The role of sexual isolation during rapid ecological divergence: Evidence for a new dimension of isolation in Rhagoletis pomonella

The pace of divergence and likelihood of speciation often depends on how and when different types of reproductive barriers evolve. Questions remain about how reproductive isolation evolves after initial divergence. We tested for the presence of sexual isolation (reduced mating between populations due to divergent mating preferences and traits) in Rhagoletis pomonella flies, a model system for incipient ecological speciation. We measured the strength of sexual isolation between two very recently diverged (~170 generations) sympatric populations, adapted to different host fruits (hawthorn and apple). We found that flies from both populations were more likely to mate within than between populations. Thus, sexual isolation may play an important role in reducing gene flow allowed by early-acting ecological barriers. We also tested how warmer temperatures predicted under climate change could alter sexual isolation and found that sexual isolation was markedly asymmetric under warmer temperatures – apple males and hawthorn females mated randomly while apple females and hawthorn males mated more within populations than between. Our findings provide a window into the early speciation process and the role of sexual isolation after initial ecological divergence, in addition to examining how environmental conditions could shape the likelihood of further divergence.     

Multiple reproductive barriers separate recently diverged sunflower ecotypes

Measuring reproductive barriers between groups of organisms is an effective way to determine the traits and mechanisms that impede gene flow. However, to understand the ecological and evolutionary factors that drive speciation, it is important to distinguish between the barriers that arise early in the speciation process and those that arise after speciation is largely complete. In this article, we comprehensively test for reproductive isolation between recently diverged (<10,000 years bp) dune and nondune ecotypes of the prairie sunflower, Helianthus petiolaris. We find reproductive barriers acting at multiple stages of hybridization, including premating, postmating–prezygotic, and postzygotic barriers, despite the recent divergence. Barriers include extrinsic selection against immigrants and hybrids, a shift in pollinator assemblage, and postpollination assortative mating. Together, these data suggest that multiple barriers can be important for reducing gene flow in the earliest stages of speciation.     

Evolution of reproductive isolation in stickleback fish

To understand how new species form and what causes their collapse, we examined how reproductive isolation evolves during the speciation process, considering species pairs with little to extensive divergence, including a recently collapsed pair. We estimated many reproductive barriers in each of five sets of stickleback fish species pairs using our own data and decades of previous work. We found that the types of barriers important early in the speciation process differ from those important late. Two premating barriers—habitat and sexual isolation—evolve early in divergence and remain two of the strongest barriers throughout speciation. Premating isolation evolves before postmating isolation, and extrinsic isolation is far stronger than intrinsic. Completing speciation, however, may require postmating intrinsic incompatibilities. Reverse speciation in one species pair was characterized by significant loss of sexual isolation. We present estimates of barrier strengths before and after collapse of a species pair; such detail regarding the loss of isolation has never before been documented. Additionally, despite significant asymmetries in individual barriers, which can limit speciation, total isolation was essentially symmetric between species. Our study provides important insight into the order of barrier evolution and the relative importance of isolating barriers during speciation and tests fundamental predictions of ecological speciation.     

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.     

Along the speciation continuum: Quantifying intrinsic and extrinsic isolating barriers across five million years of evolutionary divergence in California jewelflowers

Understanding the relative roles of intrinsic and extrinsic reproductive barriers, and their interplay within the geographic context of diverging taxa, remains an outstanding challenge in the study of speciation. We conducted a comparative analysis of reproductive isolation in California Jewelflowers (Streptanthus, s.l., Brassicaceae) by quantifying potential barriers to gene flow at multiple life history stages in 39 species pairs spanning five million years of evolutionary divergence. We quantified nine potential pre‐ and postzygotic barriers and explored patterns of reproductive isolation in relation to genetic distance. Intrinsic postzygotic isolation was initially weak, increased at intermediate genetic distances, and reached a threshold characterized by complete genetic incompatibility. Climatic niche differences were strong at shallow genetic distances, and species pairs with overlapping ranges showed slight but appreciable phenological isolation, highlighting the potential for ecological barriers to contribute to speciation. Geographic analyses suggest that speciation is not regionally allopatric in the California Jewelflowers, as recently diverged taxa occur in relatively close proximity and display substantial range overlap. Young pairs are characterized by incomplete intrinsic postzygotic isolation, suggesting that extrinsic barriers or fine‐scale spatial segregation are more important early in the divergence process than genetic incompatibilities.     

