Category Archives: Biology

Richard ffrench-Constant, Heliconius Genome Consortium Pioneer

Richard ffrench-Constant was a key member of the original Heliconius Genome Consortium which published the first butterfly genome mapped to chromosomes in 2012 (Heliconius Genome Consortium 2012).

I met Richard much earlier when he was a PhD student at Rothamsted Experimental Station (https://www.rothamsted.ac.uk/) and I was a NERC postdoctoral fellow at UCL in the late 1980s. He was an agricultural entomologist who applied molecular genetics techniques to studying insecticide resistance. One of his group’s major papers I remember was his finding that multiple different lineages of insects had evolved exactly the same amino acid substitution in “target-site” resistance to cyclodiene insecticides — i.e. dieldrin, aldrin, endrin, and BHC for example (Thompson et al. 1993). Remarkable, I think you’ll have to agree, and it demonstrates the massive power of natural selection in species (like insects) with large effective population size and consequently high per base-pair heterozygosity.

Although he started out in applied biology, like I did, he later became interested in broader issues in molecular evolution and biology of insects. He has papers on Lycaeides “blue” butterflies in California, and encouraged early members of the Heliconius Genome Consortium by funding some sequencing projects — he had a close interest in the work of Mathieu Joron on the “supergene” mimicry locus in Heliconius numata (Joron et al. 2006).

He was Cornish, and that’s where that strange name comes from. When the Falmouth (Cornwall) branch of the University of Exeter (in Devon) opened up a biology program he was awarded a Professorship there and that’s where he lived when I visited him in the late 2000-2010 period.

I have been friendly with Richard for my and his whole lives. Unfortunately, Richard’s, “his”, has been cut short. With very little warning he died of cancer last year (2025). He was 65.

I liked this wonderful obituary for Richard ffrench-Constant in “Antenna” (the Bulletin of the Royal Entomological Society), latest issue (Reynolds 2026). I’ll try to reproduce it here:

Richard ffrench-Constant, you did a great job! RIP!

James Mallet

Harvard University and Emeritus at University College London

References

Heliconius Genome Consortium. 2012. Butterfly genome reveals promiscuous exchange of mimicry adaptations among species. Nature (London) 487:94–98. https://doi.org/10.1038/nature11041

Joron, M., Papa, R., Beltrán, M., Chamberlain, N., Mavárez, J., Baxter, S.W., Bermingham, E., Humphray, S.J., Rogers, J., Beasley, H., Barlow, K., ffrench-Constant, R.H., Mallet, J., McMillan, W.O., & Jiggins, C.D. 2006. A conserved supergene locus controls colour pattern diversity in Heliconius butterflies. PLoS Biology 4:e303. https://doi.org/10.1371/journal.pbio.0040303

Reynolds, S. 2026. Obituary. Richard ffrench-Constant. 1st August 1960 – 6th јune 2026. Antenna 50:165–166

Thompson, M., Stechen, J.C., & ffrench-Constant, R.H. 1993. Conservation of cyclodiene insecticide resistance-associated mutations in insects. Insect Molecular Biology 2:149–154.

Michel Cast sent me pictures of this nice hybrid specimen from Ceara, Brazil! Thank you Michel and P. Jauffret!

Summary

In 2022, I published a ‘perspective’ piece in Evolutionary Ecology, on a hypothetical function of colouration in butterflies, and in particular in Heliconius and co-mimics.

This piece contended that:

• Heliconius butterflies are most unlikely to be aposematic,
• The similarities between species could be explained otherwise, specifically as the mimicry of intraspecific signals whose function is to attract conspecifics into night roost aggregations.

In a recent rejoinder, Dr Mallet is ‘astonished’ that an independent researcher working from literature ‘seems to have persuaded the editors and reviewers of an academic journal that his hypothesis might overturn consensus, a century and a half old, on warning colour and Müllerian mimicry’.

This characterisation is incorrect: no actual hypothesis of Müllerian mimicry in Heliconius exists, so there is no theory to overturn; the description of the complex similarities in colours is not a theory but a habit inherited from a defunct theory that rested on group selection (Müller 1879). Elsewhere, Dr Mallet describes Müllerian mimicry in Heliconius as “a venerable one hundred and sixty-year old tradition of study of a topic” (Mallet 2024); indeed, Mallet refers again and again to the age of the idea, which shows, and to its intellectual pedigree, mentioning repeatedly Poulton and Wallace: the tradition is venerable indeed. Where Dr Mallet errs is in considering that pedigree as relevant.

Dr Mallet and I both consider that extraordinary claims require extraordinary evidence. We differ in how we apply this principle: Dr Mallet considers that it would require extraordinaty evidence to overturn this idea (actually a minor upset, but Dr Mallet is a Heliconius specialist, it feels ‘Copernican’ to him) when I assessed that in Heliconius, aposematism and Müllerian mimicry would be extraordinary, that this idea is supported neither by a hypothetical mechanism, nor by any evidence, and should be rejected for now.

Given that rejection, I considered open-mindedly what could explain the puzzling rings of similarities in Heliconius, and proposed a new hypothesis, which makes easily testable predictions.

Dr Mallet’s expertise as a field biologist and Heliconius specialist is not in question. However, the theoretical conditions that allow the maintenance of traits are not his area of expertise, and he appears unaware that the conditions that would enable the maintenance of aposematism (and Müllerian mimicry) are not met in Heliconius and its co-mimics. Dr Mallet’s intuition cannot resolve such theoretical impossibilities, and his theoretical objections are mistaken and incompatible with contemporary evolutionary theory.

The hypothesis proposed in Mouy (2022) makes clear and testable predictions. As a London-based theoretician, I will not undertake field testing in Central or South America. Dr Mallet considers the hypothesis false and reminded us that good science starts by disproving false hypotheses. He is a field biologist with the capacity, and I hope he will exert his ingenuity at disproving it with contradictory observations, and would be the first to recommend abandoning the hypothesis if he does.

