All mitochondrial-encoded proteins and RNAs function through interactions with nuclear-encoded proteins, which are critical for mitochondrial performance and eukaryotic fitness. Coevolution maintains inter-genomic (i.e., mitonuclear) compatibility within a taxon, but hybridization can disrupt coevolved interactions, resulting in hybrid breakdown. Thus, mitonuclear incompatibilities may be important mechanisms underlying reproductive isolation and, potentially, speciation. Here we utilize Pool-seq to assess the effects of mitochondrial genotype on nuclear allele frequencies in fast- and slow-developing reciprocal inter-population F₂ hybrids between relatively low-divergence populations of the intertidal copepod Tigriopus californicus. We show that mitonuclear interactions lead to elevated frequencies of coevolved (i.e., maternal) nuclear alleles on two chromosomes in crosses between populations with 1.5% or 9.6% fixed differences in mitochondrial DNA nucleotide sequence. However, we also find evidence of excess mismatched (i.e., non-coevolved) alleles on three or four chromosomes per cross, respectively, and of allele frequency differences consistent with effects involving only nuclear loci (i.e., unaffected by mitochondrial genotype). Thus, our results for low-divergence crosses suggest an underlying role for mitonuclear interactions in variation in hybrid developmental rate, but despite substantial effects of mitonuclear coevolution on individual chromosomes, no clear bias favouring coevolved interactions overall.

Pool-seq count data - collected by isolating DNA from pools of fast- or slow-developing Tigriopus californicus copepodids from reciprocal hybrids lines; DNA was sequenced by whole-genome sequencing on a NovaSeq 6000; reads were mapped to a hybrid reference genome and counted using bwa; allele-specific read counts were determined with Samtools and popoolation.

Population-specific reference genomes - re-sequenced genome sequences for individual Tigriopus californicus populations; obtained by mapping whole-genome sequencing reads to the previously published San Diego, California reference sequence; population-specific consensus sequences were determined with bwa, Samtools and popoolation.

PE mtDNA sequence - consensus sequence from WGS reads from the Pescadero Beach, California Tigriopus californicus population; reads were mapped to a previously published mtDNA sequence for a population from Santa Cruz, California with CLC Genomics Workbench which was also used to determine the consensus PE sequence.

Hybrid reference genomes - assembled from previously published sequences and the PE mtDNA sequence using R.

Fixed SNP lists - list of single-nucleotide polymorphisms displaying fixed differences between pairs of Tigriopus californicus populations; determined by reciprocal whole-genome sequencing read mapping and processing with bwa, Samtools and popoolation.

All files can be opened in text editors, spreadsheet programs or the statistical software R.