Postmating–prezygotic isolation between two allopatric populations of Drosophila montana: fertilisation success differs under sperm competition

Postmating but prezygotic (PMPZ) interactions are increasingly recognized as a potentially important early‐stage barrier in the evolution of reproductive isolation. A recent study described a potential example between populations of the same species: single matings between Drosophila montana populations resulted in differential fertilisation success because of the inability of sperm from one population (Vancouver) to penetrate the eggs of the other population (Colorado). As the natural mating system of D. montana is polyandrous (females remate rapidly), we set up double matings of all possible crosses between the same populations to test whether competitive effects between ejaculates influence this PMPZ isolation. We measured premating isolation in no‐choice tests, female fecundity, fertility and egg‐to‐adult viability after single and double matings as well as second‐male paternity success (P₂). Surprisingly, we found no PMPZ reproductive isolation between the two populations under a competitive setting, indicating no difficulty of sperm from Vancouver males to fertilize Colorado eggs after double matings. While there were subtle differences in how P₂ changed over time, suggesting that Vancouver males’ sperm are somewhat less competitive in a first‐male role within Colorado females, these effects did not translate into differences in overall P₂. Fertilisation success can thus differ dramatically between competitive and noncompetitive conditions, perhaps because the males that mate second produce higher quality ejaculates in response to sperm competition. We suggest that unlike in more divergent species comparisons, where sperm competition typically increases reproductive isolation, ejaculate tailoring can reduce the potential for PMPZ isolation when recently diverged populations interbreed.     

Persistent postmating,prezygotic reproductive isolation between populations

Studying reproductive barriers between populations of the same species is critical to understand how speciation may proceed. Growing evidence suggests postmating, prezygotic (PMPZ) reproductive barriers play an important role in the evolution of early taxonomic divergence. However, the contribution of PMPZ isolation to speciation is typically studied between species in which barriers that maintain isolation may not be those that contributed to reduced gene flow between populations. Moreover, in internally fertilizing animals, PMPZ isolation is related to male ejaculate—female reproductive tract incompatibilities but few studies have examined how mating history of the sexes can affect the strength of PMPZ isolation and the extent to which PMPZ isolation is repeatable or restricted to particular interacting genotypes. We addressed these outstanding questions using multiple populations of Drosophila montana. We show a recurrent pattern of PMPZ isolation, with flies from one population exhibiting reproductive incompatibility in crosses with all three other populations, while those three populations were fully fertile with each other. Reproductive incompatibility is due to lack of fertilization and is asymmetrical, affecting female fitness more than males. There was no effect of male or female mating history on reproductive incompatibility, indicating that PMPZ isolation persists between populations. We found no evidence of variability in fertilization outcomes attributable to different female × male genotype interactions, and in combination with our other results, suggests that PMPZ isolation is not driven by idiosyncratic genotype × genotype interactions. Our results show PMPZ isolation as a strong, consistent barrier to gene flow early during speciation and suggest several targets of selection known to affect ejaculate‐female reproductive tract interactions within species that may cause this PMPZ isolation.     

Reproductive isolation among allopatric Drosophila montana populations

An outstanding goal in speciation research is to trace the mode and tempo of the evolution of barriers to gene flow. Such research benefits from studying incipient speciation, in which speciation between populations has not yet occurred, but where multiple potential mechanisms of reproductive isolation (RI: i.e., premating, postmating‐prezygotic (PMPZ), and postzygotic barriers) may act. We used such a system to investigate these barriers among allopatric populations of Drosophila montana. In all heteropopulation crosses we found premating (sexual) isolation, which was either symmetric or asymmetric depending on the population pair compared. Postmating isolation was particularly strong in crosses involving males from one of the study populations, and while sperm were successfully transferred, stored, and motile, we experimentally demonstrated that the majority of eggs produced were unfertilized. Thus, we identified the nature of a PMPZ incompatibility. There was no evidence of intrinsic postzygotic effects. Measures of absolute and relative strengths of pre‐ and postmating barriers showed that populations differed in the mode and magnitude of RI barriers. Our results indicate that incipient RI among populations can be driven by different contributions of both premating and PMPZ barriers occurring between different population pairs and without the evolution of postzygotic barriers.     

