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.     

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.     

Natural selection reinforces speciation in a radiation of neotropical rainforest plants

The importance of reinforcement, that is, natural selection that strengthens reproductive isolation between incipient species, remains controversial. We used two approaches to test for reinforcement in a species radiation of Neotropical gingers in the genus Costus. First, we conducted an intensive study of Costus pulverulentus and Costus scaber, two recently diverged species that co-occur and share hummingbird pollinators. The hummingbird pollinators transfer pollen between these Costus species, but hybrids are rarely found in nature. By performing pollinations between populations of C. pulverulentus and C. scaber from three sites across the species' geographic ranges, we find that pollen-pistil incompatibilities acting prior to fertilization have evolved only between locally sympatric populations, whereas geographically distant populations within the region of sympatry and allopatric populations remain fully interfertile. Second, we conducted a comparative study of isolating mechanisms across the genus. We find lower seed set due to pollen-pistil incompatibility between species pairs that co-occur and experience pollen transfer in nature compared to species pairs that are otherwise isolated, regardless of genetic distance. Taken together, these studies indicate that crossing barriers prevent potentially maladaptive hybridization and effectively reinforce the speciation process. Our results add to mounting evidence for reinforcement from animal studies and show that plant speciation may also involve complex mate recognition systems. Reinforcement may be particularly important in rapidly diverging lineages where ecological factors play a primary role in reproductive isolation, as may often be the case in tropical communities.     

Strength of sexual and postmating prezygotic barriers varies between sympatric populations with different histories and species abundances

The impact of different reproductive barriers on species or population isolation may vary in different stages of speciation depending on evolutionary forces acting within species and through species’ interactions. Genetic incompatibilities between interacting species are expected to reinforce prezygotic barriers in sympatric populations and lead to cascade reinforcement between conspecific populations living within and outside the areas of sympatry. We tested these predictions and studied whether and how the strength and target of reinforcement between Drosophila montana and Drosophila flavomontana vary between sympatric populations with different histories and species abundances. All barriers between D. montana females and D. flavomontana males were nearly complete, while in the reciprocal cross strong postzygotic isolation was accompanied by prezygotic barriers whose strength varied according to population composition. Sexual isolation between D. flavomontana females and D. montana males was increased in long‐established sympatric populations, where D. flavomontana is abundant, while postmating prezygotic (PMPZ) barriers were stronger in populations where this species is a new invader and still rare and where female discrimination against heterospecific males was lower. Strengthening of sexual and PMPZ barriers in this cross also induced cascade reinforcement of respective barriers between D. flavomontana populations, which is a classic signature of reinforcement process.     

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 variation and positive selection on M7 lysin, an acrosomal sperm protein in mussels (Mytilus spp.)

Successful fertilization in free-spawning marine organisms depends on the interactions between genes expressed on the surfaces of eggs and sperm. Positive selection frequently characterizes the molecular evolution of such genes, raising the possibility that some common deterministic process drives the evolution of gamete recognition genes and may even be important for understanding the evolution of prezygotic isolation and speciation in the marine realm. One hypothesis is that gamete recognition genes are subject to selection for prezygotic isolation, namely, reinforcement. In a previous study, positive selection on the gene coding for the acrosomal sperm protein M7 lysin was demonstrated among allopatric populations of mussels in the Mytilus edulis species group (M. edulis, Mytilus galloprovincialis, and Mytilus trossulus). Here, we expand sampling to include M7 lysin haplotypes from populations where mussel species are sympatric and hybridize to determine whether there is a pattern of reproductive character displacement (RCD), which would be consistent with reinforcement driving selection on this gene. We do not detect a strong pattern of RCD; neither are there unique haplotypes in sympatry nor is there consistently greater population structure in comparisons involving sympatric populations. One distinct group of haplotypes, however, is strongly affected by natural selection, and this group of haplotypes is found within M. galloprovincialis populations throughout the Northern Hemisphere concurrent with haplotypes common to M. galloprovincialis and M. edulis. We suggest that balancing selection, perhaps resulting from sexual conflicts between sperm and eggs, maintains old allelic diversity within M. galloprovincialis.     

Speciation by reinforcement

Speciation has a central place in evolution, linking genetic processes within populations to the generation of biological diversity. The formation of new species must involve the evolution of barriers to gene flow within existing species, but how these barriers arise remains a problem. In the case of prezygotic isolation it is possible that natural selection directly favours characters that decrease gene flow. Where two populations have diverged to such an extent that they produce unfit hybrids, individuals will leave more offspring if they mate within their own population. Characters increasing assortive mating will be favoured until eventually two species may result. This is the widely accepted model of speciation by reinforcement. However, recent studies suggest serious limitations on the efficacy of reinforcing selection and a lack of well-substantiated examples.     

