Journal of Biogeography (J. Biogeogr.) (2012) 39, 1058–1072

ORIGINAL Mitochondrial phylogeography of the ARTICLE Holarctic phoebus complex supports a recent refugial model for alpine butterflies Valentina Todisco1* , Paolo Gratton1,2 , Evgeny V. Zakharov3à, Christopher W. Wheat4,5, Valerio Sbordoni1 and Felix A.H. Sperling3

1Department of Biology, University of Rome ABSTRACT ‘Tor Vergata’, Via della Ricerca Scientifica 1, Aim Our study provides a description of the mitogenetic structure of alpine 00133 Rome, Italy, 2Department of Biodiversity and Molecular Ecology, Fondazione E. Mach, butterflies of the complex throughout their Holarctic 38010 San Michele all’Adige, TN, Italy, distribution. Our analyses extend and reassess population history models for 3Department of Biological Sciences, University alpine butterflies under an explicit calibration of their mitochondrial DNA of Alberta, CW405 Biological Sciences Centre, (mtDNA) substitution rate. 4 Edmonton, AB, , Department of Location Mountain ranges of the Holarctic region. Biological and Environmental Sciences, PL 65, Viikinkaari 1, 00014 University of Helsinki, Methods A fragment (824 bp) of the mitochondrial cytochrome c oxidase Helsinki, Finland, 5Centre for Ecology and subunit I (COI) gene was sequenced in 203 samples (72 locations), and combined Conservation, School of Biosciences, University with previously available COI sequences (499 samples), to obtain full coverage of of Exeter, Cornwall Campus, Penryn, Cornwall the Holarctic distribution of the P. phoebus complex. A global species distribution TR10 9EZ, UK model (SDM) was calculated by the maximum entropy (Maxent) approach, allowing assignment of samples into geographically consistent ‘operational’ units. Phylogenetic and coalescent methods were applied to describe the global mitogenetic structure and estimate population genetics parameters. Geological and palaeoecological evidence was used for internal calibration and validation of a COI substitution rate. Results Eurasian (including Alaskan) and North American populations form two distinct mitochondrial clades. The mitochondrial time to most recent common ancestor (TMRCA) of the North American clade was estimated at less than 125 ka, and the TMRCA of the Eurasian–Alaskan clade at less than 80 ka, except for a single divergent sequence from Mongolia. Pairwise divergence times between all geographical units within each continent date well within the last 100 ka, and most likely, the last 50–10 ka. Main conclusions In contrast with its currently scattered distribution within each of Eurasia and , the mitogenetic structure of the P. phoebus complex in both continents is shallow and weak, and shows no evidence of geographical structure dating back earlier than the last glacial cycle. We argue that mtDNA data are consistent with recent (Wu¨rm/Wisconsin) range expansion *Correspondence: Valentina Todisco, across each of the two continents and with persistent glacial long-range gene flow Department of Biology, University of Rome ‘Tor which ceased during the Holocene. We propose that P. phoebus may represent a Vergata’, Via della Ricerca Scientifica 1, 00133 model for Holarctic alpine invertebrates with moderate dispersal abilities in that Rome, Italy. E-mail: [email protected] its genetic structure at a continental scale reflects extensive connectivity during These authors contributed equally. the most recent glacial phases. àPresent address: Biodiversity Institute of Keywords Ontario, Canadian Centre for DNA Barcoding, University of Guelph, Guelph, ON, N1G 2W1, Alpine butterflies, coalescent, glacial cycles, Holarctic, mtDNA, Parnassius, Canada. phylogeography, substitution rates.

1058 http://wileyonlinelibrary.com/journal/jbi ª 2012 Blackwell Publishing Ltd doi:10.1111/j.1365-2699.2011.02675.x Holarctic mtDNA phylogeography of Parnassius phoebus

mitochondrial DNA (mtDNA) substitution rates may be INTRODUCTION subject to a time-dependent effect (e.g. Ho et al., 2005, 2007; Phylogeographical analyses of widely distributed taxa can Burridge et al., 2008; Henn et al., 2009), with divergence provide important insights into the combined effects of between species apparently increasing at a slower (‘phylo- historical climate change and topography on the current genetic’) rate than polymorphism within populations and genetic structure of species (e.g. Hewitt, 2000). However, divergence among recently separated populations. Explana- global assessments of population structure for species found tions for this effect include both purifying selection (e.g. across more than one continent are scarce (see Forister et al., Kivisild et al., 2006; Peterson & Masel, 2009) and oversimpli- 2004; Albach et al., 2006; Albre et al., 2008). Focus on species fication of population models (Navascue´s & Emerson, 2009). occupying alpine habitats of Europe and North America is Regardless of its causes, this effect is of paramount importance especially critical, as these species face a high risk of extinction to phylogeographical studies, as it implies that the application due to modern climate change (Descimon et al., 2006; of ‘phylogenetic’ calibrations to recent population data will Parmesan, 2006; Cochrane, 2011). result in overestimated dates (Herman & Searle, 2011). Many butterflies are habitat specialists with moderate long- range dispersal abilities. They are highly sensitive to environ- Study organisms mental change and their phylogeographical structure can be expected to closely reflect climate-driven range shifts. While Butterflies of the genus Parnassius provide an excellent model the distributions of temperate species reflect the latitudinal with which to evaluate the effects of past and present climate retreat and advance of mesophilic wooded habitats (e.g. changes on organisms in the Northern Hemisphere (Arau´ jo & Schmitt, 2007; Gratton et al., 2008), high-mountain species Luoto, 2007; Gratton et al., 2008; Todisco et al., 2010; Matter follow elevation shifts of the alpine zone. Such changes allow et al., 2011). All species in the genus are habitat specialists of range expansion or connectivity during cold/arid periods for alpine or subalpine in temperate latitudes of Eurasia alpine species, but diminish or interrupt gene flow during and western North America. They have moderate dispersal interglacial periods, including the Holocene (DeChaine & abilities (Brommer & Fred, 1999; Keyghobadi et al., 1999, Martin, 2004, 2005, 2006; Schmitt, 2007; Varga & Schmitt, 2005; Matter & Roland, 2004; Matter et al., 2004, 2009; Ross 2008; Todisco et al., 2010). et al., 2005) and their distribution and morphological vari- Under a refugial paradigm (Hewitt, 2000), climate-driven ability are well studied (Weiss, 2005). range shifts have fostered allopatric speciation in both The Parnassius phoebus complex is unique in the genus temperate and alpine taxa (Knowles, 2001; Swenson & Parnassius for having a Holarctic distribution. Scattered Howard, 2005; Carstens & Knowles, 2007). Schoville & populations occur in Eurasia in the Alps, northern Urals and Roderick (2009) integrated current views on population central and eastern , and in North America from histories of alpine species under three non-equilibrium mod- to and Colorado. Specialists have recognized five els: (1) expanding alpine archipelago, (2) alpine archipelago species, based on minor morphological or ecological features refuge, and (3) ancestral radiation with fragmentation. While and geographical isolation (Weiss, 2005) (see Fig. 1a and these models all assume that long-distance gene flow only Appendix S1 in Supporting Information): P. phoebus (Urals, occurs during cold periods, they differ in: (1) relative central and eastern Siberia, Alaska and part of Yukon), population size during cold and warm periods (bigger during P. sacerdos (Alps), P. bremeri (south-eastern Siberia, Manch- cold phases under models 1 and 3, smaller under model 2; and uria and Korea), P. ruckbeili (small range in northern China), (2) number of stable alpine refugia during warm intervals P. smintheus (North American Cordillera) and P. behrii (Sierra (several ‘sky-islands’ in models 1 and 2, or a single ‘stronghold’ Nevada, CA); each with a variable number of subspecies and in model 3). The first two models assume repeated cycles of forms (Shepard & Manley, 1998; Opler & Warren, 2003; Weiss, connection and isolation among several, distinct refugia that 2005). The typical habitats of these butterflies are alpine persist through several glacial/interglacial phases. In contrast, meadows and partially forested steppes. the ancestral radiation with fragmentation model is most All described taxa are strictly allopatric, except for P. bremeri consistent with recent range expansion during the last and P. phoebus, whose distributions overlap in a few localities glaciation followed by Holocene fragmentation. While popu- in central Siberia (Weiss, 2005; Fig. 1a). The taxonomic lation dynamics are dependent on niche-specific responses to distinctiveness of these forms is controversial (Michel et al., climate change, both the number of refugia and the intensity of 2008). Parnassius bremeri is defined by a distinctive wing gene flow are determined by regional geoclimatic characteris- pattern and it has genitalia practically identical to those of tics and can be expected to show regionally distinct phylo- P. phoebus. The existence of populations from the lower Amur geographical patterns. Basin (e.g. P. bremeri amgunensis) with intermediate characters DNA data represent the most valuable source of information suggests that gene flow may occur between these two species to assess historical models of population structure. However, (Korshunov & Gorbunov, 1995; Gorbunov, 2001). both temporal and demographic inferences are critically In our study we sequenced 824 bp of the mitochondrial dependent upon accurate estimation of nucleotide substitution genome in 105 individuals from the Eurasian–Alaskan taxa rates. Several studies have demonstrated that calibrated (P. phoebus, P. sacerdos, P. bremeri) of the Parnassius phoebus

