Journal of Biogeography (J. Biogeogr.) (2005) 32, 1943–1956

ORIGINAL Historical biogeography of two alpine ARTICLE butterflies in the : broad-scale concordance and local-scale discordance Eric G. DeChaine1* and Andrew P. Martin2

1Department of Organismic and Evolutionary ABSTRACT Biology, Harvard University, Cambridge, MA, Aim We inferred the phylogeography of the alpine butterfly meadii USA and 2Department of Ecology and Evolutionary Biology, University of Colorado, Edwards () and compared its genetic structure with that of another Boulder, CO, USA high elevation, co-distributed butterfly, smintheus Doubleday (Papilionidae), to test if the two Rocky Mountain butterflies responded similarly to the palaeoclimatic cycles of the Quaternary. Location Specimens were collected from 18 alpine sites in the Rocky Mountains of , from southern Colorado to northern Montana. Methods We sequenced 867 and 789 nucleotides of cytochrome oxidase I from an average of 19 and 20 individuals for C. meadii and P. smintheus, respectively, from each of the same 18 localities. From the sequence data, we calculated measures of genetic diversity within each population (H, h), genetic divergence

among populations (FST), and tested for geographic structure through an analysis of molecular variance (amova). Population estimates were compared against latitude and between species using a variety of statistical tests. Furthermore, nested clade analysis was implemented to infer historic events underlying the geographic distribution of genetic variation in each species. Then, we compared the number of inferred population events between species using a nonparametric Spearman’s rank correlation test. Finally, we ran coalescent simulations on each species’ genealogy to test whether the two species of fit the same model of population divergence. Results Our analyses revealed that: (1) measures of within-population diversity were not correlated with latitude for either species, (2) within-site diversity was not correlated between species, (3) within a species, nearly all populations were genetically isolated, (4) both species exhibited significant and nearly identical partitioning of genetic variation at all hierarchical levels of the amova, including a strong break between populations across the Wyoming Basin, (5) both species experienced similar cycles of expansion and contraction, although fewer were inferred for C. meadii, and (6) data from both species fit a model of three refugia diverging during the Pleistocene. Main conclusions While our findings supported a shared response of the two butterfly species to historic climate change across coarse spatial scales, a common pattern was not evident at finer spatial and temporal scales. The shared demographic history of the two species is consistent with an expanding– *Correspondence: Eric G. DeChaine, contracting archipelago model, suggesting that populations persisted across the Department of Organismic and Evolutionary geographic range throughout the climate cycles, experiencing isolation on ‘sky Biology, Harvard University, 16 Divinity Ave, Biolabs 4081, Cambridge, MA 02138, USA. islands’ during interglacial periods and becoming connected as they migrated E-mail: [email protected] down-slope during cool, wet climates.

ª 2005 Blackwell Publishing Ltd www.blackwellpublishing.com/jbi doi:10.1111/j.1365-2699.2005.01356.x 1943 E. G. DeChaine and A. P. Martin

Keywords Alpine , coalescent simulations, Colias meadii, Lepidoptera, Parnassius smintheus, Pleistocene climate cycles, Rocky Mountains, statistical phylogeog- raphy, U.S.A.

conditions across the shifting landscape (e.g. West, 1980; INTRODUCTION Coope, 1995; Johansen & Latta, 2003). During unfavourable Quaternary climate oscillations played a central role in periods, species’ distributions contracted and populations were determining the distribution of species and defining the forced to persist in a few fragmented refugia, comprising only a context for evolution (Huntley & Webb, 1989; Webb & fraction of the species’ previous range (e.g. Betancourt et al., Bartlein, 1992; Hewitt, 1996, 2000). Alternation between colder 1990). This oscillation of climatic conditions over the course of glacial periods and warmer interglacial periods (Fig. 1a) has thousands of years has promoted the cyclic expansion and created a temporal and geographic mosaic of continental ice contraction of species ranges in evolutionary time (Avise, 2000; sheets, mountain glaciers, and available habitat (Richmond, Hewitt, 2000). 1965; Winograd et al., 1997). In response to palaeoclimatic Periods of favourable climate that spurred range expansion fluctuations, species’ geographic and elevation ranges have differed among taxa depending on a species’ niche require- tracked preferred habitat and suitable environmental ments. For temperate taxa inhabiting low- to mid-elevation

(a) Palaeoclimatic record (b) Populations 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

50 T1

100 T2

150

200 T3 250

300 (c) Biogeographic regions T4 North Central South 350 T1 400 T5 Time (1000 years bp) Time 450 (d) 500 T6

550 T6

600 -3 -2 -1 0 1 2 3 4 δ18 O Interglacial Glacial

Figure 1 Hypotheses of population divergence throughout the Quaternary climate cycles. (a) The palaeoclimatic record over the last 600,000 years based on benthonic foraminifera data from SPECMAP (Imbrie et al., 1989) and Devil’s Hole in Nevada, U.S.A. (Landwehr et al., 1997). The timeline is given along the y-axis in thousands of years before present (bp). Delta 18O is given in standard deviations and serves as a proxy for past climates (low ¼ cooler and high ¼ warmer). Interglacial and glacial periods are delineated by grey and white bands, respectively. The interglacials were used to estimate divergence times (T1 through T6) in the phylogeographic models. For the models of population divergence (b, c, d), gene trees (shown in black) are constrained within the grey population trees. The null model of a single population experiencing fragmentation is shown in part b, where each population corresponds to one of the study sites (see Table 1 for populations details). The 3-refugia model, wherein the three regional populations have been determined through biogeographic and phylogeographic analyses, is shown for two of the seven different divergence time scenarios in part c and d.