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.     

THE BIOLOGY OF SPECIATION

Since Darwin published the “Origin,” great progress has been made in our understanding of speciation mechanisms. The early investigations by Mayr and Dobzhansky linked Darwin's view of speciation by adaptive divergence to the evolution of reproductive isolation, and thus provided a framework for studying the origin of species. However, major controversies and questions remain, including: When is speciation nonecological? Under what conditions does geographic isolation constitute a reproductive isolating barrier? and How do we estimate the “importance” of different isolating barriers? Here, we address these questions, providing historical background and offering some new perspectives. A topic of great recent interest is the role of ecology in speciation. “Ecological speciation” is defined as the case in which divergent selection leads to reproductive isolation, with speciation under uniform selection, polyploid speciation, and speciation by genetic drift defined as “nonecological.” We review these proposed cases of nonecological speciation and conclude that speciation by uniform selection and polyploidy normally involve ecological processes. Furthermore, because selection can impart reproductive isolation both directly through traits under selection and indirectly through pleiotropy and linkage, it is much more effective in producing isolation than genetic drift. We thus argue that natural selection is a ubiquitous part of speciation, and given the many ways in which stochastic and deterministic factors may interact during divergence, we question whether the ecological speciation concept is useful. We also suggest that geographic isolation caused by adaptation to different habitats plays a major, and largely neglected, role in speciation. We thus provide a framework for incorporating geographic isolation into the biological species concept (BSC) by separating ecological from historical processes that govern species distributions, allowing for an estimate of geographic isolation based upon genetic differences between taxa. Finally, we suggest that the individual and relative contributions of all potential barriers be estimated for species pairs that have recently achieved species status under the criteria of the BSC. Only in this way will it be possible to distinguish those barriers that have actually contributed to speciation from those that have accumulated after speciation is complete. We conclude that ecological adaptation is the major driver of reproductive isolation, and that the term “biology of speciation,” as proposed by Mayr, remains an accurate and useful characterization of the diversity of speciation mechanisms.     

Persistent postmating,prezygotic reproductive isolation between populations

Studying reproductive barriers between populations of the same species is critical to understand how speciation may proceed. Growing evidence suggests postmating, prezygotic (PMPZ) reproductive barriers play an important role in the evolution of early taxonomic divergence. However, the contribution of PMPZ isolation to speciation is typically studied between species in which barriers that maintain isolation may not be those that contributed to reduced gene flow between populations. Moreover, in internally fertilizing animals, PMPZ isolation is related to male ejaculate—female reproductive tract incompatibilities but few studies have examined how mating history of the sexes can affect the strength of PMPZ isolation and the extent to which PMPZ isolation is repeatable or restricted to particular interacting genotypes. We addressed these outstanding questions using multiple populations of Drosophila montana. We show a recurrent pattern of PMPZ isolation, with flies from one population exhibiting reproductive incompatibility in crosses with all three other populations, while those three populations were fully fertile with each other. Reproductive incompatibility is due to lack of fertilization and is asymmetrical, affecting female fitness more than males. There was no effect of male or female mating history on reproductive incompatibility, indicating that PMPZ isolation persists between populations. We found no evidence of variability in fertilization outcomes attributable to different female × male genotype interactions, and in combination with our other results, suggests that PMPZ isolation is not driven by idiosyncratic genotype × genotype interactions. Our results show PMPZ isolation as a strong, consistent barrier to gene flow early during speciation and suggest several targets of selection known to affect ejaculate‐female reproductive tract interactions within species that may cause this PMPZ isolation.     