Does a theory of aposematism exist in Heliconius?

Mouy (2022) states that none of the mechanisms hypothesised to stabilise aposematism are applicable in Heliconius; not that none has been demonstrated to apply, but that all demonstrably do not apply. This is a simple claim, which is justified in Mouy (2022), and which would be easy to refute – it just takes one plausible mechanism – and which Dr Mallet does not rebut. There is simply no theory of aposematism in Heliconius.

This is not the central claim of Mouy but is important to set the context of why we need to explore alternatives.

Dr Mallet is a field biologist and is unaware of recent developments in theory, incorrectly crediting Mouy’s work with novelty regarding the difficulty of maintaining aposematism. Fisher (1930) demonstrated a clear awareness of the problem, although the definitive analysis was not provided until Leimar et al. (1986), as cited by Mouy. This problem is frequently mentioned in introductions and abstracts of recent research[1]. Dr Mallet also shows little awareness of signalling theory and describes the proposed definition of signal[2] (not aposematism) as obscure, although, as indicated in Mouy (2022), it is a standard definition, marginally modified for the context, derived from a graduate-level textbook (Maynard Smith and Harper 2003).

The major theoretical problem of aposematism is its evolutionary stability[3]: aposematism seeks to explain the function of colours, that is, how colours are maintained in populations as warning signals to predators; this concept of biological function, what a trait ‘is for’, is central to evolutionary biology and widely relied upon (Garson 2019).

Not being able to explain how colours maintain themselves in populations as warning signals means we cannot call them aposematic, and this is a major, unresolved problem in Heliconius butterflies: as set out by Mouy (2022), all proposed hypotheses of mechanisms potentially stabilising aposematism can be rejected: there is simply no hypothesis of aposematism in Heliconius butterflies, and calling them aposematic is merely a statement of faith that these difficulties may be resolved one day.

Dr Mallet is oblivious to that problem and points to ample evidence that birds avoid these butterflies; unfortunately, this is not the question – birds will learn to recognise inedible prey regardless of how they look – and while the quicker learning of avoidance of bright colours was shown in laboratory experiments, it can only evidence the function of colours under group selection. Oblivious to that problem, Mallet assumes that Mouy really meant evolution and points out that Mouy did not put forward a rejection of the possibility of evolution (when it does reject all possibilities of maintenance). Obviously, it does not, as how traits evolved is irrelevant.

Weirdly, Mallet states that no evidence is offered after citing Mouy’s ‘None [of the potential mechanisms] are observed in Heliconius.’, although what follows offers precisely that.

Contrary to Mallet’s claim, aposematism is genuinely a problem, and finding ideas that could stabilise aposematism is genuinely a concern. This problem is common to all butterflies, as no mechanism proposed to apply appears workable, and is not apparent only to theoreticians, but also to field biologists such as Dr Kassarov, who dissented from the notion that ‘aposematism [is] a valid concept in predator-prey relationships between birds and butterflies” (Kassarov 1999, 2003). Hypotheses to explain aposematism are still regularly put forward and quietly abandoned – the latest is ‘taste-and-reject’[4] – and we do not even have a hypothesis on how butterflies can be aposematic.

The notion that Heliconius are aposematic is not a proper scientific hypothesis (a causal mechanism), merely a general direction or research. That direction is supported by the bright colours and toxicity of Heliconius: toxicity would explain brightness. This would make no sense if toxic butterflies were not more likely to be brightly coloured than non-toxic ones[5] – but this is not even the case, or at least, as reviewed in Mouy, one study contradicts the correlation, and none supports it – it works better in other insects.

In summary, Mallet offers no substantial rebuttal to the claim in Mouy (2022) that aposematism is an implausible explanation of the colouration of Heliconius, because such colourations could not be maintained for that function. I disagree with Dr Mallet’s statement that such a rejection would be a major upset; this cannot be an upset since there is not even a theory to reject and no scientific publication in the past forty years has stated that a hypothesis of aposematism in Heliconius demonstrably exists. The fact is just upsetting to some, who choose to ignore it.

Does a theory of Müllerian mimicry exist in Heliconius?

Mouy (2022) states that no hypothesis exists on the mechanisms that could stabilise the multiple overlapping mimicry rings of Heliconius butterflies and co-mimics. Again, Mouy does not claim that no hypothesis has been demonstrated, but that all have been conclusively refuted.

Again, this is a simple claim that would be easy to refute. Dr Mallet does not refute it, but contends that evidence overwhelmingly supports Müllerian mimicry – although he cannot point to an actual theory that would be supported.

Heliconius butterflies would not be famous if they did not form complex and overlapping rings of similarities – habitually described as mimicry rings, and precisely as Müllerian mimicry rings. Mouy (2022) concurs that these are mimicry rings, but does not accept that these mimicry rings have daytime predators as selective agents, or that the mechanisms through which they evolved or are somewhat similar to Müllerian mimicry.

The concept of predator-mediated mimicry[6] is not challenged in Mouy (2022); there are well-proven cases of such mimicry. Most vertebrates and some invertebrates, both predators and herbivores, will learn to recognise food from non-food. To a typical predator, non-food is a broad category, and many potential prey attempt to disguise themselves as non-food. Predator-mediated mimicry is the most common form of mimicry, but not the only, and observing similarities proves neither mimicry – colours can converge under close selective pressures – nor, obviously, predator-mediated mimicry.

Mallet emphasises, as sole evidence for predator-mediated mimicry in Heliconius and co-mimics, that these butterflies are inedible, a long-documented fact. This is beyond debate, but is not evidence of mimicry, and there are severe, well-documented, and unresolved difficulties with the notion that the convergence between these butterflies is caused by mimicry.

