Introgressive hybridization between two Iberian endemic cyprinid fish: a comparison between two independent hybrid zones

Pseudochondrostoma duriense and Achondrostoma oligolepis are two Iberian endemic cyprinid fish species that occur in sympatry over most of their distribution range and that are suspected to hybridize in nature. Here, we employed a combination of mitochondrial and microsatellite markers to explore the extent of introgressive hybridization between these fishes. Two natural hybrid zones were identified in different river basins. Introgression was bi‐directional and both hybrid zones consisted mostly of parental genotypes/phenotypes (i.e. bimodal hybrid zones). Yet, they appeared to differ in the extent and direction of introgression, which supports the view that they constitute independent outcomes of different hybridization processes probably influenced by environmental features. Several discordances were found between mtDNA and microsatellite results, suggesting that this hybridization process has complex consequences and illustrating the importance of using independent markers to define accurately the hybrid status of individuals in the presence of high levels of backcrossing.     

Negative effects of deep roadside ditches on Pelophylax porosa brevipoda dispersal and migration in comparison with Hyla japonica in a rice paddy area in Japan

In Japan, rice paddies have acted as a substitute habitat for pond-breeding frogs. However, since the 1950s, agricultural modernization has altered the rice paddy environment, and pond-breeding frog populations have been decreasing. This agricultural modernization has led to rice paddy fragmentation via roadways and the construction of deep channels. To assess the influences of habitat fragmentation, we compared the distribution of two pond-breeding frogs, a common species, Hyla japonica, and an endangered species, Pelophylax porosa brevipoda, around a deep roadside ditch. In Shiga prefecture, we selected two rice paddies along a roadway and recorded the number of frogs and their snout-vent length (SVL) at the levee of a rice paddy, ditch, bank, and adjacent roadway. In total, we identified 1,293 P. p. brevipoda and 181 H. japonica. Most P. p. brevipoda were either at the levee or ditch, and the number of this species found in the ditch was much higher than in any other location in July and October. The SVLs of P. p. brevipoda found in the ditch in June were smaller than those in October. Most H. japonica were at the levee or bank, and there were no apparent temporal or spatial patterns of distribution. Our results suggest that the ditch acts as a barrier to juveniles in early summer and to all frogs during autumn for P. p. brevipoda but not for H. japonica. For long-term conservation, it is important to study the movement patterns related to life history and rice paddy management.     

Contrasting patterns of mitochondrial DNA and microsatellite introgressive hybridization between lineages of lake whitefish (Coregonus clupeaformis); relevance for speciation

We performed a combined analysis of mitochondrial DNA (mtDNA) and microsatellite loci among lake whitefish (Coregonus clupeaformis) populations in order to assess the levels of congruence between both types of markers in defining patterns of genetic structuring, introgressive hybridization and inferring population origins in the hybrid zone of the St. John River basin. A second objective was to test the hypothesis that secondary contact between glacial lineages always resulted in the occurrence of sympatric dwarf and normal whitefish ecotypes. Fish were sampled from 35 populations and polymorphism was screened at mtDNA and six microsatellite loci for a total of 688 and 763 whitefish, respectively. Four lakes harbouring a single whitefish population of normal ecotype admixed with mtDNA haplotypes of different lineages were found. This confirmed that secondary contact between whitefish evolutionary lineages did not always result in the persistence of reproductively isolated ecotypes. Microsatellites further supported the definition of distinct glacial lineages by identifying lineage-specific allelic size groups. They also further supported the hypothesis that ecotypes originated from either a single founding lineage (sympatric divergence) or following secondary contacts between lineages (allopatric divergence), depending on the lake. In general, however, the pattern of population differentiation and introgressive hybridization observed at microsatellites was in sharp contrast with that depicted by mtDNA variation. Both factorial correspondence analysis and analysis of admixture proportion revealed a much more pronounced pattern of introgressive hybridization than depicted by mtDNA analyses. Variable levels of introgression indicated that environmental differences may be as important as the historical contingency of secondary contact in explaining the persistence of sympatric ecotypes and the differential pattern of introgressive hybridization among lakes. Whitefish populations from the St. John River basin hybrid zone represent a rare illustration of a continuum of both morphological and genetic differentiation within a given taxon, spanning from complete introgression to possibly complete reproductive isolation, depending on lakes. Thus, each lake may be viewed as a different temporal snapshot taken throughout the gradual process of speciation.     

