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Deep learning reveals genomic regions introgressed between two recurrently hybridizing lynx species.

Recently, diverged species with overlapping distributional ranges have high chances of hybridizing and if hybrids are viable, genomic material can be transferred between species in a process called introgression. To characterize the patterns and consequences of introgression in species with historically low population sizes and recent steep declines resulting in genetic erosion, we analyze the Iberian and Eurasian lynx (EL) as an illustrative and relevant case study. While genome-wide introgression was already detected, here we apply a method using a deep convolutional neural network to detect specific regions of the genome with signals of introgression in three populations of these two species. Over 6% of the genome of both Iberian lynx and ELw shows introgression from the other species, compared with only 2% in the ELs. This observation, along with the results from demographic modeling, suggests that the ELw population is genetically closest to the source of EL introgression, a probably now extinct group that coexisted with the Iberian lynx in Southern Europe and Northern Iberia until recently. As predicted by theory, introgression was generally higher in populations with smaller effective sizes and in genomic regions of high recombination. However, the Iberian lynx did not show higher overall introgression than the more abundant ELw, and coding regions introgressed as frequently as intergenic regions. Local genetic diversity is boosted approximately 3-fold in genomic windows where introgression occurs, potentially including the adaptively relevant and highly diverse MHC region of the Iberian lynx.

Animals

Extensive Introgression Failed to Erode Species Boundaries Among Multiple Sympatric Closely Related Species of Roscoea.

How species boundaries are maintained among sympatric closely related species experiencing gene flow is a puzzling question in evolutionary biology. Although introgression is commonly documented, the dynamics and gene function of introgression have rarely been explored to probe why frequent introgression does not necessarily destroy species boundaries in sympatry. In this study, we employ whole-genome resequencing data to examine introgression among five closely related species of Roscoea that coexist in a 'sky island' with seventeen distinct morphological traits. Our findings reveal that introgression has led to the phylogenomic discordance between nuclear and chloroplast genomes among these morphologically distinct species. Additionally, introgression is predominantly asymmetrical in both intensity and gene function, particularly concerning recombination. Notably, the majority of gene functions associated with introgressed loci are unrelated to reproductive processes. Our results suggest that closely related species with incomplete allele assortment can coexist despite theoretical predictions, highlighting the semipermeable nature of species boundaries as reproductive isolation develops. This provides a critical conceptual framework for understanding the interplay between introgression and species persistence. Our finding offers insights into how related sympatric species boundaries can be maintained in the face of frequent asymmetrical gene introgression.

Sympatry

Genomic introgressions from wild relatives in the wheat genome alter meiotic dynamics in inter-varietal hybrids.

The use of wild relatives to introduce original diversity in the genome of bread wheat (Triticum aestivum L.) is an interesting approach to face the challenges of sustainable agriculture and the impact of climate change on wheat production. However, the influence of these wild-species introgressions on meiosis in inter-varietal wheat hybrids remains poorly understood. We analyzed the French wheat variety Renan (Re) carrying Aegilops ventricosa (Aev)-derived 2AS/2NS and 7DL/7DvL introgressions, the reference cultivar Chinese Spring (CS), which lacks these introgressions, and their inter-varietal hybrid Chinese Spring × Renan (CSRe). This analysis combined cytogenetic approaches with the assessment of reproductive performance. Furthermore, we generated a cytological atlas of meiosis in wild tetraploid Aev, quantifying bivalent configurations and chiasma frequency. We observed a reduced pollen viability and a slight decrease in floret fertility in the hybrid CSRe. Exploration of the meiotic behavior showed that CSRe exhibited increased numbers of rod bivalents and univalents, leading to a reduced average chiasma number and frequent chromosome bridges and fragmentations, whereas the parental lines maintained stable chromosome pairing. These rearrangements indicate that homologous chromosome pairing and recombination are affected in CSRe. We applied introgression-specific oligo-Fluorescent In Situ Hybridization to localize alien segments in CSRe, providing a novel strategy to investigate the meiotic behavior of introgressed regions. The 2AS/2NS introgressed segments in CSRe were frequently located on rod bivalents or univalents, while 7DL/7DvL segments consistently formed ring bivalents. Our results provide a foundation for guiding alien gene introgression and for understanding the behavior of chromosomes with introgressions in the wheat genome.

Meiosis

Weak but repeated patterns of co-introgression of nuclear OXPHOS genes and mitochondrial DNA in Iberian wall lizards.

