Genetic Evidence for Hybrid Trait Speciation in Butterflies


Homoploid hybrid speciation is the formation of a new hybrid species without change in chromosome number. So far, there has been a lack of direct molecular evidence for hybridization generating novel traits directly involved in animal speciation. Heliconius butterflies exhibit bright aposematic color patterns that also act as cues in assortative mating. Heliconius heurippa has been proposed as a hybrid species, and its color pattern can be recreated by introgression of the H. m. melpomene red band into the genetic background of the yellow banded H. cydno cordula. This hybrid color pattern is also involved in mate choice and leads to reproductive isolation between H. heurippa and its close relatives. Here, we provide molecular evidence for adaptive introgression by sequencing genes across the Heliconius red band locus and comparing them to unlinked wing patterning genes in H. melpomene, H. cydno, and H. heurippa. 670 SNPs distributed among 29 unlinked coding genes (25,847bp) showed H. heurippa was related to H. c. cordula or the three species were intermixed. In contrast, among 344 SNPs distributed among 13 genes in the red band region (18,629bp), most showed H. heurippa related with H. c. cordula, but a block of around 6,5kb located in the 3′ of a putative kinesin gene grouped H. heurippa with H. m. melpomene, supporting the hybrid introgression hypothesis. Genealogical reconstruction showed that this introgression occurred after divergence of the parental species, perhaps around 0.43Mya. Expression of the kinesin gene is spatially restricted to the distal region of the forewing, suggesting a mechanism for pattern regulation. This gene therefore constitutes the first molecular evidence for adaptive introgression during hybrid speciation and is the first clear candidate for a Heliconius wing patterning locus.


Published in the journal: . PLoS Genet 6(4): e32767. doi:10.1371/journal.pgen.1000930
Category: Research Article
doi: 10.1371/journal.pgen.1000930

Summary

Homoploid hybrid speciation is the formation of a new hybrid species without change in chromosome number. So far, there has been a lack of direct molecular evidence for hybridization generating novel traits directly involved in animal speciation. Heliconius butterflies exhibit bright aposematic color patterns that also act as cues in assortative mating. Heliconius heurippa has been proposed as a hybrid species, and its color pattern can be recreated by introgression of the H. m. melpomene red band into the genetic background of the yellow banded H. cydno cordula. This hybrid color pattern is also involved in mate choice and leads to reproductive isolation between H. heurippa and its close relatives. Here, we provide molecular evidence for adaptive introgression by sequencing genes across the Heliconius red band locus and comparing them to unlinked wing patterning genes in H. melpomene, H. cydno, and H. heurippa. 670 SNPs distributed among 29 unlinked coding genes (25,847bp) showed H. heurippa was related to H. c. cordula or the three species were intermixed. In contrast, among 344 SNPs distributed among 13 genes in the red band region (18,629bp), most showed H. heurippa related with H. c. cordula, but a block of around 6,5kb located in the 3′ of a putative kinesin gene grouped H. heurippa with H. m. melpomene, supporting the hybrid introgression hypothesis. Genealogical reconstruction showed that this introgression occurred after divergence of the parental species, perhaps around 0.43Mya. Expression of the kinesin gene is spatially restricted to the distal region of the forewing, suggesting a mechanism for pattern regulation. This gene therefore constitutes the first molecular evidence for adaptive introgression during hybrid speciation and is the first clear candidate for a Heliconius wing patterning locus.

Introduction

Identifying the genetic mechanisms involved in speciation is an important challenge in the study of evolution [1][3]. Empirical studies have shown that species differences can be localized in just a few genomic regions [3][5], and that reproductive isolation is more easily achieved when traits causing assortative mating are also subject to natural selection [6], [7]. Such characteristics have been termed ‘magic traits’ [6] and can facilitate speciation as a side-effect of ecological divergence in the presence of ongoing gene flow [8], [9]. Likely examples of such magic traits include body size and color in sticklebacks, flowering time in edaphic plants, host shifts in phytophagous insects, color patterns in Heliconius butterflies, beak size in Darwin finches, development time in melon flies and color patterns in Hypoplectrus fish [9][15].

If ‘magic traits’ were acquired by introgression from related lineages, adaptation and speciation could proceed without the requirement for novel mutations [16], [17]. Recent studies in plants and animals have shown that introgression can provide the raw material for adaptation [18][20]. Hence, it is plausible that if introgression produces new adaptive phenotypes that also generate reproductive isolation, for example through mate choice, habitat colonization or asynchronous emergence, then hybrid speciation can occur without geographic isolation [17], [21]. We have called such a scenario ‘hybrid trait speciation’, as a special case of speciation through hybridization without a change in chromosome number or homoploid hybrid speciation (HHS) [2], [22]. Hybrid trait speciation contrasts with what we have termed mosaic genome speciation, documented in Helianthus sunflowers, where the hybrid species genome is composed of blocks derived from both parental species [23]. Rapid establishment of incompatibilities between parental and daughter species can result due to the large number of genes causing epistatic hybrid breakdown in hybrids [23]. The two scenarios contrast in their genomic signature, with hybrid trait speciation potentially involving introgression of just a few adaptively important loci into the genetic background of one of the parental species, making it much more difficult to detect using traditional approaches based on ‘neutral markers’ [21]. This is in addition to the fact that detecting hybrid speciation at the molecular level is difficult anyway, due to incomplete linage sorting and historical gene flow, which can leave similar signatures of shared variation [24].

There is evidence that hybridization has played an important role in the adaptive radiation of Heliconius butterflies [21]. Heliconius have aposematic wing coloration, mimicry between divergent species and frequent hybridization, providing an excellent opportunity to study the genetics of adaptation and speciation [25][28]. In particular, studies in the closely related species H. melpomene and H. cydno, that occur sympatrically throughout Central America and in the west Andes, show that mimicry shifts are coupled with assortative mating and lead to speciation [29]. In addition, differences in host plant use, microhabitat preferences and partial hybrid sterility also contribute to reducing genetic interchange between these species [30]. The species hybridize in both the field and the laboratory, although natural interspecific hybrids are collected at a very low rate (one in a thousand or less) [28], [30]. Nonetheless, introgression of color pattern alleles has been observed in natural hybrid zones and the same phenotypes have been recreated in experimental crosses [31], [32]. Furthermore, studies using neutral markers reveal that introgression between the species has been frequent throughout their evolutionary history [33], [34].

