Genetic drift or natural selection? Hybridization and asymmetric mitochondrial introgression in two Caribbean lizards (Anolis pulchellus and Anolis krugi)

Hybridization and gene introgression can occur frequently between closely related taxa, but appear to be rare phenomena among members of the species‐rich West Indian radiation of Anolis lizards. We investigated the pattern and possible mechanism of introgression between two sister species from Puerto Rico, Anolis pulchellus and Anolis krugi, using mitochondrial (ND2) and nuclear (DNAH3, NKTR) DNA sequences. Our findings demonstrated extensive introgression of A. krugi mtDNA (k‐mtDNA) into the genome of A. pulchellus in western Puerto Rico, to the extent that k‐mtDNA has mostly or completely replaced the native mtDNA of A. pulchellus on this part of the island. We proposed two not mutually exclusive scenarios to account for the interspecific matings between A. pulchellus and A. krugi. We inferred that hybridization events occurred independently in several populations, and determined that k‐mtDNA haplotypes harboured in individuals of A. pulchellus can be assigned to four of the five major mtDNA clades of A. krugi. Further, the spatial distribution of k‐mtDNA clades in the two species is largely congruent. Based on this evidence, we concluded that natural selection was the probable driving mechanism for the extensive k‐mtDNA introgression into A. pulchellus. Our two nuclear data sets yielded different results. DNAH3 showed reciprocal monophyly of A. pulchellus and A. krugi, indicating no effect of hybridization on this marker. In contrast, the two species shared nine NKTR alleles, probably due to incomplete lineage sorting. Our study system will provide an excellent opportunity to experimentally assess the behavioural and ecological mechanisms that can lead to hybridization in closely related taxa.     

Unexpected population genetic structure of European roe deer in Poland: an invasion of the mtDNA genome from Siberian roe deer

Introgressive hybridization is a widespread evolutionary phenomenon which may lead to increased allelic variation at selective neutral loci and to transfer of fitness‐related traits to introgressed lineages. We inferred the population genetic structure of the European roe deer (Capreolus capreolus) in Poland from mitochondrial (CR and cyt b) and sex‐linked markers (ZFX, SRY, DBY4 and DBY8). Analyses of CR mtDNA sequences from 452 individuals indicated widespread introgression of Siberian roe deer (C. pygargus) mtDNA in the European roe deer genome, 2000 km from the current distribution range of C. pygargus. Introgressed individuals constituted 16.6% of the deer studied. Nearly 75% of them possessed haplotypes belonging to the group which arose 23 kyr ago and have not been detected within the natural range of Siberian roe deer, indicating that majority of present introgression has ancient origin. Unlike the mtDNA results, sex‐specific markers did not show signs of introgression. Species distribution modelling analyses suggested that C. pygargus could have extended its range as far west as Central Europe after last glacial maximum. The main hybridization event was probably associated with range expansion of the most abundant European roe deer lineage from western refugia and took place in Central Europe after the Younger Dryas (10.8–10.0 ka BP). Initially, introgressed mtDNA variants could have spread out on the wave of expansion through the mechanism of gene surfing, reaching high frequencies in European roe deer populations and leading to observed asymmetrical gene flow. Human‐mediated introductions of C. pygargus had minimal effect on the extent of mtDNA introgression.     

MIGRATION,MITOCHONDRIA, AND THE YELLOW‐RUMPED WARBLER

Discordance between mitochondrial and nuclear DNA has been noted in many systems. Asymmetric introgression of mitochondria is a common cause of such discordances, although in most cases the drivers of introgression are unknown. In the yellow‐rumped warbler, evidence suggests that mtDNA from the eastern, myrtle warbler, has introgressed across much of the range of the western form, the Audubon's warbler. Within the southwestern United States myrtle mtDNA comes into contact with another clade that occurs in the Mexican black‐fronted warbler. Both northern forms exhibit seasonal migration, whereas black‐fronted warblers are nonmigratory. We investigated the link between mitochondrial introgression, mitochondrial function, and migration using novel genetic, isotopic, biochemical, and phenotypic data obtained from populations in the transition zone. Isotopes suggest the zone is coincident with a shift in migration, with individuals in the south being resident and populations further north becoming increasingly more migratory. Mitochondrial respiration in flight muscles demonstrates that myrtle‐type individuals have a significantly greater acceptor control ratio of mitochondria, suggesting it may be more metabolically efficient. To our knowledge this is the first time this type of intraspecific variation in mitochondrial respiration has been measured in wild birds and we discuss how such mitochondrial adaptations may have facilitated introgression.     

