Sympatric bromeliad species (Pitcairnia spp.) facilitate tests of mechanisms involved in species cohesion and reproductive isolation in Neotropical inselbergs

The roles of intra- and interspecific gene flow in speciation and species evolution are topics of great current interest in molecular ecology and evolutionary biology. Recent modelling studies call for new empirical data to test hypotheses arising from the recent shift from a 'whole-genome reproductive isolation' view to a 'genic' view of species and speciation. Particularly scarce (and thus of particular interest) are molecular genetic data on recently radiated, naturally hybridizing species in strongly structured and species-rich environments. Here, we studied four sympatric plant species (Pitcairnia spp.; Bromeliaceae) adapted to Neotropical inselbergs (isolated outcrops resembling habitat 'islands' in tropical rainforests) using nuclear and plastid DNA. Patterns of plastid DNA haplotype sharing and nuclear genomic admixture suggest the presence of both, incomplete lineage sorting and interspecific gene flow over extended periods of time. Integrity and cohesion of inselberg species of Pitcairnia are maintained despite introgression and in the face of extremely low within-species migration rates (N(e)m < 1 migrant per generation). Cross-evaluation of our genetic data against published pollination experiments indicate that species integrity is maintained by the simultaneous action of multiple prezygotic barriers, including flowering phenology, pollinator isolation and divergent mating systems. Postzygotic Bateson-Dobzhansky-Muller incompatibilities appear to contribute to isolation, as suggested by asymmetric introgression rates of single loci. Our results suggest that incomplete lineage sorting, hybridization and introgression form integral aspects of adaptive radiation in Neotropical inselberg 'archipelagos'. Inselbergs with multiple closely related co-occurring species should be of special interest to students of speciation in mountain systems, and to ongoing conservation programmes in the Atlantic Rainforest biodiversity hotspot.     

An HMM-Based Comparative Genomic Framework for Detecting Introgression in Eukaryotes

One outcome of interspecific hybridization and subsequent effects of evolutionary forces is introgression, which is the integration of genetic material from one species into the genome of an individual in another species. The evolution of several groups of eukaryotic species has involved hybridization, and cases of adaptation through introgression have been already established. In this work, we report on PhyloNet-HMM—a new comparative genomic framework for detecting introgression in genomes. PhyloNet-HMM combines phylogenetic networks with hidden Markov models (HMMs) to simultaneously capture the (potentially reticulate) evolutionary history of the genomes and dependencies within genomes. A novel aspect of our work is that it also accounts for incomplete lineage sorting and dependence across loci. Application of our model to variation data from chromosome 7 in the mouse (Mus musculus domesticus) genome detected a recently reported adaptive introgression event involving the rodent poison resistance gene Vkorc1, in addition to other newly detected introgressed genomic regions. Based on our analysis, it is estimated that about 9% of all sites within chromosome 7 are of introgressive origin (these cover about 13 Mbp of chromosome 7, and over 300 genes). Further, our model detected no introgression in a negative control data set. We also found that our model accurately detected introgression and other evolutionary processes from synthetic data sets simulated under the coalescent model with recombination, isolation, and migration. Our work provides a powerful framework for systematic analysis of introgression while simultaneously accounting for dependence across sites, point mutations, recombination, and ancestral polymorphism.     

Adaptive introgression in animals: examples and comparison to new mutation and standing variation as sources of adaptive variation

Adaptive genetic variation has been thought to originate primarily from either new mutation or standing variation. Another potential source of adaptive variation is adaptive variants from other (donor) species that are introgressed into the (recipient) species, termed adaptive introgression. Here, the various attributes of these three potential sources of adaptive variation are compared. For example, the rate of adaptive change is generally thought to be faster from standing variation, slower from mutation and potentially intermediate from adaptive introgression. Additionally, the higher initial frequency of adaptive variation from standing variation and lower initial frequency from mutation might result in a higher probability of fixation of the adaptive variants for standing variation. Adaptive variation from introgression might have higher initial frequency than new adaptive mutations but lower than that from standing variation, again making the impact of adaptive introgression variation potentially intermediate. Adaptive introgressive variants might have multiple changes within a gene and affect multiple loci, an advantage also potentially found for adaptive standing variation but not for new adaptive mutants. The processes that might produce a common variant in two taxa, convergence, trans‐species polymorphism from incomplete lineage sorting or from balancing selection and adaptive introgression, are also compared. Finally, potential examples of adaptive introgression in animals, including balancing selection for multiple alleles for major histocompatibility complex (MHC), S and csd genes, pesticide resistance in mice, black colour in wolves and white colour in coyotes, Neanderthal or Denisovan ancestry in humans, mimicry genes in Heliconius butterflies, beak traits in Darwin's finches, yellow skin in chickens and non‐native ancestry in an endangered native salamander, are examined.     

