Mitochondrial DNA Introgression Research Articles

Nuclear compensatory evolution driven by mito-nuclear incompatibilities

Mitochondrial function relies on the coordinated expression of mitochondrial and nuclear genes, exhibiting remarkable resilience despite high mitochondrial mutation rates. The nuclear compensation mechanism suggests deleterious mitochondrial alleles drive compensatory nuclear mutations to preserve mito-nuclear compatibility. However, prevalence and factors conditioning this phenomenon remain debated due to its conflicting evidence. Here, we investigate how mito-nuclear incompatibilities impact substitutions in a model for species radiation. Mating success depends on genetic compatibility (nuclear DNA) and spatial proximity. Populations evolve from partially compatible mito-nuclear states, simulating mitochondrial DNA (mtDNA) introgression. Mutations do not confer advantages nor disadvantages, but individual fecundity declines with increasing incompatibilities, selecting for mito-nuclear coordination. We find that selection for mito-nuclear compatibility affects each genome differently based on their initial state. In compatible gene pairs, selection reduces substitutions in both genomes, while in incompatible nuclear genes, it consistently promotes compensation, facilitated by more mismatches. Interestingly, high mitochondrial mutation rates can reduce nuclear compensation by increasing mtDNA rectification, while substitutions in initially compatible nuclear gene are boosted. Finally, the presence of incompatibilities accelerates species radiation, but equilibrium richness is not directly correlated to substitution rates, revealing the complex dynamics triggered by mitochondrial introgression and mito-nuclear coevolution. Our study provides a perspective on nuclear compensation and the role of mito-nuclear incompatibilities in speciation by exploring extreme scenarios and identifying trends that empirical data alone cannot reveal. We emphasize the challenges in detecting these dynamics and propose analyzing specific genomic signatures could shed light on this evolutionary process.

A reappraisal of mitochondrial DNA introgression in the Mus musculus musculus/Mus musculus domesticus hybrid zone suggests ancient North-European associations between mice and humans

ABSTRACT The house mouse is the best-studied mammal species after humans, yet our understanding of its evolutionary history remains incomplete. Here, we focused on the colonisation of Europe by two subspecies and formation of a hybrid zone between them. We carried out a large-scale study of ~7000 mice sampled across an area embracing an ~900 km long portion of the zone, supplemented with cytochrome b and D-loop sequencing of ~1200 individuals collected worldwide. We demonstrate that the course of the mtDNA contact front is intricate and highly discordant with the consensus front for autosomal and X-linked markers, suggesting that local snapshots of mtDNA introgression may be misleading when treated in isolation. While multiple unrelated M. m. domesticus haplotypes occur in the zone area, the diversity of M. m. musculus haplotypes is limited. Moreover, we uncovered a vast region of domesticus introgression into musculus territory in northern areas and showed this introgression is unlikely to be driven by positive selection. We consider two previously published hypotheses explaining this phenomenon and put forth an alternative scenario assuming associations between mice and humans in northern Europe during the Nordic Bronze Age and subsequent zone movement associated with the expansion of Balto-Slavic peoples.

Evidence for feminized genetic males in a flea beetle using newly identified X-linked markers.

The equilibrium of sex ratios in sexually reproducing species is often disrupted by various environmental and genetic factors, including endosymbionts like Wolbachia. In this study, we explore the highly female-biased sex ratio observed in the flea beetle, Altica lythri, and its underlying mechanisms. Ancient hybridization events between Altica species have led to mitochondrial DNA introgression, resulting in distinct mitochondrial haplotypes that go along with different Wolbachia infections (HT1-wLytA1, HT1*- uninfected, HT2-wLytA2, and HT3-wLytB). Notably, beetles with some haplotypes exclusively produce female offspring, suggesting potential Wolbachia-induced phenomena such as feminization of genetic males. However, the observed female bias could also be a consequence of the ancient hybridization resulting in nuclear-cytoplasmic conflicts between introgressed mtDNA and nuclear genes. Through transcriptomic analysis and the program SEX-DETector, we established markers for genotypic sex differentiation for A. lythri, enabling genetic sexing via qPCR. Our findings suggest that feminization of genetic males is contributing to the skewed sex ratios, highlighting the intricate dynamics of sex determination and reproductive strategies in this flea beetle. This study provides valuable insights into the dynamics of genetic conflicts, endosymbionts, and sex ratios, revealing the novel phenomenon of genetic male feminization in the flea beetle A. lythri.