In this study, we took advantage of the previously reported instances of mitochondrial DNA capture in the Podarcis Iberian group, a speciose group of Iberian wall lizards, to test the hypothesis that nuclear genes from the OXPHOS (Oxidative phosphorylation) chain can co-introgress with the mitochondria as an evolutionary response to mitigate the costs of mitonuclear incompatibilities. Using dense population sampling and transcriptome data, we generated capture-sequence datasets for nuclear OXPHOS chain genes (nucOXPHOS), random nuclear loci (nucControl) and the complete mitochondrial genome. Phylogenetic analyses of nuclear and mitochondrial genes confirmed two previously identified events of mitochondrial introgression in the Podarcis Iberian group and revealed two new cases. Three of these cases have led to complete local mitochondrial DNA replacements, where the introgressed mitotypes have replaced the native ones in several populations, and involve a currently unknown and presumably extinct donor species, so-called "ghost lineage". Detecting introgression from ghost lineages, whose genomes are not accessible, remains challenging. To overcome this issue, we designed or adapted several tests aimed at detecting differential signals of introgression between our nucOXPHOS and nucControl gene sets. One of these tests, based on the effects of introgression on branch lengths in phylogenetic trees, uncovered a weak but consistently significant signal of partial co-introgression of nucOXPHOS genes compared to the genomic background (represented by the nucControl gene set) in three out of four cases of mtDNA introgression.

mitochondrial introgression

Historical introgression as a driver of diversification of diploid Picris (Compositae) in the Mediterranean Basin.

The Mediterranean Basin is recognized as one of the world's most prominent biodiversity hotspots, where past climatic changes have driven range shifts, secondary contact between populations, and gene exchange. This study investigates the impact of historical introgression on the diversification of diploid members of the genus Picris (Compositae). Using nuclear and plastid genome data obtained through the Hyb-Seq approach, we assess whether introgression contributed to the evolution of the Mediterranean Picris, potentially giving rise to multiple regional endemics. We also test whether introgression was associated with the transfer of traits such as life strategy and fruit morphology, which are involved in habitat-specific adaptation. Phylogenetic network analysis revealed two major introgression events that shaped evolutionary trajectories within the genus. The earliest and most complex events involved the Turkish endemic P. campylocarpa, which hybridized with the most recent common ancestor (MRCA) of the P. cyprica-P. pauciflora lineage and with the MRCA of the B1 subclade, comprising the P. hieracioides group and the P. scaberrima-P. strigosa lineage. The latter introgression preceded shifts from iteroparity to semelparity and from heterocarpy to homocarpy, ruling out an adaptive introgression origin for these traits. Nevertheless, all detected historical introgression events contributed to the diversification of diploid Picris taxa.

Diploidy

An archaic reference-free method to jointly infer Neanderthal and Denisovan introgressed segments in modern human genomes.

Admixture between populations is a common feature of human history. Admixture events introduce new genetic variation that can fuel evolution. Characterizing the significance of admixture events on the evolution of populations across various species is of great interest to evolutionary geneticists. Local Ancestry Inference (LAI) methods infer genetic ancestry of an individual at a particular chromosomal location. Certain methods specialize in detecting archaic introgression, which consists of interbreeding between modern and archaic humans like Neanderthals and Denisovans. Most current LAI methods allow the detection of a single archaic ancestry, and post-processing may distinguish between multiple waves of introgression. These methods vary in how they choose archaic or modern reference genomes for the inference. Here, we present a new HMM-based method (DAIseg), which has the advantage of simultaneously distinguishing between multiple waves of ancient and recent admixture, using only modern human reference genomes. Simulations demonstrate that DAIseg achieves higher overall performance than state-of-the-art methods. We also apply DAIseg to Papuan populations to jointly detect Denisovan and Neanderthal introgressed segments, and identify a higher number of archaic segments than previous methods. Analysis of inferred introgressed segments, shows that we can identify evidence for two Denisovan introgression events in Papuans. Overall, on top of being able to deal with both Archaic and recent admixture, DAIseg provides a more principled approach for detecting and classifying Denisovan and Neanderthal segments which will improve downstream analysis of introgressed segments to infer the impact of archaic introgression in humans.

Denisovan

Comparative transmission genetics of introgressed chromatin in reciprocal advanced backcross populations in Gossypium (cotton) polyploids.

