Reproductive character displacement of female mate preferences for male cuticular hydrocarbons in Drosophila subquinaria

Several lines of evidence implicate sexual isolation in both initiating and completing the speciation process. Although its existence is straightforward to demonstrate, understanding the evolution of sexual isolation requires identifying the underlying phenotypes responsible so that we can determine how these have diverged. Here, we study geographic variation in female mate preferences for male sexual displays in the fly Drosophila subquinaria. Female D. subquinaria that are sympatric with its sister species D. recens discriminate strongly against both D. recens and allopatric conspecific males, whereas females from allopatric populations do not. Furthermore, female mate preferences target at least in part a suite of cuticular hydrocarbons (CHCs) in males and geographic variation in CHCs mirrors the pattern of mate discrimination. In this study, we quantify female mate preferences for male CHCs from populations that span the geographic range of D. subquinaria. We find that the direction of linear sexual selection varies significantly between populations that are sympatric versus allopatric with D. recens in a pattern of reproductive character displacement. Differences in preference partially align with existing differences in CHCs and patterns of sexual isolation, although discrepancies remain that suggest the involvement of additional traits and/or more complex, nonlinear preference functions.     

Relative abundance and the species-specific reinforcement of male mating preference in the Chrysochus (Coleoptera: Chrysomelidae) hybrid zone

Most studies of reinforcement have focused on the evolution of either female choice or male mating cues, following the long-held view in sexual selection theory that mating mistakes are typically more costly for females than for males. However, factors such as conspecific sperm precedence can buffer females against the cost of mating mistakes, suggesting that in some hybrid zones mating mistakes may be more costly for males than for females. Thus, the historical bias in reinforcement research may underestimate its frequency. In this study, we present evidence that reinforcement has driven the evolution of male choice in a hybrid zone between the highly promiscuous leaf beetles Chrysochus cobaltinus and C. auratus, the hybrids of which have extremely low fitness. In addition, there is evidence for male choice in these beetles and that male mating mistakes may be costly, due to reduced opportunities to mate with conspecific females. The present study combines laboratory and field methods to quantify the strength of sexual isolation, test the hypothesis of reproductive character displacement, and assess the link between relative abundance and the strength of selection against hybridization. We document that, while sexual isolation is weak, it is sufficient to produce positive assortative mating. In addition, reproductive character displacement was only detected in the relatively rare species. The strong postzygotic barriers in this system are sufficient to generate the bimodality that characterizes this hybrid zone, but the weak sexual isolation is not, calling into question whether strong prezygotic isolation is necessary for the maintenance of bimodality. Growing evidence that the cost of mating mistakes is sufficient to shape the evolution of male mate choice suggests that the reinforcement of male mate choice may prove to be a widespread occurrence.     

Reproductive character displacement and the genetics of gamete recognition in tropical sea urchins

Reproductive character displacement occurs when sympatric and allopatric populations of a species differ in traits crucial to reproduction, and it is commonly thought of as a signal of selection acting to limit hybridization. Most documented cases of reproductive character displacement involve characters that are poorly understood at the genetic level, and rejecting alternative hypotheses for biogeographic shifts in reproductive traits is often very difficult. In sea urchins, the gamete recognition protein bindin evolves under positive selection when species are broadly sympatric, suggesting character displacement may be operating in this system. We sampled sympatric and allopatric populations of two species in the sea urchin genus Echinometra for variation in bindin and for the mitochondrial cytochrome oxidase I to examine patterns of population differentiation and molecular evolution at a reproductive gene. We found a major shift in bindin alleles between central Pacific (allopatric) and western Pacific (sympatric) populations of E. oblonga. Allopatric populations of E. oblonga are polyphyletic with E. sp. C at bindin, whereas sympatric populations of the two species are reciprocally monophyletic. There is a strong signal of positive selection (P(N)/P(S) = 4.5) in the variable region of the first exon of bindin, which is associated with alleles found in sympatric populations of E. oblonga. These results indicate that there is a strong pattern of reproductive character displacement between E. oblonga and E. sp. C and that the divergence is driven by selection. There is much higher population structure in sympatric populations at the bindin locus than at the neutral mitochondrial locus, but this difference is not seen in allopatric populations. These data suggest a pattern of speciation driven by selection for local gamete coevolution as a result of interactions between sympatric species. Although this pattern is highly suggestive of speciation by reinforcement, further research into hybrid fitness and egg-sperm interactions is required to address this potential mechanism for character displacement.     

