The leaf beetle genus Calligrapha is one of the few examples of animals with several obligate unisexual, female-only species. Previous work showed that each one arose independently from interspecific hybridization events involving different species. However, all of them clustered in a single mtDNA clade together with some individuals of the parental bisexual species, which appeared as deeply polyphyletic in the mtDNA genealogy of the genus. The dating of these splits using a molecular clock placed them in the Quaternary and it was hypothesized that climatic change during this period may have favored range expansions and secondary contacts required for hybridization. In this work, we test this hypothesis and the origins of unisexuality in Calligrapha examining the diversity of mitochondrial (cox1) and nuclear (wingless, Wg) genes and the Bayesian continuous mtDNA phylogeography of a sample of more than 500 specimens of two bisexual species of Calligrapha at a continental scale and two unisexual species derived from them. Besides a major topological difference, whereby each bisexual species is monophyletic for Wg but paraphyletic for cox1, both gene datasets are consistent with a minimum of seven evolutionary lineages, coherent with geography and consistent with an ordered expansion to occupy their current ranges. The results also imply their survival in well-established glacial refuges during the Last Glacial Maximum (LGM). Thus, for bisexual C. multipunctata there are two main, southern and northern lineages. The southern lineage expanded its range in two evolutionary branches, to the Rocky Mountains and to the northern Mississippi and Ohio River basins, respectively. The northern lineage has one branch in the Upper Mississippi, and one that expanded west to the Pacific Northwest and east to the northeastern North Atlantic, finding refuge in both areas. These major lineages are parapatric in the Northern Great Plains, an area consistent with them having found refuge in the so-called Driftless region during the LGM. For bisexual C. philadelphica, one northern lineage expanded west from the northern Appalachians and one east and southwest along the axis of the Appalachians, and the timing of events is consistent with their persistence in glacial refugia at both sides of the main Great Lakes lobe of the Laurentide Ice Sheet. There is evidence that the northeastern North Atlantic lineages of both species hybridized at the edge of their ranges after the LGM. The additional, divergent mtDNA lineage of these species shows evidence of range expansions of two lineages, one for each species, in an area south of the Laurentide Ice Sheet, and giving origin to the unisexual species by way of hybridization with other species in the Alleghanian region after the LGM. Interestingly, the individuals of supposedly bisexual species in this clade are all females. This means that unisexuality actually predates the origin of unisexual taxa in this system and that some bisexual species in Calligrapha may be species complexes instead, with cryptic species differing in their reproductive mode.
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