Elusive origin of mitochondria shared by two sister species of Eurema butterflies

Background Wolbachia are maternally inherited arthropod endosymbionts known for their diverse effects on host reproduction, which serve to increase their prevalence in host populations. As Wolbachia spreads, the frequency of the associated mitochondrial DNA (mtDNA) haplotypes tends to increase within the host population. Two distinct Wolbachia strains, w CI and w Fem, are shared by two pierid butterfly sister species: Eurema mandarina and Eurema hecabe . The congruence of mtDNA phylogeny and Wolbachia infection status suggests hybrid introgression of mtDNA from E. hecabe to E. mandarina . This inference is based on the observation that uninfected E. mandarina have unique mtDNA haplotypes, distinct from the mtDNA haplotypes found in Wolbachia -infected E. mandarina and E. hecabe . In E. hecabe , Wolbachia infection is considered fixed, with no expectation of uninfected individuals. Results Unexpectedly, Wolbachia- free E. hecabe individuals were discovered on the Yonaguni Island of Japan. We included these individuals in the phylogenetic analyses to reassess the impact of Wolbachia infection on Eurema butterflies. The nuclear Triosephosphate isomerase gene-based phylogenetic tree formed two discrete clades corresponding to E. mandarina and E. hecabe . Contrastingly, the mitochondrial c oxidase subunit I gene-based tree consisted of three clades, Wolbachia -free E. mandarina , Wolbachia -free E. hecabe , and a clade consisting of Wolbachia -infected E. mandarina and E. hecabe , as well as two other Eurema species, Eurema ada and Eurema lacteola . Conclusions Our findings indicate that the mitochondria shared by E. mandarina and E. hecabe originate from a different species of Eurema ( E. ada , E. lacteola , or others). Although the origin was not identified, our results indicate that Wolbachia provides significant evolutionary advantages to the associated mtDNA haplotypes across multiple Eurema species, leading to a complex mtDNA phylogeny.