Increased geographic sampling suggests incomplete lineage sorting and recent introgression between Pyrrhosoma nymphula (Sulzer, 1776) and P. elisabethae Schmidt, 1948 in the Western Palearctic

We analysed COI and ITS sequences from a total of 69 European Pyrrhosoma nymphula (Sulzer, 1776) and three P. elisabethae Schmidt, 1948 to explore species boundaries and phylogeographic patterns in their Western Palearctic distributions. We found that phylogenetic and haplotype network analyses support previous results that the widespread P. nymphula and the endangered P. elisabethae are distinct species, and that P. nymphula in North Africa are distinct from P. nymphula in Europe. However, our results also suggest that neither diversification is complete as we found evidence for introgression of mitochondrial DNA between P. elisabethae and eastern Europe P. nymphula, as well as possible incomplete lineage sorting. Finally, our results indicate that while P. nymphu­la likely recolonized most of Europe from an Iberian Peninsula refugium following the Weichsel Glaciation, separate refugia possibly existed in North Africa, southern Italy, and for P. elisabethae in the southwestern Balkan.

Eco‐physiological arguments on the functional impact of Lepus timidus mitochondrial DNA introgression in Iberian hares (Lepus granatensis)

AbstractGenetic exchanges between species provide valuable opportunities to dissect the relative importance of neutral and selective processes driving introgression events and to test their functional relevance. In the northern half of the Iberian Peninsula, the Iberian hare, Lepus granatensis, presents extensive patterns of mitochondrial DNA (mtDNA) introgression from the mountain hare, L. timidus, a species that disappeared from the region after the Last Glacial Maximum. Here, we test whether mtDNA introgression affects reproductive success and body condition of Iberian hare individuals. For that end, hares (n = 149) were sampled from populations along the mtDNA introgression gradient during the 2013–2014 and 2014–2015 hunting seasons. Reproductive performance indicators, including pregnancy status and testicular weight, were recorded, while body condition was measured as kidney fat (our response variables). Predictors included individual traits (sex and age), sampling period (month and hunting season), mtDNA introgression, occurrence or burden of different endoparasites and environmental factors. Our results showed that mtDNA introgression was negatively associated with pregnancy in females and body condition in males, while a positive association was found with body condition in females. These results suggest that carrying the mountain hare mtDNA has physiological effects in Iberian hares, but that the allospecific mtDNA type may not always be beneficial for the individual. This study shows that the patterns of mtDNA introgression seen in the Iberian hare today may result from a complex balance between deleterious and beneficial individual effects.

High heterogeneity in genomic differentiation between phenotypically divergent songbirds: a test of mitonuclear co-introgression.

Comparisons of genomic variation among closely related species often show more differentiation in mitochondrial DNA (mtDNA) and sex chromosomes than in autosomes, a pattern expected due to the differing effective population sizes and evolutionary dynamics of these genomic components. Yet, introgression can cause species pairs to deviate dramatically from general differentiation trends. The yellowhammer (Emberiza citrinella) and pine bunting (E. leucocephalos) are hybridizing avian sister species that differ greatly in appearance and moderately in nuclear DNA, but that show no mtDNA differentiation. This discordance is best explained by adaptive mtDNA introgression-a process that can select for co-introgression at nuclear genes with mitochondrial functions (mitonuclear genes). To better understand these discordant differentiation patterns and characterize nuclear differentiation in this system, we investigated genome-wide differentiation between allopatric yellowhammers and pine buntings and compared it to what was seen previously in mtDNA. We found significant nuclear differentiation that was highly heterogeneous across the genome, with a particularly wide differentiation peak on the sex chromosome Z. We further investigated mitonuclear gene co-introgression between yellowhammers and pine buntings and found support for this process in the direction of pine buntings into yellowhammers. Genomic signals indicative of co-introgression were common in mitonuclear genes coding for subunits of the mitoribosome and electron transport chain complexes. Such introgression of mitochondrial DNA and mitonuclear genes provides a possible explanation for the patterns of high genomic heterogeneity in genomic differentiation seen among some species groups.