Asymmetric reproductive character displacement in male aggregation behaviour

Reproductive character displacement--the evolution of traits that minimize reproductive interactions between species--can promote striking divergence in male signals or female mate preferences between populations that do and do not occur with heterospecifics. However, reproductive character displacement can affect other aspects of mating behaviour. Indeed, avoidance of heterospecific interactions might contribute to spatial (or temporal) aggregation of conspecifics. We examined this possibility in two species of hybridizing spadefoot toad (genus Spea). We found that in Spea bombifrons sympatric males were more likely than allopatric males to associate with calling males. Moreover, contrary to allopatric males, sympatric S. bombifrons males preferentially associated with conspecific male calls. By contrast, Spea multiplicata showed no differences between sympatry and allopatry in likelihood to associate with calling males. Further, sympatric and allopatric males did not differ in preference for conspecifics. However, allopatric S. multiplicata were more variable than sympatric males in their responses. Thus, in S. multiplicata, character displacement may have refined pre-existing aggregation behaviour. Our results suggest that heterospecific interactions can foster aggregative behaviour that might ultimately contribute to clustering of conspecifics. Such clustering can generate spatial or temporal segregation of reproductive activities among species and ultimately promote reproductive isolation.     

Reproductive character displacement and signal ontogeny in a sympatric assemblage of electric fish

The reproductive signals of two or more taxa may diverge in areas of sympatry, due to selection against costly reproductive interference. This divergence, termed reproductive character displacement (RCD), is expected in species-rich assemblages, where interspecific signal partitioning among closely related species is common. However, RCD is usually documented from simple two-taxon cases, via geographical tests for greater divergence of reproductive traits in sympatry than in allopatry. We propose a novel approach to recognizing and understanding RCD in multi-species communities--one that traces the displacement of signals within multivariate signal space during the ontogeny of individual animals. We argue that a case for RCD can be made if the amount of signal displacement between a pair of species after maturation is negatively correlated to distance in signal space before maturation. Our application of this approach, using a dataset of communication signals from a sympatric Amazonian assemblage of the electric fish genus Gymnotus, provides strong evidence for RCD among multiple species. We argue that RCD arose from the costs of heterospecific mismating, but interacted with sexual selection--favoring the evolution of conspicuous male signals that not only serve for mate-choice, but which simultaneously facilitate species recognition.     

Six cryptic species on a single species of host plant: morphometric evidence for possible reproductive character displacement

Abstract 1. Diversification of some highly host‐specific herbivorous insects may occur in allopatry, without shifts in host use. Such allopatric divergence may be accelerated by sexual selection operating on courtship displays. Wing size and shape may affect visual and vibrational courtship displays in tephritid fruit flies. Geometric morphometric methods were used to examine wings of six sympatric cryptic species in the neotropical genus Blepharoneura. All six species feed on flowers of the same species of host (Gurania spinulosa), a neotropical vine in the Cucurbitaceae. Three of the fly species court and mate in close proximity on the host. Thus, courtship behaviours could serve as important reproductive isolating mechanisms. Two sets of hypotheses were tested: (i) species differ in wing shape and wing size; and (ii) species are sexually dimorphic in wing size and wing shape. Wing size differed among a few species, but wing shape differed significantly among all six species. Sexual dimorphism in wing size was found in only one species, but sexual dimorphism in wing shape was found in two of the three species known to court on the same host plant. In the two sexually dimorphic species, wing shape differed among males, but not among females. This suggests that selection for reproductive character displacement might accelerate divergence in wing shape.     

