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Introgression of wing pattern alleles and
speciation via homoploid hybridization in
Heliconius butterflies: a review of
evidence from the genome
Andrew V. Z. Brower
Review
Cite this article: Brower AVZ. 2013
Introgression of wing pattern alleles and
speciation via homoploid hybridization in
Heliconius butterflies: a review of evidence
from the genome. Proc R Soc B 280: 20122302.
http://dx.doi.org/10.1098/rspb.2012.2302
Received: 28 September 2012
Accepted: 19 November 2012
Subject Areas:
evolution, genomics, taxonomy and
systematics
Keywords:
ecological speciation, magic traits, homoploid
hybrid speciation, Müllerian mimicry,
population genomics, gene flow
Author for correspondence:
Andrew V. Z. Brower
e-mail: [email protected]
Evolution and Ecology Group, Department of Biology, Middle Tennessee State University,
Murfreesboro, TN 37132, USA
The diverse Müllerian mimetic wing patterns of neotropical Heliconius
(Nymphalidae) have been proposed to be not only aposematic signals to
potential predators, but also intra- and interspecific recognition signals that
allow the butterflies to maintain their specific identities, and which perhaps
drive the process of speciation, as well. Adaptive features under differential
selection that also serve as cues for assortative mating have been referred to
as ‘magic traits’, which can drive ecological speciation. Such traits are expected
to exhibit allelic differentiation between closely related species with ongoing
gene flow, whereas unlinked neutral traits are expected to be homogenized
to a greater degree by introgression. However, recent evidence suggests that
interspecific hybridization among Heliconius butterflies may have resulted in
adaptive introgression of these very same traits across species boundaries,
and in the evolution of new species by homoploid hybrid speciation. The
theory and data supporting various aspects of the apparent paradox of
‘magic trait’ introgression are reviewed, with emphasis on population genomic
comparisons of Heliconius melpomene and its close relatives.
1. Introduction
Neotropical Heliconius butterflies, renowned for their diverse warning
coloration, Müllerian mimicry and intraspecific geographical variation [1–5],
have become an important model system in a wide range of evolutionary and ecological disciplines. According to a keyword search in Web of Knowledge (http://
wokinfo.com), nearly 300 articles addressing aspects of Heliconius biology have
been published in the past five years. A major area of research has been ecological
and genetic aspects of speciation, with some authors arguing that the genus exemplifies Darwin’s [6] notion that the species boundary is only an arbitrary point on a
continuum [7]. Lately, Heliconius has become a focus for comparative evolutionary
genomics as well [8–14], providing a vast new source of data to address longstanding questions about the diversification of aposematic wing patterns within
and among members of the genus. This study reviews recent literature as it
bears upon a specific controversy: the hypothesis that one or more Heliconius
species in the H. melpomene–silvaniform clade have arisen as the result of
homoploid hybrid speciation (HHS). (The silvaniform clade, sister to the
H. melpomene þ H. cydno clade [15–17], includes 10 species, most of which exhibit yellow, orange and black ‘tiger’ patterns that are Müllerian mimics of one
another and of ithomiine butterflies, especially Melinaea spp. Two silvaniform
species, H. besckei and H. elevatus, are mimics of H. erato and H. melpomene forms.)
A homoploid hybrid species is a distinct evolutionary lineage resulting from
the admixture of genetic material from two (or more) hybridizing parental species.
Unlike polyploid hybrid taxa, which are chromosomally incompatible with their
progenitors, homoploid hybrid species have the same number and arrangement
of chromosomes as their parental species and so are, at least initially, reproductively compatible. To become established as distinct, true-breeding entities, the
& 2012 The Author(s) Published by the Royal Society. All rights reserved.
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Müllerian mimicry among aposematic, distasteful prey is
maintained by positive frequency-dependent stabilizing
selection [39–42]: if predators learn to avoid unpalatable,
brightly coloured butterflies by remembering an association
between an unpleasant predation event and a colour pattern,
then the more common that colour pattern is, the lower
the risk of predator attack for each individual exhibiting it.
