Persistent inter‐ and intraspecific gene exchange within a parallel radiation of caterpillar hunter beetles (Calosoma sp.) from the Galápagos

When environmental gradients are repeated on different islands within an archipelago, similar selection pressures may act within each island, resulting in the repeated occurrence of ecologically similar species on each island. The evolution of ecotypes within such radiations may either result from dispersal, that is each ecotype evolved once and dispersed to different islands where it colonized its habitat, or through repeated and parallel speciation within each island. However, it remains poorly understood how gene flow during the divergence process may shape such patterns. In the Galápagos islands, three phenotypically similar species of the beetle genus Calosoma occur at higher elevations of different islands, while lowlands are occupied by a fourth species. By genotyping all major populations within this radiation for two nuclear and three mitochondrial gene fragments and seven microsatellite markers, we found strong support that the oldest divergence separates the highland species of the oldest island from the remaining species. Despite their morphological distinctness, highland species of the remaining islands were genetically closely related to the lowland population on each island and within the same magnitude as lowland populations sampled at different islands. Repeated evolution of highland ecotypes out of the lowland species appears the most likely scenario and estimates of geneflow rates revealed extensive admixture among ecotypes within islands, as well as between islands. These findings indicate that gene exchange among the different populations and species may have shaped the phylogenetic relationships and the repeated evolution of these ecotypes.     

Island floras as model systems for studies of plant speciation: Prospects and challenges

Oceanic islands have long been called natural laboratories for studying evolution because they are geologically young, isolated, dynamic areas with diverse habitats over small spatial scales. Volcanic substrates of different ages permit the study of different stages of divergence and speciation within plant lineages. In addition to divergence, the dynamic island setting is conducive to hybridization. Discussion will focus on the potential of systematic/ecological studies, in combination with genomic data from high throughput sequencing and an ever‐increasing array of analytical techniques, for studying evolution in island plants. These studies may include: generation of highly resolved phylogenies to clarify the biogeography of speciation and whether divergence has occurred with or without gene flow; identification of the barriers to gene flow (extrinsic vs. intrinsic) of importance during divergence; documentation of historical and current hybridization events within island lineages; and elucidation of the genomic composition and ecology of hybrid populations in order to infer the evolutionary consequences of hybridization, such as the origin of stabilized homoploid hybrid species.     

Narrow hybrid zones in spite of very low population differentiation in neutral markers in an island bird species complex

Patterns of phenotypic and genic frequencies across hybrid zones provide insight into the origin and evolution of reproductive isolation. The Reunion grey white‐eye, Zosterops borbonicus, exhibits parapatrically distributed plumage colour forms across the lowlands of the small volcanic island of Reunion (Mascarene archipelago). These forms meet and hybridize in regions that are natural barriers to dispersal (rivers, lava fields). Here, we investigated the relationship among patterns of differentiation at neutral genetic (microsatellite) markers, phenotypic traits (morphology and plumage colour) and niche characteristics across three independent hybrid zones. Patterns of phenotypic divergence revealed that these hybrid zones are among the narrowest ever documented in birds. However, the levels of phenotypic divergence stand in stark contrast to the lack of clear population neutral genetic structure between forms. The position of the hybrid zones coincides with different natural physical barriers, yet is not associated with steep changes in vegetation and related climatic variables, and major habitat transitions are shifted from these locations by at least 18 km. This suggests that the hybrid zones are stabilized over natural dispersal barriers, independently of environmental boundaries, and are not associated with niche divergence. A striking feature of these hybrid zones is the very low levels of genetic differentiation in neutral markers between forms, suggesting that phenotypic divergence has a narrow genetic basis and may reflect recent divergence at a few linked genes under strong selection, with a possible role for assortative mating in keeping these forms apart.     

