PATTERNS OF VARIATION AND LINKAGE DISEQUILIBRIUM IN A FIELD CRICKET HYBRID ZONE

The distribution of multilocus genotypes found within a natural hybrid zone is determined by the sample of genotypes present when the hybrid zone first formed, by subsequent patterns of genetic exchange between the hybridizing taxa, and by drift and selection within each of the hybrid zone populations. We have used anonymous nuclear DNA restriction fragment polymorphisms (RFLPs) to characterize the array of multilocus genotypes present within a well-studied hybrid zone between two eastern North American field crickets, Gryllus pennsylvanicus and Gryllus firmus. These crickets hybridize along a zone of contact that extends from New England to Virginia. Previous studies have shown that both premating and postmating barriers exist between the two cricket species, but the absence of diagnostic morphological and allozyme markers has made it difficult to assess the consequences of these barriers for genetic exchange. Analyses based on four diagnostic anonymous nuclear markers indicate that hybrid zone populations in Connecticut contain few F₁ hybrids, and that nonrandom associations persist among nuclear gene markers, between nuclear and cytoplasmic markers, and between molecular markers and morphology. Field cricket populations within the hybrid zone are not “hybrid swarms” but consist primarily of crickets that are very much like one or the other of the parental species. Despite ample opportunity for genetic exchange and evidence for introgression at some loci, the two species remain quite distinct. Such a pattern appears to be characteristic of many natural hybrid zones.     

Searching for candidate speciation genes using a proteomic approach: seminal proteins in field crickets

In many animals, male seminal proteins influence gamete interactions and fertilization ability and are probably involved in barriers to gene flow between diverging lineages. Here we use a proteomic approach to identify seminal proteins that are transferred to females during copulation and that may be involved in fertilization barriers between two hybridizing field crickets (Gryllus firmus and Gryllus pennsylvanicus). Analyses of patterns of divergence suggest that much of the field cricket genome has remained undifferentiated following the evolution of reproductive isolation. By contrast, seminal protein genes are highly differentiated. Tests of selection reveal that positive selection is likely to be responsible for patterns of differentiation. Together, our observations suggest that some of the loci encoding seminal proteins may indeed play a role in fertilization barriers in field crickets.     

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.     

Identification and comparative analysis of accessory gland proteins in Orthoptera.

Accessory reproductive gland proteins (Acps) in Drosophila evolve quickly and appear to play an important role in ensuring the fertilization success of males. Moreover, Acps are thought to be involved in establishing barriers to fertilization between closely related species. While accessory glands are known to occur in the males of many insect groups, the proteins that are passed on to females by males during mating have not been well characterized outside of Drosophila. To gain a better understanding of these proteins, we characterized ESTs from the accessory glands of two cricket species, Allonemobius fasciatus and Gryllus firmus. Using an expressed sequence tag (EST) approach, followed by bioinformatic and evolutionary analyses, we found that many proteins are secreted and, therefore, available for transfer to the female during mating. Further, we found that most ESTs are novel, showing little sequence similarity between taxa. Evolutionary analyses suggest that cricket proteins are subject to diversifying selection and indicate that Allonemobius is much less polymorphic than Gryllus. Despite rapid nucleotide sequence divergence, there appears to be functional conservation of protein classes among Drosophila and cricket taxa.     

Influence of the Male Ejaculate on Post-Mating Prezygotic Barriers in Field Crickets