Individuals exhibiting a rare or unique colour pattern are at
a selective disadvantage because their patterns are unfamiliar
to potential predators and therefore fail to serve as aposematic
signals to deter predation. For example, a mark–release–
recapture study of Heliconius erato [43] reported a cumulative
selection coefficient of 52 per cent against novel wing pattern
phenotypes relative to endemic phenotypes on opposite
sides of an interracial hybrid zone.
Given this strong selection against novelty, one might
expect that all Müllerian mimics in a given area, and all
3. Natural hybridization and introgression
between H. cydno and H. melpomene
Heliconius cydno and H. melpomene are broadly sympatric,
geographically variable sister species in Central America
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2. Primer on selection for Müllerian mimicry
among Heliconius species
individuals of each species, would exhibit the same aposematic
pattern. The great paradox of Heliconius evolution [44,45] is that
across tropical America, multiple dissimilar Heliconius mimicry
complexes occur in sympatry, while multiple species exhibit
dramatic geographical polymorphism with numerous, different-looking allopatric or parapatric races, most spectacularly
demonstrated by the co-mimics H. melpomene and H. erato.
Unlike the more genetically integrated wing patterns of most
other butterflies [46], the various elements of Heliconius wing
patterns are encoded by genes in multiple independently segregating linkage groups [4]. While usually monomorphic and
presumably homozygous at wing pattern loci throughout
most of their ranges, different-looking geographical races
freely interbreed where they abut in narrow hybrid zones, producing viable and fertile offspring with no obvious intrinsic
pre- or post-zygotic barriers to gene flow [4,42,43]. F1 interracial hybrids (heterozygous at wing pattern loci) often appear
similar to one of the parental races owing to dominance, but
independent assortment and recombination in F2 and backcross generations result in a broad range of novel phenotypes
that are selectively disadvantageous because they do not
share the common aposematic pattern of either of their parental
forms. By contrast, loci not associated with wing patterns do
not exhibit differentiation across these intraspecific hybrid
zones [5,20,47–49].
How did the diversity of wing patterns within and among
Heliconius species evolve, given the aforementioned selection
against novelty? The traditional ‘parsimonious’ explanation
has been allopatry, often invoking isolation of local populations in Pleistocene forest refugia [3,4,50], which would
have allowed colour patterns of local mimicry complexes to
diverge by differential selection, genetic drift, or both [51].
While vicariance remains a viable explanation for neotropical
speciation events, species-level divergence of many taxa has
been argued to be too old to be explained by Pleistocene
events alone [52 –55]. Brower [5,56] inferred that ages of
divergence (at least among the more closely related races
within H. erato, H. melpomene and H. cydno) were consistent
with a Pleistocene origin (i.e. less than two Myr old), and
those age estimates have been corroborated by more recent
studies [9,57,58]. Mallet [42,49,59] argued that evidence
for the existence of Pleistocene refugia is tenuous, and that,
in any event, allopatry is unnecessary for divergence, presaging more recent discussions of ecological speciation [34].
Recent phylogenetic analysis of optix, a gene associated with
red-pigment expression in H. erato and H. melpomene [60],
suggests that the red-rayed wing patterns have recently
evolved and expanded in Amazonia, relegating races with
ancestral colour patterns to the periphery of the species’
ranges in upper Amazonian river valleys and west of the
Andes [10], a pattern predicted by Sheppard et al. [4] and
Mallet [42,49]. Again, recent large-scale molecular analyses
imply that regions of the genome not linked to colour pattern
loci show either little variation among races of H. melpomene
and H. erato, or variation correlated with geography and not
wing patterns [10,61,62].
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progenitor population of a homoploid hybrid species needs to
avoid being genetically swamped by backcrossing to one or
the other of its parental species. Because of this problem, HHS
is considered to be an unlikely mode of speciation [18,19],
particularly if the hybrid species co-occurs in space, time and
ecological requirements with one or both parental taxa: most
putative examples in plants and animals involve colonization
of novel habitats [20,21].
