Mitochondrial functions are intimately reliant on proteins and RNAs encoded in both the nuclear and mitochondrial genomes, leading to inter-genomic coevolution within taxa. Hybridization can break apart coevolved mitonuclear genotypes, resulting in decreased mitochondrial performance and reduced fitness. This hybrid breakdown is an important component of outbreeding depression and early-stage reproductive isolation. However, the mechanisms contributing to mitonuclear interactions remain poorly resolved. Here we scored variation in developmental rate (a proxy for fitness) among reciprocal F2 inter-population hybrids of the intertidal copepod Tigriopus californicus, and used RNA sequencing to assess differences in gene expression between fast- and slow-developing hybrids. In total, differences in expression associated with developmental rate were detected for 2,925 genes, whereas only 135 genes were differentially expressed as a result of differences in mitochondrial genotype. Up-regulated expression in fast developers was enriched for genes involved in chitin-based cuticle development, oxidation-reduction processes, hydrogen peroxide catabolic processes, and mitochondrial respiratory chain complex I. In contrast, up-regulation in slow developers was enriched for DNA replication, cell division, DNA damage, and DNA repair. Eighty-four nuclear-encoded mitochondrial genes were differentially expressed between fast- and slow-developing copepods, including twelve subunits of the electron transport system (ETS) which all had higher expression in fast developers than in slow developers. Nine of these genes were subunits of ETS complex I. Our results emphasize the major roles that mitonuclear interactions within the ETS, particularly in complex I, play in hybrid breakdown, and resolve strong candidate genes for involvement in mitonuclear interactions.

Developmental rate data - collected by daily monitoring naupliar (larval) development of individual Tigriopus californicus until stage 1 copepodid metamorphosis was observed

RNA-seq count data - collected by isolated RNA from pools of fast- or slow-developing Tigriopus californicus copepodids from reciprocal hybrids lines; RNA was sequenced on a NovaSeq 6000; reads were mapped to a hybrid reference genome and counted using STAR and featureCounts; differences in gene expression were tested with edgeR; gene ontology enrichments among differentially expressed genes were assessed with goseq.

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