flow, genetic drift, Pliocene-Pleistocene boundary Keywords: Chicago, IL 60637, USA. 5 Department of Ecology &Evolution, University Chicago, of 1101 E. 57th Street, 4 Patricia and PhillipFrost Museum Sci of Rosario, Carrera 24 No. 63c-69, Bogotá DC, 111221, Colombia. 3 Biology program, Faculty Natural of Sciences and Mathematics, Universidad del de Panamá. 2 Smithsonian Tropical Research Institute. Apartado 0843-03092, Panamá, República QC. 1 Department Biology,of McGillUniversity Linares This article is protected Allrights bycopyright. reserved. 10.1111/mec.12844 Acceptedof Record.versiondifferences and article Please between the cite this Version this as doi: through the copyediting, typesetting, pagination for publication accepted This articlehasbeen Article F.Arias Carlos and diversification Phylogeography of Article Article type:Original 16-Jun-2014 Date: Accepted Revised Date:14-Jun-2014 30-Jan-2014 Date: Received 3 ,

Eldredge Bermingham Eldredge cydno 1,2 , Camilo Salazar , Camilo , phylogeography, AFLPs,mtDNA, adaptive radiation, gene 2,4 2,3 and Owen McMillanW. ,

and its closest relatives: disentangling their origin Claudia Rosales and undergone full peer review buthasnot been review undergone fullpeer and ence, 3280 South Miami Avenue, Miami, FL. , 1205Ave. Dr.Penfield H3A 1B1, Montreal, and proofreading process, which may lead to 2 , MarcusR. Kronforst 2

5 , Mauricio , Mauricio among adjacent significant population structure at local scales, with strong genetic differences even ranges of the two radiations. Within probably geographic extremes of the same radiation that likely diverged from differentiation in the origin andestablishment of new adaptive forms population local and flow, gene intraspecific events, geoclimatic past of importance cydno/H.timareta Andes. Our genome-scan data indicate significant admixture among sympatric America, with asubsequent colonization of the eastern and western slopes of the this radiation originated in Central America or theNorthwestern region of South diversification that rely ongeological events in the Pliocene. The MtDNA suggest that melpomene scales. Both mtDNA and AFLP phylogenies suggest that loci, reveal acomplex history of differentiation and admixture at different geographic Our this variation to that within the closely related This article is protected Allrights bycopyright. reserved. Accepted Articlewe examine intraspecific variation across the Neogene tectonic and paleogeographic reorganizations in the generation of biodiversity, remain controversial. To test the relative importance Quaternaryof climatic change and biologists and naturalists. Yet, the underlying that factors have given rise tothis diversity The origins of the extraordinary diversitywithin the Neotropics have long fascinated Abstract Running title: [email protected] Apartado 0843-03092, Panamá,República Panamá. de Corresponding author: Carlos F.Arias. Smithsonian Tropical Research Institute.

data, which consist of both mtDNA and genome scan data from nearly 2250 AFLP prior to the Pliocene-Pleistocene boundary, consistent with hypotheses of Heliconius H. cydno and H. melpomene cydno phylogeography color pattern races. These genetic patterns highlight the H. cydno populations across the extensive geographic , both mtDNA and AFLP data indicate H. melpomene Heliconius cydno

H. timareta and radiation H. timareta . and

and compare H. cydno radiations. H. H. are

This article is protected Allrights bycopyright. reserved. Acceptedresemblance between distantly related species (i.e. between can look strikingly different (Turner 1976; Mallet and Gilbert 1995). The locally close where distantly related species often look identical and closely related species or races Geographic variation in these rings creates a complex and colorful tapestry color patterns, generating mimicry ringswhich coexist locally (Mallet and Gilbert 1995). 1965; Turner et al. 1979; Turner 1981). Most species converge ona handful of common 43 species, the group is astriking example of a modern adaptive radiation (Emsley in the generation biodiversity.of overWith 400 distinct color pattern varieties among its Quaternary climatic change and Neogene tectonic and paleogeographic reorganizations Heliconius phenotypic variation arises and accumulates in the Neotropics. In this respect, The study of recent radiations provides a unique opportunity tounderstand and test how promote speciation, the relative importance of each remains controversial (Rull 2011). 1996; Kirby et al. 2008). Despite the potential for these different geoclimatic changes to and promoting speciation (The Great American Biotic Interchange; Coates and Obando isolated plant and communities, likewise creating novel ecological opportunities years ago (MYA) changed climate and created aland corridor that joined previously Article ~3 million at ofPanama Isthmus the of closure the final that suggested mechanism third topologywith arange of new ecological conditions (Bush 1994; Elias etal. 2009). A diversification and speciation by bothisolating populations and creating anintricate during the Pliocene (Gregory-Wodzicki 2000; Hoorn et al. 2010) prompted 1969). Asecond hypothesis argued that the uplift of the Andes and its acceleration Pleistocene triggered diversification by isolating populations in forest refugia (Haffer models of high neotropical diversity proposed that climatic fluctuations during the region to major tectonic and/or climatic events (Rull 2011). For example, one of the first and, over the past four decades, a number of hypotheses have tied diversification in this dating back 150 years. Theregion has had a complex geological andclimatic history mechanisms responsible thisfor diversity havebeen the topic scientificof discussions The Neotropics contain some of the most biodiverse habitats on earth and the Introduction provide an excellent system tostudy the relative importance of

Dismorphia sp.,

Ithominae This article is protected Allrights bycopyright. reserved. Knapp and Mallet 2003; Flanagan et al. 2004; etWhinnett al. 2005; Hines et al. 2011; Neotropics has received little support as a phenomenon promoting diversification (see Pleistocene climatic changes created a series isolated of forest patches across the within a species and pattern convergence between species. However, theidea that ‘refugium/biotic’ drift model, is appealing because it can explain both pattern divergence of mimetic patterns observed today (Turner and Mallet 1996). This scenario, termed the combination of biotic (and possibly abiotic) forces leading to the geographical patchwork 1985; Turner and Mallet 1996). During periods of isolation, wing patterns diverged by a widespread populations into isolated forest refuges (Brown etal. 1974; Sheppard et al. theories hypothesized that Pleistocene climatic fluctuations separated previously geological events isolate populations promoting divergence. Some of the earliest Attempts to explain this paradox have often relied on explanations where climatic and/or Joron and Mallet 1998). yet pattern divergence is frequent (Mallet and Gilbert 1995; Turner and Mallet 1996; novel thatforms, eliminates istheforce same stabilizes existing that patterns selection sp. Acceptedexplain the origin of new phenotypes in divergent phenotypes (Mallet et al. 1998). However, natural selection cannot easily Articleselection removes non-mimetic individuals creating sharp transition zones between the maintenance of existing wing pattern diversity—strong frequency-dependent rare color patterns promotes mimicry (Müller1879). Natural selection can also explain wing differentpatterns of puzzling. Atthecore of this puzzle there is a paradox. Natural selection can explain why Nonetheless, the evolutionary processes that generate this phenotypic variation remain 2012). et al. Merrill al. 2011; et Merrill 2001; Naisbitet al. al. 2001; et Jiggins 1995; Mallet et al. 1998; Mallet et al. 2007) and interspecific signals (Mallet et al. 1998; importance of color as both intra- (Papageorgis 1975; Brown 1981; Mallet and Gilbert and its importance in speciation. Subsequently,we have learned agreat deal about the provided Darwinwith some of the most visually appealing examples of natural selection and Heliconius sp. Heliconius ) led to the original hypothesis of mimicry (Bates 1862) and Heliconius species should converge—strong selection against Heliconius . This is acomplex paradox—the

provide ageneral understanding of relative timing of diversification in this group within Here we reconstruct the history diversification of in examine variation in more species from across thelarger radiation. understand the geographic mosaic of pattern variation within how adaptive variation arises (see; Kronforst et al. 2012) and argues that to better Brower 2013 for altenative explanations). These studies are changing our ideas about consortium 2012; Kronforst et al. 2013; Martin etal. 2013) (but see Brower 2011; among closely related genome-wide studies paint an even more complex history with evidence for pervasive existingwing pattern variation (Flanagan et al. 2004; Quek et al. 2010). More recent diversify andit is argued that discordance has been interpreted as demonstrating that the two species did not co- biogeographic groups. Despite the strong phenotypic concordance, this genetic differ both in terms of the levels of standing variation and the relationship among major et al. 2004; Quek et al. 2010; Hillet al. geographic partitioning in extant genetic variation at most loci (Brower 1994; Flanagan pattern races. Previous molecular work onthe two radiations has demonstrated strong that the range of each species is composed of a nearly identical patchwork wing of species are distantly related, yet have undergone identical color pattern radiations, such et al. 1985; Brower 1994, 1996b; Flanagan et al. 2004; Hines et al. 2011). The two This article is protected Allrights bycopyright. reserved. Accepted ArticleHeliconius Most previous studies attempting toreconstruct the history diversification of among (Mallet 2010; Hines et al. 2011). particularly important in the establishment and spread of new phenotypes in this been experimentally explored in dispersal limitation and fine-scale population structure, something that has only recently occur in the absence of complete isolation (Mallet 2010). This mechanism presupposes An alternative hypothesis suggests that adaptive divergence in wingcolor patterns can Patel et al. 2011; Bennett et al. 2012; Ribas et al. 2012) (but see Brower 1994,1996b). species have focused on H. erato Heliconius Heliconius diversified first, and 2013). Genetic patterns inthe two co-mimics (Kronforst and Gilbert 2008) but may be species (The Heliconius cydno

and Heliconius H. melpomene H. melpomene Heliconius genome . Our objective is to isto objective . Our we need to “adverged” on (see Sheppard

Andes in Venezuela and Northern Colombia (Figure 1) both the Cauca and Magdelena valleys in Colombia and on the eastern slope of the from Ecuador and up into Central America as far assouthern Mexico. Itis also found in length polymorphisms (AFLPs) toconstruct a comprehensive phylogenetic and Here, we use mtDNA “barcode” gene cytochrome oxidase ( into the same mimicry ring as region (Brower 1996a; Giraldo et al. 2008; Mérot et al. 2013). Unlike Colombia to Peru and several new races have been recently identified across this geographic replacement of geographic range of diversification between biogeographic hypothesis for investigate theimportance of other mechanisms, such as hybridization and dispersal older origin thisfor diversification. Finally, we use population genetic analyses to Obando 1996) played an important role in the diversification of the group, we expect an 2000; Hoorn et al. 2010) and the closure of the Isthmus of Panama (Coates and events that occurred in this region, the final uplift of the North Andes (Gregory-Wodzicki cydno/ H. timareta/H. melpomene driven by isolation in forest refugia, we expect ayoung age (between 0-1 MYA) for likely before ~1MYA (Ravelo etal. 2004; Ribas et al. 2012). Thus, if diversification was of the isthmus of Panama. Recent climate models suggest that forest refugia were not occurred earlier in the Pliocene consistent with the final Andean uplift and/or the closure is correlated with major glacial cycles in the Pleistocene or whether diversification eleuchia iridescent blue/black with white or yellow markings andtypically mimics sympatric with Andes slopes of South and Central America. Both form a closely related complex with apartially overlapping distribution on the North This article is protected Allrights bycopyright. reserved. Accepted Articlecydno the context of the broader isvery closely to related both (see Figure 1), H. melpomene, Heliconius cydno H. cydno Heliconius timareta H. melpomene H. cydno H. cydno H. melpomene whereas and itsclose relatives radiation. In contrast, if the two major geological foundeasternthe on edgetheAndes of from H. melpomene extends along the western slopes of the Andes diversification. In particular, we test whether H. timareta and H. cydno and and is typicallyred and yellow andoften falls and H. timareta H. erato H. cydno and

. Heliconius timareta H. melpomene H. timareta radiations. Unlike (Figure 1). Co1 and ) andamplified fragment H. timareta are parapatric . The three species H. cydno and H. sapho is a isa H. timareta are broadly H. erato , which is . and The H. , H. H.

