Ancient hybridization and mtDNA introgression behind current paternal leakage and heteroplasmy in hybrid zones

Abstract

Hybridization between heterospecific individuals has been documented as playing a direct role in promoting paternal leakage and mitochondrial heteroplasmy in both natural populations and laboratory conditions, by relaxing the egg-sperm recognition mechanisms. Here, we tested the hypothesis that hybridization can lead to mtDNA heteroplasmy also indirectly via mtDNA introgression. By using a phylogenetic approach, we showed in two reproductively isolated beetle species, Ochthebius quadricollis and O. urbanelliae, that past mtDNA introgression occurred between them in sympatric populations. Then, by developing a multiplex allele-specific PCR assay, we showed the presence of heteroplasmic individuals and argue that their origin was through paternal leakage following mating between mtDNA-introgressed and pure conspecific individuals. Our results highlight that mtDNA introgression can contribute to promote paternal leakage, generating genetic novelty in a way that has been overlooked to date. Furthermore, they highlight that the frequency and distribution of mtDNA heteroplasmy can be deeply underestimated in natural populations, as i) the commonly used PCR-Sanger sequencing approach can fail to detect mitochondrial heteroplasmy, and ii) specific studies aimed at searching for it in populations where mtDNA-introgressed and pure individuals co-occur remain scarce, despite the fact that mtDNA introgression has been widely documented in several taxa and populations.

Figure 1

Map showing sampling sites of Ochthebius quadricollis and O. urbanelliae individuals. Light grey area: sympatric area between the two species (Urbanelli 2002). Photos show a specimen of O. quadricollis and a typical sea rock pool (Photo by Alessandra Spanò).

Figure 2

Chromatograms of the COI gene fragment showing two heteroplasmic positions. (A) O. quadricollis variant, (B) the sequence of the O. urbanelliae Cirella 3 heteroplasmic individual, (C) O. urbanelliae variant.

Figure 3

Phylogenetic relationships of the mtDNA haplotypes found in Ochthebius quadricollis and O. urbanelliae. Maximum likelihood (ML) tree is shown. Bootstrap values are shown above the main nodes. The haplotypes h17 and h34 (in bold) are shared between O. quadricollis and O. urbanelliae (Table 1); the haplotype h35 (in bold) was found in one O. urbanelliae individual (Table 1), but is more closely related to the O. quadricollis haplotypes.