The association between mitochondrial genetic variation and reduced colony fitness in an invasive wasp

Despite the mitochondrion's long‐recognized role in energy production, mitochondrial DNA (mtDNA) variation commonly found in natural populations was assumed to be effectively neutral. However, variation in mtDNA has now been increasingly linked to phenotypic variation in life history traits and fitness. We examined whether the relative fitness in native and invasive common wasp (Vespula vulgaris) populations in Belgium and New Zealand (NZ), respectively, can be linked to mtDNA variation. Social wasp colonies in NZ were smaller with comparatively fewer queen cells, indicating a reduced relative fitness in the invaded range. Interestingly, queen cells in this population were significantly larger leading to larger queen offspring. By sequencing 1,872 bp of the mitochondrial genome, we determined mitochondrial haplotypes and detected reduced genetic diversity in NZ. Three common haplotypes in NZ frequently produced many queens, whereas the four rare haplotypes produced significantly fewer or no queens. The entire mitochondrial genome for each of these haplotypes was sequenced to identify polymorphisms associated with fitness reduction. We found 16 variable sites; however, no nonsynonymous mutation that was clearly causing impaired mitochondrial function was detected. We discuss how detected variants may alter secondary structures, gene expression or mito‐nuclear interactions, or could be associated with nuclear‐encoded variation. Whatever the ultimate mechanism, we show reduced fitness and mtDNA variation in an invasive wasp population as well as specific mtDNA variants associated with fitness variation within this population. Ours is one of only a few studies that confirm fitness impacts of mtDNA variation in wild nonmodel populations.     

Alpine headwater streams as reservoirs of remnant populations of the Danubian clade of brown trout

1. The watercourses of Austria are mainly part of the Danube drainage system. Nonetheless, only few brown trout (Salmo trutta) populations of the Danubian phylogenetic lineage have been found in this region, not being introgressed by hatchery‐reared fish of the allochthonous Atlantic lineage. The present study was aimed at identifying further waterbodies that might harbour the non‐admixed gene pool of the Danubian clade trout. 2. In a first step, the complete mitochondrial DNA control region of 447 specimens from 20 sampling sites was sequenced to screen for the presence of specific haplotypes. In a second step, 332 individuals from 15 populations were analysed at 10 microsatellite DNA loci. 3. Although sampling concentrated on remote Alpine headwater streams, introgression of hatchery strains was more common than expected from existing stocking records. For seven populations, however, no evidence of recent hatchery trout introgression was found and it is suggested that they might represent the indigenous gene pool. 4. Populations showing no evidence of hatchery introgression displayed lower levels of intrapopulation variability and higher degrees of differentiation compared to purported admixed populations. 5. Our study demonstrates the necessity of combining different molecular markers to reveal the impact of introgression into wild populations, since at some sampling sites admixture would have remained undetected if only a single molecular technique was applied. 6. The identification and characterisation of non‐introgressed populations provides important information for setting conservation priorities and preventing further loss of genetic integrity of a unique element of the native fish fauna.     

Ancient drainage networks mediated a large‐scale genetic introgression in the East Asian freshwater snails

Biogeography and genetic variation of freshwater organisms are influenced not only by current freshwater connections but also by past drainage networks. The Seto Inland Sea is a shallow enclosed sea in Japan, but geological evidence showed that a large freshwater drainage had intermittently appeared in this area between the late Pliocene and Pleistocene. Here, we demonstrated that this paleodrainage greatly affected the genetic variation of the East Asian freshwater snails, Semisulcospira spp. We found that the mtDNA haplotypes originated in the Lake Biwa endemic Semisulcospira species at the upstream side of the paleodrainage were frequently observed in the riverine Semisulcospira species at its downstream side. The genome‐wide DNA and morphological analyses consistently showed that there was no clear evidence of nuclear introgression between the Lake Biwa endemics and riverine species. These results suggest that the large paleodrainage had facilitated mitochondrial introgression and had broadly spread the introgressed mtDNA haplotypes to its downstream region around the Seto Inland Sea. Our study highlights the role of paleodrainages in shaping the genetic variation of freshwater organisms.     

