Reproductive isolation arises during laboratory adaptation to a novel hot environment.

BACKGROUND: Reproductive isolation can result from adaptive processes (e.g., ecological speciation and mutation-order speciation) or stochastic processes such as "system drift" model. Ecological speciation predicts barriers to gene flow between populations from different environments, but not among replicate populations from the same environment. In contrast, reproductive isolation among populations independently adapted to the same/similar environment can arise from both mutation-order speciation or system drift. RESULTS: In experimentally evolved populations adapting to a hot environment for over 100 generations, we find evidence for pre- and postmating reproductive isolation. On one hand, an altered lipid metabolism and cuticular hydrocarbon composition pointed to possible premating barriers between the ancestral and replicate evolved populations. On the other hand, the pronounced gene expression differences in male reproductive genes may underlie the postmating isolation among replicate evolved populations adapting to the same environment with the same standing genetic variation. CONCLUSION: Our study confirms that replicated evolution experiments provide valuable insights into the mechanisms of speciation. The rapid emergence of the premating reproductive isolation during temperature adaptation showcases incipient ecological speciation. The potential evidence of postmating reproductive isolation among replicates gave rise to two hypotheses: (1) mutation-order speciation through a common selection on early fecundity leading to an inherent inter-locus sexual conflict; (2) system drift with genetic drift along the neutral ridges.

Selection leads to remarkable variability in the outcomes of hybridisation across replicate hybrid zones.

Hybrid zones have been viewed as an opportunity to see speciation in action. When hybrid zones are replicated, it is assumed that if the same genetic incompatibilities are maintaining reproductive isolation across all instances of secondary contact, those incompatibilities should be identifiable by consistent patterns in the genome. In contrast, changes in allele frequencies due to genetic drift should be idiosyncratic for each hybrid zone. To test this assumption, we simulated 20 replicates of each of 12 hybrid zone scenarios with varied genetic incompatibilities, rates of migration, selection and different starting population size ratios of parental species. We found remarkable variability in the outcomes of hybridisation in replicate hybrid zones, particularly with Bateson-Dobzhansky-Muller incompatibilities and strong selection. We found substantial differences among replicates in the overall genomic composition of individuals, including admixture proportions, inter-specific ancestry complement and number of ancestry junctions. Additionally, we found substantial variation in genomic clines among replicates at focal loci, regardless of locus-specific selection. We conclude that processes other than selection are responsible for some consistent outcomes of hybridisation, whereas selection on incompatibilities can lead to genomically widespread and highly variable outcomes. We highlight the challenge of mapping between pattern and process in hybrid zones and call attention to how selection against incompatibilities will commonly lead to variable outcomes. We hope that this study informs future research on replicate hybrid zones and encourages further development of statistical techniques, theoretical models and exploration of additional axes of variation to understand reproductive isolation.

Chemical species recognition in an adaptive radiation of Hawaiian Tetragnatha spiders (Araneae: Tetragnathidae).

Studies of adaptive radiations have played a central role in our understanding of reproductive isolation. Yet the focus has been on human-biased visual and auditory signals, leaving gaps in our knowledge of other modalities. To date, studies on chemical signals in adaptive radiations have focused on systems with multimodal signalling, making it difficult to isolate the role chemicals play in reproductive isolation. In this study we examine the use of chemical signals in the species recognition and adaptive radiation of Hawaiian Tetragnatha spiders by focusing on entire communities of co-occurring species, and conducting behavioural assays in conjunction with chemical analysis of their silks using gas chromatography-mass spectrometry. Male spiders significantly preferred the silk extracts of conspecific mates over those of sympatric heterospecifics. The compounds found in the silk extracts, long chain alkyl methyl ethers, were remarkably species-specific in the combination and quantity. The differences in the profile were greatest between co-occurring species and between closely related sibling species. Lastly, there were significant differences in the chemical profile between two populations of a particular species. These findings provide key insights into the role chemical signals play in the attainment and maintenance of reproductive barriers between closely related co-occurring species.

Is self-incompatibility a reproductive barrier for hybridization in a sympatric species?