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Figure 1 Geographical distribution of samples (a) and maximum likelihood (ML) phylogeny (b) of cytochrome c oxidase subunit I (COI) haplotypes for the Holarctic Parnassius phoebus complex. Symbol sizes in (a) are proportional to the number of sequence samples and different colours identify operational population units (see text for details). Pie charts at the tips of the ML tree show frequency of population units represented within each haplotype.

complex and 98 individuals from the North American taxa dix S2). One to 11 specimens, obtained from private collec- (mostly from previously unsampled north-western regions). tions or collected by the authors, were analysed for each Our data were analysed together with a large set of mtDNA sampling site. Details on extraction, amplification and sequences (496 individuals) already available from North sequencing procedures are given in Appendix S3. All sequences America (DeChaine & Martin, 2004, 2005, 2006; Schoville & were submitted to GenBank (accession numbers are given in Roderick, 2009). We used phylogenetic and coalescence Appendix S2). One sequence from each of P. acco (GenBank approaches to: (1) describe the phylogeographical structure EF473823), P. simo (EF473815), P. clodius (EF473795), P. jac- of the P. phoebus complex in Eurasia–Alaska; and (2) extend quemonti (EF473782), P. nordmanni (EF473799), P. actius and reassess population history models across the entire (EF473784) and P. nomion (EF473781), and three sequences Holarctic distribution of the complex. To do so, we derived a from P. (GU947494, GU947444, EF473775) were species-specific population-level substitution rate using cali- retrieved from GenBank and included as outgroups for brations based on palaeoecological evidence. phylogenetic analyses. Our data set was combined with published sequences for P. smintheus and P. behrii: 394 sequences (789 bp) by MATERIALS AND METHODS DeChaine & Martin (2004) and 104 sequences (930 bp) by Schoville & Roderick (2009), two sequences of P. phoebus from Samples Mongolia are included in the latter. We excluded, following An 824-bp fragment next to the 3¢ end of the mitochondrial the personal suggestion of the author Sean Schoville (Univer- cytochrome c oxidase subunit I (COI) gene was sequenced in site´ Joseph Fourier, Grenoble, France), a likely artefact 203 individuals from 72 sampling locations, including all the haplotype (FJ756826.1). The two published data sets are species of the P. phoebus complex (see Fig. 1a and Appen- nested within each other and overlap 512 bp of our new

1060 Journal of Biogeography 39, 1058–1072 ª 2012 Blackwell Publishing Ltd Holarctic mtDNA phylogeography of Parnassius phoebus sequences, forming a global alignment of 1164 bp. Different We used Maxent 3.3.2 (Phillips et al., 2006; Phillips & combinations of sequence data of different lengths were used Dudı´k, 2008; http://www.cs.princeton.edu/~shapire/Maxent) for each analysis, depending on the flexibility of available for computation of the climatic species distribution model algorithms. For ease of description we adopt the following (SDM). As ensemble model predictions may enhance the convention: A.1 data sets include fragments from all the three reliability and robustness of SDM results (Arau´ jo & New, sources, with gaps treated as missing data (1164 bp); A.2 data 2007), we computed 20 models, each of them trained with a sets include fragments from all three sources with gaps deleted randomly chosen 75% of the 128 species records, and (512 bp); A.3 data sets are made up of new sequences from this subsequently computed average values per each grid cell. The study only (824 bp); A.4 data sets are made up of fragments 25% of records omitted were used for model evaluation via from published sources only, with alignment gaps deleted AUC (area under the receiver operating characteristic curve) (789 bp). An A.1 data set was used for maximum likelihood statistics (Swets, 1988).