Investigating temporal changes in hybridization and introgression in a predominantly bimodal hybridizing population of invasive sika (Cervus nippon) and native red deer (C. elaphus) on the Kintyre Peninsula,Scotland

We investigated temporal changes in hybridization and introgression between native red deer (Cervus elaphus) and invasive Japanese sika (Cervus nippon) on the Kintyre Peninsula, Scotland, over 15 years, through analysis of 1513 samples of deer at 20 microsatellite loci and a mtDNA marker. We found no evidence that either the proportion of recent hybrids, or the levels of introgression had changed over the study period. Nevertheless, in one population where the two species have been in contact since ～1970, 44% of individuals sampled during the study were hybrids. This suggests that hybridization between these species can proceed fairly rapidly. By analysing the number of alleles that have introgressed from polymorphic red deer into the genetically homogenous sika population, we reconstructed the haplotypes of red deer alleles introduced by backcrossing. Five separate hybridization events could account for all the recently hybridized sika‐like individuals found across a large section of the Peninsula. Although we demonstrate that low rates of F1 hybridization can lead to substantial introgression, the progress of hybridization and introgression appears to be unpredictable over the short timescales.     

Persistence of sunflower crop traits and fitness in Helianthus petiolaris populations

Transgenic plants have increased interest in the study of crop gene introgression in wild populations. Genes (or transgenes) conferring adaptive advantages persist in introgressed populations, enhancing competitiveness of wild or weedy plants. This represents an ecological risk that could increase problems of weed control. Introgression of cultivar alleles into wild plant populations via crop–wild hybridisations is primarily governed by their fitness effect. To evaluate this, we studied the second generation of seven wild–crop interspecific hybrids between weedy Helianthus petiolaris and cultivated sunflower, H. annuus var. macrocarpus. The second generation comprised open‐pollinated progeny and backcrosses to the wild parent, mimicking crosses that occur in natural situations. We compared a number of morphological, life history and fitness traits. Multivariate analysis showed that the parental species H. annuus and H. petiolaris differed in a number of morphological traits, while the second hybrid generation between them was intermediate. Sunflower crop introgression lowered fitness of interspecific hybrids, but fitness parameters tended to recover in the following generation. Relative frequency of wild/weedy and introgressed plants was estimated through four generations, based on male and female parent fitness. In spite of several negative selection coefficients observed in the second generation, introgressed plants could be detected in stands of <100 weedy H. petiolaris populations. The rapid recovery of fecundity parameters leads to prediction that any trait conferring an ecological advantage will diffuse into the wild or weedy population, even if F1 hybrids have low fitness.     

Evolutionary basis of mitonuclear discordance between sister species of mole salamanders (Ambystoma sp.)

Distinct genetic markers should show similar patterns of differentiation between species reflecting their common evolutionary histories, yet there are increasing examples of differences in the biogeographic distribution of species‐specific nuclear (nuDNA) and mitochondrial DNA (mtDNA) variants within and between species. Identifying the evolutionary processes that underlie these anomalous patterns of genetic differentiation is an important goal. Here, we analyse the putative mitonuclear discordance observed between sister species of mole salamanders (Ambystoma barbouri and A. texanum) in which A. barbouri‐specific mtDNA is found in animals located within the range of A. texanum. We test three hypotheses for this discordance (undetected range expansion, mtDNA introgression, and hybridization) using nuDNA and mtDNA data analysed with methods that varied in the parameters estimated and the timescales measured. Results from a Bayesian clustering technique (structure ), bidirectional estimates of gene flow (migrate ‐n and IMa2) and phylogeny‐based methods (*beast , buck y) all support the conclusion that the discordance is due to geographically restricted mtDNA introgression from A. barbouri into A. texanum. Limited data on species‐specific tooth morphology match this conclusion. Significant differences in environmental conditions exist between sites where A. texanum with and without A. barbouri‐like mtDNA occur, suggesting a possible role for selection in the process of introgression. Overall, our study provides a general example of the value of using complimentary analyses to make inferences of the directionality, timescale, and source of mtDNA introgression in animals.     

