Discussion
The strong genetic isolation among regions (overall fixation index for COI: 0.781) indicates an extremely low dispersal rate after regional colonization, similar to the situation in Japan (Miyakawa & Mikheyev, 2015). In the Korean populations, other than the island (region F), pairwise F ST values indicated more limited dispersal in the region E population than in the other populations; the S morph is found in this region, and its mating almost always occurs in the natal nest (Table 4) (Ohkawara, Ishii, Fukushima, Yamauchi, & Heinze, 2002). The haplotype network and pairwiseF ST results indicate that populations in region E (the Korean S morph population) are closely related to populations in region H (Japan) (Figure 1 and Table 4). In the COI phylogenetic tree, we detected a migration event(s) between region E and region H (Japan) after the S morph divaricated from the L morph (Figure 2). Populations in three regions (region A, C, and D) seem to be undergoing purifying selection, although mitochondrial DNA is a neutral marker (Table 5). This may be explained by the reproductive strategy and sib-mating behavior. Selfish clonal reproduction forms strong maternal nuclear-mitochondrial bonds in gynes, and sib-mating behavior enhances paternal nuclear-mitochondrial bondage in males, similar to linkage disequilibrium. Therefore, the signature of selection on a neutral marker may reflect selection for linked loci or non-randomly associated genotypes.
Founder effect and relaxed selection may result in the loss ofWolbachia infection in some invasive ant species while exploring new habitats (Bouwma, Ahrens, De Heer, & Shoemaker, 2006; Rey, Estoup, Facon, Loiseau, Aebi, Duron, Vavre, & Foucaud, 2013; Reuter, Pedersen, & Keller, 2005; Tsutsui, Kauppinen, Oyafuso, & Grosberg, 2003). Based on the phylogenetic tree of the COI haplotypes, Wolbachiainfection is evident in the ancestral L morph, but disappeared in the S morph (Figure 2). Unlike the invasive ants, the two morphs of V. emeryi are endemic in the region we investigated. The two proposed causes are not likely to lead the loss in our case.
Instead, we suggest a positive relationship between emergence of wing formation and evolution of resistance to Wolbachia infection in the ant. Epigenetic factors may be involved in formation of the wing pattern in this ant based on L gyne production from the S morph colonies (Noh, 2014; Noh, Park, Choe, & Jeong, 2018; Okamoto, Kobayashi, Hasegawa, & Ohkawara, 2015). If that is the case, it is possible that the gene(s) responsible for the S morph formation, and the gene(s) resistant to Wolbachia infection, exhibit epistatic interactions. It will be important to investigate such gene(s) to elucidate the prevalence of Wolbachia in insects from a mechanistic evolutionary perspective. Our divergence estimates indicate that the S morph and loss of infection are evolutionarily very recent events (Figure 2). However, when interpreting these divergence data, caution is necessary because Hymenopteran insects show lineage-specific variation with respect to mitochondrial evolution (Dowton, Cameron, Austin, & Whiting, 2009). The Wolbachia bacterium is known for its manipulative effects on host reproduction (Fujii, Kubo, Ishikawa, & Sasaki, 2004; Jeong, & Suh, 2008; Stouthamer, Breeuwer, & Hurst, 1999). In particular, induction of parthenogenesis is similar to queen development in the ant V. emeryi . In the ant species,Wolbachia may not contribute to clonal production of the queen caste, since it takes place in both morphs, as is the case in W. auropunctata (Rey, Estoup, Facon, Loiseau, Aebi, Duron, Vavre, & Foucaud, 2013).
In conclusion, all L morphs, the predominant ancestral form, were infected with Wolbachia , while the rare derived S morphs were free of Wolbachia, at least in Korean populations, and were partially infected in Japanese populations in parallel with the potential evolution of Wolbachia infection resistance. To the best of our knowledge, this is the first report of an association between Wolbachia infection and host morphological characteristics.