(ML) phylogenetic reconstruction. Computation of Fu’s FS (Fu, 1997) and R (Ramos-Onsins & Rozas, 2002), and 2 Definition of operational population units mismatch parameters was performed on an A.3 data set for Eurasian–Alaskan samples and on an A.2 data set for North We used the results of the SDM analysis to divide the available American samples. An A.3 data set was used in isolation with data set into operational population units. We defined migration (IM) model (Nielsen & Wakeley, 2001) analyses of operational units as those samples collected in sites not Eurasian–Alaskan samples and A.4 for North American separated by more than 2.5 arcmin (1 pixel in our climatic samples. A.1 data sets were used for all analyses in beast model) of unsuitable habitat (Fig. 2). This distance corre- 1.5.6 (Drummond & Rambaut, 2007) (see next section). sponds to 4.63 km in latitude and to 1.7–3.6 km in longitude In all demographic analyses that follow, t indicates the in our studied range (decreasing with latitude) and it was estimated population genetics parameter and T the true time considered to represent a reasonable dispersal distance over the (in years) elapsed since each demographic event. Because the long term because single adults of P. smintheus have been P. phoebus complex is univoltine, T is also the number recorded to move for over 1.7 km (Roland et al., 2000). of generations. Phylogenetic analyses Species distribution modelling The Akaike information criterion (AIC) implemented in We obtained 230 species records for the P. phoebus complex jModeltest 0.1.1 (Posada, 2008) was used to select the best- from our own fieldwork and from the Global Biodiversity fitting model for each codon position and the ML phylogeny of Information Facility (GBIF; http://www.gbif.org). As records mtDNA haplotypes was determined in treefinder (Jobb, were strongly biased towards a few areas (Alps and North 2011). Robustness of phylogenetic inference was estimated by a America), spatial clusters were reduced via subsampling in local rearrangement (LR-ELW) approach (Strimmer & Ram- environmental space, leaving 76 records. Moreover, to further baut, 2002) on 500 replicates. Average ML distances (patristic homogenize representation of different areas, we added 52 distances) between clades were calculated in treefinder. estimated presence points in central and eastern Asia. We generated these points by approximating coordinates of type Isolation with migration model and calibration localities given in Weiss (2005) and ‘doubling’ a random set of of a COI substitution rate the Asian records within a 2-km radius (movements of more than 1.7 km have been reported by Roland et al., 2000). Parameters of the IM model (Nielsen & Wakeley, 2001) were Current data were downloaded from the WorldClim 1.4 estimated using a Markov chain Monte Carlo (MCMC) database, with a grid cell resolution of 2.5 arcmin (Hijmans approach by applying the software im (Hey & Nielsen, 2004; et al., 2005; http://www.worldclim.org) along with simulations revision of 3 May 2007) to several pairs of neighbouring describing conditions during the Last Glacial Maximum (LGM) population units. For each pair, two sets of simulations were using the Community Climate System Model (CCSM). Follow- run. In the first set (migration model), all parameters were ing Habel et al. (2010), we modelled the climatic niche as a given reasonably large uniform priors; in the second set function of 13 out of the 19 BIOCLIM variables (Busby, 1991): (no-migration model), migration parameters were set to zero, annual mean temperature (BIO1), mean monthly temperature thus assuming a ‘pure divergence’ model. The rationale for this range (BIO2), isothermality (BIO3), maximum temperature of choice is that, while the full model may estimate the initial the warmest month (BIO5), minimum temperature of the divergence of two populations, the no-migration model may coldest month (BIO6), annual temperature range (BIO7), mean be used to set a reasonable lower limit for the complete temperature of the wettest quarter (BIO8), mean temperature of cessation of gene flow. Simulations were run on the publicly the driest quarter (BIO9), annual precipitation (BIO12), available Cornell University computation facility (http:// precipitation seasonality (BIO15), precipitation of the driest cbsuapps.tc.cornell.edu) in three replicates of 107 generations quarter (BIO17), precipitation of the warmest quarter (BIO18) with a burn-in of 106 generations. Results from the three runs and precipitation of the coldest quarter (BIO19). were checked for convergence and averaged.

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Figure 2 Species distribution models (SDM) and palaeoclimate. Modelled geographical distribution of climatic niche for the Holarctic Parnassius phoebus complex obtained by Maxent analysis under current conditions (a) and community climate system model (CCSM) for 21 ka (b). Western North America is enlarged in panels (c) current conditions and (d) 21 ka. Last Glacial Maximum (LGM) ice sheet and lakes data are from Dyke et al. (2003) and CLIMAP (1981). Dots indicate sampling sites for molecular analyses (colour-coding as in Fig. 1). The full list of presence locations used for SDM analyses is available upon request to the authors. Panel (e) reports deuterium excess (deltaD) from Antarctic ice cores (EPICA Community Members, 2004) and percentage of Quercus pollen in sediments from Clear Lake, CA (Adam et al., 1981) as indicators of climate through the last 150 ka.

We used the im analysis performed on P. phoebus samples northern USA and in Canada), was covered by the maximum from Alaska and Siberia and external geological information to extent of the Cordilleran ice sheet c. 18 ka, and was not estimate a substitution rate for COI suitable for our coalescent- deglaciated until c. 12 ka (Dyke et al., 2003; Fig. 2d). This area based dating of recent evolutionary events. The posterior is in close proximity to other sampling sites in the central distribution of t = Tl (where T is the number of generations , and no significant habitat discontinuities since the populations split and l the substitution rate per gene were identified by our SDM, neither under present climate nor per generation) from the no-migration model was taken as an under CCSM reconstruction for 21 ka (Figs 2c,d). Therefore, estimate of the time since complete interruption of gene flow we assumed that genetic divergence between populations across the Bering Strait. Geological dating of the opening of the sampled north and south of the maximum glacial extent Bering Strait (c. 10 ka, US National Climatic Data Center, started as a result of the recolonization of the formerly http://www.ncdc.noaa.gov/oa/ncdc.html) was used to scale the glaciated areas. Accordingly, our expectation was that, if the model parameter into calendar years. The substitution rate calibrated rate was correct, the estimate of divergence time corresponding to the ML (peak) estimate of t was then under the IM model should be around 12 ka. calculated and applied to convert population genetics param- eters into demographic units. As butterflies of the P. phoebus Tests of demographic equilibrium and population complex are univoltine with non-overlapping generations, expansion generation time was set to 1 year. An independent geological line of evidence was used to Haplotype and nucleotide diversity were calculated using validate our calibration. The area in which a number of North DnaSP 5.0 (Librado & Rozas, 2009). Demographic equilib- American samples were collected (Rocky Mountains in the rium was tested in each operational population unit by

