DIFFERENTIAL PATTERNS OF INTROGRESSION ACROSS THE X CHROMOSOME IN A HYBRID ZONE BETWEEN TWO SPECIES OF HOUSE MICE

A complete understanding of the speciation process requires the identification of genomic regions and genes that confer reproductive barriers between species. Empirical and theoretical research has revealed two important patterns in the evolution of reproductive isolation in animals: isolation typically arises as a result of disrupted epistatic interactions between multiple loci and these disruptions map disproportionately to the X chromosome. These patterns suggest that a targeted examination of natural gene flow between closely related species at X-linked markers with known positions would provide insight into the genetic basis of speciation. We take advantage of the existence of genomic data and a well-documented European zone of hybridization between two species of house mice, Mus domesticus and M. musculus, to conduct such a survey. We evaluate patterns of introgression across the hybrid zone for 13 diagnostic X-linked loci with known chromosomal positions using a maximum likelihood model. Interlocus comparisons clearly identify one locus with reduced introgression across the center of the hybrid zone, pinpointing a candidate region for reproductive isolation. Results also reveal one locus with high frequencies of M. domesticus alleles in populations on the M. musculus side of the zone, suggesting the possibility that positive selection may act to drive the spread of alleles from one species on to the genomic background of the other species. Finally, cline width and cline center are strongly positively correlated across the X chromosome, indicating that gene flow of the X chromosome may be asymmetrical. This study highlights the utility of natural populations of hybrids for mapping speciation genes and suggests that the middle of the X chromosome may be important for reproductive isolation between species of house mice.

In recent years, theoretical models have introduced the concept that ongoing hybridization between "good" species can occur because incomplete reproductive isolation can be a selected optimum. They furthermore show that positive frequency-dependent sexual selection, which is naturally generated by some of the underlying processes that lead to assortative mating, plays a key role in the evolution of incomplete reproductive isolation. This occurs, however, through different mechanisms in sympatric versus allopatric scenarios. We investigate the evolution of incomplete reproductive isolation by sexual selection in scenarios ranging from sympatry to allopatry, to examine how these mechanisms interact. We consider an ecological scenario in which there are two habitats used during foraging and individuals can breed either within a habitat or in a common mating pool. We find that when trait divergence is maintained, sexual selection drives the evolution of choosiness in opposite ways in the common mating pool versus within each habitat. Specifically, strong choosiness is favored in the common mating pool, whereas intermediate choosiness is favored within habitat; the interaction of these forces determines whether intermediate reproductive isolation ultimately evolves in the system. We further find cases where the evolution of stronger choosiness occurs but leads to the loss of divergence. Overall, our study shows that contrasting forces on the evolution of reproductive isolation can occur in different mating areas, and we propose a new avenue for understanding the diversity in levels of reproductive isolation within and across species.

The X chromosome plays a central role in the evolution of reproductive isolation, but few studies have examined the genetic basis of X-linked incompatibilities during the early stages of speciation. We report the results of a large experiment focused on the reciprocal introgression of the X chromosome between two species of house mice, Mus musculus and M. domesticus. Introgression of the M. musculus X chromosome into a wild-derived M. domesticus genetic background produced male-limited sterility, qualitatively consistent with previous experiments using classic inbred strains to represent M. domesticus. The genetic basis of sterility involved a minimum of four X-linked factors. The phenotypic effects of major sterility QTL were largely additive and resulted in complete sterility when combined. No sterility factors were uncovered on the M. domesticus X chromosome. Overall, these results revealed a complex and asymmetric genetic basis to X-linked hybrid male sterility during the early stages of speciation in mice. Combined with data from previous studies, we identify one relatively narrow interval on the M. musculus X chromosome involved in hybrid male sterility. Only a handful of spermatogenic genes are within this region, including one of the most rapidly evolving genes on the mouse X chromosome.

