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 .