Ecological divergence plays an important role in strong but complex reproductive isolation in campions (Silene)*

New species arise through the evolution of reproductive barriers between formerly interbreeding lineages. Yet, comprehensive assessments of potential reproductive barriers, which are needed to make inferences on processes driving speciation, are only available for a limited number of systems. In this study, we estimated individual and cumulative strengths of seven prezygotic and six postzygotic reproductive barriers between the recently diverged taxa Silene dioica (L.) Clairv. and S. latifolia Poiret using both published and new data. A combination of multiple partial reproductive barriers resulted in near‐complete reproductive isolation between S. dioica and S. latifolia, consistent with earlier estimates of gene flow between the taxa. Extrinsic barriers associated with adaptive ecological divergence were most important, while intrinsic postzygotic barriers had moderate individual strength but contributed only little to total reproductive isolation. These findings are in line with ecological divergence as driver of speciation. We further found extensive variation in extrinsic reproductive isolation, ranging from sites with very strong selection against migrants and hybrids to intermediate sites where substantial hybridization is possible. This situation may allow for, or even promote, heterogeneous genetic divergence.     

Reproductive isolation driven by the combined effects of ecological adaptation and reinforcement

Recent years have seen a resurgence of interest in the process of speciation but few studies have elucidated the mechanisms either driving or constraining the evolution of reproductive isolation. In theory, the direct effects of reinforcing selection for increased mating discrimination where interbreeding produces hybrid offspring with low fitness and the indirect effects of adaptation to different environments can both promote speciation. Conversely, high levels of homogenizing gene flow can counteract the forces of selection. We demonstrate the opposing effects of reinforcing selection and gene flow in Timema cristinae walking-stick insects. The magnitude of female mating discrimination against males from other populations is greatest when migration rates between populations adapted to alternate host plants are high enough to allow the evolution of reinforcement, but low enough to prevent gene flow from eroding adaptive divergence in mate choice. Moreover, reproductive isolation is strongest under the combined effects of reinforcement and adaptation to alternate host plants. Our findings demonstrate the joint effects of reinforcement, ecological adaptation and gene flow on progress towards speciation in the wild.     

THE GENETIC ARCHITECTURE OF REPRODUCTIVE ISOLATION DURING SPECIATION‐WITH‐GENE‐FLOW IN LAKE WHITEFISH SPECIES PAIRS ASSESSED BY RAD SEQUENCING

During speciation‐with‐gene‐flow, effective migration varies across the genome as a function of several factors, including proximity of selected loci, recombination rate, strength of selection, and number of selected loci. Genome scans may provide better empirical understanding of the genome‐wide patterns of genetic differentiation, especially if the variance due to the previously mentioned factors is partitioned. In North American lake whitefish (Coregonus clupeaformis), glacial lineages that diverged in allopatry about 60,000 years ago and came into contact 12,000 years ago have independently evolved in several lakes into two sympatric species pairs (a normal benthic and a dwarf limnetic). Variable degrees of reproductive isolation between species pairs across lakes offer a continuum of genetic and phenotypic divergence associated with adaptation to distinct ecological niches. To disentangle the complex array of genetically based barriers that locally reduce the effective migration rate between whitefish species pairs, we compared genome‐wide patterns of divergence across five lakes distributed along this divergence continuum. Using restriction site associated DNA (RAD) sequencing, we combined genetic mapping and population genetics approaches to identify genomic regions resistant to introgression and derive empirical measures of the barrier strength as a function of recombination distance. We found that the size of the genomic islands of differentiation was influenced by the joint effects of linkage disequilibrium maintained by selection on many loci, the strength of ecological niche divergence, as well as demographic characteristics unique to each lake. Partial parallelism in divergent genomic regions likely reflected the combined effects of polygenic adaptation from standing variation and independent changes in the genetic architecture of postzygotic isolation. This study illustrates how integrating genetic mapping and population genomics of multiple sympatric species pairs provide a window on the speciation‐with‐gene‐flow mechanism.     