For daytime predators to be the selective agents for such overlapping mimicry rings, we predators and butterflies to segregate into separate communities. This is not found: butterflies do not segregate at all by day when birds could attack them, and birds segregate very weakly at best (Merrill et al. 2015; Mouy 2022). Only one alternative to segregated predator-prey communities has been proposed, a combination of a hypothesised predator behaviour (‘optimal sampling strategy’) and some population structure, fails; however, it ‘does not explain the existence of the coexisting mimicry rings we observe in nature’ (Aubier & Sherratt, 2015). The authors conclude that ‘more work should be done to understand how they can be generated and maintained’, without offering any research direction: the field does not even have a standing hypothesis of how Müllerian mimicry is possible.

It seems curious that Mallet writes that ‘very little effort seems to have been expended by Mouy (2022) to rebut the perceived consensus’: indeed, no effort was needed, as this is not new. The idea worked under Müller (1879) because group selection was an acceptable method of reasoning (Mallet 2010); however, it has not been valid since the rejection of group selection.  

We need a revision.

Mallet writes: ‘In science, no hypothesis is actually proved. Rather, we tend to disprove alternatives, or at least to show why the alternative hypotheses are unlikely.’. I fully subscribe to this. As shown above, aposematism and mimicry in Heliconius are extremely unlikely – and I generously treat these ideas as hypotheses; they are not even that, mere general research directions, or, as Mallet aptly described ‘traditions of study’.

This tradition of study could be justified if it looks like a proper theory could come out of it, and

it appeared that such a theory would have some explanatory value. However, no theory lurks around the corner, and there is no potential explanatory value of aposematism in butterflies.

Aposematism and Müllerian mimicry were correct explanations when they rested on the premise of group selection. These premises have been rejected conclusively over half a century ago, are research now attempts to retrofit a theory on these old ideas that do not work anymore.

Mallet (2024) alleges that a severe revision or rejection of aposematism and Müllerian mimicry in Heliconius would be a ‘major upset in evolutionary biology’. The major upset came some time ago, with Fisher and Hamilton’s gene-based approach of evolution. It so happens that all the consequences of this major upset have not been drawn yet. If it were invented today, the idea that Heliconius butterflies form Müllerian mimicry rings would be never pass peer review. We shouldn’t be sentimental about these old ideas, although be may be about the geniuses that produced them.

In summary, within the modern paradigm, no theory allows us to understand how Heliconius could be aposematic or the salient characteristic of their mimicry rings, and there is no evidence that either constitutes an adaptation to signal towards daytime predators. It is weird to keep considering Heliconius as a prime example of both.

Novel claims of Mouy (2022)

As described above, there is no novelty in the claims that aposematism and Müllerian mimicry are implausible and unsupported in Heliconius. It so happens that some scientists hope for a solution to these problems and continue to treat these ideas as if they were valid, but the ideas are not supported, and no mechanistic explanation of either exists.

However, there are some new claims:

• The function of colouration in butterflies is poorly understood, and the currently proposed explanations leave significant gaps, including bright species for which no explanation is available.

Such species, Mouy suggested, may comprise some mimicry rings in two widespread, non-toxic Eurasian butterfly families: the blues and sulphurs.

As discussed above and in Mouy (2022), there is no extant hypothesis of aposematism in butterflies, so all colours currently explained by aposematism should be considered as poorly understood. However, even assuming the issue could be resolved – unlikely – there are many other bright species for which no explanation has been offered. The purpose of Mouy (2022) is to offer an additional explanation, and to assess whether is appears consistent with observations.

Mallet does not dispute that some colours of butterflies are unexplained.

• One of the functions of bright colours in butterflies is to signal towards conspecifics to attract them and reduce predatory pressure from the dilution of predation.

Contrary to Mallet’s statement, it is not suggested that colours “evolved ‘purely as an aggregation signal’”; that quotation described the model, not the hypothesis: isolating a potential function for assessment is a common practice among theoreticians, of which Dr Mallet appears unaware.

Mouy does not claim to displace all ideas on the colouration of a wide range of insects; instead, Mouy offers a supplementary theory, and we need new explanations.

• The complex mimicry rings of Heliconius could be explained by colours constituting an aggregation signal towards conspecifics and the mimicry of this signal.

This is the central new claim: the novel idea above could explain the puzzling mimicry rings observed in Heliconius, and in particular, this hypothesis is highly consistent with the co-mimics’ tendency to roost at similar heights and occasionally together.

A substantive criticisms of Mallet (2024)

Dr Mallet’s knowledge of Heliconius is unquestionably greater than mine, and I acknowledge that expertise with respect. I can only regret, however, that one anonymous reviewer of Mouy (2022) – evidently also a Heliconius specialist – chose to focus on theory rather than observations, where their expertise could have provided constructive criticism.

In the proposedhypothesis, the primary function of colouration is to attract co-roosters.

As Dr Mallet points out, ‘we expect unprofitable prey (including aposematic prey) to be more prone to aggregate than profitable prey’, and palatable species could benefit from dilution thanks to the satiation of predators. Weirdly, Dr Mallet does not realise that it is the point: these animals do have an incentive to aggregate, and a greater incentive in gathering along similar and unpalatable individuals, hence the mimicry.

Mallet then makes a substantive point, also made in Mouy, which Mallet cites here: Heliconius are extremely similar, which seems to require that selective agents for such a level of similarity can discriminate between closely resembling individuals, and birds have a considerably better visual acuity than butterflies.

Butterflies would indeed be unlikely to tell a good from a poor mimic from a distance of more than a foot so that poor mimics would initially attract them. The question is whether, once at a short distance, allowing discrimination, they react to poor mimics. This is a valid and testable question (even if butterflies could not discriminate between close morphs, the hypothesis would be salvageable[7]).

