A Genomic Perspective on Species Delimitation

BackgroundThe evolution of species boundaries and the relative impact of selection and gene flow on genomic divergence are best studied in populations and species pairs exhibiting various levels of divergence along the speciation continuum. We studied species boundaries in Iberian barbels, Barbus and Luciobarbus, a system of populations and species spanning a wide degree of genetic relatedness, as well as geographic distribution and range overlap. We jointly analyze multiple types of molecular markers and morphological traits to gain a comprehensive perspective on the nature of species boundaries in these cyprinid fishes.ResultsIntraspecific molecular and morphological differentiation is visible among many populations. Genomes of all sympatric species studied are porous to gene flow, even if they are not sister species. Compared to their allopatric counterparts, sympatric representatives of different species share alleles and show an increase in all measures of nucleotide polymorphism (S, Hd, K, π and θ). High molecular diversity is particularly striking in L. steindachneri from the Tejo and Guadiana rivers, which co-varies with other sympatric species. Interestingly, different nuclear markers introgress across species boundaries at various levels, with distinct impacts on population trees. As such, some loci exhibit limited introgression and population trees resemble the presumed species tree, while alleles at other loci introgress more freely and population trees reflect geographic affinities and interspecific gene flow. Additionally, extent of introgression decreases with increasing genetic divergence in hybridizing species pairs.ConclusionsWe show that reproductive isolation in Iberian Barbus and Luciobarbus is not complete and species boundaries are semi-permeable to (some) gene flow, as different species (including non-sister) are exchanging genes in areas of sympatry. Our results support a speciation-with-gene-flow scenario with heterogeneous barriers to gene flow across the genome, strengthening with genetic divergence. This is consistent with observations coming from other systems and supports the notion that speciation is not instantaneous but a gradual process, during which different species are still able to exchange some genes, while selection prevents gene flow at other loci. We also provide evidence for a hybrid origin of a barbel ecotype, L. steindachneri, suggesting that ecology plays a key role in species coexistence and hybridization in Iberian barbels. This ecotype with intermediate, yet variable, molecular, morphological, trophic and ecological characteristics is the local product of introgressive hybridization of L. comizo with up to three different species (with L. bocagei in the Tejo, with L. microcephalus and L. sclateri in the Guadiana). In spite of the homogenizing effects of ongoing gene flow, species can still be discriminated using a combination of morphological and molecular markers. Iberian barbels are thus an ideal system for the study of species boundaries, since they span a wide range of genetic divergences, with diverse ecologies and degrees of sympatry.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0392-3) contains supplementary material, which is available to authorized users.

Nucleotide sequence data are being harnessed to identify species, even in cases in which organisms themselves are neither in hand nor witnessed. But how genome-wide sequence divergence maps to species status is far from clear. While gene sequence divergence is commonly used to delineate bacterial species, its correspondence to established species boundaries has yet to be explored across eukaryotic taxa. Because the processes underlying gene flow differ fundamentally between prokaryotes and eukaryotes, these domains are likely to differ in the relationship between reproductive isolation and genome-wide sequence divergence. In prokaryotes, homologous recombination, the basis of gene flow, depends directly on the degree of genomic sequence divergence, whereas in sexually reproducing eukaryotes, reproductive incompatibility can stem from changes in very few genes. Guided by measures of genome-wide sequence divergence in bacteria, we gauge how genomic criteria correspond to species boundaries in eukaryotes. In recognized species of eukaryotes, levels of gene sequence divergence within species are typically very small, averaging <1% across protein-coding regions in most animals, plants, and fungi. There are even instances in which divergence between sister species is the same or less than that among conspecifics. In contrast, bacterial species, defined as populations exchanging homologous genes, show levels of divergence both within and between species that are considerably higher. Although no single threshold delineates species, eukaryotic populations with >1% genome-wide sequence divergence are likely separate species, whereas prokaryotic populations with 1% divergence are still able to recombine and thus can be considered the same species.