Authors advocating the HHS model in Heliconius
have promoted their hypothesis effectively [8,22 –28], and
although the evidence has received little critical scrutiny,
the idea now seems to be generally accepted. For example,
a recent Nature letter [11] announcing a complete sequence
of the H. melpomene genome, asserted the following: ‘Most
species in [the H. melpomene –silvaniform] clade occasionally
hybridize in the wild with other clade members. Gene
genealogies at a small number of loci indicate introgression
between species, and one non-mimetic species, H. heurippa,
has a hybrid origin ( pp. 94 –95).’ These three statements, presented as established facts, provide the conceptual framework
for the interpretation of new data as further supporting and
extending the hybrid speciation hypothesis to additional species
such as H. timareta, and perhaps even the silvaniform H. elevatus.
However, as discussed below, there is reason to question each of
these claims. HHS is theoretically unlikely even under ideal circumstances [29,30], so if it has occurred multiple times just
within this charismatic genus of butterflies, then speciation
theory would seem to be headed for a paradigm shift.
This study briefly reviews the evolution of aposematic
wing patterns and mimicry in Heliconius, as well as mechanisms for maintaining specific identity, interspecific
hybridization, introgression and HHS, and concludes with an
alternative hypothesis to explain the origin of H. heurippa and
other Amazonian H. cydno cognates (figure 1). Since Eltringham [1], diverse Heliconius forms that interbreed and produce
hybrids have been viewed as conspecific geographical races,
and, despite debated alternatives [35–37], the biological
species concept (BSC) [38] continues to serve as the primary criterion by which Heliconius nomenclature is organized. From a
BSC perspective, it is arguable that all the taxa illustrated in
figure 1 should be considered geographical races of H. cydno.
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11a
11b
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20a
20b
20c
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13b
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Figure 1. Map of northwestern South America, illustrating diversity and distribution of geographical races of the Heliconius cydno (yellow dots) and its offshoots (red
dots). Nomenclature follows Lamas [31] except for recently described taxa; names in quotation marks are infrasubspecific and not part of formal zoological
nomenclature. See Rosser et al. [32] for detailed range information. Lettered forms are locally polymorphic. 1. H. cydno galanthus Bates, 1864; 2. H. cydno cydno
Doubleday, 1847; 3. H. cydno cordula Neustetter, 1913; 4. H. cydno gadouae Brown & Fernández, 1985; 5. H. cydno barinasensis Masters, 1973; 6. H. cydno chioneus
Bates, 1864; 7. H. cydno cydnides Staudinger, 1885; 8. H. cydno zelinde Butler, 1869; 9. H. pachinus Salvin, 1871; 10. H. cydno lisethae Neukirchen, 1995;
11a. H. cydno weymeri Staudinger, 1897; 11b. H. cydno weymeri f. ‘gustavi’; 12. H. cydno hermogenes Hewitson (1858); 13a. H. cydno alithea Hewitson, 1869;
13b. H. cydno alithea f. ‘haenschi’; 14. unnamed ‘H. cydno x H. melpomene hybrid’ (cf. [22]); 15. H. cydno wanningeri Neukirchen, 1991; 16. H. heurippa Hewitson,
1854; 17. unnamed H. cydno race (cf. [33]); 18. H. timareta florencia Giraldo, Salazar, Jiggins, Bermingham & Linares, 2008; 19. H. tristero Brower, 1996; 20a. H.
timareta timareta f. ‘richardi’; 20b. H. timareta timareta f. ‘contiguus’; 20c. H. timareta timareta Hewitson, 1867; 21. H. timareta timareta f. ‘peregrina’; 22. unnamed
species (cf. [29]); 23. H. timareta timoratus Lamas, 1998. Figure based on [3,25,32,34]. Form 14 was viewed as an interspecific hybrid based on its H. melpomene-like
wing pattern by Mavárez et al. [22], but is genetically associated at all loci examined with H. cydno and is thus hypothesized to be another unnamed H. cydno
cognate [20]. All of the ‘red’ forms except H. timareta and H. heurippa have been discovered within the last 20 years.
and trans-Andean South America [13,36]. The two species
belong to different Müllerian mimicry complexes: H. cydno
is usually bluish black with white or yellow bands on foreand hindwings (figure 1) and mimics sympatric H. sapho
and H. eleuchia, whereas H. melpomene is usually brownish
black with either red forewing bands or radiating red basal
streaks and yellow forewing bands, and mimics H. erato.