Postmating-prezygotic isolation is not an important source of selection for reinforcement within and between species in Drosophila pseudoobscura and D. persimilis

Abstract Most work on adaptive speciation to date has focused on the role of low hybrid fitness as the force driving reinforcement (the evolution of premating isolation after secondary contact that reduces the likelihood of matings between populations). However, recent theoretical work has shown that postmating, prezygotic incompatibilities may also be important in driving premating isolation. We quantified premating, postmating-prezygotic, and early postzygotic fitness effects in crosses among three populations: Drosophila persimilis, D. pseudoobscura USA (sympatric to D. persimilis ), and D. pseudoobscura Bogotá (allopatric to D. persimilis ). Interspecific matings were more likely to fail when they involved the sympatric populations than when they involved the allopatric populations, consistent with reinforcement. We also found that failure rate in sympatric mating trials depended on whether D. persimilis females were paired with D. pseudoobscura males or the reverse. This asymmetry most likely indicates differences in discrimination against heterospecific males by females. By measuring egg laying rate, fertilization success and hatching success, we also compared components of postmating-prezygotic and early postzygotic isolation. Postmating-prezygotic fitness costs were small and not distinguishable between hetero- and conspecific crosses. Early postzygotic fitness effects due to hatching success differences were also small in between-population crosses. There was, however, a postzygotic fitness effect that may have resulted from an X-linked allele found in one of the two strains of D. pseudoobscura USA. We conclude that the postmating-prezygotic fitness costs we measured probably did not drive premating isolation in these species. Premating isolation is most likely driven in sympatric populations by previously known hybrid male sterility.     

Restricted gene flow within and between rapidly diverging Neotropical plant species

Speciation involves the evolution of traits and genetic differences that contribute to reproductive isolation and the cessation of gene flow, and studying closely related species and divergent populations gives insight into how these phenomena proceed. Here, we document patterns of gene flow within and between two members of a rapid Neotropical species radiation, Costus pulverulentus and Costus scaber (Costaceae). These species co‐occur in the tropical rainforest and share pollinators, but are reproductively isolated by a series of prezygotic barriers, some of which show evidence of reinforcement at sympatric sites. Here, we genotype microsatellite markers in plants from eight sites that span the geographical range of both species, including four sympatric sites. We also genotype putative hybrids found at two sympatric sites. We find high levels of genetic isolation among populations within each species and low but detectable levels of introgression between species at sympatric sites. Putative hybrids identified by morphology are consistent with F1 or more advanced hybrids. Our results highlight the effectiveness of prezygotic isolating mechanisms at maintaining species boundaries in young radiations and provide empirical data on levels of gene flow consistent with reinforcement.     

In Situ Hybridisation Analysis of the X-Linked Genes in the Species of the Virilis Group of Drosophila

When estimating the level of DNA sequence variation within and between populations or when planning QTL analysis, it is essential to know the location of the genes under study. In the present work, five X chromosomal genes, earlier localised in Drosophila virilis and D. littoralis, were mapped by in situ hybridisation on the larval polytene chromosomes of four other virilis group species, D. a. americana, D. flavomontana, D. lacicola and D. montana. Conjugation of X chromosomes of the most interesting species pairs was studied in interspecific hybrids. Three of the marker genes were used as RFLP markers to examine the occurrence of recombination in D. flavomontana and D. montana hybrid females. The gene arrangement of all species studied, appeared to be different at the proximal end of the X chromosome, which prevented normal conjugation along the most part of the X chromosome. The data illustrating the locations of five X chromosomal marker genes are presented for D. a. americana, D. flavomontana, D. lacicola and D. montana.     

Tests of reproductive isolation among species in the <Emphasis Type="Italic">Fundulus notatus</Emphasis> (Cyprinodontiformes: Fundulidae) species complex

We assessed prezygotic (probability of spawning) and postzygotic (hatching success) reproductive isolation among the three ecologically and morphologically similar species in the Fundulus notatus species complex. We employed a multi-generation breeding experiment to test the hypotheses that karyotypic differences, body size differences, or geographic isolation among populations will increase pre or postzygotic reproductive barriers. Overall, prezygotic barriers were strong and postzygotic barriers weak in crosses of non-hybrid heterospecifics (F1 hybrid crosses) while prezygotic barriers were weaker and postzygotic barriers stronger in crosses involving hybrid individuals (F2 hybrid crosses and backcrosses). Prezygotic barriers among the two smaller species (Fundulus notatus and F. euryzonus) broke down rapidly; first generation hybrids spawned (F2 hybrid crosses and backcrosses) as frequently as parental forms in intraspecific crosses. There was no increase in postzygotic barriers among species with cytogenetic differences. There were increased prezygotic, but not postzygotic, barriers among geographically isolated populations of one species. While pure males and females were just as likely to spawn with hybrids, some types of hybrid females suffered from increased sterility, but not inviability, over hybrid males. Female sterility was only seen in hybrids with a Fundulus euryzonus parent, while other female hybrids produced viable eggs.     

