Assortative preferences and discrimination by females against hybrid male song in the grasshoppers Chorthippus brunneus and Chorthippus jacobsi (Orthoptera: Acrididae)

The grasshoppers Chorthippus brunneus and Chorthippus jacobsi are highly differentiated for male mating signals, and form a mosaic hybrid zone in northern Spain. At some sites within this zone, many hybrids are observed. At others, few hybrids are observed. Such bimodal sites may reflect recent contacts between parental genotypes, or local variation in levels of assortative mating or selection against hybrids. Playback of 12 parental and F1 male songs to 296 parental and hybrid females revealed positive assortative preferences in C. brunneus and C. jacobsi females, supporting a direct role of male mating signals in female choice. However, all female genotypic classes showed reduced responsiveness to F1 male songs. Such sexual selection against hybrids is consistent with the narrow cline width observed in the field for song characters relative to morphology. These results have implications for the genetic structure of the hybrid zone and for models of speciation by reinforcement.     

Variation in complex courtship traits across a hybrid zone between grasshopper species of the Chorthippus albomarginatus group

Closely related grasshopper species of the Chorthippus albomarginatus group are notable for their extremely complex courtship songs, accompanied by a visual display. Two species of this group, Ch. albomarginatus and Ch. oschei, were previously shown to hybridize in a wide mosaic hybrid zone in Ukraine and Moldova. In this paper, variation in five courtship song characters, one character of visual display and the number of stridulatory pegs were analysed across the hybrid zone to estimate selection against hybrids and strength of assortative mating. Comparison of cline width and position across the hybrid zone showed concordant and coincident clines in four traits, such as three song characters and one morphological character, and discordant and non‐coincident clines in two other song characters and the character of visual display. Concordance of clines in different characters suggests an equal strength of selection acting on underlying loci. Increase of variance and covariance between phenotypic traits at the cline centre could more likely result from assortative mating than from selection against hybrids. Most pairwise cases showed the highest covariance for the oschei‐like, than for the albomarginatus‐like hybrid populations. This indicates that introgression of the oschei genes into the albomarginatus genome is stronger than vice versa, and may be evidence of the movement of the hybrid zone in favour of Ch. albomarginatus. Analysis of associations between phenotype and local vegetation showed that mosaic structure of the hybrid zone is explained to a great extent by habitat–phenotype associations. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 275–291.     

SPATIAL STRUCTURE AND HABITAT VARIATION IN A GRASSHOPPER HYBRID ZONE

Abstract A hybrid zone between the grasshoppers Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae) in northern Spain has been analyzed for variation in morphology and ecology. These species are readily distinguished by the number of stridulatory pegs on the hind femur. Both sexes are fully winged and inhabit disturbed habitats throughout the study area. We develop a maximum-likelihood approach to fitting a two-dimensional cline to geographical variation in quantitative traits and for estimating associations of population mean with local habitat. This method reveals a cline in peg number approximately 30 km south of the Picos de Europa Mountains that shows substantial deviations in population mean compared with the expectations of simple tension zone models. The inclusion of variation in local vegetation in the model explains a significant proportion of the residual variation in peg number, indicating that habitat-genotype associations contribute to the observed spatial pattern. However, this association is weak, and a number of populations continue to show strong deviations in mean even after habitat is included in the final model. These outliers may be the result of long-distance colonization of sites distant from the cline center or may be due to a patchy pattern of initial contact during postglacial expansion. As well as contrasting with the smooth hybrid zones described for Chorthippus parallelus , this situation also contrasts with the mosaic hybrid zones observed in Gryllus crickets and in parts of the hybrid zone between Bombina toad species, where habitat-genotype associations account for substantial amounts of among-site variation.     

