Three types of evidence have been used in support of the hypothesis of hybrid origin of H. heurippa: the “recreation” of the heurippa phenotype by selective interbreeding of H. melpomene and H. cydno in the lab, studies of the species’ capacity to interbreed with its putative parent species, and patterns of genetic variation in sequences of mtDNA, several nuclear genes, and batteries of microsatellite loci. These will each be examined in turn.
“Re-creating” the H. heurippa phenotype in the laboratory
Mavárez et al. (2006) selected a true-breeding hybrid laboratory strain with a yellow and red banded forewing phenotype similar to that of H. heurippa, by crossing captive H. cydno cordula with H. melpomene melpomene, backcrossing F1 males to H. cydno, and selecting heurippa-like backcross individuals for subsequent matings. After an unstated number of generations, homozygous heurippa-like phenotypes were produced, which the authors claim breed true when crossed to H. heurippa (only one representative of this cross is illustrated).
Mavárez et al. indicate that there are three homologous genes shared between H. cydno and H. melpomene with allelic differences responsible for the major color pattern differences between H. cydno cordula and H. melpomene melpomene: B/b, presence/absence of a red forewing band; NN/NB, presence/absence of a yellow forewing band (heterozygotes intermediate); and Br/br, expression of the brown “forceps” on the ventral hindwing. The wild type genotype of H. cydno cordula is bbNNNNBrBr, while the wild type genotype of H. melpomene melpomene is BBNBNBbrbr, and the genotype of the heurippa-like hybrids is BBNNNNbrbr. Mavárez et al. apparently assume that the alleles making up this genotype are identical by descent with the alleles that produce the same phenotype in H. heurippa, but there are subtle differences between the phenotypes, such as the shape of the yellow band and the shape and the color of the red band, that cast some doubt upon that assumption. As has been corroborated by genome mapping efforts (e.g., Baxter et al. 2008), the genes responsible for various pattern elements are the same among divergent, mimetic Heliconius clades, albeit with different homoiologous alleles. There is thus no reason to assume that alleles resulting in a mimetic or otherwise convergent wing pattern element are IBD among all taxa that exhibit that phenotype, nor, more specifically, any reason why alleles for the expression of a red forewing patch and the masking of the brown hindwing “forceps” could not have arisen independently in H. heurippa. Since the loci responsible for the patterns are the same, selecting a hybrid strain of H. melpomene x H. cydno that looks like H. heurippa is no more evidence of the latter’s hybrid origin from those species than would a selected strain of H. erato that looks like H.heurippa be evidence that H. heurippa is descended from H. erato.
Footnote 3
Intrinsic barriers to gene exchange
There is substantial evidence that Heliconius melpomene and H. cydno are capable of mating and producing offspring in the laboratory (Brown 1981; Mallet et al. 1998; Gilbert 2003), and there is likewise mounting circumstantial evidence that the two species occasionally hybridize in the wild (Salazar 1993; Mallet et al. 1998, 2007). However, Gilbert (2003) noted that “it is possible to keep cydno and melpomene in the same 13 ft × 21 ft greenhouse for years without the occurrence of interspecific courtship or mating.” Controlled mating experiments produce the same result: Naisbit et al. (2001) and Jiggins et al. (2001b) showed, respectively, in no-choice and tetrad mating experiments in the lab that H. melpomene rosina Boisduval and H. cydno chioneus Bates from Panama are completely behaviorally isolated from one another: males of one species will not court females of the other. Mavárez et al. (2006; their Table 2) reported similar results for attempted crosses between H. melpomene melpomene and H. cydno cordula from allopatric populations on the eastern slope of the Colombian Andes: in tetrad mate choice experiments, H.
cydno and H.
melpomene never mated with one another.