Introgression is a potential source of valuable genetic variation and interspecific introgression lines are important resources for plant breeders to access novel alleles. Experimental advanced-generation backcross populations contain individuals with genomic compositions similar to those resulting from natural interspecific hybridization and provide opportunities to study the nature and transmission pattern of donor chromatin in recipient genomes. Here, we analyze transmission of donor chromatin in reciprocal backcrosses between G. hirsutum and G. barbadense. Across the genome, recurrent backcrossing in both backgrounds yielded donor chromatin at slightly higher frequencies than the Mendelian expectation in BC5F1 plants, while the average frequency of donor alleles in BC5F2 segregating families was less than expected. In the two subgenomes of polyploid cotton, the rate of donor chromatin introgression was similar. Although donor chromatin was tolerated over much of the recipient genomes, 21 regions recalcitrant to donor alleles were identified. Only limited correspondence is observed between the recalcitrant regions in the two backgrounds, suggesting the effect of species background on introgression of donor segments. Genetic breakdown was progressive, with floral abscission and seed inviability ongoing during backcrossing cycles. Regions of either high or low introgression tended to be in terminal chromosomal regions that are generally rich in both genes and crossover events, with long stretches around the centromere having limited crossover activity resulting in relatively constant low introgression frequencies. Constraints on fixation and selection of donor alleles highlights the challenges of utilizing introgression breeding in crop improvement.

Humans

Alien introgression and morpho-agronomic characterization of diploid progenies of Solanum lycopersicoides monosomic alien addition lines (MAALs) toward pre-breeding applications in tomato (S. lycopersicum).

Alien introgressions that were captured in the genome of diploid plants segregating from progenies of monosomic alien addition lines of S. lycopersicoides confer novel phenotypes with commercial and agronomic value in tomato breeding. Solanum lycopersicoides is a wild relative of tomato with a natural adaptation to a wide array of biotic and abiotic challenges. In this study, we identified and characterized diploid plants segregating from the progenies of monosomic alien addition lines (MAALs) of S. lycopersicoides to establish their potential as donors in breeding for target trait improvement in tomato. Molecular genotyping identified 28 of 38 MAAL progenies having the complete chromosome complement of the cultivated tomato parent and limited chromosome introgressions from the wild S. lycopersicoides parent. Analysis of SSR and indel marker profiles identified 34 unique alien introgressions in the 28 MAAL-derived introgression lines (MDILs) in the genetic background of tomato. Conserved patterns of alien introgressions were detected among sibs of MDILs 2, 3, 4 and 8. Across MDILs, a degree of preferential transmission of specific chromosome segments was also observed. Morphologically, the MDILs closely resembled the cultivated tomato more than S. lycopersicoides. The appearance of novel phenotypes in the MDILs that are lacking in the cultivated parent or the source MAALs indicates the capture of novel genetic variation by the diploid introgression lines that can add commercial and agronomic value to tomato. In particular, screening of representative MDILs for drought tolerance at the vegetative stage identified MDIL 2 and MDIL 11III as drought tolerant based on visual scoring. A regulated increase in stomatal conductance of MDIL 2 under drought stress indicates better water use efficiency that allowed it to survive for 7 days under 0% moisture level.

Chromosomes, Plant

Phylogenetically diverse introgression drives subtle population structure in Pacific rockfishes.

Genomic methods have shown that admixture and introgression is common across animal taxa. Pacific rockfishes, genus Sebastes, are group of commercially important species that primarily inhabit inshore, shelf, and slope habitats along the North American west coast. Among these, Copper and Quillback Rockfishes (abbreviated to Copper and Quillback) are closely related species known to hybridize, particularly within the Salish Sea in North America's Pacific Northwest. Here, we investigate genetic population structure and introgression patterns in Copper and Quillback from Alaska to California. Using whole-genome resequencing (WGS) across a broad geographic range, we seek to (1) compare population structure between these species, and (2) assess how introgression affects population structure patterns. Our analyses reveal that Copper exhibit much higher levels of population differentiation compared to Quillback, especially separating Salish Sea samples from all other populations. In contrast, Quillback populations appear to be nearly panmictic, with lower overall differentiation. Surprisingly, we detected signatures of introgression from 13 other rockfish species in Copper and 16 species in Quillback. This introgression was highly regional suggesting hybridization depended on geographic context and congener ranges. Yelloweye Rockfish introgression drives the strongest signal of regional population structure in Quillback. These findings provide novel insights into the range-wide genetic structure of these species and highlight that hybridization in Sebastes is phylogenetically broader than previously appreciated.

Journal Article

Genome-wide subgenome-resolved analysis validates chromosome 4 differentiation and prioritizes introgressed Coffea arabica accessions.