PREMISE: Barriers at different reproductive stages contribute to reproductive isolation. Self-incompatibility (SI) systems that prevent self-pollination could also act to control interspecific pollination and contribute to reproductive isolation, preventing hybridization. Here we evaluated whether SI contributes to reproductive isolation among four co-occurring Opuntia species that flower at similar times and may hybridize with each other. METHODS: We assessed whether Opuntia cantabrigiensis, O. robusta, O. streptacantha, and O. tomentosa, were self-compatible and formed hybrid seeds in five manipulation treatments to achieve self-pollination, intraspecific cross-pollination, open pollination (control), interspecific crosses or apomixis, then recorded flowering phenology and synchrony. RESULTS: All species flowered in the spring with a degree of synchrony, so that two pairs of species were predisposed to interspecific pollination (O. cantabrigiensis with O. robusta, O. streptacantha with O. tomentosa). All species had distinct reproductive systems: Opuntia cantabrigiensis is self-incompatible and did not produce hybrid seeds as an interspecific pollen recipient; O. robusta is a dioecious species, which formed a low proportion of hybrid seeds; O. streptacantha and O. tomentosa are self-compatible and produced hybrid seeds. CONCLUSIONS: Opuntia cantabrigiensis had a strong pollen-pistil barrier, likely due to its self-incompatibility. Opuntia robusta, the dioecious species, is an obligate outcrosser and probably partially lost its ability to prevent interspecific pollen germination. Given that the self-compatible species can set hybrid seeds, we conclude that pollen-pistil interaction and high flowering synchrony represent weak barriers; whether reproductive isolation occurs later in their life cycle (e.g., germination or seedling survival) needs to be determined.

Hybrid speciation driven by multilocus introgression of ecological traits.

Hybridization allows adaptations to be shared among lineages and may trigger the evolution of new species1,2. However, convincing examples of homoploid hybrid speciation remain rare because it is challenging to demonstrate that hybridization was crucial in generating reproductive isolation3. Here we combine population genomic analysis with quantitative trait locus mapping of species-specific traits to examine a case of hybrid speciation in Heliconius butterflies. We show that Heliconius elevatus is a hybrid species that is sympatric with both parents and has persisted as an independently evolving lineage for at least 180,000 years. This is despite pervasive and ongoing gene flow with one parent, Heliconius pardalinus, which homogenizes 99% of their genomes. The remaining 1% introgressed from the other parent, Heliconius melpomene, and is scattered widely across the H. elevatus genome in islands of divergence from H. pardalinus. These islands contain multiple traits that are under disruptive selection, including colour pattern, wing shape, host plant preference, sex pheromones and mate choice. Collectively, these traits place H. elevatus on its own adaptive peak and permit coexistence with both parents. Our results show that speciation was driven by introgression of ecological traits, and that speciation with gene flow is possible with a multilocus genetic architecture.

Karyotypic changes and diversification time in Epinephelidae groupers (Perciformes). Implications on reproductive isolation.

Groupers (Epinephelidae and Serranidae) have attracted special attention to fish farming, and their species offer good opportunities for successful hybridizations. Cytogenetic data allow a better understanding of the role of karyotypic diversification in the acquisition of post-zygotic reproductive isolation (RI). Thus, chromosomal analyses were performed on E. striatus (Caribbean Sea), E. coioides and E. tauvina (Indo-Pacific Region), using standard procedures and mapping of six repetitive DNA classes by the in situ hybridization. The three species have 2n=48 chromosomes. The karyotypes of E. coioides and E. striatus are composed only of acrocentric chromosomes (FN=48), while E. tauvina has 8 submetacentric chromosomes (FN=56). Heterochromatin has a preferential centromeric distribution, and the microsatellite repeats are dispersed throughout the chromosomes of all species. The 18S and 5S rDNA sites are unique but show a colocalization arrangement in E. tauvina and E. striatus. The chromosomal organization suggests that the three species still maintain a significant amount of syntenic regions. The range of the karyotype divergence and the RI levels showed low, but goes turn proportionally greater in relation to the divergence time between the parental species. The slow acquisition of postzygotic RI is consistent with the high karyotype homogeneity presented by Epinephelidae family.

Inferring the evolution of reproductive isolation in a lineage of fossil threespine stickleback, Gasterosteus doryssus.