Mavárez et al. also performed reciprocal tetrad experiments to test H. heurippa’s capacity to mate with its two hypothetical parental species. They found that neither H. cydno nor H. melpomene males will mate with H. heurippa females, that H. heurippa males will not mate with H. melpomene females, but that male H. heurippa will mate with female H. cydno. In summary, H. cydno and H. melpomene are prezygotically isolated from one another, as are H. melpomene and H. heurippa. H. cydno and H. heurippa are not prezygotically isolated. Note that all of these experiments assess the mating predilections of males, while the females employed are usually sacrificial virgins with neither the opportunity, nor apparently the will, to discriminate. Salazar et al. (2005) indicate that “sexually mature females show a low mating probability with males that are not from their own species” and “H. heurippa females show strong assortative mating when tested against both H. melpomene and H. cydno.” Thus, in addition to male courtship, it is likely that in the wild, mate choice behavior of females of these species also contributes to premating isolation.
If these prezygotically isolated species are forced to mate or happen to mate out of desperation in the insectary, what is the fate of the resultant offspring? Offspring of the H.
cydno x H.
melpomene cross are viable and partially infertile, following Haldane’s rule: heterogametic females are sterile (Linares 1989; Nijhout et al. 1990). Salazar et al. (2005) performed crosses between both of these species and H. heurippa, finding complete hybrid viability and fertility in the H.
cydno x H.
heurippa crosses, and an asymmetrical pattern of offspring fertility in the H. melpomene x H.
heurippa crosses. Male H.
melpomene x female H.
heurippa F1 females are sterile, while male H.
heurippa x female H.
melpomene F1 females are fertile (males from both crosses are viable and fertile). The patterns of pre- and postzygotic mating success described here are summarized in Fig. 2.
Mavárez et al. (2006) stated, “… the phenotype of H. heurippa reproductively isolates it from both parental species.” This is simply not the case (see Mavárez et al. 2006, Table 2). While it is true that neither H. melpomene nor H. cydno males will mate with H. heurippa females, H. heurippa males are perfectly willing and able to mate with H. cydno females (although, as noted, “mature” H. cydno females might reject attempted matings by H. heurippa males). Therefore, it is likely that H. heurippa and H. cydno remain distinct from one another in nature due solely to their geographical disjunction from one another. These authors also claimed that their study provided, “the first example of a hybrid trait causing pre-mating isolation through assortative mating.” In fact, the trait that causes (partial) isolation between H. cydno and H. heurippa is the symplesiomorphic aversive response to red wing pattern elements by H. cydno males, a behavior that also deters them from mating with H. melpomene females (Jiggins et al. 2001b). H. heurippa males share with H. cydno the positive courtship stimulus of white/yellow wing pattern elements (also a symplesiomorphy), which explains why they will pursue H. cydno females but not H. melpomene females.
Patterns of genetic differentiation
Multiple “known” loci, as well as several batteries of microsatellite and AFLP markers, have been examined to assess the genetic structure of the melpomene-cydno group and the H. heurippa hybrid speciation hypothesis. The strategy employed in this paper was to obtain and reanalyze all relevant sequence data for each gene for which a H. heurippa has been sequenced, using the parsimony algorithm as implemented in TNT (Goloboff et al. 2003) (1,000 random addition replicates; gaps encoded as a fifth character; equal weights). Sequence data were transcribed from GenBank. Terminals are labeled with individual voucher codes as reported in the GenBank annotations or corresponding publications. Note that some sequences were published on more than one occasion and are represented in GenBank with more than one accession code. Aligned data matrices for each of the genes are available as Nexus files at http://www.mtsu.edu/~abrower/datasets. Alignments were performed by eye.
Data which were unavailable for reanalysis were evaluated based upon the authors’ descriptions of them in the original publications.
Mitochondrial DNA
As noted earlier, phylogenetic analysis of mtDNA COI-COII sequences has usually placed H. heurippa in a clade with H. cydno, nested in turn within a paraphyletic H. melpomene (Brower 1996a, b; Beltrán et al. 2007; Quek et al. 2010). There has evidently been some confusion about this pattern. Bull et al. (2006), in a rather contorted argument that contradicted their own results, claimed that “mutual monophyly (of H. cydno and H. melpomene) cannot be rejected,” and Quek et al. (2010), citing Brower (1996a), stated that “early mtDNA studies indicated a sister species relationship,” despite the cited paper’s clearly stating, “… intraspecific variation in H. melpomene is complicated by the apparent paraphyly of the species with respect to H. cydno and its close relatives ….”