Heliconius melpomene’s range extends throughout Amazonia,
whereas H. cydno is largely replaced along the eastern slope
of the Andes by related forms that mimic either H. erato
and H. melpomene (e.g. H. timareta florencia, H. tristero) or exhibit ‘non-mimetic’ wing patterns with some red pattern
elements (e.g. H. heurippa, H. timareta forms).
The different wing patterns of H. melpomene and H. cydno
are thought to play a key role in maintaining their specific
identities in sympatry. Heliconius melpomene males are stimulated to court conspecific females by their red wing pattern
elements, whereas H. cydno males are stimulated by white
or yellow pattern elements and deterred by red [63–66].
The loci responsible for these wing pattern elements and
mate choice are linked, and the respective alleles of the two
species are thought to exhibit pleiotropy that promotes
discriminatory mate choice [67,68].
Gilbert [69] provided a qualitative review of decades
of experimental interspecific laboratory crosses between
H. cydno galanthus, H. melpomene rosina and H. pachinus
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4. Heliconius heurippa: homoploid
hybrid species?
A highly publicized Nature letter [22] invoked Gilbert’s
model [69] to develop a scenario for the hybrid origin of
H. heurippa, a narrowly endemic form from the eastern
slopes of the Andes in Colombia, where it is sympatric
with H. melpomene melpomene but not with any H. cydno
form. Heliconius heurippa has an H. cydno-like yellow forewing
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or ‘Bogotá’ leave much open to interpretation, and historical
collectors often had motives other than the advancement of
scientific knowledge. For example, Anton Fassl (1876–1922),
a prolific collector of Colombian Lepidoptera (including putative Heliconius interspecific hybrids) and curator at the Vienna
Natural History Museum, confessed in 1906 to stealing butterflies valued at more than $7500 (some $275 000 in 2012) from
the museum to establish his own business [82].
The rarity of wild H. cydno H. melpomene hybrids is
attested by the fact that almost none have been found by
modern Heliconius researchers: of the 66 post-1960 records,
42 are from the personal holdings of private collectors who
obtained the specimens by purchase or trade from intermediaries. For example, 28 H. cydno H. melpomene hybrids in the
database originated with either C. Farrell, L. Denhez or
J. Urbina, and wound up in the cabinets of five different amateurs. In 1998, one of these (Mallet et al. [7] co-worker Walter
Neukirchen) advertised his personal collection of Heliconius
butterflies, including 24 interspecific hybrids included in
[7], at the asking price of US $580 000 (in litt. to A.V.Z.B.).
It is not unreasonable to suspect that many of these oddities
could have been captive-reared for the butterfly trade, despite
assurances to the contrary [7].
In sum, so many of the Mallet et al. [7] records are
dubious, at least for H. cydno H. melpomene ‘hybrids’, that
this dataset must be discounted as convincing evidence of
widespread natural hybridization between those species, or
more generally for ‘the species boundary as a continuum’.
Of course, even if phenotypically intermediate specimens
did imply that interspecific hybridization has occasionally
occurred among Heliconius species, that would not constitute
evidence that introgressive gene flow has established foreign
alleles across species boundaries [18]. To support the hypothesis of interspecific gene exchange, several studies [22,83–85]
have invoked as evidence of hybridization and introgression similar alleles shared among Heliconius species in gene
genealogies of various nuclear protein-coding loci (mannose
phosphate isomerase, triose phosphate isomerase, distalless, invected, white and scalloped). A limitation of these
studies is that each compared variability among species
only at one or a few geographical localities. Brower [20]
combined data for each of these genes in global analyses
encompassing multiple geographical regions, and found
that many of the ‘introgressed’ alleles are distributed
widely among members of the H. cydno–H. melpomene
clade from throughout their geographical ranges, a pattern
explained at least as well by retention of ancestral polymorphism as by recent introgressive hybridization [86,87].