Sculpin hybrid zones: natural laboratories for the early stages of speciation

Firmly rooted as we are in the genomic era, it can seem incredible that as recently as 1974, Lewontin declared, 'we know virtually nothing about the genetic changes that occur in species formation'. To the contrary, we now know the genetic architecture of phenotypic differences and reproductive isolation between species for many diverse groups of plants, animals, and fungi. In recent years, detailed genetic analyses have produced a small but growing list of genes that cause reproductive isolation, several of which appear to have diverged by natural selection. Yet, a full accounting of the speciation process requires that we understand the reproductive and ecological properties of natural populations as they begin to diverge genetically, as well as the dynamics of newly evolved barriers to gene flow. One promising approach to this problem is the study of natural hybrid zones, where gene exchange between divergent populations can produce recombinant genotypes in situ . In such individuals, genomic variation might be shaped by introgression at universally adaptive or neutral loci, even as regions associated with local adaptation or reproductive isolation remain divergent. In Nolte et   al . (2009) , the authors take advantage of two independent, recently formed hybrid zones between sculpin species to investigate genome-wide patterns of reproductive isolation. Using a recently developed genomic clines method, the authors identify marker loci that are associated with isolation, and those that show evidence for adaptive introgression. Remarkably, Nolte et   al . (2009) find little similarity between the two hybrid zones in patterns of introgression, a fact that might reflect genetic variation within species or heterogeneous natural selection. In either case, their study system has the potential to provide insight into the early stages of speciation.     

The accumulation of reproductive barriers during speciation: postmating barriers in two behaviorally isolated species of darters (Percidae: Etheostoma)

Identifying the manner in which reproductive barriers accumulate during lineage divergence is central to establishing general principles of species formation. One outstanding question is which isolating mechanisms form the first complete barrier to gene flow in a given lineage or under a particular set of conditions. To identify these initial reproductive barriers requires examining lineages in very early stages of divergence, before multiple reproductive barriers have evolved to completion. We quantified the strength of three postmating barriers in a pair of darter species and compared these estimates to each other and to the strength of behavioral isolation (BI) reported in a previous study. Results reveal no evidence of gametic incompatibility but intermediate levels of conspecific sperm precedence and hybrid inviability. As BI is nearly complete, our analysis comparing the strength of multiple reproductive barriers implicates the evolution of mate choice as central to both the origin and maintenance of these species. Further examination of ecological isolation and hybrid sterility is necessary to determine the role of these barriers in darter speciation.     

The geography of divergence with gene flow facilitates multitrait adaptation and the evolution of pollinator isolation in Mimulus aurantiacus

Ecological adaptation is the driving force during divergence with gene flow and generates reproductive isolation early in speciation. Although gene flow opposes divergence, local adaptation can be facilitated by factors that prevent the breakup of favorable allelic combinations. We investigated how selection, genetic architecture, and geography have contributed to the maintenance of floral trait divergence and pollinator isolation between parapatric ecotypes of Mimulus aurantiacus. Combining greenhouse, field, and genomic studies, we show that sharp clines in floral traits are maintained by spatially varying selection. Although adaptation breaks down where the ecotypes co‐occur, leading to the formation of a hybrid zone, the largely non‐overlapping distributions of the ecotypes shield them from immigrant genes, facilitating divergence across most of the range. In contrast to the sharp genetic discontinuities observed across most hybrid zones, we observed a gradual cline in genome‐wide divergence and a pattern of isolation by distance across the landscape. Thus, contrary to a long period of allopatry followed by recent re‐contact, our data suggest that floral trait divergence in M. aurantiacus may have evolved with locally restricted, but ongoing gene flow. Therefore, our study reveals how the geographic distribution of an organism can contribute to the evolution of premating isolation in the early stages of divergence with gene flow.     