To clarify the current understanding of relationships implied by this gene region, all published COI–COII sequences for the melpomene-cydno complex (371ingroup exemplars, from papers cited above, as well as Beltrán et al. 2002; Kronforst et al. 2006; Giraldo et al. 2008; Chamberlain et al. 2009) were extracted from Genbank, aligned (no length variation) and analyzed via parsimony (MP). A strict consensus tree is presented in Fig. 3.
The salient features of this tree are the same as those revealed by Brower (1996a): a basal melpomene clade from the Guianas, and then a polytomy comprising melpomene clades from southeastern Brazil, Amazonia, and the trans-Andean region and a clade of cydno cognates. Thus H. melpomene in the broad sense is still “paraphyletic” with respect to H. cydno and its satellites. Note that under the Phylogenetic Species Concept of Nixon and Wheeler (1990), two such entities are indeed considered to be “sister taxa”Footnote 4 (see Brower 1999). As Brower (1996a) found, there is little resolution within each of these clades, and no obvious correspondence between haplotypes and wing-patterns or finer scale geography. H. heurippa, H. timareta, and H. tristero individuals are interspersed within the H.
cydno clade. Two “new” clades appear: a second clade of H.
melpomene individuals from French Guiana and Trinidad (which will not be discussed here), and a cluster of “H.
melpomene” haplotypes from Chirajara, Cundinamarca, Colombia and Santa Ana, Mérida, Venezuela, which is sister to the H.
cydno clade. More of these anon.
Triose phosphate isomerase
Tpi is a polymorphic, sex-linked nuclear gene, a ~530 bp region of which was developed for examining Heliconius relationships by Beltrán et al. (2002). Additional members of the H.melpomene-H. cydno complex have been sequenced by Flanagan et al. (2004), Bull et al. (2006), Kronforst et al. (2006), Dasmahapatra et al. (2007), Giraldo et al. (2008), Kronforst (2008) and Salazar et al. (2008). 186 sequences were extracted from Genbank and analyzed via MP. Unlike the mtDNA, the Tpi gene region contains a length-variable segment spanning an intron. Alignment is not trivial, and approximately 2/3 of the phylogenetically informative sites occur in the noncoding region.
Analyses of these data, either including the gaps as a fifth character state or treating them as “missing,” reveal a weakly supported clade of alleles from various H. melpomene races, embedded within a paraphyletic cluster of alleles from H. cydno races, H. timareta and H. heurippa (Fig. 4). There is no apparent correlation of the gene tree topology with finer-scale phenotypic or geographical patterns. Single alleles are shared by multiple geographical races of H. melpomene and H. cydno, and among H. heurippa and H. timareta. Alternate alleles from some individuals are highly divergent from one another (although no alleles are shared between H. cydno and H. melpomene). There is no evidence for interspecific H. melpomene—H. cydno hybridization at this locus.
Mannose-6-phosphate isomerase
Beltrán et al. (2002) also developed a region of the autosomal Mpi as a marker for examining relationships in Heliconius. The amplified region is composed of short exon sequences (9 and 13 codons) flanking a highly length-variable intron ranging from <100 to >400 bp in the current alignment. Additional sequences generated by Flanagan et al. (2004), Bull et al. (2006), Kronforst et al. (2006) and Kronforst (2008) were compiled into a matrix of 118 ingroup taxa plus five outgroups. The data exhibit long, highly similar insertions and deletions that are shared among a variety of taxa, including a 250 bp deletion shared among H. cydno chioneus and H. melpomene rosina, both from Panama, and H. melpomene melpomene from French Guiana. There is also a 65 bp insertion which occurs in several H. pachinus, H. cydno chioneus and H. cydno galanthus Bates, as well as one H. melpomene rosina.