Thus, oft-cited claims of ongoing, evolutionarily significant
gene flow between H. cydno and H. melpomene [83,84,88]
should be viewed with circumspection (as suggested by
Kronforst et al. [89]).
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from Costa Rica, and developed a model explaining the
evolution of phenotypic diversity among these species.
Gilbert argued that because wing pattern genes are homologous among Heliconius species [70] (corroborated at the
molecular level by [61,62,71–74]), evolution of wing pattern
diversity occurred in two phases. Initially, mutations produced differentiated prototypical phenotypes, but subsequently,
most of the phenotypic diversity of geographical races in
H. melpomene and H. cydno and its offshoots evolved not by
further mutation, but instead via interspecific exchange of
wing pattern alleles. Thus, for example, Gilbert hypothesized
that the presence of a marginal white band on the hindwing of
H. melpomene cythera, a feature not found in other H. melpomene
races, originated by introgression of alleles from H. cydno
alithea. In the opposite direction, he suggested that red pattern
elements in H. cydno relatives H. heurippa, H. timareta and
H. tristero originated by introgression of alleles from various
H. melpomene forms.
Although Gilbert [69] pointed out that populations of
H. melpomene and H. cydno can coexist in a greenhouse ‘for
years without occurrence of interspecific courtship or mating’
(p. 309), he argued that interspecific hybridization occurs,
rarely, in nature, citing as evidence a then-incipient database of
putative interspecific Heliconius hybrid specimens [75]. Mallet
et al. [7] compiled a more extensive list of 161 specimens in
public or private collections that they believed to be wildcaught interspecific hybrids. More than one-third of these are
hybrids between H. himera and H. erato, and not of concern
here, except to note that until Mallet ([42], p. 245) opined that
H. himera is a ‘good species’, it had been considered a geographical race or ‘semispecies’ of H. erato [3,76,77] based on the fact
that the two hybridize in nature. Mallet [35] subsequently proposed an alternative species concept to the BSC that permits
such hybridizing taxa to be viewed as specifically distinct.
Because hybridization between the parental species in nature
is a prerequisite for hybrid speciation, the dataset of Mallet et al.
[7] has become the empirical cornerstone for the Heliconius
hybrid speciation story, justifying the claim that species
boundaries within the genus form a ‘continuum’, supporting
assertions such as that ‘most species in [the melpomene-silvaniform] clade occasionally hybridize in the wild with other clade
members’ ([11], p. 94) and ‘surveys of wild-caught specimens
have revealed many instances of natural hybridization in
Heliconius’ ([28], p. 1; see also [13,78]), and fuelling more general
discussions of ecological speciation [79,80]. The keystone status
these data have taken on in subsequent work demands a detailed
re-examination of their evidentiary plausibility.
For a museum specimen to provide evidence of interspecific hybridization, we must be confident (i) of its identity (that it
is indeed an interspecific hybrid) and (ii) of its provenance (that
its label data are bona fide, ideally with a direct chain of custody
between the collector in the field and an institutional repository). Because unusual Heliconius specimens have long been
sought after by collectors and are objects of commerce [81],
the burden of proof for their acceptance as scientific evidence
must be high. Most of the 93 putative hybrid specimens representing the H. melpomene–silvaniform clade that reside in
public collections are old and of uncertain provenance.
Mallet et al. viewed antiquity itself as evidence of authenticity,
arguing that old specimens must have been wild-caught,
because captive breeding of Heliconius is a relatively recent
enterprise, but this is unconvincing in terms of either (i) or
(ii) shown above: vague locality labels such as ‘Kolumbien’
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Nadeau et al. [13] suggested that H. heurippa may instead
be the homoploid hybrid offspring of H. melpomene and
H. timareta, but that hypothesis obviates the need for an interspecific hybrid origin, because various H. timareta forms
already exhibit the red forewing pattern elements needed to
produce the H. heurippa phenotype (figure 1). Further, this
begs the question of the origin(s) of the red wing pattern
elements in H. timareta, which were themselves hypothesized
to be the results of interspecific gene flow [11,25].
