The butterfly Danaus chrysippus (L.) in East Africa comprises polyphyletic, sympatric lineages that are, despite behavioural isolation, driven to hybridization by female-biased sex ratios

Mitochondrial and nuclear DNA information was analysed among four subspecies of the African Queen butterfly, Danaus (Anosia) chrysippus sensu lato (s.l.), along with four other Danaus species drawn from all three subgenera (D. (Danaus) plexippus, D. (Salatura) genutia, D. (A.) gilippus, D. (A.) eresimus) and two outgroup species from the same tribe, Tirumala septentrionis and Amauris niavius. A mitochondrial phylogeny derived from the 12S rRNA (347 bp) and COI (537 bp) loci indicates two very distinct haplotypes for subspecies D. (A.) c. dorippus, dorippus‐1 and dorippus‐2. Interestingly, dorippus‐1, on the one hand, and all other D. (A.) chrysippus haplotypes, on the other, are the most distantly related clades within the genus and have different most recent ancestors from different subgenera, though sharing the common ancestor of the monophyletic genus. A phylogeny based on the EF1‐α nuclear locus (400 bp) shows that the two well‐separated mitochondrial lineages of dorippus are identical for this gene and reciprocally monophyletic to the other D. (A.) chrysippus lineages. Thus, nuclear and cytoplasmic phylogenies are not only discordant, but also suggest that both D. (A.) chrysippus s.l. and subspecies dorippus are polyphyletic. Paradoxically, four African subspecies, chrysippus‐orange, chrysippus‐brown, alcippus and dorippus, though substantially vicariant, hybridize extensively in East Africa wherever the ranges of two or more of them overlap. Linkage disequilibrium, and hence sexual isolation, in sympatry between colour (nuclear) genes and unlinked mitochondrial (cytoplasmic) loci is consistent across populations and therefore indicates the operation of positive natural selection. Together with data from previous experimental and field work, our results suggest that extensive hybridization occurs among once allopatric or parapatric lineages, that are now nascent species. We deduce that hybridism among lineages in sympatry is currently enforced, in the face of assortative mate choice, by a bacterial symbiont, Spiroplasma, a male‐killer that forces females in female‐biased populations to pair with heterotypic males. In discussion we emphasize that neither D. (A.) chrysippus s.l. as presently circumscribed, nor its component clades, conform to any established concept of species. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86 , 117–131.     

Genomic variation across two barn swallow hybrid zones reveals traits associated with divergence in sympatry and allopatry

Hybrid zones are geographic regions where isolating barriers between divergent populations are challenged by admixture. Identifying factors that facilitate or inhibit hybridization in sympatry can illuminate the processes that maintain those reproductive barriers. We analysed patterns of hybridization and phenotypic variation across two newly discovered hybrid zones between three subspecies of barn swallow (Hirundo rustica). These subspecies differ in ventral coloration and wing length, traits that are targets of sexual and natural selection, respectively, and are associated with genome‐wide differentiation in allopatry. We tested the hypothesis that the degree of divergence in these traits is associated with the extent of hybridization in secondary contact. We applied measures of population structure based on >23,000 SNPs to confirm that named subspecies correspond to distinct genomic clusters, and assessed coincidence between geographic clines for ancestry and phenotype. Although gene flow was ongoing across both hybrid zones and pairwise F_ST between subspecies was extremely low, we found striking differences in the extent of hybridization. In the more phenotypically differentiated subspecies pair, clines for ancestry, wing length and ventral coloration were steep and coincident, suggestive of strong isolation and, potentially, selection associated with phenotype. In the less phenotypically differentiated pair, gene flow and phenotypic variation occurred over a wide geographic span, indicative of weaker isolation. Traits associated with genome‐wide differentiation in allopatry may thus also contribute to isolation in sympatry. We discuss potentially important additional roles for evolutionary history and ecology in shaping variation in the extent hybridization between closely related pairs of subspecies.     