Examination of D-loop region and DBY gene as tools for identifying hybridisation in alpacas (Vicugna pacos) based on Polish populations

Hybridisation between alpacas (Vicugna pacos) and llamas (Lama glama) is a well-known phenomenon among alpaca breeders worldwide. Hybridisation causes poor fibre quality, which leads to economic losses. In the present study, we aimed to evaluate the d-loop region of mitochondrial DNA and the DBY gene located on chromosome Y to identify alpaca × llama hybrids. Moreover, based on these tools, the genetic diversity of alpacas in Poland was presented. We analysed the d-loop region in 235 individuals and investigated 90 pure males for the DBY gene. We generated 34 haplotypes with 446 segregation sites for the d-loop region and two haplotypes with four segregation sites for the DBY gene. Phylogenetic analysis based on the Bayesian clustering approach and a principal coordinate test showed a high level of mitochondrial DNA introgression between alpacas and llamas with a high gene flow value based on the sequence and on the haplotype data information. We found evidence of maternal line hybridisation in the d-loop region between alpacas and llamas, while this evidence was not found within the DBY gene. Moreover, the d-loop region investigation showed high levels of genetic diversity in the alpacas maintained in Poland.

Mitonuclear mismatch alters nuclear gene expression in naturally introgressed Rhinolophus bats

BackgroundMitochondrial function involves the interplay between mitochondrial and nuclear genomes. Such mitonuclear interactions can be disrupted by the introgression of mitochondrial DNA between taxa or divergent populations. Previous studies of several model systems (e.g. Drosophila) indicate that the disruption of mitonuclear interactions, termed mitonuclear mismatch, can alter nuclear gene expression, yet few studies have focused on natural populations.ResultsHere we study a naturally introgressed population in the secondary contact zone of two subspecies of the intermediate horseshoe bat (Rhinolophus affinis), in which individuals possess either mitonuclear matched or mismatched genotypes. We generated transcriptome data for six tissue types from five mitonuclear matched and five mismatched individuals. Our results revealed strong tissue-specific effects of mitonuclear mismatch on nuclear gene expression with the largest effect seen in pectoral muscle. Moreover, consistent with the hypothesis that genes associated with the response to oxidative stress may be upregulated in mitonuclear mismatched individuals, we identified several such gene candidates, including DNASE1L3, GPx3 and HSPB6 in muscle, and ISG15 and IFI6 in heart.ConclusionOur study reveals how mitonuclear mismatch arising from introgression in natural populations is likely to have fitness consequences. Underlying the processes that maintain mitonuclear discordance is a step forward to understand the role of mitonuclear interactions in population divergence and speciation.

Extensive alternative splicing triggered by mitonuclear mismatch in naturally introgressed Rhinolophus bats.

Mitochondrial function needs strong interactions of mitochondrial and nuclear (mitonuclear) genomes, which can be disrupted by mitonuclear mismatch due to mitochondrial DNA (mtDNA) introgression between two formerly isolated populations or taxa. This mitonuclear disruption may cause severe cellular stress in mismatched individuals. Gene expression changes and alternative splicing (AS) are two important transcriptional regulations to respond to environmental or cellular stresses. We previously identified a naturally introgressed population in the intermediate horseshoe bat (Rhinolophus affinis). Individuals from this population belong to R. a. himalayanus and share almost identical nuclear genetic background; however, some of them had mtDNA from another subspecies (R. a. macrurus). With this unique natural system, we examined gene expression changes in six tissues between five mitonuclear mismatched and five matched individuals. A small number of differentially expressed genes (DEGs) were identified, and functional enrichment analysis revealed that most DEGs were related to immune response although some may be involved in response to oxidative stress. In contrast, we identified extensive AS events and alternatively spliced genes (ASGs) between mismatched and matched individuals. Functional enrichment analysis revealed that multiple ASGs were directly or indirectly associated with energy production in mitochondria which is vital for survival of organism. To our knowledge, this is the first study to examine the role of AS in responding to cellular stress caused by mitonuclear mismatch in natural populations. Our results suggest that AS may play a more important role than gene expression regulation in responding to severe environmental or cellular stresses.