Phylogenetic evidence for multiple sympatric ecological diversification in a marine snail

Parallel speciation can occur when traits determining reproductive isolation evolve independently in different populations that experience a similar range of environments. However, a common problem in studies of parallel evolution is to distinguish this hypothesis from an alternative one in which different ecotypes arose only once in allopatry and now share a sympatric scenario with substantial gene flow between them. Here we show that the combination of a phylogenetic approach with life-history data is able to disentangle both hypotheses in the case of the intertidal marine snail Littorina saxatilis on the rocky shores of Galicia in northwestern Spain. In this system, numerous phenotypic and genetic differences have evolved between two sympatric ecotypes spanning a sharp ecological gradient, and as aside effect of the former have produced partial reproductive isolation. A mitochondrial phylogeny of these populations strongly suggests that the two sympatric ecotypes have originated independently several times. Building upon earlier work demonstrating size-based assortative mating as the main contributor to reproductive isolation among ecotypes, our analysis provides strong evidence that divergent selection across a sharp ecological gradient promoted the parallel divergence of body size and shape between two sympatric ecotypes. Thus, divergent selection occurring independently in different populations has produced the marine equivalent of host races, which may represent the first step in speciation.     

Mating system as a barrier to gene flow

Understanding mating system as one of reproductive isolating barriers remains important although this barrier is classified in a different sense from behavioral, ecological, and mechanical isolating barriers. Selfing enhances incipient speciation while outcrossing facilitates species integrity. Here, I study how mating system affects gene exchanges between genetically diverging species in a hybrid zone. Results show that a predominant selfing species has a greater barrier to selective gene flow than does a predominant outcrossing species. Barrier to neutral gene flow convexly changes with the selfing rate due to linkage disequilibrium, with a maximum at around intermediate selfing rate. Asymmetric transient or steady‐state barriers to neutral gene flow occur between two sides of a hybrid zone when the neutral gene is affected by its linked selective gene whose alternative alleles are adaptive to heterogeneous habitats. Selfing interacts with both a physical barrier and a density‐dependent ecological regulation (a logarithmic model) to strengthen the barriers to neutral and selective gene flow. This theory helps to interpret incipient speciation driven by selfing or to explain the asymmetric gene flow or unequal genomic mixtures between closely related species caused by their asymmetric mating systems in natural hybrid zones.     

Generic scan using AFLP markers as a means to assess the role of directional selection in the divergence of sympatric whitefish ecotypes

Under the ecological theory of adaptive radiation, adaptation and reproductive isolation are thought to evolve as a result of divergent natural selection. Accordingly, elucidating the genetic basis of these processes is essential toward understanding the role of selection in shaping biological diversity. In this respect, the number of genes that evolved by selection remains contentious. To address this issue, the pattern of genetic differentiation obtained using 440 AFLP loci was compared with that expected under neutrality in four sympatric pairs of lake whitefish ecotypes that evolved adaptive phenotypic differences associated with the exploitation of distinct ecological niches. On average, 14 loci showed restricted gene flow relative to neutral expectation, suggesting a role of directional selection on their divergence. Among all loci that are most likely under directional selection, six exhibited parallel patterns of divergence, which provided further support for the role of selection in driving their divergence. Overall, these results indicate that only a small proportion of scored AFLP loci (between 1.4% and 3.2%) might be linked to genes implicated in the adaptive radiation of lake whitefish.     

Parallel habitat specialization within the wolf spider genus Hogna from the Galápagos