Effects of water management, connectivity, and surrounding land use on habitat use by frogs in rice paddies in Japan

In Japan, rice paddies play an important role as a substitute habitat for wetland species, and support rich indigenous ecosystems. However, since the 1950s, agricultural modernization has altered the rice paddy environment, and many previously common species are now endangered. It is urgently necessary to evaluate rice paddies as habitats for conservation. Among the species living in rice paddies, frogs are representative and are good indicator species, so we focused on frog species and analyzed the influence of environmental factors on their habitat use. We found four frog species and one subspecies (Hyla japonica, Pelophylax nigromaculatus, Glandirana rugosa, Lithobates catesbeianus, and Pelophylax porosa brevipoda) at our study sites in Shiga prefecture. For all but L. catesbeianus, we analyzed the influence of environmental factors related to rice paddy structure, water management and availability, agrochemical use, connectivity, and land use on breeding and non-breeding habitat use. We constructed generalized additive mixed models with survey date as the smooth term and applied Akaike's information criterion to choose the bestranked model. Because life histories and biological characteristics vary among species, the factors affecting habitat use by frogs are also expected to differ by species. We found that both breeding and non-breeding habitat uses of each studied species were influenced by different combinations of environmental factors and that in most cases, habitat use showed seasonality. For frog conservation in rice paddies, we need to choose favorable rice paddy in relation to surrounding land use and apply suitable management for target species.     

Consequences of hybridization between Ohomopterus insulicola and O. arrowianus (Coleoptera, Carabidae) in a segmented river basin: parallel formation of hybrid swarms

A putative hybrid zone between flightless earabid beetles, (iambus (Ohomopterus) insulicola and C. (0.) arrowianus nakamurai in the Ina Valley, central Honshu. Japan, was studied using experimental hybridization and morphological analysis. The principal morphological character analysed was a functional part of the male genitalia (the eopulatory piece), which is also the key diagnostic character in taxonomy. The habitat of the earabid beetles is fragmented by the Tenryu River, which runs through the Ina Valley, and its tributaries. Ohomopterus insulicola and 0. a. nakamurai occur in the upstream and downstream areas of the river basin, respectively, and a putative hybrid swarm (0. insulicola ssp. pseiidinsulkola) is found in the boundary area on the cast side of the Tenryu River. Experimental hybridization between 0. insulicola and 0. a. nakamurai resulted in F₁ offspring that were morphologically similar to 0. i. pseudinsulicola. Pre‐zygotic reproductive isolation was incomplete, although the F₁ males had a dysfunction in sperm production and were almost sterile. Only a single F₁ pair produced F₂, Backcrosses of F₁ females of males of the parental species resulted in offspring that were similar to the parental species in genital morphology. Based on the discriminant function for the shape of the eopulatory piece resulting from the experimental hybridization, we demonstrate that similar hybrid swarms exist on both sides of the Tenryu River, but in locations 25 km apart. This demonstrates parallelism in hybridization events with similar consequences. The hybrid swarms consisted of beetles with intermediate morphological characters and did not contain obvious parental forms. The establishment of such intermediate populations may have been facilitated by selection for fertile hybrids in segregated local sites in the absence of frequent immigration of parental species. This study suggests that a segmented river basin provides an opportunity for establishing novel evolutionary lineages resulting from hybridization.     

Detection and characterization of Hyaloklossia kasumiensis (Apicomplexa: Sarcocystidae) from Nagoya Daruma pond frog,Pelophylax porosus brevipodus

The Nagoya Daruma pond frog (Pelophylax porosus brevipodus) is an endangered anuran species endemic to the semi-aquatic environment of Japan. On June 11, 2021, four of the six Nagoya Daruma pond frogs captured from the wild and kept in an aquarium died in rapid succession. Pathological and/or parasitological examinations of two of the four carcasses revealed protozoan infection in their kidneys. The pathogen identified as Hyaloklossia kasumiensis (Apicomplexa: Sarcocystidae: Hyaloklossiinae) based on their morphological and molecular characterization. This is a new host record of H. kasumiensis. Mitochondrial cox1 sequence analysis suggest that the present species detected from Nagoya Daruma pond frog belongs to the same lineage as those found in the Tokyo Daruma pond frog (P. p. porosus) from Ibaraki Prefecture, Japan. This particular lineage may be prevalent among Pelophylax porosus ssp. distributed in Japan.     