Reinforcement shapes clines in female mate discrimination in Drosophila subquinaria

Reinforcement of species boundaries may alter mate recognition in a way that also affects patterns of mate preference among conspecific populations. In the fly Drosophila subquinaria, females sympatric with the closely related species D. recens reject mating with heterospecific males as well as with conspecific males from allopatric populations. Here, we assess geographic variation in behavioral isolation within and among populations of D. subquinaria and use cline theory to understand patterns of selection on reinforced discrimination and its consequences for sexual isolation within species. We find that selection has fixed rejection of D. recens males in sympatry, while significant genetic variation in this behavior occurs within allopatric populations. In conspecific matings sexual isolation is also asymmetric and stronger in populations that are sympatric with D. recens. The clines in behavioral discrimination within and between species are similar in shape and are maintained by strong selection in the face of gene flow, and we show that some of their genetic basis may be either shared or linked. Thus, while reinforcement can drive extremely strong phenotypic divergence, the long‐term consequences for incipient speciation depend on gene flow, genetic linkage of discrimination traits, and the cost of these behaviors in allopatry.     

Geographic contrasts between pre‐ and postzygotic barriers are consistent with reinforcement in Heliconius butterflies

Identifying the traits causing reproductive isolation and the order in which they evolve is fundamental to understanding speciation. Here, we quantify prezygotic and intrinsic postzygotic isolation among allopatric, parapatric, and sympatric populations of the butterflies Heliconius elevatus and Heliconius pardalinus. Sympatric populations from the Amazon (H. elevatus and H. p. butleri) exhibit strong prezygotic isolation and rarely mate in captivity; however, hybrids are fertile. Allopatric populations from the Amazon (H. p. butleri) and Andes (H. p. sergestus) mate freely when brought together in captivity, but the female F1 hybrids are sterile. Parapatric populations (H. elevatus and H. p. sergestus) exhibit both assortative mating and sterility of female F1s. Assortative mating in sympatric populations is consistent with reinforcement in the face of gene flow, where the driving force, selection against hybrids, is due to disruption of mimicry and other ecological traits rather than hybrid sterility. In contrast, the lack of assortative mating and hybrid sterility observed in allopatric populations suggests that geographic isolation enables the evolution of intrinsic postzygotic reproductive isolation. Our results show how the types of reproductive barriers that evolve between species may depend on geography.     

Asymmetrical patterns of speciation uniquely support reinforcement in Drosophila

Understanding how often natural selection directly favors speciation, a process known as reinforcement, has remained an outstanding problem for over 70 years. Although reinforcement has been strongly criticized in the past, it is once again seen as more realistic due to the seminal discovery of enhanced prezygotic isolation among sympatric species and to a handful of well-studied examples. Nevertheless, the pattern of enhanced isolation in sympatry has alternative explanations, highlighting the need to uncover unique signatures of reinforcement to determine its overall frequency in nature. Using a novel dataset on asymmetrical prezygotic and postzygotic isolation among Drosophila species, I uncover new patterns explicitly predicted by reinforcement. Broadly, I found that almost all sympatric species had concordant isolation asymmetries, where the more costly reciprocal mating has greater prezygotic isolation relative to the less costly mating. No such patterns exist in allopatry. Using simulations, I ruled out alternative explanations and showed that concordant isolation asymmetries in sympatry are likely unique signatures of reinforcement. These results allowed me to estimate that reinforcement may impact 60-83% of all sympatric Drosophila and enhance premating isolation by 18-26%. These findings suggest that reinforcement plays a key role in Drosophila speciation.     