The evolution of strong reproductive isolation (RI) is fundamental to the origins and maintenance of biological diversity, especially in situations where geographical distributions of taxa broadly overlap. But what is the history behind strong barriers currently acting in sympatry? Using whole-genome sequencing and single nucleotide polymorphism genotyping, we inferred (i) the evolutionary relationships, (ii) the strength of RI, and (iii) the demographic history of divergence between two broadly sympatric taxa of intertidal snail. Despite being cryptic, based on external morphology, Littorina arcana and Littorina saxatilis differ in their mode of female reproduction (egg-laying versus brooding), which may generate a strong post-zygotic barrier. We show that egg-laying and brooding snails are closely related, but genetically distinct. Genotyping of 3092 snails from three locations failed to recover any recent hybrid or backcrossed individuals, confirming that RI is strong. There was, however, evidence for a very low level of asymmetrical introgression, suggesting that isolation remains incomplete. The presence of strong, asymmetrical RI was further supported by demographic analysis of these populations. Although the taxa are currently broadly sympatric, demographic modelling suggests that they initially diverged during a short period of geographical separation involving very low gene flow. Our study suggests that some geographical separation may kick-start the evolution of strong RI, facilitating subsequent coexistence of taxa in sympatry. The strength of RI needed to achieve sympatry and the subsequent effect of sympatry on RI remain open questions. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

The acquisition of floral nectar spurs is correlated with increased species diversity across multiple clades. We tested whether variation in nectar spurs influences reproductive isolation and, thus, can potentially promote species diversity using two species of Aquilegia, Aquilegia formosa and Aquilegia pubescens, which form narrow hybrid zones. Floral visitors strongly discriminated between the two species both in natural populations and at mixed-species arrays of individual flowers. Bees and hummingbirds visited flowers of A. formosa at a much greater rate than flowers of A. pubescens. Hawkmoths, however, nearly exclusively visited flowers of A. pubescens. We found that altering the orientation of A. pubescens flowers from upright to pendent, like the flowers of A. formosa, reduced hawkmoth visitation by an order of magnitude. In contrast, shortening the length of the nectar spurs of A. pubescens flowers to a length similar to A. formosa flowers did not affect hawkmoth visitation. However, pollen removal was significantly reduced in flowers with shortened nectar spurs. These data indicate that floral traits promote floral isolation between these species and that specific floral traits affect floral isolation via ethological isolation while others affect floral isolation via mechanical isolation.

Many recent studies have addressed the mechanisms operating during the early stages of speciation, but surprisingly few studies have tested theoretical predictions on the evolution of strong reproductive isolation (RI). To help address this gap, we first undertook a quantitative review of the hybrid zone literature for flowering plants in relation to reproductive barriers. Then, using Populus as an exemplary model group, we analysed genome-wide variation for phylogenetic tree topologies in both early- and late-stage speciation taxa to determine how these patterns may be related to the genomic architecture of RI. Our plant literature survey revealed variation in barrier complexity and an association between barrier number and introgressive gene flow. Focusing on Populus, our genome-wide analysis of tree topologies in speciating poplar taxa points to unusually complex genomic architectures of RI, consistent with earlier genome-wide association studies. These architectures appear to facilitate the 'escape' of introgressed genome segments from polygenic barriers even with strong RI, thus affecting their relationships with recombination rates. Placed within the context of the broader literature, our data illustrate how phylogenomic approaches hold great promise for addressing the evolution and temporary breakdown of RI during late stages of speciation. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

We ask if three decades and over 1,500 generations of divergent life-history selection on age at reproduction has resulted in the evolution of reproductive isolation (RI) between laboratory populations of Drosophila melanogaster. We tested for premating, postmating-prezygotic, and postzygotic reproductive isolation between three replicate population pairs. Large, evolved differences in body size between selection treatments suggested the potential for prezygotic barriers driven by sexual selection or physical incompatibilities between the sexes. Although a simple prediction would be preference for larger size, creating directional isolation, our results from individual mate choice trials indicate that populations from both selection treatments show a marked bias towards homotypic mate choice; indicative of prezygotic RI driven by sexual selection or sexual conflict. Hybridization between the focal populations resulted in the production of viable adult flies with intermediate size and developmental traits. We observed a suggestive but statistically nonsignificant trend of fitness decline in the F2 generation of hybrids, but no significant evidence suggesting the evolution of postmating-prezygotic or postzygotic RI. Our findings are in accord with extant literature that posits that premating RI evolves before postmating forms of RI.