Genetic divergence among invasive and native populations of Plagioscion squamosissimus (Perciformes, Sciaenidae) in Neotropical regions

The genetic divergence among invasive and native populations of Plagioscion squamosissimus from four Neotropical hydrographic basins was assessed using the hypervariable domain of the mitochondrial DNA (mtDNA) control region. Plagioscion squamosissimus is native to the neighbouring hydrographic basins of the Parnaíba and Amazon Rivers, and the latter includes the Araguaia-Tocantins drainage, but it is invasive in other basins due to introductions. The mtDNA nucleotide polymorphism supported the hypothesis that the Amazon and Parnaíba populations constitute the same species and are separated into two independent evolutionary lineages. Absence of nucleotide polymorphism was observed within and among P. squamosissimus populations invasive to the uppper and middle Paraná River basins. Nucleotide divergence was null or low comparing the Paraná invasive populations with the populations native to the Parnaíba River basin, whereas it was significantly high compared to Tocantins populations. These results ascertain that P. squamosissimus populations invasive to the upper Paraná River basin and to the middle Paraná River basin downstream of the Itaipu dam are derived from the Parnaíba River basin. The genetic data presented are potentially useful to assist further studies on P. squamosissimus taxonomic and geographic distribution, development of ecological guidelines for managing populations invasive to the upper Paraná River basin and for preservation of native fish diversity.     

Discordance Between Spatial Distributions of Y-Chromosomal and Mitochondrial Haplotypes in African Green Monkeys (Chlorocebus spp.): A Result of Introgressive Hybridization or Cryptic Diversity?

Introgressive hybridization may cause substantial discordances among phylogenies based on different genetic markers. Such discordances have been found in diverse mammal species including primates. A recent study of mitochondrial DNA (mtDNA) revealed several poly- and paraphyletic relationships in African green monkeys (Chlorocebus), suggesting contemporary and/or ancient introgressive hybridization among almost all parapatric species of the genus. However, mtDNA analyses alone do not allow us to draw conclusions concerning introgression events. In this study we analyzed two Y chromosomal (Y-chr) markers for 30 African green monkey samples and compared the resulting genetic relationships to those based on published mtDNA data. In line with the results for mtDNA, we found no Y-chr evidence of hypothesized hybridization among Chlorocebus sabaeus and C. tantalus in the northern part of the contact zone in West Africa, and we found two distinct and distantly related Y-chr haplotypes within the range of C. tantalus, suggesting possible cryptic genetic diversity rather than ancient introgressive hybridization in this species. In contrast, Y-chr data revealed monophyletic relationships within Chlorocebus pygerythrus from East Africa, suggesting that mtDNA paraphylies found in this species are most likely to be the result of ancient introgressive hybridization and subsequent cytonuclear extinction of an earlier taxon. Our results accentuate the importance of analyzing sex chromosomal data in addition to mtDNA to obtain more information on the potential outcomes of hybridization with respect to genetic and species diversity. Analysis of more diverse nuclear marker sets is needed to obtain a more complete picture of the African green monkey evolution.     

Assessment of gene flow across a hybrid zone in red-tailed chipmunks (Tamias ruficaudus)

The role of hybridization in animal speciation is controversial and recent research has challenged the long-standing criterion of complete reproductive isolation to attain species status. The speciation-with-gene-flow model posits that the genome is semi-permeable and hybridization may be a phase in the process of divergence. Here, we apply these concepts to a previously identified zone of mtDNA introgression between the two strongly morphologically differentiated subspecies of red-tailed chipmunk ( Tamias ruficaudus ) in the US Inland Northwest. Using multilocus genotype data from the southern, older contact zone, we demonstrate that neutral gene flow is unusually low between the subspecies across the Lochsa River. This is geographically congruent with the discontinuity in bacular morphology, indicating that the cline of mitochondrial DNA (mtDNA) haplotypes is displaced. Furthermore, we elucidate the evolutionary forces responsible by testing hypotheses of lineage sorting and hybridization. We determined that introgressive hybridization is the cause of mtDNA/morphology incongruence because there are non-zero levels of migration and gene flow. Although our estimate of the age of the hybrid zone has wide credibility intervals, the hybridization events occurred in the Late Pleistocene and the divergence occurred in the Middle Pleistocene. Finally, we assessed substructure within and adjacent to the hybrid zone and found that the hybrid zone constitutes a set of populations that are genetically differentiated from parental sets of populations; therefore, hybridization in this system is not likely an evolutionary sink, but has generated novel combinations of genotypes.     