(Dawson etal. 1998). All were removed and bodies preserved in a 10% dimethyl sulphoxide (DMSO) solution individuals) and 3 allopatric races of sympatric and allopatric populations (n=84 individuals), aswell as, examined the genetic variation in asubset of 10 place the respect to the size, shape, and distribution of white andyellow pattern elements. To under Lamas’ recent classification (Lamas et al. 2004). Geographic races differ with geographic race, was examined anonymousfor nuclear variation across thegenome using Geneious Pro v5.5.4. Asubset of 194 individuals, between 4-8 individuals of each GeneBank (Table 4SM) and used as outgroups. All sequences were edited and aligned Eueides amplification conditions of Elias et al. (2007). Sequences from the related genera Universidad del Rosario in Colombia while specimens from Colombian localities were stored in the collection of M. Linares at individuals of colleagues from several locations (Figure 1; Table 4SM). Our sampling included 186 Three hundred and ten adult butterflies were collected by the authors or provided by Sampling and Molecular Data. Methods This article is protected Allrights bycopyright. reserved. Acceptedsubunit 1 [ “barcode” region, specifically the 5’ half of the mitochondrial gene cytochrome oxidase different molecular markers, mtDNA and AFLPs. sequencedWe the DNeasy Kit, according to the manufacturer’s protocol. Individuals were scored twofor (see Table 4SM). Genomic DNA was extracted from thoracic tissue using aQiagen specimens were stored in collections at the Smithsonian Tropical Research Institute Articleevolution of diversity theNeotropics in processes, local population differentiation, and intraspecific hybridization in the understanding of this radiation and a context to explore the importance of geoclimatic limitation, in the diversification of the group. Our genetic data provide abroader (3 individuals) and H. cydno Co1 Heliconius cydno Heliconius , 684 base pairs (bp)]. PCR was performed using the primers and radiation into a comprehensive phylogenetic framework,we also H. cydno Heliconius numata , representing 13 out of the 14 known geographic races and H. timareta .

H. timareta H. melpomene (8 individuals) were downloaded from (n=30 individuals) (Figure 1). Wings H. melpomene ( H. t. florencia

and other races from both both from races and and H. heurippa H. timareta H. t. subsp. nov) (n= 10 10 (n=

This article is protected Allrights bycopyright. reserved. checked by eye orremoved. Toconfirm settings. Each locus was visually inspected, and those with aweak or noisy signal were establishedWe a bin width of1.0 and the other scoring parameters were left at default was used,with light smoothing turned on and all other settings left at defaults settings. within-projectand normalizationwasenabled. advanced The peak detection algorithm (PE Applied Biosystems). Only thoseranging from 50 to 500 (bp) in length were scored, (Applied Biosystems). Fragments were sized andscored using ABI GeneMapper v4.0 3730XL DNA analyzer (Applied Biosystems) with GeneScan Liz-600 size standard EcoRI_CG_FAM/MseI_CAA. Reaction products were electrophoresed on an ABI EcoRI_CG_FAM/MseI_CTG, EcoRI_CC_FAM/MseI_CAT and EcoRI_CG_FAM/MseI_CAG, EcoRI_CC_FAM/MseI_CAA, EcoRI_CG_FAM/MseI_CAC, EcoRI_CG_FAM/MseI_CAT, primer combinations were used to generate fragments: EcoRI_CC-FAM/MseI_CTA, Reagent Kit from Invitrogen and following themanufacturer’s protocol. Eight selective using AFLPs(Vos et al. 1995). AFLP data was generated using the AFLP Core selected by the Akaike Information Criterion (AIC) using ModelTest v3.8 (Posada and Inference (BI). employedWe the GTR+I+G model of nucleotide substitution, which was Phylogenetic analyses for the “barcode” region were conducted using Bayesian shared absences. Branch support was estimated with 1,000 bootstrap replicates. reduces the influence of homoplasy by relying only onshared presences as opposed to 2007). used Nei-LiWe distances to build our phylogenetic hypothesis since this method particular, the different ways bywhich taxa canshare absences (Meudt and Clarke phylogenetic hypotheses resides in the degree of homoplasy present in the data and, in (Swofford 2000). A major limitation for the use of AFLP data in the reconstruction of A neighbor-joining tree was constructed for the AFLP markers in PAUP*4.0b10 Phylogenetic and Population Genetic Analyses. combinations (i.e.separate PCR r eight individuals per taxon were independently genotyped twice for all primer Accepted2007). These analyses cantake into account phylogenetic uncertainty in both the Crandall 1998). BI analyseswere conducted in BEAST 1.6.2 (Drummond and Rambaut Article eactions, but starting same DNA). repeatability and detect r

un-to-run variation, estimator estimator gene flow. estimatedWe overall levels of genetic differentiation by calculating the average number of pair-wise differences per sequence), population differentiation, and (Schneider et al. 2000) was usedto evaluate population nucleotide diversity ( of no differentiation by permuting genotypes between populations (10 This article is protected Allrights bycopyright. reserved. of races and populations concordant with the major geographic regions in the distribution complete data set. Population-level estimates were compared between color pattern Population genetic analyseswere conducted for both was basedon genealogies sampled every 8,000steps from the two independent runs. independent runs with 40,000,000 steps were performed for each run. The final analysis (ESS) was higher than 200. To ensure that the Markov chains mixed sufficiently, two inspection of the parameter trend plots and by verifying thatthe Effective Sample Size Mixing properties and a stationary distribution of the MCMC were assessed by visual parsimonious description of the phylogeographic diffusion process (Lemey et al. 2009). selection (BSSVS) that builds a Bayes factor, a test that helps to determine the most discrete geographic states. Finally, we usedthe Bayesian stochastic search variable 2002). AContinuous-Time Markov Chain model (CTMC) was usedto reconstruct exhibits the most clock like rate among commonly used mtDNA markers (Beltrán et al. combination with aYule speciation process, since it has been found that the Guiana (FG) and Brazil (BA). The analysis was run under a strict molecular clock in Central America (CA), North-East Andes (EAN), South-East Andes (EAS), French Northern Cauca valley (CVN), Southern Cauca valley (CVS), Pacific-West Andes (PW), melpomene major geographic features in the distribution of 2013). For the latter, we established ten discrete geographic states, concordant with the Hillet al. et al. al. Quek 2010; Quek 2004; et to years (similar million per divergence states on an unknown phylogeny. Inthe former, we assumed a rate of 1.5% pairwise the date of the splits in the tree and the ancestral reconstruction of discrete geographic calibration date and the reconstruction of ancestral range states. Here, we estimated

AcceptedH. cydno, H.timareta Article Θ : Northern Magdalena valley (MGN), Southern Magdalena valley (MGS), for Wright’s F for Wright’s and ST (Weir and Cockerham 1984). testedWe the null hypothesis H. melpomene (see above). ARLEQUIN v3.5 software H. cydno, H. timareta

mt DNA andAFLP loci on the and 5 permutations). H. Co1 л ,

calculated the for after 10,000 permutations. Additionally, historical demographic parameters were since there are only two populations). The significance of the Mantel testwas assessed population expansion, bottleneck, or heterogeneity mutation of rates. Fu´s F ran 10,000 coalescent simulations. Significant D values can be due to selective effects, estimated to examine departures from a neutral model of evolution. For each case, we groupings by taxon ( races for each taxon. Locus-by-locus AMOVAS were also run over theAFLP data, with a model that contained populations on different geographic regions and individuals of all geographic regions’, ‘among races within geographic regions’, and ‘within races’, using permutations (Excoffier etal. 1992) to test howgenetic variation was structured ‘among H. c.weymeri; hermogenes effect of isolation by distance within thevalleys andregions by grouping ( taking into account the curvature of the earth. performedWe the Mantel test by taxon Geodist http://www.plantevolution.org/en/downloads.html Accessed 2013 August 12,Joli 2008). estimated with the mtDNA were mined from our ARLEQUIN analysis andgeographic distances were lead to large negative F other hand, is very sensitive topopulation demographic expansions, which generally regions. also We used an analysis ofmolecular variance (AMOVA) with 10 This article is protected Allrights bycopyright. reserved. Furthermore, we estimated F

1967; Oksanen et al. 2013). Mean F genetic divergence by performing aMantel test using the R package geographic location. furtherWe investigated the effect geographic of distance on galanthus in three major biogeographic categories (West the of Andes: AcceptedH. cydno Article generates a matrix geographic of distances from latitude and longitude data by , , H. timareta/H. heurippa H. c.zelinde , H. c.lisethae the comparison for the East Andes Co1 geo.dist H. cydno data. Tajima’s D (Tajima 1989) and Fu’s F S and values(Fu 1997). function from thescript function R from and ST H. c. alithea , H. timareta/H. heurippa between different races and among different geographic H. c. wanningeri and ST values across all populations for both AFLP and H. melpomene ; Magdalena valley: ; and Cauca valley: H. cydno

geodist ). Furthermore, we evaluated the and H. melpomene raceswas not performed (Available on: H. pachinus H. c.cydn S test (Fu 1997) were were 1997) (Fu test H. c.cydnides vegan o, H. cydno , ) and H. c. H. c. 4 (Mantel

