Perspective: Reproductive isolation caused by natural selection against immigrants from divergent habitats

The classification of reproductive isolating barriers laid out by Dobzhansky and Mayr has motivated and structured decades of research on speciation. We argue, however, that this classification is incomplete and that the unique contributions of a major source of reproductive isolation have often been overlooked. Here, we describe reproductive barriers that derive from the reduced survival of immigrants upon reaching foreign habitats that are ecologically divergent from their native habitat. This selection against immigrants reduces encounters and thus mating opportunities between individuals from divergently adapted populations. It also reduces the likelihood that successfully mated immigrant females will survive long enough to produce their hybrid offspring. Thus, natural selection against immigrants results in distinctive elements of premating and postmating reproductive isolation that we hereby dub "immigrant inviability." We quantify the contributions of immigrant inviability to total reproductive isolation by examining study systems where multiple components of reproductive isolation have been measured and demonstrate that these contributions are frequently greater than those of traditionally recognized reproductive barriers. The relevance of immigrant inviability is further illustrated by a consideration of population-genetic theory, a review of selection against immigrant alleles in hybrid zone studies, and an examination of its participation in feedback loops that influence the evolution of additional reproductive barriers. Because some degree of immigrant inviability will commonly exist between populations that exhibit adaptive ecological divergence, we emphasize that these barriers play critical roles in ecological modes of speciation. We hope that the formal recognition of immigrant inviability and our demonstration of its evolutionary importance will stimulate more explicit empirical studies of its contributions to speciation.     

Natural and sexual selection against immigrants maintains differentiation among micro‐allopatric populations

Local adaptation to divergent environmental conditions can promote population genetic differentiation even in the absence of geographic barriers and hence lead to speciation. But what mechanisms contribute to reproductive isolation among diverging populations? We tested for natural and sexual selection against immigrants in a fish species inhabiting (and adapting to) nonsulphidic surface habitats, sulphidic surface habitats and a sulphidic cave. Gene flow is strong among sample sites situated within the same habitat type, but low among divergent habitat types. Our results indicate that females of both sulphidic populations discriminate against immigrant males during mate choice. Furthermore, using reciprocal translocation experiments, we document natural selection against migrants between nonsulphidic and sulphidic habitats, whereas migrants between sulphidic cave and surface habitats did not exhibit increased mortality within the same time period. Consequently, both natural and sexual selection may contribute to isolation among parapatric populations, and selection against immigrants may be a powerful mechanism facilitating speciation among locally adapted populations even over very small spatial distances.     

Ecological differentiation and reproductive isolation of two closely related sympatric species of Oenanthe (Apiaceae)

Oenanthe conioides is a lower Elbe endemic plant species growing in the freshwater tidal zone around Hamburg (Germany). Its closest relative Oenanthe aquatica is widely distributed in Eurasia and grows in calm and shallow freshwater. The two species differ in habitat requirements but are otherwise sympatrically distributed, suggesting that ecological divergence has to be maintained in the face of gene flow. In the present study, we investigated ecological differentiation and reproductive isolation in these two species. An amplified fragment length polymorphism analysis found clear genetic differentiation between the two species implying reproductive isolation. A reciprocal transplantation experiment including artificial F₁ hybrids showed strong selection against immigrants. In the two parental habitats, the non‐native species are less fit than the native species. Hybrids are less fit in the habitat of Oe. aquatica but perform as well as the native species in the habitat of Oe. conioides. We hypothesize that selection against immigrants is the most important component of reproductive isolation between the two species, and that selection against immigrants is the result of exposure to cold and wind in the tidal habitat of Oe. conioides and to herbivory in the habitat of Oe. aquatica. These results are congruent with a role for differential ecological selection in the formation and maintenance of these two species. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 526–535.     

FREQUENCY-DEPENDENT SEED PRODUCTION AND HYBRIDIZATION RATES: IMPLICATIONS FOR GENE FLOW BETWEEN LOCALLY ADAPTED PLANT POPULATIONS

