Beyond Genes: Metabolomic Evidence Indicates Potential Species-Level Differentiation in European Wild Rabbits

The European rabbit (Oryctolagus cuniculus) plays a key ecological role in Mediterranean ecosystems, yet its populations are declining. Two subspecies, O. c. algirus and O. c. cuniculus, are present in the Iberian Peninsula and exhibit genetic, phenotypic, and ecological differences. The aim of this study was to investigate whether genetic differences between subspecies are reflected in the plasma metabolome of the European rabbit and to identify potential biomarkers for subspecies classification. Comprehensive untargeted metabolomics analyses (n = 49 wild individuals) were performed. Significant and consistent differences in the metabolomic profiles were found between subspecies. Statistically significant differences were detected in nine metabolites; three showed strong discriminatory potential, with Medicagenic acid, MG(P-18:0/0:0/0:0), and Androsta-1,4-diene-17-carboxylicacid, 17-[(ethoxycarbonyl)oxy]-11-hydroxy-3-oxo-, fluoromethyl ester showing strong potential as biomarkers. These findings demonstrate functional divergence between the subspecies, likely related to diet and ecological adaptations. They provide new molecular evidence of metabolic differentiation between subspecies, which may be relevant for understanding evolutionary divergence and could inform conservation strategies.