Post-mating prezygotic barriers to gene exchange between hybridizing field crickets

Studies of sexual selection in speciation have traditionally focused on mate preference, with less attention given to traits that act between copulation and fertilization. However, recent work suggests that post-mating prezygotic barriers may play an important role in speciation. Here, we evaluate the role of such barriers in the field crickets, Gryllus firmus and Gryllus pennsylvanicus. Gryllus pennsylvanicus females mated with G. firmus males produce viable, fertile offspring, but when housed with both species produce offspring sired primarily by conspecifics. We evaluate patterns of sperm utilization in doubly mated G. pennsylvanicus females and find no evidence for conspecific sperm precedence. The reciprocal cross (G. firmus female × G. pennsylvanicus male) produces no progeny. Absence of progeny reflects a barrier to fertilization rather than reduced sperm transfer, storage or motility. We propose a classification scheme for mechanisms underlying post-mating prezygotic barriers similar to that used for premating barriers.     

Calling song and selective phonotaxis in the field crickets,Gryllus firmus andG. pennsylvanicus (Orthoptera: Gryllidae)

The field cricket species, Gryllus firmusand G. pennsylvanicus,occur in a mosaic hybrid zone that roughly parallels the eastern slope of the Appalachian mountains in the northeastern United States. It is important to know what role, if any, the calling song plays in mate choice in sympatric and allopatric populations. In this report, we present results on the variability of calling song properties along transects across this hybrid zone. We also present the results of experiments on phonotactic selectivity of females from an allopatric population of G. firmus.The male calling song of allopatric G. firmuswas significantly slower in temporal rhythm (i. e., chirp and pulse repetition rates) and lower in pitch (i.e., dominant frequency) than that of allopatric G. pennsylvanicus.Calling song properties of males recorded in the hybrid zone varied considerably in temporal and spectral properties. In two-stimulus (choice) phonotaxis experiments, allopatric females of G. firmuspreferred synthetic calling songs with conspecific pulse repetition rates over songs that had lower and higher pulse rates. This preference persisted even when the sound pressure levels of alternative stimuli were unequal. Therefore, allopatric females of G. firmuscan discriminate between conspecific and heterospecific calling songs. Whether or not this same selectivity is present in sympatric populations remains unclear. Investigations of phonotactic selectivity in other allopatric and sympatric populations of both species are currently under way.     

Do Wolbachia infections play a role in unidirectional incompatibilities in a field cricket hybrid zone?

Two closely related field crickets, Gryllus firmus and G. pennsylvanicus, hybridize along an extensive north-south zone in the eastern United States. Crosses between G. firmus males and G. pennsylvanicus females produce viable and fertile F1, but the reciprocal cross consistently fails to produce offspring. Wolbachia, a bacterial parasite of arthropods that causes unidirectional incompatibilities in a variety of insect species, has been suggested as the cause of the observed incompatibility between G. pennsylvanicus and G. firmus. We examine the presence/absence of Wolbachia strains, defined by sequencing the ftsZ gene, in four cricket populations from the north-eastern United States. Most G. firmus individuals are infected (100% in Guilford, Connecticut; 65% in Seaside Park, New Jersey) and > 95% of those infected harbour a single strain of Wolbachia. All individuals in G. pennsylvanicus populations (Ithaca, New York; Sharon, Connecticut) are infected; the majority of individuals carry a second strain of Wolbachia, but a significant fraction carry the same strain found commonly in G. firmus. The presence of an apparently identical Wolbachia strain in crickets of both species means that some crosses between G. pennsylvanicus males and G. firmus females should be compatible. We have no evidence of such compatibility. Furthermore, if Wolbachia infections are responsible for the observed incompatibility between species, then incompatibilities must also exist within G. pennsylvanicus, because this species harbours both Wolbachia strains. Although some single pair crosses within G. pennsylvanicus do fail to produce offspring, the proportion is lower than expected if Wolbachia were responsible. Therefore, Wolbachia is unlikely to be involved in reproductive isolation between the two cricket species.     