Patterns of reproductive isolation in three angiosperm genera

Analyses among animal species have found that reproductive isolation increases monotonically with genetic distance, evolves more quickly for prezygotic than postzygotic traits, and is stronger among sympatric than allopatric species pairs. The latter pattern is consistent with expectations under the reinforcement hypothesis. To determine whether similar trends are found among plant species, patterns of reproductive isolation (postpollination prezygotic, postzygotic, and "total" isolation) in three plant genera (Glycine, Silene, Streptanthus) were examined using data from previously published artificial hybridization experiments. In Silene, all measures of reproductive isolation were positively correlated with genetic distance. In contrast, in Glycine and Streptanthus, correlations between reproductive isolation and genetic distance were weak or nonsignificant, possibly due to the influence of biologically unusual taxa, variable evolutionary forces acting in different lineages, or insufficient time to accumulate reproductive isolation. There was no evidence that postpollination prezygotic reproductive isolation evolved faster than postzygotic isolation in Glycine or Silene. We also detected no evidence for faster accumulation of postmating prezygotic isolation between sympatric than allopatric species pairs; thus we found no evidence for the operation of speciation via reinforcement. In Silene, which included six polyploid species, results suggest that changes in ploidy disrupt a simple monotonic relationship between isolation and genetic distance.     

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.     

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.     

Disentangling structural genomic and behavioural barriers in a sea of connectivity

Genetic divergence among populations arises through natural selection or drift and is counteracted by connectivity and gene flow. In sympatric populations, isolating mechanisms are thus needed to limit the homogenizing effects of gene flow to allow for adaptation and speciation. Chromosomal inversions act as an important mechanism maintaining isolating barriers, yet their role in sympatric populations and divergence with gene flow is not entirely understood. Here, we revisit the question of whether inversions play a role in the divergence of connected populations of the marine fish Atlantic cod (Gadus morhua), by exploring a unique data set combining whole‐genome sequencing data and behavioural data obtained with acoustic telemetry. Within a confined fjord environment, we find three genetically differentiated Atlantic cod types belonging to the oceanic North Sea population, the western Baltic population and a local fjord‐type cod. Continuous behavioural tracking over 4 year revealed temporally stable sympatry of these types within the fjord. Despite overall weak genetic differentiation consistent with high levels of gene flow, we detected significant frequency shifts of three previously identified inversions, indicating an adaptive barrier to gene flow. In addition, behavioural data indicated that North Sea cod and individuals homozygous for the LG12 inversion had lower fitness in the fjord environment. However, North Sea and fjord‐type cod also occupy different depths, possibly contributing to prezygotic reproductive isolation and representing a behavioural barrier to gene flow. Our results provide the first insights into a complex interplay of genomic and behavioural isolating barriers in Atlantic cod and establish a new model system towards an understanding of the role of genomic structural variants in adaptation and diversification.     

Low reproductive isolation and highly variable levels of gene flow reveal limited progress towards speciation between European river and brook lampreys

Ecologically based divergent selection is a factor that could drive reproductive isolation even in the presence of gene flow. Population pairs arrayed along a continuum of divergence provide a good opportunity to address this issue. Here, we used a combination of mating trials, experimental crosses and population genetic analyses to investigate the evolution of reproductive isolation between two closely related species of lampreys with distinct life histories. We used microsatellite markers to genotype over 1000 individuals of the migratory parasitic river lamprey (Lampetra fluviatilis) and freshwater‐resident nonparasitic brook lamprey (Lampetra planeri) distributed in 10 sympatric and parapatric population pairs in France. Mating trials, parentage analyses and artificial fertilizations demonstrated a low level of reproductive isolation between species even though size‐assortative mating may contribute to isolation. Most parapatric population pairs were strongly differentiated due to the joint effects of geographic distance and barriers to migration. In contrast, we found variable levels of gene flow between sympatric populations ranging from panmixia to moderate differentiation, which indicates a gradient of divergence with some population pairs that may correspond to alternative morphs or ecotypes of a single species and others that remain partially isolated. Ecologically based divergent selection may explain these variable levels of divergence among sympatric population pairs, but incomplete genome swamping following secondary contact could have also played a role. Overall, this study illustrates how highly differentiated phenotypes can be maintained despite high levels of gene flow that limit the progress towards speciation.     

Amount of introgression in flycatcher hybrid zones reflects regional differences in pre and post-zygotic barriers to gene exchange

Introgression is the incorporation of alleles from one species or semispecies into the gene pool of another through hybridization and backcrossing. The rate at which this occurs depends on the frequency of hybridization and the fitness of hybrids and backcrosses compared to 'pure' individuals. The collared flycatcher (Ficedula albicollis) and the pied flycatcher (F. hypoleuca) co-exist and hybridize at low to moderate frequencies in a clinal hybrid zone in Central Europe and on the islands of Gotland and Oland off the Swedish east coast. Data on hatching success suggest that hybrids are less fertile in Central Europe compared to on the islands. Direct fitness estimates using molecular markers to infer paternity are consistent with the demographic data. Applying a tag-array-based minisequencing assay to genotype interspecific substitutions and single nucleotide polymorphisms we demonstrate that the amount of introgression from the pied to the collared flycatcher is higher in the two island populations (Gotland and Oland) than in two geographically distinct areas from the Central European hybrid zone (Czech Republic and Hungary). In all areas the amount of introgression from collared to pied flycatchers is very low or seemingly absent. The different patterns of introgression are consistent with regional differences in rates of hybridization and fitness of hybrids. We suggest that barriers to gene exchange may have been partly broken down on the islands due to asymmetric gene flow from allopatry. Alternatively, or in addition, more pronounced reinforcement of prezygotic isolation in Central Europe might have increased post-zygotic isolation through hitchhiking, since genes affecting pre and post-zygotic isolation are both sex-linked in these birds. One of our genetic markers appears to introgress from pied to collared flycatchers at a much higher rate than the other markers. We discuss the possibility that the introgressed marker may be linked to a gene which is under positive selection in the novel genetic background.     