Post‐zygotic isolation in cactophilic Drosophila: larval viability and adult life‐history traits of D. mojavensis/D. arizonae hybrids

Drosophila mojavensis and Drosophila arizonae are cactophilic flies that have been used extensively in speciation studies. Incomplete premating isolation, evidence of reinforcement, and a lack of recent introgression between these species point to a potentially important role for post‐zygotic isolating barriers in this system. Other than hybrid male sterility, however, post‐zygotic isolation between D. mojavensis and D. arizonae has received little attention. In this study, we examined viability and life‐history traits of D. mojavensis/D. arizonae F₁ hybrids from sympatric crosses. Specifically, we reared hybrids and purebreds on the natural host cacti of each parental species and compared viability, development time, thorax length, and desiccation resistance between hybrids and purebreds. Interestingly, hybrid females from both crosses performed similarly or even better than purebred females. In contrast, hybrid sons of D. arizonae mothers, in addition to being sterile, had shorter average thorax length than males of both parental species, and hybrid males from both crosses had substantially lower desiccation resistance than D. mojavensis males. The probable cost to hybridization for D. mojavensis females resulting from reduced desiccation resistance of hybrid sons may have been an important selective factor in the history of reinforcement for crosses involving these females.     

Reinforcement of Gametic Isolation in Drosophila

Reinforcement, a process by which natural selection increases reproductive isolation between populations, has been suggested to be an important force in the formation of new species. However, all existing cases of reinforcement involve an increase in mate discrimination between species. Here, I report the first case of reinforcement of postmating prezygotic isolation (i.e., barriers that act after mating but before fertilization) in animals. On the slopes of the African island of São Tomé, Drosophila yakuba and its endemic sister species D. santomea hybridize within a well-demarcated hybrid zone. I find that D. yakuba females from within this zone, but not from outside it, show an increase in gametic isolation from males of D. santomea, an apparent result of natural selection acting to reduce maladaptive hybridization between species. To determine whether such a barrier could evolve under laboratory conditions, I exposed D. yakuba lines derived from allopatric populations to experimental sympatry with D. santomea, and found that both behavioral and gametic isolation become stronger after only four generations. Reinforcement thus appears to be the best explanation for the heightened gametic isolation seen in sympatry. This appears to be the first example in animals in which natural selection has promoted the evolution of stronger interspecific genetic barriers that act after mating but before fertilization. This suggests that many other genetic barriers between species have been increased by natural selection but have been overlooked because they are difficult to study.     

Reinforced postmating reproductive isolation barriers in Neurospora,an Ascomycete microfungus

Maladaptive hybridization promotes reinforcement, selection for stringent reproductive isolation barriers during speciation. Reinforcement is suspected when barriers between sympatric populations are stronger than allopatric barriers, and particularly when stronger barriers evolve in the species and sex suffering the greatest costs of hybridization. Canonically, reinforcement involves premating barriers. Selection for postmating barriers is controversial, but theoretically possible. We examined geographical patterns in reproductive isolation barriers between Neurospora crassa and Neurospora intermedia, fungi with pheromone‐mediated mate recognition and maternal care. We find that isolation is stronger between sympatric populations than allopatric populations, and stronger barriers are associated with the species (N. crassa) and mating role (maternal) suffering the greater costs of hybridization. Notably, reinforced isolation involves a postmating barrier, abortion of fruitbodies. We hypothesize that fruitbody abortion is selectively advantageous if it increases the likelihood that maternal Neurospora individuals successfully mate conspecifically after maladaptive hybrid fertilization.     

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.     

A biogeographic genetic approach for testing the role of reinforcement: the case of Drosophila pseudoobscura and D. persimilis

Abstract.— The role of reinforcement in speciation can be explained by two distinct models. In model I, two diverged populations hybridize and produce fertile hybrids that successfully backcross (hybridization with gene flow). In model II, two populations hybridize but succeeding backcrosses are unproductive (hybridization without gene flow). Using Drosophila persimilis and D. pseudoobscura , we have tested model I by comparing the extent of heterospecific introgression in sympatric versus allopatric populations. We show that certain expectations of this particular model of reinforcement, which is based on hybridization and gene flow between divergent populations after secondary contact, are not realized in these two species. The evidence consists of the similarity of genetic distances as well as proportions of unique/rare alleles between sympatric and allopatric heterospecific populations and a negative correlation between genetic distance and geographical distance between heterospecific populations, which suggests ecological differentiation. This approach in quantifying differential gene flow has important consequences to studies that compare sympatric and allopatric isolation using genetic distance. Following model I, one would expect a pattern of higher prezygotic isolation in sympatric species compared to allopatric species of the same genetic distance simply as a result of an underestimation of genetic distance due to introgression between sympatric populations. We suggest more parsimonious explanations such as reinforcement without genetic exchange (model II) and ecological differentiation, which require high levels of preexisting reproductive isolation between populations.     