Interpopulational gene flow within a species can reduce population differentiation due to genetic drift, whereas genetic exchange among taxa can impede speciation. We used allozyme data to estimate gene flow within and among geographic races and species of perennial herbs in the Ipomopsis aggregata complex (Polemoniaceae). Estimates of interpopulational gene flow within taxa from two methods (F statistics and private alleles) were correlated with one another. Gene flow among populations within each geographic race (subspecies) of I. aggregata was relatively high (Nm greater than approximately 1.0). Gene flow was also high among populations of I. arizonica and among four northern populations of I. tenuituba. However, gene flow was low (Nm less than 1.0) for I. tenuituba when a population representing subsp. macrosiphon was included. This is consistent with previous findings that subsp. macrosiphon has had an independent origin and is reproductively, as well as geographically, isolated. A recently developed model, based on hierarchical F statistics, was employed to estimate genetic exchange among taxa. Gene flow estimates were generally high among races of I. aggregata (dNmrace greater than 1.0) but were low among subspecies of I. tenuituba (dNmrace less than 1.0). Consistent with morphological evidence, estimates of interspecific gene flow were moderate between I. aggregata and I. tenuituba, which hybridize in several areas. However, contrary to morphological evidence, we estimated relatively high levels of interspecific gene flow involving I. arizonica. Our results suggest that I. arizonica has hybridized with other species without the transfer of morphological traits.(ABSTRACT TRUNCATED AT 250 WORDS)

The origin of species

Estimates of interpopulational gene flow and the levels and distribution of genetic variation in Clarkia speciosa subsp. polyantha were obtained using enzyme electrophoresis. Eight enzymes encoded by 17 loci were analyzed. Nei's mean genetic identity was 0.96, indicating little genetic divergence among populations. Gene diversity statistics also suggest little heterogeneity among populations. Interpopulational gene flow, estimated according to Slatkin (1985), was fairly high, Nm = 3.9, probably accounting for the lack of differentiation among populations.

Slatkin's method (1981) for analyzing gene flow levels is applied to eleven species of cave arthropods. This provides insights into the strength of gene flow as a force affecting the evolution of cave organisms, while serving as a test of Slatkin's method. The results show that patterns of gene flow are consistent with the dispersal abilities and the ecological requirements of each species. Troglobites in general have lower gene flow values than troglophiles or trogloxenes. However, the geology of the area, its vegetation profile, and the geographic distance among the populations considered are also important in determining the gene flow levels. Gene flow patterns in three cave species, Ptomaphagus hirtus, Neaphaenops tellkampfii tellkampfii and Hadenoecus subterraneus, are compared in detail. These species inhabit the same highly interconnected karst area in south-central Kentucky but differ in their ecological requirements. The results suggest that gene flow levels are more dependent upon the intrinsic characteristics of each species than upon the availability of routes for dispersal. Overall estimates of gene flow were coupled with a more detailed population-structure analysis in four terrestrial cave arthropods: Speonomus delarouzeei, Hadenoecus cumberlandicus, Hadenoecus subterraneus and Euhadenoecus puteanus. The results stress the need for this type of analysis for subdivided species, in which estimates of the average gene flow can produce misleading results. Moreover, they show how these types of measures are more relevant in describing historical patterns of gene exchange than in indicating current levels of gene flow.

With over 42,000 species of known crustaceans inhabiting mainly marine, but also freshwater and even terrestrial habitats, they are often key members of these diverse habitats and associated ecosystems. However, many crustaceans are either endangered or their endangered status is simply unknown because we lack critical information about these important species. Conservation genetics offer efficient tools, both molecular and analytical, to gain keen insights into population dynamics, population sizes, species boundaries, and habitat associations to inform conservation efforts based on either ecosystems and/or species. This chapter covers key concepts in conservation genetics as applied to crustacean biodiversity and provides examples of the application of these concepts in ongoing crustacean conservation efforts as case studies. We explore informative reference databases for geographic, taxonomic, and conservation information for crustaceans. We discuss sampling strategies as applied to conservation genetics and explore a diversity of molecular markers from mitochondrial DNA to anchored hybrid enrichment. We also highlight key analytical approaches for estimating population genetic parameters that help inform conservation decisions such as gene flow, genetic diversity, and phylogenetic diversity estimates. Finally, we demonstrate the application of some of these concepts through the exploration of case studies based on our own conservation genetics work in crustaceans. This chapter highlights not only the useful tools and a diversity of data types used in conservation genetics, but the extreme need for the application of these tools, data, and associated insights to the world’s crustacean diversity that is severely understudied with respect to conservation.

