A lethal mitonuclear incompatibility in complex I of natural hybrids Hybrid incompatibilities, where alleles from different species fail to interact properly in hybrids, are key reproductive barriers in speciation. This study identifies a lethal mitonuclear incompatibility in the respiratory complex I of naturally hybridizing swordtail fish species, Xiphophorus birchmanni and Xiphophorus malinche. The incompatibility involves three genes: two nuclear-encoded genes (ndufs5 and ndufa13) and a mitochondrial gene (ndufs5). Individuals homozygous for mismatched protein combinations fail to complete embryonic development or die as juveniles, while heterozygous individuals show reduced complex I function and unbalanced parental allele representation in the mitochondrial proteome. The effects of genetic interactions on survival are non-additive, highlighting the complexity of hybrid incompatibilities. The study reveals that the incompatibility has an evolutionary history, with signals of accelerated evolution and evidence of gene transfer between species via hybridization. The genes involved show signs of positive selection, and the mitochondrial genome is a hotspot for genetic incompatibilities due to high substitution rates and potential for sexually antagonistic selection. The interaction between mitochondrial and nuclear proteins in key steps of ATP synthesis increases the likelihood of coevolution between these genomes. The study uses an integrative approach to map the genetic basis and physiological effects of the incompatibility in swordtail fish. It identifies two nuclear-encoded genes that are lethal when mismatched with the mitochondrial genome of the other species. The incompatibility is associated with developmental and physiological effects, including arrested development in utero for individuals with mismatched ancestry at ndufs5 and vascular defects for those with mismatched ancestry at ndufa13. The study also shows that the incompatibility has been transferred between species via hybridization, with evidence that genes underlying the incompatibility have introgressed from X. malinche to X. cortezi. The findings highlight the role of mitonuclear interactions in hybrid incompatibilities and suggest that hybridization can transfer incompatibilities between species. The study provides new insights into the evolutionary processes that drive the emergence of hybrid incompatibilities and the mechanisms that maintain reproductive isolation between species.A lethal mitonuclear incompatibility in complex I of natural hybrids Hybrid incompatibilities, where alleles from different species fail to interact properly in hybrids, are key reproductive barriers in speciation. This study identifies a lethal mitonuclear incompatibility in the respiratory complex I of naturally hybridizing swordtail fish species, Xiphophorus birchmanni and Xiphophorus malinche. The incompatibility involves three genes: two nuclear-encoded genes (ndufs5 and ndufa13) and a mitochondrial gene (ndufs5). Individuals homozygous for mismatched protein combinations fail to complete embryonic development or die as juveniles, while heterozygous individuals show reduced complex I function and unbalanced parental allele representation in the mitochondrial proteome. The effects of genetic interactions on survival are non-additive, highlighting the complexity of hybrid incompatibilities. The study reveals that the incompatibility has an evolutionary history, with signals of accelerated evolution and evidence of gene transfer between species via hybridization. The genes involved show signs of positive selection, and the mitochondrial genome is a hotspot for genetic incompatibilities due to high substitution rates and potential for sexually antagonistic selection. The interaction between mitochondrial and nuclear proteins in key steps of ATP synthesis increases the likelihood of coevolution between these genomes. The study uses an integrative approach to map the genetic basis and physiological effects of the incompatibility in swordtail fish. It identifies two nuclear-encoded genes that are lethal when mismatched with the mitochondrial genome of the other species. The incompatibility is associated with developmental and physiological effects, including arrested development in utero for individuals with mismatched ancestry at ndufs5 and vascular defects for those with mismatched ancestry at ndufa13. The study also shows that the incompatibility has been transferred between species via hybridization, with evidence that genes underlying the incompatibility have introgressed from X. malinche to X. cortezi. The findings highlight the role of mitonuclear interactions in hybrid incompatibilities and suggest that hybridization can transfer incompatibilities between species. The study provides new insights into the evolutionary processes that drive the emergence of hybrid incompatibilities and the mechanisms that maintain reproductive isolation between species.