Asymmetry in reproductive isolation and its effect on directional mitochondrial introgression in the parapatric ground beetles <Emphasis Type="Italic">Carabus yamato</Emphasis> and <Emphasis Type="Italic">C. albrechti</Emphasis>

Speciation studies seek to clarify the origin of reproductive isolation, the various mechanisms working from mate recognition through postzygotic stages. Asymmetric effects of isolating barriers can result in asymmetrical gene introgression during interspecific hybridization. The flightless ground beetles Carabus yamato and C. albrechti are distributed parapatrically in Japan, showing repeated asymmetrical introgression of mitochondria from C. albrechti to C. yamato. This pattern suggests that reproductive isolation between these species is strong, but incomplete and asymmetric (i.e., weaker for the cross between a C. albrechti female and a C. yamato male). To test this hypothesis, we conducted interspecific mating experiments in the laboratory. The estimates of total reproductive isolation, which occurred mainly at the premating and postmating/prezygotic stages, were high (isolation index = 0.964 for C. yamato female × C. albrechti male and 0.886 for the reciprocal cross), supporting the hypothesis of strong, but incomplete isolation. However, the observed difference between the reciprocal crosses was not sufficiently large to conclude that it caused directional introgression of mitochondria. Instead, we found asymmetry in individual isolating barriers in the postmating/prezygotic stages that coincided with the prediction, perhaps resulting from morphological mismatch of heterospecific genitalia. Although this asymmetry was compensated for by an inverse asymmetry of isolation in the postzygotic stage, the contribution of these individual barriers to total isolation may change for our expectation when considering females mating with multiple heterospecific males.     

Multiple strong postmating and intrinsic postzygotic reproductive barriers isolate florally diverse species of Jaltomata (Solanaceae)

Divergence in phenotypic traits often contributes to premating isolation between lineages, but could also promote isolation at postmating stages. Phenotypic differences could directly result in mechanical isolation or hybrids with maladapted traits; alternatively, when alleles controlling these trait differences pleiotropically affect other components of development, differentiation could indirectly produce genetic incompatibilities in hybrids. Here, we determined the strength of nine postmating and intrinsic postzygotic reproductive barriers among 10 species of Jaltomata (Solanaceae), including species with highly divergent floral traits. To evaluate the relative importance of floral trait diversification for the strength of these postmating barriers, we assessed their relationship to floral divergence, genetic distance, geographical context, and ecological differences, using conventional tests and a new linear‐mixed modeling approach. Despite close evolutionary relationships, all species pairs showed moderate to strong isolation. Nonetheless, floral trait divergence was not a consistent predictor of the strength of isolation; instead this was best explained by genetic distance, although we found evidence for mechanical isolation in one species, and a positive relationship between floral trait divergence and fruit set isolation across species pairs. Overall, our data indicate that intrinsic postzygotic isolation is more strongly associated with genome‐wide genetic differentiation, rather than floral divergence.     

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.     

Both morph‐ and species‐dependent asymmetries affect reproductive barriers between heterostylous species

The interaction between floral traits and reproductive isolation is crucial to explaining the extraordinary diversity of angiosperms. Heterostyly, a complex floral polymorphism that optimizes outcrossing, evolved repeatedly and has been shown to accelerate diversification in primroses, yet its potential influence on isolating mechanisms remains unexplored. Furthermore, the relative contribution of pre‐ versus postmating barriers to reproductive isolation is still debated. No experimental study has yet evaluated the possible effects of heterostyly on pre‐ and postmating reproductive mechanisms. We quantify multiple reproductive barriers between the heterostylous Primula elatior (oxlip) and P. vulgaris (primrose), which readily hybridize when co‐occurring, and test whether traits of heterostyly contribute to reproductive barriers in unique ways. We find that premating isolation is key for both species, while postmating isolation is considerable only for P. vulgaris; ecogeographic isolation is crucial for both species, while phenological, seed developmental, and hybrid sterility barriers are also important in P. vulgaris, implicating sympatrically higher gene flow into P. elatior. We document for the first time that, in addition to the aforementioned species‐dependent asymmetries, morph‐dependent asymmetries affect reproductive barriers between heterostylous species. Indeed, the interspecific decrease of reciprocity between high sexual organs of complementary floral morphs limits interspecific pollen transfer from anthers of short‐styled flowers to stigmas of long‐styled flowers, while higher reciprocity between low sexual organs favors introgression over isolation from anthers of long‐styled flowers to stigmas of short‐styled flowers. Finally, intramorph incompatibility persists across species boundaries, but is weakened in long‐styled flowers of P. elatior, opening a possible backdoor to gene flow through intramorph pollen transfer between species. Therefore, patterns of gene flow across species boundaries are likely affected by floral morph composition of adjacent populations. To summarize, our study highlights the general importance of premating isolation and newly illustrates that both morph‐ and species‐dependent asymmetries shape boundaries between heterostylous species.     