Understanding the factors that contribute to the generation of reproductively isolated forms is a fundamental goal of evolutionary biology. Cryptic species are an especially interesting challenge to study in this context since they lack obvious morphological differentiation that provides clues to adaptive divergence that may drive reproductive isolation. Geographical isolation in refugial areas during glacial cycling is known to be important for generating genetically divergent populations, but its role in the origination of new species is still not fully understood and likely to be situation dependent. We combine analysis of 35,434 single‐nucleotide polymorphisms (SNPs) with environmental niche modeling (ENM) to investigate genomic and ecological divergence in three cryptic species formerly classified as the field vole (Microtus agrestis). The SNPs demonstrate high genomic divergence (pairwise F ST values of 0.45–0.72) and little evidence of gene flow among the three field vole cryptic species, and we argue that genetic drift may have been a particularly important mechanism for divergence in the group. The ENM reveals three areas as potential glacial refugia for the cryptic species and differing climatic niches, although with spatial overlap between species pairs. This evidence underscores the role that glacial cycling has in promoting genetic differentiation and reproductive isolation by subdivision into disjunct distributions at glacial maxima in areas relatively close to ice sheets. Future investigation of the intrinsic barriers to gene flow between the field vole cryptic species is required to fully assess the mechanisms that contribute to reproductive isolation. In addition, the Portuguese field vole (M. rozianus) shows a high inbreeding coefficient and a restricted climatic niche, and warrants investigation into its conservation status.

When two species are incompletely isolated, strengthening premating isolation barriers in response to the production of low fitness hybrids may complete the speciation process. Here, we use the sister species Drosophila subquinaria and Drosophila recens to study the conditions under which this reinforcement of species boundaries occurs in natural populations. We first extend the region of known sympatry between these species, and then we conduct a fine-scale geographic survey of mate discrimination coupled with estimates of gene flow within and admixture between species. Within D.subquinaria, reinforcement is extremely effective: we find variation in mate discrimination both against D.recens males and against conspecific allopatric males on the scale of a few kilometres and in the face of gene flow both from conspecific populations and introgression from D.recens. In D.recens, we do not find evidence for increased mate discrimination in sympatry, even where D.recens is rare, consistent with substantial gene flow throughout the species' range. Finally, we find that introgression between species is asymmetric, with more from D.recens into D.subquinaria than vice versa. Within each species, admixture is highest in the geographic region where it is rare relative to the other species, suggesting that when hybrids are produced they are of low fitness. In sum, reinforcement within D.subquinaria is effective at maintaining species boundaries, but even when reinforcing selection is strong it may not always result in a pattern of strong reproductive character displacement due to variation in the frequency of hybridization and gene flow from neighbouring populations.

Anopheles gambiae sensu stricto exists as two often-sympatric races termed the M and S molecular forms, characterized by fixed differences at an X-linked marker. Extreme divergence between M and S forms at pericentromeric "genomic islands" suggested that selection on variants therein could be driving interform divergence in the presence of ongoing gene flow, but recent work has detected much more widespread genomic differentiation. Whether such genomic islands are important in reproductive isolation or represent ancestral differentiation preserved by low recombination is currently unclear. A critical test of these competing hypotheses could be provided by comparing genomic divergence when rates of recent introgression vary. We genotyped 871 single nucleotide polymorphisms (SNPs) in A. gambiae sensu stricto from locations of M and S sympatry and allopatry, encompassing the full range of observed hybridization rates (0-25%). M and S forms were readily partitioned based on genomewide SNP variation in spite of evidence for ongoing introgression that qualitatively reflects hybridization rates. Yet both the level and the heterogeneity of genomic divergence varied markedly in line with levels of introgression. A few genomic regions of differentiation between M and S were common to each sampling location, the most pronounced being two centromere-proximal speciation islands identified previously but with at least one additional region outside of areas expected to exhibit reduced recombination. Our results demonstrate that extreme divergence at genomic islands does not simply represent segregating ancestral polymorphism in regions of low recombination and can be resilient to substantial gene flow. This highlights the potential for islands comprising a relatively small fraction of the genome to play an important role in early-stage speciation when reproductive isolation is limited.