Mallet goes on to discuss his studies of mate choice in Heliconius and suggests, towards the end, that mating cues were co-opted ‘to facilitate aggregation behaviour.’ I suppose the point he obliquely makes is that studies of mate choice are relevant for assessing the proposed hypothesis. Indeed, and if butterflies can discriminate in the context of mating, it sounds plausible that they do in the context of aggregations.

However, as shown above, the problem lies in the existence of polymorphism and a multiple mimicry ring. We know that sexual selection can drive polymorphism along geographic radiation, for instance in frogs (Yang et al. 2019), and that could well be the case in Heliconius, although Dr Mallet does not appear to support this idea (Mallet and Gilbert 1995) – Mallet is confused when indicating that I ignore these observations, since Mouy never discusses divergence, but convergence, and does not discuss sexual signals. While sexual selection cannot drive mimicry between species, let alone complex overlapping mimicry rings, aggregation signals can, and, as I show in the original paper, apparently do. Dr Mallet’s statement that ‘intraspecific preferences do sometimes make use of differences in mimetic colour patterns’ shows the hypothesis to be plausible.

Besides being a plausible explanation of the colouration and convergence of colours in Heliconius, this hypothesis can plausibly explain divergences, which Dr Mallet considers unexplained, and the recurrent evolution of overlapping mimicry rings. A ‘magic trait’ is a trait subject to both diversifying disruptive selection and assortative mating, which could produce polymorphism, radiation, and speciation (Rueffler et al. 2006; Servedio et al. 2011).

The proposed selection for attracting co-roosting mates would constitute a disruptive selection as it requires one signal per roosting habitat, and roosting habitats will change with localities: Heliconius would indeed ‘exploits a continuously varying resource’ of roosting sites, as required to cause diversification (Rueffler et al. 2006). As noted by Mallet, the colour and patterns of Heliconius’s wings are subject to assortative mating – such colours are not required to keep the species apart, which he points out they do not.

A short reply to methodological criticisms of Mouy (2022)

Besides the remarks to which I replied above, Dr Mallet makes a few notes on the methods in Mouy.

• Reliance on crowd-sourced photographs

Mouy (2022) tests the plausibility of the idea that butterflies’ colours serve to attract one another by assessing if they do attract one another. This is based on the analysis of a large amount of enthusiast photographs, almost entirely in the United Kingdom[8], Mallet (2024) apparently resents the intrusion of amateurs (and independent scientists) and describes these as ‘some blurry photos on a British butterfly amateur website’; I let readers judge that the photographs published on UK Butterflies are all of excellent quality and correctly classified.

Mallet points to resource patchiness as explaining aggregations, but this entirely fails to address the finding: bright butterflies are 8 times more likely to be found in groups than dull ones. Incidentally, in many cases, aggregations (e.g., collective puddling) are manifestly not the result of resource scarcity; many butterflies may congregate densely on a small fraction of a river bank when plenty of other access are available.

• Modelling itself

Mallet (2024) correctly identifies the only claim of the model: In some circumstances, brighter colours can be beneficial if they attract both predators and prey. This model assumes some dilution effect, which is expected to be present in all species, albeit stronger in toxic species, as mentioned in Mouy (2022) and Mallet (2024). Unfortunately, noting that some of the assumptions are similar to those of the models of aposematism, Mallet (2024) then criticises the model for not being a model of aposematism[9] and searches in vain for a “recognisable model of populations or their evolution over time”, which is indeed not present.

Dr Mallet is clearly unaware of game-theoretic modelling: it is banal not to model populations explicitly, and it is habitual to single out one particular type of interaction in models, ignoring others, which says nothing about the existence of those other interactions.

Dr Mallet also apparently overlooked the caption of Figure 2 and is befuddled by negative changes in predation risk (having probably overlooked the term ‘change’). Unsurprisingly, the extensive explanations in the appendix, which attracted no criticism for clarity, are unclear to Dr Mallet, who did not consider it appropriate to reach out to me for clarifications.

Conclusion

Mallet expresses his astonishment that my research was published in an academic publication. In the ensuing confusion, Dr Mallet read statements that were not part of the research and failed to understand those that were.

Indeed, it is not common for a non-academic to publish in a reputable scientific journal a piece challenging a ‘venerable one hundred and sixty-year old tradition of study’ – and since an additional five – which should alert Mallet to the potential intrinsic merit of the pieces and brittleness of his belief system.

The ideas defended by Mallet (2024), regarding aposematism and Müllerian mimicry in Heliconius butterflies, are not scientific hypotheses; they ceased to be hypotheses when the theoretical framework underlying them – group selection – was superseded by a gene-based view of evolution and evolutionary game theory. This last subject is almost mathematics, and I cannot fault Dr Mallet for being unaware of the fine points of this speciality. However, the repeated mentions of the antiquity of these ideas, and of their excellent pedigree, show an unwelcome sentimentality that apparently prevents Dr Mallet from letting these ideas go.

Whether the alternative hypothesis I am offering has any merit is moot: if we had only one theory and abandoned it, we would only be following the excellent example of Darwin, who, on several occasions, gave up hypotheses without having a replacement, despite the anxiety this caused him (Loehle 1987).

I do not think, however, that this alternative should be buried already: Dr Mallet’s long reply addresses none of the substantive points made in Mouy (2022). Dr Mallet’s hostility to my hypothesis is most welcome, as he will certainly now put his ingenuity as a field scientist to work and attempt to disprove it.

Acknowledgement: I am grateful to Dr Mallet for publishing his commentary on my original work, as I had invited him to do, and for offering me the opportunity to respond on Heliconius.org without editorial control.

Note: This post was hastily written in February 2024 as a quick reply to Dr Mallet’s post and later edited for clarity and concision, with the last edit in November 2025.