THE ROLE OF INTROGRESSIVE HYBRIDIZATION IN THE EVOLUTION OF THE GILA ROBUSTA COMPLEX (TELEOSTEI: CYPRINIDAE)

Abstract The extent and impact of introgressive hybridization was examined in the Gila robusta complex of cyprinid fishes using mitochondrial DNA (mtDNA) sequence variation. Lower Colorado River basin populations of G. robusta, G. elegans, and G. cypha exhibited distinct mtDNAs, with only limited introgression of G. elegans into G. cypha. The impact of hybridization was significant in upper Colorado River basin populations; most upper basin fishes sampled exhibited only G. cypha mtDNA haplotypes, with some individuals exhibiting mtDNA from G. elegans. The complete absence of G. robusta mtDNA, even in populations of morphologically pure G. robusta, indicates extensive introgression that predates human influence. Analysis of the geographic distribution of variation identified two distinctive G. elegans lineages; however, the small number of individuals and localities sampled precluded a comprehensive analysis. Analysis of haplotype and population networks for G. cypha mtDNAs from 15 localities revealed low divergence among haplotypes; however, significant frequency differences among populations within and among drainages were found, largely attributable to samples in the Little Colorado River region. This structure was not associated with G. cypha and G. robusta, as morphotypes from the same location are more similar than conspecific forms in other locations. This indicates that morphological and mtDNA variation are affected by different evolutionary forces in Colorado River Gila and illustrates how both hybridization and local adaptation can play important roles in evolution.     

Genetic diversity and phylogenetic origin of brown trout Salmo trutta populations in eastern Balkans

The study focuses on the phylogenetic origin and genetic diversity of brown trout in the eastern part of the Balkan Peninsula. It further aims to reveal the impact of human-mediated transfers and stocking with non-indigenous trout on the populations in this area. For these purposes, mtDNA control region and microsatellite variation of 204 individuals from 16 populations were analysed. The results indicate that mtDNA haplotypes from the lower Danube basin and southern Black Sea basins differ substantially from a subclade of the Danubian lineage consisting of haplotypes found so far in the most of the Danube basin and in the Caspian and Aral Sea basins. Considering also the results of demographic analyses, this study evidences a complex evolutionary history of brown trout in the southern and western parts of the Black Sea basin. In the Aegean Sea basin, a high frequency of the central haplotype of Adriatic mtDNA lineage has been found. The other Adriatic lineage haplotypes found in this basin differ from the central haplotype by one mutational step only, indicating a recent evolution of the Adriatic lineage in the Aegean Sea basin. Substantial genetic differentiation among populations and basins was revealed. The hybridization with Atlantic brown trout was indicated in both sea basins, but especially in the Danube basin. Compared to other European regions, it can be inferred that the introgression of exogenous brown trout in the eastern Balkan populations is rather low.     

Introgressive hybridization off chars of the genus Salvelinus as inferred from mitocchondrial DNA variation

Nucleotide sequence analysis of the mitochondrial cyttochrome b gene in the genus Salvelinus revealed hybrids with S. leucomaenis in S. malma malma populations ofthe Northern Okhotsk Sea basin. Hybrids of S. m. malma and S. m. krascheninnikovi were found in S. m. malma populations from the Northern Okhotsk Sea basin and Kamchatka. The findings testify to a secondary contact and mtDNA transfer between these species and forms. It was assumed that introgressive hybridization took place both long ago and relatively recently and occurred in one direction: from S. leucomaenis or S. m. krascheninnikovi to S. m. malma.     