RAPID EVOLUTION OF REPRODUCTIVE ISOLATION BETWEEN INCIPIENT OUTCROSSING AND SELFING CLARKIA SPECIES

A major goal of speciation research is to understand the processes involved in the earliest stages of the evolution of reproductive isolation (RI). One important challenge has been to identify systems where lineages have very recently diverged and opportunities for hybridization are present. We conducted a comprehensive examination of the components of RI across the life cycle of two subspecies of Clarkia xantiana, which diverged recently (ca. 65,000 bp). One subspecies is primarily outcrossing, but self‐compatible, whereas the other is primarily selfing. The subspecies co‐occur in a zone of sympatry but hybrids are rarely observed. Premating barriers resulted in nearly complete isolation in both subspecies with flowering time and pollinator preference (for the outcrosser over the selfer) as the strongest barriers. We found that the outcrosser had consistently more competitive pollen, facilitating hybridization in one direction, but no evidence for pollen–pistil interactions as an isolating barrier. Surprisingly, postzygotic isolation was detected at the stage of hybrid seed development, but in no subsequent life stages. This crossing barrier was asymmetric with crosses from the selfer to outcrosser most frequently failing. Collectively, the results provide evidence for rapid evolution of multiple premating and postzygotic barriers despite a very recent divergence time.     

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.     

The lesser wanderer butterfly, Danaus petilia (Stoll 1790) stat. rev. (Lepidoptera: Danainae), reinstated as a species

Abstract  The lesser wanderer butterfly, Danaus ( Anosia ) chrysippus petilia (Stoll) (Lepidoptera: Danainae), has been treated as a subspecies for the last 100 years. New mitochondrial DNA sequence data for D. petilia , in conjunction with allozyme, structural, morphometric and pattern characters, constitute a compelling case for its specific rank. The holotype of D. petilia has never been located and, as the type location is uncertain, a neotype is designated. Fresh material was collected and examined for this project. Danaus petilia and D. chrysippus have been separated at Lydekker's Line for an estimated 1.1 million years, and they remain interfertile. However, as an allopatric taxon, diagnosable from D. chrysippus , D. petilia merits specific status under the phylogenetic species concept. The following pairs of D. chrysippus subspecies are considered to be synonyms, the first member of each pair having priority: chrysippus  +  aegyptius (subspecies), klugii  +  infumata (hybrid phenotypes) and orientis  + liboria (subspecies).     

The strength of reproductive isolation in two hybridizing food-deceptive orchid species

Reproductive isolation is of fundamental importance for maintaining species boundaries in sympatry. In orchids, the wide variety of pollination systems and highly diverse floral traits have traditionally suggested a prominent role for pollinator isolation, and thus for prezygotic isolation, as an effective barrier to gene flow among species. Here, we examined the nature of reproductive isolation between Anacamptis morio and Anacamptis papilionacea, two sister species of Mediterranean food-deceptive orchids, in two natural hybrid zones. Comparative analyses of the two hybrid zones that are located on soils with volcanic origin and have different and well-dated ages consistently revealed that all hybrid individuals were morphologically and genetically intermediate between the parental species, but had strongly reduced fitness. Molecular analyses based on nuclear ITS1 and (amplified fragment length polymorphism) AFLP markers clearly showed that all examined hybrids were F1 hybrids, and that no introgression occurred between parental species. The maternally inherited plastid DNA markers indicated that hybridization between A. morio and A. papilionacea was bidirectional, as confirmed by the molecular analysis of seed families. The genetic architecture of the two hybrid zones suggests that the two parental species easily and frequently hybridize in sympatry as a consequence of partial pollinator overlap but that strong postzygotic barriers reduce hybrid fitness and prevent gene introgression. These results corroborate that chromosomal divergence is instrumental for reproductive isolation between these food-deceptive orchids and suggest that hybridization is of limited importance for their diversification.     

Speciation in Progress? A Continuum of Reproductive Isolation in <Emphasis Type="Italic">Drosophila Bipectinata</Emphasis>

Incipient species in the early stages of divergence can provide crucial information about the genetic basis of reproductive isolation and the evolutionary forces that promote speciation. In this report, we describe two subspecies of Drosophila bipectinata that show a continuum of reproductive isolation. Crosses between strains of the same subspecies produce fully fertile offspring. At the same time, each subspecies harbors extensive variation for the degree of reproductive isolation from the other subspecies. The percentage of fertile hybrid males varies from 0 to 90%, depending on the origin of parental strains, indicating that the genes responsible for hybrid sterility are not fixed within either subspecies, or even within local populations. Reproductive isolation is non-transitive, so that the extent of hybrid sterility depends on the particular combination of strains. The two subspecies show little or no evidence of genetic differentiation at three nuclear loci, suggesting that they diverged very recently or continue to experience significant levels of gene flow. A hybrid zone between the two subspecies may exist in New Guinea and Northeastern Australia.     