Mitochondrial DNA duplication, recombination, and introgression during interspecific hybridization

mtDNA recombination events in yeasts are known, but altered mitochondrial genomes were not completed. Therefore, we analyzed recombined mtDNAs in six Saccharomyces cerevisiae × Saccharomyces paradoxus hybrids in detail. Assembled molecules contain mostly segments with variable length introgressed to other mtDNA. All recombination sites are in the vicinity of the mobile elements, introns in cox1, cob genes and free standing ORF1, ORF4. The transplaced regions involve co-converted proximal exon regions. Thus, these selfish elements are beneficial to the host if the mother molecule is challenged with another molecule for transmission to the progeny. They trigger mtDNA recombination ensuring the transfer of adjacent regions, into the progeny of recombinant molecules. The recombination of the large segments may result in mitotically stable duplication of several genes.

Positive selection on two mitochondrial coding genes and adaptation signals in hares (genus Lepus) from China

BackgroundAnimal mitochondria play a central role in energy production in the cells through the oxidative phosphorylation (OXPHOS) pathway. Recent studies of selection on different mitochondrial OXPHOS genes have revealed the adaptive implications of amino acid changes in these subunits. In hares, climatic variation and/or introgression were suggested to be at the origin of such adaptation. Here we looked for evidence of positive selection in three mitochondrial OXPHOS genes, using tests of selection, protein structure modelling and effects of amino acid substitutions on the protein function and stability. We also used statistical models to test for climate and introgression effects on sites under positive selection.ResultsOur results revealed seven sites under positive selection in ND4 and three sites in Cytb. However, no sites under positive selection were observed in the COX1 gene. All three subunits presented a high number of codons under negative selection. Sites under positive selection were mapped on the tridimensional structure of the predicted models for the respective mitochondrial subunit. Of the ten amino acid replacements inferred to have evolved under positive selection for both subunits, six were located in the transmembrane domain. On the other hand, three codons were identified as sites lining proton translocation channels. Furthermore, four codons were identified as destabilizing with a significant variation of Δ vibrational entropy energy between wild and mutant type. Moreover, our PROVEAN analysis suggested that among all positively selected sites two fixed amino acid replacements altered the protein functioning. Our statistical models indicated significant effects of climate on the presence of ND4 and Cytb protein variants, but no effect by trans-specific mitochondrial DNA introgression, which is not uncommon in a number of hare species.ConclusionsPositive selection was observed in several codons in two OXPHOS genes. We found that substitutions in the positively selected codons have structural and functional impacts on the encoded proteins. Our results are concordantly suggesting that adaptations have strongly affected the evolution of mtDNA of hare species with potential effects on the protein function. Environmental/climatic changes appear to be a major trigger of this adaptation, whereas trans-specific introgressive hybridization seems to play no major role for the occurrence of protein variants.

Phylogeny of Late Pleistocene and Holocene Bison species in Europe and North America

Species of the genus Bison, along with many other vertebrate taxa, represented an important part of Pleistocene megafauna. Following the Quaternary extinction event, however, only the American bison (Bison bison) and the European bison or wisent (Bison bonasus) survived to present times. The phylogeny of Bison species from the Late Pliocene to the end of the Middle Pleistocene is still uncertain. In comparison, the availability of genetic data has greatly improved our knowledge of Late Pleistocene and Holocene bison. In recent years, the phylogenetic approach has provided new details and insights into the evolution and phylogeny of the genus, such as i) Bison bison evolved from central North American bison populations and, contrary to earlier assumptions, was not the result of mixing with Beringian bison; ii) in Europe, the Late Pleistocene ancestors of Bison bonasus occupied the same areas as coeval Bison priscus; iii) admixture events between the genera Bos and Bison led to the introgression of Bos mitochondrial DNA into extant Bison bonasus, but the exact evolution of the latter remains unknown.