Occasionally, novel color pattern variants produced through hybridization appear to have produced stable hybrid populations. The best studied example is Heliconius heurippa, found in the eastern slopes of Colombian Andes, which has a color pattern that can be recreated in the laboratory through crosses between H. c. cordula and H. m. melpomene, the races of the melpomene group adjacent to its current geographical range [35], [36]. H. heurippa is abundant and its color pattern is stable along several hundred kilometers of the Andean slopes, although is not mimetic with any other species [30], [37]. This wing pattern stability across a broad geographic area contrasts with the transient production of hybrid forms in narrow Heliconius hybrid zones [25].

Surprisingly, only three generations of crosses are needed to obtain a homozygous H. heurippa like color pattern [35]. Two tightly linked loci controlling the red forewing band (B allele, hereafter HmB) and the absence of brown forceps marks in the ventral surface (br allele) from linkage group 18 are introgressed into an H. c. cordula genetic background that includes the yellow forewing band allele at the HmN locus on linkage group 15. The resultant pattern contributes to reproductive isolation through assortative mating, and therefore plays a direct role in speciation [35]. In particular, mating experiments revealed strong pre-mating mating isolation between H. heurippa and H. melpomene (≈90%) and between H. heurippa and H. cydno (≈75%). Furthermore, assays with wing models showed that H. heurippa males use the combined red and yellow bands to discriminate females [35]. Even first-generation backcross hybrids between H. m. melpomene and H. c. cordula, resembling H. heurippa, showed a strong preference for their own color pattern over that of either parental species, implying that mate preference can be established directly through hybridization [38]. This result implies, first that assortative mating preferences would facilitate the initial establishment of a homozygous hybrid color pattern by increasing the probability that early generation hybrids mate among themselves. Second, once the new hybrid population was established, it would immediately possess the assortative mating preferences that generate partial reproductive isolation from the parental species. Thus, H. heurippa is an excellent candidate for speciation through adaptive introgression [38].

Despite extensive support for the hybrid origin of H. heurippa from biogeography, crosses, mate choice experiments and mathematical simulations [39], molecular evidence for a hybrid origin is inconclusive. Neutral markers reveal extensive gene flow between all three species, H. heurippa, H. melpomene and H. cydno [40]. To definitively test the hybrid origin hypothesis we need to study the loci controlling the adaptive traits responsible for speciation, in this case wing patterns [21], [40]. Here, we take advantage of the recent cloning of the HmB locus controlling the red forewing band of H. melpomene, in order to carry out such a test [41], [42].

Results

Previous work on H. heurippa has used a panel of largely intronic markers and shown evidence for ongoing gene flow between H. heurippa and its close relatives [40]. Here, we directly addressed the adaptive introgression hypothesis by sampling 18 contigs based on 24 amplicons representing 18,629bp across the HmB locus for ten individuals of each species (60 alleles; Table S1) [41], [42]. In addition, we improved our broader genome sampling by developing a larger panel of unlinked molecular markers based on single copy genes with large exons. We analyzed around 15,000 contigs assembled from H. melpomene GSS (BAC-end) sequences, 484 of which showed strong homology with single copy genes in B. mori. From these unigenes, 27 had exons longer than 700 bp (Table S2). Additionally, we used two previously published markers, CAD and GAPDH [43]. Thus, we used a set of 29 markers that were putatively distributed in at least 17 of the 21 chromosomes in H. m. melpomene (Table S2). Sequences of these markers were obtained for the same ten individuals per species used in the HmB locus analysis (60 sequences per gene).

Among the 29 loci sampled from across the genome, most SNP polymorphisms were shared among the three species and therefore did not associate H. heurippa with one or other of the two parental species. Only 8 SNPs (over six genes) were fixed polymorphisms shared by H. heurippa and H. c. cordula relative to H. m. melpomene (Figure 1A), consistent with previous data from mitochondrial genes that related H. heurippa with H. cydno [40], while no SNPs were fixed in H. heurippa and H. m. melpomene relative to H. c. cordula (Figure 1A).

Relative likelihood of association between <i>H. heurippa</i> and <i>H. m. melpomene</i> versus <i>H. c. cordula</i> for (A) genes unlinked to color pattern and (B) across the <i>HmB</i> red color pattern locus.
Fig. 1. Relative likelihood of association between H. heurippa and H. m. melpomene versus H. c. cordula for (A) genes unlinked to color pattern and (B) across the HmB red color pattern locus.
Likelihood values are plotted for each variable position, where positive likelihood values indicate a SNP position at which H. heurippa and H. m. melpomene are more similar, and negative values a position at which H. heurippa shows a stronger association with H. c. cordula. Fixed sites are indicated by dotted lines showing a likelihood value of 244 for a complete association of H. heurippa with H. m. melpomene and −244 for that with H. c. cordula. Colors represent different coding regions. The majority of unlinked SNPs (634) show shared polymorphism among the three species (240> ΔLnL >−240). At unlinked loci, H. heurippa and H. c. cordula shared fixed polymorphism at only 8 SNPs whereas H. heurippa and H. m. melpomene did not share any fixed polymorphism. For the HmB locus, sequenced BAC clones are indicated above the gene annotation [41].

Our prediction derived from the adaptive introgression hypothesis was that within the HmB locus there should be a region introgressed from H. melpomene into the H. heurippa genome. This prediction was upheld. From nearly 19Kb of sequence analyzed across the HmB locus, there was a 6,493 bp region corresponding to the 3′ end of a putative kinesin gene (hereafter, 3′ kinesin) showing a strong association between H. heurippa and H. m. melpomene (Figure 1B). Across the remaining HmB region there was either shared variation among the three species, unique polymorphisms in one of the three species, or nearly fixed changes unique to H. heurippa. In the case of two genetic markers, kin_2 and sdp, H. heurippa was most strongly associated with H. c. cordula (Figure 1B).