Within most island archipelagos, such as the Galápagos, similar ecological gradients are found on geographically isolated islands. Species radiations in response to these ecological gradients may follow different scenarios being (i) a single habitat specialization event followed by secondary colonization of each ecotype on the different islands or (ii) repeated and parallel habitat specialization on each island separately. This latter scenario has been considered less likely as gene flow might hamper such ecotypic differentiation. At least for the Galápagos, the extent to which this process is involved in species radiations remains yet poorly understood. Within the wolf spider genus Hogna, seven species are described that can be divided into three different ecotypes based on general morphology and habitat preference i.e. species that inhabit the pampa vegetation in the highlands, species that occur in coastal dry habitats and one generalist species. Comparison of the species phylogeny based on one mitochondrial (COI) and one nuclear (28S) gene fragment convincingly demonstrates that ‘pampa’ and ‘coastal dry’ species evolved in parallel on the islands Santa Cruz and San Cristóbal. Despite the observation that allozymes analysis indicated that each species forms a distinct genetic cluster, phylogenetic divergence within these species complexes was very low and paraphyletic and most likely due to hybridization rather than incomplete lineage sorting, as demonstrated for the Santa Cruz species complex. This suggests that within‐island speciation occurred under low levels of gene flow. Species phylogeny in general did not follow the progression of island emergence as a molecular clock analysis suggested that island endemic species may have diverged after as well as before the emergence of the islands. This represents the first clear example of parallel and within‐island speciation because of habitat specialization on the Galápagos and that such divergence most likely occurred under historic gene flow.     

Heterogeneous genomic differentiation between walking-stick ecotypes: "isolation by adaptation" and multiple roles for divergent selection

Genetic differentiation can be highly variable across the genome. For example, loci under divergent selection and those tightly linked to them may exhibit elevated differentiation compared to neutral regions. These represent "outlier loci" whose differentiation exceeds neutral expectations. Adaptive divergence can also increase genome-wide differentiation by promoting general barriers to neutral gene flow, thereby facilitating genomic divergence via genetic drift. This latter process can yield a positive correlation between adaptive phenotypic divergence and neutral genetic differentiation (described here as "isolation-by-adaptation"). Here, we examine both these processes by combining an AFLP genome scan of two host plant ecotypes of Timema cristinae walking-sticks with existing data on adaptive phenotypic divergence and ecological speciation in these insects. We found that about 8% of loci are outliers in multiple population comparisons. Replicated comparisons between population-pairs using the same versus different host species revealed that 1-2% of loci are subject to host-related selection specifically. Locus-specific analyses revealed that up to 10% of putatively neutral (nonoutlier) AFLP loci exhibit significant isolation-by-adaptation. Our results suggest that selection may affect differentiation directly, via linkage, or by facilitating genetic drift. They thus illustrate the varied and sometimes nonintuitive contributions of selection to heterogeneous genomic differentiation.     

Immigrant inviability produces a strong barrier to gene flow between parapatric ecotypes of Senecio lautus

Speciation proceeds when gene exchange is prevented between populations. Determining the different barriers preventing gene flow can therefore give insights into the factors driving and maintaining species boundaries. These reproductive barriers may result from intrinsic genetic incompatibilities between populations, from extrinsic environmental differences between populations, or a combination of both mechanisms. We investigated the potential barriers to gene exchange between three adjacent ecotypes of an Australian wildflower to determine the strength of individual barriers and the degree of overall isolation between populations. We found almost complete isolation between the three populations mainly due to premating extrinsic barriers. Intrinsic genetic barriers were weak and variable among populations. There were asymmetries in some intrinsic barriers due to the origin of cytoplasm in hybrids. Overall, these results suggest that reproductive isolation between these three populations is almost complete despite the absence of geographic barriers, and that the main drivers of this isolation are ecologically based, consistent with the mechanisms underlying ecological speciation.     

The contribution of post-copulatory mechanisms to incipient ecological speciation in sticklebacks

Ecology can play a major role in species diversification. As individuals are adapting to contrasting habitats, reproductive barriers may evolve at multiple levels. While pre-mating barriers have been extensively studied, the evolution of post-mating reproductive isolation during early stages of ecological speciation remains poorly understood. In diverging three-spined stickleback ecotypes from two lakes and two rivers, we observed differences in sperm traits between lake and river males. Interestingly, these differences did not translate into ecotype-specific gamete precedence for sympatric males in competitive in vitro fertilization experiments, potentially owing to antagonistic compensatory effects. However, we observed indirect evidence for impeded development of inter-ecotype zygotes, possibly suggesting an early stage of genetic incompatibility between ecotypes. Our results show that pre-zygotic post-copulatory mechanisms play a minor role during this first stage of ecotype divergence, but suggest that genetic incompatibilities may arise at early stages of ecological speciation.     