Chromosome 4 introgression in Timor hybrid-derived Coffea arabica is established, but the robustness of accession prioritization and the relative strength of cultivated-introgressed differentiation across the canephora-derived (sgC) and eugenioides-derived (sgE) subgenomes remained unclear under explicit subgenome filtering. We reanalyzed public genomic resources from 44 coffee accessions using strict contig-level subgenome filtering, Arabica-only population-structure analysis, SNP-panel sensitivity testing, genome-wide differentiation scans, permutation testing, and direct sequence alignment. Population structure and accession rankings were stable across marker densities and random seeds, and the same six introgressed references were retained throughout. Chromosome 4 ranked first in both subgenomes, with a strong sgC signal and a markedly weaker sgE signal; independent baseline-panel permutation tests supported both chromosome 4-associated signals. Direct alignment supported correspondence to the expected chromosome 4 pseudomolecules while showing incomplete source coverage and unresolved exact boundaries. Alignment-supported blocks contained 88 sgC and 62 sgE provisional defense-, signaling-, and regulatory-associated annotations. These results provide a genome-wide, quantitatively validated framework for prioritizing introgressed germplasm and candidate chromosome 4 regions for phenotype-linked coffee research without implying equivalent introgression, exact liftover, or causal resistance genes.

Coffea arabica

Comparing Neanderthal introgression maps reveals core agreement but substantial heterogeneity.

Statistical methods to identify Neanderthal ancestry in modern human genomes rest on varying assumptions and inputs. Nonetheless, most studies of introgression use only a single method to define Neanderthal ancestry. Due to a lack of "ground truth," we have a limited understanding of the accuracy, comparative strengths and weaknesses, and the sensitivity of downstream conclusions for these methods. Here, we performed large-scale comparisons of genome-wide introgression maps from 12 representative Neanderthal introgression detection algorithms. These span methods that consider archaic and human reference genomes not from Africa (ArchaicSeeker2, CRF, DICAL-ADMIX), only archaic genomes (S*, Sprime, HMM, SARGE, ARGWeaver-D), only human reference genomes, including from Africa (IBDmix), or simulated data (ArchIE). Our results highlight a core set of regions predicted by nearly all methods, as well as substantial heterogeneity in commonly used Neanderthal introgression maps. Furthermore, we find that downstream analyses may result in different conclusions depending on the map used. Thus, we recommend careful consideration of map(s) chosen for an analysis and support the use of multiple maps to ensure robustness of conclusions. We make integrated prediction sets available, enabling further understanding of Neanderthal introgression's legacy on modern humans.

Journal Article

Genomic Footprints of Historical Introgression Between Ancient Lineages of Wild Oryza AA-Genome Species With Widely Separated Contemporary Distributions.

Phylogenetic incongruence is increasingly recognized as pervasive, yet the extent to which reticulate evolution occurs between groups separated by substantial geographical distances and deep phylogenetic divergence remains poorly characterized. In the Oryza AA-genome group-a model for plant speciation and domestication-the traditional bifurcation model posits that Australian Oryza meridionalis and African Oryza longistaminata occupy basal branches, distinct from the more recently diversified monophyletic clade comprising Asian and other African lineages, including major cultivars. However, recent evidence from endogenous viral sequences has hinted at unexpected genetic relatedness between African O. longistaminata and Asian Oryza sativa, which are geographically and phylogenetically distant. Here, we conducted a genome-wide survey across 11 Oryza species to systematically identify genomic regions exhibiting phylogenetic incongruence. Widespread phylogenetic discordance was observed, notably involving genomic segments in which O. longistaminata showed phylogenetic proximity to Asian species, contradicting their established deep divergence. To distinguish between introgression and incomplete lineage sorting, we performed four-taxon ABBA-BABA tests, which provided statistical support for introgression. Furthermore, divergence time estimates for these incongruent regions were younger than the species divergence times, suggesting historical introgression between the ancestors of lineages that are currently separated by vast geographical distances. Systematic assessments indicated that potential analytical artifacts, such as compositional bias and substitution saturation, were unlikely to explain the observations. These convergent lines of evidence suggest that ancient introgression had occurred between currently geographically separated and evolutionarily divergent Oryza lineages, leaving detectable footprints across their modern genomes.

Oryza

KCFtools: rapid alignment-free method for introgression screening and GWAS using k-mer profiles.