Theory predicts that closely related species that have arisen in
allopatry will exhibit genetic divergence across broad regions
of the genome, whereas species that evolve in parapatry or
sympatry with ongoing gene flow will tend to exhibit divergence only in ‘islands’ surrounding those loci that establish
and maintain specific differences by selection (and regions
closely linked to them) [95–98]. Nadeau et al. [12] have
argued that Heliconius of the H. melpomene –silvaniform
clade exhibit the second type of pattern, based on slidingwindow estimates of FST, with relatively narrow peaks of
differentiation representing genomic regions associated with
wing patterns. However, genomic genealogies of the same
Heliconius species appear to exhibit the first pattern, yielding
trees that consistently reflect the traditional ‘species phylogeny’, except for specific gene regions associated with
expression of mimetic wing patterns that cluster according
to those patterns and are incongruent with the ‘species phylogeny’ [11,13,14]. These interpretations of the data appear to
contradict one another: if large portions of the genomes of
these species are not genetically differentiated from one
another, then what characters consistently support the standard groups in the various phylogenetic analyses? The
phylogenetic evidence for wing pattern allele introgression in
the absence of more widespread gene flow of ostensibly ‘neutral’ parts of the genome seems highly counterintuitive, given
that it is precisely those phenotypes that are under strong selection and have been implicated in the establishment and
maintenance of species boundaries [12,34,63,64,89]. Rather
than being the only gene regions that flow between hybridizing
species, wing pattern alleles should be highly resistant to introgression, as the putative ‘magic traits’ that establish species
boundaries in the first place. How might this be explained?
There are three means by which an adaptive phenotype
can be shared among species. The most parsimonious of
these is common ancestry. As discussed earlier, however,
the rampant geographical variation within and among
Heliconius species indicates that the diversity of wing patterns
cannot be explained parsimoniously as the simple result of
descent without abundant homoplasy [5]. The classic alternative, recognized by Bates [99], is independent evolution of
similar features via convergence: for example, wing patterns
shared between ithomiine and dismorphiine butterflies.
Subtly different from convergence is parallelism, in which
phenotypes arise independently in related lineages using
homologous genetic and developmental architectures,
termed ‘homoiology’ by Plate [100]. As understanding of
the developmental mechanisms regulating butterfly wing
patterns has grown [46], patterns once considered convergent
have come to be seen as parallelisms, to a greater or lesser
degree. Within Heliconius, the genetic architecture responsible
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5. New phylogenomic evidence of introgression
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band with a distal red border, a phenotype reproduced in the
laboratory by successive generations of selective backcrossing
between H. cydno cordula and H. melpomene melpomene [22].
The authors asserted that H. heurippa exhibits novel prezygotic barriers that prevent backcrossing to either of its putative
parental species, and, based on DNA sequence and microsatellite data, that it has a ‘hybrid genome’.
Brower [20] re-evaluated the evidence supporting
the H. heurippa HHS hypothesis, and pointed out several
difficulties in addition to the weak ‘support’ from the ambiguous gene genealogies mentioned earlier. To briefly
summarize, the prezygotic barriers separating H. heurippa
from H. melpomene and H. cydno are not novel, but rather
are ancestral features stemming from the mate recognition
cues that allow the two ‘parental’ species to avoid interspecific mating where they occur in sympatry [27,63,66,78].
Heliconius heurippa males will mate with H. cydno females
[65], suggesting that the only barrier to gene flow between
those species is allopatry. Likewise, there is no intrinsic postzygotic loss of fitness in H. heurippa H. cydno crosses. By
contrast, H. melpomene is prezygotically and partially postzygotically isolated from H. heurippa and H. cydno. The
preponderance of the genetic markers, including mtDNA
[20,36], nuclear SNPs [8] and the weight of evidence
from recent genomic comparisons [11,13,14], show that
H. heurippa is more closely related to H. cydno than it is to
H. melpomene. Because H. cydno H. melpomene hybrid
F1 females are sterile, the HHS scenario requires F1 males to
repeatedly backcross to H. cydno in order to obtain this homogeneous genetic background, while somehow retaining the
H. melpomene alleles responsible for the red forewing band
and unmarked ventral hindwing.
