ECOLOGICAL GENETICS OF A MOSAIC HYBRID ZONE: MITOCHONDRIAL,NUCLEAR, AND REPRODUCTIVE DIFFERENTIATION OF CRICKETS BY SOIL TYPE

We investigated the effects that habitat variation has on the structure and dynamics of a hybrid zone between two closely related crickets in Connecticut. A collecting protocol was developed in which crickets were sampled from characteristic habitats on either side of the hybrid zone and from two distinct habitat types within the zone. Presumptive pure Gryllus pennsylvanicus were sampled from fields in northwestern Connecticut and represent “inland” populations. “Pure” Gryllus firmus were sampled from beaches along the coast and represent the “coastal” populations. Crickets from within the hybrid zone were sampled from two different soil types: the “loam” populations from loamy soils and the “sand” populations from sandy soils. Moreover, an attempt was made to identify closely adjacent sand and loam localities to determine the scale of habitat variation and its possible effects on hybrid-zone structure. In general, there was little variation in morphological traits or in allozyme and mtDNA genotype frequencies among localities from within each of the four habitat types. Between each of the closely situated sand and loam localities within the hybrid zone, however, there were very significant differences in each of these sets of markers. In addition, crickets from hybrid-zone populations were tested for reproductive isolation. The asymmetric outcome of hybrid crosses that exists across the zone (Harrison, 1983) also exists on a finer ecological scale within the zone. Thus, this hybrid zone is a mosaic of strikingly differentiated populations. The dynamics of hybrid zones with mosaic structures are discussed in contrast to the traditional clinal models. The data are also discussed in light of the semipermeable nature of species boundaries. The extent to which a species boundary is permeable varies not only from one genetic marker to the next, but also with the ecological and geographic context of species interaction.     

GENETIC VARIATION IN CRICKET CALLING SONG ACROSS A HYBRID ZONE BETWEEN TWO SIBLING SPECIES

The sibling ground crickets Allonemobius fasciatus and A. socius meet along a mosaic hybrid zone at ≈ 40°N latitude in eastern North America. In this paper we report the findings of a genetic analysis of calling-song variation within and among six cricket populations sampled along a transect through the hybrid zone in southern New Jersey. We compared aspects of the calling song of both wild-caught and laboratory-reared crickets to test the hypothesis that population differences in song observed in the wild were genetically based. We found significant, species-level differences in all aspects of the calling song, and these differences persisted even after a generation of common-garden rearing in the laboratory, supporting the hypothesis that interspecific variation observed in the wild largely reflects genetic differentiation between the two taxa. A discriminant function analysis indicated that individual crickets could be assigned to the proper taxon with less than 10% error, supporting the premise that calling song could be used by female crickets as a mechanism for species recognition. One population, collected from within the hybrid zone and containing significant numbers of hybrid individuals, was intermediate in its calling song, presumably reflecting this population's mixed genetic makeup. In this hybrid zone population, song phenotype was highly correlated to a hybrid index score generated using species-specific alleles at four diagnostic allozyme markers, suggesting a multigenic basis to calling-song variation in these crickets as well as linkage disequilibrium between markers and song. Based on an analysis of laboratory-reared full-sib families, broad-sense heritabilities for calling-song characteristics were generally significant in the two A. socius populations, whereas many components of song showed no significant family effects in the three A. fasciatus populations. The genotypically mixed, hybrid zone population showed very high heritabilities for most calling-song components, which likely reflect the influence of interspecific gene flow on genetic variation for quantitative traits.     

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.     