A hybrid zone between Chorthippus parallelus parallelus and Chorthippus parallelus erythropus (Orthoptera: Acrididae): behavioural characters

Two subspecies of the grasshopper Chorthippus parallelus have ranges that abut in the Pyrenees. Males produce two types of song—calling song and courtship song. We have examined the use and structure of these songs in males from nine sites near Mont-Louis, Pyrénées-Onentales, France. These sites were previously used to identify the position of the hybrid zone on morphological characters. The subspecies differ in the use and structure of both types of song. Clines in these characters correspond in position with the morphological cline. At least one character may be relevant to mate choice but none of the characters show evidence of reinforcement.     

Premating barriers to gene exchange and their implications for the structure of a mosaic hybrid zone between Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae)

Many hybrid zones contain a deficit of hybrid genotypes relative to expectations from tension zone models. This is often associated with separation of parental genotypes into distinct habitats (mosaicism), but sometimes parentals can be found co-occuring in the same local population (bimodality). In both cases, prezygotic isolation may play an important role in determining the genotypic composition of the zone. Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae) meet and form a complex hybrid zone in northern Spain. Analysis of stridulatory peg numbers reveals partial spatial and seasonal isolation in a 25 km2 area of the zone: C. jacobsi phenotypes predominate in June and July and are present in both valley and mountain habitats; C. brunneus phenotypes predominate in August and are restricted to valley habitats, always in sympatry with C. jacobsi. Strong assortative mating was observed in laboratory mating experiments. Spatial, seasonal and behavioural isolation combine to produce strong premating isolation in the study area. These results suggest a role for premating isolation in maintaining both the mosaic structure and bimodality of this hybrid zone.     

A hybrid zone between Chorthippus parallelus parallelus and Chorthippus parallelus erythropus (Orthoptera: Acrididae): morphological and electrophoretic characters

Two subspecies of the grasshopper Chorthippus parullelus have distributions that abut in the Pyrenees. We have studied morphological and electrophoretic variation in II populations near Mont Louis, Pyrénées-Orientales, France. The two subspecies differ in several morphological characters and at one enzyme locus, esterase-2, and in this area they hybridize forming a cline in morphology less than 5 km wide. Examination of museum specimens suggests that this hybrid zone extends for most of the length of the Pyrenees, possibly breaking down in the extreme west. As the two subspecies are known to differ in mating behaviour this hybrid zone is well suited to the study of reinforcement.     

Concordance of genetic and phenotypic characters across a sapsucker hybrid zone

Hybridization has presented a challenge for taxonomists and conservation biologists, since hybridizing forms could be stable evolutionary entities or ephemeral forms that are blending together. However, hybrid zones also provide a unique opportunity for evolutionary biologists who study the interaction between gene flow and reproductive isolation in speciation. Three forms of woodpeckers (sapsuckers; genus Sphyrapicus) in North America that are mostly geographically separated but hybridize with each other where they come into contact present a remarkable system for the study of hybridization. We provide the first comprehensive analysis of phenotypic and genetic variation across a hybrid zone between two of these forms, the red‐breasted Sphyrapicus ruber and yellow‐bellied S. varius sapsuckers. The objective was to infer whether selection maintains the differences between forms. Our analysis of eight morphometric and 20 plumage traits, and two molecular markers showed clear differences between the forms and roughly concordant clinal variation across a narrow hybrid zone. Thirty percent of sampled birds in the hybrid zone had mixed west/east genotypes at the genetic markers examined. The center of the genetic cline was located 20 km west of the crest of the Rocky Mountains. The width of the zone was 122 km, narrower than would be expected under neutral blending given reasonable estimates of the age of the zone and individual dispersal distances. Heterozygote deficit and cytonuclear disequilibrium at the centre of the hybrid zone suggested nonrandom mating or limited hybridization. Given these patterns and lack of evidence for habitat segregation we conclude that this hybrid zone is maintained by selection, most likely in the form of hybrid inferiority. This study provides an illustrative example of extensive hybridization between stable entities, providing additional evidence against the historical practice of treating hybridizing forms as members of the same species.     