The HmB locus was a significant outlier relative to the rest of the genome. We calculated a likelihood statistic that estimated the relationship of H. heurippa to the parental species, where positive values indicate sites linking H. heurippa with H. m. melpomene and negative values sites where H. heurippa is similar to H. c. cordula. The distribution of mean likelihood values across unlinked loci gives a distribution of expected values for the genome. Comparison of the 3′ kinesin region showed values that fell outside this distribution derived from unlinked markers (Figure 2; p<0.05), demonstrating that this genetic region has a significantly stronger association with H. melpomene than any other region of the H. heurippa genome. The kin_2 region was also an outlier, but with a significantly stronger association with H. cydno (Figure 2; p<0.05), implying that the kinesin gene is in fact a chimera derived from two parental species. As H. heurippa is most closely allied to H. c. cordula, we analyzed linkage disequilibrium (LD) for these two species combined. Across the HmB region, the highest r2 values (p<0.001) were observed within the 3′ kinesin and nearby genes, with LD decaying in surrounding regions, suggesting a haplotype structure across the kinesin gene resulting from strong selection on wing pattern (Figure 3). Nonetheless, consistent with the wing patterning alleles being relatively ancient, there was no evidence for a reduction in diversity or deviation from neutrality that might indicate a recent selective sweep at 3′ kinesin (Table 1). Similarly, there was no evidence for adaptive amino acid substitution at this locus, with Ka/Ks ratios not significantly greater than 1 (p>0.05; Figure S1) and McDonald-Kreitman tests showing no deviation from neutrality (p>0.05). H. heurippa also had five private amino acid substitutions not observed in the other two species (Figure S2). Only one amino acid replacement was shared between the red-banded species, H. heurippa and H. m. melpomene, representing a putative causative site for this phenotype (Figure S2).

Distribution of average likelihood values at SNPs in unlinked and <i>HmB</i> linked loci.
Fig. 2. Distribution of average likelihood values at SNPs in unlinked and HmB linked loci.
The average of likelihood values for each unlinked marker and for 1,000 bp windows in the HmB region was calculated. This histogram shows the distribution of these values. Dotted lines represent the 95% two-tailed interval for unlinked genes. The asterisk over the bars indicates those 1,000 bp windows showing average values that lie outside the unlinked genes distribution (p<0,005). Positive values outside that distribution correspond to 3′ kinesin whereas negative values are those in kin_2.

<i>HmB</i> linkage disequilibrium analysis.
Fig. 3. HmB linkage disequilibrium analysis.
Pairwise estimates of linkage disequilibrium (r2) among 199 SNPs in the HmB locus (those with a rare allele frequency less than 10% were excluded) for combined H. c. cordula and H. heurippa population samples. Physical distance between sites is shown in the adjacent map.

Tab. 1. Species nucleotide diversity for each locus.
Species nucleotide diversity for each locus.
s indicate significance: * p<0.05; n.a.: not applicable due to lack of polymorphism.

An alternative hypothesis to adaptive introgression is that the H. heurippa pattern might be ancestral. However we could rule this out by reconstructing a rooted genealogy of the 3′ kinesin with either nucleotides or amino acid sequences (Figure 4B; data not shown for AA). Gene genealogies for kin_2, sdp and 3′ kinesin confirmed the results obtained with the SNP association tests (Figure 4). In the 3′ kinesin, H. heurippa was monophyletic and branched from within the H. m. melpomene clade, with H. c. cordula an outgroup to both species (Figure 4B, Table 2). In contrast, genomic regions surrounding 3′ kinesin showed H. heurippa alleles more closely related to H. c. cordula (Figure 4A and 4C; Table 2). Thus, the SNP analysis, LD patterns and gene genealogies revealed that a clearly delimited genomic portion of the HmB region closely relates H. heurippa with H. m. melpomene. This result is in contrast to the rest of the genome where no other gene showed a similar association.

Gene genealogies for 3′ <i>kinesin</i> and its flanking regions.
Fig. 4. Gene genealogies for 3′ kinesin and its flanking regions.
(A) sorting nexin (sdp); (B) 3′ kinesin; and (C) 5′ kinesin partial sequence (kin_2). Filled color circles represent alleles of each species. The 3′ kinesin tree is rooted with H. numata as outgroup (black). Numbers above and below the branches are bootstrap support values for likelihood and parsimony analyses respectively. (B) shows H. heurippa most closely related to H. m. melpomene, while (A,C), the genomic regions surrounding 3′ kinesin, show H. heurippa alleles more closely related to H. c. cordula. A similar tree topology was obtained from an amino acid alignment (data not shown).

Tab. 2. Net divergence among populations.
Net divergence among populations.
c: H. c cordula; m: H. m. melpomene; h: H. heurippa.

When the genealogy of 3′ kinesin was used to estimate divergence times, we found that H. heurippa alleles were derived from H. m. melpomene around 0.43 Mya (0.12–0.84) ago, subsequent to the splitting of the H. c. cordula/H. m. melpomene alleles at 2.82 Mya (1.03–5.22) ago. A coalescent expansion model [44], [45] (SSD>0.05 in all the cases) similarly suggested that H. heurippa haplotypes radiated more recently (∼0.385 Mya; 0.176–1.428) than either H. m. melpomene or H. c. cordula (∼2.055 Mya (1.175–4.219) and ∼2.745 Mya (1.584–4.489) respectively), giving a confirmation of the relative ages of the alleles found in each species, independent of tree topology. Thus, the H. heurippa 3′ kinesin alleles diverged subsequent to the split between H. m. melpomene and H. c. cordula at this locus.

To examine the role of the putative kinesin gene in specification of wing pattern, we visualized spatial localization of kinesin transcripts in developing wings using in situ hybridization. In two red-banded forms H. melpomene cythera and H. melpomene rosina, a probe from exon 13 of the 3′ kinesin showed localization to the distal portion of the developing wing in early pupal stages (72–96 hrs after pupation; Figure 5A; Figure S3). No such spatial localization was seen either in individuals of H. cydno that do not express a red band phenotype (Figure 5B), nor in H. melpomene forewings treated with riboprobes for a different gene (Figure S3). This spatial localization suggests a model for pattern specification whereby the kinesin gene interacts with another as yet unidentified gene product to specify proximal and distal boundaries of the forewing band (Figure 5C), leading to upregulation of pigmentation genes such as cinnabar [46].

Expression pattern of <i>kinesin</i> in forewings.
Fig. 5. Expression pattern of kinesin in forewings.
Of (A) H. m. cythera and (B) H. cydno. A similar expression pattern to that present in (A) is also observed in H. m. rosina forewings (Figure S3), consistent with the red band phenotype of these two races. The lack of any localized kinesin expression in H. cydno forewings is consistent with the absence of a red band in this species. (C) Model of how kinesin expression (K, solid line), might interact with an unknown gene (X, dotted line) to regulate forewing red band expression.