Gene flow between genetically distinct plant populations can have significant evolutionary consequences. It can increase genetic diversity, create novel gene combinations, and transfer adaptations from one population to another. This study addresses the roles of frequency-dependent selection and mating system in gene exchange between two subspecies of Gilia capitata (Polemoniaceae). Long-distance migrants are likely to be rare in new habitats, and the importance of immigrant frequency to fitness, gene exchange, and ultimately introgression, has not been explored. To test for the importance of frequency in migration, a field experiment was conducted in which artificial populations (arrays) composed of different mixtures of the two subspecies were placed in the home habitats of both. Female function (seed production) and a portion of male function (hybridization rate) were compared for the two subspecies to assess the potential for gene exchange and introgression between them. Individual fitness (through both hybridization and seed production) for the inland subspecies varied with its frequency as an immigrant at the coastal site. Rare immigrants produced fewer seeds and fathered fewer hybrid offspring. In contrast, both forms of reproductive function were frequency independent for the coastal subspecies when it was an immigrant at the inland site. Seed production was high and insensitive to frequency, and immigrants from the coast never successfully fertilized the inland subspecies' seeds. To control for the effects of frequency-dependent pollinator behavior in the field, hand crosses were performed in the greenhouse using a range of pollen mixtures. The greenhouse experiment demonstrated that cross-fertilization is possible in only one direction, that cross-pollination in the other direction is only partially successful, and that pollen from the coastal subspecies has a strong negative effect on seed production by the inland subspecies. Experimental pollen supplementation in the field verified both the unilateral incompatibility and the negative effect of coastal pollen on inland plant seed production observed in the greenhouse. Contrasts between field array and greenhouse results suggest that pollinator behavior and other ecological factors act to exaggerate reproductive barriers between the two subspecies. In this system, immigrant frequency interacts with reproductive biology and pollinator ecology to enhance gene flow between the populations in one direction, while restricting gene establishment and introgression in the other direction.     

Divergent sexual selection via male competition: ecology is key

Sexual selection and ecological differences are important drivers of speciation. Much research has focused on female choice, yet the role of male competition in ecological speciation has been understudied. Here, we test how mating habitats impact sexual selection and speciation through male competition. Using limnetic and benthic species of threespine stickleback fish, we find that different mating habitats select differently on male traits through male competition. In mixed habitat with both vegetated and open areas, selection favours two trait combinations of male body size and nuptial colour: large with little colour and small with lots of colour. This matches what we see in reproductively isolated stickleback species, suggesting male competition could promote trait divergence and reproductive isolation. In contrast, when only open habitat exists, selection favours one trait combination, large with lots of colour, which would hinder trait divergence and reproductive isolation. Other behavioural mechanisms in male competition that might promote divergence, such as avoiding aggression with heterospecifics, are insufficient to maintain separate species. This work highlights the importance of mating habitats in male competition for both sexual selection and speciation.     

WEAK CROSSABILITY BARRIER BUT STRONG JUVENILE SELECTION SUPPORTS ECOLOGICAL SPECIATION OF THE HYBRID PINE PINUS DENSATA ON THE TIBETAN PLATEAU

Determining how a new hybrid lineage can achieve reproductive isolation is a key to understanding the process and mechanisms of homoploid hybrid speciation. Here, we evaluated the degree and nature of reproductive isolation between the ecologically successful hybrid species Pinus densata and its parental species P. tabuliformis and P. yunnanensis. We performed interspecific crosses among the three species to assess their crossability. We then conducted reciprocal transplantation experiments to evaluate their fitness differentiation, and to examine how natural populations representing different directions of introgression differ in adaptation. The crossing experiments revealed weak genetic barriers among the species. The transplantation trials showed manifest evidence of local adaptation as the three species all performed best in their native habitats. Pinus densata populations from the western edge of its distribution have evolved a strong local adaptation to the specific habitat in that range; populations representing different directions of introgressants with the two parental species all showed fitness disadvantages in this P. densata habitat. These observations illustrate that premating isolation through selection against immigrants from other habitat types or postzygotic isolation through selection against backcrosses between the three species is strong. Thus, ecological selection in combination with endogenous components and geographic isolation has likely played a significant role in the speciation of P. densata.     

GENETIC DIFFERENTIATION AND SELECTION AGAINST MIGRANTS IN EVOLUTIONARILY REPLICATED EXTREME ENVIRONMENTS

We investigated mechanisms of reproductive isolation in livebearing fishes (genus Poecilia) inhabiting sulfidic and nonsulfidic habitats in three replicate river drainages. Although sulfide spring fish convergently evolved divergent phenotypes, it was unclear if mechanisms of reproductive isolation also evolved convergently. Using microsatellites, we found strongly reduced gene flow between adjacent populations from different habitat types, suggesting that local adaptation to sulfidic habitats repeatedly caused the emergence of reproductive isolation. Reciprocal translocation experiments indicate strong selection against immigrants into sulfidic waters, but also variation among drainages in the strength of selection against immigrants into nonsulfidic waters. Mate choice experiments revealed the evolution of assortative mating preferences in females from nonsulfidic but not from sulfidic habitats. The inferred strength of sexual selection against immigrants (RI_s) was negatively correlated with the strength of natural selection (RI_m), a pattern that could be attributed to reinforcement, whereby natural selection strengthens behavioral isolation due to reduced hybrid fitness. Overall, reproductive isolation and genetic differentiation appear to be replicated and direct consequences of local adaptation to sulfide spring environments, but the relative contributions of different mechanisms of reproductive isolation vary across these evolutionarily independent replicates, highlighting both convergent and nonconvergent evolutionary trajectories of populations in each drainage.     