BARRIERS TO GENE EXCHANGE BETWEEN CLOSELY RELATED CRICKET SPECIES. II. LIFE CYCLE VARIATION AND TEMPORAL ISOLATION

The closely related field crickets Gryllus firmus and Gryllus pennsylvanicus are known to hybridize in a zone of contact that extends over more than 800 km from the Blue Ridge in Virginia to southern Connecticut. Here I present evidence that the extent of temporal reproductive isolation of the two cricket species varies along the length of the zone. Adults of G. firmus and G. pennsylvanicus occur synchronously in Connecticut but G. firmus matures significantly later than G. pennsylvanicus along the Blue Ridge in Virginia. Variation in the extent of temporal isolation is a consequence of intrinsic differences in the egg-to-adult development time of G. firmus from the two localities. In laboratory rearing experiments, there is little variation in development time among crickets from G. pennsylvanicus populations ranging from Vermont to Virginia. However, G. firmus from Virginia takes significantly longer to mature than G. firmus from Connecticut. Comparisons between species from neighboring localities show that lowland Virginia G. firmus take much longer to develop and are larger as adults than montane Virginia G. pennsylvanicus. In Connecticut, crickets of the two species differ very little in development time and body size. Patterns of variation in development time and body size are compared with similar data for frogs along one of the same environmental gradients and for field crickets along similar gradients elsewhere. Although G. firmus and G. pennsylvanicus are temporally isolated in Virginia, adults of the two species do occur together. Examination of Esterase genotypes of field-inseminated females and their progeny from a mixed population on the Blue Ridge suggests that pre-mating barriers other than temporal isolation are not effective in preventing gene exchange.     

A fine-scale spatial analysis of the mosaic hybrid zone between Gryllus firmus and Gryllus pennsylvanicus

Abstract.— The pattern of character variation within a hybrid zone, the hybrid zone structure, has been used to infer the processes that maintain hybrid zones. Unfortunately it is difficult to infer process from structure alone because many different processes can produce the same pattern of character variation. Mosaic hybrid zones may be maintained by exogenous selection in a heterogeneous environment and/or endogenous selection against hybrid individuals; habitat preference, premating isolating barriers and/or fertility selection can also contribute. The spatial scale at which a hybrid zone is sampled affects its apparent structure; a hybrid zone may appear clinal at one scale and mosaic at another. Here, we sample the mosaic hybrid zone between two field crickets, Gryllus firmus and G. pennsylvanicus , at a scale that spans the boundaries between individual soil-habitat patches. From our analysis, we find that at fine scales, the mosaic hybrid zone resolves into a set of steep clines across patch boundaries. Both morphological and molecular traits exhibit sharp and generally concordant clines. However, clines for mitochondrial DNA and one anonymous nuclear marker are clearly displaced as a result of current hybridization or past introgression (the "ghost of hybridization past"). Thus, scale is important for the structure of this and probably other hybrid zones. The extremely sharp, concordant clines across patch boundaries indicate that the cricket hybrid zone is undoubtedly structured by selection. However, the detailed mechanisms responsible for the maintenance of the hybrid zone–whether endogenous selection against hybrids, exogenous selection by the environment, and/or behavioral preferences for mates or habitats– remain to be elucidated. Determining these mechanisms will depend on closer inspection of the organisms themselves and their interactions, as is the case for all hybrid zones.     

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 biogeography and host relations of a parasitoid fly

Successful geographic range expansion by parasites and parasitoids may also require host range expansion. Thus, the evolutionary advantages of host specialization may trade off against the ability to exploit new host species encountered in new geographic regions. Here, we use molecular techniques and confirmed host records to examine biogeography, population divergence, and host flexibility of the parasitoid fly, Ormia ochracea (Bigot). Gravid females of this fly find their cricket hosts acoustically by eavesdropping on male cricket calling songs; these songs vary greatly among the known host species of crickets. Using both nuclear and mitochondrial genetic markers, we (a) describe the geographical distribution and subdivision of genetic variation in O. ochracea from across the continental United States, the Mexican states of Sonora and Oaxaca, and populations introduced to Hawaii; (b) demonstrate that the distribution of genetic variation among fly populations is consistent with a single widespread species with regional host specialization, rather than locally differentiated cryptic species; (c) identify the more‐probable source populations for the flies introduced to the Hawaiian islands; (d) examine genetic variation and substructure within Hawaii; (e) show that among‐population geographic, genetic, and host song distances are all correlated; and (f) discuss specialization and lability in host‐finding behavior in light of the diversity of cricket songs serving as host cues in different geographically separate populations.     

