Evolution of premating reproductive isolation among conspecific populations of the sea rock-pool beetle Ochthebius urbanelliae driven by reinforcing natural selection

How natural selection might be involved in speciation remains a fundamental question in evolutionary biology. When two or more species co-occur in the same areas, natural selection may favor divergence in mating traits. By acting in sympatric but not allopatric populations, natural selection can also affect mate choice within species and ultimately initiate speciation among conspecific populations. Here, we address this potential effect in the sea rock-pool beetles Ochthebius quadricollis and O. urbanelliae. The two species, which inhabit the Mediterranean coasts, co-occurr syntopically in an area along the Italian Tyrrhenian coast and completed reproductive isolation by reinforcement. In this article, through mating trials under laboratory conditions between conspecific populations, we found in O. quadricollis no deviations from random mating. Conversely, in O. urbanelliae, we found a clear pattern of premating isolation between the reinforced populations sympatric with O. quadricollis and those nonreinforced allopatric. This pattern is consistent with the view that natural selection, which completed the reproductive isolation between the two species in sympatry, led incidentally also to partial premating reproductive isolation (I(PSI) estimator from 0.683 to 0.792) between conspecific populations of O. urbanelliae. This case study supports an until recently underappreciated role of natural selection resulting from species interactions in initiating speciation.     

Genetic divergence and reproductive isolation in the Ochthebius (Calobius) complex (Coleoptera: Hydraenidae)

The reproductive isolation in hydrenid beetles of the Ochthebius complex was studied by analysing gene exchange in natural populations of O. quadricollis, Ochthebius sp. A and O. brevicollis steinbuehleri collected along the Mediterranean coasts. The ranges of these three species are largely allopatric, but sympatric areas were detected between contiguous taxa, ie, O. quadricollis and Ochthebius sp. A; Ochthebius sp. A and O. b. steinbuehleri. Three levels of reproductive isolation and genetic divergence were observed. One level involves extensive intraspecific genetic divergence within the biological species O. quadricollis, Ochthebius sp. A and O. brevicollis, associated with both physical barriers (eg, sea and sand stretches) and the low dispersal capacity of larvae and adults. The finding of transitional samples between the most differentiated population groups should indicate, however, that there is still some gene flow between the populations of the three taxa. Another level is found between Ochthebius sp. A and O. b. steinbuehleri, whose gene pools appear to be fairly distinct in spite of the fact that reproductive isolation is still incomplete: in their few syntopic sites, some F1 hybrids appeared indeed to have lower fitness, since backcrosses or recombinant genotypes were never observed. The final level in the evolution of reproductive isolation (full reproductive isolation) has been achieved by the species O. quadricollis and Ochthebius sp. A. No F1 or F(n) hybrids, nor backcrosses were found in their sympatric areas. The relative importance of ecological factors and evolutionary forces in the prevention of gene exchange between taxa are discussed.     

Strong assortative mating between allopatric sticklebacks as a by-product of adaptation to different environments

Speciation involves the evolution of reproductive isolation between populations. One potentially important mechanism is the evolution of pre- or postzygotic isolation between populations as a by-product of adaptation to different environments. In this paper, we tested for assortative mating between allopatric stickleback populations adapted to different ecological niches. Our experimental design controlled for interpopulation interactions and non-adaptive explanations for assortative mating. We found that prezygotic isolation was surprisingly strong: when given a choice, the majority of matings occurred between individuals from similar environments. Our results indicate that the by-product mechanism is a potent source of reproductive isolation, and likely contributed to the origin of sympatric species of sticklebacks.     

REINFORCEMENT OF STICKLEBACK MATE PREFERENCES: SYMPATRY BREEDS CONTEMPT

Detailed studies of reproductive isolation and how it varies among populations can provide valuable insight into the mechanisms of speciation. Here we investigate how the strength of premating isolation varies between sympatric and allopatric populations of threespine sticklebacks to test a prediction of the hypothesis of reinforcement: that interspecific mate discrimination should be stronger in sympatry than in allopatry. In conducting such tests, it is important to control for ecological character displacement between sympatric species because ecological character divergence may strengthen prezygotic isolation as a by-product. We control for ecological character displacement by comparing mate preferences of females from a sympatric population (benthics) with mate preferences of females from two allopatric populations that most closely resemble the sympatric benthic females in ecology and morphology. No-choice mating trials indicate that sympatric benthic females mate less readily with heterospecific (limnetic) than conspecific (benthic) males, whereas two different populations of allopatric females resembling benthics show no such discrimination. These differences demonstrate reproductive character displacement of benthic female mate choice. Previous studies have established that hybridization between sympatric species occurred in the past in the wild and that hybrid offspring have lower fitness than either parental species, thus providing conditions under which natural selection would favor individuals that do not hybridize. Results are therefore consistent with the hypothesis that female mate preferences have evolved as a response to reduced hybrid fitness (reinforcement), although direct effects of sympatry or a biased extinction process could also produce the pattern. Males of the other sympatric species (limnetics) showed a preference for smaller females, in contrast to the inferred ancestral preference for larger females, suggesting reproductive character displacement of limnetic male mate preferences as well.     