Discussion

Homoploid hybrid speciation has been considered controversial in animals. We here provide the first molecular support for this hypothesis derived from sequence analysis of a gene region directly implicated in controlling a hybrid trait. H. heurippa was originally proposed as a hybrid species based on its unusual color pattern. The main evidence in support of this hypothesis are crossing experiments demonstrating experimental introgression of the H. m. melpomene red color forewing band into the H. c. cordula genomic background [35]. Such experiments demonstrate a plausible route for the origin of H. heurippa, and make a clear prediction: the region controlling the red forewing band should show a pattern of introgression from H. melpomene into H. heurippa. Here, we provide support for this hypothesis at a molecular level, by demonstrating a 6.5Kb region in the HmB locus that is introgressed from H. melpomene into H. heurippa.

The majority of SNPs (634) sampled in 29 coding genes, located on 17 of the 21 H. melpomene linkage groups, showed shared polymorphism among the three species (Figure 1A). H. heurippa and H. c. cordula shared fixed polymorphism relative to H. m. melpomene at only 8 SNPs, and there were no fixed SNPs in H. heurippa and H. m. melpomene relative to H. c. cordula (Figure 1A). This agrees with previous genetic data showing extensive allele sharing in the nuclear genome between the three species, but H. heurippa somewhat closer to H. c. cordula [40]. As we have argued previously [40], these data do not strongly support a hybrid speciation scenario, but are more consistent with either recent gene flow among the three species or shared ancestral polymorphism.

Here we have taken advantage of the recent cloning of HmB, the key locus underlying the speciation of H. heurippa. Our hypothesis derived from previous crossing experiments and sequence surveys is that the H. heurippa genome is most closely related to H. cydno, but with the introgression of the red forewing band, controlled by HmB, from H. melpomene. Here we directly test this hypothesis by sampling markers across the 721 Kb HmB locus [41]. From 13 genes evaluated in this region (comprising 24 molecular markers), we found a 6,493 bp region, corresponding to the 3′ end of the kinesin locus, where H. heurippa is strongly related to H. melpomene (Figure 1B). The likelihood values for species relationships at this locus differs significantly from that seen among unlinked genes, implying that this relationship cannot be explained by chance (Figure 2). The high long-range LD at 3kinesin relative to the H. c. cordula genetic background is also expected under the introgression hypothesis (Figure 3). The pattern is comparable to that seen across the same region in a Heliconius melpomene hybrid zone, where long-range LD is observed between sites showing significant genotype-by-phenotype association [41]. A rather surprising observation is that across the HmB locus there is also shared variation between the three species, at sites interspersed between those generating a strong phylogenetic signal. These could be due to gene flow and recombination subsequent to speciation, recurrent mutations or alternatively a hybrid founding event for H. heurippa that transferred significant polymorphism from the parents to the hybrid species. In the data, there was no marked difference in the transition/transversion ratio among fixed and shared polymorphisms, which might be indicative of recurrent mutation (data not shown).

An alternative hypothesis to be considered is that H. heurippa pattern might be ancestral and have given rise to H. melpomene and H. cydno lineages that inherited different aspects of the ancestral wing pattern. However, this is not supported by the rooted gene genealogy for the 3′ kinesin that shows H. heurippa monophyletic, forming a well supported and derived clade within H. melpomene (Figure 4B). Furthermore, none of the dating approaches showed H. heurippa older than the other two species. Other genomic regions have shown a genealogical pattern whereby H. heurippa was similarly nested within an H. cydno clade, also arguing that H. heurippa is not an ancestral taxon [47].

In addition, kinesin in situ hybridizations on developing wing tissue (72–96h post-pupation) showed localized gene expression in the distal region of the wing, supporting a likely functional role in specification of the proximal boundary of the forewing band. In combination with previous analyses showing parallel differences in expression levels of the kinesin gene between color pattern races of both H. melpomene and H. erato [41], [48], these data strongly suggest that a regulatory change in the kinesin gene is functionally required for pattern determination. The kinesin gene appears to be chimeric, with the 3′ region derived from H. m. melpomene and the 5′ end more strongly related to H. c. cordula. Since crossing experiments suggest that introgression of the HmB allele from H. melpomene is sufficient to generate the H. heurippa pattern, the implication is that the functionally important sites are located at the 3′ end of the gene. We have identified one amino acid change and 11 synonymous changes shared between red-banded H. melpomene and H. heurippa, representing candidate functional sites for red band specification.

We also observed five amino acid differences between H. melpomene versus H. heurippa (Figure S2), perhaps reflecting adaptive change subsequent to formation of H. heurippa, although there was no significant evidence for selection on the locus (Figure S1). Perhaps more likely, these changes may represent fixation of nearly-neutral variation due to a population bottleneck during the origin of H. heurippa.

The implication of this gene in phenotypic control at HmB is also consistent with previous population genetic analysis of phenotypic races of H. melpomene, which showed a region of high genetic differentiation corresponding to a genomic region including the kinesin [41]. Members of the kinesin superfamily (KIFs) are key players in cellular functioning and morphology that interact with cargo molecules such as proteins, lipids or nucleic acids [49], [50]. In both vertebrates and invertebrates, kinesin molecules are implicated in pigment transport, however in Heliconius melpomene upregulation of pigment pathway genes occurs later in development relative to the localized kinesin expression observed here. This would suggest a likely upstream role in scale cell fate specification, rather than pigmentation per se.

Although adaptive introgression has recently been demonstrated at a molecular level, for example between species in the genus Senecio [19], H. heurippa is unusual in the fact that the hybrid trait contributes directly to reproductive isolation and hence speciation. We have also recently demonstrated that first generation backcross hybrids resembling H. heurippa also exhibit mate preferences very similar to that of wild H. heurippa. This implies that mate preferences could also have been produced by introgression in addition to color pattern [38]. A possible mechanism for this is suggested by the recent demonstration of a genetic association between the red band and male preference for red mates, in interspecific hybrids between H. m. rosina and H. c. chioneus (Merrill et al. pers. comm.). Thus, the derived color pattern and mate preferences of H. heurippa could potentially have arisen from introgression of the same gene or tightly linked genes.

