Characterizing a lethal mitonuclear incompatibility in naturally hybridizing Xiphophorus swordtails

The evolution of reproductive barriers is the first step in the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of “hybrid incompatibilities,” where alleles derived from two different species no longer interact properly in hybrids. Theory predicts that hybrid incompatibilities may be more likely to arise at rapidly evolving genes and that incompatibilities involving multiple genes should be common, but there has been sparse empirical data to evaluate these predictions. Here, we describe a mitonuclear incompatibility involving three genes in physical contact within respiratory Complex I of naturally hybridizing swordtail fish species. Individuals homozygous for mismatched protein combinations fail to complete embryonic development or die as juveniles, while those heterozygous for the incompatibility have reduced Complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. We find that the impacts of different genetic interactions on survival are non-additive, highlighting subtle complexity in the genetic architecture of hybrid incompatibilities. Finally, we document the evolutionary history of the genes involved, showing signals of accelerated evolution and the first case of an incompatibility transferred between species via hybridization.

Methods
This project was based genomic DNA samples collected from live fish in Hidalgo, Mexico and lab populations in Stanford, California, followed by low-coverage sequencing and local ancestry inference using an HMM-based approach. The resulting ancestry information was used for QTL and admixture mapping to identify mito-nuclear associations, and estimation of selection on particular genotype combinations. We also genotyped embryos dissected from in utero after performing developmental staging, respirometry measurements, and morphometrics. We isolated mitochondria from adult heterozygotes, then used these samples to perform respirometry using an Oroboros O2K respirometer, and a Parallel Reaction Monitoring proteomics experiment. Using inferred protein sequences from the relevant species, we performed in silico modeling in RaptorX and MODELLER of Complex I genes of interest, analyses of evolutionary rates using codeml, and tested for historical introgression using a combination of phylogentics on PacBio mitochondrial genomes and simulations of ILS vs. gene flow. Further details are available in the online-only methods and supplemental information of the associated manuscript.