With only a few absolute geographic barriers in marine environments, the factors maintaining reproductive isolation among marine organisms remain elusive. However, spatial structuring in breeding habitat can contribute to reproductive isolation. This is particularly important for marine organisms that migrate to use fresh- or brackish water environments to breed. The Japanese Gasterosteus stickleback species, the Pacific Ocean three-spined stickleback (G.aculeatus) and the Japan Sea stickleback (G.nipponicus) overwinter in the sea, but migrate to rivers for spawning. Although they co-occur at several locations across the Japanese islands, they are reproductively isolated. Our previous studies in Bekanbeushi River showed that the Japan Sea stickleback spawns in the estuary, while the Pacific Ocean stickleback mainly spawns further upstream in freshwater. Overall genomic divergence was very high with many interspersed regions of introgression. Here, we investigated genomic divergence and introgression between the sympatric species in the much shorter Tokotan River, where they share spawning sites. The levels of genome-wide divergence were reduced and introgression was increased, suggesting that habitat isolation substantially contributes to a reduction in gene flow. We also found that genomic regions of introgression were largely shared between the two systems. Furthermore, some regions of introgression were located near loci with a heterozygote advantage for juvenile survival. Taken together, introgression may be partially driven by adaptation in this system. Although, the two species remain clearly genetically differentiated. Regions with low recombination rates showed especially low introgression. Speciation reversal is therefore likely prevented by barriers other than habitat isolation.

Ecological speciation, the origin of new species via divergent natural selection, is one of the most fundamental and unresolved processes in evolution. Although the evidence for adaptation of organisms to their environment is abundant, the role of ecological selection in mediating species formation remains controversial. This knowledge-gap arises in part from a scarcity of experimental evidence linking environmental selection to the creation of reproductive barriers that ultimately lead to species formation. An experimental framework to investigate ecological speciation consists in studying the genetic architecture of adaptive traits and reproductive isolation in interbreeding populations adapted to contrasting environments. In my thesis I explored the genomics of ecological speciation in plants using the Senecio lautus species complex, a diverse group of plants that have adapted to a broad array of environment across Australia. It has been suggested that local adaptation to different environments will lead to genetic divergence and speciation only if genomic regions controlling adaptive traits are not exchanged between organisms adapted to different niches. This will happen if adaptive genes also mediate reproductive isolation, thus making migration between environments difficult, or leading to poor survival of recombinants. This model of ecological speciation creates testable predictions on the genomic architecture of adaptation: Firstly, genomic divergence between incipient species is expected to be heterogeneous, where a few genomic regions display outlier differentiation. Secondly one expects divergent regions to contain genes affecting fitness in natural environments. Thirdly, these “genomic islands of speciation” will also contain loci controlling adaptive traits and reproduction. In my thesis I tested these predictions using divergent populations of plants from the S. lautus species complex. I used a combination of genomic and ecological approaches to: (i) Describe patterns of genomic differentiation between natural populations across Australia and made inferences about the forces that generated these patterns. Specifically, I tested the repeated and independent evolution of forms to coastal environments and analyzed whether genomic divergence was more heterogeneous between parapatric than allopatric populations. (ii) Demonstrate experimentally that divergent genomic regions contain genes controlling differential survivorship between environments. (iii) Detect QTLs associated to environmentally selected traits and associate their location to genomic regions of high differentiation between parapatric populations. In combination, these experiments were used to test the role of ecology in creating and maintaining the reproductive barriers that ultimately lead to plant speciation. A phylogenomic study of a continental collection of S. lautus populations showed that these plants have a monophyletic origin and evolved rapidly colonizing a broad array of environments. Importantly, populations adapted to adjacent but contrasting coastal environments, appeared as sister groups in phylogenetic analyses of thousands of loci, which suggests that these environments have been colonized repeatedly, and possibly in the presence of gene flow. To explore this hypothesis in further detail I analysed genetic differentiation between the genomes of populations. Our results revealed that genomic divergence was less heterogeneous between allopatric than parapatric populations, where a few genomic regions showed high differentiation while the rest of the genome was very similar. Additionally, genomic differentiation between some of these adjacent populations was related the magnitude of the differences between the environments that they inhabit, suggesting that divergence between them occurred in the face of gene flow. I investigated the evolutionary role of highly differentiated genomic regions through a combination of techniques that allowed us to connect genotype, phenotype and fitness. Firstly, I showed that these regions are enriched in coding mutations and associations to environmental variables, which suggest that they contain functionally important genes. However, patterns of divergence varied considerably across natural populations indicating that genomic divergence followed complex and divergent trajectories. Interestingly, functional analyses of divergent genes suggest that natural selection could have targeted different genes participating in the similar processes. By mapping loci involved in the control of fitness and convergent morphological traits across replicate populations I was able to demonstrate that divergent genomic regions contain adaptive and reproductive genes. Additionally I showed that genomic regions involved in adaptive trade-offs, have diverged repeatedly between environments, which supports their importance in mediating parapatric divergence. Overall my results provide genomic and functional evidence for a model of ecological speciation where natural selection creates divergence between the genomes of locally adapted organisms. These results also show that natural selection can have a complex genetic basis but create predictable patterns, especially at higher scales of biological organization.