‘Classic’ homoploid hybrids are expected to exhibit
mosaic genomes composed of blocks of DNA from the two
parental species [90,91]. Mallet [23] and Jiggins et al. [26],
recognizing that the H. heurippa’s widespread genomic affinity to H. cydno does not fit that model, have relaxed their
concepts of HHS by suggesting that only a few ‘magic traits’
(i.e. alternative alleles subject to divergent selection that are
pleiotropically associated with alleles promoting reproductive
isolation [79,92,93]) are necessary. Wing patterns, under selection for both mimicry and mate recognition [63], are the
obvious phenotypic traits that might lead to such selectiondriven ecological speciation [94], and so the genomic search
is on for chromosomal segments correlated with (and, ideally,
causally regulating) differential expression of those characters
and corresponding mate choice. Salazar et al. [8] suggested
that a region at the 30 end of the kinesin gene exhibits the
pattern of differentiation expected of such a feature, but
Brower [20] argued that while shared SNPs in the gene
region are consistent with introgression between H. heurippa
and H. melpomene, they are mostly silent substitutions invisible
to selection, and so do not represent the ‘smoking gun’. It is
interesting to contemplate how, under the Mavárez et al. [22]
model, selection could favour H. heurippa’s ‘non-mimetic’ phenotype, particularly in the initial stages when it would be rare,
given that more common combinations of alleles (including the
all-heterozygous F1 phenotype) that would cause it to bear a
closer resemblance to its sympatric ‘parent’ (H. melpomene)
would seem to be favoured by selection for mimetic resemblance. The HHS scenario seems fundamentally at odds with
the frequency-dependent selective basis of Müllerian mimicry
described earlier.
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6. Paradigms and paradoxes
Recent publications raise a number of new conundrums
regarding the evolution and diversification of Heliconius butterflies. How can wing mimetic pattern alleles flow from one
species to another (and apparently be the only gene regions
that do so), when it has been shown that wing patterns are
perhaps the key adaptations responsible for intrinsic maintenance of species boundaries by mate choice? How can
wing pattern alleles spread from one species to another
when such introgression does not occur across intraspecific
hybrid zones in geographically differentiated species in
which there are no barriers to interracial hybridization? How
can ‘non-mimetic’ phenotypes arise and become fixed as a
result of interspecific gene flow when there is a strong selective
advantage to phenotypic conformity due to Müllerian mimicry? And, once again, if wing patterns are promiscuously
shared across species boundaries, then why has this not led
to fixation of a single, shared aposematic pattern, which
would represent a stable, selectively advantageous global
optimum for all Heliconius butterflies?
With particular regard to the H. cydno cognates on the
eastern slopes of the Andes (figure 1), Brower [20] hypothesized that mutations producing red wing pattern elements
in peripheral H. cydno populations that have dispersed over
the Andes where their otherwise sympatric co-mimics
do not occur might be at a selective advantage. Given the
underlying homology of the genetic architecture and the plesiomorphic occurrence of red pigment production in the
clade, such mutations seem like a plausible mechanism,
and would explain the existence of ‘non-mimetic’ forms (as
evolving towards, rather than away from, locally dominant
mimicry rings). Allowing that evolutionarily significant interspecific hybridization could have taken place, shared red
wing pattern elements could also be explained by simple
introgression and selection, if the introgressing alleles were
uniformly advantageous in the novel genetic background
[112–114]. Perhaps rare H. cydno immigrants with no comimics could interbreed with more common endemic
H. melpomene. But this does not explain why the preponderance of genetic markers in these ‘hybrids’ imply H. cydno
ancestry—one would expect backcrosses to the more
common parental form [20]. Nor are non-mimetic phenotypes explained by the introgression hypothesis: if selection
for mimicry drives the process of introgression, then phenotypes resulting from introgressed alleles should be identical
to those of the species from which they came. In addition,
the absence of introgressed neutral loci (e.g. microsatellites
6
Proc R Soc B 280: 20122302
hypothesis, and could also support the traditional, alternative
hypothesis of convergent natural selection for shared wing patterns among species. A similar problem occurs with use of the
statistical program IM [107] and linkage-disequilibrium tests
[108] to infer interspecific gene flow [14]. These methods also
assume neutrality of tested loci, and empirical and simulation
studies [109–111] have shown that violations of the model
assumptions can result in spurious estimates of population parameters such as rates of introgression. The bottom line is that
when the traits of interest are under selection, as genes responsible for wing patterns manifestly are, then inferences drawn
from coalescent-based methods for inferring gene flow that
assume neutrality may be unreliable.