Nuclear markers,mitochondrial DNA and male secondary sexual traits variation in a newt hybrid zone (Triturus vulgaris x T. montandoni)

The smooth and the Montandon's newts (Triturus vulgaris and T. montandoni) are genetically similar sister species with highly divergent male secondary sexual traits involved in complex courtship behaviour. Their parapatric ranges overlap at moderate elevations in the Carpathian Mountains where they hybridize readily. Here we present a detailed study of genetic and morphological variation in populations from the area of sympatry. Analysis of variation at seven nuclear markers, mtDNA and male sexual secondary traits was complemented with an ecological survey of breeding sites characteristics. Extensive hybridization was revealed with back-cross individuals similar to either parental species predominating among hybrids. The hybrid zone exhibited a mosaic pattern: the genetic composition of the populations was correlated only weakly with their geographical position. No association with habitat type was found. Departures from Hardy-Weinberg proportions, significant linkage disequilibria and bimodal distribution of genotypes suggest strongly that assortative mating is an important factor shaping the genetic composition of hybrid populations. The pattern of cytonuclear disequilibria did not indicate much asymmetry in interspecific matings. Changes in the frequency of nuclear markers were highly concordant, whereas mtDNA showed much wider bidirectional introgression with 14% excess of T. montandoni haplotype. We argue that the mosaic structure of the newt hybrid zone results mainly from stochastic processes related to extinction and recolonization. Microgeographical differences in mtDNA introgression are explained by historical range shifts. Since morphologically intermediate males were underrepresented when compared to hybrid males identified by genetic markers, sexual selection acting against the morphological intermediates is implied. We discuss the implications of these findings in the context of reinforcement of prezygotic isolation in newts.     

Molecular, morphological, and biogeographic resolution of cryptic taxa in the Greenside Darter Etheostoma blennioides complex

The systematic identity and genetic divergence of cryptic taxa and morphological subspecies in the Greenside Darter Etheostoma blennioides complex are analyzed from mitochondrial and nuclear DNA sequence data, along with morphological characters. We sequenced the mtDNA cytochrome b gene and control region and is the nuclear S7 intron 1 for 345 Greenside Darters from 19 locations across their distribution including areas of sympatry and allopatry, in comparison to putative sister species and relatives. Results provide the first genetic evidence that E. gutselli is a separate species and is the sister species of E. blennius, which together with E. rupestre comprise the sister group to the Greenside Darter complex; separating approximately 4.0 mya. MtDNA results show that the complex comprises 6 clades and supports only the morphological subspecies Etheostoma blennioides blennioides, distinguished by theta(ST)=0.94, approximately 1.7 my, scale counts, and ventral squamation. The former E. b. pholidotum and E. b. newmanii are polyphyletic and are invalid taxa, together comprising 5 differentiated clades that diverged approximately 0.90-1.7 mya. Nuclear DNA results recover some of the mtDNA clades, which are distinguished morphologically by subtle meristic count differences. Populations of E. b. blennioides genetically diverge, with diversity increasing to the southwest; attributed to restricted gene flow and genetic isolation with geographic distance. Samples of the former E. b. pholidotum from the Great Lakes/Wabash River clade are less divergent, with diversity increasing to the southwest, reflecting allopatric fragmentation and isolation by distance.     

Speciation through evolution of sex-linked genes

Identification of genes involved in reproductive isolation opens novel ways to investigate links between stages of the speciation process. Are the genes coding for ecological adaptations and sexual isolation the same that eventually lead to hybrid sterility and inviability? We review the role of sex-linked genes at different stages of speciation based on four main differences between sex chromosomes and autosomes; (1) relative speed of evolution, (2) non-random accumulation of genes, (3) exposure of incompatible recessive genes in hybrids and (4) recombination rate. At early stages of population divergence ecological differences appear mainly determined by autosomal genes, but fast-evolving sex-linked genes are likely to play an important role for the evolution of sexual isolation by coding for traits with sex-specific fitness effects (for example, primary and secondary sexual traits). Empirical evidence supports this expectation but mainly in female-heterogametic taxa. By contrast, there is clear evidence for both strong X- and Z-linkage of hybrid sterility and inviability at later stages of speciation. Hence genes coding for sexual isolation traits are more likely to eventually cause hybrid sterility when they are sex-linked. We conclude that the link between sexual isolation and evolution of hybrid sterility is more intuitive in male-heterogametic taxa because recessive sexually antagonistic genes are expected to quickly accumulate on the X-chromosome. However, the broader range of sexual traits that are expected to accumulate on the Z-chromosome may facilitate adaptive speciation in female-heterogametic species by allowing male signals and female preferences to remain in linkage disequilibrium despite periods of gene flow.     