Genomics of speciation and introgression in Princess cichlid fishes from Lake Tanganyika

How variation in the genome translates into biological diversity and new species originate has endured as the mystery of mysteries in evolutionary biology. African cichlid fishes are prime model systems to address speciation‐related questions for their remarkable taxonomic and phenotypic diversity, and the possible role of gene flow in this process. Here, we capitalize on genome sequencing and phylogenomic analyses to address the relative impacts of incomplete lineage sorting, introgression and hybrid speciation in the Neolamprologus savoryi‐complex (the ‘Princess cichlids’) from Lake Tanganyika. We present a time‐calibrated species tree based on whole‐genome sequences and provide strong evidence for incomplete lineage sorting in the early phases of diversification and multiple introgression events affecting different stages. Importantly, we find that the Neolamprologus chromosomes show centre‐to‐periphery biases in nucleotide diversity, sequence divergence, GC content, incomplete lineage sorting and rates of introgression, which are likely modulated by recombination density and linked selection. The detection of heterogeneous genomic landscapes has strong implications on the genomic mechanisms involved in speciation. Collinear chromosomal regions can be protected from gene flow and harbour incompatibility genes if they reside in lowly recombining regions, and coupling can evolve between nonphysically linked genomic regions (chromosome centres in particular). Simultaneously, higher recombination towards chromosome peripheries makes these more dynamic, evolvable regions where adaptation polymorphisms have a fertile ground. Hence, differences in genome architecture could explain the levels of taxonomic and phenotypic diversity seen in taxa with collinear genomes and might have contributed to the spectacular cichlid diversity observed today.     

Genome Patterns of Selection and Introgression of Haplotypes in Natural Populations of the House Mouse (Mus musculus)

General parameters of selection, such as the frequency and strength of positive selection in natural populations or the role of introgression, are still insufficiently understood. The house mouse (Mus musculus) is a particularly well-suited model system to approach such questions, since it has a defined history of splits into subspecies and populations and since extensive genome information is available. We have used high-density single-nucleotide polymorphism (SNP) typing arrays to assess genomic patterns of positive selection and introgression of alleles in two natural populations of each of the subspecies M. m. domesticus and M. m. musculus. Applying different statistical procedures, we find a large number of regions subject to apparent selective sweeps, indicating frequent positive selection on rare alleles or novel mutations. Genes in the regions include well-studied imprinted loci (e.g. Plagl1/Zac1), homologues of human genes involved in adaptations (e.g. alpha-amylase genes) or in genetic diseases (e.g. Huntingtin and Parkin). Haplotype matching between the two subspecies reveals a large number of haplotypes that show patterns of introgression from specific populations of the respective other subspecies, with at least 10% of the genome being affected by partial or full introgression. Using neutral simulations for comparison, we find that the size and the fraction of introgressed haplotypes are not compatible with a pure migration or incomplete lineage sorting model. Hence, it appears that introgressed haplotypes can rise in frequency due to positive selection and thus can contribute to the adaptive genomic landscape of natural populations. Our data support the notion that natural genomes are subject to complex adaptive processes, including the introgression of haplotypes from other differentiated populations or species at a larger scale than previously assumed for animals. This implies that some of the admixture found in inbred strains of mice may also have a natural origin.     

GENE FLOW AND SPECIES DELIMITATION: A CASE STUDY OF TWO PINE SPECIES WITH OVERLAPPING DISTRIBUTIONS IN SOUTHEAST CHINA

Species delimitation detected by molecular markers is complicated by introgression and incomplete lineage sorting between species. Recent modeling suggests that fixed genetic differences between species are highly related to rates of intraspecific gene flow. However, it remains unclear whether such differences are due to high levels of intraspecific gene flow overriding the spread of introgressed alleles or favoring rapid lineage sorting between species. In pines, chloroplast (cp) and mitochondrial (mt) DNAs are normally paternally and maternally inherited, respectively, and thus their relative rates of intraspecific gene flow are expected to be high and low, respectively. In this study, we used two pine species with overlapping geographical distributions in southeast China, P. massoniana and P. hwangshanensis, as a model system to examine the association between organelle gene flow and variation within and between species. We found that cpDNA variation across these two pine species is more species specific than mtDNA variation and almost delimits taxonomic boundaries. The shared mt/cp DNA genetic variation between species shows no bias in regard to parapatric versus allopatric species’ distributions. Our results therefore support the hypothesis that high intraspecific gene flow has accelerated cpDNA lineage sorting between these two pine species.     