1944 Journal of Biogeography 32, 1943–1956, ª 2005 Blackwell Publishing Ltd Historical biogeography of Rocky Mountain butterflies environments, distributions are larger now, in the current (400,000 yr bp) and establishes a baseline for predicting the interglacial, than during previous glacial periods when species effects of future climate change. were restricted to a few, distant refugia of suitable habitat (Webb & Bartlein, 1992; Comes & Kadereit, 1998; Ayoub & MATERIALS AND METHODS Riechert, 2004; Dobes et al., 2004). By contrast, glacial periods probably connected the alpine tundra and permitted broader Mead’s Sulphur, Colias meadii, inhabits alpine tundra and distributions of high elevation species, while warm interglacials subalpine steppe of the Rocky Mountains from southern caused the fragmentation of habitat and upslope contraction of Colorado, where the effects of interglacial habitat fragmenta- species’ ranges into ‘sky islands’ (Elias, 1996; Hewitt, 2000). tion are most severe, to northern Montana (Fig. 2a; Scott, Because species responded individually to historic climate 1986; Nabokov, 2000), which was completely covered by the changes, the detection of general phylogeographic patterns has Cordilleran and Laurentide ice sheets (Richmond, 1965). proven difficult, even within a habitat-type (Delcourt & Colias meadii is not known from the Rockies of mid-Montana, Delcourt, 1991; FAUNMAP Working Group et al., 1996; nor in the Wyoming Basin (Ferris & Brown, 1981; Opler et al., Comes & Kadereit, 1998; Taberlet et al., 1998). 1995), which, due to its low elevation and xeric habitat, acts as Alpine species could have persisted across an extensive a significant barrier to dispersal of some high elevation latitudinal range of the Rocky Mountain cordillera throughout (Noonan, 2001; DeChaine & Martin, 2004). P. smintheus, the several glacial–interglacial climate cycles by shifting elevation. Rocky Mountain , shares the range of C. meadii, but is The alpine habitat in the Rocky Mountains is highly dissected more broadly distributed (Scott, 1986). Both species of and widely-dispersed, comprising an archipelago of high Lepidoptera are strong flyers, but exhibit short-distance elevation sky islands (Ku¨chler, 1985; Fig. 2a). Many popula- dispersal as revealed through population analyses using genetic tions of alpine and plants are isolated on mountain markers (Johnson, 1977; Watt et al., 1996; Keyghobadi et al., tops in the current interglacial and probably exhibit different 1999; DeChaine & Martin, 2004). demographic characteristics (Golden & Bain, 2000; Knowles, 2000, 2001; Masta, 2000; DeChaine & Martin, 2004, 2005). In Specimen collection contrast, the Rocky Mountain alpine zone was relatively contiguous during glacial periods, except for a major break Specimens of C. meadii and P. smintheus were collected from associated with the Wyoming Basin (Pewe, 1983; Elias, 1996; eighteen sites throughout their range (Fig. 3a). The number Fig. 2b). Interconnection during the glacial periods could have of sampling locations within a region (north, south, and facilitated genetic and demographic cohesion within a geo- central Rockies; Brouillet & Whetstone, 1993) was propor- graphic region. tional to the estimated relative abundance of populations of How did climate cycles of the Quaternary affect the C. meadii within that region (Fig. 2a). Seven of the sites were demographic histories of wide-ranging, high elevation species within national parks: Glacier National Park (permit no. in the Rocky Mountains? And, did species respond similarly or GLAC-2001-SCI-0020), Yellowstone National Park (permit independently to the glacial cycles? If different species shared a no. YELL-2001-SCI-0212), Grand Teton National Park (permit common demographic response to palaeoclimatic cycles, then no. GRTE-2001-SCI-0009), and Rocky Mountain National geographic partitioning of genetic variation and historic Park (permit no. ROMO-2001-SCI-0037), while the remain- inferences of population divergence will be correlated between ing eleven sampling locations were on public lands. From the species. To address these questions, we inferred the each population, ten to twenty-nine individuals were cap- historical biogeography of the alpine butterfly Colias meadii tured with a hand-net, stored in glassine envelopes, transported Edwards (Pieridae) throughout its range in the Rocky Moun- on dry ice and stored at )80 C at the University of tains using mtDNA and compared the results from C. meadii Colorado, Boulder. with data from a co-distributed species, Parnassius smintheus Doubleday (Papilionidae; DeChaine & Martin, 2004) and with Molecular techniques patterns described for other species (Britten & Brussard, 1992; Noonan, 1992, 1999; Reiss et al., 1999; Nice & Shapiro, 2001). The cytochrome oxidase I (COI) gene from the mitochondrial Moreover, we tested specific models of Pleistocene divergence genome was employed for comparative population genetic for both species, based on the biogeography of the Rocky analyses because of its rapid rate of evolution and its wide use in Mountains and previous inferences for P. smintheus (DeChaine studies of phylogeography (Caterino et al., 2000). & Martin, 2004): a null hypothesis of fragmentation in a single, Following DNA extraction from thorax tissue using DNeasy widespread ancestral population and a 3-refugia model Tissue Extraction Kits (Qiagen, Valencia, CA, USA), we (partitioned into the northern, central, and southern Rockies), amplified and sequenced a portion of COI, using specific with possible divergence times ranging from 50,000 (most primers. The C. meadii primers were: CmF: (5¢-GAG- recent warm period) to 1,700,000 (pre-Pleistocene) yr bp TATCGTCGAGGTATTCC-3¢) and CmR: (5¢-GCAGGAA- (Fig. 1). Our comparative approach yielded support for a CTGGATGAACAG-3¢). Products from PCR were cleaned general model of how high elevation organisms have respon- using the Wizard PCR Preps (Promega; Madison, WI, USA). ded to climate change over the past four glacial cycles Sequencing products were generated from the PCR amplicons

Journal of Biogeography 32, 1943–1956, ª 2005 Blackwell Publishing Ltd 1945 E. G. DeChaine and A. P. Martin

(a) Interglacial 49 N MT

47 Latitude (°N)

45 WY C

43 ID W 41 CO

S 39

UT 37

Figure 2 The distribution of alpine habitat (b) Glacial in the Rocky Mountains during interglacial 49 and glacial periods and the known distribu- MT tion of Colias meadii Edwards. The grey shaded regions represent the distribution of 47 alpine habitat during (a) an interglacial, using the current distribution of alpine hab- Latitude (°N) itat (from Ku¨chler, 1985) and (b) a glacial period, given a 1000 m drop in elevation 45 WY (Pewe, 1983). The Wyoming Basin (W) is labelled. Elevation profiles for each period are on the right. The transect for the elevation 43 profiles basically follows the Continental ID Divide from the San Juan Range in southern W Colorado to Glacier National Park in Mon- 41 tana, but incorporates study sites and other CO locations that emphasize changes in eleva- tion. Latitude and elevation data were obtained from topographic atlases of the 39 region (DeLorme, 1998a–d). In part a, the known distribution of C. meadii (Ferris & UT Brown, 1981; Opler et al., 1995) is shaded in