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.     

Hybrid queen butterflies from the cross Danaus chrysippus (L.) × D. gilippus (Cramer):confirmation of species status for the parents and further support for Haldane's Rule

A cross between queen butterflies of the Palaeotropical species Danaus chrysippus and the Neotropical D. gilippus was achieved with difficulty in both directions. Only one progeny ( N  = 70) was reared comprising sterile males and inviable females in a precisely 1 : 1 ratio. Both prezygotic and postzygotic barriers to gene flow are strong. The result supports Haldane's Rule, to which we propose a minor amendment. The F₁ hybrids were intermediate for background colour between the brown (genotype BB ) of gilippus and orange (genotype bb ) of chrysippus . Most F₁ pattern characters were also intermediate. In polymorphic chrysippus populations, because Bb heterozygotes are brown, or nearly so, we suggest the B allele may have evolved towards dominance in sympatry. Hybrid males show positive heterosis for body size. The close similarity of male genitalia between the allopatric, genetically distant species chrysippus  and gilippus , compared to their divergence between gilippus and its largely sympatric sister species eresimus , suggest that reinforcement of sexual isolation or reproductive character displacement have evolved in sympatry.  © 2002 The Linnean Societyof London, Biological Journal of the Linnean Society , 2002, 76 , 535–544.     

Patterns of hybridization and population genetic structure in the terrestrial orchids Liparis kumokiri and Liparis makinoana (Orchidaceae) in sympatric populations

We investigated the potential for gene flow and genetic assimilation via hybridization between common and rare species of the terrestrial orchid genus Liparis, focusing specifically on sympatric and allopatric populations of the common Liparis kumokiri and the rare Liparis makinoana. We utilized analyses of genetic diversity, morphology, and the spatial distributions of individuals and genotypes to quantify the dynamics of interspecific gene flow at within- and among-population scales. High levels of allozyme genetic diversity (HE) were found in populations of the rare L. makinoana (0.317), whereas the common L. kumokiri (N = 1744 from 14 populations) revealed a complete lack of variation. This contrast may reflect different breeding systems and associated rates of genetic drift (L. makinoana is self-incompatible, whereas L. kumokiri is self-compatible). At the two known sympatric sites, individuals were found that recombined parental phenotypes, possessing floral characteristics of L. kumokiri and vegetative characteristics of L. makinoana. These putative hybrids were the only individuals found segregating alleles diagnostic of both parental species. Analysis of these individuals indicated that hybrid genotypes were skewed towards L. kumokiri and later generation recombinants of L. kumokiri at both sympatric sites. Furthermore, Ripley's bivariate L(r) statistics revealed that at one site these hybrids are strongly spatially clustered with L. kumokiri. Nonetheless, the relatively low frequency of hybrids, absence of ongoing hybridization (no F1s or first generation backcrossess), and strong genetic differentiation between morphologically 'pure' parental populations at sympatric sites (FST = 0.708-0.816) indicates that hybridization was not an important bridge for gene flow. The results from these two species suggest that natural hybridization has not played an important role in the diversification of Liparis, but instead support the view that genetic drift and limited gene flow are primarily responsible for speciation in Liparis. Based on genetic data and current status of the species, implications of the research for conservation are considered to provide guidelines for appropriate conservation and management strategies.     

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.     

Hybridization in closely related Rhododendron species: half of all species‐differentiating markers experience serious transmission ratio distortion

An increasing number of studies of hybridization in recent years have revealed that complete reproductive isolation between species is frequently not finalized in more or less closely related organisms. Most of these species do, however, seem to retain their phenotypical characteristics despite the implication of gene flow, highlighting the remaining gap in our knowledge of how much of an organism's genome is permeable to gene flow, and which factors promote or prevent hybridization. We used AFLP markers to investigate the genetic composition of three populations involving two interfertile Rhododendron species: two sympatric populations, of which only one contained hybrids, and a further hybrid‐dominated population. No fixed differences between the species were found, and only 5.8% of the markers showed some degree of species differentiation. Additionally, 45.5% of highly species‐differentiating markers experienced significant transmission distortion in the hybrids, which was most pronounced in F1 hybrids, suggesting that factors conveying incompatibilities are still segregating within the species. Furthermore, the two hybrid populations showed stark contrasting composition of hybrids; one was an asymmetrically backcrossing hybrid swarm, while in the other, backcrosses were absent, thus preventing gene flow.