References

Fisher R (1930) The Genetical Theory of Natural Selection. The Clarendon Press

Garson J (2019) What Biological Functions Are and Why They Matter, 1st edn. Cambridge University Press

Hämäläinen L, Thorogood R (2020) The signal detection problem of aposematic prey revisited: integrating prior social and personal experience. Phil Trans R Soc B 375:20190473. https://doi.org/10.1098/rstb.2019.0473

Kassarov L (1999) Are Birds Able To Taste And Reject Butterflies Based On “Beak Mark Tasting”? A Different Point Of View. Behav 136:965–981. https://doi.org/10.1163/156853999501676

Kassarov L (2003) Are Birds the Primary Selective Force Leading to Evolution of Mimicry and Aposematism in Butterflies? An Opposing Point of View. Behav 140:433–451. https://doi.org/10.1163/156853903322127922

Kitamura T, Imafuku M (2015) Behavioural mimicry in flight path of Batesian intraspecific polymorphic butterfly Papilio polytes. Proc R Soc B 282:20150483. https://doi.org/10.1098/rspb.2015.0483

Leimar O, Enquist M, Sillén-Tullberg B (1986) Evolutionary Stability of Aposematic Coloration and Prey Unprofitability: A Theoretical Analysis. The American Naturalist 128:469–490

Loehle C (1987) Hypothesis Testing in Ecology: Psychological Aspects and the Importance of Theory Maturation. The Quarterly Review of Biology 62:397–409. https://doi.org/10.1086/415619

Mallet J (2024) Colour patterns in Lepidoptera evolved mainly as signals to predators. Heliconius are indeed Müllerian mimics. Reply to Mouy (2022). In: Heliconius.org

Mallet J (2010) Group selection and the development of the biological species concept. Phil Trans R Soc B 365:1853–1863. https://doi.org/10.1098/rstb.2010.0040

Mallet J, Gilbert LE (1995) Why are there so many mimicry rings? Correlations between habitat, behaviour and mimicry in Heliconius butterflies. Biological Journal of the Linnean Society 55:159–180. https://doi.org/10.1111/j.1095-8312.1995.tb01057.x

Mallet J, Joron M (1999) Evolution of Diversity in Warning Color and Mimicry: Polymorphisms, Shifting Balance, and Speciation. Annu Rev Ecol Syst 30:201–233. https://doi.org/10.1146/annurev.ecolsys.30.1.201

Maynard Smith J, Harper DM (2003) Animal signals, Nouvelle éd. Oxford university press, Oxford

Merrill RM, Dasmahapatra KK, Davey JW, et al (2015) The diversification of Heliconius butterflies: what have we learned in 150 years? J of Evolutionary Biology 28:1417–1438. https://doi.org/10.1111/jeb.12672

Mouy H (2022) Colours as aggregation signals in Lepidoptera: Are Heliconius Müllerian mimics? Evolutionary Ecology 36:341–353. https://doi.org/10.1007/s10682-022-10183-8

Müller F (1879) Ituna and Thyridia: a remarkable case of mimicry in butterflies. Transactions of the Entomological Society of London 20–29

Páez E, Valkonen JK, Willmott KR, et al (2021) Hard to catch: experimental evidence supports evasive mimicry. Proc R Soc B 288:20203052. https://doi.org/10.1098/rspb.2020.3052

Rueffler C, Van Dooren TJM, Leimar O, Abrams PA (2006) Disruptive selection and then what? Trends in Ecology & Evolution 21:238–245. https://doi.org/10.1016/j.tree.2006.03.003

Servedio MR, Doorn GSV, Kopp M, et al (2011) Magic traits in speciation: ‘magic’ but not rare? Trends in Ecology & Evolution 26:389–397. https://doi.org/10.1016/j.tree.2011.04.005

Yang Y, Servedio MR, Richards-Zawacki CL (2019) Imprinting sets the stage for speciation. Nature 574:99–102. https://doi.org/10.1038/s41586-019-1599-z

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[1] As an example, the first line of the abstract of Hämäläinen and Thorogood (2020) states ‘Ever since Alfred R. Wallace suggested brightly coloured, toxic insects warn predators about their unprofitability, evolutionary biologists have searched for an explanation of how these aposematic prey evolve and are maintained in natural populations.’ .

[2] Mouy (2022): In the context of aposematism, a signal is defined as (derived from Smith & Harper, 2003): “A visible structure which alters predators behaviours, which evolved or failed to disappear because of that effect and which is effective because predators either evolved or learned a response to that signal.”

Compare with Maynard Smith and Harper (2003), page 3: “any act or structure which alters the behaviour of other organisms, which evolved because of that effect, and which is effective because the receiver’s response has also evolved”.

There are material differences. In particular, Mouy (2022) excludes acts, else, e.g., gazelle stotting would be an aposematic act, and the definition of aposematism would be even more haphazard than it already is. It also makes allowance for the signal receptor to have not evolved but learned to respond to the signal.

[3] A strategy is an ESS if it cannot be displaced by another strategy that would be initially rare.

In the case of a Heliconius erato with red patches on the wings, for instance, the red patch could be marginally smaller or marginally duller. Unless a marginally less bright individual would sustain come costs for that reduced signal, signalling will eventually collapse through the invasion of progressively weaker signals.

As outlined in Mouy (2022), all hypotheses put forward on how that collapse can be prevented can be rejected.

[4] Taste-and-reject proposes that the warning signals of aposematic butterflies is stable as birds, after catching a toxic butterfly, would reject it.