Genomic divergence and introgression among three Populus species

Understanding the genetic basis of population divergence and adaptation is an important goal in population genetics and evolutionary biology. However, the relative roles of demographic history, gene flow, and/or selective regime in driving genomic divergence, climatic adaptation, and speciation in non-model tree species are not yet fully understood. To address this issue, we generated whole-genome resequencing data of Liquidambar formosana and L. acalycina, which are broadly sympatric but altitudinally segregated in the Tertiary relict forests of subtropical China. We integrated genomic and environmental data to investigate the demographic history, genomic divergence, and climatic adaptation of these two sister species. We inferred a scenario of allopatric species divergence during the late Miocene, followed by secondary contact during the Holocene. We identified multiple genomic islands of elevated divergence that mainly evolved through divergence hitchhiking and recombination rate variation, likely fostered by long-term refugial isolation and recent differential introgression in low-recombination genomic regions. We also found some candidate genes with divergent selection signatures potentially involved in climatic adaptation and reproductive isolation. Our results contribute to a better understanding of how late Tertiary/Quaternary climatic change influenced speciation, genomic divergence, climatic adaptation, and introgressive hybridization in East Asia's Tertiary relict flora. In addition, they should facilitate future evolutionary, conservation genomics, and molecular breeding studies in Liquidambar, a genus of important medicinal and ornamental values.

Diverse geographical modes and mechanisms of speciation are known, and individual speciation genes have now been identified. Despite this progress, genome-wide outcomes of different evolutionary processes during speciation are less understood. Here, we integrate ecological and spatial information, mating trials, transplantation data and analysis of 86 130 single nucleotide polymorphisms (SNPs) in eight populations (28 pairwise comparisons) of Timema cristinae stick insects to test the effects of different factors on genomic divergence in a system undergoing ecological speciation. We find patterns consistent with effects of numerous factors, including geographical distance, gene flow, divergence in host plant use and climate, and selection against maladaptive hybridization (i.e. reinforcement). For example, the number of highly differentiated ‘outlier loci’, allele-frequency clines and the overall distribution of genomic differentiation were recognizably affected by these factors. Although host use has strong effects on phenotypic divergence and reproductive isolation, its effects on genomic divergence were subtler and other factors had pronounced effects. The results demonstrate how genomic data can provide new insights into speciation and how genomic divergence can be complex, yet predictable. Future work could adopt experimental, mapping and functional approaches to directly test which genetic regions are affected by selection and determine their physical location in the genome.