MOTIVATION: In the era of multiple genome references, researchers often align sequencing reads against distinct assemblies or even multiple references simultaneously. This enables applications such as the detection of introgressed segments or highly variable genomic regions, which are especially prevalent in large-genome crop species such as lettuce or wheat. However, these applications come at the cost of increased computational burden, inconsistencies in mapping methods, and reduced reproducibility across studies. To address these limitations, we developed KCFtools, a Java-based toolkit that identifies the presence and absence of k-mers in nonoverlapping genomic or transcriptomic windows by comparing query and reference genomes. This alignment-free approach enables the efficient computation of an identity score for each window, thereby facilitating robust detection of introgressed or variable regions across genomes. RESULTS: We systematically evaluated the performance and accuracy of the k-mer-based method implemented in KCFtools, benchmarking it against conventional single nucleotide variation-based introgression detection pipelines. Our results demonstrate that KCFtools effectively captures introgressed segments and structurally diverse regions, even in species with fragmented or highly divergent reference genomes. In addition, we extended KCFtools to generate genotype matrices from k-mer variation tables. These matrices are compatible with genome-wide association studies software and allow the identification of loci associated with phenotypic traits. We showcase the utility of this approach by detecting known and novel associations for downy mildew resistance in lettuce, underscoring the pipeline's potential for high-resolution, reference-agnostic population genetic analysis. AVAILABILITY AND IMPLEMENTATION: https://github.com/sivasubramanics/kcftools.

Software

Pervasive hybridization and introgression in Diervilleae (Caprifoliaceae).

Diervilleae (Caprifoliaceae) is a horticulturally important lineage with striking floral diversity and a long history of interspecific crossing, suggesting reticulate evolution. We integrated nuclear SNPs and whole plastome data to reconstruct a phylogenomic backbone for the tribe and to identify hybrids, cultivated accessions, and introgression among lineages. Nuclear and plastid phylogenies consistently recover Weigela and Diervilla as reciprocally monophyletic and resolve four major lineages within Weigela, providing a reproducible framework for revising sectional limits and species boundaries. Cultivated accessions form a well supported clade sister to W. florida and show predominantly W. florida ancestry while retaining contributions from multiple wild lineages, consistent with recurrent crossing, backcrossing, and selection. Analyses of wild populations reveal recurrent hybrids and enable plausible parental combinations to be inferred. Tests across the genome further indicate strong evidence for historical introgression across Diervilleae, with the strongest signals involving W. middendorffiana, W. maximowiczii, and Diervilla. Fossil evidence, divergence time estimation, and paleodistribution modelling together suggest range expansion during the Miocene and Pliocene followed by climate driven contraction, providing a spatiotemporal context for episodic contact, introgression, and the East Asia-North America disjunction.

Hybridization, Genetic

Pan-Genome Analysis Reveals Local Adaptation to Climate Driven by Introgression in Oak Species.

The genetic base of local adaptation has been extensively studied in natural populations. However, a comprehensive genome-wide perspective on the contribution of structural variants (SVs) and adaptive introgression to local adaptation remains limited. In this study, we performed de novo assembly and annotation of 22 representative accessions of Quercus variabilis, identifying a total of 543,372 SVs. These SVs play crucial roles in shaping genomic structure and influencing gene expression. By analyzing range-wide genomic data, we identified both SNPs and SVs associated with local adaptation in Q. variabilis and Quercus acutissima. Notably, SV-outliers exhibit selection signals that did not overlap with SNP-outliers, indicating that SNP-based analyses may not detect the same candidate genes associated with SV-outliers. Remarkably, 29%-37% of candidate SNPs were located in a 250 kb region on chromosome 9, referred to as Chr9-ERF. This region contains 8 duplicated ethylene-responsive factor (ERF) genes, which may have contributed to local adaptation of Q. variabilis and Q. acutissima. We also found that a considerable number of candidate SNPs were shared between Q. variabilis and Q. acutissima in the Chr9-ERF region, suggesting a pattern of repeated selection. We further demonstrated that advantageous variants in this region were introgressed from western populations of Q. acutissima into Q. variabilis, providing compelling evidence that introgression facilitates local adaptation. This study offers a valuable genomic resource for future studies on oak species and highlights the importance of pan-genome analysis in understating mechanism driving adaptation and evolution.

Quercus

Detecting Introgression in Shallow Phylogenies: How Minor Molecular Clock Deviations Lead to Major Inference Errors.