Darwin attributed the absence of species transitions in the fossil record to his hypothesis that speciation occurs within isolated habitat patches too geographically restricted to be captured by fossil sequences. Mayr's peripatric speciation model added that such speciation would be rapid, further explaining missing evidence of diversification. Indeed, Eldredge and Gould's original punctuated equilibrium model combined Darwin's conjecture, Mayr's model and 124 years of unsuccessfully sampling the fossil record for transitions. Observing such divergence, however, could illustrate the tempo and mode of evolution during early speciation. Here, we investigate peripatric divergence in a Miocene stickleback fish, Gasterosteus doryssus. This lineage appeared and, over approximately 8000 generations, evolved significant reduction of 12 of 16 traits related to armour, swimming and diet, relative to its ancestral population. This was greater morphological divergence than we observed between reproductively isolated, benthic-limnetic ecotypes of extant Gasterosteus aculeatus. Therefore, we infer that reproductive isolation was evolving. However, local extinction of G. doryssus lineages shows how young, isolated, speciating populations often disappear, supporting Darwin's explanation for missing evidence and revealing a mechanism behind morphological stasis. Extinction may also account for limited sustained divergence within the stickleback species complex and help reconcile speciation rate variation observed across time scales.

The succession of ecological divergence and reproductive isolation in adaptive radiations.

Adaptive radiation is a major source of biodiversity but the way in which known components of ecological opportunity, ecological differentiation, and reproductive isolation underpin such biodiversity patterns remains elusive. Much is known about the evolution of ecological differentiation and reproductive isolation during single speciation events, but exactly how those processes scale up to complete adaptive radiations is less understood. Do we expect complete reproductive barriers between newly formed species before the ecological differentiation continues, or does proper species formation occur much later, long after the ecological diversification? Our goal is to improve our mechanistic understanding of adaptive radiations by analyzing an individual-based model that includes a suite of mechanisms that are known to contribute to biodiversity. The model includes variable biogeographic settings, ecological opportunities, and types of mate choice, which makes several different scenarios of an adaptive radiation possible. We find that evolving clades tend to exploit ecological opportunities early whereas reproductive barriers evolve later, demonstrating a decoupling of ecological differentiation and species formation. In many cases, we also find a long-term trend where assortative mating associated with ecological traits is replaced by sexual selection of neutral display traits as the primary mechanism for reproductive isolation. Our results propose that reticulate phylogenies are likely common and stem from initially low reproductive barriers, rather than the previously suggested idea of repeated hybridization events between well-separated species.

Yosemite toad (Anaxyrus canorus) transcriptome reveals interplay between speciation genes and adaptive introgression.

Genomes are heterogeneous during the early stages of speciation, with small 'islands' of DNA appearing to reflect strong adaptive differences, surrounded by vast seas of relative homogeneity. As species diverge, secondary contact zones between them can act as an interface and selectively filter through advantageous alleles of hybrid origin. Such introgression is another important adaptive process, one that allows beneficial mosaics of recombinant DNA ('rivers') to flow from one species into another. Although genomic islands of divergence appear to be associated with reproductive isolation, and genomic rivers form by adaptive introgression, it is unknown whether islands and rivers tend to be the same or different loci. We examined three replicate secondary contact zones for the Yosemite toad (Anaxyrus canorus) using two genomic data sets and a morphometric data set to answer the questions: (1) How predictably different are islands and rivers, both in terms of genomic location and gene function? (2) Are the adaptive genetic trait loci underlying tadpole growth and development reliably islands, rivers or neither? We found that island and river loci have significant overlap within a contact zone, suggesting that some loci are first islands, and later are predictably converted into rivers. However, gene ontology enrichment analysis showed strong overlap in gene function unique to all island loci, suggesting predictability in overall gene pathways for islands. Genome-wide association study outliers for tadpole development included LPIN3, a lipid metabolism gene potentially involved in climate change adaptation, that is island-like for all three contact zones, but also appears to be introgressing (as a river) across one zone. Taken together, our results suggest that adaptive divergence and introgression may be more complementary forces than currently appreciated.

Do genetic loci that cause reproductive isolation in the lab inhibit gene flow in nature?