37 1000 1500 2000 3000 4000 2500 3500 4500 black over the current alpine distribution and Distribution of Colias meadii the northern (N), central (C) and southern Distribution of alpine hatitat Elevation (m) (S) regions are labelled. with a Thermo Sequenase DYEnamic Direct Cycle Sequencing AlignIR v2.0 software (Li-Cor, Inc.) and aligned with Clustal X Kit with 7-deaza-dGTP (Amersham Biosciences, Piscataway, (Thompson et al., 1997; Strasbourg, France). A total of 867 NJ, USA) and nested primers. The nested primers for C. meadii and 789 nucleotides of COI were sequenced from an average were CmNF: (5¢-AACGGAGCAGGAACAGGATG-3¢) and of 19 and 20 individuals for C. meadii and P. smintheus CmNR: (5¢-GGGTAATCTGAATATCGACG-3¢). Forward (DeChaine & Martin, 2004), respectively, from 18 alpine and reverse strands were sequenced on a Li-Cor 4200 automa- populations. ted sequencer (Li-Cor, Inc, Lincoln, NE, USA). Reactions for PCR, cycle sequencing, and thermal cycler profiles, along Population genetic analyses with the molecular methods used to collect mitochondrial sequence data for P. smintheus, are as previously described Estimates of within and among population genetic variation (DeChaine & Martin, 2004). Sequences were checked on were calculated for C. meadii and P. smintheus and then

1946 Journal of Biogeography 32, 1943–1956, ª 2005 Blackwell Publishing Ltd Historical biogeography of Rocky Mountain butterflies

(a) Colias meadii (b) Parnassius smintheus Private Private Alleles Alleles 1 2 MT12 MT G1 CG1 3 G5 3 CG3 Y1 CY1 Y4 CY2 Y7 CY14 Y8 CY15 CC2 R4 6 5 6 CC5 5 R5 4 WY4 WY 7 CC8 7 R6 8 CC9 8 R7 CC15 C1 ID CC16 ID C2 CC17 C4 CC27 C6 C7 9 CO9 CO C12 10 10 C13 12 11 12 11 13 13 C21 14 14 C22 C23 15 17 15 17 C28 16 16 18 18 C29 UT UT C31

Figure 3 Collection sites and haplotype distributions. Sample sites are shown as circles and population numbers correspond to those in Table 1. (a) Colias meadii Edwards. (b) Parnassius smintheus Doubleday (modified from DeChaine & Martin, 2004). Pie charts show the proportions of haplotypes within each population. The distributions of wide-ranging haplotypes are denoted by colour, while private alleles are white. Note that C. meadii and P. smintheus do not share any haplotypes regardless of overlap in colours between the two species. See Table 1 for the frequency of private alleles within each population.

contrasted between the two species to test for evidence of to an analysis of molecular variance (amova) with three similarities in demographic history. For each population, hierarchical levels: among regions, among populations within haplotype diversity (H) and three measures of h, a genealogical each region, and within populations. For the amova, regions estimate determined with the fluctuate 1.4 software package were defined as the north (populations 1–3), central (popu- (Kuhner et al., 1995), and two others based on the average lations 4–8) and southern (populations 9–18) Rocky Mountain within-population pair-wise sequence divergence (p) and the physiographic provinces, following Brouillet & Whetstone number of segregating sites (S) were calculated on arlequin (1993). All calculations were performed using arlequin v.2.0 v.2.0 (Schneider et al., 2000). All measures of genetic diversity, (Schneider et al., 2000). H and h, were regressed against latitude and significance was assessed using a Mantel test. To test for similarities between Historical inferences based on nested clade analysis species at the population level, values of H from the two species were compared using a Wilcoxon sign rank test and Population processes underlying the geographic distribution of values of h were contrasted using a t-test following log- genetic variation in both species were inferred from intraspe- transformation of the data. We also compared values of h for cific genealogies. For both species, intraspecific phylogenies each population between species using a nonparametric were estimated using statistical parsimony with the aid of the Spearmann rank correlation. Finally, Tajima’s D tests of tcs (acronym for Templeton, Crandall, and Sing from neutrality (Tajima, 1989) were performed on data for both Templeton et al., 1992) software package (Clement et al., species by comparing hp and hS within each population. While 2000). From the genealogies, population processes were significantly negative D-values are evidence of a recent inferred through nested clade analysis (NCA; Templeton et al., population bottleneck (Rogers, 1995), inferences must be 1995) implemented using GeoDis (Posada et al., 2000) and the viewed with caution due to the potential for biases to be inference key in Templeton (1998). Through NCA, phylo- introduced by sampling error and population structure genetic clades at all depths, or levels, within the tree are tested (Hammer et al., 2003). to determine if there is a significant match between genealogy The degree of genetic divergence, or isolation, at different and geography. Although widely used, inferences gleaned geographic scales was assessed using FST, as is commonly done through NCA must be interpreted with caution because the in studies of population structure (Avise, 2004). Matrices of analysis is unable to statistically examine alternative scenarios pair-wise population FST values for each species were com- (Knowles & Maddison, 2002). For this reason, we lumped pared using a Mantel test, to evaluate patterns of population inferences into two categories: (1) expansion events that differentiation between the species. To test for genetic included contiguous range expansion and long distance structure at various geographic scales, we subjected the data dispersal, and (2) fragmentation events that could have