Most of the authors working with Mpi have viewed the sharing of alleles among these species as evidence for current interspecific gene flow. How hybridization explains the distribution of the 250 bp deletion in specimens from Central America and French Guiana (more than 2,000 km apart) is not clear. Further, when one takes into account the phylogenetic distribution of alleles present in heterozygous individuals, the pattern suggests that the diversity of alleles is nearly panmictic, not only among members of the H.
cydno-H.
melpomene group, but also among outgroup H. hecale (Fabricius) and H. ethilla (Godart). In regards to the “hybrid speciation” question, the alleles from the single H. heurippa sampled for this gene fall into two distinct clades (Fig. 5), each of which contains alleles from both H. cydno and H. melpomene. Given that the patterns of change in the Mpi marker are incoherent with respect to species boundaries and biogeography and because the variation is evidently not driven by single nucleotide substitutions, it seems premature to ascribe much evidentiary value to phylogenetic interpretations of this gene region.
Distal-less
Kronforst et al. (2006) and Mavárez et al. (2006) have examined two sets of sequences from the autosomal developmental gene Dll, each study interpreting the distribution of alleles to support the hypothesis of interspecific gene flow. Kronforst’s specimens were all Costa Rican, and Mavárez’s were all from the eastern slopes of the Andes in Colombia and Venezuela. Again, the bulk of informative variation in this gene region comes from a length-variable intron. Data from both these studies comprising 99 ingroup alleles were combined and analyzed via MP with gap characters from the intron included as a fifth character state. Mavárez et al. (2006) reported “no allele sharing between H. cydno and H. melpomene, whereas the H. heurippa genome appears as an admixture, sharing allelic variation with both putative parental species.” The tree based on the combined dataset (Fig. 6; see also Kronforst et al. 2007) complicates the interspecific hybridization scenario by revealing nearly identical alleles shared not only among sympatric H. melpomene and H. cydno, but also between Costa Rican and eastern Colombian H. melpomene, and between Costa Rican H. cydno galanthus and H. heurippa. Such a pattern suggests that ancestral polymorphism could also explain the maintenance of allelic diversity across the ranges of these taxa. It seems that further sampling of this locus from the geographical diversity of H. melpomene and H. cydno forms will be necessary before the extent and mode of gene flow throughout the clade may be clearly discerned.
Invected
The data for this gene have the same provenances as the Dll data, and were also invoked by their respective authors as evidence of the origin of H. heurippa via interspecific hybridization. Once again, most informative variation in the inv fragment comes from a length-variable intron. The two data sets were combined into a matrix of 67 ingroup alleles × 479 bp and analyzed via MP with gap characters from the intron included as a fifth character state. The combined tree (Fig. 7; see also Kronforst et al. 2007) exhibits near reciprocal monophyly of H.
cydno-group alleles and H.
melpomene alleles, but several H. heurippa alleles and one H. pachinus allele cluster among the H. melpomene alleles. This pattern represents the most convincing evidence of hybridization from any of the genes discussed so far. However, it is again evident that very closely-related alleles are distributed between Venezuela and Costa Rica, suggesting not only sympatric interspecific hybridization, but also extreme long-distance dispersal or some other mechanism that results in homologous (or convergent) intron variation among remote areas. Further, no H. heurippa allele is identical to any H. melpomene allele, and one of the H. heurippa alleles is sister taxon to all H. melpomene alleles save one, suggesting that if these alleles were acquired via hybridization, the hybridization events happened long ago and on several separate occasions. As in Dll, the entire picture of allelic diversity at this locus may not be represented by the limited geographical samples examined to date.
White
Sequences of the ommochrome biosynthesis gene w for 37 ingroup alleles were obtained from Kronforst et al. (2006) and Salazar et al. (2008), representing the same taxa as Dll and inv. Again, the aligned region is short (434 bp) and contains a length-variable intron. Parsimony analysis resulted in a consensus tree (Fig. 8) with all species’ alleles polyphyletic with respect to one another, and no obvious geographical structure. If the distribution of alleles among species at this locus is due to hybridization, then H. cydno- H.
melpomene group members are exchanging alleles not only among themselves, but also with the outgroup H. hecale.