Species limits in birds: Australian perspectives on interrelated challenges of allopatry, introgression of mitochondrial DNA, recent speciation, and selection

Abstract Four main challenges that can underpin ongoing, intransigent debates about species limits in birds are reviewed: allopatry (population subdivision vs. speciation), geographically widespread introgression of mitochondrial DNA (mtDNA), recent speciation, and selection. Examples from birds of the Australian region show how these challenges, their interplay, and the molecular-phenotypic discordance they generate can clarify or mislead species limits. Examples of how phylogenetic frameworks help reject or retain hypotheses of species limits under these challenges are given. Although mtDNA’s strengths and limitations are well known, an underappreciated limitation of mtDNA is geographically widespread introgression that homogenizes mtDNA diversity across species, subspecies, or population boundaries and across hundreds of kilometers. The resulting discordance between mtDNA and phenotype can be profound. If undetected, the setting of species limits and evolutionarily significant units are misled. An example shows how recent genomic analyses can detect and solve the problem. Other examples concern legacy mtDNA-only datasets. These are often essentially unfinished studies leaving residual uncertainty in species limits. Examples illustrate when the possibility of large-scale introgression across species boundaries needs to be considered, and how genomic scale data offer solutions. Researchers must carefully parse 3 questions: has there been introgression of mtDNA and, if so, which population genetics-based driver has caused introgression, and do species limits need altering? Understanding of allopatry, mtDNA introgression, recent speciation, and selection must be properly integrated if species limits are to be robustly understood and applied with maximum benefit in downstream applications such as conservation and management.

Genetic structure, morphological variation, and gametogenic peculiarities in water frogs ( Pelophylax ) from northeastern European Russia

The edible frog, Pelophylax esculentus, is a hybrid form that reproduces via clonal propagation of only one of the parental genomes through generations of hybrids while the genome of other parental species is eliminated during gametogenesis. Such reproductive ability requires hybrids to coexist with one of the parental species or rarely both parental species causing the formation of so-called population systems. Population systems and reproductive biology of water frogs from the east of the range remained partially unexplored. In this study, we investigated the distributions, population systems, genetic structure, types of gametes, and morphological variability of water frogs of the genus Pelophylax from the northeastern parts of their ranges (Mari El Republic and adjacent territories, Russia). We examined 1,337 individuals from 68 localities using morphological traits combined with DNA flow cytometry and a multilocus approach (fragments of a nuclear and two mitochondrial genes). We revealed five types of population systems: “pure” populations of the parental P. ridibundus (R) and P. lessonae (L), mixed populations of parental species (R-L) along and with their hybrids (R-E-L), as well as mixed populations of P. lessonae and P. esculentus (L-E). However, the “pure” hybrid (E) and the mixed P. ridibundus and P. esculentus (R-E) population systems were not found. All hybrids studied by DNA flow cytometry were diploid. Analysis of gametogenesis showed that the majority of hybrid males, as well as hybrid females from the L-E system, produced gametes with the P. ridibundus genome. However, in the R-E-L system, hybrid females were usually sterile. The reproduction of hybrids in such systems is primarily based on crosses of P. esculentus males with P. lessonae females. Molecular analysis showed the presence of mitochondrial and nuclear DNA introgression of the Anatolian marsh frog (P. cf. bedriagae) into both P. ridibundus and P. esculentus. The observations of alleles and haplotypes of P. cf. bedriagae in P. ridibundus and P. esculentus individuals from the same localities suggest de novo formation of local hybrids. However, the presence of the Balkan marsh frog (P. kurtmuelleri) haplotypes in local hybrids supports the hypothesis regarding the migration of old hemiclonal lineages from glacial refugia. Finally, the diagnostic value of various morphological characteristics was discussed.

Phylotranscriptomics reveals the complex evolutionary and biogeographic history of the genus Tsuga with an East Asian-North American disjunct distribution

The disjunct distribution between East Asia and North America is one of the best established biogeographic patterns. A robust phylogeny is fundamental for understanding the biogeographic histories of taxa with this distribution pattern. Tsuga (hemlock) is a genus of Pinaceae with a typical intercontinental disjunct distribution in East Asia and eastern and western North America, and its phylogeny has not been completely reconstructed in previous studies. In this study, we reconstructed a highly resolved phylogeny of Tsuga using 881 nuclear genes, 60 chloroplast genes and 23 mitochondrial genes and explored its biogeographic and reticulate evolutionary history. The results of phylogenetic analysis, molecular dating and ancestral area reconstruction indicate that Tsuga very likely originated from North America in the late Oligocene and dispersed from America to East Asia via the Bering Land Bridge during the middle Miocene. In particular, we found complex reticulate evolutionary pattern among the East Asian hemlock species. T. sieboldii possibly originated from hybridization with the ancestor of T. chinensis from mainland China and T. forrestii as the paternal donor and the ancestor of T. diversifolia and T. ulleungensis as the maternal donor. T. chinensis (Taiwan) could have originated by hybridization together with T. sieboldii and then evolved independently after dispersal to the Taiwan Island, subsequently experiencing mitochondrial DNA introgression with T. chinensis from mainland China. Moreover, our study found that T. chinensis from western China is more closely related to T. forrestii than to T. chinensis from eastern China. The nonmonophyletic T. chinensis needs taxonomic reconsideration.