Post-copulatory interactions between males and females involve highly coordinated, complex traits that are often rapidly evolving and divergent between species. Failure to produce and deposit eggs may be a common post-mating prezygotic barrier, yet little is known about what prevents the induction of egg-laying between species. The field crickets, Gryllus firmus and G. pennsylvanicus are isolated by a one-way reproductive incompatibility; G. pennsylvanicus males fail to fertilize G. firmus eggs or to induce normal egg-laying in G. firmus females. We use experimental crosses to elucidate the role of accessory gland-derived vs. testis-derived components of the G. firmus male ejaculate on egg-laying in conspecific and heterospecific crosses. Using surgical castrations to create ‘spermless’ males that transfer only seminal fluid proteins (SFPs) we test whether G. firmus male SFPs can induce egg-laying in conspecific crosses and rescue egg-laying in crosses between G. pennsylvanicus males and G. firmus females. We find G. firmus SFPs induce only a small short-term egg-laying response and that SFPs alone cannot explain the normal induction of egg-laying. Gryllus firmus SFPs also do not rescue the heterospecific cross. Testis-derived components, such as sperm or prostaglandins, most likely stimulate egg-laying or act as transporters for SFPs to targets in the female reproductive tract. These results highlight the utility of experimental approaches for investigating the phenotypes that act as barriers between species and suggest that future work on the molecular basis of the one-way incompatibility between G. firmus and G. pennsylvanicus should focus on divergent testis-derived compounds or proteins in addition to SFPs.     

GENEALOGICAL DISCORDANCE AND PATTERNS OF INTROGRESSION AND SELECTION ACROSS A CRICKET HYBRID ZONE

In recently diverged species, ancestral polymorphism and introgression can cause incongruence between gene and species trees. In the face of hybridization, few genomic regions may exhibit reciprocal monophyly, and these regions, usually evolving rapidly under selection, may be important for the maintenance of species boundaries. In animals with internal fertilization, genes encoding seminal protein are candidate barrier genes. Recently diverged hybridizing species such as the field crickets Gryllus firmus and G. pennsylvanicus , offer excellent opportunities to investigate the origins of barriers to gene exchange. These recently diverged species form a well-characterized hybrid zone, and share ancestral polymorphisms across the genome. We analyzed DNA sequence divergence for seminal protein loci, housekeeping loci, and mtDNA, using a combination of analytical approaches and extensive sampling across both species and the hybrid zone. We report discordant genealogical patterns and differential introgression rates across the genome. The most dramatic outliers, showing near-zero introgression and more structured species trees, are also the only two seminal protein loci under selection. These are candidate barrier genes with possible reproductive functions. We also use genealogical data to examine the demographic history of the field crickets and the current structure of the hybrid zone.     

Structure of a mosaic hybrid zone between the field crickets Gryllus firmus and G. pennsylvanicus

Hybrid zones provide insight into the nature of species boundaries and the evolution of barriers to gene exchange. Characterizing multiple regions within hybrid zones is essential for understanding both their history and current dynamics. Here, we describe a previously uncharacterized region of a well‐studied hybrid zone between two species of field crickets, Gryllus pennsylvanicus and G. firmus. We use a combination of mitochondrial DNA sequencing, morphological data, and modeling of environmental variables to identify the ecological factors structuring the hybrid zone and define patterns of hybridization and introgression. We find an association between species distribution and natural habitat; Gryllus pennsylvanicus occupies natural habitat along forest edges and natural clearings, whereas G. firmus occupies more disturbed areas in agricultural and suburban environments. Hybridization and introgression occur across patch boundaries; there is evidence of substantial admixture both in morphological characters and mtDNA, over a broad geographic area. Nonetheless, the distribution of morphological types is bimodal. Given that F₁ hybrids are viable and fertile in the lab, this suggests that strong pre‐zygotic barriers are operating in this portion of the hybrid zone.     

Insight into the roles of selection in speciation from genomic patterns of divergence and introgression in secondary contact in venomous rattlesnakes