Scalloped
Sequences of a region of this putative developmental gene representing 46 ingroup alleles were obtained from Kronforst et al. (2006) and Salazar et al. (2008), representing the same taxa as the preceding three genes. The aligned region is 555 bp and contains a length-variable intron. Parsimony analysis resulted in a consensus tree (Fig. 9) with all species’ alleles polyphyletic with respect to one another, and no obvious geographical structure.
Wingless and elongation factor 1-alpha
Sequence data for these genes have been useful for a wide variety of higher-level studies of butterfly relationships (e.g., Brower 2000a; Wahlberg et al. 2009), but have proven to exhibit little variability within or among closely-related Heliconius species (Brower and Egan 1997; Beltrán et al. 2007). 33 wg sequences and 31 Ef-1 alpha sequences representing the H. cydno-H. melpomene group were obtained from Genbank, aligned and analyzed, but resulted in largely unresolved polytomies (data not shown).
Microsatellite and AFLP data
Microsatellites and AFLP data are the allozymes of the twenty-first century, and their use as a source of evidence to infer population structure has resurrected many of the dubious phenetic approaches that were superceded by cladistics in the late 1970s. Unlike sequences of known genes, these markers are “anonymous,” their putative homology is not testable, and they cannot be compared across studies in the manner performed for the loci discussed above. Their inscrutability is further exacerbated by the unfortunate tendency by researchers who employ them to not make available the raw data for critical reexamination. Thus, the would-be intersubjective corroborator is relegated to deciphering recondite graphical summaries and discursive interpretations as a means of evaluating these “data.” Several of the following studies did not examine H. heurippa, but the patterns found among other Heliconius species bear upon the interpretation of the H. heurippa story.
Mavárez et al. (2006) examined 12 microsatellite loci from five populations of H. cydno and H. melpomene and one population of H. heurippa using Structure 2.1 (Pritchard et al. 2000), a Bayesian model that simultaneously infers how many clusters of similar multilocus genotypes exist in the data and assigns individuals to those clusters. They reported that the “best model” specified three clusters, allowed for admixture and independent estimations of allele frequencies. They did not report whether or not the “best model” was significantly more likely than any other.Footnote 5 Nor do they mention the interesting phenomenon that the degree of inferred admixture among H. heurippa, H. melpomene and H. cydno appears to be highest between the populations of the latter two that are the most geographically remote (Pipeline Road, Panama) from the range of H. heurippa. In sum, the methods used to analyze these data were so inadequately described that the results are indeterminate. Their diagram (their Fig. 1) shows individuals of the three species organized into blocks of three different colors, suggesting that H. cydno, H. melpomene and H. heurippa are different from one another. But how different? Are these differences nested?
Mavárez et al. (2006, their supplementary Fig. 4) conducted a separate, also inadequately–described, microsatellite analysis of 36 H. melpomene melpomene, 44 H. cydno cordula and 9 putative cydno x melpomene hybrids from San Cristóbal, Venezuela. The microsatellite profiles of these “hybrid” specimens in Structure are apparently identical to those of H. cydno cordula (see epigram #1), suggesting under the hybrid speciation hypothesis that the only alleles from H. melpomene they have retained from their interbreeding are specifically those responsible for their “hybrid” wing patterns.
Jesús Mavárez (pers. comm., Nov. 2005) reported results of a to-date unpublished Factorial Analysis of Correspondence based on microsatellite variation at 12 loci for the two specimens of H. tristero known at that time,Footnote 6 compared to H. melpomene and H. cydno from Panama, Colombia and Venezuela. H. tristero clustered within H. cydno and not within H. melpomene, and no H. melpomene individual ever fell within the H. cydno cluster. He also indicated that his results show that H. pachinus and H. timareta are also closely related to H. cydno.
Kronforst et al. (2006, 2007) examined a large data set of AFLP loci to assess interspecific gene flow among Costa Rican H. pachinus, H. cydno galanthus and H. melpomene rosina. They found (2006) “multiple instances of mixed ancestry in all three species,” but concluded (2007) based on the same data that all their analyses “supported a close genetic relationship between H. pachinus and H. cydno and none suggested a genetic contribution from H. melpomene in the origin of H. pachinus.” They drew an important distinction between hybridization as an incidental process and hybridization as a source of evolutionarily significant genetic variation, ruling out the latter in H. pachinus, but invoked the inv and Dll data discussed above to suggest that H. heurippa may have a hybrid origin.