S , on the, on races and The best estimate of the number and nature of clusters. transformation step. used AIC We to assess the best-supported model, and therefore explained above. In all analyses, 100 principal components were retained in the data two same comparisons the for in STRUCTURE) (as clusters possible of range wide successive K-means clustering with increasing number of clusters ( number clustersof was assessed using the function package for the Rsoftware (http://adegenet.r groups that minimize the latter. This analysis was implemented in the variation into a between group and a within-group component, and attempts to find analysis (PCA) asaprior step to a Discriminant Analysis (DA). DA partitions genetic methodological approach that relies on data transformation using Principal Component 2005). also ranWe a Discriminant Analysis Principal of Components (DAPC), a This article is protected Allrights bycopyright. reserved. among individuals in 2 comparisons: 1) the complete data collection, loci. (Pritchard etal. our v2.1.4 on estimated AFLP 2000) admixture STRUCTURE We A Bayesian model–based clustering algorithm was implemented in the program Accepted Articleraces). Each run consisted 10of for the first comparison (all data) and between 1and 15 for the second ( comparing thelikelihood ratios in ten independent runs for ancestry(Falushetal. determined 2003). We thenumber ancestral of clusters, in the different populations are likely tobesimilar, probably dueto migration or shared suggested by the authors (Pritchard et al. 2000). This model assumes that frequencies ancestry) with theoption of correlated allele frequencies between populations, as alone. Both analyses were run under an admixture model (individuals may have mixed melpomene and H. timareta K was calculated using the / H. heurippa 5 iterations, after ashort burn-in period of 10 ) and 2) the color pattern races of -forge.r-project.org/; Jombart 2010). The ad hoc ad

find.clusters statistic K values between 1 and 30 Δ , which runs K K (Evanno et al. ). We covered a covered ). We H. cydno adegenet H. cydno H. cydno 4 iterations. ,

H. K

, by

high support assister to the collection individualsof including all One the in phylogeny. places two different in falling showed that 0.01075 and H. melpomene diversity for mtDNA differed between the species, with ahigher nucleotide diversity for This article is protected Allrights bycopyright. reserved. cydno Although closely related, we observed no shared mitochondrial haplotypes between H. cydno timareta of Ecuador, nearly all Colombian/North Ecuadorian Withinbackdrop the of the (MGN) races and southern Cauca valley (CVS); and c) athird, including northern Magdalena valley races (PW), both from Northern Magdalena valley (MGN);b) asecond, that combines Pacific-West Andes (EAN) races, including some haplotypes from largely concordant with major geographic regions: a) one formed by Northeastern westward splinting trend (Figure 2a). For melpomene lineage of 100% support in posterior pr melpomene examinedWe mtDNA (n= 303) and AFLP (n=194) variation from across the Phylogenetic andpopulation genetic analysis Results

AcceptedH. t. florencia Article and

lineage fell in an apparently derived lineage with individuals of collected from Central America and theMagdalena valley(see Figure 2a). H. melpomene H. timareta races from Brazil andFrench Guiana branching later and following a radiations, including a number of H. c.chioneus H. cydno π ( = 0.01131 respectively). Phylogenetic analyses (BI) for the and π H. c. cydnides = 0.01974) and similar estimates for H. pachinus H. t. and . occurs on the eastern slopes theof Andes, with subsp. nov from Southern Colombia (n=12). The remaining H. melpomene from Central America (CA), and H. melpomene obability (BI)for species monop H. cydno H. timareta collected in Costa Rica (except one; n=7), and a portion from the northern Cauca valley (CVN; Figure 2a). H. timareta clade. This group contains a geographically mixed individuals thatwe sampled were paraphyletic, H. cydno and and were reciprocally monophyletic. There was H. H. cydno individuals (n=11) collected from

timareta , three well supported clades were H. timareta

H. c.cydno radiations, the south H. cydno subspecies. Nucleotide H. c. weymer hyly (Figure 2a).A basal lineage has moderately and and and H.timareta H. c. wanningeri H. heurippa i from the i from H. Co1 H. cydno gene ( π and H. = / H. , H. number of haplotypes were shared between them (Figure 1SM). Regional F cydno cydno, H. melpomene andH. timareta There was strong genetic structure in both mtDNA and AFLP markers across all Brower 2011; Brower 2013). gainedcolor pattern elements from al. melpomene shared with individuals showed strong signatures of admixture with more than 50% of their genome timareta 3), whereas Andes showed an admixture pattern with and clear signal of admixture was detected between sympatric populations of three, which perfectly corresponded to the three radiations (Figure 3). Interestingly, a Over theentire dataset, the best estimate for the number of distinct populations ( H. t.florencia group (Figure 2b) that contained all but two ( this radiation (Quek et al. 2010). Similarly, the AFLPsshowed a third well-supported splitting in awestward trend (Figure 2b), a pattern observed in previous AFLP the Hierarchical analyses (AMOVA) on both mtDNA and AFLPs in varied from 0.008 to 0.955 for mtDNA and 0.051 to 0.435 for AFLPs (Table 1SM). This article is protected Allrights bycopyright. reserved. distinguishes geographyracialand designation. inseen As the mtDNA, AFLPtree clearly stronger resolution of the three radiations, clustering them by species designation, identifiedWe 2257 polymorphic AFLP loci between these taxa. These data showed

Accepted (2013), suggesting that Article H. melpomene H. cydno/H. timareta races were genetically structured from each other, even at the populations (EAS; green cluster; Figure 3). Moreover, two and H. melpomene H. melpomene from Colombia) Colombia) from H. melpomene H. cydno basal lineages occur farther tothe east with more derived lineages , which has been proposed as a species between . For instance, , fell within the within the , fell H. heurippa populations from eastern Andes were admixed with (one and H. timareta H. t.florencia H. cyndo H. melpomene populations (Figure 1SM). In particular, all is one of several cases where H. melpomene H. timareta specimens (Figure 2b). H. cydno (Figure 2b). However unlike the mtDNA tree, H. timareta and one (CA, PW, EAN; blue cluster; Figure Figure cluster; blue EAN; PW, (CA, (Pardo-Diaz etal. 2012) (but see lineage, consistent with Nadeau

populations western from the H. t. subsp. nov from Colombia and subsp. nov).Finally, H. cydno H. timareta Co1 H. timareta showed that H. melpomene locus where a ST H.

values trees of

H. H. has K H. ) was et

0.05; Figure 2SM). In contrast, > 0.05; Figure 2SM), while distance (IBD) in mtDNA (r This article is protected Allrights bycopyright. reserved. melpomene correlation between geographic distance and genetic divergence between geographic locations, most likely due to current gene flow. In fact, there was strong suggests thatsome regions the of genome are shared between species atspecific melpomene. regions of the genome that maintain differences between significantly associated with geography butnot taxa. This implies that there are specific cydno locus AMOVA found that 268 loci (11.8%) were significantly associated with taxon ( flow between resultsThese suggest thatdifferences among variation explained by geographic regions (mtDNA:~1%; AFLP: ~1%; Table 2SM). ~12%) and within races (mtDNA:~23%; AFLP: ~87%), with asmall proportion of the explained by variation among races within geographic regions (mtDNA:~76%; AFLP: timareta (AFLP) of the variation occurring between geographic regions (Table 2SM). In 44.75% (mtDNA) and 8.47% (AFLP) of the total, with only 17.52% (mtDNA) and 3.97% within races, whereas variation among races within geographic regions accounted for nearly 37.73% (mtDNA) and 87.56% (AFLP) of the total variation was due tovariation association between geographic and genetic distance (r AFLP Accepted(Table 2SM). These results suggest differences in current and past patterns of gene between the regions, with close to72% of the variation due to variation within races between geographic regions, whereas AFLP data suggested that just 12% occurs ArticleAFLP markers. mtDNA data suggested that nearly 87%of the total variation was AMOVA analysis on regions, but both had a minor contribution to overall genetic variance. Incontrast, geographical regions are asimportant as differences observed between geographic =0.104, p>0.05; Figure 2SM). Aseparate analyses of IBD for , H.timareta , similar to , similar populations at both AFLPs and mtDNA (r However, thestrong association of some loci with geography and not taxa H. melpomene and H. cydno, H. melpomene H. melpomene M, mtDNA M, H. cydno populations. Consistent with theseresults, alocus-by- genetic variation atboth mtDNA and AFLP data was better H. timareta =0.415, p<0.05; Figure 2SM), but not AFLP data (r showed a discordant pattern between mtDNA and populations showed significant isolation by ) butnot with geography, and 54 loci (2.3%) were populations did not showed significant H. cydno

M, mtDNA M, and M, mtDNA M, H. cydno, H.timareta H. timareta =0.536, r =0.536, =0.634, r H. cydno M, AFLP M, races within M, AFLP M, races by H. =0.811, P < =0.558, P H. and H. H. M, M, continuous sampling strategy (r p>0.05; Figure 3SM), but strong IBD was detected in the CV region with amore This article is protected Allrights bycopyright. reserved. geographic distance and genetic divergence on the Andes (rWest geographic region (West Andes, MG, and CV) showed noclear correlation between only exceptions were: a) with eight clusterscorresp and identified eleven groups (Figure 4SM). These clusters were sorted by phenotype, contained individuals from pachinus C11, and C12; Figure 4) were perfectly assigned to phenotypic color pattern races ( (DAPC) gave abestestimate of 13 for geography. The admixture pattern in the discriminant analysis of principal components them (Figure 2b). Assignment tests of these races showed clear correspondence with largely into phenotypic groups and by geography, butwithout clear resolution between Phylogenetic reconstruction of the AFLP data clustered individuals of (Arias etal. 2012). AFLP Divergence time estimates and demographic analysis were notdistinguishable (Pale-Blue cluster; Figure 4SM). cluster (Pale-Green cluster, Figure 4SM) and b) barinasensis comprised specimens from group of c. cydno from individuals from different races: two of the clusters (C7 and C8) included individuals and Acceptedbased on ageneral mole estimatedWe the time of divergence between Article =0.077, P > 0.05; Figure 3SM) andMG regions (r H. c.cordula H. cydnides , H. c. galanthus , H. c.zelinde, H. c.zelinde, H. c.alithea (Figure analysis 4).The STRUCTURE and ; Figure 4). theWhereas, other six clusters were composed of H. c.weymeri