On the systematics and phylogeography of eight-barbel loaches the genus Lefua (Cobitoidea: Nemacheilidae): mtDNA typing of L. pleskei

Comparative analysis of personal sequence data for the mtDNA control region (926 to 928 bp) from eight-barbel loaches inhabiting eight localities in the Amur River basin (4) and the Sea of Japan (4) and the GeneBank/NCBI data for the Lefua individuals from the other regions of the world showed that eight-barbel loaches from Primorskii krai water basins were marked by a specific group of mtDNA haplotypes. This finding is considered as supporting the species status of L. pleskei. Genetic distances within L. pleskei are small (on average 0.355) and close to those within L. nikkonis (on average 0.48%). The distances between this species pair are the least (on average 2.15%) among all other pairs compared. In MP, ML, and Bayesian trees, L. pleskei and L. nikkonis haplotypes formed a common clade with high statistically significant support. In all tree variants, L. costata mtDNA haplotypes were located out of the group of interest. A clade consisting of highly diverged lineages of Lefua sp. and L. echigonia haplotypes occupied even more independent position. The mtDNA haplotypes of L. pleskei and L. costata from the Amur River basin were evolutionary young and derived from the haplotypes found in these species from the Sea of Japan (L. pleskei) or the Yellow Sea (L. costata) basins. It is thereby suggested that both species rather recently migrated into the Amur River system. According to the molecular clock data, basal diversification of the eight-barbel loach lineages took place at the end of middle Miocene (about 11 to 12 Myr ago), while divergence of L. pleskei and L. costata ancestral forms probably occurred approximately, 5 Myr ago. Since all main lineages of eight-barbel loaches were found in the Sea of Japan basin (continental coastline and the islands), the divergence order and dispersal patterns of the Lafua species might have been largely determined by the geological development pattern of this water body and the adjacent territories.     

Hybridization and skua phylogeny

The close similarity in mitochondrial DNA (mtDNA) between Pomarine and (North Atlantic) Great skuas, Stercorarius pomarinus and Stercorarius skua, has several possible explanations. Two of the main alternatives are stochastic lineage sorting of mtDNA and introgressive hybridization between S. pomarinus and S. skua. Here, a new probabilistic approach to testing these alternatives using different phylogenies based on external morphology, mtDNA and ectoparasites leads to rejection of the lineage sorting and corroboration of the hybridization hypothesis. If great skuas are a monophyletic group, as indicated by the present cladistic analysis based on morphology and by other evidence, the available molecular genetic data are as expected if mtDNA has been transferred from S. pomarinus to S. skua by introgressive hybridization. Further evidence from several unlinked regions of nuclear DNA is crucial for critical testing of the alternative hypotheses on skua evolution.     

Hybridization and introgression in New World red mangroves, Rhizophora (Rhizophoraceae)

? Premise of the study: Hybridization is common in both animals and plants and can lead to a diverse array of outcomes ranging from the generation of new ecotypes or species to the breakdown of morphological differences. Here, we explore the extent of hybridization in the three currently recognized New World Rhizophora species-R. mangle, R. racemosa, and the putative hybrid species R. harrisonii. ? Methods: We assayed variation across the three recognized Rhizophora species using two noncoding chloroplast (cpDNA), two flanking microsatellite regions (FMRs), and six microsatellite loci. ? Key results: Gene genealogies of cpDNA and FMRs showed a strong phylogeographic break across the Central American Isthmus, but little relationship to recognized species boundaries. Instead, individuals collected in the same ocean basin and classified as R. mangle and R. racemosa by morphological characteristics were more closely related to each other than with similar looking individuals collected in the other ocean basin. Nonetheless, there were low, yet significant differences at microsatellite loci among co-occurring populations of R. mangle and R. racemosa in both ocean basins, suggesting that two taxonomic groups coexist. However, we found no genetic evidence that R. harrisonii was a hybrid species. Rather, R. harrisonii appears to represent a morphotype produced by ongoing hybridization and backcrossing between R. mangle and R. racemosa. ? Conclusions: Our data support ancient and persistent introgressive hybridization among new world Rhizophora and argue for a full revision of the systematic relationships of the group based on much finer morphological, ecological, and genetic analyses.     

Molecules and morphology: evidence for introgression of mitochondrial DNA in Dalmatian cyprinids

In one population of Scardinius dergle, mitochondrial DNA was observed originating from Squalius tenellus. Scardinius dergle shared all diagnostic morphological characters and similarities at a highly variable nuclear region with the genus Scardinius. While crosses and backcrosses most likely resulted in the loss of morphologically diagnostic Squalius-features, maternal inheritance of mtDNA fixed their diagnostic substitutions. Anthropogenic influences resulting from dam building in 1962 are suspected to be the initial force for the hybridization events. However, because hybridization took place despite both lineages being only very distantly related (p=11.2%), we conclude that introgressive hybridization events can be seen as generally possible among leuciscine cyprinids.     