Protoplast-fusion-derived Solanum cybrids: application and phylogenetic limitations

Summary We established interspecific Solanum cybrids in order to study the intrageneric nuclear-organelle compatibility and the introgression of advantageous plasmone-coded breeding traits into potato. Cybridization was performed by the donor-recipient protoplast-fusion procedure. We found that the plastomes of S. chacoense, S. brevidens, and S. etuberosum could be transferred into the cybrids having S. tuberosum nuclear genomes; chondriome components were likewise transferred from the former species into these cybrids. The combination with S. chacoense as organelle donor and potato as recipient resulted in green fertile plants with potato morphology. By using S. etuberosum as an organelle donor and potato as recipient, male-sterile cybrid plants, most of them having pigmentation abnormalities, were obtained. The combination of S. brevidens with potato resulted in palegreen (almost albino) regenerants. The latter albino plantlets had both the chloroplast DNA and the mitochondrial DNA of the donor (S. brevidens) and did not survive the transfer into the greenhouse. An immediately applicative result of this study is the de novo establishment of male-sterile plants in a potato cultivar. Such plants should be useful as seed parents in the production of hybrid, true-potato seeds.     

Introgression of mtDNA in Urosaurus lizards: historical and ecological processes

Introgression of mtDNA appears common in animals, but the implications of acquiring a novel mitochondrial genome are not well known. This study investigates mito‐genome introgression between the lizard species Urosaurus graciosus, a thermal specialist, and U. ornatus, a species that occupies a wider range of thermal environments. As ectotherms, their metabolic rate is strongly influenced by the thermal environment; with mitochondria being linked to metabolic rates, overall energy budgets could be impacted by introgression. I use mitochondrial gene trees, inferred from Bayesian analyses of Cyt‐B and ND1 gene sequences, along with morphology and microsatellites from nineteen populations of these two species to address if the direction and location of mito‐nuclear discordance match predictions of introgression resulting from past population expansions. MtDNA is expected to move from resident species into expanding or invading species. Second, does having a heterospecific form of mitochondria impact body size, a trait strongly associated with fitness? Multiple independent introgression events of historic origin were detected. All introgression was unidirectional with U. ornatus‐type mtDNA found in U. graciosus parental type individuals. This result was consistent with population expansions detected in U. graciosus but not U. ornatus. Females with heterospecific mtDNA were significantly smaller than homospecific forms, and heterospecific males had a different relationship of body mass to body length than those with homospecific mtDNA. These changes indicate a potential selective disadvantage for individuals with heterospecific mitochondria and are consistent with the theoretical expectation that deleterious alleles are more likely to persist in expanding populations.     

Phylogeography and lineage‐specific patterns of genetic diversity and molecular evolution in a group of North American skinks

Geography influences the evolutionary trajectory of species by mediating opportunities for hybridization, gene flow, demographic shifts and adaptation. We sought to understand how geography and introgression can generate species‐specific patterns of genetic diversity by examining phylogeographical relationships in the North American skink species Plestiodon multivirgatus and P. tetragrammus (Squamata: Scincidae). Using a multilocus dataset (three mitochondrial genes, four nuclear genes; a total of 3455 bp) we discovered mito‐nuclear discordance, consistent with mtDNA introgression. We further tested for evidence of species‐wide mtDNA introgression by using comparisons of genetic diversity, selection tests and extended Bayesian skyline analyses. Our findings suggest that P. multivirgatus acquired its mitochondrial genome from P. tetragrammus after their initial divergence. This putative species‐wide mitochondrial capture was further evidenced by statistically indistinguishable substitution rates between mtDNA and nDNA in P. multivirgatus. This rate discrepancy was observed in P. multivirgatus but not P. tetragrammus, which has important implications for studies that combine mtDNA and nDNA sequences when inferring time since divergence between taxa. Our findings suggest that by facilitating opportunities for interspecific introgression, geography can alter the course of molecular evolution between recently diverged lineages.     