The genetic dissection of reproductive barriers between diverging lineages provides enticing clues into the origin of species. One strategy uses linkage analysis in experimental crosses to identify genomic locations involved in phenotypes that mediate reproductive isolation. A second framework searches for genomic regions that show reduced rates of exchange across natural hybrid zones. It is often assumed that these approaches will point to the same loci, but this assumption is rarely tested. In this perspective, we discuss the factors that determine whether loci connected to postzygotic reproductive barriers in the laboratory are inferred to reduce gene flow in nature. We synthesize data on the genetics of postzygotic isolation in house mice, one of the most intensively studied systems in speciation genetics. In a rare empirical comparison, we measure the correspondence of loci tied to postzygotic barriers via genetic mapping in the laboratory and loci at which gene flow is inhibited across a natural hybrid zone. We find no evidence that the two sets of loci overlap beyond what is expected by chance. In light of these results, we recommend avenues for empirical and theoretical research to resolve the potential incongruence between the two predominant strategies for understanding the genetics of speciation.

Genomic islands of speciation harbor genes underlying coloration differences in a pair of Neotropical seedeaters.

Incomplete speciation can be leveraged to associate phenotypes with genotypes, thus providing insights into the traits relevant to the reproductive isolation of diverging taxa. We investigate the genetic underpinnings of the phenotypic differences between Sporophila plumbea and Sporophila beltoni. Sporophila beltoni has only recently been described based, most notably, based on differences in bill coloration (yellow vs. black in S. plumbea). Both species are indistinguishable through mtDNA or reduced-representation genomic data, and even whole-genome sequencing revealed low genetic differentiation. Demographic reconstructions attribute this genetic homogeneity to gene flow, despite divergence in the order of millions of generations. We found a narrow hybrid zone in southern Brazil where genetically, yet not phenotypically, admixed individuals appear to be prevalent. Despite the overall low genetic differentiation, we identified 3 narrow peaks along the genome with highly differentiated SNPs. These regions harbor 6 genes, one of which is involved in pigmentation (EDN3) and is a candidate for controlling bill color. Within the outlier peaks, we found signatures of resistance to gene flow, as expected for islands of speciation. Our study shows how genes related to coloration traits are likely involved in generating prezygotic isolation and establishing species boundaries early in speciation.

The extent of introgression between incipient Clarkia species is determined by temporal environmental variation and mating system.

Introgression is pervasive across the tree of life but varies across taxa, geography, and genomic regions. However, the factors modulating this variation and how they may be affected by global change are not well understood. Here, we used 200 genomes and a 15-y site-specific environmental dataset to investigate the effects of environmental variation and mating system divergence on the magnitude of introgression between a recently diverged outcrosser-selfer pair of annual plants in the genus Clarkia. These sister taxa diverged very recently and subsequently came into secondary sympatry where they form replicated contact zones. Consistent with observations of other outcrosser-selfer pairs, we found that introgression was asymmetric between taxa, with substantially more introgression from the selfer to the outcrosser. This asymmetry was caused by a bias in the direction of initial F1 hybrid formation and subsequent backcrossing. We also found extensive variation in the outcrosser's admixture proportion among contact zones, which was predicted nearly entirely by interannual variance in spring precipitation. Greater fluctuations in spring precipitation resulted in higher admixture proportions, likely mediated by the effects of spring precipitation on the expression of traits that determine premating reproductive isolation. Climate-driven hybridization dynamics may be particularly affected by global change, potentially reshaping species boundaries and adaptation to novel environments.

Divergent dynamics of sexual and habitat isolation at the transition between stick insect populations and species.

Speciation is often viewed as a continuum along which populations diverge until they become reproductively-isolated species. However, such divergence may be heterogeneous, proceeding in fits and bursts, rather than being uniform and gradual. We show in Timema stick insects that one component of reproductive isolation evolves non-uniformly across this continuum, whereas another does not. Specifically, we use thousands of host-preference and mating trials to study habitat and sexual isolation among 42 pairs of taxa spanning a range of genomic differentiation and divergence time. We find that habitat isolation is uncoupled from genomic differentiation within species, but accumulates linearly with it between species. In contrast, sexual isolation accumulates linearly across the speciation continuum, and thus exhibits similar dynamics to morphological traits not implicated in reproductive isolation. The results show different evolutionary dynamics for different components of reproductive isolation and highlight a special relevance for species status in the process of speciation.

Speciation across biomes: Rapid diversification with reproductive isolation in the Australian delicate mice.