Several cases of animal homoploid hybrid species have been recently proposed, such as Rhagoletis sp., Lycaeides sp., Cottus gobio group, cichlid fishes, Xiphophorus clemensiae and Pogonomyrmex sp. [17], [51] where ecological divergence, sexual selection or both promote reproductive isolation of a hybrid taxon. However, to our knowledge, this is the first time that molecular evidence for introgression has been established for an adaptive trait that also contributes directly to reproductive isolation and hence speciation. We feel that our results therefore represent the most convincing molecular evidence to date for homoploid hybrid speciation in animals. Similar molecular evidence also supports the hybrid origin of sunflower species in the genus Helianthus, although the pattern is very different in this case. Hybrid sunflower genomes are a mosaic of genomic blocks inherited from one or other parent [23], in contrast to H. heurippa which shares polymorphism with both parental species across most of the genome. Although mathematical simulation has suggested that the origin of H. heurippa probably involved an initial period of allopatry, during which the hybrid pattern became established [39], the contemporary genetic pattern supports our model of ‘hybrid trait speciation’ whereby localized introgression of key traits can promote the origin of hybrid species [21].

Materials and Methods

Specimen collection and DNA isolation

Butterflies were collected from Colombia and Venezuela: H. m. melpomene in Morcote (5°37′0.52″N; 72°18′0″W, Casanare-Colombia) and Chirajara (4°12′48″ N; 73°47′70″W, Cundinamarca-Colombia), H. c. cordula in San Cristobal (7°47′56″N; 72°11′56″W, Merida-Venezuela) and H. heurippa in Buenavista (4°10′30″N; 73°40′41″W, Meta –Colombia). Wings of 10 individuals of each species were removed and stored in glassine envelopes and are lodged in the Natural History Museum of the Universidad de los Andes. The bodies were preserved in 20% DMSO-0.25M EDTA salt saturated solution. DNA was isolated with DNeasy Blood & Tissue Kit (QIAGEN) following manufacturer's instructions. Quality of genomic DNA was confirmed by visualisation in a 0.8% agarose gel.

Development, amplification, and sequencing of genetic markers

Single copy large exons

In order to develop new coding markers in Heliconius, we designed primers to amplify single copy large exon markers that were widely distributed across the Heliconius genome. We used the Basic Local Alignment Search Tool via nucleotide (BLASTN) to compare Heliconius melpomene genomic raw reads against unique genes (unigenes) of Bombyx mori [52]. Those unigenes showing homology with H. melpomene were subjected to BLASTN against B. mori whole genome shotgun (WGS) to reveal the location of introns [53]. Additionally, the program Spidey, freely available at http://www.ncbi.nlm.nih.gov/spidey/, was employed to confirm this information [43], [54]. With this knowledge in hand, introns and exons limits were determined in the H. melpomene contigs. To estimate the genomic location of selected markers we used the tool SilkMap [55], to locate genes on the silkworm chromosomes. Given highly conserved synteny between B. mori and H. melpomene [53], we could infer the putative chromosome location of markers in Heliconius. Identified H. melpomene contigs containing exons longer than 700 bp and located on different chromosomes were selected for PCR primer design using Primer 3 v.0.3.0 [56]. Two previously reported single copy large exons were also included [43]. We performed all PCR reactions in a 10 µL reaction volume containing 1× PCR buffer, 2.5 mM MgCl2, 200µM dNTPs, 1 µM each primer, 0.5 U Taq polymerase (QIAGEN) and 30–40 ng of DNA. The PCR cycling profile was 95°C for 5 min, 30 cycles of 95°C for 30 s, Tm for 45 s (see Table S2 for Tm values for each locus), 72°C for 1 min and final extension at 72°C during 15 min. Two microlitres of the PCR reaction were visualised in a 1% agarose gel to verify the success of PCR. The amplicons were cleaned up by ExoSAP-IT (USB Corp., Cleveland, OH). The BigDye Terminator Cycle Sequencing kit (PerkinElmer Applied Biosystems, USA) was used for direct sequencing using 24 cycles of denaturation at 96°C for 10 s, annealing at 50°C for 5 s, and extension at 60°C for 4 min. Sequencing reactions were cleaned up with Sephadex G50 (SIGMA) and analyzed with an ABI 3130xl DNA genetic analyzer (Applied Biosystems, USA).

Markers in the HmB locus region

A candidate region where the HmB locus is located in H. m. melpomene was previously described and annotated [41], [42]. It comprises seven BAC clones with a total length of around 721 kb [41], [42]. Here, we developed 24 PCR amplicons located in BAC clones AEHM-28L23, AEHM-21P16 and AEHM-27I5 (Table S1). PCR and clean up conditions were as described above. Direct sequencing was possible for 8 markers comprising only coding regions (Table S1). However, due to the presence of introns with considerable indel variation, the other 16 markers were cloned before sequencing (Table S1). PCR products were ligated into the pGEM-T easy vector (Promega). Five or more positive clones per individual were selected, re-amplified and again purified with ExoSAP-IT (USB Corp., Cleveland, OH) to identify distinct alleles. Direct sequencing of clones was performed as described above.

Sequence processing

Gene sequences were read, edited and aligned with Sequencher v4.6 (Gene Codes Corporation). For sequences resulting from direct sequencing, haplotypes reconstruction was conducted using DNAsp v4.90.1 [57] implementing the algorithm provided in PHASE [58]. Basically, PHASE assigns a probability of the correct inference of haplotype phase at every heterozygous position. PHASE simulations were repeated eight times for each locus, four without recombination and four with recombination. Each simulation was run with 5,000 iterations. In all cases, the most common output inferring haplotypes with >95% confidence was accepted. In the case of cloned products, aligned sequences of each individual were compared to discard PCR errors and false alleles caused by recombination in the cloning reaction. Purified alignments were translated to protein and checked for stop codons in MacClade v4.08 [59]. Sequences were deposited in GenBank under accession numbers GQ985506–GQ988326.

Genetic analysis

SNP association statistic

Allele frequencies were calculated for each single nucleotide polymorphism (SNP) with rare allele frequency greater than 10% in the total sample of 60 haplotypes. With these numbers, a likelihood statistic was calculated to indicate the degree to which the H. heurippa SNP frequency was similar to H. m. melpomene relative to H. c. cordula.Where hi and hj are the number of H. heurippa individuals with i or j nucleotide respectively at a particular SNP position. The frequency of the nucleotide i or j in H. m. melpomene is represented by pim and pjm, respectively, and that in H. c. cordula by pic and pjc. A positive value reflects H. heurippa being more similar to H. m. melpomene, while a negative value indicates association with H. c. cordula. Shared polymorphism at similar frequency in the three species, unique polymorphism in any one of the three species or a private allele in H. heurippa all give a value near to zero. For unlinked markers the average of these values was calculated to give a frequency distribution for the genomic background. Then, average values were similarly calculated in 1,000 bp windows across the HmB region and compared to the frequency distribution for unlinked markers. In order to determine if average likelihood values in the HmB region differ significantly from those in the genomic background, a confidence interval of 95% was established over the distribution of values for unlinked markers, by computing the 2.5 and 97.5 percentiles. Furthermore, using RSXL Excel [60], the statistical significance of outlier values was determined by resampling with replacement the likelihood averages for all 1,000 bp windows, with 40,000 repetitions.