Social selection parapatry in Afrotropical sunbirds

The extent of range overlap of incipient and recent species depends on the type and magnitude of phenotypic divergence that separates them, and the consequences of phenotypic divergence on their interactions. Signal divergence by social selection likely initiates many speciation events, but may yield niche‐conserved lineages predisposed to limit each others’ ranges via ecological competition. Here, we examine this neglected aspect of social selection speciation theory in relation to the discovery of a nonecotonal species border between sunbirds. We find that Nectarinia moreaui and Nectarinia fuelleborni meet in a ～6 km wide contact zone, as estimated by molecular cline analysis. These species exploit similar bioclimatic niches, but sing highly divergent learned songs, consistent with divergence by social selection. Cline analyses suggest that within‐species stabilizing social selection on song‐learning predispositions maintains species differences in song despite both hybridization and cultural transmission. We conclude that ecological competition between moreaui and fuelleborni contributes to the stabilization of the species border, but that ecological competition acts in conjunction with reproductive interference. The evolutionary maintenance of learned song differences in a hybrid zone recommend this study system for future studies on the mechanisms of learned song divergence and its role in speciation.     

Reproductive character displacement of female,but not male song discrimination in an avian hybrid zone

Divergence of male sexual signals and female preferences for those signals often maintains reproductive boundaries between closely related, co‐occurring species. However, contrasting sources of selection, such as interspecific competition, can lead to weak divergence or even convergence of sexual signals in sympatry. When signals converge, assortative mating can be maintained if the mating preferences of females diverge in sympatry (reproductive character displacement; RCD), but there are few explicit examples. Pied flycatchers (Ficedula hypoleuca) are sympatric with collared flycatchers (F. albicollis) on the Baltic island of Öland, where males from both species compete over nestboxes, their songs converge, and the two species occasionally hybridize. We compare song discrimination of male and female pied flycatchers on Öland and in an allopatric population on the Swedish mainland. Using field choice trials, we show that male pied flycatchers respond similarly to the songs of both species in sympatry and allopatry, while female pied flycatchers express stronger discrimination against heterospecific songs in sympatry than in allopatry. These results are consistent with RCD of song discrimination of female pied flycatchers where they co‐occur with collared flycatchers, which should maintain species assortative mating despite convergence of male sexual signals.     

Sexual selection on phenotypic traits in a hybrid zone of Littorina saxatilis (Olivi)

Step clinal transitions in inherited character(s) between genetically distinct populations are usually referred to as hybrid zones. An example is found in the population of the intertidal snail Littorina saxatilis in Galicia (NW Spain). We studied the shape of the overall fitness surface for sexual selection in this hybrid zone, and the position of hybrids and pure morphs on this surface. We found that sexual divergent selection acted on a combination of phenotypic traits separating the pure morphs, and therefore that sexual selection contributed to morph differentiation. The average fitness of hybrids as a group was not significantly different from that of the pure morphs, but they did show divergent sexual selection in some traits. These results are in agreement with a model of divergent selection favouring both the pure morph as well as those hybrids most resembling each morph. The finding of divergent selection is remarkable because quadratic selection gradients are usually weak in nature.     

Spatial variation in gene flow across a hybrid zone reveals causes of reproductive isolation and asymmetric introgression in wall lizards*

Hybrid zones provide insights into the evolution of reproductive isolation. Sexual selection can contribute to the evolution of reproductive barriers, but it remains poorly understood how sexual traits impact gene flow in secondary contact. Here, we show that a recently evolved suite of sexual traits that function in male-male competition mediates gene flow between two lineages of wall lizards (Podarcis muralis). Gene flow was relatively low and asymmetric in the presence of exaggerated male morphology and coloration compared to when the lineages share the ancestral phenotype. Putative barrier loci were enriched in genomic regions that were highly differentiated between the two lineages and showed low concordance between the transects. The exception was a consistently low genetic exchange around ATXN1, a gene that modulates social behavior. We suggest that this gene may contribute to the male mate preferences that are known to cause lineage-assortative mating in this species. Although female choice modulates the degree of reproductive isolation in a variety of taxa, wall lizards demonstrate that both male-male competition and male mate choice can contribute to the extent of gene flow between lineages.     