H. c.galanthus and H. c.chioneus onding to eight different H.c. zelinde H. c. weymeri H. c.chioneus M, mtDNA M, cular to clock(similar Quek et al. 2010; Hillet al. 2013) ; theC2 cluster was formed by a combination of , H. c. hermogenes =0.387, r =0.387, and and K , . Seven of the clusters (C1, C5, C6,C9, C10 H. c. galanthus , H. c.cydno H. c.chioneus H. c.cydno samples; and finally the cluster C13 H. melpomene M, AFLP M, H. c.cydnides H. cydno

produced nearlyident M, mtDNA M, =0.516, P > 0.05; Figure 3SM) , ; cluster C3 was formed by a , H. c. cordula and H. c. lisethae individuals; cluster C4 color pattern races. The =0.592, r H. c. cydno and and M, mtDNA H. cydno/H. timareta H. c.weymeri and M, AFLP M, , H. cydno H. c. wanningeri, thatone form H. =0.464, r ical results, =0.216, races that M, H. H.

hermogenes [Maximum State Credibility (MSC)= MG 32% + CVN 22% + CA 16% = 70%; Figure 2a], 2a], Figure 16% =70%; +CA 22% +CVN 32% MG Credibility (MSC)= State [Maximum H. cydno/H.timareta nodes by color labeling following aBayesian approach. This strategy suggested that the annotated thetree brancheswith the most cydno respectively. Within and significantly negative F regions, North East Andes (EAN) and the Pacific Andes (PW),West we observed high and Fu’s F 5SM), consistent with recently expanding populations. In the case of populations showed narrow(3-7 mutations), smooth and unimodal distributions (Figure (Figure 5SM), consistent with old and stationary populations. In contrast, EAN and PW presented wide (9-12 mutations), irregular and multimodal mismatch distributions reflected in the shape of their mismatch distributions. The CA, MG and CV regions negative values for this statistic (Table 3SM). The difference in these two regions was This article is protected Allrights bycopyright. reserved. Demographic history assessed through two different neutrality tests, Tajima’s D (D ( diversities of the Magdalena and Cauca valley, andCentral America have the highest nucleotide CVS, MGS) revealed MSC levels < of 5% each (Figure 2a). Notably, the northern parts while theNortheastern Andes showed aMSC of 22%. The remaining regions (PW, cydno/H.timareta clade occurred 1.3 MYA (1.9%, 1.6-1 MYA). Haplotype diversitywas similar between at least 2MYA (3%, 2.4-1.6 MYA), while the first detectable split in Likewise, MYA). (2.5-1.7 years ago million ~2.1 least minimum age for the origin the of group near the Pliocene-Pleistocene boundary, at among Using this calibration and based on our mtDNA data, the mean pairwise divergence al. 2004). (Quek et years million per divergence pairwise rate 1.5% of a assumed that Acceptedlargely consistent with neutral processes (Table 3SM). For Article followedby H. melpomene S π , yielded slightly similar results. Variation within most regions andraces was followed by ) (Table 3SM). and H. c.cydnides H. cydno clade likely originated in the northwestern part of South America H. melpomene and H. c.lisethae S H. cydno/H.timareta values, with some races within these regions also showing races, the highest nucleotide diversity ( and , with 21.8%and 18.3% unique haplotypes, H. c. weymeri and probable range states of their descendent H. c. barinasensis H. c. clade was 3.2%, which places the . The lowest. The

H. melpomene H. cydno (Table 3SM). furtherWe π was for for was H. cydno/H.timareta in two geographic started diversifying π H. melpomene ) was for ) was for H. c. H. c. T ) , the H. presented a narrow (4 mutations) and unimodal distribution, as expected in arecent multimodal distributions, consistent with stable and old populations. However, PW between the regions, with CA, EASand FG showingwide (10-15 mutations) and 3SM). Mismatch distributions were generally congruent with differences observed race in the EAS region displaying anegative andsignificant value for this statistic (Table regions or races presented significant negative F negative significant presented races or regions expanding population (Figure 5SM). Finally, in This article is protected Allrights bycopyright. reserved. 2011, 2013 and Mallet blog: http://www. (Pardo-Diaz etal. 2012; The melpomene melpomene accelerated adaptation and speciation. Indeed, important source of evolutionary novelty by providing thegenetic raw material bothfor known tooccur (Mallet etal. 2007). This hybridization is increasingly recognized asan Hybridization among members of the significant negative F Pacific Andes (PW)West and the South East Andes (EAS) region, exhibited high and Acceptedradiations Interspecific gene flow andspecies boundaries amongrecent Heliconius natural selection that have been playin geological processes and local population differentiation within the context of strong demographic analyses suggest acomplex interplay among interspecific gene flow, related species, radiation and place our emerging genetic patterns within thecontext of the two closely presentWe the first comprehensive genetic examination of the Discussion Article old, stable populations (Table 3SM, Figure 5SM). distribution thefor EAN region waswide (15 mutations) and bimodal, consistent with

through the adaptive introgression of in many places across its range, was able to phenotypically track H. melpomene S values, where Heliconius and H. H. m.melpomene melpomene

g out over thepast ~2 million years. timareta. genome consortium 2012; but see Brower heliconius.org/2013/in H. timareta H. timareta Overall, our phylogeographic and S / values. In fact, the mismatch H. melpomene cydno

from Colombia was the only / silvaniform none of thegeographic , which mimics trogression- Heliconius cydno color pattern alleles alleles pattern color (MCS) clade is browers- H. H.

This article is protected Allrights bycopyright. reserved. Accepted isolation. timareta between pattern region, seems to have been retained (Nadeau et al. 2013). Genetic differences scale. Rather most of the nuclear variation from previously (Nadeau et al. 2013), this event did not lead to genomic admixture on a large tested experimentally in Colombia (Linares, unpublished). As has been pointed out timareta of origin the newly discovered and geographically proximal melpomene melpomene heurippa of races fewer associated DNA (RAD) sequencing analysis, which surveyed more nucleotide sites but diversification. This placement of timareta ArticleAlthough Our data suggest that them among differences heurippa discrimination between H. melpomene al. 2008). The genomic region that controls red color pattern variation introgressed from via hybridization between trait speciation (see Jiggins et al. 2008), et al. 2004; Mavárez et al. 2006; Melo et al. 2009). In one of the best examples of hybrid patterns play in mimicry andmating behavior in part-ii/). Moreover, hybridization canpromote speciation due to the dual role that color criticisms-part-i/ and http://www.heliconius.org/2013/introgression-browers-criticisms- mtDNA lineage, our AFLP data clearly place races (Figure 1SM), a pattern that suggests some level of reproductive reproductive of level some suggests that pattern a 1SM), (Figure races subsp. nov. race was discovered and is presently in the process of being H. heurippa clusters with racesof wing color pattern (red plus yellow bands) is in part caused by phenotypic H. heurippa H. heurippa into a . Thus, in this case, adaptive introgression from a red banded form of (Salazar etal. 2010; Pardo-Diaz etal. 2012) and strong mating H. cydno H. timareta . This possibility was suggested five years ago when the and ’s mtDNA haplotype falls on a derived branch of the H. heurippa H. melpomene

and (Melo et al. 2009; Salazar etal. 2010; Pardo-Diaz etal. 2012). H. timareta H. cydno H. timareta race with aphenotype very similar to H. timareta H. heurippa is more likely anortheastern race of and are greater than those observed among , H. cydno H. melpomene H. heurippa H. (Nadeau2013). et al. Inbothstudies, , to the exclusion of both was also found in a recent restriction Heliconius H. timareta and lab-created hybrids with a H. t.

has been proposed to have arisen H. heurippa subsp. nov form, likely led to the (Mavárez etal. 2006; Salazar et (Jiggins et al. 2001; Jiggins Jiggins 2001; al. et (Jiggins , save that around the color within the H. cydno H. cydno H. timareta H. cydno H. timareta and , perhaps H. H. H. H. H. H. . / H.

was notan F this case, knowledge of the genetic basis of color pattern suggests that this individual as much as 20% of their genome coming from the individual possessed a possible thatthe subsp. nov. Sympatric mtDNA haplotype, but largely possessed a from a mating between a female H. melpomene 2013). Similar to our AFLP data, these studies showed that sympatric species pairs of genome sequencing data (see Kronforst et al. 2013; Martin et al. 2013; Nadeau et al. consistent with recent explorations of evidence admixture for with either m. nanna admixture (~20%). In contrast, none of the three allopatric races of malleti melpomene recurrent gene flow among sympatric In addition to the above individual examples, there was also general evidence for backcrossing with either postzygotic reproductive barriers between This article is protected Allrights bycopyright. reserved. 50% of their genome shared with two species. Although our AFLPdata distinguished the three species radiations, there were There is clear genetic evidence for hybridization and admixture among thethree melpomene subsp. nov individual, in contrast, is easily distinguishable from the co-occurring populations. The genome scan data suggest that al.

Accepted in prep), but when matings occur, F Article H. timareta and , H. m. thelxiopeia H. m. plesseni race based on its wing color pattern. This individual had a (Colombia), 1 , but rather some later generation hybrid. There are strong pre- and and individuals that showed strong signatures of admixture with more than H. t. florencia H. cydno/ H. timareta racessympatric with H. melpomene H. timareta parental type (Sanchez H. m. vulcanus ] in the eastern Andes slopes showed a similar pattern of and individual was incorrectly identified. However, this H. m. melpomene H. t. florencia H. melpomene mtDNA haplotype and was more likely aF H. timareta genomic admixture based on RAD andwhole Heliconius melpomene and 1 males are fertile, viable and capable of and had patterns of admixture consistent with H. timareta H. t. florenciaH. t. H. t. H. timareta H. m. rosina or with a subsp. nov wing pattern phenotype. In – one et al. H. cydno H. melpomene (French Guiana)] demonstrate any H. cydno

H. melpomene in prep). prep). in and H. t. florenciaH. t. [ are perfect mimics and it is ] sympatric with H. m. ecuadorensis, H. m. (Figure 3). These data are H. melpomene gene pool (Figure 3). and races[ H. cydno/timareta H. melpomene and one male. male. The H. melpomene H. m. H. cydno (Sanchez 1 H. t. hybrid H. t. H. have [ H. et

cydno/timareta/heurippa This article is protected Allrights bycopyright. reserved. AcceptedH. melpomene as displacing the ancestral western slopes of the Andes and ultimately recolonized Central America, perhaps eastern slopes of the Andes and into the Magdalena and Cauca valleys, across tothe genealogy suggests thatthe lineage evolved in Central America and spread down the this case, the proximal position of the bulk of individual of mtDNA genealogywith 2006). The remaining individuals of galanthus the Pacific coast of Costa Rica, which is historically known tohybridizewith the geographically distant cydno particular, the difficult to reconcile based on geographic proximity or asimple colonization history. In 2011). Viewed in this light, the mtDNA data indicate a complicated relationship that is Articleet al. 2013) provide strong support for adistinct monophyletic maximum likelihood phylogeny constructed using aligned RAD sequence data (Nadeau that they are geographic extensions of the same radiation. Our data and a recent Giventhe allopatric distribution of H. cydno diversificationthe of mtDNA data, retain useful information about the potential origin and timing of Even within thecontext substantial of hybridization, our genetic data, particularly the Center oforiginand diversification behavior andecology. largely cluster sympatric taxa by recognized species boundaries based on morphology, substantial contemporary gene flow. Despite the ongoing gene flow, thegenetic data H. timareta / H. timareta / H. timareta (also from Costa Rica; comparably seeFigure 2 and 4a; Kronforst et al. H. pachinus, acquired color pattern alleles through introgression and began mimicking H. timareta ; whereas, mtDNA lineage (Figure 2). Most fall on a basal mtDNA branch with group is less than that seen across the H. cydno H. heurippa, lineage (Figure 2 and 3) (but see Brower 1996a, b; Brower H. pachinus and individuals cluster in two different regions theof broader H. cydno H. pachinus radiation. Overall thelevel ofmtDNA variation within the H. cydno wanningeri H. cydno largely phenotypic tracked variation in distantly H. timareta

. Along the way, thelineage that we now recognize H. cydno

. Heliconius pachinus and H. pachinus

galanthus cluster in a derived position theof H.timareta from the upper Magdalena valley. In

from Central America, one individuals on our mtDNA , it istempting to speculate is an incipient species from H. melpomene H.