Giraldo et al. (2008) used microsatellite data in Structure to sort an unspecified number of individuals of H. melpomene malleti Lamas and H. florencia Giraldo et al.Footnote 7 into populations, and found five individuals with microsatellite profiles containing markers assigned to multiple species that they interpreted to be hybrids. As in Mavárez et al. (2006), there is no way to assess the robustness of this interpretation from the data as presented.
Chamberlain et al. (2009) examined microsatellites from samples of H. cydno alithea, H. cydno galanthus and H. pachinus to look for possible genetic differences between yellow and white polymorphic forms of the former. They didn’t find any, but interestingly, when they ran Structure on their data allowing admixture, as in the studies reviewed above, all of their H. cydno galanthus specimens appear as “hybrids” (sharing alleles from allopatric H. cydno alithea and H. pachinus!) with an equal or higher degree of admixture than most of the “hybrid” specimens discussed above (their Fig. 3b).
Most recently, Quek et al. (2010) have used 3,186 AFLP loci to examine geographical structure among populations of H. melpomene throughout its range. Their NJ phenogram for these data is largely congruent with trees inferred from mtDNA (e.g., Fig. 3), although bootstrap values indicate rather high instability of relationships among most races and geographical areas. Specimens from “Colombia” represent three different geographical areas—the Pacific slope, the Magdalena Valley and the eastern slope of the Andes, and it would have been a surprise if they did form a “clade.” Quek et al. also performed a Structure analysis of the same data, which as in the studies discussed above revealed “admixture” between various sampled individuals, including H. cydno from Panama and H. melpomene from southeastern Brazil.
So, to summarize these various Structure analyses, it seems that when one wants to find admixture to support one’s hypothesis, then one can do so. Or when one wants to ignore admixture to support one’s hypothesis, then one can do so. It is indeed an excellent type of evidence that is so compliant to the desired interpretations of the researcher.
In general, the available genetic evidence support a limited degree of shared polymorphism between H. melpomene and H. cydno and its various offshoots. Kronforst et al. (2006) summed up the pattern nicely: “Our results indicate that the three Heliconius species [Costa Rican H. cydno, H. pachinus and H. melpomene] studied here hybridize frequently enough to leave recognizable evidence of admixture and introgression but not enough to erode species boundaries.” However, studies limited to populations of different species from a single geographical region may reveal shared alleles, but these patterns cannot be interpreted as the necessary result of recent hybridization when the same or closely-related alleles are also shared among remotely allopatric populations, implying additional historical processes besides exchange of alleles among sympatric relatives. The patterns are also made difficult to interpret by the fact that most of the informative variation in the genes studied occurs in length-variable intron regions. It is clear that these length variants do not evolve one nucleotide at a time, but the “gap characters” are treated as though they did (as in the above reanalyses) or ignored altogether as missing data. It is extremely doubtful that either of these approaches adequately captures the character state transformations as they have occurred, but until more is known about the tempo and mode of intron evolution, there does not seem to be a more defensible alternative. Again, when the assumptions of the analytical approach are not met by the patterns of variation in the data, one is treading on thin epistemological ice to interpret the results as an accurate reflection of history.
Thirty years ago, Turner et al. (1979) found a high degree of shared polymorphism among Heliconius species using allozymes, and argued that the genes related to wing patterns must be decoupled or behave independently from the remainder of the genome. This pattern has been corroborated by recent genomic studies (Baxter et al. 2010), who found limited signatures of selection around specific loci associated with wing pattern elements. It therefore may not be possible to obtain a clear understanding of the evolution of mimetic phenotypes in these butterflies until we are able to examine gene genealogies for the genes that are responsible for the wing pattern elements themselves. I predict that the allele producing a red band on the forewing of H. heurippa will not be homologous (IBD) to that of sympatric H. melpomene melpomene, a pattern that would lay to rest the H. heurippa HHS hypothesis.