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calculating FS (Fu, 1997) and R2 (Ramos-Onsins & Rozas, priate burn-in was determined by eye and set up in Tracer 2002) statistics, which have been shown to be sensitive to 1.5. signals of population expansion (Ramos-Onsins & Rozas, 2002). Arlequin 3.0 (Excoffier et al., 2005) and DnaSP 5.0 RESULTS were employed to compute FS and R2, respectively, and test their statistical significance by simulating random samples Species distribution modelling (10,000 replicates) under the null hypothesis of selective neutrality and constant population size using coalescent We used the Maxent algorithm to develop a climate-based algorithms (both modified from Hudson, 1990). P-values SDM for the P. phoebus complex. The average test AUC for the for the two statistics were obtained as the proportion of replicate runs was 0.897 (SD = 0.023), indicating that our simulated values smaller than or equal to the observed values model output was of good quality (Swets, 1988). Analyses of (a = 0.05). the relative contribution of the variables to the model revealed Expected mismatch distributions and parameters of sudden that the ‘annual mean temperature’ had the highest explan- expansion s =2lT were calculated using Arlequin 3.0 by a atory power, followed by the ‘isothermality’ and ‘maximum generalized least-squares approach (Schneider & Excoffier, temperature of the warmest month’. 1999), under models of pure demographic expansion and Mapped outputs of the Maxent analyses are shown in spatial expansion (Ray et al., 2003; Excoffier, 2004). The Fig. 2. As a threshold between suitable and unsuitable condi- probability of the data under the given model was assessed by tions from continuous Maxent output we applied the lowest the goodness-of-fit test implemented in Arlequin 3.0. 10% training presence (Liu et al., 2005), corresponding to a Parameter confidence limits were calculated in Arlequin 3.0 logistic output probability of 0.34 in our analysis. Projecting through a parametric bootstrap (1000 simulated random the model onto the CCSM scenario (Fig. 2b,d) describing samples). conditions for 21 ka (LGM) suggests a much more continuous and broader potential distribution across Eurasia–Alaska and less markedly enlarged suitable habitat in western North Estimation of time to most recent common ancestor America (Fig. 2d), indicating that most areas currently occu- and reconstruction of demographic histories pied by the P. phoebus complex were connected at that time by We used the software beast 1.5.6 (Drummond & Rambaut, corridors of suitable habitat. 2007) to estimate time to most recent common ancestor (TMRCA) of major mitochondrial clades under a MCMC Definition of operational population units Bayesian approach. Both a constant size coalescent tree-prior and a flexible Bayesian skyline tree-prior, accounting for Results from the SDM analysis were used to identify opera- changes in population size, were applied in separate analyses. tional regional ‘populations’, by grouping those samples The substitution rate was not sampled and fixed to 1. collected within a single large-scale area of suitable habitat. Parameters were subsequently converted into demographic In the Eurasian section (including areas occupied by P. phoebus units by the calibrated rate obtained in the im analyses. In in Alaska and Yukon) four regional populations were identi- order to transfer the rate calibration (obtained on the fied (Fig. 1a): Alps (n = 26), Urals (n = 8), Alaska (n = 23) Siberian–Alaskan A.3 data set, see ‘Samples’ in Materials and and Siberia (n = 49). Four isolated samples from Sakhalin Methods) across different data sets, we compared results from Island, Kamchatka and six from far-eastern Siberia were separate beast analyses as detailed in Appendix S3.2. More- included in the Siberian unit. Samples of P. bremeri were over, as the im software (used to calibrate the substitution rate) distinguished from Siberian samples of P. phoebus only for the only implements the HKY (Hasegawa–Kishino–Yano; Haseg- presentation of phylogenetic analyses. In the North American awa et al., 1985) model for nucleotide evolution, and does not section (Fig. 1a), we distinguished populations from Sierra allow for partitioning into codon positions, we checked the Nevada (P. behrii), Klamath Mountains (northern California consistency of results across substitution models by perform- and southern Oregon) and southern (S) Rocky Mountains ing separate beast analyses (under a constant tree-prior) (populations south-east of the Wyoming Basin). Two locations under the codon-partitioned model indicated by AIC and an in the Klamath Mountains were lumped together with other unpartitioned HKY model. samples in the same region despite being separated by c.13km beast 1.5.6 was also used to explore the demographic of predicted unsuitable habitat. Another continuous-habitat histories of population units by running the MCMC analysis stretch included all sampling sites in northern and central under the Bayesian skyline tree-prior (piecewise, with 10 (NC) Rocky Mountains and samples from northern Coast intervals) and using Tracer 1.5 (Drummond & Rambaut, Range in State, USA and , 2007) to reconstruct the Bayesian skyline plot (BSP; Shapiro Canada. This unit was split into separate northern (N) and et al., 2006). All beast simulations were run on the Cornell central (C) units in some analyses, with the N Rocky Mountain University computation facility (http://cbsuapps.tc.cornell. ‘population’ including those sampling sites that were covered edu). Each model was run three times independently for 107 by the maximum extent of Cordilleran ice sheet c.18ka to 3 · 107 generations. Convergence was checked and appro- (Fig. 2d).

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Within the North American clade, a basal position is Phylogenetic reconstruction occupied by some haplotypes found in populations of The combined data set including our new sequences and P. smintheus from the Klamath Mountains, which also carry published sequences of P. behrii and P. smintheus (DeChaine & other clustered haplotypes, closely related to or shared with Martin, 2004; Schoville & Roderick, 2009; see Fig 1b), spans samples from the Rocky Mountains. Parnassius behrii is 1164 bp and includes 178 unique haplotypes. The AIC divided into two clades, but the analysis does not support its indicated GTR+I+G, GTR and GTR+I+G as the best-fitting monophyly. A large, weakly defined haplogroup is private to models for codon positions 1, 2 and 3, respectively. The ML the S Rocky Mountains. All other haplotypes found across the reconstruction (Fig. 1b) returned very few robust nodes. Rocky Mountains are connected by very short branches (one Nonetheless, the topology confirmed that P. ruckbeili (Michel or two substitutions) and their relationships are not resolved. et al., 2008) is a highly differentiated taxon and suggests No clear phylogeographical signal is detectable among reciprocal monophyly of Eurasian–Alaskan and North Amer- Eurasian–Alaskan haplotypes, as none of the recognized taxa, ican populations, with the monophyly of the North American nor any of the regional populations, carries one or more lineage supported by LR-ELW > 90. The average evolutionary distinctive clades. However, some genetic structure does exist, distance between Eurasian–Alaskan and North American as Alpine (P. sacerdos), Alaskan and Ural populations contain haplotypes was 0.030 (SD 0.006). mostly private haplotypes, while more extensive sharing

Figure 3 Isolation with migration (IM) analyses on 10 pairs of neighbouring operational population units of the Parnas- sius phoebus complex. Marginal posterior distributions of IM parameters, estimated on five pairs of neighbouring population units. Population size parameters are given only for the no-migration model. Parameters are scaled by the substitution rate (0.086 substi- ) tutions/site Myr 1) calibrated by assuming complete interruption of gene flow between Alaska and Siberia 10,000 years ago (see text). The validation point assumes divergence of N Rocky Mountain popula- tions from C Rocky Mountain populations after deglaciation 12,000 years ago (see text).