Rapid evolution of prezygotic barriers in non‐territorial damselflies

A central question in evolutionary biology concerns the accumulation of reproductive barriers during speciation. However, separating the reproductive barriers that have led to speciation from those that have secondarily accumulated (i.e. after initial divergence) is a widely recognized problem. Ideal candidate species for overcoming this problem are young species, where time for additional barriers to accrue has been limited. In the present study, we add to previous studies investigating the strength of reproductive barriers between the parapatric damselflies Ischnura elegans and Ischnura graellsii by quantifying seven prezygotic barriers between the allopatric pairs of I. elegans and Ischnura genei, as well as I. graellsii and I. genei. Specifically, we measured four premating (temporal, sexual, mechanical _I, and mechanical _II) and three postmating (oviposition success, fecundity, and fertility) barriers using experimental approaches and, for first time, we investigated the mechanisms causing mechanical isolation, which is the strongest reproductive barrier in ischnurans. The findings of the present study support the notion that premating barriers are generally strong and contribute significantly to total reproductive isolation in young lineages (65–98%), although they never solely lead to complete isolation. Asymmetry was generally stronger in premating than in postmating barriers, and was driven mostly through asymmetry in mechanical isolation, which is caused by morphological divergence of secondary sexual appendages. We found that barriers act multiplicatively in all species combinations tested, with the exception of sexual isolation, which was not detected. Our results are consistent with a recent allopatric speciation scenario driven by differences in male anal appendages, either impeding copulation or affecting female preferences. Taken together, the results from this and previous studies in diverse odonate genera suggest that premating barriers have evolved rapidly in ischnuran damselflies and, although reproductive isolation in ischnurans is more commonly the result of several barriers acting together, morphological divergence of secondary sexual appendages appears to be a common factor facilitating premating isolation in this group. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 485–496.     

The relationship between postmating reproductive isolation and reinforcement in Phlox

The process of speciation involves the accumulation of reproductive isolation (RI) between diverging lineages. Selection can favor increased RI via the process of reinforcement, whereby costs to hybridization impose selection for increased prezygotic RI. Reinforcement results in phenotypic divergence within at least one taxon, as a result of costly hybridization between sympatric taxa. The strength of selection driving reinforcement is determined by the cost of hybridization and the frequency of hybridization. We investigated the cost of hybridization by quantifying postmating RI barriers among Phlox species that comprise one of the best‐studied cases of reinforcement. We determined if the strength of RI differs among lineages that have and have not undergone reinforcement, how much variability there is within species in RI, and whether RI is associated with phylogenetic relatedness. We found high RI for the species that underwent phenotypic divergence due to reinforcement; however, RI was also high between other species pairs. We found extensive variability in RI among individuals within species, and no evidence that the strength of RI was associated with phylogenetic relatedness. We suggest that phenotypic divergence due to reinforcement is associated with the frequency of hybridization and introgression, and not the cost of hybridization in this clade.     