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.     

Inheritance of song and stridulatory peg number divergence between Chorthippus brunneus and C. jacobsi, two naturally hybridizing grasshopper species (Orthoptera: Acrididae)

Knowledge of the genetic basis of divergence in mating signal characters that contribute to reproductive isolation is critical to understanding speciation. Here, we describe a semi-automated system for characterizing grasshopper acoustic signals. We used this system to study the genetic basis of divergence in three male calling song components [echeme (EL), syllable (SL) and phrase (PL) lengths] between Chorthippus brunneus and C. jacobsi, two species of grasshoppers that hybridize in northern Spain. We also studied the number of pegs in the stridulatory file. For all characters, additive effects accounted for most of the genetic differentiation between species. However, the three song components also showed small but significant epistatic effects. No sex linkage was detected. Wright-Castle-Lande estimates of the minimum numbers of genetic factors underlying song and peg number divergence were low: peg number (n(e)=5.87+/-5.84), SL (n(e)=2.37+/-4.79) and PL (n(e)=0.87+/-0.86). On the other hand, EL appeared to be controlled by many genes. These results suggest that divergence in SL and PL might be driven by sexual selection whereas EL might not be under selection. This is consistent with experimental results on female song preference in related species. However, the fact that few factors appear to underlie the differences in peg number is surprising. Peg number is not closely related to song characteristics. It often varies between closely related grasshopper species and it has been assumed to be a neutral character. The biometrical approaches used here tend to underestimate the number of factors influencing a trait but provide valuable background for subsequent quantitative trait loci analyses.     

Habitat association and seasonality in a mosaic and bimodal hybrid zone between Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae)

Understanding why some hybrid zones are bimodal and others unimodal can aid in identifying barriers to gene exchange following secondary contact. The hybrid zone between the grasshoppers Chorthippus brunneus and C. jacobsi contains a mix of allopatric parental populations and inter-mingled bimodal and unimodal sympatric populations, and provides an ideal system to examine the roles of local selection and gene flow between populations in maintaining bimodality. However, it is first necessary to confirm, over a larger spatial scale, previously identified associations between population composition and season and habitat. Here we use cline-fitting of one morphological and one song trait along two valley transects, and intervening mountains, to confirm previously identified habitat associations (mountain versus valley) and seasonal changes in population composition. As expected from previous findings of studies on a smaller spatial scale, C. jacobsi dominated mountain habitats and mixed populations dominated valleys, and C. brunneus became more prevalent in August. Controlling for habitat and incorporating into the analysis seasonal changes in cline parameters and the standard errors of parental trait values revealed wider clines than previous studies (best estimates of 6.4 to 24.5 km in our study versus 2.8 to 4.7 km in previous studies) and increased percentage of trait variance explained (52.7% and 61.5% for transects 1 and 2 respectively, versus 17.6%). Revealing such strong and consistent patterns within a complex hybrid zone will allow more focused examination of the causes of variation in bimodality in mixed populations, in particular the roles of local selection versus habitat heterogeneity and gene flow between differentiated populations.     

Phenotypic variation and covariation indicate high evolvability of acoustic communication in crickets

Studying the genetic architecture of sexual traits provides insight into the rate and direction at which traits can respond to selection. Traits associated with few loci and limited genetic and phenotypic constraints tend to evolve at high rates typically observed for secondary sexual characters. Here, we examined the genetic architecture of song traits and female song preferences in the field crickets Gryllus rubens and Gryllus texensis. Song and preference data were collected from both species and interspecific F1 and F2 hybrids. We first analysed phenotypic variation to examine interspecific differentiation and trait distributions in parental and hybrid generations. Then, the relative contribution of additive and additive‐dominance variation was estimated. Finally, phenotypic variance–covariance ( P ) matrices were estimated to evaluate the multivariate phenotype available for selection. Song traits and preferences had unimodal trait distributions, and hybrid offspring were intermediate with respect to the parents. We uncovered additive and dominance variation in song traits and preferences. For two song traits, we found evidence for X‐linked inheritance. On the one hand, the observed genetic architecture does not suggest rapid divergence, although sex linkage may have allowed for somewhat higher evolutionary rates. On the other hand, P matrices revealed that multivariate variation in song traits aligned with major dimensions in song preferences, suggesting a strong selection response. We also found strong covariance between the main traits that are sexually selected and traits that are not directly selected by females, providing an explanation for the striking multivariate divergence in male calling songs despite limited divergence in female preferences.     

