Kannangara, J.; Connallon, T.; Nogueira Alves, A.; Dowling, D.

Emerging evidence suggests mtDNA haplotypes contribute to fitness variation and local adaptation, with studies highlighting the potential for directional selection, imposed by distinct thermal environments, and negative frequency-dependent selection to shape spatial patterns of mtDNA haplotype diversity. However, the interplay between these different forms of selection remains unexplored. To address this, we conducted experimental evolution using Drosophila melanogaster populations derived from opposite ends of a latitudinal cline in Australia (Melbourne and Townsville), exposing them to contrasting thermal environments and varying starting frequencies of two mtDNA haplotypes (A1 and B1) that each segregate at high frequencies in natural populations. We paired this approach with population genetic simulations to estimate selection and its influence on haplotype evolutionary trajectories. We found that mtDNA haplotype frequencies were influenced by complex interactions among temperature, starting frequency, and nuclear genomic background (Melbourne, Townsville, or admixed). Simulations supported directional selection as a driver of haplotype frequencies in populations evolving at 17 degrees Celsius in the Melbourne background, and otherwise selection was predominantly balancing and shaped by negative frequency-dependent fitness effects of the haplotypes. Finally, our results suggest that the thermal environment is not the sole environmental variable shaping mitochondrial evolutionary dynamics and point to a possible role for mito-nuclear epistasis.