This hypothesis comes from a creative effort to find stabilising mechanisms (they are really a problem) and the observation (later shown to be unsound) that in museum collections, butterflies of species assumed to be inedible had more beak marks than those of other species. Until then, beak marks were used as a proxy for predatory pressure, so this, obviously didn’t reconcile well, as those ‘aposematic’ species should be under a much weaker pressure. Hence a motivated U-turn: beak mark became evidence of a reduced predatory pressure.

Kassarovs’ objections that colours are largely irrelevant received recent confirmatory evidence (Páez et al. 2021), and another showed mimicry in flight patterns not colours, also consistent with Kassarovs’ objections (Kitamura and Imafuku 2015). This last study engages with Kassarov’s arguments but the rebuttal is peculiarly ineffective: authors suggest that ‘aposematic coloration [may evolve] for protection against predation in the early morning’. That a study which is neither about colouration nor about aposematism dedicates a significant fraction of its discussion to defending aposematism and Müllerian mimicry against its own findings is very currious.

[5] Although aposematism does not require toxicity, only unprofitability, if toxicity was one cause of unprofitability among many, a correlation would still be found. Similarly, if we knew only who is smoking brand X of cigarettes, although brand X cigarettes are only one among many of lung cancer, we could find a very strong correlation between smoking brand X cigarettes and subsequently developing lung cancer. The existence of other brands of cigarettes would not affect correlation, unless everyone smoked one brand or another. But if all butterflies were defended, what woud be the point of signalling at all?

[6] I use ‘predator-mediated’ rather than ‘Batesian’ as many authors consider that ‘Batesian’ mimicry must be for aposematic signals, when predator-mediated mimicry can be of any colour or pattern, including cryptic.

[7] The most interesting characteristic of the Heliconius mimicry rings is their membership, and the weird overlaps in membership between groups of species depending on locality. For instance, in one locality, species {A, B, C} may form an obvious group, and {D, E, F} another group, while in another {A, B, D} and {C, E, F} look similar, and still in another place, it is {A, E, F} and {B, C, D}. This cannot be explained by predator-mediated mimicry. If mimicry membership could be explained, this would be a huge progress. As noted repeatedly, Heliconius butterflies and many of their co-mimics are unpalatable, and avian predators clearly have some awareness of their unpalatability and rarely attack these. If would, then, not seem absurd that, given mimicry rings which would have evolved as aggregation signals, further convergence would be selected for by avian predators and similarities improved.

[8] Hence, unlilely to show any heterospecific aggregation of American species, a defect pointed by Mallet (2024), but this was never the point.

[9] Mallet: “dilution effect is involved in all hypotheses of aposematism and Müllerian mimicry, as well as of aggregation under conditions of predation, this requires modelling frequency-dependent or “number-dependent” selection (Mallet and Joron 1999), both of which are conspicuously absent from the authors’ mathematical model”

Jacques Mallet[1] @eratosignis

Colour patterns in Lepidoptera evolved mainly as signals to predators. Heliconius are indeed Müllerian mimics. Reply to Mouy (2022).

Abstract

A recent paper in the journal Evolutionary Ecology by Henri Mouy, an independent researcher and theoretician who gives his address as “City of Westminster, London, UK,” argues that aposematism (warning colour) and Müllerian mimicry are problematic hypotheses. Instead, he claims that the bright colours and apparent colour pattern mimicry act primarily as an intraspecific or interspecific communication device to enable aggregation. In this critique, I show that the long-standing understanding that aposematic and mimetic colour patterns communicate directly with predators is much more likely than Mouy’s aggregation hypothesis.

Figure 1. Heliconius erato (above), and H. melpomene (below), a pair of Müllerian co-mimics from different sites in Ecuador and Northern Peru. Each species gains protection from the other’s unpalatability. Within any site, the two species are excellent co-mimics, but major geographic differences in colour pattern have evolved within each species. This geographic diversity is extraordinary by temperate zone standards: the region (about 600km x 300km) of the Andean foothills from which all these forms within each species can be found is less extensive than New England or Great Britain.

Introduction

As a tropical field biologist and geneticist who specializes on the evolution and genomics of Heliconius butterflies (Fig. 1), I try to keep up with the literature. This is becoming an increasingly difficult task, with at least ~50 Heliconius publications per year. While engaged in this activity, I came across a paper by one Henri Mouy titled “Colours as aggregation signals in Lepidoptera: Are Heliconius Müllerian mimics?” The answer to the question in the title, is an emphatic “no!” according to the author. To my surprise, Mouy claims to unseat Heliconius as an example both of aposematic (i.e. warning) coloration, and of Müllerian mimicry (Mouy, 2022), ideas that have been held by evolutionary biologists since their proposals by Bates and Wallace 160 years ago. I therefore read further.

Briefly, Mouy carries out a cursory citizen science analysis of some blurry photos on a British butterfly amateur website. The data, according to Mouy, show that brightly coloured butterflies aggregate more than cryptic or “semi-cryptic” butterflies (Mouy, 2022: his Fig. 1). These data apparently suggest to the author the idea that bright coloration in butterflies (or perhaps all Lepidoptera) evolved “purely as an aggregation signal.”

Then Mouy constructs a mathematical model that purports to show how bright colours can evolve to cause intraspecific attraction to aggregations, where the benefit of the aggregation can outweigh the cost in terms of increased conspicuousness to predators.

Having by now convinced himself that bright coloration in butterflies evolved as an intraspecific signal to enhance aggregation (or maybe in Lepidoptera in general), rather than primarily as a signal to predators, for example, aposematism, including as advertisement for escape ability (Mallet & Singer, 1987), Mouy then moves on to suggest that the reason for close similarity among brightly coloured Heliconius species, usually assumed to be due to Müllerian mimicry, may be incorrect. In support of his aggregation signalling hypothesis for mimetic coloration, he cites my own paper on nocturnal gregarious roosting and mimicry rings in Costa Rican Heliconius (Mallet & Gilbert, 1995), as well as more recent papers on gregarious roosting in Heliconius by Susan Finkbeiner and others (Finkbeiner et al., 2012, Finkbeiner et al., 2014, Salcedo, 2011).