Parallel introgression and selection on introduced alleles in a native species

As humans cause the redistribution of species ranges, hybridization between previously allopatric species is on the rise. Such hybridization can have complex effects on overall fitness of native species as new allelic combinations are tested. Widespread species introductions provide a unique opportunity to study selection on introgressed alleles in independent, replicated populations. We examined selection on alleles that repeatedly introgressed from introduced rainbow trout (Oncorhynchus mykiss) into native westslope cutthroat trout (Oncorhynchus clarkii lewisi) populations in western Canada. We found that the degree of introgression of individual single nucleotide polymorphisms from the invasive species into the native is correlated between independent watersheds. A number of rainbow trout alleles have repeatedly swept to high frequency in native populations, suggesting parallel adaptive advantages. Using simulations, we estimated large selection coefficients up to 0.05 favoring several rainbow trout alleles in the native background. Although previous studies have found reduced hybrid fitness and genome‐wide resistance to introgression in westslope cutthroat trout, our results suggest that some introduced genomic regions are strongly favored by selection. Our study demonstrates the utility of replicated introductions as case studies for understanding parallel adaptation and the interactions between selection and introgression across the genome. We suggest that understanding this variation, including consideration of beneficial alleles, can inform management strategies for hybridizing species.     

Cloning of neuronal mtDNA variants in cultured cells by synaptosome fusion with mtDNA-less cells

Synaptosome cybrids were used to confirm the presence of heteroplasmic mtDNA sequence variants in the human brain. Synaptosomes contain one to several mitochondria, and when fused to mtDNA-deficient (ρ°) mouse or human cell lines result in viable cybrid cell lines. The brain origin of mouse synaptosome cybrid mtDNAs was confirmed using sequence polymorphisms in the mtDNA COIII, ND3 and tRNA^Arg genes. The brain origin of the human synaptosome cybrids was confirmed using a rare mtDNA MboI polymorphism. Fusion of synaptosomes from the brain of a 35-year-old woman resulted in 71 synaptosome cybrids. Sequencing the mtDNA control region of these cybrid clones revealed differences in the number of Cs in a poly C track between nucleotide pairs (nps) 301 and 309. Three percent of the cybrid clones had mtDNAs with 10 Cs, 76% had nine, 18% had eight and 3% had seven Cs. Comparable results were obtained by PCR amplification, cloning and sequencing of mtDNA control regions directly from the patient’s brain tissue, but not when the control region was amplified and cloned from a synaptosome cybrid homoplasmic for a mtDNA with nine Cs. Thus, we have clonally recovered mtDNA control region length variants from an adult human brain without recourse to PCR, and established the variant mtDNAs within living cultured cells. This confirms that some mtDNA heteroplasmy can exist in human neurons, and provides the opportunity to study its functional significance.     

Characterization of cybrid cell lines containing mtDNA from Huntington's disease patients.

Electron transport chain (ETC) dysfunction may arise from mitochondrial genetic, nuclear genetic, or toxic etiologies. Cytoplasmic hybrid (cybrid) systems can help distinguish between these possibilities by facilitating expression of suspect mitochondrial DNA (mtDNA) within a nuclear and environmentally controlled context. Perpetuation of ETC dysfunction in cybrids is consistent with an mtDNA pathogenesis while defect correction is not. We previously used cybrids to screen sporadic Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis patients for mtDNA mutation with positive results. To further address the fidelity of these experiments, we created and characterized cybrids expressing mtDNA from persons with Huntington's disease (HD), an autosomal dominant, nuclear DNA-determined disorder in which mitochondrial ETC functioning is abnormal. On ETC, oxidative stress, and calcium homeostasis assays HD cybrid lines were indistinguishable from control cybrid lines. These data support the use of the cybrid technique for mtDNA mutation screening in candidate diseases.     