Investigating secondary contact of historically isolated lineages can provide insight into how selection and drift influence genomic divergence and admixture. Here, we studied the genomic landscape of divergence and introgression following secondary contact between lineages of the Western Diamondback Rattlesnake (Crotalus atrox) to determine whether genomic regions under selection in allopatry also contribute to reproductive isolation during introgression. We used thousands of nuclear loci to study genomic differentiation between two lineages that have experienced recent secondary contact following isolation, and incorporated sampling from a zone of secondary contact to identify loci that are resistant to gene flow in hybrids. Comparisons of patterns of divergence and introgression revealed a positive relationship between allelic differentiation and resistance to introgression across the genome, and greater‐than‐expected overlap between genes linked to lineage‐specific divergence and loci that resist introgression. Genes linked to putatively selected markers were related to prominent aspects of rattlesnake biology that differ between populations of Western Diamondback rattlesnakes (i.e., venom and reproductive phenotypes). We also found evidence for selection against introgression of genes that may contribute to cytonuclear incompatibility, consistent with previously observed biased patterns of nuclear and mitochondrial alleles suggestive of partial reproductive isolation due to cytonuclear incompatibilities. Our results provide a genome‐scale perspective on the relationships between divergence and introgression in secondary contact that is relevant for understanding the roles of selection in maintaining partial isolation of lineages, causing admixing lineages to not completely homogenize.     

Multiple barriers to gene exchange in a field cricket hybrid zone

Data on patterns of variation within hybrid zones, combined with studies of life history, mate choice, and hybrid performance, allow estimates of the contribution of different pre-zygotic and post-zygotic barriers to reproductive isolation. We examine the role of behavioural barriers to gene exchange in the maintenance of a hybrid zone between North American field crickets Gryllus firmus and Gryllus pennsylvanicus . We consider these barriers in the context of previous studies that documented temporal and ecological isolation and a one-way post-mating incompatibility (i.e. G. firmus females do not produce offspring when they mate only with heterospecific males). Based on no-choice mating experiments in the laboratory, we demonstrate strong behavioural pre-mating barriers between the two species, but no apparent fecundity or fertility costs for G. firmus females when they mate with both conspecific and heterospecific males. Furthermore, we show that G. firmus females do not discriminate between hybrids and conspecifics, whereas G. pennsylvanicus females do. This observation could explain the asymmetric allele introgression observed in the hybrid zone. We also document a failure of heterospecific males to induce normal oviposition in G. firmus females, which may be due to rapid evolution of accessory gland proteins and may serve as an additional barrier to gene exchange.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 390–402.     

Molecular dissection of nuptial gifts in divergent strains of Ostrinia moths

Seminal fluid proteins (SFPs) produced in the male accessory glands and ejaculatory duct are subject to strong sexual selection, often evolve rapidly and therefore may play a key role in reproductive isolation and species formation. However, little is known about reproductive proteins for species in which males transfer ejaculate to females using a spermatophore package. By combining RNA sequencing and proteomics, we characterize putative SFPs, identify proteins transferred in the male spermatophore and identify candidate genes contributing to a one‐way gametic incompatibility between Z and E strains of the European corn borer moth Ostrinia nubilalis. We find that the accessory glands and ejaculatory duct secrete over 200 highly expressed gene products, including peptidases, peptidase regulators and odourant‐binding proteins. A comparison between Ostrinia strains reveals that accessory gland and ejaculatory duct sequences with hormone degradation and peptidase activity are among the most extremely differentially expressed. However, most spermatophore peptides lack reproductive tissue bias or canonical secretory signal motifs and aproximately one‐quarter may be produced elsewhere before being sequestered by the male accessory glands during spermatophore production. In addition, most potential gene candidates for postmating reproductive isolation do not meet standard criteria for predicted SFPs and almost three‐quarters are novel, suggesting that both postmating sexual interactions and gametic isolation likely involve molecular products beyond traditionally recognized SFPs.     