Phylogeographic studies of continental clades, especially when combined with palaeoclimate modelling, provide powerful insight into how environment drives speciation across climatic contexts. Australia, a continent characterized by disparate modern biomes and dynamic climate change, provides diverse opportunity to reconstruct the impact of past and present environments on diversification. Here, we use genomic-scale data (1310 exons and whole mitogenomes from 111 samples) to investigate Pleistocene diversification, cryptic diversity, and secondary contact in the Australian delicate mice (Hydromyini: Pseudomys), a recent radiation spanning almost all Australian environments. Across northern Australia, we find no evidence for introgression between cryptic lineages within Pseudomys delicatulus sensu lato, with palaeoclimate models supporting contraction and expansion of suitable habitat since the last glacial maximum. Despite multiple contact zones, we also find little evidence of introgression at a continental scale, with the exception of a potential hybrid zone in the mesic biome. In the arid zone, combined insights from genetic data and palaeomodels support a recent expansion in the arid specialist P. hermannsburgensis and contraction in the semi-arid P. bolami. In the face of repeated secondary contact, differences in sperm morphology and chromosomal rearrangements are potential mechanisms that maintain species boundaries in these recently diverged species. Additionally, we describe the western delicate mouse as a new species and recommend taxonomic reinstatement of the eastern delicate mouse. Overall, we show that speciation in an evolutionarily young and widespread clade has been driven by environmental change, and potentially maintained by divergence in reproductive morphology and chromosome rearrangements.

Temperature and sex shape reproductive barriers in a climate change hotspot.

Climate change is altering species ranges and reproductive interactions in existing ranges, offering species new scope to mate and hybridize. The outcomes will depend on how environmental factors shape reproductive barriers across life stages, yet this is rarely assessed across the environments that species encounter in nature. We assess prezygotic and postzygotic barriers, and their dependence on temperature and parental sex, in species of a reef-building tubeworm (Galeolaria) from a fast-warming biodiversity hotspot in southern Australia. By replicating pure and reciprocal hybrid crosses across 5 temperatures spanning species' thermal ranges, we estimate thermal tolerance curves (defining niches) for crosses and reproductive isolation at each temperature. By also replicating crosses at 3 life stages, we partition the contributions of prezygotic barriers at fertilization, postzygotic barriers at embryogenesis, and postzygotic barriers at larval development to reproductive isolation. We show that barriers are weaker at fertilization and embryogenesis, but stronger and more temperature sensitive at larval development, as species diverge in thermal niche. Asymmetry of barriers between parental sexes, moreover, suggests a complex interplay between niche differentiation and maternal inheritance. Our findings point to a key role for temperature in reproductive isolation, but also challenges for predicting the fate of isolation in future climates.

The evolution of seminal fluid gene expression and postmating reproductive isolation in Drosophila.

Seminal fluid protein (Sfp) genes show, in general, a higher rate of sequence divergence than genes from other categories, which is often attributed to forms of postcopulatory sexual selection or sexual conflict. Recently, the relaxation of selective constraints has been proposed as an alternative explanation for the rapid sequence evolution of Sfps and other genes with sex-limited expression. The expression of Sfp genes is a likely target of selection, but the evolution of differences in their expression levels is less understood. Here, we explore both polymorphism and divergence in Sfp gene expression between Drosophila melanogaster and Drosophila simulans, how selection might have influenced their expression, and whether changes in expression might trigger the evolution of reproductive isolating barriers. In our analysis, Sfp genes showed higher divergence, but not higher polymorphism, in expression than the average male reproductive glands gene. Sfp genes with reproductive-tissue-specific expression were enriched for both directional and stabilizing selection, while relaxed selection was the predominant mode of evolution among Sfp genes with any other nonreproductive tissue-specific or nontissue-specific expression. The knockdown of single genes known to affect intraspecific sperm competition, and with patterns of expression divergence and polymorphism suggestive of directional selection, was not enough to break down postmating reproductive isolation barriers between species. Our results identify the expression of male-specific Sfp genes as an enriched target of selection and suggest a complex molecular relationship between postcopulatory sexual selection on a single gene's expression and its effect on the onset of speciation.

Coevolutionary Interactions between Sexual and Habitat Isolation during Reinforcement.