rspb.royalsocietypublishing.org
for various discrete wing pattern components is homologous
at the molecular level across the genus [61,62,71,72,101], and
phenotypic differences result from differential expression
controlled by as-yet-unidentified regulatory factors.
At a finer scale, phenotypes shared within and among
Heliconius species may have arisen ‘independently’ from
this underlying architecture, may have been preserved as
ancestral polymorphisms by selection, or may be the result
of horizontal transfer via hybridization (Gilbert’s ‘toolbox’
[69]). There is no doubt that some instances of common
mimetic patterns have evolved intrinsically without hybridization. For example, if it is not the ancestral pattern of the
genus (cf. [4]), then the red-rayed phenotype must have
evolved at least four times among species or clades that are
reproductively isolated from one another (H. xanthocles
clade; H. melpomene –silvaniform clade; H. erato; H. demeter;
plus additional times in related heliconiine genera Neruda
and Eueides). Within the H. melpomene –silvaniform clade,
the red-rayed pattern occurs as an apparently derived state
in H. melpomene, H. timareta and H. elevatus, and recent phylogenomic analyses have revealed shared groups of DNA
polymorphisms that imply horizontal transfer of these alleles
from H. melpomene to the others [11,14]. These trees ostensibly
refute the independent origin hypothesis proposed by
Brower [20].
It is easy to be intimidated by the overwhelming quantity
of data and elaborate analyses presented in genomics
publications, and to accept the authors’ interpretations of
their results at face value. However, third-generation sequencing and comparative population/phylogenomics are still
in their infancy, and there are issues both of data quality
and analytical rigour that raise concerns [102,103]. The
H. melpomene genome paper [11] includes comparative
sequence data for chromosomal regions of interest for nine
H. melpomene –silvaniform clade species, comprising alignments more than 1.7 million bp long (K. Dasmahapatra
2012, unpublished data). Unfortunately, these data matrices
contain an enormous amount of missing data. For instance,
one of the 10 kb blocks of the N/Yb region providing phylogenetic support for the introgression hypothesis contained an
average of more than 53 per cent ‘N’s per taxon, with some
taxa missing more than 70 per cent. To be sure, 30 per cent
of 1.7 million bp is a lot of data, and phylogenetic analyses
of various walk segments do yield the published topologies.
However, eyeball scrutiny of the nucleotide sites that provide
support for grouping of the taxa by wing pattern reveals
that they are rife with ambiguity: there is not a single fixed
character state difference (sensu [104,105]) supporting
the tidy assortment of individuals by wing pattern in the
480–490 kb segment of the N/Yb region, and only one in
the 580–590 kb segment. Published trees [11,13,14] do not
make clear how many or what kind of characters support
these patterns, nor what models were used to produce
the trees.
The statistic Dasmahapatra et al. [11] used to infer introgression is Patterson’s D, a measure of bias from an expected equal
distribution of homoplasy in a four-taxon comparison. They
observed that D levels appear significantly high in chromosomes 15 and 18, where wing pattern-regulating genes B/D
and N/Yb, respectively, reside. However, the test assumes neutrality, and its authors [106] noted that ‘natural selection could
potentially bias D statistics’ (p. 2250). It therefore seems that this
evidence does not unequivocally support the introgression
Downloaded from http://rspb.royalsocietypublishing.org/ on June 11, 2017
genome now provides a vast frontier for extending this knowledge. The criticisms presented this review are intended to
provoke further, more detailed and extensive studies of both
comparative genomics and natural history in this outstanding
system. Undoubtedly, surprises remain to be revealed.
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