Variation in complex mating signals in an “island” hybrid zone between Stenobothrus grasshopper species

Two grasshopper species Stenobothrus rubicundus and S. clavatus were previously shown to meet in a narrow hybrid zone on Mount Tomaros in northern Greece. The species are remarkable for their complex courtship songs accompanied by conspicuous movements of antennae and wings. We analyzed variations in forewing morphology, antenna shape, and courtship song across the hybrid zone using a geographic information system, and we documented three contact zones on Mount Tomaros. All male traits and female wings show abrupt transitions across the contact zones, suggesting that these traits are driven by selection rather than by drift. Male clines in antennae are displaced toward S. clavatus, whereas all clines in wings are displaced toward S. rubicundus. We explain cline discordance as depending on sexual selection via female choice. The high covariance between wings and antennae found in the centers of all contact zones results from high levels of linkage disequilibria among the underlying loci, which in turn more likely results from assortative mating than from selection against hybrids. The covariance is found to be higher in clavatus‐like than rubicundus‐like populations, which implies asymmetric assortative mating in parental‐like sites of the hybrid zone and a movement of the hybrid zone in favor of S. clavatus.     

Steep,coincident, and concordant clines in mitochondrial and nuclear‐encoded genes in a hybrid zone between subspecies of Atlantic killifish,Fundulus heteroclitus

Steep genetic clines resulting from recent secondary contact between previously isolated taxa can either gradually erode over time or be stabilized by factors such as ecological selection or selection against hybrids. We used patterns of variation in 30 nuclear and two mitochondrial SNPs to examine the factors that could be involved in stabilizing clines across a hybrid zone between two subspecies of the Atlantic killifish, Fundulus heteroclitus. Increased heterozygote deficit and cytonuclear disequilibrium in populations near the center of the mtDNA cline suggest that some form of reproductive isolation such as assortative mating or selection against hybrids may be acting in this hybrid zone. However, only a small number of loci exhibited these signatures, suggesting locus‐specific, rather than genomewide, factors. Fourteen of the 32 loci surveyed had cline widths inconsistent with neutral expectations, with two SNPs in the mitochondrial genome exhibiting the steepest clines. Seven of the 12 putatively non‐neutral nuclear clines were for SNPs in genes related to oxidative metabolism. Among these putatively non‐neutral nuclear clines, SNPs in two nuclear‐encoded mitochondrial genes (SLC25A3 and HDDC2), as well as SNPs in the myoglobin, 40S ribosomal protein S17, and actin‐binding LIM protein genes, had clines that were coincident and concordant with the mitochondrial clines. When hybrid index was calculated using this subset of loci, the frequency distribution of hybrid indices for a population located at the mtDNA cline center was non‐unimodal, suggesting selection against advanced‐generation hybrids, possibly due to effects on processes involved in oxidative metabolism.     