Differential introgression of a female competitive trait in a hybrid zone between sex‐role reversed species

Mating behavior between recently diverged species in secondary contact can impede or promote reproductive isolation. Traditionally, researchers focus on the importance of female mate choice and male–male competition in maintaining or eroding species barriers. Although female–female competition is widespread, little is known about its role in the speciation process. Here, we investigate a case of interspecific female competition and its influence on patterns of phenotypic and genetic introgression between species. We examine a hybrid zone between sex‐role reversed, Neotropical shorebird species, the northern jacana (Jacana spinosa) and wattled jacana (J. jacana), in which female–female competition is a major determinant of reproductive success. Previous work found that females of the more aggressive and larger species, J. spinosa, disproportionately mother hybrid offspring, potentially by monopolizing breeding territories in sympatry with J. jacana. We find a cline shift of female body mass relative to the genetic center of the hybrid zone, consistent with asymmetric introgression of this competitive trait. We suggest that divergence in sexual characteristics between sex‐role reversed females can influence patterns of gene flow upon secondary contact, similar to males in systems with more typical sex roles.     

Hybridization following recent secondary contact results in asymmetric genotypic and phenotypic introgression between island species of Myzomela honeyeaters

Hybridization and introgression can have important evolutionary consequences for speciation, especially during early stages of secondary contact when reproductive barriers may be weak. Few studies, however, have quantified dynamics of hybridization and introgression in systems in which recent natural dispersal across a geographic barrier resulted in secondary contact. We investigated patterns of hybridization and introgression between two Myzomela honeyeaters (M. tristrami and M. cardinalis) that recently achieved secondary contact on Makira in the Solomon Islands. Hybridization in this system was hypothesized to be a byproduct of conspecific mate scarcity during early stages of colonization. Our research, however, provides evidence of ongoing hybridization more than a century after secondary contact. Mitochondrial sequencing revealed strongly asymmetric reproductive isolation that is most likely driven by postzygotic incompatibilities rather than prezygotic behavioral barriers. Nuclear introgression was observed from the native species (M. tristrami) to the colonizing species (M. cardinalis). Nuclear introgression in the reverse direction is almost exclusively limited to birds that are phenotypically M. tristrami but possess M. cardinalis mitochondrial haplotypes, consistent with introgression of plumage‐related alleles into the genomic background of M. cardinalis. These results provide unique insight into the dynamics and consequences of hybridization and introgression during early stages of secondary contact.     

The genomics of speciation: investigating the molecular correlates of X chromosome introgression across the hybrid zone between Mus domesticus and Mus musculus

Understanding the genetic details of reproductive isolation is a key goal in the study of speciation. Hybrid zones, geographical regions where two species meet and exchange genes, can provide insight into the genetic basis of reproductive isolation. This is especially true in species with mapped molecular markers because patterns of gene flow can be compared among different genomic regions. Even greater insight can be obtained in species with complete genome sequences because gene identity, gene number and other features of interest can be assessed for genomic regions with different patterns of introgression. Here, we review recent studies on the well-characterized hybrid zone between Mus domesticus and M. musculus , including a detailed survey of patterns of introgression for 13 markers on the X chromosome. We then compare levels of introgression for these 13 regions to a number of genomic attributes inferred from the complete sequence of the X chromosome, with two purposes. First, we identify candidate genes for reproductive isolation by finding genes that map to an X-linked region of reduced introgression and that are only expressed in the male germ line or that show high rates of protein evolution in comparison with rat. Second, we ask whether patterns of gene flow are correlated with recombination rate, gene density, base composition, CpG island density, mutation rate and the rate of protein evolution, as might be expected if many genes contribute to reproductive isolation. We identify seven candidate genes for reproductive isolation between M. domesticus and M. musculus , and our analyses reveal no general correlations between levels of introgression and other measured sequence characteristics. These results underline the utility of the house mouse as a model system for the study of speciation.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 523–534.     