Linkage disequilibrium analysis

When new polymorphisms are introduced by recent introgression at some time after species divergence, this should increase levels of linkage disequilibrium (LD) above the background within the region involved. We analyzed LD across the HmB locus for combined samples of H. heurippa and H. c. cordula. This calculation was made among 199 SNPs from the 24 genetic markers sampled in the HmB linked region. These SNPs were those where the minor allele frequency was greater than 10% (from 60 alleles) and thus, were considered as informative for the LD analysis. The LD computation was executed using the software MIDAS [61], which considers the distance between pairwise markers and does not assume that the genotypic phases are known. The resulting LD among the SNPs was visualized with the R package LDheatmap [62] plotting the r2 estimates versus physical distance.

Net divergence, nucleotide diversity, and protein evolution

For all the loci net divergence, fixed differences, shared polymorphisms and nucleotide diversity (π) were estimated with SITES [63]. Additionally, we calculated the number of substitutions per site for synonymous sites (Ks) and non-synonymous sites (Ka) in pairwise comparisons among the three species. The ratio Ka/Ks was determined in order to detect protein evolution in DNAsp v4.90.1 [57]. McDonald-Kreitman test [64] was also performed in DNAsp v4.90.1.

Gene genealogies and time of introgression

In order to confirm the species relationships, genealogical topologies were reconstructed for three fragments within the HmB region, rooted for the 3′ kinesin (6,493 pb) using H. numata as outgroup and unrooted for 5′ kinesin partial sequence (kin_2; 1,100 pb) and sorting nexin (sdp; 402 pb) (Figure 4). Maximum Parsimony analysis was carried out in PAUP*v4.0b10 [65] using a heuristic search with TBR branch swapping; bootstrap values were calculated with 5,000 replicates using the same search conditions. Modeltest v3.7 [66] was used to determine the most appropriate model for nucleotide substitution based on corrected Akaike information criterion (AICc). For the 3′ kinesin data set Modeltest identified the HKY+I+G model, for 5′ kinesin the K81uf+I+G and for nexin the K80+I. Likelihood reconstructions were also made in PAUP*v4.0b10 [65] based on selected evolutionary models. Heuristic search and bootstrapping were carried out as for parsimony.

The 3′ kinesin genealogy was used to enforce a molecular clock hypothesis. When the likelihoods were compared, constant rate evolution was rejected (x2 = 96.92, df = 48; P<0.001). Then a Bayesian framework, implemented in BEAST v1.4.8 [67], was employed to obtain an approximate time for the 3′ kinesin introgression. We applied the HKY+I+G model of evolution with four rate categories and assumed a relaxed lognormal clock. Based on the calibration proposed by Wahlberg et al. for Nymphalidae, with Heliconius and Eueides diverged from their common ancestor 18.4 Mya [68]. This date was used as a prior for a probabilistic calibration to determine the splitting time between H. cydno and H. melpomene alleles and between H. heurippa and H. melpomene alleles. The rest of the parameters were sampled keeping the default prior distributions. Two independent runs were implemented, with 50 million steps and burn-ins of 5,000,000. Tracer v1.4 was used to combine runs and observe parameter convergence [69]. Divergence time standard deviations were calculated from 95% confidence/credibility intervals using a normal approximation. We also computed the time of 3′ kinesin introgression under the assumption of species expansion. To perform this calculation, we first tested the fit of the observed mismatch distribution to the theoretical expectation as implemented in Arlequin v. 3.0 [70]. The calculations were made with a neutral mutation rate of ∼2.99×10−10 per base per generation for this region and 10 generations per year.

Expression analysis

kinesin RNA in situ hybridizations were performed on H. m. cythera, H. m. rosina and H. cydno 72 to 96h pupal forewings. The specific races involved in the rest of the study were not available as live tissue for this analysis. A 303bp region of exon 13 in the H. melpomene kinesin gene was cloned into the vector pSPT19 (linearised with NheI). RNA probes were prepared with the DIG RNA labeling kit (SP6/T7) (Roche, Cat. 11 175 025 910) according to the manufacturer's instructions. Tissue fixation and in situ hybridization were carried out following a procedure modified from Ramos and Monteiro, 2007 [71].

Supporting Information

Attachment 1

Attachment 2

Attachment 3

Attachment 4

Attachment 5


Zdroje

1. KirkpatrickM

RavignéM

2002 Speciation by natural and sexual selection: models and experiments. Am Nat 159 S22 S35 doi:10.1086/338370

2. CoyneJA

OrrHA

2004 Speciation Sunderland, Mass. Sinauer Associates

3. NosilP

FunkDJ

Ortiz-BarrientosD

2009 Divergent selection and heterogeneous genomic divergence. Mol Ecol 18 375 402 doi:310.1111/j.1365-1294X.2008.03946.x

4. Ortiz-BarrientosD

CountermanBA

NoorMA

2004 The genetics of speciation by reinforcement. PLoS Biol 2 e416 doi:410.1371/journal.pbio.0020416

5. TurnerTL

HahnMW

NuzhdinSV

2005 Genomic islands of speciation in Anopheles gambiae. PLoS Biol 3 e285 doi:210.1371/journal.pbio.0030285

6. GavriletsS

2004 Fitness Landscapes and the Origin of Species: Monographs in Population Biology vol. 41;

LevinSA

HornHS

Princeton, NJ Princeton University Press

7. BolnickD

FitzpatrickBM

2007 Sympatric speciation models and empirical evidence. Annu Rev Ecol Evol Syst 38 459 487 doi:410.1146/annurev.ecolsys.1138.091206.095804

8. OttoSP

ServedioMR

NuismerSL

2008 Frequency-dependent selection and the evolution of assortative mating. Genetics 179 2091 2112 doi:2010.1534/genetics.2107.084418

9. ChamberlainNL

HillRI

KapanDD

GilbertLE

KronforstMR

2009 Polymorphic butterfly reveals the missing link in ecological speciation. Science 326 847 850 doi:810.1126/science.1179141