Journal of Biogeography 32, 1943–1956, ª 2005 Blackwell Publishing Ltd 1947 E. G. DeChaine and A. P. Martin occurred through past fragmentation or restricted gene flow, as divergence time (T/2Ne). The program was run, using the defined by Templeton (1998). To test whether similar HKY. finite sites model, three times for each species to evaluate demographic events occurred at the same time within both convergence for each parameter. From the results, the effective species, we contrasted the number of significant expansion and population size (Ne) was estimated using a 2.3% sequence fragmentation events within a clade level (depth of phylogeny) divergence per million years for COI in Lepidoptera (Brower, between species using the nonparametric Spearman’s correla- 1994). tion test. Coalescent simulations, incorporating the species’ geneal- ogy and estimate of Ne, were performed on mesquite 1.05 (Maddison & Maddison, 2003) to test how well the observed Testing phylogeographic hypotheses using coalescent data fit each model of divergence. To test the null simulations hypothesis, whether fragmentation in a widespread ancestral Objective tests of the scenarios resulting from the population population could explain the observed genealogy, 100 gene genetic and nested clade approaches were performed in order trees were simulated by coalescence within the null model to evaluate support for the phylogeographic models (Knowles (Fig. 1b). First, a set of gene matrices was simulated for & Maddison, 2002). Coalescent simulations of genealogies each of the hypothetical divergence times [where the constrained within models of population divergence provide a branch length ¼ the number of generations, and 1 genera- powerful means of assessing how well observed genetic tion ¼ 1 year for these species of Lepidoptera (Scott, 1986)], patterns fit phylogeographic hypotheses (Knowles, 2001; using the species-specific model of DNA substitution Carstens et al., 2005). We tested two general models of determined by MODELTEST 3.6 (Posada & Crandall, population divergence to determine whether observed patterns 1998) and the estimate of Ne. Trees were then reconstructed in C. meadii and P. smintheus were consistent with the null from the simulated gene matrices in PAUP* 4.10b hypothesis of fragmentation from a single, ancestral popula- (Swofford, 2003). The resulting gene trees were contained tion (Fig. 1b) or differentiation in three refugia and if within the 3-refugia model with the corresponding diver- divergence times were concordant between the two species. gence time (Fig. 1a,c,d) and the amount of discordance, as Under the alternative hypothesis (Fig. 1c,d), populations of measured by S, the minimum number of sorting events alpine butterflies were isolated into 3-refugia, corresponding to required to produce the genealogy within a given model of the northern, central, and southern biogeographic provinces of divergence (Slatkin & Maddison, 1989), was determined. The the Rocky Mountains (Fig. 2a; Brouillet & Whetstone, 1993). S-value for the observed tree constrained within the The alternative model was developed from phylogeographic 3-refugia model was compared to the distribution of patterns for P. smintheus (DeChaine & Martin, 2004) and S-values from the simulations to determine if the observed patterns of genetic diversity inferred for C. meadii in this study genealogy could have been generated under the null model. (see Results). Because high elevation taxa are expected to A similar approach was adopted to test the 3-refugia model experience population fragmentation during warm, intergla- over a range of different divergence times. One hundred gene cials, the seven divergence times that were tested for each matrices were simulated under the chosen model of DNA model correspond to interglacials over the last 600,000 years substitution and constrained within the population history (Fig. 1a; T1 ¼ 50,000, T2 ¼ 100,000, T3 ¼ 200,000, T4 ¼ predicted by the 3-refugia model for a given time of 300,000, T5 ¼ 400,000, T6 ¼ 500,000 yr bp) and include a divergence. Trees were reconstructed in PAUP* 4.10b (Swof- pre-Pleistocene date (1,700,000 yr bp). ford, 2003) and the S-distribution from the simulated gene In order to determine the probability that a species’ trees constrained by the 3-refugia model was compared with genealogy was generated under each hypothesis of population that of the observed tree for each divergence time (T1 through divergence, we first estimated the genealogy and population T7). In so doing, we were able to test whether the observed size (Ne) for a species and then evaluated how well the genealogies were consistent with the 3-refugia model and observed data fit the expectations generated by coalescent estimate a range of times during which divergence could have simulations of each model. modeltest 3.6 (Posada & occurred. Crandall, 1998) was employed to evaluate models of DNA substitution for both species individually and to select the RESULTS model that best fitted the data based on the Akaike Information Criterion (AIC). The best-fit model for a species The COI gene was highly polymorphic in both species. In was used to generate two genealogies in paup* 4.10b C. meadii, 35 variable sites defined 52 unique haplotypes (Swofford, 2003), one with and one without a molecular clock (Genbank accession numbers DQ105804 through DQ105855); enforced. The two trees were compared with a Likelihood by contrast, 53 variable sites defined 74 haplotypes in Ratio Test (LRT; Felsenstein, 1988) to determine if the data P. smintheus (DeChaine & Martin, 2004). For both species, were consistent with a molecular clock. all polymorphisms occurred at synonymous nucleotide posi- The sequence data were also subjected to additional tions. The observed genetic variation illustrates the sensitivity population genetic analyses in MDIV (Nielsen & Wakeley, of this marker to population processes and its utility in 2001), which simultaneously estimates h (¼ 2Nel) and intraspecific phylogenetic analyses of insects.

1948 Journal of Biogeography 32, 1943–1956, ª 2005 Blackwell Publishing Ltd Historical biogeography of Rocky Mountain butterflies

sampled from a single locality (Table 1; Fig. 3), implying Population genetics isolation among sky islands (Slatkin, 1985). In addition, only a Estimates of within population genetic diversity varied widely few haplotypes were common among physiographic provinces among sites within a species and were not correlated between in P. smintheus and only one (CC16) in C. meadii (Figs 3 & 4). species. The average haplotype diversity (H) was significantly Overall, these patterns of haplotype diversity imply geographic greater in P. smintheus than in C. meadii (Wilcoxon sign rank isolation of populations in both species. test, P ¼ 0.045), with estimates of H ranging from 0.00 to 0.89 Similarly, the three-level analyses of molecular variance and from 0.59 to 0.91 in C. meadii and P. smintheus, respectively (amova) revealed significant partitioning of genetic variation (Table 1). In contrast, estimates of h did not differ between the at all spatial scales (Table 2). This result, combined with species, C. meadii (h mean ¼ 0.00307) and P. smintheus significant genetic divergence based on pair-wise population

(h mean ¼ 0.00331), based on a paired t-test (h t ¼ 0.35, comparisons using FST and in plots of the normalized pair-wise P ¼ 0.73), but ranged widely in both species (Table 1). For both FST values [FST/(1 ) FST)] against the log of distance (C. meadii species, estimates of h were highly variable among collecting r2 ¼ 0.05, P < 0.005; P. smintheus r2 ¼ 0.14, P < 0.001), localities at similar latitudes and were not correlated with implies a history of restricted gene flow for both species. latitude (for C. meadii h r2 ¼ 0.11, P ¼ 0.18; for P. smintheus However, the magnitudes and patterns of pair-wise population h r2 ¼ 0.08, P ¼ 0.24). Importantly, in a comparison of within- genetic divergence were not similar between species (Mantel test, site diversity, values of h were not correlated between the two P < 0.01), a result that reflects lack of significant correlation in lepidopterans based on Spearman’s rank correlation test levels of within population genetic variation between species.

(rs ¼ 153, P > 0.5). Tests of neutrality based on Tajima’s D The percent of variation explained at each hierarchical level of showed that nearly all populations in both species were stable, the amova analyses were nearly identical between species but that in C. meadii, the Sundance Mountain population (Table 2), suggesting that gene flow, and thus dispersal barriers, (population 9) probably experienced a recent contraction. are similar between the species. But, the regional pattern of The geographic distribution of haplotypes suggests little sequence diversity differed between C. meadii and P. smintheus. gene flow among populations at local and regional scales. In C. meadii, levels of variation, as measured by h, were greater in Populations of both species harboured a relatively high and the north whereas P. smintheus exhibited more variation in the variable frequency of private alleles, or haplotypes that were south than in the central and northern Rockies (Table 1). These