Frequency dependent natural selection during character displacement in sticklebacks

Abstract We know little about how natural selection on a species is altered when a closely related species consuming similar resources appears in its environment. In a pond experiment with threespine sticklebacks I tested the prediction that divergent natural selection between competitors is frequency-dependent, changing with the distribution of phe-notypes in the environment. Differential growth and survival of phenotypes in a target stickleback population were contrasted between two treatments. In one treatment an offshore zooplankton feeder (the limnetic stickleback species) was added to the same pond as the target. In the other treatment I added the benthic stickleback instead, a species adapted to feeding on invertebrates from sediments and inshore vegetation. The target population was ecologically and morphologically intermediate with phenotypic variance artificially inflated by hybridization. Growth rates of phenotypes within the target population differed between treatments as predicted by character displacement. The impact of adding a second species always fell most heavily on those phenotypes in the target population resembling the added species most closely. However, those individuals in the target population that most resembled the added species did not experience reduced survival. Instead, consistent survival differences between populations suggested the presence of an inshore –offshore gradient in mortality risk. These results provide further support for the hypothesis of character displacement in sympatric sticklebacks. They suggest that displacement along the resource gradient also led to divergence in vulnerability to agents of mortality, probably including predation.     

Existence of a pattern of reproductive character displacement in Homobasidiomycota but not in Ascomycota

Generally, stronger reproductive isolation is expected between sympatric than between allopatric sibling species. Such reproductive character displacement should predominantly affect premating reproductive isolation and can be due to several mechanisms, including population extinction, fusion of insufficiently isolated incipient species and reinforcement of reproductive isolation in response to low hybrid fitness. Experimental data on several taxa have confirmed these theoretical expectations on reproductive character displacement, but they are restricted to animals and a few plants. Using results reported in the literature on crossing experiments in fungi, we compared the degree and the nature of reproductive isolation between allopatric and sympatric species pairs. In accordance with theoretical expectations, we found a pattern of enhanced premating isolation among sympatric sibling species in Homobasidiomycota. By contrast, we did not find evidence for reproductive character displacement in Ascomycota at similar genetic distances. Both allopatric and sympatric species of Ascomycota had similarly low levels of reproductive isolation, being mostly post-zygotic. This suggests that some phylogeny-dependent life-history trait may strongly influence the evolution of reproductive isolation between closely related species. A significant correlation was found between degree of reproductive isolation and genetic divergence among allopatric species of Homobasidiomycota, but not among sympatric ones or among Ascomycota species.     

Genetic and phenotypic variation across a hybrid zone between ecologically divergent tree squirrels (Tamiasciurus)

A hybrid zone along an environmental gradient should contain a clinal pattern of genetic and phenotypic variation. This occurs because divergent selection in the two parental habitats is typically strong enough to overcome the homogenizing effects of gene flow across the environmental transition. We studied hybridization between two parapatric tree squirrels (Tamiasciurus spp.) across a forest gradient over which the two species vary in coloration, cranial morphology and body size. We sampled 397 individuals at 29 locations across a 600‐km transect to seek genetic evidence for hybridization; upon confirming hybridization, we examined levels of genetic admixture in relation to maintenance of phenotypic divergence despite potentially homogenizing gene flow. Applying population assignment analyses to microsatellite data, we found that Tamiasciurus douglasii and T. hudsonicus form two distinct genetic clusters but also hybridize, mostly within transitional forest habitat. Overall, based on this nuclear analysis, 48% of the specimens were characterized as T. douglasii, 9% as hybrids and 43% as T. hudsonicus. Hybrids appeared to be reproductively viable, as evidenced by the presence of later‐generation hybrid genotypes. Observed clines in ecologically important phenotypic traits—fur coloration and cranial morphology—were sharper than the cline of putatively neutral mtDNA, which suggests that divergent selection may maintain phenotypic distinctiveness. The relatively recent divergence of these two species (probably late Pleistocene), apparent lack of prezygotic isolating mechanisms and geographic coincidence of cline centres for both genetic and phenotypic variation suggest that environmental factors play a large role in maintaining the distinctiveness of these two species across the hybrid zone.     