Reconstructing asymmetrical reproductive character displacement in a periodical cicada contact zone

Selection against costly reproductive interactions can lead to reproductive character displacement (RCD). We use information from patterns of displacement and inferences about predisplacement character states to investigate causes of RCD in periodical cicadas. The 13-year periodical cicada Magicicada neotredecim exhibits RCD and strong reproductive isolation in sympatry with a closely related 13-year species, Magicicada tredecim. Displacement is asymmetrical, because no corresponding pattern of character displacement exists within M. tredecim. Results from playback and hybridization experiments strongly suggest that sexual interactions between members of these species were possible at initial contact. Given these patterns, we evaluate potential sources of selection for displacement. One possible source is 'acoustical interference', or mate-location inefficiencies caused by the presence of heterospecifics. Acoustical interference combined with the species-specificity of song pitch and preference appears to predict the observed asymmetrical pattern of RCD in Magicicada. However, acoustical interference does not appear to be a complete explanation for displacement in Magicicada, because our experiments suggest a significant potential for direct sexual interactions between these species before displacement. Another possible source of selection for displacement is hybrid failure. We evaluate the attractiveness of inferred hybrid mating signals, and we examine the viability of hybrid eggs. Neither of these shows strong evidence of hybrid inferiority. We conclude by presenting a model of hybrid failure related to life cycle differences in Magicicada.     

Complex epistasis and the genetic basis of hybrid sterility in the Drosophila pseudoobscura Bogota-USA hybridization.

We analyzed the genetic basis of postzygotic isolation between the Bogota and USA subspecies of Drosophila pseudoobscura. These subspecies diverged very recently (perhaps as recently as 155,000 to 230,000 years ago) and are partially reproductively isolated: Bogota and USA show very little prezygotic isolation but form sterile F1 males in one direction of the hybridization. We dissected the basis of this hybrid sterility and reached four main conclusions. First, postzygotic isolation appears to involve a modest number of genes: we found large chromosome regions that have no effect on hybrid fertility. Second, although apparently few in number, the factors causing hybrid sterility show a remarkably complex pattern of epistatic interaction. Hybrids suffer no hybrid sterility until they carry the "right" allele (Bogota vs. USA) at at least four loci. We describe the complete pattern of interactions between all chromosome regions known to affect hybrid fertility. Third, hybrid sterility is caused mainly by X-autosomal incompatibilities. Fourth, hybrid sterility does not involve a maternal effect, despite earlier claims to the contrary. In general, our results suggest that fewer genes are required for the appearance of hybrid sterility than implied by previous studies of older pairs of Drosophila species. Indeed, a maximum likelihood analysis suggests that roughly 15 hybrid male steriles separate the Bogota and USA subspecies. Only a subset of these would act in F1 hybrids.     

RANDOM MATING IN THE NORTHERN FLICKER HYBRID ZONE: IMPLICATIONS FOR THE EVOLUTION OF BRIGHT AND CONTRASTING PLUMAGE PATTERNS IN BIRDS

The Red-shafted and Yellow-shafted Flickers hybridize in a narrow zone on the western Great Plains of North America. The two subspecies are markedly different in six plumage traits. Plumage phenotypes were scored for the male and female of 125 mated pairs from the hybrid zone. Correlation analyses revealed no tendency towards pairing of similar phenotypes. A binomial test was performed to determine whether flickers that have parental phenotypes and are presumably immigrants rather than hybrids tend to choose mates similar to themselves. This test again indicated random mating in the hybrid zone. These data complement earlier reports in providing an additional test of alternative hybrid zone theories. Random mating along with evidence of high reproductive success in hybrids suggests that reinforcement of premating reproductive isolation is not occurring and that speciation will not result from the dynamics of the hybrid zone. At least some of the plumage traits seem to be important in the integrated courtship and territorial behaviors of flickers. Since these plumage differences have no significance in mate choice, it is inferred that they evolved in an intrasexual selection context rather than in intersexual or species-recognition contexts. Selection gradients appear to be important in restricting hybridization to a narrow zone. The gradients could result from ecological selection or from sexual selection.     

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.     