Table 1 Sampling and distribution of genetic variation in Colias meadii Edwards and Parnassius smintheus Doubleday

nH hhp hS %p

Locality CPCPC P CPCPC PPrivate alleles in C. meadii

1 Gunsight Pass 10 27 0.89 0.67 0.0064 0.0009 3.76 1.91 4.10 1.81 60 4 CG6, CG7, CG8, CG10 2 Triple Divide Pass, 22 21 0.65 0.85 0.0020 0.0029 4.43 2.28 2.47 1.95 9 24 CG4 3 Dawson Pass 17 13 0.70 0.85 0.0028 0.0045 4.44 2.63 3.25 2.66 17 24 CG5, CG9 4 Hyndman Pk 13 32 0.59 0.80 0.0011 0.0026 0.95 1.72 0.97 1.78 38 48 CI1, CI2 5 Mt. Washburn 20 27 0.89 0.80 0.0073 0.0030 6.02 1.79 4.79 1.81 40 7 CY4, CY8, CY9, CY13, CY16, CY17, CY18 6 Amethyst Mt. 26 20 0.88 0.75 0.0052 0.0014 4.91 1.85 3.41 1.41 39 30 CY3, CY7, CY10, CY11, CY12, CY19 7 Moose Pass 12 20 0.48 0.91 0.0022 0.0063 4.36 2.67 2.98 2.27 0 53 n/a 8 Static Pk 12 15 0.62 0.59 0.0027 0.0013 4.95 1.18 3.31 0.92 8 7 CY5 9 Sundance Mt. 19 13 0.45 0.81 0.0017 0.0033 1.09 2.17 2.29 2.00 5 16 10 Long’s Pk 17 35 0.84 0.88 0.0039 0.0053 4.85 3.54 3.55 3.08 0 32 n/a 11 Quandary Pk 19 10 0.84 0.69 0.0049 0.0005 4.40 0.91 3.72 0.71 16 0 CC22, CC28 12 Maroon Bells 20 10 0.70 0.73 0.0022 0.0025 1.12 1.67 1.81 1.77 18 40 CC4, CC13, CC14 13 Mt. Elbert 16 15 0.40 0.83 0.0010 0.0024 1.60 1.85 1.21 1.54 25 33 CC26 14 Mt. Shavano 25 21 0.72 0.83 0.0025 0.0043 3.66 2.08 2.12 2.50 12 71 CC10, CC30 15 American Basin 20 33 0.36 0.88 0.0025 0.0045 1.62 3.77 2.82 2.96 10 27 CC20, CC29 16 San Luis Pk 29 28 0.78 0.75 0.0026 0.0045 3.75 2.70 2.29 2.54 66 24 CC11, CC12, CC19, CC21 17 Humboldt Pk 12 13 0.00 0.73 0.0000 0.0044 0.00 2.98 0.00 2.65 0 8 n/a 18 Iron Nipple 19 10 0.74 0.80 0.0043 0.0050 4.39 3.12 3.15 2.73 42 27 CC18, CC23 Average 19 20 0.64 0.79 0.0031 0.0033 3.35 2.28 2.68 2.06 22.5 26.4 Among regions Northern 50 65 0.77 0.79 0.0037 0.0028 4.52 2.24 3.27 2.14 60 48 Central 83 121 0.87 0.90 0.0037 0.0029 5.43 2.24 2.96 1.70 100 55 Southern 196 188 0.82 0.96 0.0026 0.0036 4.67 4.46 2.30 2.28 85 84

Collection sites, numbers of individuals surveyed, haplotype diversity (H), genetic diversity (h), and percentage of private alleles (%p) for both species are shown. The private alleles from each population of C. meadii are given, while those for P. smintheus were previously published (DeChaine & Martin, 2004). Abbreviations are as follows: C ¼ C. meadii,P¼ P. smintheus.

Journal of Biogeography 32, 1943–1956, ª 2005 Blackwell Publishing Ltd 1949 E. G. DeChaine and A. P. Martin

Table 2 Tests of genetic subdivision using a Species Source n d.f. SS Variance % three level amova; among regions, among populations within regions, and within pop- Colias meadii Edwards Among Regions 3 2 198.2 0.90 25.9 ulations Among Populations 18 15 245.3 0.82 23.4 Within Populations 325 307 541.2 1.76 50.7 Total 325 324 984.7 3.48

Parnassius smintheus Among Regions 3 2 185.1 0.67 27.3 Doubleday Among Populations 18 15 200.8 0.60 24.4 Within Populations 374 356 424.4 1.19 48.3 Total 374 373 810.3 2.47

Regions were defined as the northern, central, and southern Rocky Mountain biogeographic provinces following Brouillet & Whetstone (1993). Values for the sum of squares based on pair- wise distances (SS), the variance components (variance), and the percentage of variation at each hierarchical level (%) are shown. analyses of differentiation indicate that genetic diversity is phylogenies (Wares et al., 2001). Colias meadii exhibited a structured in both species, but that similarities between species geographically structured genealogy (Fig. 4a), including a depend on the geographic scale of the analyses. southern clade (3-1), a central clade (3-3), and a clade harbouring haplotypes from all regions (3-7). Northern haplotypes in C. meadii were derived from two or three Inferences from nested clade analyses separate transitions (CG1, CG10, and CG3) from the central The intraspecific phylogenies revealed a pattern of genetic and southern Rockies. A similar history is apparent in the divergence associated with known geographic provinces in the genealogy of P. smintheus (Fig. 4b), with a southern clade (4-1), Rocky Mountains (Fig. 4). We did not determine the ancestral a central clade (4-2) with one transition to the south, and a root, and thus geographic origin, for either species, but we were clade including haplotypes from all regions (4-3), with two able to make inferences of geographic transitions from the switches to the north. In general, deeper clades (i.e. clade levels

(a) Colias meadii (b) Parnassius smintheus

4-3 G6 G5 CG6 3-7 CG4 CG8 G3 G7 CG5 G9 CG7 G4 G8 CG1 CG10 4-2 CG9 Y1 Y13 Y12 Y4 CG3 CC15 3-3 Y3 Y8 CY13 CY3 Y5 CC16 I2 Y9 Y7 CY4 Y11 CY11 CY1 R8 CY19 CY17 G1 Y6 CC11 Y17 Y16 Y10 R6 CI2 I3 I4 CY7 R7 CY16 Y14 CC18 R9 CY14 I1 CY5 CY2 CY15 R12 R11 G2 CY12 CY8 CY18 R4 R5 CY9 CI1 CY10 3-4 Y15 R3 3-1 R2 R1 C34 CC4 C30 R10 C33 CC12 CC21 C28 C29 C32 CC29 CC23 4-1 CC27 C18 CC19 C4 C17 C15 C16 C23 CC13 C9 C13 C2 C11 C6 C1 CC9 CC2 CC8 CC17 C24 C25 CC30 CC22 C7 CC28 C12 CC20 CC14 C10 C14 C27 CC26 C31 C21 C19 C8 C3 C5 CC10 C26

C22 C20

Haplotype location Haplotype frequency North Central South Shared Unsampled 1 2-5 6-10 11-15 15-2021-50 51+

Figure 4 TCS-inferred mtDNA genealogies of sampled haplotypes for (a) Colias meadii Edwards and (b) Parnassius smintheus Doubleday. Haplotypes are shown as labelled circles, with the size of the circle indicating the relative frequency of each haplotype. Circle shading denotes the geographic region from which the haplotype was sampled; pie charts designate the frequency of a haplotype within each region. Each line connecting haplotypes represents a one-step mutational change between the haplotypes. Boxes delineate major NCA-clades referred to in the text.