HYBRIDIZATION,NATURAL SELECTION,AND EVOLUTION OF REPRODUCTIVE ISOLATION: A 25‐YEARS SURVEY OF AN ARTIFICIAL SYMPATRIC AREA BETWEEN TWO MOSQUITO SIBLING SPECIES OF THE Aedes mariae COMPLEX

Natural selection can act against maladaptive hybridization between co‐occurring divergent populations leading to evolution of reproductive isolation among them. A critical unanswered question about this process that provides a basis for the theory of speciation by reinforcement, is whether natural selection can cause hybridization rates to evolve to zero. Here, we investigated this issue in two sibling mosquitoes species, Aedes mariae and Aedes zammitii, that show postmating reproductive isolation (F1 males sterile) and partial premating isolation (different height of mating swarms) that could be reinforced by natural selection against hybridization. In 1986, we created an artificial sympatric area between the two species and sampled about 20,000 individuals over the following 25 years. Between 1986 and 2011, the composition of mating swarms and the hybridization rate between the two species were investigated across time in the sympatric area. Our results showed that A. mariae and A. zammitii have not completed reproductive isolation since their first contact in the artificial sympatric area. We have discussed the relative role of factors such as time of contact, gene flow, strength of natural selection, and biological mechanisms causing prezygotic isolation to explain the observed results.     

The effects of heterospecific mating frequency on the strength of cryptic reproductive barriers

Heterospecific mating frequency is critical to hybrid zone dynamics and can directly impact the strength of reproductive barriers and patterns of introgression. The effectiveness of post‐mating prezygotic (PMPZ) reproductive barriers, which include reduced fecundity via heterospecific matings and conspecific sperm precedence, may depend on the number, identity and order of mates. Studies of PMPZ barriers suggest that they may be important in many systems, but whether these barriers are effective at realistic heterospecific mating frequencies has not been tested. Here, we evaluate the strength of cryptic reproductive isolation in two leaf beetles (Chrysochus auratus and C. cobaltinus) in the context of a range of heterospecific mating frequencies observed in natural populations. We found both species benefited from multiple matings, but the benefits were greater in C. cobaltinus and extended to heterospecific matings. We found that PMPZ barriers greatly limited hybrid production by C. auratus females with moderate heterospecific mating frequencies, but that their effectiveness diminished at higher heterospecific mating frequencies. In contrast, there was no evidence for PMPZ barriers in C. cobaltinus females at any heterospecific mating frequency. We show that integrating realistic estimates of cryptic isolation with information on relative abundance and heterospecific mating frequency in the field substantially improves our understanding of the strong directional bias in F1 production previously documented in the Chrysochus hybrid zone. Our results demonstrate that heterospecific mating frequency is critical to understanding the impact of cryptic post‐copulatory barriers on hybrid zone structure and dynamics, and that future studies of such barriers should incorporate field‐relevant heterospecific mating frequencies.     

Sympatry, allopatry and sexual isolation between Drosophila mojavensis and D. arizonae

Populations of the North American cactophilic fruitfly Drosophila mojavensis and its sibling species D. arizonae exist both in sympatry and in allopatry. Females of D. arizonae, regardless of their population of origin, are effectively completely isolated behaviorally from D. mojavensis males. On the other hand, females of D. mojavensis from the sympatric populations in Sonora, Mexico exhibit significantly stronger premating isolation from D. arizonae males than do D. mojavensis females from allopatric populations from the Baja California peninsula. Earlier studies interpreted these limited observations as support for reinforcement. Since the time of those studies, additional allopatric populations of D. mojavensis have been collected from southern California and from Santa Catalina Island, off the coast of southern California. Here, we tested the prediction that if sympatry is in fact associated with increased isolation in D. mojavensis, these additional allopatric populations also should show, relative to the sympatric ones, less isolation from D. arizonae. Our results are consistent with this prediction and suggest that isolation is in fact stronger in sympatry.