10. BoughmanJW

2001 Divergent sexual selection enhances reproductive isolation in sticklebacks. Nature 411 944 948 doi:910.1038/35082064

11. SavolainenV

AnstettM-C

LexerC

HuttonI

CJJ

2006 Sympatric speciation in palms on an oceanic island. Nature 441 210 213 doi:210.1038/nature04566

12. BerlocherSH

FederJL

2002 Sympatric speciation in phytophagous insects: moving beyond controversy? Ann Rev Entomol 47 773 815 doi:710.1146/annurev.ento.1147.091201.145312

13. PodosJ

2001 Correlated evolution of morphology and vocal signal structure in Darwin's finches. Nature 409 185 188 doi:110.1038/35051570

14. MiyatakeT

2002 Pleiotropic effect, clock genes, and reproductive isolation. Popul Ecol 44 201 207 doi:210.1007/s101440200023

15. PueblaO

BerminghamE

GuichardF

WhitemanE

2007 Colour pattern as a single trait driving speciation in Hypoplectrus coral reef fishes. Proc R Soc Lond B 274 1265 1271 doi:1210.1098/rspb.2006.0435

16. SeehausenO

2004 Hybridization and adaptive radiation. Trends Ecol Evol 19 198 207 doi:110.1016/j.tree.2004.1001.1003

17. MalletJ

2007 Hybrid speciation. Nature 446 279 283 doi:210.1038/nature05706

18. FederJL

BerlocherSH

RoetheleJB

DambroskiH

SmithJJ

2003 Allopatric genetic origins for sympatric host-plant shifts and race formation in Rhagoletis. Proc Nat Acad Sci U S A 100 10314 10319 doi:10310.11073/pnas.1730757100

19. KimM

CuiM-L

CubasP

GilliesA

LeeK

2008 Regulatory genes control a key morphological and ecological trait transferred between species. Science 322 1116 1119 doi:1110.1126/science.1164371

20. AndersonTM

vonHoldtBM

CandilleSI

MusianiM

GrecoC

2009 Molecular and evolutionary history of melanism in north american gray wolves. Science 323 1339 1343 doi:1310.1126/science.1165448

21. JigginsCD

SalazarC

LinaresM

MavárezJ

2008 Hybrid trait speciation and Heliconius butterflies. Philos Trans R Soc Lond B 363 3047 3054 doi:3010.1098/rstb.2008.0065

22. ArnoldML

2006 Evolution through genetic exchange Oxford, UK Oxford University Press

23. UngererMC

BairdSJE

PanJ

RiesebergLH

1998 Rapid hybrid speciation in wild sunflowers. Proc Nat Acad Sci U S A 95 11757 11762

24. MengC

KubatkoLS

2009 Detecting hybrid speciation in the presence of incomplete lineage sorting using gene tree incongruence: a model. Theor Popul Biol 75 35 45 doi:10.1016/j.tpb.2008.1010.1004

25. MalletJ

McMillanWO

JigginsCD

1998 Mimicry and warning colour at the boundary between races and species.

HowardDJ

BerlocherSH

Endless Forms: Species and Speciation New York Oxford University Press 390 403

26. BaxterSW

JohnstonSE

JigginsCD

2009 Butterfly speciation and the distribution of gene effect sizes fixed during adaptation. Heredity 102 57 65 doi:10.1038/hdy.2008.1109

27. JoronM

PapaR

BeltránM

ChamberlainN

MavárezJ

2006 A conserved supergene locus controls colour pattern diversity in Heliconius butterflies. PLoS Biol 4 e303 doi:310.1371/journal.pbio.0040303

28. MalletJ

BeltránM

NeukirchenW

LinaresM

2007 Natural hybridization in Heliconiine butterflies: the species boundary is a continuum. BMC Evol Biol 7 28 doi:10.1186/1471-2148-1187-1128

29. JigginsCD

NaisbitRE

CoeRL

MalletJ

2001 Reproductive isolation caused by colour pattern mimicry. Nature 411 302 305 doi:310.1038/35077075

30. JigginsCD

2008 Ecological speciation in mimetic butterflies. Bioscience 58 541 548 doi:510.1641/B580610

31. GilbertLE

2003 Adaptive novelty through introgression in Heliconius wing patterns: evidence for shared genetic “tool box” from synthetic hybrid zones and a theory of diversification.

BoggsCL

WattWB

EhrlichPR

Ecology and Evolution Taking Flight: Butterflies as Model Systems Chicago University of Chicago Press 281 318

32. LinaresM

1989 Adaptive microevolution through hybridization and biotic destruction in the neotropics. PhD thesis. University of Texas, Austin, TX

33. BullV

BeltránM

JigginsCD

McMillanWO

BerminghamE

2006 Polyphyly and gene flow between non-sibling Heliconius species. BMC Biol 4 11 doi:10.1186/1741-7007-1184-1111

34. KronforstM

YoungLG

BlumeLM

GilbertLE

2006 Multilocus analyses of admixture and introgression among hybridizing Heliconius butterflies. Evolution 60 1254 1268 doi:1210.1111/j.0014-3820.2006.tb01203.x

35. MavárezJ

SalazarC

BerminghamE

SalcedoC

JigginsCD

2006 Speciation by hybridization in Heliconius butterflies. Nature 441 868 871 doi:810.1038/nature04738

36. SalazarC

JigginsCD

AriasCF

ToblerA

BerminghamE

2005 Hybrid incompatibility is consistent with a hybrid origin of Heliconius heurippa Hewitson from its close relatives, Heliconius cydno Doubleday and Heliconius melpomene Linnaeus. J Evol Biol 18 247 256 doi:210.1111/j.1420-9101.2004.00839.x

37. MalletJ

2009 Rapid speciation, hybridization and adaptive radiation in the Heliconius melpomene group.

ButlinR

BridleJ

SchluterD

Speciation and Patterns of Diversity Sheffield Cambridge University Press 177 194

38. MeloMC

SalazarC

JigginsCD

LinaresM

2009 Assortative mating preferences among hybrids offers a route to hybrid speciation. Evolution 63 1660 1665 doi:1610.1111/j.1558-5646.2009.00633.x

39. Duenez-GuzmanEA

MavarezJ

VoseMD

GavriletsS

2009 Case studies and mathematical models of ecological speciation. 4. Hybrid speciation in butterflies in a jungle. Evolution 63 2611 2626 doi:2610.1111/j.1558-5646.2009.00756.x