1950 Journal of Biogeography 32, 1943–1956, ª 2005 Blackwell Publishing Ltd Historical biogeography of Rocky Mountain butterflies

4 or 5) included haplotypes from broad geographic distribu- cies: A ¼ 0.2984, C ¼ 0.1400, G ¼ 0.1433, T ¼ 0.4183; pin- tions, while haplotypes in the more derived clades (lower var ¼ 0.8439; gamma shape parameter ¼ 0.9071; rA-C ¼ 1, clade number) were geographically restricted. The genealogies rA-G ¼ 3.3568, rA-T ¼ 0.3184, rC-G ¼ 0.3184, rC-T ¼ corroborate the amova findings of geographic structure. 3.3568, rG-T ¼ 1). These models were used to generate A nested clade analysis (NCA) of the intraspecific geneal- intraspecific phylogenies with and without enforcing a ogies revealed that the two species were affected similarly by molecular clock. Because the LRT failed to reject the clock in historic events (the Spearman’s r ¼ 0.93, P < 0.001). The both species (C. meadii LRT ¼ 63.08 df ¼ 50, P > 0.10; geographic distributions of the major clades illustrate the P. smintheus LRT ¼ 79.2, df ¼ 73, P > 0.25), the rate of cycles of expansion and fragmentation experienced by both 2.3% substitutions per million years (Brower, 1994) was species (Fig. 5). NCA of C. meadii uncovered evidence for applied to MDIV (Nielsen & Wakeley, 2001) estimates of h eight clades that experienced fragmentation or restricted gene (¼ 2Nel) to calculate Ne (C. meadii h ¼ 8.1, Ne ¼ 203,099; flow and three clades that underwent contiguous range P. smintheus h ¼ 11.1, Ne ¼ 305,836). Multiple data analyses expansion or long-distance colonization (Table 3; Fig. 5a). on MDIV (Nielsen & Wakeley, 2001) converged on similar Fewer clades were inferred to have undergone demographic estimates for each parameter. The intraspecific genealogies and events in C. meadii than for P. smintheus, which experienced estimates of Ne were incorporated into the testing of 11 fragmentations and seven expansions (Table 3; Fig. 5b), a phylogeographic hypotheses. result that stems, in part, from the greater number of distinct The single and 3-refugia hypotheses were tested with haplotypes in P. smintheus than in C. meadii. Note that for coalescent simulations in MESQUITE 1.05 (Maddison & both species haplotypes from northwestern Colorado cluster Maddison, 2003). We computed S-values for C. meadii with the central and northern clades as opposed to the (S ¼ 10) and P. smintheus (S ¼ 6) by constraining their southern clades. Moreover, both species showed evidence for genealogies within the 3-refugia model. The null model of a recent and more ancient range expansions and a series of single ancestral population could be rejected for both species fragmentation events and restricted gene flow across the three for all time periods tested (P < 0.01). Not only did the two lowest levels of the trees. alpine butterflies fit the 3-refugia model, but the range of divergence times was nearly identical for both species. For the 3-refugia model, C. meadii, divergence times ranging from Tests of the phylogeographic hypotheses 100,000 to 400,000 yr bp (T2 P ¼ 0.25, T3 P ¼ 0.30, T4 In preparation for testing the phylogeographic hypotheses, P ¼ 0.10, T5 P ¼ 0.07) could not be rejected, but divergence genealogies and population parameters were inferred for both times at T1 (P ¼ 0.02), T6 (P ¼ 0.01), and T7 (P < 0.01) were species. Results from the modeltest 3.6 (Posada & Crandall, all rejected. Likewise, for P. smintheus, population divergence 1998) analyses revealed that the best model of substitution times of 100,000 to 300,000 yr bp (T2 P ¼ 0.30, T3 P ¼ 0.22, for C. meadii corresponded to a TrN + I + G model (AIC ¼ T4 P ¼ 0.07) could not be rejected for the 3-refugia model, 3199.3228; nucleotide frequencies: A ¼ 0.3121, C ¼ 0.1278, but more recent and later divergence times were rejected (T1 G ¼ 0.1437, T ¼ 0.4164; pinvar ¼ 0.8526; gamma shape P < 0.01, T5 P ¼ 0.03, T6 P < 0.01, T7 P < 0.01). Thus, a parameter ¼ 0.2283; rA-C ¼ 1, rA-G ¼ 17.0302, rA-T ¼ 1, 3-refugia model with population divergence occurring between rC-G ¼ 1, rC-T ¼ 9.4366, rG-T ¼ 1) and P. smintheus fit a 100,000 to 300,000 yr bp is strongly supported for both species K81uf + I + G model (AIC ¼ 3423.8008; nucleotide frequen- by the coalescent simulations.

(a) Colias meadii (b) Parnassius smintheus

1 MT 1 MT 2 2 3 3

6 6 WY 5 WY 4 5 4 Figure 5 Geographic patterns of NCA- 7 7 inferences for (a) Colias meadii Edwards and 8 8 (b) Parnassius smintheus Doubleday. Ellipses ID ID designate clades that experienced expansion (solid lines, white interior) or fragmentation 9 CO 9 CO 10 10 12 12 (dashed lines, grey interior) as inferred 11 11 through NCA. The thickness of the ellipse 13 13 14 14 lines corresponds to clade depth with thicker 15 17 15 17 16 16 lines representing deeper clades. For simpli- UT 18 UT 18 city, not all clades that exhibited significant geographic structure are shown. A complete list of all NCA-inferences is given in Table 3. Expansion Fragmentation

Journal of Biogeography 32, 1943–1956, ª 2005 Blackwell Publishing Ltd 1951 E. G. DeChaine and A. P. Martin