Patterns of reproductive isolation in Mediterranean deceptive orchids

The evolution of reproductive isolation is of central interest in evolutionary biology. In plants, this is typically achieved by a combination of pre- and postpollination mechanisms that prevent, or limit, the amount of interspecific gene flow. Here, we investigated and compared two ecologically defined groups of Mediterranean orchids that differ in pollination biology and pollinator specificity: sexually deceptive orchids versus food-deceptive orchids. We used experimental crosses to assess the strength of postmating prezygotic, and postzygotic reproductive isolation, and a phylogenetic framework to determine their relative rates of evolution. We found quantitative and qualitative differences between the two groups. Food-deceptive orchids have weak premating isolation but strong postmating isolation, whereas the converse situation characterizes sexually deceptive orchids. Only postzygotic reproductive isolation among food-deceptive orchids was found to evolve in a clock-like manner. Comparison of evolutionary rates, within a common interval of genetic distance, showed that the contribution of postmating barriers was more relevant in the food-deceptive species than in the sexually deceptive species. Asymmetry in prezygotic isolation was found among food-deceptive species. Our results indicate that postmating barriers are most important for reproductive isolation in food-deceptive orchids, whereas premating barriers are most important in sexually deceptive orchids. The different rate of evolution of reproductive isolation and the relative strength of pre- and postmating barriers may have implication for speciation processes in the two orchid groups.     

Introgression despite substantial divergence in a broadcast spawning marine invertebrate

Understanding the relationship between reproductive isolation and time since divergence is critical to our understanding of speciation. One group for which we know little about the relationship between hybridization/introgression and time since divergence is the marine broadcast spawners. Here, we investigate the distribution of closely related cryptic species of marine broadcast spawners (Type A and B Ciona intestinalis) in areas of potential sympatry to determine whether these two types occur together and if so, whether they show evidence of hybridization and introgression. Then we combine our data with other studies to investigate general patterns of reproductive isolation versus divergence in marine broadcast spawners. We found that Type A and B C. intestinalis occurred sympatrically in 2007, and that 21 individuals show evidence of introgression in sympatry (out of approximately 500). Type A and B C. intestinalis are 12.4% divergent at mitochondrial COI (mtCOI), and in comparison with other marine broadcast spawning species at mtCOI, these two types may be near the upper limit of the range of divergence values in which introgression is still possible. However, introgression at divergence levels similar to those found in Ciona does exist, prompting questions about the strength of postmating prezygotic reproductive barriers in marine broadcast spawners.     

Divergence and reproductive isolation in the early stages of speciation

To understand speciation we need to identify the factors causing divergence between natural populations. The traditional approach to gaining such insights has been to focus on a particular theory and ask whether observed patterns of reproductive isolation between populations or species are consistent with the hypothesis in question. However, such studies are few and they do not allow us to compare between hypotheses, so often we cannot determine the relative contribution to divergence of different potential factors. Here, I describe a study of patterns of phenotypic divergence and premating and postmating isolation between populations of the grasshopper Chorthippus parallelus. Information on the phylogeographic relationships of the populations means that a priori predictions from existing hypotheses for the evolution of reproductive isolation can be compared with observations. I assess the relative contributions to premating isolation, postmating isolation and phenotypic divergence of long periods of allopatry, adaptation to different environments and processes associated with colonisation (such as population bottlenecks). Likelihood analysis reveals that long periods of allopatry in glacial refugia are associated with postmating reproductive isolation, but not premating isolation, which is more strongly associated with colonisation. Neither premating nor postmating isolation is higher between populations differing in potential environmental selection pressures. There are only weak correlations between patterns of genetic divergence and phenotypic divergence and no correlation between premating and postmating isolation. This suggests that the potential of a taxon to exercise mate choice may affect the types of factor that promote speciation in that group. I discuss the advantages and disadvantages of the general approach of simultaneously testing competing hypotheses for the evolution of reproductive isolation.     

Role of seed germination in adaptation and reproductive isolation in Arabidopsis lyrata

Seed germination is an important developmental and life history stage. Yet, the evolutionary impact of germination has mainly been studied in the context of dormancy, or for its role in reproductive isolation between species. Here, we aim to examine multiple consequences of genetic divergence on germination traits between two Arabidopsis lyrata subspecies: ssp. petraea (Eurasia) and ssp. lyrata (North America). Postdormancy germination time, a potentially adaptive trait, showed differentiation between the populations, and quantitative trait loci (QTL) mapping revealed that the trait variation is mainly controlled by two antagonistic loci. These QTL areas contain several candidate genes with known function in postdormancy germination in A. thaliana. The sequence variation of three genes was consistent with differential selection, and they also included fixed nonsynonymous substitutions with potential to account for the phenotypic differentiation. We further show that the divergence between the subspecies has led to a slight but significant reduction in hybrid germination proportions, indicating incipient reproductive isolation. Comparison of reciprocal F₁ and F₂ progenies suggests that Bateson–Dobzhansky–Muller incompatibilities likely act through uniparentally inherited factors. Examination of genomewide transmission ratio distortion further revealed that cytonuclear interactions cause substantial pregermination inviability in the hybrids. These results confirm that seed germination has adaptive potential beyond the dormancy stage and that hybrid seed inviability can be one of the first reproductive barriers to arise during divergence.     