GENETIC EVIDENCE FOR REPRODUCTIVE ISOLATION AND MULTIPLE ORIGINS OF SYMPATRIC TROPHIC ECOTYPES OF WHITEFISH (COREGONUS)

We assessed variation in mitochondrial DNA (mtDNA) by restriction fragment length polymorphism (RFLP) analysis and in nuclear genes by allozyme analysis among sympatric pairs of limnetic and benthic ecotypes of whitefish (Coregonus) coexisting in three lakes of southern Yukon to address three evolutionary questions regarding their origins. Are sympatric low and high gill-raker count ecotypes genetically differentiated? Are they issued from monophyletic or polyphyletic evolutionary events? If they are polyphyletic in origins, did they originate from multiple allopatric speciation events or intralacustrine radiation? Our results corroborated previous genetic and ecological studies of these ecotypes, indicating that they represent genetically distinct reproductive units, and therefore refuting the hypothesis of phenotypic polymorphism within a single population. However, the amount of gene flow between ecotypes varied among lakes, correlating with the extent of morphological differentiation and the potential for premating reproductive isolation. The results indicated a polyphyletic origin of ecotypes whereby each of them have been expressed independently more than once. In the two lakes of Squanga Creek drainage, the existence of sympatric pairs was best explained by the secondary contact of two monophyletic whitefish groups that evolved in allopatry during the last glaciation events. In Dezadeash L. of Alsek R. drainage, our results could not verify either sympatric or allopatric (or microallopatric) origin of ecotypes. Regardless of the mode of speciation involved in their origins, these sympatric whitefish populations provided further evidence that Pleistocene glaciation events created conditions favoring rapid divergence and phenotypic differentiation among northern freshwater fishes.     

The role of different reproductive barriers during phenotypic divergence of isopod ecotypes

The question of how diverging populations become separate species by restraining gene flow is a central issue in evolutionary biology. Assortative mating might emerge early during adaptive divergence, but the role of other types of reproductive barriers such as migration modification have recently received increased attention. We demonstrate that two recently diverged ecotypes of a freshwater isopod (Asellus aquaticus) have rapidly developed premating isolation, and this isolation barrier has emerged independently and in parallel in two south Swedish lakes. This is consistent with ecological speciation theory, which predicts that reproductive isolation arises as a byproduct of ecological divergence. We also find that in one of these lakes, habitat choice acts as the main barrier to gene flow. These observations and experimental results suggest that migration modification might be as important as assortative mating in the early stages of ecological speciation. Simulations suggest that the joint action of these two isolating barriers is likely to greatly facilitate adaptive divergence, compared to if each barrier was acting alone.     

Genetic association of physically unlinked islands of genomic divergence in incipient species of Anopheles gambiae

Previous efforts to uncover the genetic underpinnings of ongoing ecological speciation of the M and S forms of the African malaria vector Anopheles gambiae revealed two centromere‐proximal islands of genetic divergence on X and chromosome 2. Under the assumption of considerable ongoing gene flow between M and S, these persistently divergent genomic islands were widely considered to be ‘speciation islands’. In the course of microarray‐based divergence mapping, we discovered a third centromere‐associated island of divergence on chromosome 3, which was validated by targeted re‐sequencing. To test for genetic association between the divergence islands on all three chromosomes, SNP‐based assays were applied in four natural populations of M and S spanning West, Central and East Africa. Genotyping of 517 female M and S mosquitoes revealed nearly complete linkage disequilibrium between the centromeres of the three independently assorting chromosomes. These results suggest that despite the potential for inter‐form gene flow through hybridization, actual (realized) gene flow between M and S may be substantially less than commonly assumed and may not explain most shared variation. Moreover, the possibility of very low gene flow calls into question whether diverged pericentromeric regions—characterized by reduced levels of variation and recombination—are in fact instrumental rather than merely incidental to the speciation process.     