REPRODUCTIVE CHARACTER DISPLACEMENT OF MALE STICKLEBACK MATE PREFERENCE: REINFORCEMENT OR DIRECT SELECTION?

Reproductive isolation can evolve between species as a byproduct of adaptation to different niches, through reinforcement, and by direct selection on mating preferences. We investigated the potential role of direct selection in the reproductive isolation between sympatric species of threespine sticklebacks. Each sympatric pair consists of a small-bodied limnetic species and large-bodied benthic species. We compared the mate preferences and courtship behavior of males from one sympatric limnetic population and two allopatric populations. We used limnetic-like allopatric populations to control for the effects of ecological character displacement and adaptation to different niches on mate preferences. The sympatric limnetic males preferred the small limnetic females, whereas the allopatric limnetic-like males preferred the large benthic females, suggesting that adaptation to the limnetic niche does not automatically confer a preference for small limnetic females. This reproductive character displacement of male preference is consistent with the predictions of both reinforcement and direct selection on mate preferences. To test for direct selection, we assessed a prediction of one proposed mechanism: predation by benthic females on eggs guarded by limnetic males. The allopatric males come from populations in which there is no egg predation. Sympatric limnetic males were more aggressive toward benthic females than toward limnetic females, whereas the allopatric limnetic-like males did not treat the two types of females differently. The contrast in male behavior suggests that egg predation has shaped male preferences. Direct selection is potentially more effective than indirect selection via reinforcement, and it is likely that it has been important in building up reproductive isolation between limnetic and benthic sticklebacks.     

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.     

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.     

Reproductive isolation between sympatric and allopatric Brazilian populations of Lutzomyia longipalpis s.l. (Diptera: Psychodidae)

Lutzomyia longipalpis s.l., the main vector of Leishmania chagasi in Latin America, is a species complex although the exact number of siblings is yet unknown. In Brazil, the siblings differ in male copulatory courtship songs and pheromones that most certainly act as pre-zygotic reproductive barriers. Here we analysed the reproductive isolation between three allopatric and two sympatric populations of Lu. longipalpis s.l. from Brazil. The results indicate a strong copulatory and pre-mating isolation between the three allopatric populations. In addition, the results also indicate a stronger pre-mating isolation between the two sympatric siblings than between the three allopatric ones, suggesting a role for reinforcement in the speciation of the Lu. longipalpis s.l. complex.     

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.     

昆虫同域物种形成机制及检验

从生物学、生态和遗传的角度阐述昆虫同域物种形成过程中涉及到的可能性机制。昆虫同域种的分化与作用于同域初始种群的歧化选择密切相关,歧化选择间接导致种群生态特征和遗传特征的分化,促进同域近缘种群间的生殖隔离。同域物种形成的过程中涉及到性状替换、性选择、同型交配等机制。寄主专化型多见于昆虫同域种的分化过程中,一般以植食性昆虫为主。有关昆虫同域物种形成的检验机制有多种,归纳起来主要包括同型交配的检验、遗传漂流的量化、遗传分化程度和连锁不平衡(LD)的检测、杂交后代适合度的估算等。目前发现在许多昆虫种类中存在同域物种形成的可能性,但是有关其隔离机制并没有得到充分的解释。     

Complex histories of repeated gene flow in Cameroon crater lake cichlids cast doubt on one of the clearest examples of sympatric speciation

One of the most celebrated examples of sympatric speciation in nature are monophyletic radiations of cichlid fishes endemic to Cameroon crater lakes. However, phylogenetic inference of monophyly may not detect complex colonization histories involving some allopatric isolation, such as double invasions obscured by genome‐wide gene flow. Population genomic approaches are better suited to test hypotheses of sympatric speciation in these cases. Here, we use comprehensive sampling from all four sympatric crater lake cichlid radiations in Cameroon and outgroups across Africa combined with next‐generation sequencing to genotype tens of thousands of SNPs. We find considerable evidence of gene flow between all four radiations and neighboring riverine populations after initial colonization. In a few cases, some sympatric species are more closely related to outgroups than others, consistent with secondary gene flow facilitating their speciation. Our results do not rule out sympatric speciation in Cameroon cichlids, but rather reveal a complex history of speciation with gene flow, including allopatric and sympatric phases, resulting in both reproductively isolated species and incipient species complexes. The best remaining non‐cichlid examples of sympatric speciation all involve assortative mating within microhabitats. We speculate that this feature may be necessary to complete the process of sympatric speciation in nature.     