Reproductive character displacement shapes a spatially structured petal color polymorphism in Leavenworthia stylosa

Character displacement is a potentially important process driving trait evolution and species diversification. Floral traits may experience character displacement in response to pollinator‐mediated competition (ecological character displacement) or the risk of forming hybrids with reduced fitness (reproductive character displacement). We test these and alternative hypotheses to explain a yellow‐white petal color polymorphism in Leavenworthia stylosa, where yellow morphs are spatially associated with a white‐petaled congener (Leavenworthia exigua) that produces hybrids with complete pollen sterility. A reciprocal transplant experiment found limited evidence of local adaptation of yellow color morphs via increased survival and seed set. Pollinator observations revealed that Leavenworthia attract various pollinators that generally favor white petals and exhibit color constancy. Pollen limitation experiments showed that yellow petals do not alleviate competition for pollination. Interspecific pollinator movements were infrequent and low hybridization rates (～0.40–0.85%) were found in each morph, with natural rates likely being lower. Regardless, hybridization rates were significantly higher in white morphs of L. stylosa, yielding a small selection coefficient of s = 0.0042 against this phenotype in sympatry with L. exigua. These results provide support for RCD as a mechanism contributing to the pattern of petal color polymorphism in L. stylosa.     

Cross‐decades stability of an avian hybrid zone

Hybrid zones are particularly valuable for understanding the evolution of partial reproductive isolation between differentiated populations. An increasing number of hybrid zones have been inferred to move over time, but in most such cases zone movement has not been tested with long‐term genomic data. The hybrid zone between Townsend's Warblers (Setophaga townsendi) and Hermit Warblers (S. occidentalis) in the Washington Cascades was previously inferred to be moving from northern S. townsendi southwards towards S. occidentalis, based on plumage and behavioural patterns as well as a 2000‐km genetic wake of hermit mitochondrial DNA (mtDNA) in coastal Townsend's Warblers. We directly tested whether hybrid zone position has changed over 2–3 decades by tracking plumage, mtDNA and nuclear genomic variation across the hybrid zone over two sampling periods (1987–94 and 2015–16). Surprisingly, there was no significant movement in genomic or plumage cline centres between the two time periods. Plumage cline widths were narrower than expected by neutral diffusion, consistent with a ‘tension zone’ model, in which selection against hybrids is balanced by movement of parental forms into the zone. Our results indicate that this hybrid zone is either stable in its location or moving at a rate that is not detectable over 2–3 decades. Despite considerable gene flow, the stable clines in multiple phenotypic and genotypic characters over decades suggest evolutionary stability of this young pair of sister species, allowing divergence to continue. We propose a novel biogeographic scenario to explain these patterns: rather than the hybrid zone having moved thousands of kilometres to its current position, inland Townsend's met coastal Hermit Warbler populations along a broad front of the British Columbia and Alaska coast and hybridization led to replacement of the Hermit Warbler plumage with Townsend's Warbler plumage patterns along this coastline. Hence, hybrid zones along British Columbia and Alaska moved only a short distance from the inland to the coast, whereas the Hermit Warbler phenotype appears stable in Washington and further south. This case provides an example of the complex biogeographic processes that have led to the distribution of current phenotypes within and among closely related species.     

Female mating preference functions predict sexual selection against hybrids between sibling species of cichlid fish

The evolutionary outcome of interspecific hybridization, i.e. collapse of species into a hybrid swarm, persistence or even divergence with reinforcement, depends on the balance between gene flow and selection against hybrids. If female mating preferences are open-ended but sign-inversed between species, they can theoretically be a source of such selection. Cichlid fish in African lakes have sustained high rates of speciation despite evidence for widespread hybridization, and sexual selection by female choice has been proposed as important in the origin and maintenance of species boundaries. However, it had never been tested whether hybridizing species have open-ended preference rules. Here we report the first experimental test using Pundamilia pundamilia, Pundamilia nyererei and their hybrids in three-way choice experiments. Hybrid males are phenotypically intermediate. Wild-caught females of both species have strong preferences for conspecific over heterospecific males. Their responses to F1 hybrid males are intermediate, but more similar to responses to conspecifics in one species and more similar to responses to heterospecifics in the other. We suggest that their mate choice mechanism may predispose haplochromine cichlids to maintain and perhaps undergo phenotypic diversification despite hybridization, and that species differences in female preference functions may predict the potential for adaptive trait transfer between hybridizing species.     