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

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

Genetic detection of sperm from forced copulations between sympatric populations of Poecilia reticulata and Poecilia picta

The reproductive success of the sneaky mating tactic is difficult to determine in fish with internal fertilization. We approached this problem by developing an assay that allows the recovery of sperm DNA from the reproductive tract of females. This assay was used to test whether sperm transfer occurs between sympatric populations of the guppy, Poecilia reticulata , and its putative sister species, Poecilia picta . Six hundred females of both species from two sympatric sites in Trinidad were collected, and then the contents of the females' reproductive tracts were analysed using reciprocally diagnostic microsatellite markers. Conspecific sperm occurred in approximately 86% of females, whereas heterospecific sperm were found in only 4% of females. Because females of either species do not mate willingly with heterospecific males, the results indicate that sneaky mating results in the transfer of sperm. The data are consistent with the idea that sexual conflict can result in differences in the strength of behavioural isolation between the sexes, and they suggest that behavioural isolation is unlikely to have driven speciation between the guppy and P. picta .  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 397–402.     

Wolbachia plays no role in the one-way reproductive incompatibility between the hybridizing field crickets Gryllus firmus and G. pennsylvanicus

Wolbachia are cytoplasmically inherited alpha-proteobacteria that can cause cytoplasmic incompatibility (CI) in insects. This incompatibility between sperm and egg is evident when uninfected females mate with infected males. Wolbachia-driven reproductive incompatibilities are of special interest because they may play a role in speciation. However, the presence of Wolbachia does not always imply incompatibility. The field crickets Gryllus firmus and G. pennsylvanicus exhibit a very clear unidirectional incompatibility and have been cited as a possible example of Wolbachia-induced CI. Here, we conduct curing experiments, intra- and interspecific crosses, cytological examination of Wolbachia in testes and Wolbachia quantifications through real-time PCR. All of our data strongly suggest that Wolbachia are not involved in the reproductive incompatibility between G. firmus and G. pennsylvanicus.     

Males,but not females,contribute to sexual isolation between two sympatric species of <Emphasis Type="Italic">Gambusia</Emphasis>

Reproductive isolation restricts genetic exchange between species. Various pre- and post-mating barriers, such as behavior, physiology and gametic incompatibility, have been shown to evolve in sympatry. In certain scenarios, isolation can be asymmetrical, where species differentially prefer conspecifics. We examined sexual isolation via conspecific mate preference between Gambusia affinis and G. geiseri in both sexes. To investigate male contribution to sexual isolation, we compared the number of mating attempts (gonopodial thrusts) directed at either a conspecific or a heterospecific female, in both species. We also examined sperm priming and expenditure in males in the presence of conspecific or heterospecific females. We then measured female preference for either a conspecific or heterospecific male, in both species. We found that males of both species preferred to mate with conspecific females, but showed no difference in sperm production or expenditure between conspecific and heterospecific females. Females of both species did not prefer conspecific over heterospecific males. Our results suggest that sexual isolation might be mediated by male mate choice in this system and not female choice, suggesting that there is asymmetrical reproductive isolation between the sexes in G. affinis and G. geiseri, but symmetrical species isolation.     

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

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

SEX DIFFERENCES IN MATE RECOGNITION AND CONSPECIFIC PREFERENCE IN SPECIES WITH MUTUAL MATE CHOICE

Sexual isolation is often assumed to arise because choosy females recognize and reject heterospecific males as mates. Yet in taxa in which both males and females are choosy, males might also recognize and reject heterospecific females. Here, we asked about the relative contribution of the sexes to the strong sexual isolation found in limnetic–benthic species pairs of threespine sticklebacks, which show mutual mate choice. We asked whether males and females of the two species recognize conspecifics and also prefer to mate with them. We found evidence for mate recognition by both sexes but only females prefer conspecifics. The nature of male courtship depended on which species of female they were courting, indicating that males recognized conspecific females and differentiated them from heterospecifics. However, males courted both species of females with equal vigor and changed courtship in a manner that would increase the chance of mating with heterospecifics. Females both recognized conspecifics and strongly preferred them. They responded very little to heterospecific male courtship and almost never mated with them. Therefore, males are likely to undermine sexual isolation, but females uphold it. Despite mutual mate choice and mate recognition in both sexes, females are primarily responsible for sexual isolation in these taxa.     

