Mitochondrial DNA Indicates Late Pleistocene Divergence of Populations of Heteronympha merope, an Emerging Model in Environmental Change Biology Melanie Norgate 1. , Jay Chamings 1. , Alexandra Pavlova 1 , James K. Bull 1 , Neil D. Murray 2 , Paul Sunnucks 1 * 1 School of Biological Sciences and Australian Centre for Biodiversity, Monash University, Clayton, Victoria, Australia, 2 Department of Genetics, La Trobe University, Bundoora, Victoria, Australia Abstract Knowledge of historical changes in species range distribution provides context for investigating adaptive potential and dispersal ability. This is valuable for predicting the potential impact of environmental change on species of interest. Butterflies are one of the most important taxa for studying such impacts, and Heteronympha merope has the potential to provide a particularly valuable model, in part due to the existence of historical data on morphological traits and glycolytic enzyme variation. This study investigates the population genetic structure and phylogeography of H. merope, comparing the relative resolution achieved through partial DNA sequences of two mitochondrial loci, COI and ND5. These data are used to define the relationship between subspecies, showing that the subspecies are reciprocally monophyletic. On this basis, the Western Australian subspecies H. m. duboulayi is genetically distinct from the two eastern subspecies. Throughout the eastern part of the range, levels of migration and the timing of key population splits of potential relevance to climatic adaptation are estimated and indicate Late Pleistocene divergence both of the Tasmanian subspecies and of an isolated northern population from the eastern mainland subspecies H. m. merope. This information is then used to revisit historical data and provides support for the importance of clinal variation in wing characters, as well as evidence for selective pressure acting on allozyme loci phosphoglucose isomerase and phosphoglucomutase in H. merope. The study has thus confirmed the value of H. merope as a model organism for measuring responses to environmental change, offering the opportunity to focus on isolated populations, as well as a latitudinal gradient, and to use historical changes to test the accuracy of predictions for the future. Citation: Norgate M, Chamings J, Pavlova A, Bull JK, Murray ND, et al. (2009) Mitochondrial DNA Indicates Late Pleistocene Divergence of Populations of Heteronympha merope, an Emerging Model in Environmental Change Biology. PLoS ONE 4(11): e7950. doi:10.1371/journal.pone.0007950 Editor: Amit Singh, University of Dayton, United States of America Received September 1, 2009; Accepted October 28, 2009; Published November 24, 2009 Copyright: ß 2009 Norgate et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Australian Research Council grant DP0772837 (http://www.arc.gov.au/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: paul.sunnucks@sci.monash.edu.au . These authors contributed equally to this work. Introduction Butterflies are one of the most important taxa for studying the effects on species of environmental change due to their sensitivity to temperature and their history of extensive population monitoring [1]. The study of butterflies has provided convincing evidence of the impact of forces such as climate change on range distribution and extinction risk [2,3]. Since many butterflies are relatively convenient research organisms, they have also been used for several key studies that identify proximal factors to which organisms respond under environmental change [4,5]. The Australian endemic butterfly, Heteronympha merope, is an exceptional study organism for this purpose because research in the late 1970s provided historical records of wing morphology and allozyme allele variation over the entire species range from field sites located near weather stations [6,7]. Heteronympha merope is common and widespread over 20 degrees latitude (Figure 1) and has several congenerics of limited habitat tolerance, including some of conservation concern [8]. Isolated populations at the extremes of the wide latitudinal distribution and three geographically isolated subspecies (Figure 1) provide useful comparisons within the species. Heteronympha merope thus offers the potential to investigate adaptive responses to environmental change across an environmental gradient as well as in the context of barriers to dispersal. Investigation of H. merope wing morphology over its entire range in the late 1970s identified a north-south cline associated with winter humidity and an east-west cline associated with yearly rainfall [7]. Indirect evidence suggests that these patterns are the result of natural selection rather than stochastic processes (e.g. the combination of characters contributing to each cline are independently inherited), but it remains possible that factors such as isolation by distance contribute substantially to the clinal variation. Knowledge of the population genetic structure would resolve this issue by providing information on gene flow throughout the range as a point of comparison with traits that may be under selective pressure. The only available information on H. merope population genetic structure comes from three allozymes [6]. These allozymes display fairly uniform frequencies throughout the contiguous distribution PLoS ONE | www.plosone.org 1 November 2009 | Volume 4 | Issue 11 | e7950