomes.     

Genetic structure of two Prosopis species in Chaco areas: A lack of allelic diversity diagnosis and insights into the allelic conservation of the affected species

The Gran Chaco is the largest continuous region of the South American dry forest, spanning Argentina, Paraguay, Bolivia, and Brazil. Prosopis rubriflora and Prosopis ruscifolia are typical tree species of chaquenian area forests, which have been subjected to continuous fragmentation caused by cattle raising. This study evaluated P. rubriflora and P. ruscifolia in areas with varying levels of disturbance. We investigated the contemporary genetic diversities of both species in areas with distinct anthropogenic disturbances. Even with a lower heterozygote frequency, disturbed areas can provide important storage for alleles, allowing the maintenance of diversity. The genetic diversity of P. rubriflora was surprisingly similar to that of P. ruscifolia (H_e = 0.59 and H_e = 0.60, respectively) even with very different distribution ranges of both species. However, P. ruscifolia exhibited a higher intrapopulation fixation index than P. rubriflora. P. rubriflora showed evidence of bottlenecking in 64% of the sampled areas, while P. ruscifolia showed such evidence in 36% of the sampled areas. Additionally, P. rubriflora had two distinct populations due to its disjunctive geographic distribution, whereas P. ruscifolia had a single population that exhibited few signs of population structure in some areas, possibly due to the main pollinators presenting a short range of dispersion. Our results suggest that 42 Chaco areas should be conserved to retain the minimum of 500 individuals necessary to maintain genetic diversity for 100–1,000 generations. This study improves our understanding of these two Prosopis species and provides information for the conservation of their genetic diversities.     

Monophyly of Wolbachia pipientis genomes within Drosophila melanogaster: geographic structuring,titre variation and host effects across five populations

Wolbachia pipientis is one of the most widely studied endosymbionts today, yet we know little about its short‐term adaptation and evolution. Here, using a set of 91 inbred Drosophila melanogaster lines from five populations, we explore patterns of diversity and recent evolution in the Wolbachia strain wMel. Within the D. melanogaster lines, we identify six major mitochondrial clades and four wMel clades. Concordant with past studies, the Wolbachia haplotypes contain an overall low level of nucleotide diversity, yet they still display geographic structuring. Using Bayesian analysis informed with demographic estimates of colonization times, we estimate that all extant D. melanogaster mitochondrial haplotypes coalesce to a Wolbachia‐infected ancestor approximately 2200 years ago. Finally, we measure wMel titre within the infected flies and find that titre varies across populations, an effect attributable to host genetic factors. This demonstration of local phenotypic divergence suggests that intraspecific host genetic variation plays a key role in shaping this model symbiotic system.