Integrating Bayesian genomic cline analyses and association mapping of morphological and ecological traits to dissect reproductive isolation and introgression in a Louisiana Iris hybrid zone

Hybrid zones provide unique opportunities to examine reproductive isolation and introgression in nature. We utilized 45,384 single nucleotide polymorphism (SNP) loci to perform association mapping of 14 floral, vegetative and ecological traits that differ between Iris hexagona and Iris fulva, and to investigate, using a Bayesian genomic cline (BGC) framework, patterns of genomic introgression in a large and phenotypically diverse hybrid zone in southern Louisiana. Many loci of small effect size were consistently found to be associated with phenotypic variation across all traits, and several individual loci were revealed to influence phenotypic variation across multiple traits. Patterns of genomic introgression were quite heterogeneous throughout the Louisiana Iris genome, with I. hexagona alleles tending to be favoured over those of I. fulva. Loci that were found to have exceptional patterns of introgression were also found to be significantly associated with phenotypic variation in a small number of morphological traits. However, this was the exception rather than the rule, as most loci that were associated with morphological trait variation were not significantly associated with excess ancestry. These findings provide insights into the complexity of the genomic architecture of phenotypic differences and are a first step towards identifying loci that are associated with both trait variation and reproductive isolation in nature.     

Use of introgression lines and zonal mapping to identify RAPD markers linked to QTL

RAPD primers were identified as giving parent-specific bands when screened with a set of introgression lines containing introgressed regions of Lycopersicon pennellii that encompass 5 quantitative trait loci affiliated with the production and composition of acylsugars, compounds associated with insect resistance. Primers giving L. pennellii introgression specific bands were zonally mapped to identify bands affiliated with the quantitative trait target and flanking regions using subsets of 7 to 16 F2 individuals which contained small overlapping segments (zones) of the L. pennellii genome spanning those regions. Seventeen RAPD primers, agt-related primers, and an agt clone were then used in mapping the complete F2 population of 144 individuals. This work resulted in the identification of RAPD markers for three of the 5 quantitative trait loci and the construction of an integrated RAPD/RFLP genomic map for tomato (Lycopersicon esculentum x L. pennellii LA716) of 111 RAPD and 8 acylglucose transferase related markers added to a framework map of 150 RFLP markers.     

TESTING MITOCHONDRIAL CAPTURE AND DEEP COALESCENCE IN AMAZONIAN CICHLID FISHES (CICHLIDAE: CICHLA)

Hybridization and introgression have important consequences in evolution, such as increasing the genetic diversity and adaptive potential of a species. One of their most conspicuous footprints is discordance among gene trees or between genes and phenotypes. However, most studies that report introgression fail to disprove the null hypothesis that genetic incongruence may result from stochastic sorting of ancestral allelic polymorphisms. In the case of ancient introgression, these two processes may be especially difficult to distinguish topologically, but they make different predictions about the patterns of coalescence among loci. Here we apply three methods, molecular dating, multispecies coalescent models, and gene tree simulation under coalescence, to compare these two hypotheses that explain the polyphyletic mtDNA of the butterfly peacock bass, Cichla orinocensis. In comparison with a species tree based on 20 unlinked nuclear loci, we determined that mtDNA divergences were too recent to be explained by ancestral polymorphism. Similarly, coalescent species tree branches were significantly shorter when putative introgressed mtDNA was incorporated, and simulations showed the mtDNA topology to be unlikely under lineage sorting only. We conclude that introgression approximately 1.5 million years ago resulted in capture by C. orinocensis of an mtDNA lineage ancestral to the modern subspecies C. oc. monoculus.     