Flickers of speciation: Sympatric colour morphs of the arc‐eye hawkfish,Paracirrhites arcatus,reveal key elements of divergence with gene flow

One of the primary challenges of evolutionary research is to identify ecological factors that favour reproductive isolation. Therefore, studying partially isolated taxa has the potential to provide novel insight into the mechanisms of evolutionary divergence. Our study utilizes an adaptive colour polymorphism in the arc‐eye hawkfish (Paracirrhites arcatus) to explore the evolution of reproductive barriers in the absence of geographic isolation. Dark and light morphs are ecologically partitioned into basaltic and coral microhabitats a few metres apart. To test whether ecological barriers have reduced gene flow among dark and light phenotypes, we evaluated genetic variation at 30 microsatellite loci and a nuclear exon (Mc1r) associated with melanistic coloration. We report low, but significant microsatellite differentiation among colour morphs and stronger divergence in the coding region of Mc1r indicating signatures of selection. Critically, we observed greater genetic divergence between colour morphs on the same reefs than that between the same morphs in different geographic locations. We hypothesize that adaptation to the contrasting microhabitats is overriding gene flow and is responsible for the partial reproductive isolation observed between sympatric colour morphs. Combined with complementary studies of hawkfish ecology and behaviour, these genetic results indicate an ecological barrier to gene flow initiated by habitat selection and enhanced by assortative mating. Hence, the arc‐eye hawkfish fulfil theoretical expectations for the earliest phase of speciation with gene flow.     

Ancient islands and modern invasions: disparate phylogeographic histories among Hispaniola's endemic birds

With its large size, complex topography and high number of avian endemics, Hispaniola appears to be a likely candidate for the in situ speciation of its avifauna, despite the worldwide rarity of avian speciation within single islands. We used multilocus comparative phylogeography techniques to examine the pattern and history of divergence in 11 endemic birds representing potential within-island speciation events. Haplotype and allele networks from mitochondrial ND2 and nuclear intron loci reveal a consistent pattern: phylogeographic divergence within or between closely related species is correlated with the likely distribution of ancient sea barriers that once divided Hispaniola into several smaller paleo-islands. Coalescent and mitochondrial clock dating of divergences indicate species-specific response to different geological events over the wide span of the island's history. We found no evidence that ecological or topographical complexity generated diversity, either by creating open niches or by restricting long-term gene flow. Thus, no true within-island speciation appears to have occurred among the species sampled on Hispaniola. Divergence events predating the merging of Hispaniola's paleo-island blocks cannot be considered in situ divergence, and postmerging divergence in response to episodic island segmentation by marine flooding probably represents in situ vicariance or interarchipelago speciation by dispersal. Our work highlights the necessity of considering island geologic history while investigating the speciation-area relationship in birds and other taxa.     

Selection on hybrids of ecologically divergent ecotypes of a marine snail: the relative importance of exogenous and endogenous barriers

Unravelling the form of selection acting on hybrids of ecotypes undergoing ecological speciation is essential to understand the mechanisms behind the evolution of reproductive isolation in the face of gene flow. Shell phenotype is known to be affected by natural selection and is involved in the fitness of the marine snail Littorina saxatilis. Here, we studied the association between shell traits and fitness in hybrids in order to determine the relative role of exogenous and endogenous selection in this hybrid zone of L. saxatilis. We show that directional selection is the predominant mode of selection among hybrids. We also show its heterogeneity, affecting different shell traits, within populations at the level of the microhabitat. Therefore, endogenous selection mechanisms are most probably lacking in this hybrid zone and exogenous barriers (pre‐ and post‐zygotic) are possibly one of the main forces behind the evolution of barriers to gene flow between these ecologically divergent ecotypes. This study shows how this barrier might represent an important type of reproductive isolation within ecological speciation, and this should be taken into account in future studies of speciation in hybrid zones. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 391–400.