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.     

Evolution of postzygotic reproductive isolation in a guild of deceptive orchids

The evolution of reproductive barriers is of central importance for speciation. Here, we investigated three components of postzygotic isolation-embryo mortality, hybrid inviability, and hybrid sterility-in a group of food-deceptive Mediterranean orchids from the genera Anacamptis, Neotinea, and Orchis. In these orchids, pollinator-mediated isolation is weak, which suggests that postpollination barriers exist. Based on crossing experiments and a literature survey, we found that embryo mortality caused complete reproductive isolation among 36.3% of the species pairs, and hybrid inviability affected 55.6% of the potentially hybridizing species pairs. Hybrid sterility was assessed experimentally for seven species pairs. A strong reduction of fertility in all investigated hybrids was found, together with clear differences between male and female components of hybrid sterility. Postzygotic isolation was found to evolve gradually with genetic divergence, and late postzygotic isolation (i.e., hybrid inviability and sterility) evolved faster than embryo mortality, which is an earlier postzygotic isolation stage. These results reveal that intrinsic postzygotic isolation strongly contributes to maintaining species boundaries among Mediterranean food-deceptive orchids while establishing a prominent role for these reproductive barriers in the early stage of species isolation.     

THE ACCUMULATION OF REPRODUCTIVE INCOMPATIBILITIES IN AFRICAN CICHLID FISH

The rate at which different components of reproductive isolation accumulate with divergence time between species has only been studied in a limited, but growing, number of species. We measured premating isolation and hybrid inviability at four different ontogenetic stages from zygotes to adults in interspecific hybrids of 26 pairs of African cichlid species, spanning the entire East African haplochromine radiation. We then used multiple relaxed molecular clock calibrations to translate genetic distances into absolute ages to compare evolutionary rates of different components of reproductive isolation. We find that premating isolation accumulates fast initially but then changes little with increasing genetic distance between species. In contrast, postmating isolation between closely related species is negligible but then accumulates rapidly, resulting in complete hybrid inviability after 4.4/8.5/18.4 million years (my). Thus, the rate at which complete intrinsic incompatibilities arise in this system is orders of magnitude lower than rates of speciation within individual lake radiations. Together these results suggest divergent ecological adaptations may prevent populations from interbreeding and help maintain cichlid species diversity, which may be vulnerable to environmental degradation. By quantifying the capacity to produce viable hybrids between allopatric, distantly related lineages our results also provide an upper divergence time limit for the “hybrid swarm origin” model of adaptive radiation.     

Gametic selection,developmental trajectories,and extrinsic heterogeneity in Haldane's rule

Deciphering the genetic and developmental causes of the disproportionate rarity, inviability, and sterility of hybrid males, Haldane's rule, is important for understanding the evolution of reproductive isolation between species. Moreover, extrinsic and prezygotic factors can contribute to the magnitude of intrinsic isolation experienced between species with partial reproductive compatibility. Here, we use the nematodes Caenorhabditis briggsae and C. nigoni to quantify the sensitivity of hybrid male viability to extrinsic temperature and developmental timing, and test for a role of mito‐nuclear incompatibility as a genetic cause. We demonstrate that hybrid male inviability manifests almost entirely as embryonic, not larval, arrest and is maximal at the lowest rearing temperatures, indicating an intrinsic‐by‐extrinsic interaction to hybrid inviability. Crosses using mitochondrial substitution strains that have reciprocally introgressed mitochondrial and nuclear genomes show that mito‐nuclear incompatibility is not a dominant contributor to postzygotic isolation and does not drive Haldane's rule in this system. Crosses also reveal that competitive superiority of X‐bearing sperm provides a novel means by which postmating prezygotic factors exacerbate the rarity of hybrid males. These findings highlight the important roles of gametic, developmental, and extrinsic factors in modulating the manifestation of Haldane's rule.     