Table 3 Inferences of historical processes in Colias meadii central, and southern provinces, which implies a shared history Edwards and Parnassius smintheus Doubleday of restricted gene flow among the regions. Moreover, palaeo- climatic changes of the Quaternary likely influenced genetic Clade Inferred event divergence in the two species in a similar way, through Colias meadii repeated cycles of down-slope population expansion and 4-1 Contiguous range expansion upslope contraction. But, across more limited geographic 4-2 Long distance colonization scales and over shorter time periods, population parameters 4-3 Contiguous range expansion are not correlated between the species. Overall, the demogra- 3-1 Restricted gene flow with isolation-by-distance phic histories of these two alpine butterflies are marked by 3-3 Restricted gene flow with isolation-by-distance broad-scale concordance and local-scale disparities. 3-7 Past fragmentation Clear large-scale geographic patterns of genetic divergence 2-1 Restricted gene flow with isolation-by-distance were shared by both species. For instance, C. meadii and 2-4 Contiguous range expansion P. smintheus exhibited a strong genetic break across the 2-9 Restricted gene flow with isolation-by-distance 2-13 Past fragmentation Wyoming Basin, a region of low elevation and dry habitat that 1-1 Restricted gene flow with isolation-by-distance lies on the Continental Divide at approximately 42 N and 1-18 Restricted gene flow with isolation-by-distance separates the southern from the central Rockies (Fig. 2a; Parnassius smintheus Brouillet & Whetstone, 1993). A similar pattern was evident 5-1, total Past fragmentation for other high elevation Lepidoptera (Britten & Brussard, 1992; 4-1 Contiguous range expansion Nice & Shapiro, 2001) and Coleoptera (Noonan, 1992, 1999; 4-2 Long distance colonization Reiss et al., 1999) suggesting that this region is a major 3-1 Restricted gene flow with isolation-by-distance biogeographic boundary for alpine insects. But, the tighter 3-2 Past fragmentation association between populations in northwestern Colorado 3-6 Contiguous range expansion (numbers 9, 10, and 11) with northern populations (Figs 4 & 5) 3-7 Restricted gene flow with isolation-by-distance as opposed to other southern populations suggests a secondary 2-2 Restricted gene flow with isolation-by-distance 2-3 Restricted gene flow with isolation-by-distance colonization of the south from the central and/or northern 2-6 Past fragmentation populations for both species. Although less distinct, a genetic 2-7 Past fragmentation break was detected in both species between the most northern 2-13 Past fragmentation collection sites from the previously glaciated area of Montana 2-17 Contiguous range expansion and the central Rocky Mountains in Wyoming and Idaho. This 1-9 Long distance colonization part of the Rocky Mountains is also marked by lower elevation 1-28 Restricted gene flow with isolation-by-distance and reduced alpine habitat (Fig. 2a, elevation profile). Genetic 1-32 Past fragmentation divergence across regions was most pronounced in C. meadii,in 1-41 Long distance colonization which only one haplotype was found in more than one Summary geographic region (Fig. 3a; haplotype CC16). For the few P. smintheus haplotypes that were widespread, frequencies Parnassius differed dramatically among the three regions. Moreover, the Colias meadii smintheus amova revealed that about 26% of the genetic variation Clade level EFEFsampled was distributed among regions in both species. This distribution of haplotype diversity suggests that, within both Fifth 0 0 0 1 species, genetic divergence among regions is due to restricted Fourth 2 0 3 0 gene flow across low-elevation geographic barriers. Third 0 3 1 3 Demographic histories inferred for C. meadii and Second 1 3 1 5 P. smintheus were similar throughout the climate cycles of First 0 2 2 2 the Quaternary. Both species showed evidence of multiple cycles of population expansion and fragmentation over a period defined by the 5-level nested cladograms. Moreover, the DISCUSSION cycles of population expansion and contraction were correla- Analysis of mitochondrial DNA sequences from eighteen sky ted between the species with respect to clade depth (level), even islands spanning about 12 of latitude in the Rocky Mountain though the number of demographic events was greater in cordillera revealed an intriguing pattern of genetic divergence P. smintheus. These results suggest that both species may have for the co-distributed alpine butterflies, C. meadii and responded to climate cycles similarly. P. smintheus. Populations of both species are geographically Testing phylogeographic hypotheses through coalescent structured at all spatial scales and both species probably simulations provided strong support for concordance between persisted across most of the latitudinal range throughout the the biogeographic histories of the two butterflies. Data from glacial cycles. At the scale of biogeographic regions, popula- both species were consistent with divergence among three tions of both species are relatively isolated within north, refugia occurring during the Pleistocene, from 100,000 to

1952 Journal of Biogeography 32, 1943–1956, ª 2005 Blackwell Publishing Ltd Historical biogeography of Rocky Mountain butterflies

400,000 yr bp, a period including three to four major glacial– alpine systems have been performed on European plants and interglacial cycles (Winograd et al., 1997). Because gene arthropods, where mountain ranges that run along an east- divergence times generally overestimate population divergence west axis were mostly covered by ice during glacial periods. times (Edwards & Beerli, 2000), the more ancient divergences This implies that in Europe, alpine species were forced to (T4 and T5) with low statistical support (P < 0.1) probably do persist in a few isolated refugia during glacial periods, as has not accurately reflect the ages of population divergence, but do been shown for several taxa (Taberlet et al., 1998; Stehlik, provide a maximum confidence limit for the inferences. 2000; Garnier et al., 2004; Schmitt & Hewitt, 2004). The Moreover, since high elevation populations are expected to disjunct pattern of glacial refugia in Europe lies in stark have diverged during interglacial periods when the alpine contrast to the hypothetical wide distribution of alpine tundra was fragmented and xeric, low elevation regions like the habitat in the Rocky Mountains during glacial periods Wyoming Basin were impassable, the populations of alpine (Fig. 2b), and underscores the need for a phylogeographic butterflies in the northern, central, and southern regional model that accounts for the prevalence of alpine habitat refugia likely diverged during one of the more recent throughout the Quaternary climate cycles along the north- interglacials at approximately 100,000 or 200,000 yr bp (T2 south spanning cordillera. or T3). These findings, like those for alpine grasshoppers in the DeChaine & Martin (2004) hypothesized that populations of Rocky Mountains (Knowles, 2001), suggest that the palaeocli- alpine and sub-alpine organisms have existed on an expanding matic cycles of the Quaternary promoted divergence among and contracting archipelago of sky islands, along the Rocky high elevation insects. Mountain cordillera. The Rocky Mountains are one of the Despite evidence for shared history across large spatial scales longest terrestrial mountain ranges on the planet, and are and over the last few hundred thousand years, there is little marked by topographic and climatological heterogeneity, evidence for a common demographic pattern when the abrupt changes in elevation, resources, and a mosaic of alpine analyses were focused at the level of individual localities. For habitat. For inhabitants of the Rocky Mountain alpine tundra, instance, estimates of genetic diversity for C. meadii and we imagine a dynamic process, as did Hewitt (1996), in which P. smintheus were not correlated. A similar pattern of a species is subdivided into small, isolated populations discordance between species was evident from the pair-wise (contraction phase) punctuated by periods when populations