H. cydno radiation. H. This article is protected Allrights bycopyright. reserved. MYA [(1.6 -1 MYA)]. Earlier studies, also using mtDNA and other nuclear markers, cydno/H. timareta Heliconius melpomene Time ofdiversification populations at the local scale. correlated with geography but not , consistent with current gene flow between colonization scenarios. Thus,locus-by-locus AMOVAs found that some loci were highly flow and do not provide the genealogical resolution needed to tease apart different 2b). In general, our AFLP data are more likely revealing contemporary patterns of gene relationships between races and/or geographic regions are not well supported (Figure timareta not obviously supported by our AFLP data. AFLP data clustered the northwest Andes or Central American origin ofthe However, al. 2013). (Hillet origin this supports America in Central haplotypes mtDNA In the for origination northwest of the Andes and later southward expansion has been proposed consistent with recent colonization and population expansion. Asimilar pattern of Northeastern Andes and the Pacific-West region showed mismatch distributions Cauca valleys, consistent with older and stationary populations. In contrast, the analysis found broad and multimodal curves for Central America, the Magdalena and probability aNortheastern for Andes origin northwestern part of South America and Central America, relative to a 22% posterior the for probability posterior 70% a suggests tree mtDNA The variation. of levels extant patterns. This scenario is also supported by theancestral area reconstruction and by haplotype tree and because contemporary gene flow is likely obscuring historical present mtDNA data, as there is little support for the internal branches within the mtDNA related Accepteddetectable split within the Pliocene-Pleistocene boundary, ~2.1 m Article H. erato H. erato H. sapho races mainly by phenotype and secondarily by geographic region, but the based on mtDNA and nuclear variation (Quek et al. 2010; Hill et al. 2013). al. 2013). Hillet 2010; et al. (Quek variation nuclear and on mtDNA based case, and as seen in our data, the presence ancientof and diverse and most likely started diversifying during thePleistocene [at least ~1.3 H. eleuchia H. melpomene and H. cydno/ . The exact order of colonization is notclear from the occurred around the same period. In contrast, H. timareta . In addition, the mismatch distribution illion years ago (2.5-1.7 MYA), andthe first populations began to diverge around

H. cydno/H. timareta H. cydno/H. H. cydno and radiation is H. H. colonization and re-colonization of Central America, perhaps facilitated by the closure of closure of by the facilitated perhaps America, ofCentral re-colonization and colonization clustered with Pacific races. This observed pattern suggests acomplex pattern of the North Andes (2-5 MYA; Hoorn et al. 1995; Gregory-Wodzicki 2000; Ochoa et al. Panama (~3 MYA; Coates and Obando 1996; Kirby etal. 2008) and the final uplifting of with thePliocene-Pleistocene boundary. Two events, the closure of the Isthmus of MYA) are roughly correct, diversification may betied to geoclimatic events associated This article is protected Allrights bycopyright. reserved. galanthus related to races from different geographic basins. In our analyses, above). Moreover, the other two races in Central America appeared more closely lineage may have evolved in Central America and spread into South America (see as an ancient lineage in the important role of both events. First, the placement of habitats that could promote diversification. Different lines of evidence suggest an Central America and South America, whereas the emergence of the Andes created new 2012) are particularly noteworthy. The former likely increased connectivity between If our time estimates for thesplit between certainly violated in multilocus species-tree approach, estimated that the divergence between (2012), using the Simonsen et al. (2011) Papilioninae calibration and a studies in that we used alarger sample racesof within within thePleistocene (1.5 MYA; Beltrán et al. 2002). Our study differs from those proposed that the differentiation between Accepted Articlegene flow (Knowles 2009; McCormack et al. 2009) - an assumption that is almost methods assume that the incomplete lineage sorting observed is not caused by current events appear much more recent than they actually are. This is because species-tree tree estimation methods in the presence of ongoing gene flow canmake some splitting H. melpomene Charleston and Hoyal recently, More 2004). et al. (Quek years million per divergence toQuekclock (similar et al. 2010; Hillet al. a different estimate of divergence per lineage per time. Here we used ainsect molecular was more closely related to eastern offorms occurred during the Pleistocene (around ~ 0.6 MYA). However, species- Heliconius H. cydno/H. timareta butterflies. H. melpomene H. cydno/H. timareta 2013) that assumes a rate of 1.5% pairwise diversification suggests that the

H. pachinus H. pachinus H. melpomene H. cydno and H. cydno and , while from Central America Heliconius c. H. melpomene and occurred earlier H. c.chioneus H. cydno H. cydno (~2.1 and and

al. 2008; Elias et al. 2009; Santos et al. 2009; Hoorn et al. 2010; Chaves etal. 2011). other animal groups (Jiggins etal. 2006; Wahlberg andFreitas 2007; Silva-Brandão et 2000 m). Coincident diversification with the rise of the Andes has been observed in higher altitudinal ranges in the understories of forests in the Andes (between 500 to with tropical lowland habitats (0 to 1000 m), while races of This article is protected Allrights bycopyright. reserved. geographic scale. The clustering we observe could be the result of discrete sampling four well-defined clusters, implying prominent population differentiation at avery small Within Genetic differentiation am differentiation. accurately estimate branching order, colonization history, and the timing of complex. Panama and the final rise of the Andes in the evolution of we observe derived taxa showed highland distributions (Hall 2005). This is exactly thepattern that elevation gradient, where old basal species are distributed in the lowlands andyounger riodinid butterfly related genera of Ithomiine butterflies was correlated with elevation range. Similarly, the For example, Elias et al. (2009) found that the origin and diversification of two closely melpomene Presently there are clear altitudinal differences between the distribution of the northern Andes (Van der Hammen et al. 1973; Hooghiemstra and Ran 1994). composition from tropical lowland to a montane forest type during the late Pliocene in the Isthmus of Panama. Additionally, palynological datasuggest achange in plant Acceptedgradients separating them. Yet, both STRUCT less than ~500 Km (Figure 1), with noclear geographic barriers or environmental Magdalena valley there are four different color pattern races of among geographic regions but significant local population differentiation. Notably, in the differences among adjacent races. Similarly, AFLPdata revealed widespread gene flow scales. Although major mtDNA lineages are spread widely, there are strong genetic Article Heliconius

However

and and our results suggest a mixed role of both the closing the of Isthmus of Ithomiola H. timareta species, our data suggested strong differences among races at local , more comprehensive genomic sampling will berequired to more shows aclear pattern parapatric of speciation across an ong Heliconiusong populations / H. cydno . Races of URE and DAPC assigned individuals to H. melpomene

H. H. timareta

cydno/H. timareta are more associated H. cydno / H. cydno in arange of H. occupy This article is protected Allrights bycopyright. reserved. higher resolution data, a reality given growinggenomic resources. likely will require and isneeded radiations adaptive ofrecent genomes the across understanding of how these processes (historical and contemporary) are playing out formation and spread of new variation (sensu Rius and Darling 2014). Abetter (Loh et al. 2013). For stickl lakes newly within formed radiations rapid that facilitated variation genetic important suggesting animportant role for these populations as aconduit for functionally within isolate rift lakes. River populations, in contrast, showed admixed genomes, former case, there is strong genetic differentiation among ecologically divergent forms and marine and freshwater stickleback populations (Schluter and Conte 2009). In the interplay appears to underlie diversification of African rift lake cichlids (Loh et al. 2013) these two local processes are strongly entwined with past geoclimatic events. Asimilar potent source for the origin andspread of new adaptive variation. As our data suggest, The coupling of strong population structure and interspecific hybridization provides a Mallet 2010). exactly thetype of population structure that would promote local differentiation (see species across ageographic transect in Costa Rica (Kronforst and Gilbert 2008). This is pattern of strong isolation by distance was observed in several different valley showed strong isolation by distance (Figure 3SM; Arias et al. 2012). Asimilar with this expectation, continuous sampling of individuals of from a population exhibiting continuous variation (see Pritchard et al. 2000). Consistent Acceptedforms. In particular, these case studies highlight the importance of hybridization in the between historical and contemporary processes in the generation of new adaptive by geoclimatic events. These findings, together with our study, underscore the interplay created environments freshwater novel to adaption rapid facilitates example fish cichlid alleles to be maintained in low frequency in marine populations, which similar to the between freshwater and marine populations. This gene flows allows freshwater adapted parallel evolution in freshwater adapted forms that relies on recurrent gene flow Article ebacks, Schluter and Conte (2