Ants exhibit asymmetric hybridization in a mosaic hybrid zone

Research on hybridization between species provides unparalleled insights into the pre‐ and postzygotic isolating mechanisms that drive speciation. In social organisms, colony‐level incompatibilities may provide additional reproductive barriers not present in solitary species, and hybrid zones offer an opportunity to identify these barriers. Here, we use genotyping‐by‐sequencing to sequence hundreds of markers in a hybrid zone between two socially polymorphic ant species, Formica selysi and Formica cinerea. We characterize the zone, determine the frequency of hybrid workers, infer whether hybrid queens or males are produced and investigate whether hybridization is influenced by colony social organization. We also compare cuticular hydrocarbon profiles and aggression levels between the two species. The hybrid zone exhibits a mosaic structure. The asymmetric distribution of hybrids skewed towards F. cinerea suggests a pattern of unidirectional nuclear gene flow from F. selysi into F. cinerea. The occurrence of backcrossed individuals indicates that hybrid queens and/or males are fertile, and the presence of the F. cinerea mitochondrial haplotype in 97% of hybrids shows that successful F1 hybrids will generally have F. cinerea mothers and F. selysi fathers. We found no evidence that social organization contributes to speciation, because hybrids occur in both single‐queen and multiple‐queen colonies. Strongly differentiated cuticular hydrocarbon profiles and heightened interspecific aggression further reveal that species recognition cues are both present and perceived. The discovery of fertile hybrids and asymmetrical gene flow is unusual in ants, and this hybrid zone will therefore provide an ideal system with which to investigate speciation in social insects.     

EVOLUTIONARY IMPLICATIONS OF DIVERGENT CLINES IN AN AVIAN (MANACUS: AVES) HYBRID ZONE

Abstract A previous study of the hybrid zone in western Panama between white‐collared (Manacus candei) and golden‐collared manakins (M. vitellinus) documented the unidirectional introgression of vitellinus male secondary sexual traits across the zone. Here, we examine the hybrid zone in greater genetic and morphological detail. Statistical comparisons of clines are performed using maximum‐likelihood and nonparametric bootstrap methods. Our results demonstrate that an array of six molecular and two morphometric markers agree in cline position and width. Clines for male collar and belly color are similar in width to the first eight clines, but are shifted in position by at least five cline widths. The result is that birds in intervening populations are genetically and morphometrically very like parental candei, but males have the plumage color of parental vitellinus. Neither neutral diffusion nor nonlinearity of color scales appear to be viable explanations for the large cline shifts. Genetic dominance of vitellinus plumage traits is another potential explanation that will require breeding experiments to test. Sexual selection remains a plausible explanation for the observed introgression of vitellinus color traits in these highly dimorphic, polygynous, lek‐mating birds. Two other clines, including a nondiagnostic isozyme locus, are similar in position to the main cluster of clines, but are broader in width. Thus, introgression at some loci is greater than that detected with diagnostic markers. Assuming that narrow clines are maintained by selection, variation in cline width indicates that selection is not uniform throughout the genome and that diagnostic markers are under more intense selective pressure. The traditional focus on diagnostic markers in studies of hybrid zones may therefore lead to underestimates of average introgression. This effect may be more pronounced in organisms with low levels of genetic divergence between hybridizing taxa.     

Fine‐scale geographic patterns of gene flow and reproductive character displacement in Drosophila subquinaria and Drosophila recens

When two species are incompletely isolated, strengthening premating isolation barriers in response to the production of low fitness hybrids may complete the speciation process. Here, we use the sister species Drosophila subquinaria and Drosophila recens to study the conditions under which this reinforcement of species boundaries occurs in natural populations. We first extend the region of known sympatry between these species, and then we conduct a fine‐scale geographic survey of mate discrimination coupled with estimates of gene flow within and admixture between species. Within D. subquinaria, reinforcement is extremely effective: we find variation in mate discrimination both against D. recens males and against conspecific allopatric males on the scale of a few kilometres and in the face of gene flow both from conspecific populations and introgression from D. recens. In D. recens, we do not find evidence for increased mate discrimination in sympatry, even where D. recens is rare, consistent with substantial gene flow throughout the species’ range. Finally, we find that introgression between species is asymmetric, with more from D. recens into D. subquinaria than vice versa. Within each species, admixture is highest in the geographic region where it is rare relative to the other species, suggesting that when hybrids are produced they are of low fitness. In sum, reinforcement within D. subquinaria is effective at maintaining species boundaries, but even when reinforcing selection is strong it may not always result in a pattern of strong reproductive character displacement due to variation in the frequency of hybridization and gene flow from neighbouring populations.