Five questions on ecological speciation addressed with individual‐based simulations

We use an individual‐based simulation model to investigate factors influencing progress toward ecological speciation. We find that environmental differences can quickly lead to the evolution of substantial reproductive barriers between a population colonizing a new environment and the ancestral population in the old environment. Natural selection against immigrants and hybrids was a major contributor to this isolation, but the evolution of sexual preference was also important. Increasing dispersal had both positive and negative effects on population size in the new environment and had positive effects on natural selection against immigrants and hybrids. Genetic divergence at unlinked, neutral genetic markers was low, except when environmental differences were large and sexual preference was present. Our results highlight the importance of divergent selection and adaptive divergence for ecological speciation. At the same time, they reveal several interesting nonlinearities in interactions between environmental differences, sexual preference, dispersal and population size.     

Demography of source—sink populations and the evolution of ecological niches

Summary The demography of populations living in variable environments is an important factor molding the evolution of ecological niches, for it determines the relative strength of selection pressures on adaptations to different habitats. Here I consider a coarse-grained environment consisting of two habitat types and investigate how the selection pressure on reproductive success in different habitats depends on their quality and frequency and the dispersal pattern. The results suggest that selection on adaptations to optimal habitats will usually be stronger than on adaptations to poor habitats and the ecological niche will thus tend to be an evolutionarily conservative character. It is because under the habitat choice or limited dispersal that seem to prevail in natural populations, more individuals encounter the better habitat than would be expected solely on the basis of its relative area. This bias results in reduced selection pressure on reproductive success in the poorer habitat. With habitat choice or limited dispersal, selection pressure on reproductive success in the poorer habitat may exceed that on reproductive success in the better habitat only if the poorer habitat is much more frequent in the environment than the better habitat and the difference in their quality is not large.     

Counteracting selective regimes and host preference evolution in ecotypes of two species of walking-sticks

The evolution of ecological specialization has been a central topic in ecology because specialized adaptations to divergent environments can result in reproductive isolation and facilitate speciation. However, the order in which different aspects of habitat adaptation and habitat preference evolve is unclear. Timema walking-stick insects feed and mate on the host plants on which they rest. Previous studies of T. cristinae ecotypes have documented divergent, host-specific selection from visual predators and the evolution of divergent host and mate preferences between populations using different host-plant species (Ceanothus or Adenostoma). Here we present new data that show that T. podura, a nonsister species of T. cristinae, has also formed ecotypes on these host genera and that in both species these ecotypes exhibit adaptive divergence in color-pattern and host preference. Color-pattern morphs exhibit survival trade-offs on different hosts due to differential predation. In contrast, fecundity trade-offs on different hosts do not occur in either species. Thus, host preference in both species has evolved before divergent physiological adaptation but in concert with morphological adaptations. Our results shed light onto which traits are involved in the initial stages of ecological specialization and ecologically based reproductive isolation.     

Ecological selection maintains cytonuclear incompatibilities in hybridizing sunflowers

Despite the recent renaissance in studies of ecological speciation, the connection between ecological selection and the evolution of reproductive isolation remains tenuous. We tested whether habitat adaptation of cytoplasmic genomes contributes to the maintenance of reproductive barriers in hybridizing sunflower species, Helianthus annuus and Helianthus petiolaris. We transplanted genotypes of the parental species, reciprocal F1 hybrids and all eight possible backcross combinations of nuclear and cytoplasmic genomes into the contrasting xeric and mesic habitats of the parental species. Analysis of survivorship across two growing seasons revealed that the parental species' cytoplasms were strongly locally adapted and that cytonuclear interactions (CNIs) significantly affected the fitness and architecture of hybrid plants. A significant fraction of the CNIs have transgenerational effects, perhaps due to divergence in imprinting patterns. Our results suggest a common means by which ecological selection may contribute to speciation and have significant implications for the persistence of hybridizing species.     