009) proposed a model for H. cydno along the Cauca Heliconius

This article is protected Allrights bycopyright. reserved. Brower, A. 1996a. A new mimetic species of Amazon of the fauna insect toan Contributions 1862. H. Bates, Sala Rosales, C. C. F., C. Arias, References the Ministerio de Ambiente, Vivienda y Desarrollo Territorial (Colombia). (DEB-1316037). For the genetic access permit number RGE0027-LAM3483, we thank Fondo FIUR from Universidad del Rosario. M. Kronforst was funded by NSF grant Salazar received support from Universidad del Rosario. M. Linares was funded by McGill C. University. and NEO-STRI Family, Max Family, Binz Levinson the Colfuturo, 640279) and IOS(1052541) to C.F.WOM. Arias was supported by fellowships from workwas carried out and funded. This project was also funded by NSF grant (DEB- acknowledge the Smithsonian Tropical Research Institute (STRI), where the laboratory and three anonymous reviewers valuable for comments on the manuscript. We R. to Gillespie grateful are the manuscript. of We versions earlyon comments useful thankWe C.Jiggins and t members of Acknowledgements Brower, A. 1996b. Parallel racef 1996b. A. Brower, Brower, A.1994. Rapid morphological and radiation convergence refug Neotropical 2012. Willis. K. and Bhagwat, S. K., Bennett, V. Bull, C. Jiggins, M., Beltrán, Accepted butterfl Heliconius in speciation Hybrid 2011. Z. V. A. Brower, Article Molecular Ecology 21:5778-5794. 21:5778-5794. Ecology Molecular 2012. Sharp genetic discontinuity across aunimodal Zool. J. Linn. Soc. 116:317-332. 116:317-332. Soc. Linn. J. Zool. Sourtheastern Colombia, revealed by analysis or mitochondrial D 91:6491-64 USA ofSciences, Academy National ofthe Proceedings butterfly 1214. 19:2176-2190. Evolution and Biology Molecular discordance atthespeciesbou Heliconidae. Transactions of the the evidence. Genetica 139:589-609. 139:589-609. Genetica evidence. the 50:195-221. butterflies: a phylogenetic hypot Heliconius erato zar, J. Castaño, E.Bermingham W. McMillan, E. Bermingham, a ormation andtheevolutionof inferredfrompatternsofm ndary: gene in genealogies Linnean Society ofLondon23:4 hesis frommitochondrialDNAs he McMillan and Bermingham Laboratory for Heliconius

(: ), from , M. Linares, and W. O. McMillan. Heliconius nd J. Mallet. 2002. Phylogenetic Phylogenetic 2002. Mallet. J. nd itochondrial evolution. DNA ia. The Holocene 22:1207- Holocene The ia. ies? A review ies? Areview and critique of mimicry in mimicry valley. Lepidoptera: Lepidoptera: valley. Heliconius among races ofthe among hybrid zone. hybridzone. 95-566. 95-566. equences. Evolution equences. NA sequences. sequences. NA 95. 95. Heliconius butterflies.

This article is protected Allrights bycopyright. reserved. an alleles pattern ofwing Introgression 2013. Z. V. A. Brower, Brown, K., P. Sheppard, and J. Turner. 1974. Quaternary refugia Quaternary 1974. Turner. J. and Sheppard, P. K., Brown, Bush, M. 1994. Amazonian speciation: a necessarily complex mode complex necessarily a speciation: Amazonian 1994. M. Bush, of biology The 1981. S. K. Brown, Elias, M., M. Joron, K. Willmott, K. Silva-Brandao, V. Kaiser, Elias, M., R.Hill, K.Willmott, K.Dasmahapatra, A. Brower, J. evolutionary Bayesian BEAST: 2007. Rambaut. A. and A. Drummond, pre 1998. Field Jacobs. K. D. and Raskoff, K.A. N., M. Dawson, of the evolution geological The 1996. Obando. J. and A. Coates, Diversificati 2011. Smith. B. T. and T. Weir, J. A., J. Chaves, Gregory-Wodzicki, K. 2000. Uplift history of the Central and No and Central ofthe history Uplift 2000. K. Gregory-Wodzicki, ag ofmutations ofneutrality tests Statistical 1997. Y.-X. Fu, ofmol Analysis 1992. Quattro. J. and Smouse, P. L., Excoffier, numb the Detecting 2005. Goudet. J. and Regnaut, S. G., Evanno, in Speciation G.1965. M. Emsley, AcceptedGiraldo, N.,C.Salazar, C. Jiggi Flanagan, N., A. Tobler, A. Davison, O. Pybus, D. Kapan, S. Pla ofp Inference 2003. Pritchard. J. and Stephens, M. D., Falush, Article evidence from race formation in evidence fromraceformation 280. Sciences B:Biological Society Royal ofthe Proceedings hybridization inHeliconiusbutterflies: a review ofevidence f Entomology 26:427-456. B187:369-378. ofLondon, Society butterflies. Proceedings of the Royal Society of London, B274: London, of Society Royal ofthe Proceedings butterflies. Limited performance of DNAbarcoding inadiverse community of 7:214. Biology Evolutionary BMC trees. 7:145-152. Biology Marine Molecular for DNAanalyses. tissue invertebrate 20:4564-4576. Ecology Molecular uplift. follows Andean 21:5-17. Biogeography diversification inclearwing bu Outofthe 2009. Jiggins. C. and Freitas, A. Brower, A. Uribe, tropical America. TheUniversity 21–56 Pp. States of America 101:9704. 101:9704. ofAmerica States using the software structure: a geographic distribution. Zoologica, New York 50:191-254. Geological Society of America Bulletin 112:1091. 112:1091. Bulletin ofAmerica Society Geological same dress: 147:915-925. Genetics selection. backgroud and hitchhiking Heliconius i mimicry ofMüllerian demography Historical 2004. McMillan. W. 164:1567-1587. multilocus genotype linked data: lociand correlated allelefre 131:479-491. Genetics data. restriction metric distancesamongDNAhaplo butterflies. Proceedings of the in Heliconius J. Jackson, A. Budd, and A. Coat andA. Jackson, Budd, J. A. ns, E. Bermingham, and M. Lina cryptic species.BMC Evol Heliconius Heliconius tterflies. Molecular Ecology 18: Molecular Ecology tterflies. simulation study. Molecular Eco Molecular simulation study. of Chicago Press., Chicago. ofChicago Press., Heliconius types: Applicationtohumanmit (Lep., Nymphalidae): morphology and morphology Nymphalidae): (Lep., and related genera. AnnualReview of National AcademyofScienceso butterflies. Proceedings of the Royal es, eds. Evolution andenviron Evolution eds. es,

utionary Biology 8:324. on in C. L. Gom Arias, ainst population growth, Mallet, and C. Jiggins. 2007. 2007. Jiggins. C. and Mallet, nas, M. Linares, D. Heckel, and res. 2008. Two sisters in the the in Two sisters 2008. res. ecular variance inferredvariance fromecular opulation opulation structure using servation ofmarine d speciation via homoploid Central American Central Isthmus. Adelomyia rthern Andes: a review. review. a Andes: rthern intropical America: er ofclustersindividuals Andes: patterns of Andes: patterns l. Journalof l. rom the genome. genome. the rom quencies. Genetics analysisbysampling 1716-1729. 1716-1729. 2881-2889. logy 14:2611-2620. and Biotechnology and Biotechnology n theneotropical hummingbirds tropical tropical ez Piñerez, S. ochondrial DNA f the United ment in This article is protected Allrights bycopyright. reserved. in patterns speciation Montane 2005. J. Hall, Science birds. forest amazonian in Speciation 1969. J. Haffer, Hooghiemstra, H. and E. T. H. Ran. 1994. Late and Middle Pleist and Middle Late 1994. Ran. H. E.T. and H. Hooghiemstra, Hines, H. M., B. A. Counterman, R. Papa, P. A. D. Moura, M. Z. Cryptic 2013. Kronforst. R. M. and Gilbert, E. L. I., R. Hill, Hoyal, J. and M. Charleston. 2012. Phylogenetic codivergence su codivergence Phylogenetic 2012. Charleston. M. and J. Hoyal, Hoorn, C., F. P. Wesselingh, H. ter Steege, M. A. Bermudez, A. Hoorn, C., J. Guerrero, G. A. Sarmiento, and M. A. Lorente. 199 Jiggins, C., R. Naisbit, R. Coe, and J. Mallet. 2001. Reproduct 2001. Mallet. J. and R. Coe, R. Naisbit, C., Jiggins, 2006 Bermingham. E. and Willmott, K. R. Mallarino, C., Jiggins, Jiggins, C., C. Estrada, and A. R Kirby, M. X., D. S. Jones, and B. J. MacFadden. 2008. Lower Mio Lower 2008. MacFadden. J. B. and Jones, D. S. X., M. Kirby, or paradox mimicry: 1998. in Diversity Mallet. J. and M. Joron, components: ofprincipal analysis Discriminant 2010. Jombart. Jiggins, C.,C. Salazar, M. Linar Accepted ofphylo trees: methods species Estimating 2009. L. L. Knowles, 300:71 Science Refugia? Refuting 2003. Mallet. J. and S. Knapp, Article National Academy of Sciences 108:19666-19671. 108:19666-19671. ofSciences Academy National gene redefinesthemimetichistoryof 20 Mcmillan. O. andW. Kronforst, M. R. Jiggins, D. C. Reed, D. in amimetic 272:2457-2466. Sciences B:Biological Society Riodinidae): are they moving consistently up intheworld? Proc 211-246 211-246 m II(2e158 Funza record pollen Colombia; in development forest Science 330:927-931. 330:927-931. Science cli throughAndean uplift, time: Hooghiemstra, J. Lundberg, T. Stadler, T. Särkinen, and A. Anto Sanchez-Meseguer, C. L. Anderson, J. P. Figueiredo, C. Jaramill 23:237-240. for changingdrainage patterns inMiocene northern SouthAmeric Amsterdam, Netherlands. mimetic Nymphalidae). Evolution 60:1454-1466. 60:1454-1466. Evolution Nymphalidae). speciation and wing pattern cha isolation in & Evolution 13:461-466. 13:461-466. Evolution & 363:3047. Sciences Heliconius 411:302-305. Nature mimicry. pattern is incongruence across genes. Systematic Biology 58:463-467. Biology 58:463-467. Systematic genes. across incongruence is 3:e2791. Panama canalanditsbearing on analysis ofgenetically structure Heliconius in butterflies. Philosophical Transactions oftheRoyal Society B R.Bonnefille,and H. Hooghiemstra, eds. Pollenand Climate. E Heliconius melpomene Heliconius butterflies. PLoS ONE 7:e36464. 7:e36464. ONE PLoS butterflies. es, and J. Mavarez. 2008. Hybr 2008. Mavarez. J. and es, odrigues. 2004. Mimicry andth Mimicry 2004. odrigues. butterfly. Molecular Ecology 22:2760-2770. 22:2760-2770. Ecology Molecular butterfly. mate change, landscapemate change, evolutio nge inneotropical Ithomia butte d populations. BMC Genetics1 the Central American peninsula.the Central American . Journal of Evolutionary Biology 17:680-691. 17:680-691. Biology ofEvolutionary Journal . Ithomiola Heliconius