Marker-Assisted Introgression in Backcross Breeding Programs

The efficiency of marker-assisted introgression in backcross populations derived from inbred lines was investigated by simulation. Background genotypes were simulated assuming that a genetic model of many genes of small effects in coupling phase explains the observed breed difference and variance in backcross populations. Markers were efficient in introgression backcross programs for simultaneously introgressing an allele and selecting for the desired genomic background. Using a marker spacing of 10-20 cM gave an advantage of one to two backcross generations selection relative to random or phenotypic selection. When the position of the gene to be introgressed is uncertain, for example because its position was estimated from a trait gene mapping experiment, a chromosome segment should be introgressed that is likely to include the allele of interest. Even for relatively precisely mapped quantitative trait loci, flanking markers or marker haplotypes should cover ~10-20 cM around the estimated position of the gene, to ensure that the allele frequency does not decline in later backcross generations.     

Detecting adaptive trait introgression between Iris fulva and I. brevicaulis in highly selective field conditions

The idea that natural hybridization has served as an important force in evolutionary and adaptive diversification has gained considerable momentum in recent years. By combining genome analyses with a highly selective field experiment, we provide evidence for adaptive trait introgression between two naturally hybridizing Louisiana Iris species, flood-tolerant Iris fulva and dry-adapted I. brevicaulis. We planted reciprocal backcross (BC1) hybrids along with pure-species plants into natural settings that, due to a flooding event, favored I. fulva. As expected, I. fulva plants survived at much higher rates than I. brevicaulis plants. Backcross hybrids toward I. fulva (BCIF) also survived at significantly higher rates than the reciprocal backcross toward I. brevicaulis (BCIB). Survivorship of BCIB hybrids was strongly influenced by the presence of a number of introgressed I. fulva alleles located throughout the genome, while survivorship in the reciprocal BCIF hybrids was heavily influenced by two epistatically acting QTL of opposite effects. These results demonstrate the potential for adaptive trait introgression between these two species and may help to explain patterns of genetic variation observed in naturally occurring hybrid zones.     

Morphology and nuclear markers reveal extensive mitochondrial introgressions in the Iberian Wall Lizard species complex

Mitochondrial markers are still often used alone to identify evolutionary units, despite widespread evidence for processes such as incomplete lineage sorting or introgressive hybridization that may blur past population history. The combination of mitochondrial DNA data with other sources of information (morphology, nuclear genes) is a powerful tool to reveal when and why mitochondrial markers are potentially misleading. In this study, we evaluate the performance of mtDNA markers to unravel the evolutionary history of Spanish lizards from the Podarcis hispanicus species complex. We first uncover several cases of discordance between morphological and mitochondrial data in delimitation of taxa. To assess the origin of these discordances, we analysed the same populations using several independent nuclear loci. Both morphological and nuclear markers identified the same three evolutionary units in the region, while mitochondrial data revealed four deeply divergent lineages. We suggest here that the most likely scenario to explain this discordance is ancient mitochondrial introgression originating from a fourth evolutionary unit presently absent from the study area. Notably, this resulted in a complete replacement of the original lineage in a large part of the distribution of one of the taxa investigated. We discuss the potential evolutionary scenarios leading to this complete mitochondrial replacement and suggest why the previous studies have failed to recover the correct history of this species complex.     

Scale and direction of adaptive introgression between black cottonwood (Populus trichocarpa) and balsam poplar (P. balsamifera)

Introgression can introduce novel genetic variation at a faster rate than mutation alone and result in adaptive introgression when adaptive alleles are maintained in the recipient genome over time by natural selection. A previous study from our group demonstrated adaptive introgression from Populus balsamifera into P. trichocarpa in a target genomic region. Here we expand our local ancestry analysis to the whole genome of both parents to provide a comprehensive view of introgression patterns and to identify additional candidate regions for adaptive introgression genomewide. Populus trichocarpa is a large, fast‐growing tree of mild coastal regions of the Pacific Northwest, whereas P. balsamifera is a smaller stature tree of continental and boreal regions with intense winter cold. The species hybridize where they are parapatric. We detected asymmetric patterns of introgression across the whole genome of these two poplar species adapted to contrasting environments, with stronger introgression from P. balsamifera to P. trichocarpa than vice versa. Admixed P. trichocarpa individuals contained more genomic regions with unusually high levels of introgression (19 regions) and also the largest introgressed genome fragment (1.02 Mb) compared with admixed P. balsamifera (nine regions). Our analysis also revealed numerous candidate regions for adaptive introgression with strong signals of selection, notably related to disease resistance, and enriched for genes that may play crucial roles in survival and adaptation. Furthermore, we detected a potential overrepresentation of subtelomeric regions in P. balsamifera introgressed into P. trichocarpa and possible protection of sex‐determining regions from interspecific gene flow.     