PATTERNS OF HYBRIDIZATION IN THE PIRIQUETA CAROLINIANA COMPLEX IN CENTRAL FLORIDA: EVIDENCE FOR AN EXPANDING HYBRID ZONE

We have identified a broad zone of hybridization between two morphologically and ecologically distinct herbaceous perennial taxa (morphotypes) within the Piriqueta caroliniana complex, which extends more than 300 km across the central Florida peninsula. Phylogeographic analyses indicate that the caroliniana morphotype has been present in north and central Florida since the early Pleistocene and that the viridis morphotype has immigrated into southern Florida much more recently. We examine the distribution of diagnostic morphological characters and nuclear genetic markers to assess the extent and patterns of introgression in this system. The results are consistent with the hypothesis that this hybrid zone has expanded north in recent history as viridis alleles have introgressed into regions that were previously occupied by populations of caroliniana. Genetic markers diagnostic for caroliniana have consistently high frequencies across the hybrid zone, whereas markers for viridis are extremely variable among populations with frequency reversals in adjacent populations. The latter pattern is probably the result of the combined stochastic effects of dispersal and drift on viridis alleles as they introgressed northward. Additional evidence for the recent expansion of this hybrid zone comes from patterns of variation for morphological and genetic markers. As expected for an expanding hybrid zone, within-population morphological variation was greatest toward the advancing front of introgression and levels of genetic variation for neutral diagnostic markers were greatest in the region of initial contact and lower in areas of recent expansion. The observed patterns of variation suggest that at least some hybrid genotypes have high fitnesses, which has led to the expansion of the hybrid zone via the displacement of parental genotypes in central Florida.     

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.     

Introgressive hybridization and morphological transgression in the contact zone between two Mediterranean Solea species

Hybrid zones provide natural experiments where new combinations of genotypes and phenotypes are produced. Studying the reshuffling of genotypes and remodeling of phenotypes in these zones is of particular interest to document the building of reproductive isolation and the possible emergence of transgressive phenotypes that can be a source of evolutionary novelties. Here, we specifically investigate the morphological variation patterns associated with introgressive hybridization between two species of sole, Solea senegalensis and Solea aegyptiaca. The relationship between genetic composition at nuclear loci and individual body shape variation was studied in four populations sampled across the hybrid zone located in northern Tunisia. A strong correlation between genetic and phenotypic variation was observed among all individuals but not within populations, including the two most admixed ones. Morphological convergence between parental species was observed close to the contact zone. Nevertheless, the samples taken closest to the hybrid zone also displayed deviant segregation of genotypes and phenotypes, as well as transgressive phenotypes. In these samples, deviant body shape variation could be partly attributed to a reduced condition index, and the distorted genetic composition was most likely due to missing allelic combinations. These results were interpreted as an indication of hybrid breakdown, which likely contributes to postmating reproductive isolation between the two species.     

Genetic and phenotypic variation in a colourful treefrog across five geographic barriers

Aim Understanding the patterns and processes underlying phenotype in a polytypic species provides key insights into microevolutionary mechanisms of diversification. The red‐eyed treefrog, Agalychnis callidryas, exhibits strong regional differentiation in colour pattern, corresponding to five admixed mitochondrial DNA clades. We evaluated spatial diversity patterns across multiple, putative barriers to examine the fine‐scale processes that mediate phenotypic divergence between some regions while maintaining homogeneity between others. Location We examined patterns of phenotypic diversification among 17 sites that span five putative biogeographic barriers in lower Central America (Costa Rica and Panama). Methods We tested the extent to which genetic distance (F_ST) derived from six multilocus nuclear genotypes covaried with measures of phenotypic distance (leg coloration) within and between biogeographic regions. We used linear regression analyses to determine the role of geographic and genetic factors in structuring spatial patterns of phenotypic diversity. Results The factors that best explained patterns of phenotypic diversity varied among biogeographic regions. We identified one geographic barrier that impeded gene exchange and resulted in concordant phenotypic divergence across the Continental Divide, isolating Caribbean and Pacific populations. Across Caribbean Costa Rican populations, one barrier structured phenotypic but not genetic diversity patterns, indicating a role for selection. In other regions, the putative barriers had no determining effect on either genetic or leg colour structure. Main conclusions The processes mediating the distribution and diversification of colour pattern in this polytypic, wide‐ranging treefrog varied among biogeographic regions. Spatially varying selection combined with the isolating effects of geographic factors probably resulted in the patchy distribution of colour diversity across Costa Rican and Panamanian populations.     