Speciation in sympatric species of land snails from the genus Trochulus (Gastropoda, Hygromiidae)

AbstractThe identification and designation of land snail species in the genus Trochulus on the basis of shell characteristics are problematic because of their great phenotypic plasticity. Some genetic analyses have proved inconclusive, with much variation within populations and apparent gene flow among them. We examined this issue by morphometric and molecular approaches on the morphologically similar species T. coelomphala, T. hispidus and T. striolatus, co‐occurring in the Alpenvorland of Germany. While these species differed in shell and reproductive system morphology, there were forms that turned out intermediate in shell characters between T. coelomphala and T. hispidus but had genital morphology similar to T. coelomphala. Phylogenetic analysis, however, showed that these forms clustered neither with T. coelomphala nor T. hispidus but are sister to T. striolatus from the same region, which suggests that they evolved by way of sympatric speciation. Further, these analyses suggest that T. coelomphala diverged within T. hispidus; a crossing experiment indicated that they were interfertile. Expanding the study to include all available Trochulus sequences enabled us to infer evolutionary relationships between them and showed that T. hispidus is polyphyletic. Some Trochulus samples of one nominal species were grouped within others. The combination of phenotypic plasticity and possible mitochondrial DNA introgression illustrates the complex nature of evolutionary processes and the need for caution in the application of traditional taxonomic practice.

Genome-wide data reveal discordant mitonuclear introgression in the intermediate horseshoe bat (Rhinolophus affinis)

Closely related taxa often exhibit mitonuclear discordance attributed to introgression of mitochondrial DNA (mtDNA), yet few studies have considered the underlying causes of mtDNA introgression. Here we test for demographic versus adaptive processes as explanations for mtDNA introgression in three subspecies of the intermediate horseshoe bat (Rhinolophus affinis). We generated sequences of 1692 nuclear genes and 13 mitochondrial protein-coding genes for 48 individuals. Phylogenetic reconstructions based on 320 exon sequences and 2217 single nucleotide polymorphisms (SNPs) both revealed conflicts between the species tree and mtDNA tree. These results, together with geographic patterns of mitonuclear discordance, and shared identical or near-identical mtDNA sequences, suggest extensive introgression of mtDNA between the two parapatric mainland subspecies. Under demographic hypotheses, we would also expect to uncover traces of ncDNA introgression, however, population structure and gene flow analyses revealed little nuclear admixture. Furthermore, we found inconsistent estimates of the timing of population expansion and that of the most recent common ancestor for the clade containing introgressed haplotypes. Without a clear demographic explanation, we also examined whether introgression likely arises from adaptation. We found that five mtDNA genes contained fixed amino acid differences between introgressed and non-introgressed individuals, including putative positive selection found in one codon, although this did not show introgression. While our evidence for rejecting demographic hypotheses is arguably stronger than that for rejecting adaptation, we find no definitive support for either explanation. Future efforts will focus on larger-scale resequencing to decipher the underlying causes of discordant mitonuclear introgression in this system.

Variability of Fragments of Nuclear Brca1 Gene, Exon 11, and Mitochondrial Cox1 Gene in House Mice Mus musculus

