Historical specimens reveal past relationships and current conservation status of populations in a declining species: the regal fritillary butterfly NUSHA KEYGHOBADI, 1 DARIA KOSCINSKI, 1 JASON D. WEINTRAUB 2 and DINA M. FONSECA 3 1 Department of Biology, Western University, London, ON, Canada, 2 Department of Entomology, Academy of Natural Sciences, Philadelphia, PA, USA and 3 Department of Entomology, Rutgers University, New Brunswick, NJ, USA Abstract. 1. The regal fritillary butterfly, Speyeria idalia Drury 1773, was once widespread across eastern North America, but has declined significantly and rapidly over the past half-century. Although more stable in the western portion of its range, only two populations survive east of the Great Lakes, one in eastern Pennsylvania and the other in Virginia. 2. Previous studies have found that the remnant Pennsylvania population is geneti- cally differentiated from populations in the west, and have suggested the designation of separate eastern and western subspecies. However, the historical pattern of genetic variation from which the current distinctness of the Pennsylvania population has arisen was not known, nor was the relationship with the remnant Virginia population. 3. We amplified and sequenced two mitochondrial loci (COI ⁄ II and ND4) from preserved specimens to infer historical patterns of genetic variation in this species, and we used non-lethally obtained tissue samples to assess the relationship of the two eastern remnant populations. 4. We found very consistent patterns between the two loci. Both had a very shal- low haplotype network with few mutations separating most haplotypes. At both loci, we observed distinct groups of haplotypes in the western and far eastern (i.e. New England) portions of the range; a region of transition was centred on Ohio, western Pennsylvania, and the Virginias, where both groups, and intermediate haplotypes, were represented. 5. Importantly, the extant Virginia population shared haplotypes with western populations of S. idalia and not with the extant Pennsylvania population. We discuss the implications of this result for the taxonomy and translocations ⁄ introductions of the species. Key words. Ancient DNA, conservation genetics, mtDNA, phylogeography, re- introduction, Speyeria idalia, translocation. Introduction Assessing the distinctness of populations or groups of popula- tions within a species is often a critical step in conservation plan- ning (Green, 2005). Such distinct units, for which various definitions and assessment criteria have been proposed (Moritz, 1994; Vogler & Desalle, 1994; Waples, 1995; Paetkau, 1999), form the basis for deciding where and how conservation efforts will be expended. Understanding the relationships among sub- specific units is also particularly important for specific conserva- tion activities such as the establishment of movement corridors, translocations, or re-introductions into formerly occupied habi- tat (Moritz, 1999; Storfer, 1999). Regardless of the definition or criteria chosen to define subspecific units for conservation, appropriate and informed Correspondence: Nusha Keyghobadi, Department of Biology, Western University, 1151 Richmond St., London, ON N6A 5B7, Canada. E-mail: nkeyghob@uwo.ca Insect Conservation and Diversity (2013) 6, 234–242 doi: 10.1111/j.1752-4598.2012.00208.x Ó 2012 The Authors 234 Insect Conservation and Diversity Ó 2012 The Royal Entomological Society