Formation of a recent hybrid zone offers insight into the geographic puzzle and maintenance of species boundaries in musk turtles

Speciation is the result of an accumulation of reproductive barriers between populations, but pinpointing the factors involved is often difficult. However, hybrid zones can form when these barriers are not complete, especially when lineages come into contact in intermediate or modified habitats. We examine a hybrid zone between two closely related riverine turtle species, Sternotherus depressus and S. peltifer, and use dual‐digest RAD sequencing to understand how this hybrid zone formed and elucidate genomic patterns of reproductive isolation. First, the geographical extent and timing of formation of the hybrid zone is established to provide context for understanding the role of extrinsic and intrinsic reproductive isolating mechanisms in this system. The strength of selection on taxon‐specific contributions to maintenance of the hybrid zone is then inferred using a Bayesian genomic cline model. These analyses identify a role for selection inhibiting introgression in some genomic regions at one end of the hybrid zone and promoting introgression in many loci at the other. When selective pressures necessary to generate outliers to the genomic cline are considered with the geographical and temporal context of this hybrid zone, we conclude that habitat‐specific selection probably limits introgression from S. depressus to S. peltifer in the direction of river flow. However, selection is mediating rapid, unidirectional introgression from S. peltifer to S. depressus, which is probably facilitated by anthropogenic habitat alteration. These findings indicate a potentially imminent threat of population‐level genomic extinction for an already imperiled species due to ongoing human‐caused habitat alteration.     

Introgression between invasive and native blue mussels (genus Mytilus) in the central California hybrid zone

The ecological and genetic factors determining the extent of introgression between species in secondary contact zones remain poorly understood. Here, we investigate the relative importance of isolating barriers and the demographic expansion of invasive Mytilus galloprovincialis on the magnitude and the direction of introgression with the native Mytilus trossulus in a hybrid zone in central California. We use double‐digest restriction‐site‐associated DNA sequencing (ddRADseq) to genotype 1337 randomly selected single nucleotide polymorphisms and accurately distinguish early and advanced generation hybrids for the first time in the central California Mytilus spp. hybrid zone. Weak levels of introgression were observed in both directions but were slightly more prevalent from the native M. trossulus into the invasive M. galloprovincialis. Few early and advanced backcrossed individuals were observed across the hybrid zone confirming the presence of strong barriers to interbreeding. Heterogeneous patterns of admixture across the zone of contact were consistent with the colonization history of M. galloprovincialis with more extensive introgression in northern localities furthest away from the putative site of introduction in southern California. These observations reinforce the importance of dynamic spatial and demographic expansions in determining patterns of introgression between close congeners, even in those with high dispersal potential and well‐developed reproductive barriers. Our results suggest that the threat posed by invasive M. galloprovincialis is more ecological than genetic as it has displaced, and continues to displace the native M. trossulus from much of central and southern California.     

Premating reproductive barriers between hybridising cricket species differing in their degree of polyandry

Understanding speciation hinges on understanding how reproductive barriers arise between incompletely isolated populations. Despite their crucial role in speciation, prezygotic barriers are relatively poorly understood and hard to predict. We use two closely related cricket species, Gryllus bimaculatus and G. campestris, to experimentally investigate premating barriers during three sequential mate choice steps. Furthermore, we experimentally show a significant difference in polyandry levels between the two species and subsequently test the hypothesis that females of the more polyandrous species, G. bimaculatus, will be less discriminating against heterospecific males and hence hybridise more readily. During close-range mating behaviour experiments, males showed relatively weak species discrimination but females discriminated very strongly. In line with our predictions, this discrimination is asymmetric, with the more polyandrous G. bimaculatus mating heterospecifically and G. campestris females never mating heterospecifically. Our study shows clear differences in the strength of reproductive isolation during the mate choice process depending on sex and species, which may have important consequences for the evolution of reproductive barriers.     

Development of microsatellite markers for the wood cricket, Nemobius sylvestris (Orthoptera: Gryllidae)

Thirty-four novel microsatellite markers developed for wood cricket (Nemobius sylvestris) were tested and optimized. Twenty-five microsatellite loci were polymorphic, exhibiting between two and nine alleles. Observed heterozygosities ranged from 0.038 to 0.925. The microsatellites were also tested in a species belonging to another genus of the Gryllidae family (Gryllus bimaculatus). Two markers produced clear banding patterns with the expected product size. These markers will be used to study the effects of forest fragmentation on genetic connectivity using wood cricket as a model species.