To clarify genetic differences between subspecies of the house mouse Mus musculus, their distribution, and hybridization, we first conducted a comparative analysis of variability of nucleotide sequences of fragments of the nuclear gene Brca1, exon 11 (2331 bp), and mitochondrial gene Cox1 (1260 bp) in 40 house mice from West and East Europe, Transcaucasia, Siberia, and Central and South Asia. Brca1 genotypes were divided into five main groups, which differed in a number of fixed substitutions. Genotypes of each group are characteristic for the certain geographical region and the following subspecies: M. m. musculus, M. m. domesticus, M. m. castaneus, and M. m. wagneri together with M. m. gansuensis; a fifth group corresponds to an unidentified subspecies or a distinct genetic form of M. musculus from India (Sikkim State). Besides the homozygous specimens, we revealed mice, which were heterozygous for all diagnostic loci simultaneously; these specimens were determined as hybrid. Hybrid mice were mainly found in the zones of contact of subspecies, but in some cases, quite far from one of the parent subspecies (possibly, due to transportation). In two hybrid mice (from Bakhtiari Province of Iran and Transbaikalia of Russia), unique Brca1 haplotypes were detected. It cannot be ruled out that, at least partly, they may be characteristic of the M. m. bactrianus and M. m. gansuensis subspecies, respectively. Thus, the results of the study showed that the nuclear Brca1 gene is a promising molecular genetic marker for the analysis of variability, differentiation, and hybridization of house mice as well for subspecific identification of M. musculus specimens. Despite more rapid evolution of the Cox1 gene, it is not well suited for discrimination of M. m. musculus, M. m. wagneri, M. m. gansuensis specimens and Transcaucasian representatives of M. m. domesticus due to introgression and long-term maintenance of foreign mitochondrial DNA in populations. However, Cox1 gene analysis (along with the diagnostics of animals by nuclear DNA) may be useful for estimation of population differences in M. m. castaneus and M. m. domesticus subspecies.

Reproductive characteristics of two Triturus species (Amphibia: Caudata)

During three consecutive years, we compared the reproductive characteristics and oviposition dynamics of two crested newt species, Triturus ivanbureschi and T. macedonicus. These two well-defined species are of special interest because of complex interactions at their contact zone, which include hybridization, species replacement and asymmetric mitochondrial DNA introgression. In common garden experiments, females were introduced to conspecific males, to males of other species and to hybrid males. We monitored the total number and size of the deposited eggs, as well as the dynamics and duration of oviposition during three consecutive years. The number of deposited eggs of Triturus species was much higher than previously reported. Triturus macedonicus lay more eggs that were of larger size in comparison to T. ivanbureschi. Also, the onset of T. macedonicus oviposition was considerably delayed compared to T. ivanbureschi. These are fundamental data, important for understanding complex species interactions in their contact zones.

Non-native and hybrid in a changing environment: conservation perspectives for the last Italian red-legged partridge (Alectoris rufa) population with long natural history

The ever-increasing biotic homogenization - especially when associated with introgressive hybridization - raises concern for the reduction of the spatial component of diversity in wildlife worldwide. Nonetheless, there is a growing attention to the potential conservation value of hybridization in fastening the adaptive evolutionary responses to rapidly changing selective pressures. Under these premises, we investigated the genetic affinity of the red-legged partridge (Alectoris rufa) population inhabiting Elba Island (Italy) in the context of the overall species phylogeography and particularly of the nominate subspecies it is traditionally ascribed to. Although notoriously hybrid with the congeneric A. chukar, this island population is of undisputedly value because of its long natural history and self-sustainability. As such, its adaptive conservation management calls for a comprehensive knowledge including the assessment of its geographic origin. For this purpose, 110 fecal samples were collected across Elba, genotyped at their joint Cytochrome-b and Control Region genes (2,249 characters), and compared with 149 conspecifics from all over the species distribution range. We confirmed a widespread A. chukar mitochondrial DNA introgression in Elba partridges, whereas their expected formal assignment to the nominate subspecies from Italy and France was rejected, since these turned out to be closely related to conspecifics from the Iberian Peninsula. This counterintuitive result found support in a large variety of literary sources and compelling evidences from personal testimonies revealing recent intense management with farm-reared birds of Spanish origin. Although the nativeness of Elba partridges was disproved, we advise local authorities to keep warranting the ongoing conservation efforts - and especially restore the connectivity between the western and eastern island sub-populations - as this resource may still be conceived as the ultimate repository for part of the otherwise extinct Italian A. r. rufa genome. Indeed, the admixture with conspecifics from the Iberian Peninsula does not necessarily mean that the entirety of the native nuclear genome of Elba partridges has been wiped out. Furthermore, these latter represent an interesting case study in conservation biology to investigate the possible role played by introgressive hybridization in the adaptation to recent land use and vegetation cover changes associated with rural abandonment in an insular yet heavily anthropized context.