Recent theoretical and algorithmic advances in introgression detection, coupled with the growing availability of genome-scale data, have highlighted the widespread occurrence of interspecific gene flow across the tree of life. However, current methods largely depend on the molecular clock assumption-a questionable premise given empirical evidence of substitution rate variation across lineages. While such rate heterogeneity is known to compromise gene flow detection among divergent lineages, its impact on closely related taxa at shallow evolutionary timescales remains poorly understood, likely because these taxa are often assumed to adhere to a molecular clock. To address this gap, we combine theoretical analyses and simulations to evaluate the robustness of widely used site pattern methods (D-statistic and HyDe) to rate variation across phylogenetic timescales. Our results demonstrate that both methods exhibit high sensitivity to even minor deviations from the molecular clock at shallow timescales, complementing previous findings at deeper scales. Specifically, in young phylogenies (with an age of 3 × 105 generations) with small population sizes, weak (17% difference) and moderate (33% difference) rate variation can inflate false-positive rates up to 35% and 100%, respectively, using site pattern counts from a 500 Mb genome. Employing a more distant outgroup intensifies these spurious signals. Our study demonstrates that summary tests for introgression are pervasively vulnerable to minor rate variations and underscores the critical need for advanced methodologies to disentangle genuine introgression from false signals generated by rate heterogeneity.

Phylogeny

Ancient Introgression Explains Mitochondrial Genome Capture and Mitonuclear Discordance Among South American Collared Tropidurus Lizards.

Mitonuclear discordance-evolutionary discrepancies between mitochondrial and nuclear DNA phylogenies-can arise from various factors, including introgression, incomplete lineage sorting, recent or ancient demographic fluctuations, sex-biased dispersal asymmetries, among others. Understanding this phenomenon is crucial for accurately reconstructing evolutionary histories, as failing to account for discordance can lead to misinterpretations of species boundaries, phylogenetic relationships, and historical biogeographic patterns. We investigate the evolutionary drivers of mitonuclear discordance in the Tropidurus spinulosus species group, which contains nine species of lizards inhabiting open tropical and subtropical environments in South America. Using a combination of population genetic and phylogenomic approaches applied to mitochondrial and nuclear data, we identified different instances of gene flow that occurred in ancestral lineages of extant species. Our results point to a complex evolutionary history marked by prolonged isolation between species, demographic fluctuations, and potential episodes of secondary contact with genetic admixture. These conditions likely facilitated mitochondrial genome capture while diluting signals of nuclear introgression. Furthermore, we found no strong evidence supporting incomplete lineage sorting or natural selection as primary drivers of the observed mitonuclear discordance. Therefore, the unveiled patterns are most consistent with neutral demographic processes, coupled with ancient mitochondrial introgression, as the main factors underlying the mismatch between nuclear and mitochondrial phylogenies in this system. Future research could further explore the role of other demographic processes, such as asymmetric sex-biased dispersal, in shaping these complex evolutionary patterns.

Animals

Introgression among maternal lineages inferred from complete mitogenomes and molecular dating helps resolve phylogeography of European roe deer.

BACKGROUND: The European roe deer (Capreolus capreolus) is one of the most widespread ungulates in Europe, with a phylogeographic structure mainly shaped by Pleistocene glacial cycles and secondary contacts with the Siberian roe deer (C. pygargus). METHODS: We sequenced 52 complete mitogenomes of C. capreolus from Slovenia, Poland and France, and combined them with 24 publicly available sequences of C. capreolus and C. pygargus, yielding an alignment of 76 genomes representing 59 haplotypes (42 from C. capreolus and 17 from C. pygargus). Phylogeographic structure was assessed using a median-joining network, and divergence times were estimated using a time-calibrated Bayesian phylogeny based on mitochondrial coding regions, incorporating published ancient C. pygargus mitogenomes. We additionally screened mitochondrial protein-coding genes for selection. RESULTS: The haplotype network recovered the three major European roe deer clades (Eastern, Central, and Western) and detected Central-clade haplotypes in France. Two Polish haplotypes (Cp9 and Cp10), detected in C. capreolus, clustered within the C. pygargus mitochondrial lineage, supporting mitochondrial introgression. Time-calibrated phylogenies placed introgressed haplotypes within established C. pygargus lineages. Selection analyses provided limited evidence for episodic positive selection restricted to a small number of codons. CONCLUSIONS: Whole mitogenomes improve resolution of roe deer phylogeography and reveal introgressed maternal lineages, while time-calibrated phylogenies and selection tests add evolutionary context for interpreting mtDNA diversity in genus Capreolus.

Animals