ORIGINAL ARTICLE Comparative historical biogeography of Plateumaris leaf beetles (Coleoptera: Chrysomelidae) in Japan: interplay between fossil and molecular data Teiji Sota 1 * and Masakazu Hayashi 2 INTRODUCTION DNA sequence data provide a good opportunity to explore the past biogeographical events of organisms (Avise, 2000). Additionally, although usually far from abundant for most organisms, fossil records play an essential role in the dating of molecular phylogenies and in validating various inferences from molecular data (e.g. Page & Holmes, 1998; Kidwell & 1 Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606- 8502, Japan and 2 Hoshizaki Green Foundation, Izumo 691-0076, Japan *Correspondence: Teiji Sota, Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan. E-mail: sota@terra.zool.kyoto-u.ac.jp ABSTRACT Aim In an attempt to use molecular and fossil data interactively in historical biogeography, we studied the phylogeography of five Plateumaris leaf beetles in Japan using mitochondrial cytochrome oxidase subunit I (COI) sequence data to explore interspecific differences in phylogeographical patterns and estimate the timings of colonization and geographical differentiation. Location A total of 461 beetles from five species on Hokkaido, Honshu and Kyushu islands of Japan were analysed with 117 beetles from three conspecies and two congeners from the mainland (Russia, including Sakhalin; Korea; Mongolia; Belgium; France; hereafter, the continent). Methods Using the sequence data from a 750-bp portion of the COI gene, we studied the phylogeny of COI haplotypes, intraspecific population differentiation using analysis of molecular variance and the Mantel test, and intraspecific phylogeography using nested clade analysis. In addition, divergence times between the continental and Japanese lineages, as well as among the various Japanese lineages, were estimated using a Bayesian approach with node constraints based on fossil records of extant species. Results Three widely distributed species showed different degrees of geographical differentiation corresponding to their different colonization history in Japan. Bayesian estimates of divergence time revealed that one of two endemic species, which originated before the late Pliocene, attained intraspecific differentiation through the Pliocene and Pleistocene, whereas another endemic species has been confined in one locality, and three non- endemic species colonized Japan after the mid-Pleistocene. Main conclusions Molecular analyses of an insect group with relatively abundant fossil data can contribute greatly to the understanding of diverse biogeographical histories of related species in a region. Bayesian estimates of divergence time could be used to assess the variable evolutionary rates of the COI gene, and may be applied to other related insect species. Keywords Cytochrome oxidase, divergence time estimation, donaciine beetles, Japan, molecular clock, nested clade analysis, phylogeography, wetland. Journal of Biogeography (J. Biogeogr.) (2007) 34, 977–993 ª 2007 The Authors www.blackwellpublishing.com/jbi 977 Journal compilation ª 2007 Blackwell Publishing Ltd doi:10.1111/j.1365-2699.2006.01672.x