EVOLUTION OF COURTSHIP SONG AND REPRODUCTIVE ISOLATION IN THE DROSOPHILA WILLISTONI SPECIES COMPLEX: DO SEXUAL SIGNALS DIVERGE THE MOST QUICKLY?

Reproductive isolation increases with genetic distance between species. Although sexual selection may drive divergence of sexual signals and traits, causing rapid evolution of sexual isolation, quantitative data supporting this idea are rare. We examine the rates of divergence of a species-specific courtship signal, sexual isolation, and postmating isolation in the Drosophila willistoni group. Both types of isolation increase with genetic distance and postmating isolation is the most strongly correlated with genetic divergence, suggesting this has the least variable divergence rate. Song divergence is not correlated with genetic divergence. Homoplasy in song pattern results in poorly resolved phylogenies that are different from molecular phylogenies. Song evolves more quickly than sexual isolation, which evolves more quickly than postmating isolation.     

Divergence in style length and pollen size leads to a postmating‐prezygotic reproductive barrier among populations of Silene latifolia

A central tenet of speciation research is the need to identify reproductive isolating barriers. One approach to this line of research is to identify the phenotypes that lead to reproductive isolation. Several studies on flowering plants have shown that differences in style length contribute to reproductive isolation between species, leading us to consider whether style length could act as a reproductive barrier among populations of a single species. This could occur if style length varied sufficiently and pollen size covaried with style length. Populations of Silene latifolia exhibit variation in flower size, including style length, that is negatively correlated with annual precipitation. We show that this divergence in style length has a genetic basis and acts as a reproductive barrier: males from small‐flowered populations produced relatively small pollen grains that were poor at fertilizing ovules when crossed to females from large‐flowered populations, leading to a significant reduction in seed production. Manipulating the distance pollen tubes had to travel revealed that this failure was purely mechanical and not the result of other incompatibilities. These results show that style length acts as a postmating‐prezygotic reproductive barrier and indicate a potential link between ecotypic differentiation and reproductive isolation within a species.     

Premating, not postmating, barriers drive genetic dynamics in experimental hybrid populations of the endangered Sonoran topminnow

The timing and pattern of reproductive barrier formation in allopatric populations has received much less attention than the accumulation of reproductive barriers in sympatry. The theory of allopatric speciation suggests that reproductive barriers evolve simply as by-products of overall genetic divergence. However, observations of enhanced premating barriers in allopatric populations suggest that sexual selection driven by intraspecific competition for mates may enhance species-specific signals and accelerate the speciation process. In a previous series of laboratory trials, we examined the strength of premating and postmating barriers in an allopatric species pair of the endangered Sonoran topminnow, Poeciliopsis occidentalis and P. sonoriensis. Behavioral observations provided evidence of asymmetrical assortative mating, while reduced brood sizes and male-biased F(1) sex ratios suggest postmating incompatibilities. Here we examine the combined effects of premating and postmating barriers on the genetic makeup of mixed populations, using cytonuclear genotype frequencies of first- and second-generation offspring. Observed genotype frequencies strongly reflect the directional assortative mating observed in behavioral trials, illustrating how isolating barriers that act earlier in the reproductive cycle will have a greater effect on total reproductive isolation and may be more important to speciation than subsequent postmating reproductive barriers.     

Multilocus test for introgression between the cactophilic species Drosophila mojavensis and Drosophila arizonae

Information obtained from laboratory studies regarding the efficacy of barriers to gene flow (reproductive isolation) between species is often incomplete or misleading, so detailed genetic analyses are needed to determine whether hybridization and introgression occur in nature. Previous laboratory studies of the cactophilic species Drosophila mojavensis and Drosophila arizonae suggest that reproductive isolation is incomplete and that gene flow may occur in sympatry. We sampled 18 nuclear and one mitochondrial loci from multiple populations of D. arizonae and D. mojavensis to test for the signature of recent or historic gene flow between these two species. We located chromosomal regions that were inverted between these species and analyzed those regions independently of others. Statistical tests for introgression using all loci or only collinear loci failed to reject expectations of an isolation model. Further tests using average nucleotide differences between species and phylogenetic analyses also failed to find support for introgression between D. mojavensis and D. arizonae. Additional ecological and behavioral studies of these species in their natural habitats are required to explain why the signature of gene flow was not detected at the DNA sequence level in populations when laboratory studies suggest such gene flow should be possible.