Mitochondrial introgression and incomplete lineage sorting through space and time: phylogenetics of crotaphytid lizards

We investigate the roles of mitochondrial introgression and incomplete lineage sorting during the phylogenetic history of crotaphytid lizards. Our Bayesian phylogenetic estimate for Crotaphytidae is based on analysis of mitochondrial DNA sequence data for 408 individuals representing the 12 extant species of Crotaphytus and Gambelia. The mitochondrial phylogeny disagrees in several respects with a previously published morphological tree, as well as with conventional species designations, and we conclude that some of this disagreement stems from hybridization-mediated mitochondrial introgression, as well as from incomplete lineage sorting. Unidirectional introgression of Crotaphytus collaris (western collared lizard) mitochondria into C. reticulatus (reticulate collared lizard) populations in the Rio Grande Valley of Texas has resulted in the replacement of ancestral C. reticulatus mitochondria over approximately two-thirds of the total range of the species, a linear distance of approximately 270 km. Introgression of C. collaris mitochondria into C. bicinctores (Great Basin collared lizard) populations in southwestern Arizona requires a more complex scenario because at least three temporally separated and superimposed introgression events appear to have occurred in this region. We propose an "introgression conveyor" model to explain this unique pattern of mitochondrial variation in this region. We show with ecological niche modeling that the predicted geographical ranges of C. collaris, C. bicinctores, and C. reticulatus during glacial maxima could have provided enhanced opportunities for past hybridization. Our analyses suggest that incomplete lineage sorting and/or introgression has further confounded the phylogenetic placements of additional species including C. nebrius, C. vestigium, C. insularis, C. grismeri, and perhaps G. copei. Despite many independent instances of interspecific hybridization among crotaphytid lizards, the species continue to maintain morphological and geographic cohesiveness throughout their ranges.     

De novo genetic variation associated with retrotransposon activation, genomic rearrangements and trait variation in a recombinant inbred line population of Brassica napus derived from interspecific hybridization with Brassica rapa

Interspecific hybridization is a significant evolutionary force as well as a powerful method for crop breeding. Partial substitution of the AA subgenome in Brassica napus (AⁿAⁿCⁿCⁿ) with the Brassica rapa (A^rA^r) genome by two rounds of interspecific hybridization resulted in a new introgressed type of B. napus (A^rA^rCⁿCⁿ). In this study, we construct a population of recombinant inbred lines of the new introgressed type of B. napus. Microsatellite, intron‐based and retrotransposon markers were used to characterize this experimental population with genetic mapping, genetic map comparison and specific marker cloning analysis. Yield‐related traits were also recorded for identification of quantitative trait loci (QTLs). A remarkable range of novel genomic alterations was observed in the population, including simple sequence repeat (SSR) mutations, chromosomal rearrangements and retrotransposon activations. Most of these changes occurred immediately after interspecific hybridization, in the early stages of genome stabilization and derivation of experimental lines. These novel genomic alterations affected yield‐related traits in the introgressed B. napus to an even greater extent than the alleles alone that were introgressed from the A^r subgenome of B. rapa, suggesting that genomic changes induced by interspecific hybridization are highly significant in both genome evolution and crop improvement.     

Dynamics of introgressive hybridization assessed by SNP population genomics of coding genes in stocked brook charr (Salvelinus fontinalis)

Salmonid fishes rank among species being most severely affected by introgressive hybridization as a result of a long tradition of stocking with hatchery-reared conspecifics. The objectives of this study were (i) to evaluate the genetic consequences of stocking and resulting introgression rates on the genetic integrity of natural populations of brook charr, (ii) to identify genomic regions potentially associated with adaptation to natural and artificial rearing environments, and (iii) to test the null hypothesis that introgression from domesticated brook charr into wild populations is homogeneous among loci. A total of 336 individuals were sampled from nine lakes, which were stocked at different intensities with domestic fish. Individuals were genotyped at 280 SNPs located in transcribed regions and developed by means of next-generation sequencing. As previously reported with microsatellites, we observed a positive relationship between stocking intensity and genetic diversity among stocking groups, and a decrease in population differentiation. Individual admixture proportions also increased with stocking intensity. Moreover, genomic cline analysis revealed 27 SNPs, seven of which were also identified as outliers in a genome scan, which showed an introgression rate either more restricted or enhanced relative to neutral expectations. This indicated that selection, mainly for growth-related biological processes, has favored or hampered the introgression of genomic blocks into the introgressed wild populations. Overall, this study highlights the usefulness of investigating the impact of stocking on the dynamics of introgression of potentially adaptive genetic variation to better understand the consequences of such practice on the genomic integrity of wild populations.     