Assessment of mate preference in the house mouse with reference to investigations on assortative mating

This study analysed patterns of mate choice based on different types of laboratory test, and points out the advantages of combined methods to assess mate preference. We compared dyadic encounters and two-way choice tests involving a pair of mice or their urinary signature. Preference was assessed in males and females of the two European subspecies of the house mouse Mus musculus that share a secondary hybrid zone. A preference was deduced from directional choice or behavioural discrimination in favour of a mouse. Our results show discrepancies between the outcomes of the different types of test, which we discuss in terms of the quality of the stimuli involved, and of physical access to the information carried by those stimuli. Despite variation, our results indicate that M. m. domesticus did not show a preference, but could discriminate between stimuli of the two subspecies and tended to direct sexual behaviour assortatively. In contrast, both male and female M. m. musculus showed positive discrimination and preference for potential mates and signals carried by their own subspecies. Additionally, our results confirm that subspecies informative signals are present in mouse urine and suggest that they may be molecules of low volatility, necessitating contact for preference to be displayed.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 461–471.     

Female alpine newts (Triturus alpestris) mate initially with males signalling fertility benefits

Gametic asymmetry implies that females invest more per gamete than males do and thus sperm is considered to be a relatively cheap resource. However, contrary to this classic view, sperm has been shown to be frequently in short supply; hence, selection favouring females that mate for fertility benefits should occur. For this reason, we determined whether males signalling fertility are preferred by female newts of the species Triturus alpestris . We performed paired female–male trials using unmated and previously inseminated females to determine potential criteria for female interest in a courting male, to establish what factors lead to successful mating and to assess the importance of female choice for direct and indirect benefits. We found that female interest in any potential mate and mating success decreased once mating had occurred. Furthermore, we detected an increase in spermatophore deposition rate and rapid spermatophore transfer in encounters that resulted in a successful mating. The results obtained indicate that female alpine newts are attracted to males showing signs of relatively high fertility and that females exhibit a decreased propensity to mate once initial sperm reserves have been acquired. Our results support the theory of initial female choice for fertility benefits.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 483–491.     

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.     

Effects of body size on selectivity for mating cues in different sensory modalities

We investigated the effects of body mass on the selectivity of female mating preferences in two different sensory channels (acoustic and chemical) using the field cricket, Gryllus integer. We found that body mass affected female selectivity for acoustic cues: larger females were more selective than smaller females for long‐distance calls of males. In contrast, body mass did not affect selectivity for chemical cues of males, which are assessed at close range. Nevertheless, we observed selectivity for these cues. Finally, selectivity for acoustic cues was not correlated with selectivity for chemical cues. These results suggest that energetic concerns may influence mating decisions made at a distance and that the effects of body mass on mating decisions are not necessarily shared across different sensory modalities. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 160–168.     

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.     

Intraguild interactions promote assortative mating and affect sexual attractiveness in a phytophagous fly

Changes in acoustic and substrate-borne sexual signals in phytophagous insects associated with host plant shifts are known to have the potential to promote assortative mating, reproductive isolation and speciation. In this article, we ask whether the switch between pure herbivory and intraguild predation (IGP), which is common amongst phytophagous insects, has similar potential. Male flies in the genus Lipara (Diptera: Chloropidae) search for females by vibrating reed stems and waiting for a reply. By kleptoparasitizing other phytophagous species in the genus (a form of IGP), Lipara rufitarsis can increase its nonsexual fitness considerably. We looked at the impact of IGP on the timing of hatching, body size and attractiveness of male calls in L. rufitarsis . L. rufitarsis males that had engaged in IGP hatched significantly earlier than purely phytophagous flies and were significantly larger, but their calls were less likely to elicit responses from females during playback experiments. We conclude that, although behavioural observations of females provided no evidence of 'like preferring like', changes in phenology associated with IGP are likely to promote assortative mating in this system. The general preference of females for the calls of smaller males is a phenomenon worthy of further study: it may have no adaptive significance, or it may indicate that mating with large males is associated with a fitness cost.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 171–180.     

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.     