Viability selection on overwintering eggs in a field cricket mosaic hybrid zone

Habitat segregation allows recently diverged taxa to minimize competition and maximize fitness. Consequently, the overall distribution of a species in part will be determined by interactions with their close relatives. Two recently diverged but hybridizing field crickets, Gryllus firmus and G. pennsylvanicus , form a mosaic hybrid zone in Connecticut in which each species is associated with a specific soil type: G. firmus is found on sand soils and G. pennsylvanicus is found on loam soils. Both species produce diapausing eggs, which spend the winter in the ground. We investigate how viability selection on overwintering eggs in different soils influences the distributions of the two species, habitat partitioning between them, and the structure of the mosaic hybrid zone. Our results suggest that selection on eggs by soil types is not important in determining the success of crickets on different soils. However, winter climate has a strong effect on egg viability, and variation in egg survivorship over different winter climate regimes likely plays an important role in determining the distribution of these cricket species and the position of the hybrid zone.     

Species‐specific habitat preferences do not shape the structure of a crested newt hybrid zone (Triturus cristatus x T. carnifex)

Reproductive isolation barriers maintain the integrity of species by preventing interspecific gene flow. They involve temporal, habitat or behavioral isolation acting before fertilization, and postzygotic isolation manifested as hybrid mortality or sterility. One of the approaches of how to study reproductive isolation barriers is through the analysis of hybrid zones. In this paper, we describe the structure of a hybrid zone between two crested newt species (Triturus cristatus and T. carnifex) in the southern part of the Czech Republic using morphological, microsatellite, and mitochondrial (mtDNA) markers. Specifically, we tested the hypothesis that the structure of the hybrid zone is maintained by species‐specific habitat preferences. Comparing the genetic structure of populations with geographical and ecological parameters, we found that the hybrid zone was structured primarily geographically, with T. cristatus‐like populations occurring in the northeast and T. carnifex‐like populations in the southwest. Despite T. cristatus tending to occur in deeper ponds and T. carnifex on localities with more shading, the effect of both ecological parameters on the structure of the zone was minimal. Next, we corroborated that T. carnifex individuals and some hybrids possess mtDNA of T. dobrogicus, whose nuclear background was not detected in the studied hybrid zone. Hybridization between T. carnifex and T. dobrogicus (resulting in unidirectional mtDNA introgression) had to predate subsequent formation of the hybrid zone between T. cristatus and T. carnifex. Populations of crested newts in the southern part of the Czech Republic thus represent a genetic mosaic of nuclear and mitochondrial genomes of three species.     

GENETIC POPULATION STRUCTURE AND MATING SYSTEM IN CHONDRUS CRISPUS (RHODOPHYTA)1

Chondrus crispus Stackh. has been intensely studied, yet no study to date has elucidated its population structure or mating system despite many populations in which there was a haploid bias and lack of male gametophytes. Therefore, 12 nuclear microsatellite loci were identified in this red alga. Microsatellite markers were developed and tested against a panel of specimens collected from two shore levels at two sites in Brittany, France: Pointe de Primel and Pointe de la Jument, Concarneau. Single locus genetic determinism was verified at eight polymorphic loci, as only one band was observed for haploid genotypes, whereas one or two bands were observed for diploids. These markers enabled the detection of unique genotypes within sampled populations, indicating that very few fronds shared the same multilocus genotype. This finding suggests that asexual reproduction was not the prevailing mode of reproduction. In addition, we explored the hierarchical population structure showing that gene flow is restricted at small spatial scales (<50 m) between upper and lower Chondrus‐range populations within a shore. Sexual reproduction predominated in the populations of C. crispus studied, but probably due to fine‐scale spatial substructuring, inbreeding was also significant. In conclusion, this study reveals that fine‐scale genetic variation is of major importance in C. crispus, suggesting that differences between microhabitats should be essential in understanding evolutionary processes in this species.     