Ecological divergence and habitat isolation between two migratory forms of Japanese threespine stickleback (Gasterosteus aculeatus)

When two closely related species migrate to divergent spawning sites, divergent use of spawning habitats can directly reduce heterospecific mating. Furthermore, adaptations to divergent spawning habitats can promote speciation as a by‐product of ecological divergence. Here, we investigated habitat isolation and ecological divergence between two anadromous forms of threespine stickleback (Gasterosteus aculeatus), the Japan Sea and Pacific Ocean forms. In several coastal regions of eastern Hokkaido, Japan, these forms migrate to the same watershed to spawn. Our field surveys in a single watershed revealed that segregation of distinct spawning sites between the two forms was maintained within the watershed across multiple years. These spawning sites diverged in salinity and predator composition. Morphological and physiological divergence between the forms also occurs in the direction predicted by ecological differences between the spawning sites. Our data indicate that migration into divergent spawning habitats can be an important mechanism contributing to speciation and phenotypic divergence in anadromous fishes.     

Ecological divergence promotes the evolution of cryptic reproductive isolation

Speciation can involve the evolution of 'cryptic' reproductive isolation that occurs after copulation but before hybrid offspring are produced. Because such cryptic barriers to gene exchange involve post-mating sexual interactions, analyses of their evolution have focused on sexual conflict or traditional sexual selection. Here, we show that ecological divergence between populations of herbivorous walking sticks is integral to the evolution of cryptic reproductive isolation. Low female fitness following between-population mating can reduce gene exchange between populations, thus acting as a form of cryptic isolation. Female walking sticks show reduced oviposition rate and lower lifetime fecundity following between-population versus within-population mating, but only for mating between populations using different host-plant species. Our results indicate that even inherently sexual forms of reproductive isolation can evolve as a by-product of ecological divergence and that post-mating sexual interactions do not necessarily evolve independently of the ecological environment.     

Adaptation with gene flow across the landscape in a dune sunflower

Isolation by adaptation increases divergence at neutral loci when natural selection against immigrants reduces the rate of gene flow between different habitats. This can occur early in the process of adaptive divergence and is a key feature of ecological speciation. Despite the ability of isolation by distance (IBD) and other forms of landscape resistance to produce similar patterns of neutral divergence within species, few studies have used landscape genetics to control for these other forces. We have studied the divergence of Helianthus petiolaris ecotypes living in active sand dunes and adjacent non-dune habitat, using landscape genetics approaches, such as circuit theory and multiple regression of distance matrices, in addition to coalescent modelling. Divergence between habitats was significant, but not strong, and was shaped by IBD. We expected that increased resistance owing to patchy and unfavourable habitat in the dunes would contribute to divergence. Instead, we found that landscape resistance models with lower resistance in the dunes performed well as predictors of genetic distances among subpopulations. Nevertheless, habitat class remained a strong predictor of genetic distance when controlling for isolation by resistance and IBD. We also measured environmental variables at each site and confirmed that specific variables, especially soil nitrogen and vegetation cover, explained a greater proportion of variance in genetic distance than did landscape or the habitat classification alone. Asymmetry in effective population sizes and numbers of migrants per generation was detected using coalescent modelling with Bayesian inference, which is consistent with incipient ecological speciation being driven by the dune habitat.     

Asymmetric isolating barriers between different microclimatic environments caused by low immigrant survival

Spatially variable selection has the potential to result in local adaptation unless counteracted by gene flow. Therefore, barriers to gene flow will help facilitate divergence between populations that differ in local selection pressures. We performed spatially and temporally replicated reciprocal field transplant experiments between inland and coastal habitats using males of the common blue damselfly (Enallagma cyathigerum) as our study organism. Males from coastal populations had lower local survival rates than resident males at inland sites, whereas we detected no differences between immigrant and resident males at coastal sites, suggesting asymmetric local adaptation in a source–sink system. There were no intrinsic differences in longevity between males from the different environments suggesting that the observed differences in male survival are environment-dependent and probably caused by local adaptation. Furthermore, the coastal environment was found to be warmer and drier than the inland environment, further suggesting local adaptation to microclimatic factors has lead to differential survival of resident and immigrant males. Our results suggest that low survival of immigrant males mediates isolation between closely located populations inhabiting different microclimatic environments.     

Divergence in coloration and ecological speciation in the Anolis marmoratus species complex

Adaptive divergence in coloration is expected to produce reproductive isolation in species that use colourful signals in mate choice and species recognition. Indeed, many adaptive radiations are characterized by differentiation in colourful signals, suggesting that divergent selection acting on coloration may be an important component of speciation. Populations in the Anolis marmoratus species complex from the Caribbean island of Guadeloupe display striking divergence in the colour and pattern of adult males that occurs over small geographic distances, suggesting strong divergent selection. Here we test the hypothesis that divergence in coloration results in reduced gene flow among populations. We quantify variation in adult male coloration across a habitat gradient between mesic and xeric habitats, use a multilocus coalescent approach to infer historical demographic parameters of divergence, and examine gene flow and population structure using microsatellite variation. We find that colour variation evolved without geographic isolation and in the face of gene flow, consistent with strong divergent selection and that both ecological and sexual selection are implicated. However, we find no significant differentiation at microsatellite loci across populations, suggesting little reproductive isolation and high levels of contemporary gene exchange. Strong divergent selection on loci affecting coloration probably maintains clinal phenotypic variation despite high gene flow at neutral loci, supporting the notion of a porous genome in which adaptive portions of the genome remain fixed whereas neutral portions are homogenized by gene flow and recombination. We discuss the impact of these findings for studies of colour evolution and ecological speciation.     