Extrapair paternity and mate choice in a chickadee hybrid zone

The dynamics of hybrid zones are likely to be influenced greatlyby patterns of mate choice, including "cryptic" choice mediatedthrough extrapair copulations. To understand changes in hybridzones over time and space, a detailed examination of matingpatterns and correlates is needed. We studied the role of extrapairfertilizations (EPFs) in the breeding biology of hybridizingblack-capped and Carolina chickadees in southeastern Pennsylvaniaover 4 years, using microsatellite DNA markers. We detectedextrapair offspring (EPO) in 56% of 90 broods examined; theseaccounted for at least 26% of 477 offspring. Chickadees do notappear to use EPFs to reduce costs of heterospecific pairing:EPFs were no more likely to occur in genetically dissimilar(heterospecific) social pairs than in pairs where social mateswere genetically similar. However, females paired with black-capped–likemales were more likely to have EPO. Females that acquired EPFsdid not obtain these from males genetically similar to themselves;instead, all females, regardless of their genotype or that oftheir social mate, tended to prefer Carolina-like males as extrapairpartners. There was no relationship between the presence ofEPO and hatching or fledging success. High rates of extrapairpaternity and apparent female preference for Carolina-like malessuggest that mate choice is an important influence in ongoingnorthward movement of this hybrid zone.     

Seasonal and individual variation in gregarine parasite levels in the field crickets Gryllus veletis and G.pennsylvanicus

ABSTRACT. 1. Gregarines, debilitating protozoan gut parasites, were monitored in two species of field cricket, Gryllus veletis (Alexander and Bigelow) and G.pennsylvanicus Burmeister, in northern Michigan in 1983 and 1984.
2. Gregarines were found in 31–51% of G.veletis individuals and 50–70% of G.pennsylvanicus.
3. Males were more heavily infected than females in G. veleris , but G.pennsylvanicus males and females had nearly equal infection levels; this disparity resulted in an overall lower infection rate for G. veletis.
4. Both species had fewer gregarines in 1983 than in 1984, possibly due to the adverse effect of drier conditions in 1983 on development of parasite cysts in the soil.
5. Levels of infection were not constant throughout the breeding season for either cricket species.
6. Body size was not correlated with parasite load in either G. veletis or G.pennsylvanicus.
7. Adult age was unrelated to gregarine level in G. veferis , whereas G.pennsylvanicus showed a positive correlation between adult age and parasite load during two non-consecutive weeks of the breeding season.     

Seasonal monogamy and multiple paternity in a wild population of a territorial reptile (tuatara)

Investigating the mating system of a population provides insight into the evolution of reproductive patterns, and can inform conservation management of threatened or endangered species. Combining behavioural and genetic data is necessary to fully understand the mating system and factors affecting male reproductive success, yet behavioural data are often difficult to collect for threatened species. In the present study, we use behavioural data and paternity analyses to characterize the mating system of a high density population of a long-lived, ancient reptile (tuatara, Sphenodon punctatus ). We further investigate the phenotypic traits (including body size, body condition, tail length, and ectoparasite load) that affect male reproductive success. Our behavioural data reflect a seasonally monogamous system with low levels of polyandry and polygyny that are consistent with male mate guarding. Male reproduction is highly skewed (only 25–30% of males are successful), and body size is the primary predictor of male reproductive success. Based on the genetic data, multiple paternity was found in only 8% of clutches, and the results of the paternity analyses showed monandrous clutches from socially polyandrous females. Our behavioural and genetic results revealed complexities in female mating patterns that support the potential for cryptic female choice or sperm competition. This warrants further experimental investigation into the mechanisms underlying reptile fertilization and the disparities between social and genetic polyandry in wild populations.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 161–170.     

Direct and indirect analysis of the fitness of Chrysochus (Coleoptera: Chrysomelidae) hybrids

The chrysomelid beetles Chrysochus auratus and C. cobaltinus form a narrow hybrid zone in western North America. We used a combination of direct and indirect analyses to examine the fitness of Chrysochus hybrids. For the direct analyses, we compared the mating frequency, longevity, fecundity and fertility of hybrid females and parentals. For the indirect approach, we tested predictions of multilocus genotype frequencies at a focal site in the hybrid zone, based on the frequencies of mating combinations during the previous generation. Hybrid females produced fewer eggs than did parentals and the eggs they produced in the lab failed to hatch, in contrast to those of parental females. In addition, contrary to predictions that 15.8% of the individuals at the focal site would have multilocus genotypes other than those expected of parentals or F₁ individuals, we found no such genotypes at this site. This hybrid zone appears to be an example of a classic tension zone, with endogenous selection against hybrid individuals. We discuss the implications of low hybrid fitness for the evolution of premating barriers in this system, and argue that the integration of direct and indirect approaches is a powerful means of assessing the relative fitness of hybrids, particularly for species in which mate choices are easy to observe in the field.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 273–286.