1064 Journal of Biogeography 39, 1058–1072 ª 2012 Blackwell Publishing Ltd Holarctic mtDNA phylogeography of Parnassius phoebus occurs between Siberian populations of P. phoebus and P. bremeri. )

Isolation with migration model and calibration 015 ) of a COI substitution rate )( ) 0 (0–0.000043) (0–0.000008) (0.000005–0.000028) 0 (0–0.000044) m pop1 to pop2 Parameters of the IM model were estimated for all neighbour- ing pairs of operational population units with more than 20 sequence samples. Results are shown in Fig. 3 and Table 1. The )( results for the Sierra Nevada–Klamath Mountains analysis are ) )( ) (0–0.000054) (0–0.000064) not presented, as a nearly identical sample set was previously m pop2 to pop1 analysed by Schoville & Roderick (2009). Analyses returned

closed marginal posterior distributions within the priors for all ulation pair, one incorporating a migration parameters of the no-migration models. However, in a few complex. Highest posterior estimates and 90%

cases the data were not sufficient to properly estimate all ) symbol indicates parameters whose value or HPD )

parameters of the migration model. )( The highest marginal posterior density of the divergence ) ). The ( time parameter (t) for the Alaska–Siberia split under the no- 1 e ) N migration model was at 0.715 [90% highest posterior density ancestral (HPD) 0.325–1.145]. Assuming a divergence time of 10 ka Parnassius phoebus (see above), this estimate corresponds to a substitution rate ) of 0.086 (0.039–0.139) substitutions/site Myr 1. All diver- gence estimates under the migration models (initial diver- gence) fall between 16 and 32 ka, with the exception of the divergence time between populations from the N Rocky Mountains and C Rocky Mountains The latter was estimated e N at 11.6 (90% HPD 5.6–23.8) ka, closely matching the pop2 expectations of post-glacial recolonization (12 ka, see above), and providing an independent validation of the calibrated substitution rate. Estimates under the no-migration model

(complete interruption of gene flow) span between 8 and , forward in time, in individuals generation m 25 ka (Table 1). ) 62,521 (10,916–390,011) 18,194 ( ) Demographic analyses and estimation of TMRCA e N Fu’s FS and Rozas & Ramon-Onsins’ R2 statistics were pop1 calculated for all operational population units containing at ) and migration rates ( least 20 sequences (Table 2). The null hypothesis of constant e N population size could be rejected, according to the FS statistic, for populations from Siberia, the S Rocky Mountains and the ) 69,860 (32,791–141,145) 226,687 (163,956–317,933) 92,671 (43,563–398,406) 0 (0–0.000018) 0.000001 ) 223,289 ( ) N and C Rocky Mountains (bold in Table 2), but the more ) conservative R2 statistic only indicates significant disequilib- Divergence time (years ago) rium for the N and C Rocky Mountain populations taken 10,000 (4586–16,158) 334,437 (128,019–1,350,648) 169,699 (48,627–1,118,428) 47,966 (14,886–126,255) together. Mismatch distribution of all groups with significant

FS was examined under the sudden expansion model (Table 2). Goodness-of-fit tests did not show significant deviations from expected distributions, so that the parameter s =2lT could be used to estimate the time (T) elapsed since population expansion (Table 2). Applying our calibrated substitution rate, all demographic expansions are traced to 18–38 ka [95% confidence interval (CI) spanning from 8 to 51 ka] (Table 2, Fig. 4). The posterior median TMRCA of Eurasian–Alaskan popu- Results from isolation with migration (IM) analyses on 10 pairs of neighbouring operational population units of the lations under a constant size tree-prior was estimated at 188 (95% HPD 103–297) ka under the partitioned model and at SiberiaSiberia Alps Alps no mig mig 14,605 (9102–22,649) 22,367 320,255 (7550–53,694) (123,476–1,235,112) 112,766 (60,443–296,800) 228,239 (83,898–1,027,524) 31,575 (9522–88,709) 62,247 (26,162–139,830) 6515 (501–134,818) 0.000014 could not be reliably estimated due to insufficient data. Calibration time is underlined. Two different models were applied in the analysis of each pop parameter (mig) and one that did not (no mig). NC Rocky MtsNC Rocky Mts S Rocky Mts S Rocky Mts no mig mig 13,258 (9256–18,594) 31,602 195,613 (20,596–46,445) (131,925–280,530) 150,122 (98,565–216,843) 322,989 (232,006–441,267) 78,346 259,301 (36,225–162,590) (183,482–350,284) 31,170 (4212–110,359) 0 (0–0.00001) 0.000 Table 1 pop1 pop2 Model HPD (in parentheses) of divergence times, population sizes ( C Rocky MtsC Rocky Mts N Rocky Mts N Rocky Mts no mig mig 8588 (5086–14,592) 11,590 (5587–23,764) 213,783 (124,106–356,305) 172,147 (104,890–313,068) 48,842 (23,220–90,477) 34,429 (15,213–71,261) 23,576 (6672–76,065) 17,348 (1335–68,948) 0.000013 Klamath MtsKlamath Mts NC Rocky Mts NC Rocky Mts no mig mig 25,265 (15,593–41,108) 81,265 29,101 (35,643–163,956) ( 238,093 (169,659–326,487) 89,503 (34,058–232,073) Alaska Siberia mig 16,299 ( 135 (95% HPD 80–191) ka under the HKY model. The Alaska Siberia no mig

Journal of Biogeography 39, 1058–1072 1065 ª 2012 Blackwell Publishing Ltd V. Todisco et al.

Table 2 Nucleotide and haplotype diversity, tests of demographic equilibrium and mismatch analysis for nine regional population units of the Holarctic Parnassius phoebus complex.