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.     

RIVERSCAPE GENETICS IDENTIFIES REPLICATED ECOLOGICAL DIVERGENCE ACROSS AN AMAZONIAN ECOTONE

Ecological speciation involves the evolution of reproductive isolation and niche divergence in the absence of a physical barrier to gene flow. The process is one of the most controversial topics of the speciation debate, particularly in tropical regions. Here, we investigate ecologically based divergence across an Amazonian ecotone in the electric fish, Steatogenys elegans. We combine phylogenetics, genome scans, and population genetics with a recently developed individual‐based evolutionary landscape genetics approach that incorporates selection. This framework is used to assess the relative contributions of geography and divergent natural selection between environments as biodiversity drivers. We report on two closely related and sympatric lineages that exemplify how divergent selection across a major Amazonian aquatic ecotone (i.e., between rivers with markedly different hydrochemical properties) may result in replicated ecologically mediated speciation. The results link selection across an ecological gradient with reproductive isolation and we propose that assortative mating based on water color may be driving the divergence. Divergence resulting from ecologically driven selection highlights the importance of considering environmental heterogeneity in studies of speciation in tropical regions. Furthermore, we show that framing ecological speciation in a spatially explicit evolutionary landscape genetics framework provides an important first step in exploring a wide range of the potential effects of spatial dependence in natural selection.     

Sexual isolation promotes divergence between parapatric lake and stream stickleback

Speciation can be initiated by adaptive divergence between populations in ecologically different habitats, but how sexually based reproductive barriers contribute to this process is less well understood. We here test for sexual isolation between ecotypes of threespine stickleback fish residing in adjacent lake and stream habitats in the Lake Constance basin, Central Europe. Mating trials exposing females to pairings of territorial lake and stream males in outdoor mesocosms allowing for natural reproductive behaviour reveal that mating occurs preferentially between partners of the same ecotype. Compared to random mating, this sexual barrier reduces gene flow between the ecotypes by some 36%. This relatively modest strength of sexual isolation is surprising because comparing the males between the two ecotypes shows striking differentiation in traits generally considered relevant to reproductive behaviour (body size, breeding coloration, nest size). Analysing size differences among the individuals in the mating trials further indicates that assortative mating is not related to ecotype differences in body size. Overall, we demonstrate that sexually based reproductive isolation promotes divergence in lake–stream stickleback along with other known reproductive barriers, but we also caution against inferring strong sexual isolation from the observation of strong population divergence in sexually relevant traits.     

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.     

Both Geography and Ecology Contribute to Mating Isolation in Guppies

Local adaptation to different environments can promote mating isolation – either as an incidental by-product of trait divergence, or as a result of selection to avoid maladaptive mating. Numerous recent empirical examples point to the common influence of divergent natural selection on speciation based largely on evidence of strong pre-mating isolation between populations from different habitat types. Accumulating evidence for natural selection''s influence on speciation is therefore no longer a challenge. The difficulty, rather, is in determining the mechanisms involved in the progress of adaptive divergence to speciation once barriers to gene flow are already present. Here, we present results of both laboratory and field experiments with Trinidadian guppies (Poecilia reticulata) from different environments, who do not show complete reproductive isolation despite adaptive divergence. We investigate patterns of mating isolation between populations that do and do not exchange migrants and show evidence for both by-product and reinforcement mechanisms depending on female ecology. Specifically, low-predation females discriminate against all high-predation males thus implying a by-product mechanism, whereas high-predation females only discriminate against low-predation males from further upstream in the same river, implying selection to avoid maladaptive mating. Our study thus confirms that mechanisms of adaptive speciation are not necessarily mutually exclusive and uncovers the complex ecology-geography interactions that underlie the evolution of mating isolation in nature.     