FST values. This independent biogeographic response of species are large and span vast geographic ranges (expansion phase). to climate cycles has been repeatedly demonstrated through But, specific to the north-south running mountain ranges, palynological records (West, 1980; Whitlock & Bartlein, 1997), local habitat is available throughout the climate cycles, fossil insect assemblages (Coope, 1995; Elias, 1996) and obviating the need for long distance migration or glacial molecular phylogeographic studies (Taberlet et al., 1998; refugia. Rather, it is during interglacials, like the current Stewart & Lister, 2001; Kropf et al., 2003). For C. meadii and conditions, that populations contracted and fragmented as P. smintheus, the lack of correlation detected from pair-wise they moved up in elevation with the warming climate. comparisons of the species is probably not due to stochastic Throughout these contraction periods, populations were variance associated with the coalescent process (Edwards & isolated and underwent genetic divergence [as shown for Beerli, 2000) because broad-scale genetic patterns correspond Melanoplus grasshoppers in the central and northern Rockies to geographic barriers and indicate a common cause. Rather, (Knowles, 2000)], and many populations probably went local extinction and re-colonization dynamics, which greatly extinct. With the return of cool, wetter climates, the alpine influence the magnitude of within-population genetic varia- and sub-alpine habitats expanded and became connected, as tion and the genetic divergence among populations (Slatkin, did populations of high elevation organisms. Throughout 1977; Pannell, 2003), may be responsible for the differences expansion periods, gene flow was more extensive, and habitats observed between the species. Our findings corroborate in which populations had gone extinct during the interglacials previous records showing that, at fine geographic scales, the were probably re-colonized. The current study and those of individual response of a species governs population size, spatial DeChaine & Martin (2004, 2005) provide empirical evidence structure, and genetic structure, and depends on the magni- for repeated cycles of population expansion and contraction tude of climate change (Webb, 1987; Bennett, 1990; Davis & within mountain blocks along a vast latitudinal range of the Shaw, 2001). Rocky Mountains. Similarity of large-scale geographic patterns and inferences The distribution of genetic variation in C. meadii and of population expansions and fragmentation over the last P. smintheus is undoubtedly due to a combination of historic 400,000 years suggests a general model for alpine and sub- and current ecological factors. At broad geographic and alpine biogeography for the Rocky Mountains south of the temporal scales, the two species exhibited concordant patterns Cordilleran Ice sheet. The basis for this model was outlined of genetic divergence, but at finer scales, demographic histories by Hewitt (1996): the hypothetical response of high elevation were not correlated between the species. Inferences from our taxa to the shifting environment associated with climate study suggest that the general response of alpine taxa in the cycles is to track alpine habitat by migrating up and down in Rocky Mountains to palaeoclimatic oscillations of the elevation, rather than dispersing over great distances. As with Quaternary fits an expanding–contracting archipelago model. Hewitt’s (1999, 2000) work, most phylogeographic studies of According to this hypothesis, populations persisted across the

Journal of Biogeography 32, 1943–1956, ª 2005 Blackwell Publishing Ltd 1953 E. G. DeChaine and A. P. Martin latitudinal range of the Rockies throughout the climate cycles Brouillet, L. & Whetstone, R.D. (1993). Climate and physiog- by expanding down-slope during glacial periods, and con- raphy: flora of North America (ed. by Flora of North tracting upslope into a fragmented archipelago of sky islands America North of Mexico Editorial Committee). Oxford during warm, interglacials. While the palaeoclimatic cycles of University Press, New York. the Quaternary governed the general distribution of genetic Brower, A.V.Z. (1994) Rapid morphological radiation and diversity in high elevation taxa, biological characteristics convergence among races of the butterfly Heliconius erato peculiar to each species probably promoted different patterns inferred from patterns of mitochondrial DNA evolution. of genetic variation between the species on finer spatial and Proceedings of the National Academy of Science, 91, 6491–6495. temporal scales as biologically relevant environmental variables Carstens, B.C., Degenhardt, J.D., Stevenson, A.L. & Sullivan, J. changed independently; sometimes in concert, at other times (2005) Accounting for coalescent stochasticity in testing in opposition (Jackson & Overpeck, 2000). Further examina- phylogeographical hypotheses: modelling Pleistocene tion of data for other Rocky Mountain alpine taxa will help to population structure in the Idaho giant salamander determine the generality of our findings and identify the Dicamptodon aterrimus. Molecular Ecology, 14, 255–265. potential effects of future climate change on diversity at high Caterino, M.S., Cho, S. & Sperling, F.A.H. (2000) The current elevations. state of insect molecular systematics: a thriving Tower of Babel. Annual Review of Entomology, 45, 1–54. Clement, M., Posada, D. & Crandall, K.A. (2000) TCS: a ACKNOWLEDGEMENTS computer program to estimate gene genealogies. Molecular Permission to collect butterflies was generously provided by Ecology, 9, 1657–1659. Glacier National Park (permit no. GLAC-2001-SCI-0020), Comes, H.P. & Kadereit, J.W. (1998) The effect of Quaternary Yellowstone National Park (permit no. YELL-2001-SCI-0212), climatic changes on plant distribution and evolution. Trends Grand Teton National Park (permit no. GRTE-2001-SCI- in Plant Science, 3, 432–438. 0009), and Rocky Mountain National Park (permit no. Coope, G.R. (1995) Insect faunas in ice age environments: why ROMO-2001-SCI-0037). The work was funded by the National so little extinction? Extinction rates (ed. by J.H. Lawton and Science Foundation, the University of Colorado, the Beverly R.M. May), pp. 55–74. Oxford University Press, Oxford. Sears Graduate Student Grants, the John W. Marr Ecology Davis, M.B. & Shaw, R.G. (2001) Range shifts and adaptive re- Fund, the Indian Peaks Wilderness Association, Canon- sponses to Quaternary climate change. Science, 292, 673–679. National Parks Scholarships, the Edna Bailey Sussman Fellow- DeChaine, E.G. & Martin, A.P. (2004) Historic cycles of ship, the Colorado Mountain Club Academic Fellowship, and fragmentation and expansion in Parnassius smintheus the Southern Rockies Ecosystem Project. For help collecting (Papilionidae) inferred using mitochondrial DNA. specimens, we thank Gerald DeChaine, Mathew Burt, and Evolution, 58, 113–127. Thomas Walla. We also thank Deane Bowers, William DeChaine, E.G. & Martin, A.P. 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