butterflies (Lepidoptera: butterflies. Proceedings of the genetic and wing pattern diversity ive isolationcaused by colour id trait speciationand Cardoso, M. Linares, J. Mallet, R. Linares, R. J. M. Mallet, Cardoso, Mora, J. Sevink, J. Sanmartín, I. Mora, A. 5. Andeantectonicsasacause 5. 165:131-137. 165:131-137. e evolution ofpre-matinge evolution cene stratigraphy along the paradigm? Trends inEcology . Thephylogenetic pattern of -72. -72. ocene climaticchange and genetic analysis when there pports coevolution of pports a newmethodforthe 11. Wing patterning o, D. Riff, F. R. Negri, H. nelli. 2010. Amazonia Amazonia 2010. nelli. 1:94. 1:94. n, and biodiversity. eedings ofeedings the Royal rflies (Lepidoptera: PLoS ONE core interval). core Pp. a. Geology : Biological lsevier, lsevier, This article is protected Allrights bycopyright. reserved. Multil Gilbert. and L. L. Young, Blume, 2006. L. M., Kronforst, of genetics population The 2008. Gilbert. L. and M. Kronforst, Kronforst, M. R., M. E. B. Hanse Kronforst, M. R., G. S. Barsh, A. Kopp, J. Mallet, A. Monteiro, Mallet, J. 2010. Shift happens! Shifting balance evolut the balance and Shifting happens! Shift 2010. J. Mallet, Ca M. Callaghan, M. C. G., Lamas, Mallet, J., M. Beltrán, W. Neukirchen, and M. Linares. 2007. Na 2007. Linares. M. and W. Neukirchen, Beltrán, M. J., Mallet, Swo R. Roberts, R. B. Muenzel, Bezault, M. F. E., E. Y.-H. Loh, Lemey, P., A. Rambaut, A. J. Drummond, and M. A. Suchard. 2009. Mantel, N. 1967. The detection of disease clustering and a gene a and clustering ofdisease detection The 1967. N. Mantel, warn and Mimicry 1998. Jiggins. C. and W. McMillan, J., Mallet, W J.and L. Gilbert. 1995. Mallet, Mavárez, J., C. Salazar, E. Bermingham, C. Salcedo, C. Jiggins, Martin, S. H., K. K. Dasmahapatra McCormack, J. E., H. Huang, and L. L. Knowles. 2009. Maximum li Maximum L.Knowles. H. Huang,2009. and L. E., J. McCormack, Accepted2009. Assorta Linares. M. and Jiggins, C. C. Salazar, M., Melo, Article 275:493-500. 275:493-500. Heliconius Pigment Cell&Melanoma Research 25:411-433. nature’s tapestry:thegenetics Rosenblum, C. J. Schneider, and H. E. Hoekstra. 2012. Unravelin introgression amonghybridizing 2004. Atlas of Neotropical Lepido ofNeotropical Atlas 2004. 5:666–677. Reports Cell ofspeciation. architecture H P.Kapan, Mullen. S. and 2013. colour and mimicry. Ecological Entomology 35:90-104. 35:90-104. Entomology Ecological mimicry. colour and Biology Molecular Cichlids. Va Genetic ofShared Origins 2013. Streelman. T. J. and Kocher, Di Palma, Howe, K. F. E. Lindbla 5:e1000520. Comput Biol PLoS roots. its finds Gainesville, Florida. Papilionoidea. Association forTr Heliconiine butterflies: the spec Forms: Speciesand Speciation. 390-403 Pp. species. and races between 55:159-180. Society Linnean habitat, behaviour and mimicry in Biology 7:28. speciation with gene flow in Genome- 2013. Jiggins. D. C. and Mallet, J. Manica, A. Blaxter, 27:209-220. Research Cancer Speciation by hybridization in hybrids offers aroute hybrids offers Biology 58:501-508. Systematic design. sampling and history species trees: how accuracy of phylogenetic inference depends u butterflies. ProceedingsSociety of B:the Royal Biological Sc to hybrid to speciation.Evolution 63:1660- n, N. G. Crawford, J. R. Gallan J. Crawford, G. n, N. hy are there so many mimicry hy arethereso sagrande, O. Mielke, T. Pyrcz, T. Mielke, O. sagrande, , N. J.Nadeau, C. Salazar, J. and Evolution 30:906-917. 30:906-917. Evolution and Heliconius Heliconius of diversityand d-Toh, J. Hey, O. O. Hey, Seehausen, W. J. d-Toh, ies boundary asacontinuum.B ybridization rev ybridization opical Lepidoptera ⁄ Scientii Heliconius ptera. Checklist: Part 4A. Hes ptera. Checklist:Part4A. Heliconius butterflies. Genome Research 23:1817-1828. 23:1817-1828. Research Genome butterflies. in butterflies. Nature 441:868-871. 441:868-871. Nature butterflies. D. Howard, and S. Berlocher, eds. Endless butterflies. Evolution 60:1254-1268. 60:1254-1268. Evolution butterflies. butterflies. BiologicalJournalofthe convergencein

eals theevolvi S. P. Mullen, M. Protas, E. B. R. Walters, F. Simpson, M. t, W. Zhang, R. J. Kulathinal, D. D. W. D. Kulathinal,t, D. Zhang, J. R. fford, M. Barluenga, C. E. Kidd, A. and M. Linares. 2006. 2006. Linares. M. and tive matingpreferences among ocus analysesofadmixtureand R.Robbins, and A. Viloria. ion ofdiversity inwarning tural hybridization in mimetic diversity in ing colour attheboundarying colour rings? Correlations between between Correlations rings? ralized regression approach. ralized regression kelihood estimatesof Bayesian phylogeography wide evidence for evidence wide c Publishers, Salzburger, T. D. D. T. Salzburger, animalpigmentation. ng genomic perioidea— riation inAfrican 1665. 1665. g the thread of MC Evolutionary Evolutionary MC pon the divergence iences This article is protected Allrights bycopyright. reserved. 1879. F. Müller, Merrill, R. M., Z. Gompert, L. M. Dembeck, M. R. Kronforst, W. Mérot, C., J. Mavárez, A. Evin, K. K. Dasmahapatra, J. Mallet, Nadeau, N. J., S. H. Martin, K. S. K. H. Martin, J., N. Nadeau, or latest forgotten Almost 2007. Clarke. C. A. and M. H. Meudt, Merrill, R. M., R. W. R. Wallban Naisbit, R., C. Jiggins, and J. Mallet. 2001. Disruptive sexual Disruptive 2001. Mallet. and J. C. R., Jiggins, Naisbit, Quek, S.-P., B. Counterman, P. A of Inference 2000. Donnelly. P. and Stephens, M. J., Pritchard, mod the testing Modeltest: 1998. Crandall. A. K. and D. Posada, Patel, S., J. D. Weckstein, J. S. Ochoa, D., C. Hoorn, C. Jaramillo, G. Bayona, M. Parra, and F. AcceptedPardo-Diaz, C., C. Salazar, S. W. Baxter, C. Merot, W. Figueire Ameri butterflies. neotropical in Mimicry 1975. C. Papageorgis, R. Blanchet, Kindt, F. P. P.G. R. J., Minch Legendre, Oksanen, Article 109:830-847. 109:830-847. species (Lepidoptera: Genetic differentiation without mimicry inapair ofhybr shift of Sciences 107:7365-7370. 107:7365-7370. of Sciences Baxter, M. L. Blaxter, J. Mallet, Transactions oftheEntomologica analyses and advances. Trends 279:4907-4913. Sciences B:Biological Society Royal cue. mating a on selection ecological Disruptive 2012. Jiggins. 65:1489–1500. Evolution butterflies. 2011. Matepreference across th 826. 826. divergence and geneacross abutterfly flow radiation. Molecula divergent histories of convergent butterflies. Proceedings of t in radiations comimetic Dissecting 2010. Kronforst. M. and 155:945-959. Genetics data. genotype multilocus 14:817-818. Bioinformatics 58:105-115. Evolution and Phylogenetics Molecular Pleistocene. Constant rate of diversification 1854. Proceedings of the RoyalSociety of London Series B-Biological contributes tospeciation between Earth Sciences 39:157-169. 39:157-169. Sciences Earth Colombia: Evidence from three paly phase oftropical lowland condit diversification of in acros Introgression Adaptive 2012. Jiggins. D. C. and McMillan, Package. Communit vegan: 2013. Wagner. H. and Stevens, H. H. M. Solymos, Heliconius IItuna Butterflies. PLoS Genet 8:e1002752. 8:e1002752. Genet PLoS Butterflies. and Pteroglossus araçaris IThyridia L. L. Patané, J.Bates, M. andA M. Kozak, C.Salazar, K.Das Nymphalidae). Biological Journal oftheL k, V. Bull, P. C. A. J A. Salazar, C. P. Bull, V. k, lbuquerque de Moura, M. Cardoso M. deMoura, lbuquerque ; caseofm a remarkable and C. D. Jiggins. 2013. Geno Jiggins. 2013. andD. C. doesnotsupport anincreasei in Plant Science 12:106-117. 12:106-117. Science Plant in e speciationcontinuum inaclad ions intheaxialzoneofE l Society of London 1879:xx-xxi ofLondon l Society Heliconius cydno nological records. Journalof nological records. (AVES: Ramphastidae) intheneotropics:

. Aleixo. 2011. Temporal and spatial spatial and Temporal 2011. Aleixo. . . Mallet, M. Stevens, and C. D. mahapatra, J. W. Davey, S. W. W. mahapatra, S. J. Davey, W. selectionagainsthybrids and G. Lamas, and M. Joron. 2013. 2013. Joron. and M. Lamas, G. in, R. B. O'Hara, G. L. Simpson, P. De la Parra. 2012. The final final The 2012. Parra. la De do-Ready, M. Joron, W. O. W. O. Joron, M. do-Ready, imicry inbutterflies. population structureusing O. McMillan, and C. D. D. andC. Jiggins. McMillan, O. practice? AFLPapplications, el of DNA substitution. el ofDNA can Scientist 63:522-532. 63:522-532. Scientist can Heliconius melpomene , C. Marshall, W. McMillan, McMillan, W. Marshall, C. , me-wide patterns of Heliconius Proceedings of the astern Cordillera of Cordillera astern he NationalAcademy idizing n radiation during the s Species Boundaries Sciences 268:1849- Sciences innean Society r Ecology 22:814- r Ecology South American South e of mimetic x. x. y Ecology y Ecology Heliconius reveals .