Hybrid zones, where phenotypically distinct populations interbreed, should expand or contract until reaching a balance between selection and dispersal. Few studies examine multiple contacts within one species complex to compare how their extent changes over time. Black-crested and Tufted Titmice (Baeolophus atricristatus and B. bicolor) hybridize extensively within a narrow zone in Texas and southwestern Oklahoma. In Texas, hybridization has been occurring for several thousands of years, while evidence suggests the southwestern Oklahoma contact is more recent, beginning within the past century. We quantify plumage and morphology of the two species across both the younger and older hybrid zones and compare the current and historical extent of phenotypic variation in the older Texas contact with that in the younger Oklahoma contact. Variation in plumage between species is similar in the younger and older contacts, while overlap in morphological characters is broader in the older contact. Recently and historically surveyed transects in the older zone have similar cline widths, indicating selection, at least on crest and forehead plumage, has reached equilibrium with dispersal over the time periods involved (comparing both the historically surveyed data from 1955 vs. the recently surveyed data from the 2000s in Texas). In the recently surveyed younger Oklahoma contact, cline width is narrower, indicating potential for expansion if it follows the course of the older contact. This temporal complexity should make this species complex a productive system for future work, using plumage and additional traits such as song and genetics, on the relative influences of both natural and sexual selection on the evolution of reproductive isolation.

Hybrid zones between closely related species or subspecies provide useful settings for studying the genetic architecture of speciation. Using markers distributed throughout the mouse genome, we use a hybrid zone between two recently diverged species of house mice (Mus musculus and Mus domesticus) as a natural mapping experiment to identify genomic regions that may be involved in reproductive isolation. Using cline analysis we document a nearly 50-fold variation in level of introgression among markers. Some markers have extremely narrow cline widths; these genomic regions may contribute to reproductive isolation. Biological processes associated with these narrow clines include physiological and immune responses to the environment as well as physiological and behavioral aspects of reproduction. Other autosomal markers exhibit asymmetrically broad clines, usually with high frequencies of M. domesticus alleles on the M. musculus side of the hybrid zone. These markers identify genome regions likely housing genes with alleles that are spreading from one species to the other. Biological processes associated with these wide clines include cell signaling, olfaction, and pheromone response. These processes play important roles in survival and reproduction, and associated genes are likely targets of selection. Patterns of linkage disequilibrium in the center of the hybrid zone suggest that isolation may be caused by multiple epistatic interactions between sets of genes. These data highlight the complex genetic architecture underlying speciation even at early stages of divergence and point to some of the biological processes that may govern this architecture.

We possess limited understanding of how speciation unfolds in the most species-rich region of the planet-the Amazon basin. Hybrid zones provide valuable information on the evolution of reproductive isolation, but few studies of Amazonian vertebrate hybrid zones have rigorously examined the genome-wide underpinnings of reproductive isolation. We used genome-wide genetic datasets to show that two deeply diverged, but morphologically cryptic sister species of forest understorey birds show little evidence for prezygotic reproductive isolation, but substantial postzygotic isolation. Patterns of heterozygosity and hybrid index revealed that hybrid classes with heavily recombined genomes are rare and closely match simulations with high levels of selection against hybrids. Genomic and geographical clines exhibit a remarkable similarity across loci in cline centres, and have exceptionally narrow cline widths, suggesting that postzygotic isolation is driven by genetic incompatibilities at many loci, rather than a few loci of strong effect. We propose Amazonian understorey forest birds speciate slowly via gradual accumulation of postzygotic genetic incompatibilities, with prezygotic barriers playing a less important role. Our results suggest old, cryptic Amazonian taxa classified as subspecies could have substantial postzygotic isolation deserving species recognition and that species richness is likely to be substantially underestimated in Amazonia.

We developed a Bayesian genomic cline model to study the genetic architecture of adaptive divergence and reproductive isolation between hybridizing lineages. This model quantifies locus-specific patterns of introgression with two cline parameters that describe the probability of locus-specific ancestry as a function of genome-wide admixture. 'Outlier' loci with extreme patterns of introgression relative to most of the genome can be identified. These loci are potentially associated with adaptive divergence or reproductive isolation. We simulated genetic data for admixed populations that included neutral introgression, as well as loci that were subject to directional, epistatic or underdominant selection, and analysed these data using the Bayesian genomic cline model. Under many demographic conditions, underdominance or directional selection had detectable and predictable effects on cline parameters, and 'outlier' loci were greatly enriched for genetic regions affected by selection. We also analysed previously published genetic data from two transects through a hybrid zone between Mus domesticus and M.musculus. We found considerable variation in rates of introgression across the genome and particularly low rates of introgression for two X-linked markers. There were similarities and differences in patterns of introgression between the two transects, which likely reflects a combination of stochastic variability because of genetic drift and geographic variation in the genetic architecture of reproductive isolation. By providing a robust framework to quantify and compare patterns of introgression among genetic regions and populations, the Bayesian genomic cline model will advance our understanding of the genetics of reproductive isolation and the speciation process.

