Conserv Genet (2015) 16:703–716 DOI 10.1007/s10592-014-0694-9 RESEARCH ARTICLE The phylogeographic history of the threatened Diana fritillary, Speyeria diana (Lepidoptera: Nymphalidae): with implications for conservation Carrie N. Wells • Peter B. Marko • David W. Tonkyn Received: 2 April 2014 / Accepted: 30 December 2014 / Published online: 20 January 2015 Ó Springer Science+Business Media Dordrecht 2015 Abstract The Diana fritillary, Speyeria diana (Cramer but, rather, appear to have vanished. Our results highlight 1777) (Lepidoptera: Nymphalidae), is a North American the value of incorporating genetic data from preserved endemic butterfly that disappeared from low elevation sites specimens when investigating the phylogeographic history throughout its range in the twentieth century. It now per- and conservation status of a threatened species. sists in two geographically isolated mountainous regions, with an 800 km disjunction. Using mitochondrial cyto- Keywords Genetic differentiation Á Gene flow Á Range chrome oxidase II DNA sequences from museum and field- collapse Á Historical specimens Á Climate change Á Butterfly sampled specimens, we found greater mtDNA diversity and more widespread differentiation among eastern popu- lations than western ones. In addition, using coalescent- Introduction based population divergence models we dated the earliest splitting of eastern and western populations at least Habitat connectivity is important for maintaining genetic 20,000 years ago, during the Last Glacial Maximum. variation in natural populations. Restrictions in range and Therefore, the recent range collapse across the center of the fragmentation of habitat can lead to an overall decrease in historical species distribution may have exacerbated an available habitat and reductions in population size for the ancient genetic differentiation between eastern and western species. The ecological and evolutionary consequences of populations. Finally, the loss of lowland haplotypes and the habitat fragmentation are typically most pronounced in relatively large variation among local populations suggests small populations, which are expected to hold less genetic that dispersal is low and lowland populations did not move variation than the system as a whole (Broquet et al. 2010). to higher elevations, perhaps in response to climate change The impacts of habitat fragmentation will also depend on how much migration is maintained among remaining pat- ches. When populations are both small and isolated, they Electronic supplementary material The online version of this become even more vulnerable to extinction from demo- article (doi:10.1007/s10592-014-0694-9) contains supplementary graphic and genetic processes, including a reduced ability material, which is available to authorized users. to respond evolutionarily to random and directional chan- C. N. Wells (&) ges in the environment (Frankham et al. 2002). Department of Biological Sciences, University of North Carolina Habitat loss and population fragmentation have caused at Charlotte, 235E Woodward Hall, Charlotte, NC 28213, USA several North American butterfly species from the fritillary e-mail: [email protected] genus Speyeria to become threatened with extinction over P. B. Marko the past 200 years (Hammond and McCorkle 1983; Wil- Department of Biology, University of Hawaii, 2538 McCarthy liams 2002; Cech and Tudor 2005). The Diana fritillary, Mall, Honolulu, HI 96822, USA Speyeria diana (Cramer), is an example, having disap- peared from large portions of its former distribution. His- D. W. Tonkyn Department of Biological Sciences, Clemson University, torically, this species was distributed across the 132 Long Hall, Clemson, SC 29634, USA southeastern US, ranging from coastal Virginia, up to the 123 704 Conserv Genet (2015) 16:703–716 Ohio River Valley, and west to Arkansas and Missouri structure in the threatened Diana fritillary, and to use those (Opler and Krizek 1984; Moran and Baldridge 2002). Over patterns to reconstruct the demographic history of this the past century, S. diana populations have disappeared species using coalescent methods. Given that S. diana was entirely from the Ohio River Valley, and coastal habitat in continuously distributed across the southeastern US a little North Carolina and Virginia (Cech and Tudor 2005). The over a century ago, we ask if there is measurable genetic result is a geographic disjunction of close to 800 km differentiation across the present range and, if so, did it between remaining eastern populations in the southern arise concurrent with the recent range collapse (Wells and Appalachian Mountains from Georgia to Virginia, and Tonkyn 2014). We present our interpretation of these western populations in the Ozark and Ouachita Mountains