)2 Population Data set (bp) nHh p (· 10 ) FS R2 ss(5%) s (95%) T (ka) T (5%) T (95%)

Eurasia Alps A.3 (824) 26 13 0.935 ± 0.024 0.410 ± 0.027 )2.04 0.14 – – – – – – Alaska A.3 (824) 23 10 0.893 ± 0.040 0.331 ± 0.039 )0.49 0.3 – – – – – – Siberia A.3 (824) 47 19 0.885 ± 0.035 0.350 ± 0.046 )9.45 0.12 2.82 1.3 4.23 19.9 8.0 29.8 North America Klamath Mts A.2 (512) 37 10 0.847 ± 0.031 0.526 ± 0.076 )1.36 0.11 – – – – – – Sierra Nevada A.2 (512) 76 11 0.743 ± 0.039 0.804 ± 0.024 1.24 0.14 – – – – – – S Rockies A.2 (512) 171 29 0.913 ± 0.010 0.504 ± 0.026 )15.46 0.06 2.71 1.51 3.61 38.5 21.4 51.2 NC Rocky Mts A.2 (512) 308 35 0.831 ± 0.012 0.303 ± 0.015 )27.71 0.03 1.57 1.33 1.75 22.3 18.9 24.8 C Rocky Mts A.2 (512) 133 17 0.760 ± 0.029 0.247 ± 0.023 )10.17 0.04 1.29 1.04 1.61 18.3 14.8 22.9 N Rocky Mts A.2 (512) 119 15 0.785 ± 0.019 0.253 ± 0.015 )7.64 0.05 1.46 1.16 1.8 20.7 16.5 25.6

N, number of sequences; H, number of unique haplotypes; h, haplotype diversity (±SD); p, nucleotide diversity (±SD); FS, Fu’s FS statistic; R2, Rozas and Ramon-Onsins’ R2 statistic; s, sudden demographic expansion parameter, with 5% and 95% confidence limits; T, true time since population expansion, from s =2lT (where l is the substitution rate per gene). Significantly small values of FS and R2 are in bold.

discrepancy between the two models is due to a single between North American and Eurasian–Alaskan sequences is divergent published sequence from Mongolia (FJ756817.1). estimated at c. 3.0%. As nucleotide divergence between the two The posterior median TMRCA of the remaining sequences was lineages is considerable, the fast population-based rate, estimated at 69 (95% HPD 42–105) ka under the partitioned calibrated from the most recent opening of the Bering Strait, model and at 67 (95% HPD 42–99) ka under the HKY model, is probably not appropriate, and a conventional ‘phylogenetic’ ) thus demonstrating that the substitution model only affected rate for COI of c. 0.01–0.017 substitutions/site Myr 1 (Brower, estimation of that single long branch. The BSP tree-prior 1994; Quek et al., 2004; Papadopoulou et al., 2010) may be analysis was therefore only run under the simpler HKY model. tentatively assumed. Under this assumption, the colonization The estimated posterior median TMRCA of the Eurasian– of North America by P. phoebus may have occurred about 1.5– Alaskan clade under BSP was 122 (95% HPD 75–180) ka and 0.8 Ma, similar to the estimates of Vila et al. (2011) for other 52 (95% HPD 30–80) ka when FJ756817.1 was excluded. The cold-tolerant butterflies. TMRCA of the North American clade was estimated at 86 Our samples of P. smintheus from Yukon and British (95% HPD 59–125) ka under the constant-size partitioned Columbia share their haplotypes with samples from the model, 84 (95% HPD 59–116) ka under the constant-size HKY northern (Washington and Montana). This model and 76 (95% HPD 39–97) ka under the BSP HKY result indicates that this taxon expanded northwards following model. the retreat of glaciers after the LGM (Fig. 2c,d). The North The BSP calculated for regional ‘population’ samples American ice sheets therefore acted as a massive geographical containing more than 20 sequences are shown in Fig. 4. barrier between P. smintheus and P. phoebus during the last Most of the plots do not provide clear support for population glacial period. Although this geographical barrier has not been size change, as the 95% HPD of mitochondrial effective size in existence since at least 11 ka (Dyke et al., 2003), all samples overlaps the time interval spanned by the reconstructions. from the near-contact zone of P smintheus and P. phoebus in

However, all samples with significant FS statistics show a Yukon (Fig. 1b) displayed mtDNA consistent with their trend of recent growth, with a start that approximately morphology, with no evidence of hybridization, thus suggest- coincides with the estimated expansion from mismatch ing that reproductive isolation may exist among the two analysis (Fig. 4). lineages. Within the two continents, none of the recognized taxa corresponds to a single monophyletic clade (Fig. 1b), although DISCUSSION P. behrii shares no haplotypes with P. smintheus and most haplotypes carried by Alpine populations ascribed to P. sacerdos Mitochondrial DNA phylogeography of the Parnassius are private to this area. On the other hand, P. bremeri shares phoebus complex four out of its seven haplotypes with populations of P. phoebus Our data, which present a large new sample from Eurasia and from Siberia and Alaska (Fig. 1b). Our results thus indicate Alaska, confirm the monophyly of North American taxa that evolutionary divergence among nominal taxa within the (P. smintheus and P. behrii; Schoville & Roderick, 2009) and P. phoebus complex is very recent (as already noted by suggest that Eurasian–Alaskan sequences also represent a Schoville & Roderick, 2009; for P. smintheus and P. behrii). In monophyletic lineage. The mean nucleotide divergence particular, P. bremeri is even less mitogenetically distinct than

1066 Journal of Biogeography 39, 1058–1072 ª 2012 Blackwell Publishing Ltd Holarctic mtDNA phylogeography of Parnassius phoebus

other regional populations ascribed to P. phoebus (e.g. from Alaska or Urals), despite its wing pattern and antennae being so distinctive that it has long been regarded as a separate species (Weiss, 2005). Our result is consistent with the findings of populations with intermediate characters between P. phoebus and P. bremeri (Korshunov & Gorbunov, 1995; Gorbunov, 2001) in supporting very weak neutral genetic differentiation of P. bremeri from P. phoebus. On the other hand, more detailed data on the distribution of genetic and morphological variation would be needed to appraise the sharpness of the cline in morphological characters and the forces that shaped it.