Evolutionary relationships and reproductive isolating mechanisms in the rice frog (Fejervarya limnocharis) species complex from Sri Lanka, Thailand, Taiwan and Japan, inferred from mtDNA gene sequences, allozymes, and crossing experiments

The rice frog (Fejervarya limnocharis) species complex is widely distributed, from India to Japan, and most prevalently in Southeast Asia. Conspicuous morphological variation has been reported for this species complex throughout its distribution range. In the present study, we used mtDNA gene sequence and allozyme analyses to infer evolutionary affinities within this species complex using eight populations (Sri Lanka; Bangkok and Ranong in Thailand; Taiwan; and Hiroshima, Okinawa, Ishigaki and Iriomote in Japan). We also conducted crossing experiments among four populations from Japan, Thailand, and Sri Lanka in order to find out more about the reproductive isolating mechanisms that might exist among the East, Southeast, and South Asian populations of this species complex. The crossing experiments revealed that the Sri Lanka population is reproductively isolated from the Hiroshima, Bangkok, and Ranong populations by complete hybrid inviability, and that the Bangkok population may be reproductively isolated from the Hiroshima population by partial hybrid inviability. Thus, it is not unreasonable to regard the Sri Lanka population as a species separated from F. limnocharis. The mtDNA and allozyme data showed that the Ranong population is most closely related to the Bangkok population in nuclear genome, but more similar to the Okinawa and Taiwan populations in mtDNA genome. The present, preliminary survey may raise questions about the species status of these particular populations and also about the nature of the biological species concept.     

Evaluating the genetic status of a Great Basin endemic minnow: the relict dace (<Emphasis Type="Italic">Relictus solitarius</Emphasis>)

The relict dace (Relictus solitarius) is an endemic minnow restricted to isolated spring systems within Butte, Goshute, Ruby and Steptoe valleys in the eastern Nevada portion of the Great Basin. It is the only native fish in these basins, but speckled dace (Rhinichthys osculus) have been introduced into some localities in Ruby Valley and, based on morphology, the two species are believed to have hybridized in those areas. We used DNA sequence data from one mitochondrial gene (cytochrome b) and one nuclear intron (S7) to determine if putative relict dace populations in Ruby Valley share mtDNA haplotypes with speckled dace, or show signs of hybridization. Results of our analyses show that 11 of 16 Ruby Valley populations sampled contain only speckled dace, and one speckled dace population exhibited signs of limited introgressive hybridization. The remaining five Ruby Valley populations that were sampled contain relict dace that remain genetically distinct from speckled dace. The integrity of the Ruby Valley relict dace populations should be actively protected, particularly in the area surrounding Ruby Lake where speckled dace have been widely introduced.     

Recent introgressive hybridization revealed by exclusive mtDNA transfer from Oreochromis leucostictus (Trewavas, 1933) to Oreochromis niloticus (Linnaeus, 1758) in Lake Baringo, Kenya

Nuclear DNA and mtDNA polymorphisms were surveyed in various species of East African Oreochromis . In Lake Baringo, where only Oreochromis niloticus baringoensis is present, alien mtDNA haplotypes were observed, apparently the result of introgressive hybridization with Oreochromis leucostictus . This introgression is not accompanied by any substantial or recorded transfer of nuclear genes into O. n. baringoensis .     

Negligible nuclear introgression despite complete mitochondrial capture between two species of chipmunks

The idea that species boundaries can be semipermeable to gene flow is now widely accepted but the evolutionary importance of introgressive hybridization remains unclear. Here we examine the genomic contribution of gene flow between two hybridizing chipmunk species, Tamias ruficaudus and T. amoenus. Previous studies have shown that ancient hybridization has resulted in complete fixation of introgressed T. ruficaudus mitochondrial DNA (mtDNA) in some populations of T. amoenus, but the extent of nuclear introgression is not known. We used targeted capture to sequence over 10,500 gene regions from multiple individuals of both species. We found that most of the nuclear genome is sorted between these species and that overall genealogical patterns do not show evidence for introgression. Our analysis rules out all but very minor levels of interspecific gene flow, indicating that introgressive hybridization has had little impact on the overall genetic composition of these species outside of the mitochondrial genome. Given that much of the evidence for introgression in animals has come from mtDNA, our results underscore that unraveling the importance introgressive hybridization during animal speciation will require a genome‐wide perspective that is still absent for many species.