Sympatric reinforcement of reproductive barriers between <Emphasis Type="Italic">Neotinea tridentata</Emphasis> and <Emphasis Type="Italic">N. ustulata</Emphasis> (Orchidaceae)

Reinforcement is the process by which selection favors traits that decrease mating between two incipient species in response to costly mating or the production of maladapted hybrids, causing the evolution of greater reproductive isolation between emerging species. I have studied a pair of orchids, Neotinea tridentata and N. ustulata, to examine the level of postmating pre- and post-zygotic isolating mechanisms that maintain these species, and the degree to which the boundary may still be permeable to gene flow. In this study, I performed pollen tube growth rate experiments and I investigated pre- and post-zygotic barriers by performing hand pollination experiments in order to evaluate fruit set, embryonate seed set and seed germination rates by intra- and interspecific crosses. Fruit set, the percentage of embryonate seeds and germinability of interspecific crosses were reduced compared to intraspecific pollinations, showing significant differences between sympatric and allopatric populations. While in allopatric populations the post-pollination isolation index ranged between 0.40 and 0.11, in sympatric populations orchid pairs showed total isolation due to post-pollination prezygotic barriers, guaranteed at the level of pollen–stigma interactions. Indeed, in sympatric populations, pollen tubes reached the ovary after 24 h in only 8 out of 45 plants; in the remaining cases, the pollen tubes did not enter the ovary, and thus no fruit set occurred. This pair of orchids is characterized by postmating pre-zygotic reproductive isolation in sympatric populations that prevents the formation of hybrids. This mechanism of speciation, starting in allopatry and triggering the reinforcement mechanisms of reproductive isolation in secondary sympatry, is the most likely explanation for the pattern of evolutionary transitions found in this pair of orchids.     

Female Preference for Sympatric vs. Allopatric Male Throat Color Morphs in the Mesquite Lizard (Sceloporus grammicus) Species Complex

Color polymorphic sexual signals are often associated with alternative reproductive behaviors within populations, and the number, frequency, or type of morphs present often vary among populations. When these differences lead to assortative mating by population, the study of such polymorphic taxa may shed light on speciation mechanisms. We studied two populations of a lizard with polymorphic throat color, an important sexual signal. Males in one population exhibit orange, yellow, or blue throats; whereas males in the other exhibit orange, yellow, or white throats. We assessed female behavior when choosing between allopatric and sympatric males. We asked whether females discriminated more when the allopatric male was of an unfamiliar morph than when the allopatric male was similar in coloration to the sympatric male. We found that female rejection of allopatric males relative to sympatric males was more pronounced when males in a pair were more different in throat color. Our findings may help illuminate how behavioral responses to color morph differences between populations with polymorphic sexual signals contribute to reproductive isolation.     

INCIPIENT REPRODUCTIVE ISOLATION BETWEEN TWO SYMPATRIC MORPHS OF THE INTERTIDAL SNAIL LITTORINA SAXATILIS

The study of speciation in recent populations is essentially a study of the evolution of reproductive isolation mechanisms between sub-groups of a species. Prezygotic isolation can be of central importance to models of speciation, either being a consequence of reinforcement of assortative mating in hybrid zones, or a pleiotropic effect of morphological or behavioral adaptation to different environments. To suggest speciation by reinforcement between incipient species one must at least know that gene flow occurs, or have recently occurred, and that assortative mating has been established in the hybrid zone. In Galician populations of the marine snail Littorina saxatilis, two main morphs appear on the same shores, one on the upper-shore barnacle belt and the other in the lower-shore mussel belt. The two morphs overlap in distribution in the midshore where hybrids are found together with pure forms. Allozyme variation indicates that the two parental morphs share a common gene pool, although within shores, gene flow between morphs is less than gene flow within morphs. In this study, we observed mating behavior in the field, and we found that mating was not random in midshore sites, with a deficiency of heterotypic pairs. Habitat selection, assortative mating, and possibly sexual selection among females contributed to the partial reproductive isolation between the pure morphs. Sizes of mates were often positively correlated, in particular, in the upper shore, indicating size-assortative mating too. However, this seemed to be a consequence of nonrandom microdistributions of snails of different sizes. Because we also argue that the hybrid zone is of primary rather than secondary origin, this seems to be an example of sympatric reproductive isolation, either established by means of reinforcement or as a by-product to divergent selection acting on other characters.     