A reappraisal of refugial models

Although precise molecular dating from a single mtDNA locus is not feasible (Howell et al., 2008), we have shown that a fast ) COI substitution rate (0.086 substitutions/site Myr 1)is consistent with at least two recent, datable evolutionary events in the P. phoebus complex (i.e. the interruption of gene flow due to the opening of the Bering Strait and the post-glacial colonization of a sector of the Rocky Mountains). This calibrated rate is c. 4–20-fold faster than the conventional ‘phylogenetic’ rates that are still widely applied in phylogeo- graphical studies of (e.g. Hammouti et al., 2010; Schoville et al., 2011). In fact, our estimated rate is remarkably similar to the rate estimated by Gratton et al. (2008) from similarly recent events in P. mnemosyne (median 0.096), as well as that adopted by Schoville & Roderick (2009) in a previous study on P. smintheus/behrii and by Todisco et al. (2010) in P. apollo. Assuming the validity of our calibration, all divergences between regional population pairs date between 10 and 35 ka (up to 70 ka including confidence intervals), that is, to well after the last interglacial period (c. 130 ka; Fig. 2e). A similar figure (48 ka, 90% HPD 21–87 ka) was also estimated by Schoville & Roderick (2009) for the divergence time between P. behrii and P. smintheus (mostly samples from the Klamath Mountains). The oldest divergence estimates involve the southernmost North American populations: Sierra Nevada and S Rocky Mountains (under the migration model, Fig. 3). Moreover, the mitochondrial TMRCA of all North American populations dates back to no more than 125 ka. Schoville & Roderick (2009) reported a much higher estimate of 220 (96– Figure 4 Demographic reconstructions. Bayesian skyline plots 638) ka for the TMRCA of North American populations. (BSPs) for nine regional population units within the Parnassius While our results fall within their confidence interval, the phoebus complex: mean (thick lines), median (thin lines) and 95% results are not directly comparable since their TMRCA highest posterior density (HPD) interval (grey areas). BSPs interrupt estimate was based on concatenated pseudo-haplotypes from at median time to most recent common ancestor (TMRCA), except mtDNA and nuclear data as well as the assumption of a for Siberia, whose median TMRCA falls outside the graph limits. uniform posterior distribution of substitution rate from 0.029 Vertical lines indicate estimated value (solid) and 95% confidence ) to 0.210 substitutions/site Myr 1 (cf. in Gratton et al., 2008, limits (dashed) of the mismatch expansion parameter s for those this interval was reported as the 95% HPD of a sharp populations with significantly negative FS. A deuterium excess (deltaD) curve (proportional to global temperatures) from ice core posterior). data (EPICA, 2004) is presented for comparison. [Correction added The estimated Eurasian–Alaskan TMRCA is higher (c. 100– after online publication, 23 April 2012: Some vertical, dashed lines 200 ka, according to different substitution models). However, indicating 95% confidence limits and some thick and thin lines re- this is due to a single sequence from Mongolia (FJ756817.1). In presenting mean and median were missing from the original figure.] contrast, the TMRCA of the remaining Eurasian–Alaskan

Journal of Biogeography 39, 1058–1072 1067 ª 2012 Blackwell Publishing Ltd V. Todisco et al. sequences is estimated at less than 100 ka. If multiple refugial Schoville & Roderick (2009), although under different areas surviving through several glacial/interglacial cycles had approaches, contrasted the multiple-refugia model with the existed – as argued by DeChaine & Martin (2004, 2005, 2006) same model of the range-expansion process, which they refer and postulated by Schoville & Roderick (2009) – divergence to as the ‘ancestral radiation’ model (sensu Knowles, 2001). In between regions should have started before (or during) the last this model, any single sampling site of a phylogeographical interglacial period, and the mitochondrial MRCAs should date study is regarded as the product of a completely independent to a time before it. We find no evidence of such scenarios in colonization from a single ancestral source and does not our data from North America. For Eurasia–Alaska, an old exchange migrants with any other site, irrespective of its divergence of a single haplotype in Mongolia implies that some geographical location. Consistently, a key prediction of this polymorphism, presumably pre-dating the last interglacial, model is that neither isolation by distance nor any regional survived in P. phoebus populations of central Asia. However, geographical structure should be observed. As both these we did not find divergent lineages with distinct centres of features are indeed present in the mtDNA data of the North distribution, which could have been evidence of separate American populations, DeChaine & Martin (2005, 2006) and refugia. The deep mtDNA divergence is confined to a single Schoville & Roderick (2009) discarded the ‘ancestral radiation’ locality (Khangai Mountains in central Mongolia), which also hypothesis and considered multiple refugia to be supported by harbours a sequence belonging to the main Eurasian–Alaskan the data. mtDNA clade. This could imply either: (1) that a separate, In contrast, we argue that the model of ‘ancestral radiation’ ancient refuge existed in central Asia, and was later invaded by presented above is not a realistic approximation of a recent newcomers carrying the main Eurasian–Alaskan mtDNA clade; range expansion. Because a single adult Parnassius will not or (2) that the area is a long-standing refuge and a probable usually travel more than a few kilometres (Roland et al., 2000) source for the recolonization of the whole Eurasian–Alaskan a range expansion across several thousand kilometres should range. New data from Mongolia would be needed to clarify this take (at least) several hundred generations. During this topic. However, under both hypotheses (1) and (2), all process, each new empty area will be colonized from the remaining mtDNA diversity found from the Alps through neighbouring ‘leading edge’ (Hewitt, 1996). In the course of Alaska would result from long-range dispersal from a single this process, geographical structure may arise as genetic source during the Wurm/Wisconsin glacial phase. lineages may ‘surf’ to different frequencies in different regions Therefore, we argue that no clear evidence of multiple (e.g. Excoffier & Ray, 2008; Petit, 2011). Moreover, popula- refugia older than 100 ka can be recovered in the mitogenetic tions in closer spatial proximity are likely to exchange more structure of the P. phoebus complex. Indeed, all evolutionary migrants than populations at the opposite ends of an events that left traces in the mtDNA diversity within each of expansion front (Ibrahim et al., 1996), so that a pattern of North America and Eurasia–Alaska occurred within the isolation by distance should be observed. Therefore, a com- Wisconsin/Wu¨rm glacial cycle. On both continents, the pletely random distribution of genetic diversity over space dispersal of mtDNA genomes across the current range of the cannot be regarded as an expectation of a recent range species complex occurred between 100 and 10 ka (most likely expansion, and the presence of some geographical structure at after c. 50 ka). This scenario is consistent with the recon- a regional level is not, per se, evidence of multiple refugia. structed distribution of the climatic niche under LGM conditions (Fig. 2). During this period, the potential distribu- Patterns of genetic structure in North America tion of the species extended nearly continuously over most of and Eurasia–Alaska the central-eastern European lowlands, southern Siberia, Beringia and present western United States, with the significant The different geography of the two continental masses exception of most of the Great Basin. We thus argue that probably played a significant role in shaping the current mtDNA data indicate that population history of the P. phoebus distribution of mtDNA diversity. In fact, despite greater complex in both continents is better interpreted as a recent geographical separation among extant populations in Eurasia– range expansion during the late cold phases of the Wisconsin/ Alaska than in North America, geographical structure is Wu¨rm glacial cycle (50–18 ka; Fig. 2e) followed by Holocene apparent in the latter and almost absent in the former. During fragmentation, rather than as a long history of stable refugia the LGM, the North American ice sheets covered the present undergoing several cycles of fragmentations and connection. Canadian provinces of Alberta and British Columbia and the Previous studies, although recognizing that the observed northern areas of Montana (Fig. 2d). Our SDM analysis shallow, ‘star-like’, phylogeny was consistent with a recent predicts that corridors of suitable climate circled the arid range expansion (Schoville & Roderick, 2009), discarded this central basin and connected all main mountain ranges, partly hypothesis in favour of a multiple-refugia model deeper in covered by glaciers. These corridors may have been almost time. This focus upon older events derives from an assumption continuous north of the central basin, while they were more of a slow ‘phylogenetic’ substitution rate (Brower, 1994), scattered in the south (Fig. 2d). This palaeoclimatic framework which suggested to DeChaine & Martin (2004) that the suggests a reasonable scenario for the recent population history coalescence of mtDNA lineages was very old (c. 350 ka). of P. smintheus/behrii. The distinctive features of the genetic Moreover, both DeChaine & Martin (2005, 2006) and structure of these populations are the homogeneity of northern

