Origins and diversity of Exmoor ponies: A mitogenome framework for assessing maternal lineage diversification in endangered pony breeds

In response to growing concerns over human-driven biodiversity loss, rewilding efforts increasingly employ semi-wild herbivores to restore ecosystems. The Exmoor pony is often cited as an ancient breed retaining primitive traits suited for such projects, yet its genetic history remains underexplored. We combined studbook records, mitochondrial DNA (mtDNA) D-loop, and whole mitogenome sequences to evaluate maternal lineage diversity for improved management recommendations and the phylogenetic placement of Exmoor ponies among modern and ancient breeds. We also assessed whether data from multiple breeds already available based on D-loop sequences could be organised into a useful framework for resolving the complex history of modern horse breeds. We identified eight mtDNA haplotypes among 88 Exmoor pony samples, representing all extant maternal lines. While phylogenetic trees based on D-loop sequences lacked resolution, clustering published sequences to a mitogenome-derived haplogroup framework allowed assessment of the distribution and relationships of lineages across diverse horse populations, including ancient DNA samples and Przelwalskis horses. The most basal lineages of Exmoors were traced back to the Pleistocene, supporting their ancient origin and status as a primitive population, with a possible ancestral role in the development of some modern breeds. The founder lineages were found to be polyphyletic, with more derived haplogroups representing subsequent mixture with other domestic horse breeds. Although nuclear genome sequencing is needed to fully assess levels of admixture, we identified new haplotypes, along with rare or basal haplotypes that could be prioritised for conservation in rewilding studies. Our results highlight the potential for broad testing of hypotheses about origins and relationships among horse breeds using short regions of DNA mapped onto a more robust phylogenetic framework resolved through comparison of whole mitogenomes.