Interactions between the sexes: new perspectives on sexual selection and reproductive isolation

Understanding the processes underlying the origin of new species is a fundamental problem in evolutionary research. Whilst it has long been recognised that closely related taxa often differ markedly in reproductive characteristics, only relatively recently has sexual selection been evoked as a key promoter of speciation through its ability to generate reproductive isolation (RI). Sexual selection potentially can influence the probability that individuals from the same or different populations will reproduce successfully since it shapes precisely those traits involved in mating and reproduction. If reproductive characters diverge along different trajectories, then sexual selection can impact on the evolution of reproductive barriers operating both before and after mating. In this perspective, we consider some new advances in our understanding of the coevolution of male and female sexual signals and receptors and suggest how these developments may provide heretofore neglected insights into the mechanisms by which isolating barriers may emerge. Specifically, we explore how selfish genetic elements (SGEs) can mediate pre- and post-copulatory mate choice, thereby influencing gene flow and ultimately population divergence; we examine evidence from studies of intracellular sperm–egg interactions and propose that intracellular gametic incompatibilities may arise after sperm entry into the egg, and thus contribute to RI; we review findings from genomic studies demonstrating rapid, adaptive evolution of reproductive genes in both sexes and discuss whether such changes are causal in determining RI or simply associated with it; and finally, we consider genetic, developmental and functional mechanisms that might constrain reproductive trait diversification, thereby limiting the scope for reproductive barriers to arise via sexual selection. We hope to stimulate work that will further the understanding of the role sexual selection plays in generating RI and ultimately speciation.     

Body size evolution simultaneously creates and collapses species boundaries in a clade of scincid lizards

Speciation is generally viewed as an irreversible process, although habitat alterations can erase reproductive barriers if divergence between ecologically differentiated species is recent. Reversed speciation might also occur if geographical contact is established between species that have evolved the same reproductive isolating barrier in parallel. Here, we demonstrate a loss of intrinsic reproductive isolation in a clade of scincid lizards as a result of parallel body size evolution, which has allowed for gene flow where large-bodied lineages are in secondary contact. An mtDNA phylogeny confirms the monophyly of the Plestiodon skiltonianus species complex, but rejects that of two size-differentiated ecomorphs. Mate compatibility experiments show that the high degree of body size divergence imposes a strong reproductive barrier between the two morphs; however, the strength of the barrier is greatly diminished between parallel-evolved forms. Since two large-bodied lineages are in geographical contact in the Sierra Nevada Mountains of California, we were also able to test for postzygotic isolation under natural conditions. Analyses of amplified fragment length polymorphisms show that extensive gene exchange is occurring across the contact zone, resulting in an overall pattern consistent with isolation by distance. These results provide evidence of reversed speciation between clades that diverged from a common ancestor more than 12Myr ago.     

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.     

Divergence before the host shift? Prezygotic reproductive isolation among three varieties of a specialist fly on a single host plant

1. Although divergence via host‐plant shifting is a common theme in the speciation of some phytophagous insects, it is not clear whether host shifts are typically initiators of speciation or if they instead contribute to divergence events already in progress. While host shifts appear to be generally associated with speciation events for flies in the genus Strauzia, three sympatric varieties of the sunflower fly [Strauzia longipennis (Wiedemann)] co‐occur on the same host plant in the Midwestern United States and may have evolved reproductive barriers without a host shift. 2. The strength of two prezygotic reproductive barriers was compared among the three S. longipennis varieties: one barrier that is often associated with divergent ecological selection (allochronic isolation), and another that is more likely to be independent of ecological selection (pre‐copulatory sexual isolation). The presence and relative strength of each barrier between fly varieties were evaluated using microsatellites, no choice mating experiments, studies of allochronic isolation, and field collection data. 3. Evidence for both allochronic isolation and pre‐copulatory sexual isolation was detected between the three varieties of S. longipennis. The measure of isolation calculated for each barrier between the three varieties was lower than measures calculated between different species of Strauzia found on different hosts, suggesting that subsequent host shifts may increase the degree of reproductive isolation. For Strauzia and other specialist insects, some reproductive isolation may evolve prior to, and indeed may facilitate, host shifts.