The evolution of placentation is predicted to intensify intergenomic conflicts between mothers and offspring over optimal levels of maternal investment by providing offspring opportunities to manipulate mothers into allocating more resources. Parent-offspring conflicts can result in the evolution of reproductive isolation among populations when conflicts resolve in different ways. Postzygotic reproductive isolation is hypothesized to evolve more rapidly following the evolution of placentation due to the predicted increase in conflict. We tested this hypothesis by performing interpopulation crosses within placental and nonplacental species of Poeciliopsis to determine if the relationship between genetic distance and measures of postzygotic reproductive success differed as function of reproductive mode. We did not observe any differences in offspring viability or sterility among crosses. Offspring size declined rapidly as a function of interpopulation genetic distance within the placental species, but not among our nonplacental species. The decrease in offspring size in the placental species was beyond normal variation, likely representing a major fitness cost, consistent with the prediction that negative epistatic interactions are evolving more quickly among populations in our placental species than the nonplacental species. We discuss how our results support the role parent-offspring conflicts play in the evolution of reproductive isolation and reproductive mode.

Gene flow tends to impede the accumulation of genetic divergence. Here, we determine the limits for the evolution of postzygotic reproductive isolation in a model of two populations that are connected by gene flow. We consider two selective mechanisms for the creation and maintenance of a genetic barrier: local adaptation leads to divergence among incipient species due to selection against migrants, and Dobzhansky–Muller incompatibilities (DMIs) reinforce the genetic barrier through selection against hybrids. In particular, we are interested in the maximum strength of the barrier under a limited amount of local adaptation, a challenge that many incipient species may initially face. We first confirm that with classical two-locus DMIs, the maximum amount of local adaptation is indeed a limit to the strength of a genetic barrier. However, with three or more loci and cryptic epistasis, this limit holds no longer. In particular, we identify a minimal configuration of three epistatically interacting mutations that is sufficient to confer strong reproductive isolation.This article is part of the theme issue ‘Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers’.

Speciation, that is, the evolution of reproductive barriers eventually leading to complete isolation, is a crucial process generating biodiversity. Recent work has contributed much to our understanding of how reproductive barriers begin to evolve, and how they are maintained in the face of gene flow. However, little is known about the transition from partial to strong reproductive isolation (RI) and the completion of speciation. We argue that the evolution of strong RI is likely to involve different processes, or new interactions among processes, compared with the evolution of the first reproductive barriers. Transition to strong RI may be brought about by changing external conditions, for example, following secondary contact. However, the increasing levels of RI themselves create opportunities for new barriers to evolve and, and interaction or coupling among barriers. These changing processes may depend on genomic architecture and leave detectable signals in the genome. We outline outstanding questions and suggest more theoretical and empirical work, considering both patterns and processes associated with strong RI, is needed to understand how speciation is completed. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

The evolution of reproductive isolation in the presence of gene flow is supported by theoretical models but rarely by data. Empirical support might be gained from studies of parallel hybrid zones between interbreeding taxa. We analysed gene flow over two hybrid zones separating ecotypes of Littorina saxatilis to test the expectation that neutral genetic markers will show site-specific differences if barriers have evolved in situ. Distinct ecotypes found in contrasting shore habitats are separated by divergent selection and poor dispersal, but hybrid zones appear between them. Swedish islands formed by postglacial uplift 5000 years ago provide opportunities to assess genetic structure in a recently evolved system. Each island houses a discrete population containing subpopulations of different ecotypes. Hybrid zones between ecotypes may be a product of ecological divergence occurring on each island or a consequence of secondary overlap of ecotypes of allopatric origin that have spread among the islands. We used six microsatellite loci to assess gene flow and genetic profiles of hybrid zones on two islands. We found reduced gene flow over both hybrid zones, indicating the presence of local reproductive barriers between ecotypes. Nevertheless, subpopulations of different ecotypes from the same island were genetically more similar to each other than were subpopulations of the same ecotype from different islands. Moreover, neutral genetic traits separating the two ecotypes across hybrid zones were site-specific. This supports a scenario of in situ origin of ecotypes by ecological divergence and nonallopatric evolution of reproductive barriers.