Assessment of the role of Wolbachia in mtDNA paraphyly and the evolution of unisexuality in Calligrapha (Coleoptera: Chrysomelidae)

Each unisexual species of Calligrapha has an independent hybrid origin, but all cluster in a single mtDNA clade with some individuals of parental bisexual species, deeply polyphyletic for mtDNA. This pattern was suggestive of a selective sweep which, together with mtDNA taxonomic incongruence and the evolution of unisexuality in Calligrapha, led to hypothesize that Wolbachia might be responsible. I tested this hypothesis studying the correlation between diversity of Wolbachia and well-established mtDNA lineages in a sample of >500 specimens of two species of Calligrapha at a continental scale and two derived unisexual species. Wolbachia is highly prevalent (83.4%) in the sample and fifteen new supergroup-A strains of the bacteria were characterized, belonging to three main classes. wCallA occupies the whole species ranges, while wCallB and wCallC are narrowly parapatric, with 71.6% of beetles carrying double infections of wCallA with another sequence class. When wCallB and wCallC are sympatric, they infect beetles with highly divergent mtDNAs. Association tests and Bayesian inference of ancestral character states of bacterial diversity on the mtDNA genealogy show that the main mtDNA lineages of Calligrapha have specific types of infection, but they shift according to expectations based on cytoplasmic incompatibility between wCallB and wCallC types, suggesting that the symbionts hitchhike with the host and are not responsible for selective mtDNA sweeps. The latter and the fact that individuals in the unisexual clade are uninfected or infected by the widespread wCallA type indicate that Wolbachia does not manipulate reproduction, hence dissociating from unisexuality in Calligrapha.