The unusual inheritance pattern of the courtship songs in closely related grasshopper species of the Chorthippus albomarginatus-group (Orthoptera: Gomphocerinae)

Sibling species of the Chorthippus albomarginatus-group are exceptional among all European grasshopper species because they have an extremely elaborate courtship behaviour. Here, we present a genetic analysis of the courtship song differences between two closely related grasshopper species Ch. albomarginatus and Ch. oschei. Measurements of seven courtship song characters and one parameter of courtship visual display were compared among parentals, F(1) and F(2) hybrids, and backcrosses. Variation in one morphological character, the number of stridulatory pegs, was studied as well. The distributions of song phenotypes were more consistent with a type-III genetic architecture, which involves complementary or duplicate pairs of loci. We suggest that this type of architecture may provide the novel elements of the hybrid songs, even more complex than the parental ones, which may offer a new starting point for sexual selection. One parameter of the visual display appeared to be controlled by a single locus, but differences in other characters of visual display could be explained by a more complex inheritance pattern. Inheritance of the peg number differences was consistent with a simple polygenic additive model. Sex linkage and/or maternal effects were not detected for any trait. We found a disproportionate influence of the Ch. albomarginatus parent on most hybrid song characteristics and the visual display, and this may support a previously suggested idea that Ch. albomarginatus is an ancestral form and Ch. oschei is a derived species.     

STRUCTURE OF AN ASYMMETRIC HYBRID ZONE BETWEEN TWO WATER STRIDER SPECIES (HEMIPTERA: GERRIDAE: LIMNOPORUS)

The water stricter species Limnoporus dissortis and L. notabilis hybridize across a broad zone in western Canada. Body length and alleles at four allozyme loci show a steep cline along the east slope of the Rocky Mountains in western Alberta, while in central British Columbia the parental phenotypes coexist without merging fully. One sex-linked locus shows little introgression, while there is apparently considerable gene flow at three autosomal loci. Although the hybrid zone has characteristics of a broad tension zone, the spatial distribution of introgression suggests that habitat patchiness and differential habitat associations of the two species also contribute to the pattern of hybridization. Asymmetry in interspecific mating success and incompatibilities of sex chromosomes with each other or with cytoplasmic factors appear to account for the occurrence of L. dissortis genotypes within the range of L. notabilis, and the lack of L. notabilis genotypes within the range of L. dissortis. The genetic structure of this hybrid zone supports the importance of sex-linked traits in maintaining the integrity of species, while its spatial structure suggests that extrinsic habitat features can combine with intrinsic genetic incompatibilities to produce complex hybrid interactions.     

Genotypic dispersion and covariance between relatives under linkage disequilibrium

A random mating diploid population under linkage disequilibrium is considered. In the case of two diallelic loci, the problem about condition and joint distributions of genotypes of relatives being in arbitrary genetic relations is solved. Formulae of the partitioning of genotypic variance and covariance between relatives with respect to a polygenic character are inferred (in the case of many characters - of genotypic covariance matrix).     

Transitions in cuticular composition across a hybrid zone: historical accident or environmental adaptation?

Two subspecies of the grasshopper Chorthippus parallelus form a hybrid zone in the Pyrenees. Transitions across the zone, including changes in mating signals and reproductive isolation have been intensively studied. Cuticular pheromones have been identified as likely mate recognition signals. Since the major role of the cuticle is in waterproofing, environmental adaptation of cuticular composition has the interesting potential to generate assortative mating as an incidental by-product. We describe the pattern of variation in cuticular hydrocarbon blend in four transects through the hybrid zone. We find no evidence for a previously observed displaced cline in one blend component. There were differences between subspecies but these varied among transects and were small compared with variation between transects. We examined environmental variation within one transect and found a correlation between vegetation and cuticular composition, suggesting that environment influences the constitution of the cuticle, and hence natural selection may interact with mating signals in this species.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78 , 193−201.     