Finally, in sections labelled “Discussion on Heliconius” and “Discussion”, the author strengthens his opposition to accepted theories of aposematism and Müllerian mimicry. In particular, the divergence of mimicry colour patterns between different regions, even within the same species, somehow suggests to the author that aggregation signalling rather than Müllerian mimicry is the cause of colour pattern convergence. The author goes on to argue, without any evidence I could find in his paper, that resemblance by the viceroy butterfly (Limenitis archippus) to monarchs (Danaus plexippus) and queens (Danaus gilippus) evolved “to attract them” (i.e. to attract monarchs, queens and viceroys together). As it happens, I have published a more mainstream view of viceroy mimicry (Mallet, 2001). In the final two sentences of his paper, Mouy argues that the well-known mimicry among unpalatable lycid beetles (Bocak & Yagi, 2010) “may need a reconstruction as well.”

The hypothesis that, to Mouy, explains almost all bright coloration in Lepidoptera and other insects, and also the apparent mimicry between species of Heliconius and other butterflies and beetles is as follows: instead of bright coloration advertising unpalatability to predators, it is an adaptation that advertises aggregations to conspecifics, and these aggregations reduce predation. In my critique below, I attempt to follow Mouy’s logic and sequence of sections.

Association of bright colour with gregariousness, according to Mouy

Mouy’s data extracted from online photographs when plotted against a gregariousness dimension (his Fig. 1) reveal several things. I have cursorily reviewed some of the photos on the cited amateur website.

Firstly, most of these photographs from an British amateur website of butterflies show bright or “semi-bright” coloration. This is hardly surprising, as even British butterflies are relatively conspicuous, and bright colours make butterflies a popular subject for amateur photographers.

Secondly, the majority of photographs show single butterflies, with only a few showing groups of individuals of the same species. Some photos, for instance of monarch butterflies (Danaus plexippus), which are well known to roost in winter aggregations in California and Mexico, do show genuine aggregations. However, for the majority of their life cycle, and indeed during their reproductive cycle, monarchs are notably solitary. Furthermore, there are no images of co-mimics Danaus gilippus and Limenitis archippus aggregating with D. plexippus; heterospecific aggregation is as far as I know unknown behaviour for these latter two species. Other photos, such as a photo with a number of Painted Lady butterflies (Vanessa cardui), clearly shows a large group feeding on a stand of garden nectar plants, presumably due to resource patchiness in a suburban environment rather than to any inherent tendency of this butterfly to aggregate. Mouy discusses similar aggregations also with respect to mud puddling in many butterfly species.

Thus the photos do include a few genuine aggregations, but also some that are clearly examples of concentration due to resource patchiness (such as nectar, or puddling sites). If there is a minor tendency for brighter coloured species to aggregate, there is nothing in these data that indicates colour signalling as the cause of aggregation.

Mathematical model: conspicuous intraspecific signalling supposedly evolves to enhance aggregation

The model proposed by Mouy “determines under which conditions signalling for aggregation through an increase in conspicuousness results in a net reduction in predation.” The model apparently predicts “a reduction in predation when the increase in conspicuousness to co-aggregators is not matched by a proportional increase in conspicuousness to predators.” To paraphrase the verbal result the author I think is reaching for, an increase in conspicuousness to predators can evolve if the benefit of signalling to conspecifics to form an aggregation (which is argued to reduce predation) outweighs the increased cost due to conspicuousness to the predators (which could increase predation). However, it is most unclear whether this actually models the aggregation signalling hypothesis.

The author argues rather vaguely for a “dilution effect” of aggregation. However, while a kind of dilution effect is involved in all hypotheses of aposematism and Müllerian mimicry, as well as of aggregation under conditions of predation, this requires modeling frequency-dependent or “number-dependent” selection (Mallet & Joron, 1999), both of which are conspicuously absent from the authors’ mathematical model. The current model subsumes the whole of this frequency-dependent complexity into a constant parameter, D_pred , “the increase in the probability of detection to predators.” The results are then displayed in a graph of change in overall “predation risk” (Mouy 2022: his Fig. 2), although how time is factored into predation risk is not discussed in the main paper. The author argues that “predation is reduced as a result of signalling when the result is below zero.” The axes are unlabeled, and it is not quite clear what “the result” is, although the reader infers that the x axis refers to time, and the y axis to “the result”, which varies from -20% to +20%; this presumably means some measure of predation risk. I would have thought that a 0% predation risk would be optimal, but according to the author a negative risk percentage is even better, though it is somewhat unclear what a negative predation risk would be.

In the modelling section of the main paper the author refers repeatedly to details in online Appendices 4, 5 and 6. In those appendices I found various scantily documented mathematical formulae, more graphs with unlabeled axes, some python code, and extensive tables of results. However, there was very little in the way of clear explanation. I was able to take away the idea that the author somehow models prey numbers or density by means of encounter rate over time, but not really how the “dilution effect” is supposed to work or what the benefit of aggregation was supposed to be. The mathematical model presented here is not a recognizable model of populations or their evolution over time.

Heliconius: shared aggregation signal or Müllerian mimicry?

