mtDNA and nuclear discordance
Culicoides albertensis , C. sonorensis , and C. variipennis have a considerable amount of genome-wide differentiation (Fig. 1); however, there was no clear differentiation of the COI gene (Fig. 5). In fact, several individuals of C. albertensis andC. variipennis shared identical haplotypes. Multiple studies have shown a high degree of genetic similarity in mtDNA between C. sonorensis and C. variipennis (Hopken, 2016; Jewiss-Gaines, Barelli, & Hunter, 2017; Shults, 2015), though it was proposed that this was due to misidentifications. As all of the individuals included in our mitochondrial haplotype analysis from the current study were identified to species using the SNP data, this lack of mitochondrial separation has an underlying biological source. Ongoing hybridization with “leaky” pre-zygotic isolation, or a semipermeable species boundaries, has been shown to produce mitochondrial introgression without detectable nuclear DNA introgression (Chan & Levin, 2005; Harrison, 1990). This is likely due to the fact the mitochondrial genome is independent of the nuclear genome and thus unlinked to the genes contributing to reproductive isolation (Harrison, 1989). This does not appear to be the case throughout the entire complex though as hybridization was also found between C. sonorensis and C. occidentalis and the mtDNA from these two species was highly divergent.
In addition to the convergence of a single haplogroup by three species,C. occidentalis was found to have two distinct haplogroups based on geography (Fig. 5). The mean percent divergence between C. occidentalis from California (CABL) and C. occidentalis from the other collection sites (BC-NV-UT) was equal to the divergence between the other species in the complex (Table 3). This high level of differentiation within C. occidentalis could be due to geographic isolation alone; however, endosymbionts have also been shown to significantly increase mitochondrial diversity in the presence of geographic structure (Ballard, Chernoff, & James, 2002; Behura, Sahu, Mohan, & Nair, 2001). Naturally occurring endosymbionts have been found in Culicoides midges, including C. sonorensis (Covey et al., 2020; Pagès, Muñoz-Muñoz, Verdún, Pujol, & Talavera, 2017), and recently, a Cardinium sp. was linked to mitochondrial divergence of C . imicola (Pilgrim et al., 2021). Further screening is needed to determine the diversity and abundance of endosymbionts infecting Culicoides midges, though the possibility remains that these could be playing a role in the phylogeographical structure ofC. occidentalis .