Santos, J., L.Coloma, K. Summers Schneider, S., D. Roessli, and L. Excoffier. 2000. Arlequin Ver Arlequin 2000. Excoffier. L. and Roessli, D. S., Schneider, spe and ecological Genetics 2009. Conte. L. G. and D. Schluter, Salazar, C., C. Jiggins, J. Taylor, M. Kronforst, and M. Linare C. Pardo- Baxter, S. C., Salazar, Rull, V. 2011. Neotropical biodiversity: timingpotential d and intraspecifi is important How 2014. Darling. A. J. and M. Rius, Ribas, C. C., A. Aleixo, A. C. R. Nogueira, C. Y. Miyaki, and J This article is protected Allrights bycopyright. reserved. mut neutral the fortesting method Stadistical 1989. F. Tajima, parsimony using analysis Phylogenetic PAUP*. 2000. D. Swofford, Simonsen, T. J., E. V. Zakharov, M. Djernaes, A. M. Cotton, R. Sheppard, P., J. Turner , K. Brown, W. Benson, and M. Singer. 1 Ravelo, A. C., D. H. Andreasen, M. Lyle, A. Olivarez Lyle, and Co 2004. Pierce. E. N. and Itino, T. Davies, J. S. S.-P., Quek, AcceptedThe Silva-Brandão, K.L., N. Wahlber Article Heliconius National Academy of Sciences. National AcademyofSciences. 7:1000056. BIOLOGY amphibian diversity is primarily latederived MioceneAnde from genealogical history of 6:e1000930. Genetics PLoS in speciation trait for hybrid evidence Genetic 2010. Jiggins. & Evolution 26:508-513. success ofcolonisingpopulations? 689. three millionyears. Proceedings ofB: the RoyalBiolog Society palaeobiogeographic model for biotic diversification within Ama 429:263-267. climate shiftscaused by gradual 58:554-570. Cladistics 27:113-137. 27:113-137. Cladistics with specialreference to theenigmatic genera Teinopalpusand of Papilioni times divergence and Phylogenetics 2011. Sperling. Switzerland. Geneva. population genetics data analysi of mimicry adaptations among species. Nature 487:94-98. 487:94-98. Nature species. among adaptations of mimicry 123:585-595. Genetics polymorhism. 46:515-53 Evolution and Phylogenetics Molecular use. host plant of thetribe Acraeini (Lepidopte re Phylogenetic 2008. Freitas. L. A. V. and Lees, C. D. Paluch, 308:433-613. Sciences Biological B: Society Royal of the evolution ofMuellerian mimicry in (Formicidae: Myrmici mutualism: stemtextureand th genome consortium. 2012. Butterf 2012. consortium. genome nae) inhabitants of nae) Diaz, G. Wu, A. Surridge, M. L M. Surridge, A. Wu, G. Diaz, Heliconius heurippa g, R. B. Francini, A. M. L. Aze L. M. Francini, A. B. R. g, , J. Caldwell, R. Ree, and D. andD. Ree, R. Caldwell, J. , e evolution of host usein e evolution of host ra, Nymphalidae, ) global cooling in thePliocene global coolingin s. Genetics and Biometry Labora Trends in Ecology &Evolutio TrendsinEcology Heliconius Macaranga ly genome reveals promiscuous ly genomereveals . BMC Evolutionary8:132. Biology butterflies. Philosophical Transactions

. Cracraft. 2012. A 2012. Cracraft. . diversification inanant-plant s. 2008. Gene flow and the the flow and Gene s. 2008. inares, Bermingham, inares, andC. E. M. W. Wara. 2004. Regional Regional 2004. Wara. W. M. I. Vane-Wright, and F. A.H. Cannatella. 2009. Amazonian Amazonian 2009. Cannatella. redo-Espin, K. S. Brown Jr, M. (Euphorbiaceae). Evolution .2000: A software for Asoftware .2000: c genetic admixture tothe ciation. Proceedings ofthe rivers. Trends in Ecology Ecology in Trends rivers. 985. Genetics and the the and Genetics 985. ation hypothesis by DNA Crematogaster (*and other methods). (*andothermethods). lationships of butterflies Heliconius epoch. Nature ical Sciences 279:681- Sciences ical and the evolution of nae (Lepidoptera) n 29:233-242. 29:233-242. n Meandrusa. zonia over thepast tory. University of an lineages. PLoS 1. 1. butterflies.

exchange This article is protected Allrights bycopyright. reserved. subdivis in convergence and radiation Adaptive 1976. J. Turner, Turner, J. and J. Mallet. 1996. D andJ. 1996. Mallet. J. Turner, Van der Hammen, T., J. H. Werner, and H. Van Dommelen. 1973. Pa 1973. Dommelen. Van H. and Werner, J. H. T., Hammen, der Van 1979. Contras Eanes. F. W. and Johnson, S. M. G., R. J. Turner, in evolution and Adaptation 1981. G. R. J. Turner, manuscript withmanuscript input C.S,from M.L,M.R.K and E.B. specimens and aided in interpretation of the results. C.F.A and wroteW.O.M the data and C.F.A, C. S and analyzedW.O.M the data. M.R.K and M.L contributed C.F.A, E.B and designedW.O.M the project. C.F.A and C.R generated all molecular Box Author Contributions 10.5061/dryad.b5d6b. data, Co1 Data access Weir, B. S. and C. C. Cockerham. ra of and Colonization 2007. Freitas. L. V. A. and N. Wahlberg, Vos, P.,R. Hogers, M. Bleeker, Accepted Mallari R. N. Herrera, Willmot, K. Zimmermann, M. A., Whinnett, Article “barcode” sequences have been deposited in GenBank (see Table 4SM). AFLP Co1 58:297-308. 58:297-308. Heliconius National Academy of Sciences USA 76:1924-1928. 76:1924-1928. USA ofSciences Academy National same genome: and Allozymes adaptive change in a Annual ReviewofEcology 351:835-845. Sciences B:Biological Society Royal butterflies?and Bioticdrift theshifting bance. Philosophical the ColombianEastern Cordillera upheaval oftheNorthern Andes: Phylogenetics and Evolution 44:1257-1272. 44:1257-1272. Evolution and Phylogenetics butterflies inthesubtribe Phyci fingerprinting. NucleicAcids Research 23:4407-4414. technique new a AFLP: 1995. Zabeau. and M. Kuiper, M. Peleman, Review of Palaeobotany Sciences 272:2525-2533. 272:2525-2533. Sciences Amazonian butterfly ‘su time divergence variable Strikingly 2005. Mallet. J. and Lamas, 38:1358-1370. Evolution structure. alignment and additional data files have been submitted to DRYAD entry doi: (Lepidoptera: Nymphalidae). Zool M. Rijans, M. Van T. Ho M. de Lee, id forestdrive islands thedi ture zone’. Proceedings of the Royal Soc and Palynology 16. 16. andPalynology 1984. Estimating F-statistics nd Systematics 12:99-121. 12:99-121. Systematics nd odina (Lepidoptera: Nymphalida A study of the Pliocene andLow Pliocene ofthe A study and the early evolutionofits andtheearly Heliconius ogical Journal oftheLinneanogical

Heliconius versity of warningly coloured coloured of warningly versity : a defense of neo-Darwinism. rnes, Frijters, A. J. Pot, J. ted modes ofevolutionted inthe modes for the analysis ofpopulation diation inSouthAmerica by ions ofthe butterflygenus no, G.no, Joron, Simpson, M. F. lynological record ofthe record lynological . Proceedings of the Transactionsofthe s inferred across an High-Andean biota. iety B: Biologicaliety B: e). Molecular Molecular e). er Quaternary of for DNA Society This article is protected Allrights bycopyright. reserved. three radiations, Figure 1. melpomene H. m. plesseni Southeastern Andes and is formed by individuals of and Orange, Southeastern Andes (EAS). The EASclade represents races from the Pink, Pacific Andes (PW);West Purple, French Guiana (FG); Dark Blue, Brazil (BA); Bright Blue, Northern Cauca valley (CVN); Pale Blue, Southern Cauca valley (CVS); Northern Magdalena valley (MGN); Pale Brown, Southern Magdalena valley (MGS); defined, Yellow, Northeastern Andes (EAN); Green, Central America (CA); Dark Brown, probability greaterthan 50%.Color code fo AFLP markers. Numbers over branches represent bootstrap support for branches with a with aprobability greaterthan 50%. b) Ne particular node. Numbers over branches represent posterior probabilities for branches deeper nodes, pies are color coded with the credibility maximum st phylogenetic reconstruction for the “barcode” mtDNA region. Branches on the tree are Figure 2. Phylogenetic trees for the Andes. races not collected in this study: races, and Green melpomene tropical Central and South America. The map presents sample localities for Figure 3. Population assignment tests for timareta proportion of color on the ba the software Structure 2.1.4. Each vertical bar corresponds to one individual. The adegenet clusters and its phenotypic classification using the function races. Figure 4.DiscriminantAnalysis ofPrincipal Components (DAPC) for French Guiana (FG) and Brazil (BA). Magdalena valley (MG), Northeastern Andes (EAN), Southeastern Andes (EAS), regions: Central America (CA), Pacific AndesWest (PW), Cauca valley (CV), classification. Gray-horizontal bars on the bottom of figure follows thegeographic different clusters. Black-horizontal bars on the top and bottom of figure show phenotypic Accepted Southern Cauca valley (CVS); and Pink, Pacific Andes (PW).West Magdalena valley (MGS); Bright Blue, Northern Cauca valley (CVN); Pale Blue, America (CA); Dark Brown, Northern Magdalena valley (MGN); Pale Brown, Southern regions displayed on the map: Yellow, Northeastern Andes (EAN); Green, Central Article The squares show thenumber of individuals assigned to the different genotypic . ; Jombart Bar plots showing Bayesian assignment probabilities for three clusters from clusters from three for probabilities assignment Bayesian showing plots Bar Heliconius cydno and , H. timareta and H. cydno H. timareta H. timareta H. m. bellula H. m. et al. presented with the posterior prob 2010). The color of the squares corresponds to the geographic and , H. melpomene races are presented in Figure 1. r represents an individual’s a radiation and closely related taxa. and H. heurippa from the Colombian East Andes. Location of of Location Andes. East Colombian the from H. c. gadouae H. heurippa Heliconius cydno . Pink ighbor-joining phylogenet , and ate reconstruction from BEAST v1.6. For llows thegeographic regions previously H. melpomene . An asterisk denotes the only distributed on the Northeast slopes of H. cydno H. timareta

H. m. malleti m. H. and related species. races, Blue abilities for abilities each statefor at that ssignment probability tothe encompasses much of of encompasses much find.clusters , H. cydno , The range Thethe of H. m. ecuadorensis ic reconstruction for H. melpomene (Rpackage: and H. cydno a) Bayesian H. cydno H. cydno H. H.

,

, H. This article is protected Allrights bycopyright. reserved. Accepted Article

This article is protected Allrights bycopyright. reserved. Accepted Article

This article is protected Allrights bycopyright. reserved. Accepted Article