VARIABLE PROGRESS TOWARD ECOLOGICAL SPECIATION IN PARAPATRY: STICKLEBACK ACROSS EIGHT LAKE-STREAM TRANSITIONS

Divergent selection between contrasting habitats can sometimes drive adaptive divergence and the evolution of reproductive isolation in the face of initially high gene flow. "Progress" along this ecological speciation pathway can range from minimal divergence to full speciation. We examine this variation for threespine stickleback fish that evolved independently across eight lake-stream habitat transitions. By quantifying stickleback diets, we show that lake-stream transitions usually coincide with limnetic-benthic ecotones. By measuring genetically based phenotypes, we show that these ecotones often generate adaptive divergence in foraging morphology. By analyzing neutral genetic markers (microsatellites), we show that adaptive divergence is often associated with the presence of two populations maintaining at least partial reproductive isolation in parapatry. Coalescent-based simulations further suggest that these populations have diverged with gene flow within a few thousand generations, although we cannot rule out the possibility of phases of allopatric divergence. Finally, we find striking variation among the eight lake-stream transitions in progress toward ecological speciation. This variation allows us to hypothesize that progress is generally promoted by strong divergent selection and limited dispersal across the habitat transitions. Our study thus makes a case for ecological speciation in a parapatric context, while also highlighting variation in the outcome.     

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.     

Adaptation and the origin of species

As reflected in the title of his masterwork On the Origin of Species, Darwin proposed that adaptation is the primary mechanism of speciation. On this, Darwin was criticized for his neglect of reproductive isolation, his lack of appreciation for the role of geographic barriers, his failure to distinguish varieties from species, and his typological species concept. Two developments since Darwin, the biological species concept of Ernst Mayr and the methods of Coyne and Orr for estimating the contribution of different barriers to the total reproductive isolation, provide a framework for reconciling Darwin's view on the primacy of adaptation in speciation with later proposals that emphasize reproductive isolation. A review of the few studies that have estimated the contributions of multiple isolating barriers suggests that habitat isolation and other barriers that operate before hybrid formation are much stronger than intrinsic postzygotic isolation. In light of these data, I suggest that Darwin's focus on adaptation in the origin of species was essentially correct, a conclusion that calls for future studies that explore the links between adaptation and speciation, in particular, ecogeographic isolating barriers that result from adaptive divergence in habitat use. The recent revival in thinking about ecological factors and adaptive divergence in the origin of species echoes Darwin's much-criticized "principle of divergence" and suggests that the emerging views from today's naturalists are not so different from those espoused by Darwin some 150 years ago.     

Thermal adaptation and ecological speciation

Ecological speciation is defined as the emergence of reproductive isolation as a direct or indirect consequence of divergent ecological adaptation. Several empirical examples of ecological speciation have been reported in the literature which very often involve adaptation to biotic resources. In this review, we investigate whether adaptation to different thermal habitats could also promote speciation and try to assess the importance of such processes in nature. Our survey of the literature identified 16 animal and plant systems where divergent thermal adaptation may underlie (partial) reproductive isolation between populations or may allow the stable coexistence of sibling taxa. In many of the systems, the differentially adapted populations have a parapatric distribution along an environmental gradient. Isolation often involves extrinsic selection against locally maladapted parental or hybrid genotypes, and additional pre- or postzygotic barriers may be important. Together, the identified examples strongly suggest that divergent selection between thermal environments is often strong enough to maintain a bimodal genotype distribution upon secondary contact. What is less clear from the available data is whether it can also be strong enough to allow ecological speciation in the face of gene flow through reinforcement-like processes. It is possible that intrinsic features of thermal gradients or the genetic basis of thermal adaptation make such reinforcement-like processes unlikely but it is equally possible that pertinent systems are understudied. Overall, our literature survey highlights (once again) the dearth of studies that investigate similar incipient species along the continuum from initial divergence to full reproductive isolation and studies that investigate all possible reproductive barriers in a given system.