Molecular Ecology (2005) 14, 3457–3470 doi: 10.1111/j.1365-294X.2005.02669.x © 2005 Blackwell Publishing Ltd Blackwell Publishing, Ltd. Phylogeographic patterns of Hawaiian Megalagrion damselflies (Odonata: Coenagrionidae) correlate with Pleistocene island boundaries STEVE JORDAN,* CHRIS SIMON,* DAVID FOOTE † and RONALD A. ENGLUND ‡ *Department of Ecology and Evolutionary Biology, Box U-3043, University of Connecticut, Storrs, CT 06269-3043, †Kilauea Field Station, Pacific Island Ecosystems Research Center, US Geological Survey, PO Box 44, Hawaii National Park, HI 96718, ‡Hawaii Biological Survey, Bishop Museum, 1525 Bernice Street, Honolulu, HI 96817 Abstract The Pleistocene geological history of the Hawaiian Islands is becoming well understood. Numerous predictions about the influence of this history on the genetic diversity of Hawaiian organisms have been made, including the idea that changing sea levels would lead to the genetic differentiation of populations isolated on individual volcanoes during high sea stands. Here, we analyse DNA sequence data from two closely related, endemic Hawaiian damselfly species in order to test these predictions, and generate novel insights into the effects of Pleistocene glaciation and climate change on island organisms. Megalagrion xanthomelas and Megalagrion pacificum are currently restricted to five islands, including three islands of the Maui Nui super-island complex (Molokai, Lanai, and Maui) that were connected during periods of Pleistocene glaciation, and Hawaii island, which has never been subdivided. Maui Nui and Hawaii are effectively a controlled, natural experiment on the genetic effects of Pleistocene sea level change. We confirm well-defined morphological species boundaries using data from the nuclear EF-1α gene and show that the species are reciprocally monophyletic. We perform phylogeographic analyses of 663 base pairs (bp) of cytochrome oxidase subunit II (COII) gene sequence data from 157 individuals representing 25 populations. Our results point to the importance of Pleistocene land bridges and historical island habitat availability in maintaining inter-island gene flow. We also propose that repeated bottlenecks on Maui Nui caused by sea level change and restricted habitat availability are likely responsible for low genetic diversity there. An island analogue to northern genetic purity and southern diversity is proposed, whereby islands with little suitable habitat exhibit genetic purity while islands with more exhibit genetic diversity. Keywords: phylogeography, Pleistocene, sea level change Received 27 February 2005; revision accepted 9 June 2005 Introduction Enormous advances have been made in deciphering the evolutionary and demographic processes that shaped continental biotas during the Pleistocene (e.g. Taberlet et al. 1998; Hewitt 2000; Good & Sullivan 2001; Johnson 2002). In contrast, the effects of Pleistocene climate change on island biogeography have received much less attention (but see Chown 1990; Ruedi et al . 1998; Buckley et al . 2001; Arensburger et al . 2004). This may be due to the lack of direct glacial effects on many of the world’s biotically rich islands. This subject merits more attention, however, because glacially mediated sea level variation during the Pleistocene was on the order of 120 m (Matthews 1990) and declines in sea level dramatically altered the size and shape of islands. In the Indonesian archipelago, for example, over 1.53 million km 2 of new land was available to terrestrial organisms at maximum glaciation [∼17 000 years ago (ka)] and details of the topography and river systems in this Correspondence: Steve Jordan, Present address: Department of Biology, Bucknell University, Lewisburg, PA 17837. Fax: +1 (570) 577–3537; E-mail: sdjordan@bucknell.edu