1068 Journal of Biogeography 39, 1058–1072 ª 2012 Blackwell Publishing Ltd Holarctic mtDNA phylogeography of Parnassius phoebus populations (Fig. 1b) and the increase of genetic uniqueness Our analyses, using an explicit calibration of recent COI south- and westward. The most likely dating of mitochondrial substitution rates, show that stable refugial models are largely TMRCA at c. 80 (50–120) ka and of the initial divergence of inconsistent with available data for the P. phoebus complex in P. behrii at c. 50 (20–90) ka (Schoville & Roderick, 2009) both North America and Eurasia–Alaska. More generally, we indicate the cold interval peaking c. 65 ka (Fig. 2e) as the most argue that the mitogenetic structure of the P. phoebus complex likely context for an initial spread across western North may serve as a model for the effect of Pleistocene climate America. Milder conditions between 55 and 30 ka (Fig. 2e) fluctuations on cold-adapted, moderately vagile invertebrates may have caused the isolation of Sierra Nevada populations, of the Holarctic region. while the onset of the LGM probably allowed enhanced A striking feature of the observed mitogenetic structure in connectivity and dispersal across the rest of the range. The im Eurasia–Alaska and in North America is the shallow conti- analyses, in particular, indicate ‘initial’ divergence among nental-scale phylogeny, associated with very weak geographical P. smintheus populations 30–20 ka and possible gene flow until structure (although with the partial exception of marginal c. 13 ka, suggesting that genetic connection between present populations in Sierra Nevada and southern Colorado) and refugial areas in the Klamath Mountains and Rocky Mountains high regional haplotypic diversity, contrasting with the gradually ceased with rising temperature at the Pleistocene/ currently highly fragmented distribution. This pattern con- Holocene boundary. trasts with that of several mesophile butterflies (e.g. Gratton, Demographic analyses are less straightforward, and under- 2006; Wahlberg & Saccheri, 2007; Gratton et al., 2008), that line differences among regional areas. Genetic signals of show highly structured mitogenetic diversity, despite fairly demographic expansion between 40 and 20 ka are evident in continuous current distributions. These data are consistent the S Rocky Mountains (Fig. 4), while N and C Rocky with a general model in which genetic structures of specialized Mountain populations show signals of later increase, consis- organisms with moderate dispersal abilities reflect ‘average’ tent with Holocene expansion into formerly glaciated areas Quaternary climate much more than their present distribution. (Fig. 2c,d). In particular, very recent (c. 10 ka) divergence of N In fact, climate similar to the present has been very uncommon Rocky Mountain populations from C Rocky Mountain pop- in the last 500 kyr, while much colder conditions were largely ulations (Fig. 3) is consistent with the former originating from dominant (Fig. 2e). Consequently, we argue that further the latter during post-glacial expansion, providing indepen- studies on the genetic structure of alpine butterflies at a dent support for the validity of our rate calibration. IM continental scale may reveal additional general properties of analyses also show that gene flow across the Wyoming Basin the distribution of genetic diversity in the Holarctic. did not cease before 20 ka, and most likely about 13 ka. These results therefore suggest that the importance of this depression ACKNOWLEDGEMENTS as a long-term barrier for alpine butterflies (DeChaine & Martin, 2004, 2005, 2006; Schoville & Roderick, 2009) may This work was supported by a Natural Sciences and Engineer- have been overestimated. ing Research Council of Canada Discovery Grant to F.A.H.S. In Eurasia–Alaska, no geographically restricted clades exist, and by a Tor Vergata University Grant to V.S. The authors are indicating that movement of genes was significant across the grateful to all who contributed by providing butterfly samples whole continent until very recent times. The SDM indeed and in particular to Gian Cristoforo Bozano, Costantino Della shows that a huge stretch of almost continuous suitable habitat Bruna, Ken Davenport, Alessandro Floriani, Enrico Gallo, Jiri existed from central Europe to Eastern Siberia and Beringia Klir, James Kruse, Kristine Mazzei, Paul Opler, Kenelm Philip, c. 21 ka. Our results therefore corroborate the view that the Umberto Nardelli and Carol Yoon. We thank Eric DeChaine east–west orientation of most Euro-Asiatic mountain ranges, and Sean Schoville for sharing their original data, as well as which determined isolation of temperate taxa in multiple Andy Warren and the Butterflies of America group for four refugia (Hewitt, 2000), may have, conversely, favoured the images. genetic homogeneity of cold-tolerant species (see Forister et al., 2004; Albach et al., 2006). REFERENCES

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Proceedings of the Royal statistical data analyses; E.V.Z. on preliminary DNA work; Society B: Biological Sciences, 269, 137–142. C.W.W. on collecting samples; V.S. on coordinating the Italian Swenson, N.G. & Howard, N.J. (2005) Clustering of contact team and assembling samples; and F.A.H.S. on assembling zones, hybrid zones, and phylogeographic breaks in North samples and coordinating the overall study. America. The American Naturalist, 166, 581–591. Swets, K. (1988) Measuring the accuracy of diagnostic systems. Science, 240, 1285–1293. Editor: Melodie McGeoch

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