FURTHER GENETIC ANALYSES OF A HYBRID ZONE BETWEEN LEOPARD FROGS (RANA PIPIENS COMPLEX) IN CENTRAL TEXAS

Additional animals were collected from a previously described hybrid zone between two species of leopard frogs. The material now includes 1,054 specimens collected over a five-year period. The new and the original data were reanalyzed using two multilocus techniques: an analysis of linkage disequilibrium (Hill, 1975); and an analysis of the variance of the distribution of individual heterozygosities (Brown et al., 1980). An analysis of cline shape (Barton and Hewitt, 1983) also was performed. The zone appears to be stable in position. There are major year-to-year changes in allele frequencies, but these do not show concordant patterns between localities. This is probably the result of local stochastic factors. The shapes and position of clines at five loci are similar. Multilocus analysis shows the greatest disequilibrium values in the center of the zone. The ratio of observed to expected variance of individual heterozygosity is also largest in the central localities. Analysis of a single cohort during a three-month period that included both the larval and post-metamorphic stages shows a strong shift in genotypes. The froglet population had more animals with parental genotypes and fewer hybrid combinations. This probably is a result of differential mortality due to developmental incompatibilities when hybrids begin to metamorphose. We compare the usefulness of the two multilocus analytical techniques and make recommendations about collecting procedures for future studies of hybrid zones.     

Inferences from a rapidly moving hybrid zone

Anartia fatima and Anartia amathea (Lepidoptera: Nymphalidae) are sister taxa whose ranges abut in a narrow hybrid zone in eastern Panama. At the center of the zone, hybrids are abundant, although deviations from Hardy-Weinberg and linkage disequilibria are strong, due in part to assortative mating. We measured differences across the zone in four wing color-pattern characters, three allozyme loci, and mitochondrial haplotype. Wing pattern, allozyme, and mitochondrial clines were coincident (i.e., had the same positions) and concordant (i.e.. all markers had similar cline shapes, about 28 km wide). Repeated samples demonstrated that the hybrid zone has been moving eastwards at an average rate of 2.5 km/year over the past 20 years, accompanied by an equivalent movement of the mtDNA cline. No introgression of mtDNA haplotypes were found in the 'wake" of the moving cline, as might be expected for a neutral marker. The concordance of morphological and mtDNA clines between 1994 and 2000, in spite of hybrid zone movement, suggests strong epistasis between the mitochondrial genome and nuclear loci. Cline movement is achieved mainly by pure fatima immigrating into amathea populations; hybrids had little effect, and were presumably outcompeted by fitter pure fatima genotypes. This movement can be explained if random dispersal of 7-19 km x gen(1/2) is coupled with a competitive advantage to A. fatima genomes of 2-5%. Hybrid zone motion is equivalent to Phase III of Wright's shifting balance. Hybrid zone movement has rarely been considered likely in the past, but our results show that it may be more important in biogeography and evolution than generally realized.     

A dispersal-dependent zone of introgressive hybridization between weakfish, Cynoscion regalis, and sand seatrout, C. arenarius, (Sciaenidae) in the Florida Atlantic

Five diagnostic codominant nuclear DNA markers and a diagnostic mitochondrial DNA marker were used to survey weakfish (Cynoscion regalis) and sand seatrout (C. arenarius), with particular focus on heretofore uncharacterized juvenile populations along the Florida (FL) Atlantic coast. Geographic and reproductive ranges of weakfish and sand seatrout were shown to overlap on the Atlantic coast along north and central FL. An active bidirectional zone of introgressive hybridization exists between these taxa, centered in the St Johns River, FL. Strong patterns of Hardy-Weinberg, linkage, and cytonuclear disequilibrium and a bimodal hybrid index distribution were observed for juvenile cohorts in the zone center, coupled with narrow (～240 km) concordant clines. Parental forms had disparate habitat preferences; hybrid forms occurred predominantly in intermediate habitats. All genetic data were consistent with the hypothesis that the C. arenarius-C. regalis hybrid zone is maintained by a dynamic equilibrium between continued interspecific gene flow and one or more opposing forces. Cytonuclear analyses indicated that parental forms mate assortatively in the zone but that mate recognition was imperfect. Ethological mating dynamics are likely stabilized by some form of endogenous or exogenous postfertilization selection against hybrids such that parental taxa will likely continue to evolve independently.