population genetic data, and discuss the implications for of Arkansas and Oklahoma (Wells and Tonkyn 2014) conservation practices of this threatened fritillary species. (Fig. 1). Studies based solely on mtDNA may not accurately Nothing is known about the genetic consequences of represent the entire phylogeographic history of populations recent population extinctions across most of the range of S. and species, yet they remain a useful first step in phylog- diana, or the phylogeographic background against which eographic analyses, particularly for studies involving these extinctions have occurred. To address this, we char- ancient DNA or poorly preserved museum specimens acterized the population genetic structure of S. diana (Avise 2000). Our study involved extracting DNA from a throughout its range by sequencing a 549-bp segment of number of old, often degraded, museum specimens, and the cytochrome oxidase II (COII) mitochondrial gene from because the successful amplification of full-length DNA field-sampled and museum S. diana specimens collected sequences from dried and pinned insect specimens pre- over the past century. The purpose of our study is to served over 10 years is generally lower than that of fresh describe patterns of genetic variation and population samples (Hajibabaei et al. 2006), we relied on mtDNA data Fig. 1 Distributional data for Speyeria diana specimens documented circles represent specimens collected from 1777 to 1960 (N = 881); in Wells and Tonkyn (2014) showing the two geographically black circles represent S. diana specimens collected from 1961 to separated population groups: the southern Appalachian Mountains 2010 (N = 2,517) (see Wells and Tonkyn 2014; for a complete in the east, and the Ozark and Ouachita Mountains in the west. Open description of these data) 123 Conserv Genet (2015) 16:703–716 705 Fig. 2 Collection sites for Speyeria diana.N= sample size for 2) (elev.: 889 m), Arkansas-3 (AR-3) (elev.: 740 m), and Oklahoma private haplotypes. Eastern samples were taken from Georgia (GA) (OK) (elev.: 724 m); Museum samples were collected to represent (elevation: 996 m), South Carolina (SC) (elev.: 914 m), Tennessee-1 extirpated populations (indicated with dagger) from Virginia-2 (VA- (TN-1) (elev.: 1,304 m), Tennessee-2 (TN-2) (elev.: 1,450 m), 2) (elev.: 50 m), Indiana (IN) 121 m, and Ohio (OH) (elev.: 172 m). Tennessee-3 (TN-3) (elev.: 540 m), North Carolina (NC) (elev.: See Table 1 for sources of all museum samples. The size of each pie 914 m), and Virginia-1 (VA-1) (elev.: 1,150 m); Western samples graph, indicating haplotype distribution, represents the sample size of were taken from Arkansas-1 (AR-1) (elev.: 775 m), Arkansas-2 (AR- each population to produce reliable sequence data for our analysis. While representing the western range. Adult butterflies were cap- the addition of additional unlinked markers would greatly tured with a handheld net and non-lethally sampled by improve the ability to infer the population history, we removal of a single posterior tarsus. Tarsi were preserved in could not amplify other loci described for the related S. 95 % ethanol and stored at -20 °C. idalia (Williams et al. 2003), and consider a mitochondrial locus to be an appropriate marker for our phylogeographic Museum specimens study. Samples from natural history museums and other collections were obtained to represent extirpated portions of the species’ Methods former distribution, including coastal Virginia (1900–1920), Indiana (1928–1934), and Ohio (1911–1930), all popula- Field sampling tions that were extirpated by the 1950s (Table 1). Specimens (stored under a variety of conditions) were dried and pinned During the summers of 2006–2009, we sampled 11 S. diana prior to removal of a single rear tarsus for DNA extraction. populations across the species’ entire current range (Fig. 2). These include seven populations from the southern Appa- DNA extraction lachian Mountains, from Georgia to Virginia, representing the eastern range and four populations, including the Ozark The DNeasy kit (Qiagen, Inc., Valencia, CA) was used to and Ouachita Mountains of Arkansas and Oklahoma, and extract DNA from fresh samples of S. diana, from Mount Magazine, the highest point in Arkansas (839 m), approximately half of each tarsal segment. Each sample 123 706 Conserv Genet (2015) 16:703–716 Table 1 Mitochondrial DNA population diversity indices for S. diana populations from fresh and museum specimens Population County/region NPCOII haplotypes (count) Source of mtDNA Georgia (GA) Fannin/Blue Ridge Mountains 13 2 H2 (10), H6 (3) Fresh 2006–2008 North Carolina (NC) Transylvania/ Blue Ridge Mountains 18 5 H2 (12), H9, H10 (3), H11 (2) H12 (2) Fresh 2006–2009 South Carolina (SC) Greenville/Blue Ridge Escarpment 11 3