40. SalazarC

JigginsCD

TaylorJ

KronforstM

LinaresM

2008 Hybrid speciation and the genealogical history of Heliconius heurippa. BMC Evol Biol 8 132 doi:110.1186/1471-2148-1188-1132

41. BaxterSW

NadeauN

MarojaL

WilkinsonP

CountermanB

2009 Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in the Heliconius melpomene clade. PLoS Genet 6(2) e1000794 doi:1000710.1001371/journal.pgen.1000794

42. BaxterSW

PapaR

ChamberlainN

HumphraySJ

JoronM

2008 Convergent evolution in the genetic basis of Müllerian mimicry in Heliconius butterflies. Genetics 180 1567 1577 doi:1510.1534/genetics.1107.082982

43. WahlbergN

WheatCW

2008 Genomic outposts serve the phylogenomic pioneers: designing novel nuclear markers for genomic DNA extractions of Lepidoptera. Syst Biol 57 231 242 doi:210.1080/10635150802033006

44. ExcoffierL

2004 Patterns of DNA sequence diversity and genetic structure after a range expansion: lessons from the infinite-island model. Mol Ecol 13 853 864 doi:810.1046/j.1365-1294X.2003.02004.x

45. SchneiderS

ExcoffierL

1999 Estimation of past demographic parameters from the distribution of pairwise differences when the mutation rates vary among sites: Application to human mitochondrial DNA. Genetics 152 1079 1089

46. FergusonLC

JigginsCD

2009 Shared and divergent expression domains on mimetic Heliconius wings. Evol Dev 11 498 512 doi:410.1111/j.1525-1142X.2009.00358.x

47. BeltránM

JigginsCD

BrowerAV

BerminghamE

MalletJ

2007 Do pollen feeding, pupal-mating and larval gregariousness have a single origin in Heliconius butterflies? Inferences from multilocus DNA sequence data. Biol J Linn Soc 92 19

48. CountermanBA

Araujo-PerezF

HinesHM

BaxterSW

MorrisonCM

2009 Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in Heliconius erato. PLoS Genet 6(2) e1000796 doi:1000710.1001371/journal.pgen.1000796

49. MikiH

OkadaY

HirkawaN

2005 Analysis of the kinesin superfamily: insights into structure and function. Trends Cell Biol 15 467 476 doi:410.1016/j.tcb.2005.1007.1006

50. TekotteH

DavisI

2002 Intracellular mRNA localization: motors move messages. Trends Genet 18 636 642 doi:610.1016/S0168-9525(1002)02819-02816

51. MavárezJ

LinaresM

2008 Homoploid hybrid speciation in animals. Mol Ecol 17 4181 4185 doi:4110.1111/j.1365-4294X.2008.03898.x

52. PapanicolaouA

Gebauer-JungS

BlaxterM

McMillanWO

JigginsCD

2008 ButterflyBase: a platform for lepidopteran genomics. Nucleic Acids Res 36 d582 d587 doi:510.1093/nar/gkm1853

53. PringleEG

BaxterSW

PapanicolaouWA

LeeSF

JigginsCD

2007 Synteny and chromosome evolution in the Lepidoptera: evidence from mapping in Heliconius melpomene. Genetics 177 417 426 doi:410.1534/genetics.1107.073122

54. MitaK

KasaharaM

SasakiS

NagayasuY

YamadaT

2004 The genome sequence of silkworm, Bombyx mori. DNA Res 11 27 35

55. WangJ

XiaQ

HeX

DaiM

RuanJ

2005 SilkDB: a knowledgebase for silkworm biology and genomics. Nucleic Acids Res 33 399 402 doi:310.1093/nar/gki1116

56. RozenS

SkaletskyH

2000 Primer3 on the WWW for general users and for biologist programmers.

KrawetzS

MisenerS

Bioinformatics Methods and Protocols: Methods in Molecular Biology Totowa, NJ Humana Press 365 386

57. RozasJ

Sánchez-DelBarrioJC

MesseguerX

RozasR

2003 DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19 2496 2497 doi:2410.1093/bioinformatics/btg2359

58. StephensM

SmithN

DonnellyP

2001 A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68 978 989 doi:910.1086/319501

59. MaddisonWP

MaddisonDR

2001 MacClade version 4.02: analysis of phylogeny and character evolution Sunderland, Massachusetts Sinauer Associates

60. BlankS

SeiterC

BruceP

Resampling stats in excel. 2 ed Arlington, Virginia Resampling stats, Inc.

61. GauntTR

RodriguezS

ZapataC

DayIN

2006 MIDAS: software for analysis and visualisation of interallelic disequilibrium between multiallelic markers. BMC Bioinformatics 7 227 doi:210.1186/1471-2105-1187-1227

62. ShinJ-H

BlayS

McNeneyB

GrahamJ

2006 LDheatmap: An R function for graphical display of pairwise linkage disequilibria between single nucleotide polymorphims. J Statist Soft 16 1 19

63. HeyJ

WakeleyJ

1997 A coalescent estimator of the population recombination rate. Genetics 145 833 846

64. McDonaldJH

KreitmanM

1991 Adaptive protein evolution at the Adh locus in Drosophila. Nature 351 1111 1117 doi:1110.1038/351652a351650

65. SwoffordDL

2003 PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). 4. ed Sunderland, Massachusetts Sinauer Associates

66. PosadaD

CrandallKA

1998 Modeltest: testing the model of DNA substitution. Bioinformatics 14 817 818 doi:810.1093/bioinformatics/1014.1099.1817

67. DrummondAJ

HoSYW

PhillipsMJ

RambautA

2006 Relaxed phylogenetics and dating with confidence. PLoS Biol 4 e88 doi:10.1371/journal.pbio.0040088

68. WahlbergN

LeneveuJ

KodandaramaiahU

PenaC

NylinS

2009 Nymphalid butterflies diversify following near demise at the Cretaceous/Tertiary boundary. Proc Biol Sci 276 4295 4302 doi:4210.1098/rspb.2009.1303

69. RambautA

DrummondAJ

2007 Tracer v1.4, Available from http://beast.bio.ed.ac.uk/Tracer

70. ExcoffierL

LavalG

SchneiderS

2005 Arlequin ver. 3.0: An integrated software package for population genetics date analysis. Evol Bioinform Online 1 47 50

71. RamosD

MonteiroA

2007 In situ protocol for butterfly pupal wings using riboprobes. J Vis Exp 208 doi:210.3791/3208

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