Understanding the cause of reproductive barriers that demarcate species (e.g. hybrid lethality and sterility), is essential for understanding the process of speciation. We have recently discovered a lethal incompatibility between two distinct wild-isolates (JU1825 and NIC59) of Caenorhabditis nouraguensis. Embryonic death seems to be the result of a maternally inherited cytoplasmic factor from each strain being incompatible with a recessive nuclear gene from the other. To preclude the possibility that maternally inherited endosymbiotic bacteria cause the incompatibility, we have treated both strains with tetracycline for nine generations and shown that hybrid death is unaffected. We hypothesize that the maternally inherited factor is the mitochondrial genome and that mitochondrial dysfunction underlies hybrid death. We performed Illumina sequencing on these two strains and have generated the first genomic assemblies of C. nouraguensis. We have mapped the nuclear incompatibility loci to single chromosomes by bulk sequencing of viable F2 hybrids. We are currently genotyping introgression lines to more finely resolve these loci. Additionally, the NIC59 and JU1825 mitochondrial genomes differ by 95 SNPs and a 1 bp indel, with strong candidate polymorphisms in the cysteine tRNA gene, 16s rRNA gene and non-synonymous changes in the nd-1 and
cox-1 genes (subunits of complex I and IV of the electron transport chain). Interestingly, NIC59 has a heteroplasmic 184 bp deletion that is predicted to result in a 122 a.a. C-terminal truncation of the nd-5 gene (subunit of complex I), reminiscent of a heteroplasmic deletion in the same gene seen in C. briggsae. We are also characterizing the mechanism of hybrid embryonic lethality. Incompatible hybrid embryos exhibit gross defects during the lima-bean stage and fail to undergo proper epidermal enclosure, resulting in ventral explosion and death. This phenotype is similar to C. elegans mutants with deficiencies in actin cytoskeletal regulation and cell-cell adhesion. Therefore, we will assay these cellular processes in inviable hybrids. .