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.     

Reproductive isolating barriers between colour‐differentiated populations of an African annual killifish,Nothobranchius korthausae (Cyprinodontiformes)

Allopatric populations separated by vicariance events are expected to evolve reproductive isolating mechanisms as a result of disparate selection pressures and genetic drift. The appearance of reproductive isolating mechanisms may vary across taxa with differences in the opportunity for mate choice, and may be asymmetrical. In addition, premating barriers may be affected by individual mating experience. We used choice and no‐choice experiments to investigate reproductive isolation between two allopatric (island and mainland) and colour‐differentiated populations of an African annual fish, Nothobranchius korthausae. Assortative mating under experimental conditions was limited and asymmetrical. Preference for sympatric males was only expressed in nonvirgin females from one population. Virgin fish from both populations mated indiscriminately. No difference in the number of eggs laid, fertilization rate and hatching success was detected in no‐choice experiments. All mating combinations produced viable offspring and no postmating barriers were detected in terms of the performance and fertility of F1 hybrids. Overall, we found little evidence for significant reproductive isolation, which is in contrast with the related killifish taxa in which assortative mating can be strong, even among allopatric populations with no colour differentiation. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 62–72.     

Experimental evolution of mating discrimination in budding yeast

Assortative mating, when individuals of similar phenotypes mate, likely plays a key role in preventing gene flow during speciation. Reinforcement occurs when two previously geographically separated (allopatric) groups meet after having evolved partial postzygotic isolation; they are selected to evolve or enhance assortative mating to prevent costly intergroup matings that produce only maladaptive or sterile hybrids. Studies in Drosophila have shown that the genetic architectures of mating discrimination could differ significantly with or without reinforcement, suggesting that the evolution of assortative mating may be more complicated than expected. To study the evolution of assortative mating, we evolved mating discrimination in populations of the budding yeast, Saccharomyces cerevisiae. After 36 cycles of selection, these cells are five times more likely to mate with each other than to their ancestors, despite detectable one-way gene flow between the selected and reference populations. Several individual cultures evolved mating discrimination by changing their mating kinetics, with some mating more rapidly and others more slowly than the ancestral population. Genetic analysis indicates that multiple mutations have accumulated to produce the altered mating preference. Our results show that subtle details of mating behavior can play an important role in the evolution of reproductive isolation.     

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.     

Population differentiation in plethodontid salamanders: divergence of allozymes and sexual compatibility among populations of Desmognathus imitator and D. ochrophaeus (Gaudata: Plethodontidae)

Plethodontid salamanders of the genus Desmognathus exhibit varying levels of genetic differentiation among and within both allopatric and sympatric taxa. This provides excellent opportunities to study population differentiation and speciation. Two morphologically similar species in this genus, D. imitator and D. ochrophaeus, are genetically well-differentiated from one another and occur in sympatry with no evidence of hybridization and introgression. We report that the degree of sexual isolation between these two species is very high, regardless of whether the populations under comparison are allopatric or sympatric with one another. Neither reinforcement nor reproductive character displacement are required to explain the evolution of sexual incompatibility in sympatry. Sexual behaviour apparently diverges while populations are allopatric with one another. Preliminary study indicates that D. imitator consists of populations among which there may be significant sexual isolation in the absence of detectable genetic differentiation.     

Phenotypic divergence but not genetic distance predicts assortative mating among species of a cichlid fish radiation

The hypothesis of ecological divergence giving rise to premating isolation in the face of gene flow is controversial. However, this may be an important mechanism to explain the rapid multiplication of species during adaptive radiation following the colonization of a new environment when geographical barriers to gene flow are largely absent but underutilized niche space is abundant. Using cichlid fish, we tested the prediction of ecological speciation that the strength of premating isolation among species is predicted by phenotypic rather than genetic distance. We conducted mate choice experiments between three closely related, sympatric species of a recent radiation in Lake Mweru (Zambia/DRC) that differ in habitat use and phenotype, and a distantly related population from Lake Bangweulu that resembles one of the species in Lake Mweru. We found significant assortative mating among all closely related, sympatric species that differed phenotypically, but none between the distantly related allopatric populations of more similar phenotype. Phenotypic distance between species was a good predictor of the strength of premating isolation, suggesting that assortative mating can evolve rapidly in association with ecological divergence during adaptive radiation. Our data also reveals that distantly related allopatric populations that have not diverged phenotypically, may hybridize when coming into secondary contact, e.g. upon river capture because of diversion of drainage systems.