Cytonuclear discordance and the species status of Myotis myotis and Myotis blythii (Chiroptera)

We used both highly variable mitochondrial and nuclear loci to investigate the large mouse‐eared bat species complex in the Western Palaearctic to clarify their systematic position. Although mitochondrial lineages show no species segregation and some haplotypes are shared between Myotis myotis and Myotis blythii sensu lato, Bayesian clustering methods based on multilocus genotypes indicate highly concordant nuclear and morphological species assignment. These multilocus, nuclear analyses detected only a single putative F₁ hybrid in the extensive areas of sympatry sampled, thus confirming the biological species status of M. myotis and M. blythii s.l. We propose that the strong cytonuclear discordance in these species complex results from a combination of prior spatial isolation of the two species in different glacial refugia, followed by a succession of mitochondrial introgression events that occurred during the eastward and westward expansions of M. myotis and of M. blythii, respectively. The nuclear markers further indicate the presence of a notable genetic discontinuity within M. myotis that broadly separates populations into an eastern and a western component with an overlap zone in the Balkans. This eastern and western discontinuity is also apparent in the mitochondrial lineages with the D haplogroup largely confined to samples found in Thrace and Asia Minor. None of these genetic discontinuities correspond to the distribution of the two commonly recognized M. myotis subspecies (myotis and macrocephalicus). We also show that distinct morphological subspecies within M. blythii (oxygnathus, omari, risorius and lesviacus) in Europe and the near‐East do not correlate with significant evolutionarily units, whether identified by mitochondrial or nuclear data and thus only represent local morphological variants with little taxonomic relevance.     

Detection and pattern of interspecific hybridization between Gliricidia sepium and G. maculata in Meso-America revealed by PCR-based assays

Gliricidia sepium provides a variety of products important for rural communities in tropical countries. Native populations in Meso-America currently form an important source of seed for distribution to farmers, but concerns centre on mechanisms which may lead to their genetic erosion, including anthropogenic dispersal and subsequent introgression from the related species, G. maculata. Populations of Gliricidia were examined genetically using approaches based on the polymerase chain reaction to test for interspecific hybridization and introgression between G. sepium and G. maculata. Analysis involved 13 RAPD and two RFLP-PCR markers which were identified to have species-diagnostic distributions. Data from both approaches corresponded and indicated three locations where multilocus genotypes were consistent with an hybrid origin. Data at one of these sites was consistent with introgression following hybridization. The hybrid origin of populations was supported by the intermediate geographical location of these sites to ‘pure’ populations of each species. Analysis of maternally inherited organellar DNA, which involved the detection of SSCPs in mitochondrial DNA amplification products, allowed further delineation of genetic structure among Gliricidia populations. Mitochondrial data indicated a high degree of organelle differentiation between sampled locations and identified G. sepium- and G. maculata-diagnostic haplotypes. This data supported the interpretation of genetic structure based on RAPDs and RFLP-PCR. In addition, cytonuclear analysis allowed the directionality of gene transfer during the formation of hybrid populations to be described. Despite evidence for the occurrence of interspecific hybridization and introgression in Gliricidia, important resource populations of G. sepium on the Pacific coast appear to have retained their genetic integrity. Implications in terms of the conservation and utilization of genetic resources within the genus are discussed.     

hybridlab (version 1.0): a program for generating simulated hybrids from population samples

We present the computer program hybridlab 1.0 for simulating intraspecific hybrids from population samples of nuclear genetic markers such as microsatellites, allozymes or SNPs (single nucleotide polymorphisms). The program generates a user‐specified number of multilocus F1 hybrid genotypes between any pair of potentially hybridizing populations included in a standard input‐file of multilocus genotypes for population genetic analysis. This simple, user‐friendly program has a wide range of applications for studying natural and artificial hybridization; in particular, for evaluating the statistical power for individual assignment of parental and hybrid individuals. An example of application for Atlantic cod populations is given.     

Stuck in the middle with you: close concordance between geographical clines in a cricket hybrid zone

Students of speciation have long recognized that hybridization between populations does not affect all parts of the genome in the same way (Key 1968, Bazykin 1969, Wu 2001, Nosil et al. 2009). For example, divergence is expected to be high at loci involved in Dobzhansky–Muller incompatibilities or at loci under divergent natural selection, while those that are effectively neutral should show only weak divergence. Studies that examine geographical clines at divergent loci in a hybrid zone can be particularly powerful, as here one can estimate how net selection is affecting each locus (Payseur 2010). An excellent example of this approach appears in this issue (Larson et al. 2014) for a hybrid zone between the crickets Gryllus firmus and Gryllus pennsylvanicus in the eastern United States.