Estimating the species tree for Hawaiian Schiedea (Caryophyllaceae) from multiple loci in the presence of reticulate evolution

Schiedea (Caryophyllaceae) is a monophyletic genus of 34 species, all endemic to the Hawaiian Islands, that arose from a single colonization, providing one of the best examples of adaptive radiation in Hawai'i. Species utilize a range of habitats and exhibit a variety of growth forms and transitions in breeding systems from hermaphroditism toward dimorphism or autogamy. Our study included the most thorough sampling to date: 2-5 individuals per species and 4 independent genetic partitions: eight plastid and three low-copy nuclear loci (9217bps), allowing a three-locus BEST species tree. Despite incomplete resolution at the tips, our results support monophyly for each extant species. Gene trees revealed several clear cases of cytonuclear incongruence, likely created by interspecific introgression. Conflict occurs at the divergence of section Alphaschiedea as well as at the tips. Ages inferred from a BEAST analysis allow an original colonization onto either Nihoa or Kauaì and inform some aspects of inter-island migrations. We suggest that several hard polytomies on the species tree are biologically realistic, signifying either nearly simultaneous speciation or historical introgressive hybridization. Based on inferred node ages that exceed expected coalescent times, we propose that undetected nuclear introgression may play a larger role than incomplete lineage sorting in sections Schiedea and Mononeura.     

Next-generation hybridization and introgression

Hybridization has a major role in evolution-from the introgression of important phenotypic traits between species, to the creation of new species through hybrid speciation. Molecular studies of hybridization aim to understand the class of hybrids and the frequency of introgression, detect the signature of ancient hybridization, and understand the behaviour of introgressed loci in their new genomic background. This often involves a large investment in the design and application of molecular markers, leading to a compromise between the depth and breadth of genomic data. New techniques designed to assay a large sub-section of the genome, in association with next-generation sequencing (NGS) technologies, will allow genome-wide hybridization and introgression studies in organisms with no prior sequence data. These detailed genotypic data will unite the breadth of sampling of loci characteristic of population genetics with the depth of sequence information associated with molecular phylogenetics. In this review, we assess the theoretical and methodological constraints that limit our understanding of natural hybridization, and promote the use of NGS for detecting hybridization and introgression between non-model organisms. We also make recommendations for the ways in which emerging techniques, such as pooled barcoded amplicon sequencing and restriction site-associated DNA tags, should be used to overcome current limitations, and enhance our understanding of this evolutionary significant process.     

Nuclear loci and coalescent methods support ancient hybridization as cause of mitochondrial paraphyly between gadwall and falcated duck (Anas spp.)

Many species have mitochondrial DNA lineages that are phylogenetically intermixed with other species, but studies have rarely tested the cause of such paraphyly. In this study, we tested two hypotheses that could explain mitochondrial paraphyly of Holarctic gadwalls (Anas strepera) with respect to Asian falcated ducks (A. falcata). First, hybridization could have resulted in falcated duck mitochondrial DNA (mtDNA) introgressing into the gadwall gene pool. Second, gadwalls and falcated ducks could have diverged so recently that mtDNA lineages have not sorted to reciprocal monophyly. We used coalescent analyses of three independent loci to distinguish between these two hypotheses. Two lines of evidence support introgression. First, analyses of the three loci combined show that some introgression is necessary to explain current genetic diversity in gadwalls. Second, we generated alternative predictions regarding time since divergence estimated from mtDNA: falcated ducks and gadwalls would have diverged between 65,000 and 700,000 years before present (ybp) under the introgression hypothesis and between 11,000 and 76,000 ybp under the incomplete lineage sorting hypothesis. The two independent nuclear introns indicated that these species diverged between 210,000 and 5,200,000 ybp, which did not overlap the predicted time for incomplete lineage sorting. These analyses also suggested that ancient introgression ( approximately 14,000 ybp) has resulted in the widespread distribution and high frequency of falcated-like mtDNA (5.5% of haplotypes) in North America. This is the first study to use a rigorous quantitative framework to reject incomplete lineage sorting as the cause of mitochondrial paraphyly.