Exploring mitonuclear interactions in the regulation of cell physiology: Insights from interspecies cybrids

Brown hares (Lepus europaeus) and mountain hares (Lepus timidus) frequently hybridize in regions where their range overlaps, producing fertile offspring and enabling gene flow between the species. Despite this, no hybrid species has emerged, suggesting that hybrid backcrosses may incur fitness costs. One potential mechanism for such costs involves the interactions between mitochondrial and nuclear gene products, where incompatibilities between species-specific alleles may reinforce species barriers and lead to hybrid breakdown. However, direct experimental evidence for this hypothesis remains limited. In this study, we used fibroblasts derived from skin biopsies of wild-caught hares to generate cytoplasmic hybrid (cybrid) cell lines, wherein mitochondria and mtDNA from one species were transferred to mitochondria-depleted cells of the other species, creating novel mitonuclear gene combinations while preserving the original diploid nuclear background. Employing a range of techniques – including transcriptomics, metabolomics, microscopy, and respirometry – we explored the consequences of mitochondrial transfer between these hare species. Our results reveal that in the studied species mitonuclear incompatibilities exhibit strong effects on cellular fitness but are limited to specific genotypes. We propose mechanisms of cellular-level incompatibility and their potential consequences for interspecific hybrids, offering new insights into the complexity of mitonuclear interactions.