When a clonal genome finds its way back to a sexual species: evidence from ongoing but rare introgression in the hybridogenetic water frog complex

Besides several exceptions, asexual metazoans are usually viewed as ephemeral sinks for genomes, which become ‘frozen’ in clonal lineages after their emergence from ancestral sexual species. Here, we investigated whether and at what rate the asexuals are able to introgress their genomes back into the parental sexual population, thus more or less importantly affecting the gene pools of sexual species. We focused on hybridogenetic hybrids of western Palaearctic water frogs (Pelophylax esculentus), which originate through hybridization between P. ridibundus and P. lessonae, but transmit only clonal ridibundus genome into their gametes. Although usually mating with P. lessonae, P. esculentus may upon mating with P. ridibundus or another hybrid produce sexually reproducing P. ridibundus offspring with the introgressed ex‐clonal genome. We compared the rate of nuclear amplified fragment length polymorphism (AFLP) and mitochondrial introgression in two types of populations, that is, those where P. ridibundus occurs in isolation and those where it lives with the hybridogens. Although significant differentiation (Φpt) between sexual and clonal ridibundus genomes suggested limited gene flow between sexuals and hybridogens, a non‐negligible (~5%) proportion of P. ridibundus bore introgressed mtDNA and AFLP markers. Whereas transfer of mtDNA was exclusively unidirectional, introgression of nuclear markers was bidirectional. The proportion of introgressed P. ridibundus was highest in syntopic populations with P. esculentus, proving an ongoing and site‐specific interspecific genetic transfer mediated by hybridogenetic hybrids. It turns out that asexual hybrids are not just a sink for genes of sexual species, but may significantly influence the genetic architecture of their sexual counterparts.     

Evolutionary neutrality of mtDNA introgression: evidence from complete mitogenome analysis in roe deer

Introgressive hybridization offers a unique platform for studying the molecular basis of natural selection acting on mitogenomes. Most of the mtDNA protein‐coding genes are extremely conserved; however, some of the observed variations have potentially adaptive significance. Here, we evaluated whether the evolution of mtDNA in closely related roe deer species affected by widespread mtDNA introgression is neutral or adaptive. We characterized and compared 16 complete mitogenomes of European (Capreolus capreolus) and Siberian (C. pygargus) roe deer, including four of Siberian origin introgressed into European species. The average sequence divergence of species‐specific lineages was estimated at 2.8% and varied across gene classes. Only 21 of 315 fixed differences identified in protein‐coding genes represented nonsynonymous changes. Only three of them were determined to have arisen in the C. pygargus lineage since the time to the most recent common ancestor (TMRCA) of both Capreolus species, reflecting a decelerated evolutionary ratio. The almost four‐fold higher d_N/d_S ratio described for the European roe deer lineage is constrained by overall purifying selection, especially pronounced in the ND4 and ND5 genes. We suggest that the highly divergent C. capreolus lineage could have maintained a capability for genomic incorporation of the well‐preserved and almost ancestral type of mtDNA present in C. pygargus. Our analyses did not indicate any signs of positive selection for Siberian roe deer mtDNA, suggesting that the present widespread introgression is evolutionarily neutral.     

Genomic consequences of a recent three‐way admixture in supplemented wild brown trout populations revealed by local ancestry tracts

Understanding the evolutionary consequences of human‐mediated introductions of domesticated strains into the wild and their subsequent admixture with natural populations is of major concern in conservation biology. However, the genomic impacts of stocking from distinct sources (locally derived vs. divergent) on the genetic integrity of wild populations remain poorly understood. We designed an approach based on estimating local ancestry along individual chromosomes to provide a detailed picture of genomic admixture in supplemented populations. We used this approach to document admixture consequences in the brown trout Salmo trutta, for which decades of stocking practices have profoundly impacted the genetic make‐up of wild populations. In southern France, small local Mediterranean populations have been subject to successive introductions of domestic strains derived from the Atlantic and Mediterranean lineages. To address the impact of stocking, we evaluate the extent of admixture from both domestic strains within populations, using 75,684 mapped SNPs obtained from double‐digested restriction site‐associated DNA sequencing. Then, the chromosomal ancestry profiles of admixed individuals reveal a wider diversity of hybrid and introgressed genotypes than estimated using classical methods for inferring ancestry and hybrid pedigrees. In addition, the length distribution of introgressed tracts retained different timings of introgression between the two domestic strains. We finally reveal opposite consequences of admixture on the level of polymorphism of the recipient populations between domestic strains. Our study illustrates the potential of using the information contained in the genomic mosaic of ancestry tracts in combination with classical methods based on allele frequencies for analysing multiple‐way admixture with population genomic data.