Dynamics in the evolution of sexual traits: losses and gains,radiation and convergence in yellow wagtails (Motacilla flava)

We analyse patterns of genetic diversity and song complexity in the Palaearctic yellow wagtail (Motacilla flava), a highly polytypic species complex. Mitochondrial and nuclear DNA show that the complex is polyphyletic, despite parallel plumage variation in western and eastern clades. In the western clade there is genetic structure among southern subspecies, haplotype diversity decreases with latitude, and northern subspecies show evidence of bottlenecking and rapid expansions, as expected from isolation in glacial refugia followed by postglacial colonization. However, northern subspecies, which have more divergent male plumages, lack genetic structure and sing simpler songs. Loss of song complexity and evolution of plumage in founder populations are consistent with the Kaneshiro model, which posits that variation among species is a consequence of founder-induced shifts in female preference leading to loss of ancestral male sexual traits. Our results suggest possible postglacial founder-effect mechanisms for the morhological diversification of the yellow wagtail complex.     

Hybrids between Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae) do not show endogenous postzygotic isolation

Studies of prezygotic and postzygotic isolation in Drosophila have shown in general that species in sympatry tend to evolve prezygotic barriers earlier than do species in allopatry. However, postzygotic barriers tend to evolve at the same evolutionary rate in both sympatric and allopatric species. In contrast to these observations, the grasshoppers Chorthippus parallelus parallelus and C. p. erythropus show complete hybrid male sterility but only limited prezygotic isolation after an estimated 0.5 millions years of divergence. Like their congeners, C. brunneus and C. jacobsi form a hybrid zone where their ranges meet in northern Spain. However, the hybrid zone is mosaic and bimodal and, in contrast to the high levels of postzygotic isolation between C. parallelus subspecies, these two species showed no significant reduction in hybrid fitness in F₁ or backcross generations relative to the parental generations. The level of prezygotic isolation in laboratory tests was comparable to that between C. parallelus subspecies. These results suggest that endogenous postzygotic isolation does not play an important role in the reproductive isolation between C. brunneus and C. jacobsi , or in determining the structure of the hybrid zone. Exogenous postzygotic isolation may be present and should be tested in future studies.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 195–203.     

The sequence of the acidic repeat protein (arp) gene differentiates venereal from nonvenereal Treponema pallidum subspecies, and the gene has evolved under strong positive selection in the subspecies that causes syphilis

Despite the completion of the Treponema pallidum genome project, only minor genetic differences have been found between the subspecies that cause venereal syphilis (ssp. pallidum) and the nonvenereal diseases yaws (ssp. pertenue) and bejel (ssp. endemicum). In this paper, we describe sequence variation in the arp gene which allows straightforward differentiation of ssp. pallidum from the nonvenereal subspecies. We also present evidence that this region is subject to positive selection in ssp. pallidum, consistent with pressure from the immune system. Finally, the presence of multiple, but distinct, repeat motifs in both ssp. pallidum and Treponema paraluiscuniculi (the pathogen responsible for rabbit syphilis) suggests that a diverse repertoire of repeat motifs is associated with sexual transmission. This study suggests that variations in the number and sequence of repeat motifs in the arp gene have clinical, epidemiological, and evolutionary significance.     

Revealing the mechanisms of sexual isolation in a case of sympatric and parallel ecological divergence

Two ecotypes of a marine intertidal snail (Littorina saxatilis), living at different microhabitats and shore levels, have evolved in sympatry and in parallel across the Galician rocky shore. These ecotypes differ in many traits (including size) due to differential adaptation. They meet, mate assortatively, and partially hybridize at the mid shore where the two microhabitats overlap. The partial sexual isolation observed is claimed to be a side‐effect of the size differences between ecotypes combined with a size assortative mating found in most populations of this species. We investigated this hypothesis using three complementary experimental approaches. First, we investigated which of the different shell variables contributed most to the variation in individual sexual isolation in the field by using two new statistics developed for that purpose: (1) pair sexual isolation and (2) r_i, which is based on the Pearson correlation coefficient. We found that size is the most important trait explaining the sexual isolation and, in particular, the males appear to be the key sex contributing to sexual isolation. Second, we compared the size assortative mating between regions: exposed rocky shore populations from north‐westwern Spain (showing incomplete reproductive isolation due to size assortative mating) and protected Spanish and Swedish populations (showing size assortative mating but not reproductive isolation between ecomorphs). Most of the variation in size assortative mating between localities was significantly explained by the within‐population level of variation on size. Third, we performed a laboratory male choice experiment, which further suggested that the choice is made predominantly on the basis of size. These results confirm the mechanism proposed to explain the sexual isolation in the Galician hybrid zone and thus support this case as a putative example of parallel incipient speciation. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94 , 513–526.