Molecular Determinants of Speciation, Adaptation and Interspecies Compatibility: Lessons Learned from Cell Lines

Evolution has shaped the diversity of life on Earth, driving the adaptation of organisms to their environments through natural selection. Species evolved unique strategies to survive, and their ability to persist and thrive in a wide range of environmental conditions, from extreme colds to deep-sea pressures, relies on mitochondrial efficiency. Amongst other functions, mitochondria regulate energy production, integrate metabolic pathways, generate reactive oxygen species (ROS) and produce heat. Organismal homeostasis depends on intrinsic interconnection and coordinated response between mitochondrial and nuclear encoded gene products. For most species, mitochondria together with its genome are uniparentally inherited, meaning that hybridization between closely related species facilitates the gene flow and mitochondria crosses the species barrier. This may generate genetic conflicts or conversely lead to adaptive introgression or even hybrid speciation. Nowadays in Nordic regions the cold-acclimated mountain hare (Lepus timidus) competes for the same habitat with the brown hare (Lepus europaeus) whose expansion to North is facilitated by climate change. Remarkably, these species recurrently hybridize, but molecular phenotypes of the species, which enable their adaptations, and the fitness of hybrid animals are largely unknown.

The goal of this work is to characterize molecular features between closely related yet ecologically distinct hare species, focusing on adaptive traits that distinguish their life-history strategies. Using skin fibroblast lines derived from the wild animals sampled in Finland and cytoplasmic hybrid (cybrid) cell lines generated from them, I applied various methods, including respirometry, flow cytometry and microscopy, to uncover several molecular mechanisms that underpin species differentiation, metabolic adaptability and regulation of mitochondrial functions. Results showed that mountain hare fibroblasts are characterized by elevated mitochondrial membrane potential accompanied with increased glycerol 3-phosphate and creatine metabolism, reduced cell growth and migration rates compared to the brown hare fibroblasts. Furthermore, silencing mitochondrial glycerol 3-phosphate dehydrogenase (GPD2) promoted growth and migration in mountain hare cells and reduced mitochondrial temperature and mitochondrial membrane potential in both species. Microscopy using temperature sensitive probe (Mito Thermo Yellow) revealed that mitochondria of cold-adapted mountain hare potentially function at lower temperatures than in the brown hare, demonstrating for the first time mitochondrial temperature difference between mammalian species. These findings highlight the trade-offs between metabolic, bioenergetic and thermogenic functions in mitochondria to support animal adaptation in its natural environment.

Additionally, mitonuclear compatibility between the hare species was assessed using cybrid cell lines. Cybrids characterized by profound gene expression changes some of which may have been caused by their generation process, emphasizing the need for caution as cybrids are extensively used in research for modelling and studying of various human pathologies. Results showed that incompatibilities between mitochondrial and nuclear genomes had strong individual features and primary manifested in decreased protein levels of Complexes I and III, reduced mitochondrial respiration and membrane potential, diminished cell growth and migration and increased production of ROS.

This work demonstrates that cells retain species-specific molecular features, offering a valuable perspective for understanding and comparing wild animal physiology. It also suggests that in interfertile species, mitonuclear incompatibilities emerge under specific genetic backgrounds. This research deepens our knowledge about metabolic adaptations in animals, trait differences between species and their evolutionary significance, underscoring the potential of in vitro approaches for animal replacement. It also contributes to a better understanding of the impact of hybridization in hares, which may facilitate the development of appropriate conservation strategies. Moreover, the present work could set a precedent for future research using cultured cells from wild-life species, including endangered species, for studying extreme adaptations in traits like longevity, deep diving and hibernation, leading to novel biomedical applications. From the medical perspective, a deeper insight into mitonuclear compatibility has the potential to enhance the success rates of in vitro fertilizations and to aid the development of mitochondria replacement therapies, as well as more effective approaches for the diagnosis and treatment of mitochondrial disorders.