Speciation often involves the evolution of multiple genetic-based barriers to gene flow (i.e., "coupling"). However, barriers may exhibit a diversity of evolutionary interactions during speciation. These dynamics are important in reinforcement, where selection may favor different prezygotic isolating barriers to avoid maladaptive hybridization. Here we study the interaction between evolution of sexual and habitat isolation. We first review the empirical literature where both barriers were explicitly considered, and then develop a population genetic model of reinforcement. Most studies of both sexual and habitat isolation were found in phytophagous insect systems. In 76% of these studies, both barriers coevolved; the remaining cases either showed only habitat isolation (21%) or only sexual isolation (3%). Our two-allele genetic mechanism model of each barrier also found that these often coevolved, but habitat isolation was generally more effective during reinforcement. Depending on the fitness of hybrids (e.g., Dobzhansky-Muller incompatibilities) and initial migration rate, these barriers could either facilitate, curtail, or have no effect on each other. This indicates that basic parameters will alter the underlying evolutionary dynamics, and thus the nature of "speciation coupling" will be highly variable in natural systems. Finally, we studied initially asymmetrical migration rates and found that populations with higher initial emigration evolved stronger habitat isolation, while populations that initially received more immigrants exhibited stronger sexual isolation. These results are in line with observations in some empirical studies, but more data is needed to test their generality.

How Important Is Sexual Isolation to Speciation?

A central role for sexual isolation in the formation of new species and establishment of species boundaries has been noticed since Darwin and is frequently emphasized in the modern literature on speciation. However, an objective evaluation of when and how sexual isolation plays a role in speciation has been carried out in few taxa. We discuss three approaches for assessing the importance of sexual isolation relative to other reproductive barriers, including the relative evolutionary rate of sexual trait differentiation, the relative strength of sexual isolation in sympatry, and the role of sexual isolation in the long-term persistence of diverging forms. First, we evaluate evidence as to whether sexual isolation evolves faster than other reproductive barriers during the early stages of divergence. Second, we discuss available evidence as to whether sexual isolation is as strong or stronger than other barriers between closely related sympatric species. Finally, we consider the effect of sexual isolation on long-term species persistence, relative to other reproductive barriers. We highlight challenges to our knowledge of and opportunities to improve upon our understanding of sexual isolation from different phases of the speciation process.

Genome sequences and population genomics reveal climatic adaptation and genomic divergence between two closely related sweetgum species.

Understanding the genetic basis of population divergence and adaptation is an important goal in population genetics and evolutionary biology. However, the relative roles of demographic history, gene flow, and/or selective regime in driving genomic divergence, climatic adaptation, and speciation in non-model tree species are not yet fully understood. To address this issue, we generated whole-genome resequencing data of Liquidambar formosana and L. acalycina, which are broadly sympatric but altitudinally segregated in the Tertiary relict forests of subtropical China. We integrated genomic and environmental data to investigate the demographic history, genomic divergence, and climatic adaptation of these two sister species. We inferred a scenario of allopatric species divergence during the late Miocene, followed by secondary contact during the Holocene. We identified multiple genomic islands of elevated divergence that mainly evolved through divergence hitchhiking and recombination rate variation, likely fostered by long-term refugial isolation and recent differential introgression in low-recombination genomic regions. We also found some candidate genes with divergent selection signatures potentially involved in climatic adaptation and reproductive isolation. Our results contribute to a better understanding of how late Tertiary/Quaternary climatic change influenced speciation, genomic divergence, climatic adaptation, and introgressive hybridization in East Asia's Tertiary relict flora. In addition, they should facilitate future evolutionary, conservation genomics, and molecular breeding studies in Liquidambar, a genus of important medicinal and ornamental values.

The role of divergent host use and geography in the evolution of habitat isolation and sexual isolation among sister species of Belonocnema gall wasps.

Ecology and geography can play important roles in the evolution of reproductive isolation across the speciation continuum, but few studies address both at the later stages of speciation. This notable gap in knowledge arises from the fact that traditional ecological speciation studies have predominantly focused on the role of ecology in initiating the speciation process, while many studies exploring the effect of geography (e.g., reinforcement) concentrate on species pairs that lack divergent ecological characteristics. We simultaneously examine the strength of habitat isolation and sexual isolation among three closely related species of Belonocnema gall-forming wasps on two species of live oaks, Quercus virginiana and Q. geminata, that experience divergent selection from their host plants and variable rates of migration due to their geographic context. We find that the strength of both habitat isolation and sexual isolation is lowest among allopatric species pairs with the same host plant association, followed by allopatric species with different host plant associations, and highest between sympatric species with different host-plant associations. This pattern suggests that divergent selection due to different host use interacts with geography in the evolution of habitat isolation and sexual isolation during the later stages of speciation of Belonocnema wasps.