This section, as well as Mouy’s following section (Discussion on Heliconius), and the final discussion are the main parts of the paper (Mouy, 2022) that deal with aposematism (warning colour) and mimicry. He classifies aposematism somewhat obscurely as “a visible structure which alters predators [sic] behaviours, which evolved or failed to disappear because of that effect and which is effective because predators either evolved or learned a response to that signal.” However, in his online Appendix 2, it becomes clear that the author does understand that aposematism is a theory of warning signals that communicate to the predator some sort of prey unprofitability. Apparently the author regards the theory of aposematism unlikely: “As reviewed in online appendix 2, the maintenance of an aposematic signal requires either peak-shift on the part of predators (or another indistinguishable mechanism), or kin-selection (Leimar et al., 1986), or potentially some other enabling mechanism such as innate aversion or neophobia. None are observed in Heliconius.” Mouy gives no evidence for this latter statement. In fact, the maintenance of aposematism has never been seen as a problem by most other authors since Wallace (1867); it acts as a mutualism between predator and prey that avoids damage to either, and is particularly advantageous to the prey. The maintenance of aposematism once established is easy to understand because all individuals in a local prey population benefit from the shared signal to warn the mostly educated predators in a community away.

It is the origin of aposematism that is sometimes seen as problematic: it might involve “peak shift,” “kin selection” or “some other enabling mechanism.” Rare conspicuous individuals among majority less conspicuous individuals could suffer greater initial predation costs because the predators perhaps do not recognize the new signal of an unprofitable species. Nonetheless, I personally believe that warning colours often, and perhaps mostly evolved via individual selection. The various routes that seem likely are colour pattern enhancement (including behavioural “peak shift”), receiver bias, and of course mimicry (Mallet & Singer, 1987, Mallet & Joron, 1999, Ruxton et al., 2018). Alternative explanations could involve a generalization of the population structure model “kin selection,” “kin-founding” or “shifting balance” models, but these are more controversial (Mallet, 2010).

Apart from his dismissive statement that “Heliconius are not even particularly colourful by butterfly standards,” I cannot find any other cited evidence why Mouy might reject the prevailing and well-accepted idea that Heliconius are aposematic. Heliconius colour patterns include crimson bands, matte black, yellow bars, orange rays, stripey contrasts, and sometimes iridescent blue which flashes in the sunlight (Fig. 1). In contrast to Mouy, I guess most people who have seen Heliconius would agree that they are among the most brightly coloured butterflies!

Müllerian mimicry

So much for aposematism. But Heliconius are classically viewed also as Müllerian mimics. In fact, Müllerian mimicry was a major component of Bates’ (1862) original hypothesis of mimicry, especially between Heliconius and ithomiine butterflies, and among different species of Heliconius. Bates argued that rare Heliconius mimicked commoner species of ithomiines (which he called “Danaoid Heliconidae.” A little later, a German émigré in Brazil, Fritz Müller proposed almost certainly the first mathematical model of Darwinian fitness under natural selection (Müller, 1879) in his argument for what is now termed Müllerian mimicry. He could have applied the hypothesis to local Brazilian species of Heliconius, but he focused on a different pair of species of Danaini and Ithomiini. Today, Heliconius is widely used in examples of Müllerian mimicry (Mallet & Joron, 1999, Mallet, 2001, Futuyma & Kirkpatrick, 2017, Ruxton et al., 2018).

Of course, a venerable one hundred and sixty-year old tradition of study of a topic does not necessarily mean that the prevailing view is correct, but it ought at least make the author of any alternative hypothesis take some care when attempting to disprove previous work. Yet very little effort seems to have been expended by Mouy (2022) to rebut the perceived consensus.

Mouy’s aggregation signalling hypothesis

Mouy’s alternative to aposematism and Müllerian mimicry is his own “aggregation signalling hypothesis”. According to Mouy, classical understanding of signalling to predators fails. Instead, conspicuous colours and close mimicry among species enables signalling within and between species of prey in order to aggregate. Aggregation is beneficial because of a dilution effect that reduces per capita predation.

The more probable explanation of aggregations is a reverse of this logic. In an aggregation of a particular size, unprofitable prey will suffer lower per capita mortality than an aggregation of more profitable prey because predators are satiated by fewer unpalatable than palatable individuals. Therefore, if predation is likely, we expect unprofitable prey (including aposematic prey) to be more prone to aggregate than profitable prey. Aggregation can also benefit even palatable individuals via satiation. This is also an old idea: one of the most famous proponents was the “selfish herd” hypothesis of Bill Hamilton (Hamilton, 1971), but it is older than that. Poulton spent many years collating examples of gregariousness in aposematic butterflies (Poulton, 1890, Jones, 1930, Poulton, 1931c, Poulton, 1931a, Poulton, 1931b, Poulton, 1933).

How could the extraordinarily accurate “mimicry” among Heliconius species evolve? Mouy (2022) argues not only that conspicuous colour patterns are examples of adaptations that originated as aggregation signals within species, but also that the tight similarity (mimicry) among species is favoured in signalling to attract other species to their aggregations. Here Mouy cites my own work on gregarious roosting in a community of Heliconius (Mallet & Gilbert, 1995) in support of aggregation signalling (Mouy, 2022): we found heterospecific aggregations of Heliconius species that preferentially (although not only) involved co-mimics, and we did conclude that “it therefore seems likely that individuals actively select co-mimics as roost-mates.”

However, it is most unlikely that aposematism and mimicry is primarily an aggregation signal, for a variety of reasons:

1) As Mouy admits, “birds have a much better visual acuity than butterflies … Modelling how a butterfly … would be seen by conspecifics and predators, it is found that the complex patterns are usually not resolvable by conspecifics, but that the whole-body coloration are still resolvable, and thus can function as an intraspecific signal. Predators have a considerably better acuity, can resolve the shape of butterflies at a distance and are likely attracted to butterfly wing shapes.” This view of insect vision is generally accepted, and argues against Mouy’s aggregation signalling hypothesis. Insects have excellent colour vision, motion detection and contrast perception. But insect vision is highly pixellated, and insects do not have good shape perception, compared with vertebrate vision. Yet mimicry among Heliconius species is so accurate that even experts are sometimes fooled, and fail to distinguish the species — almost all major butterfly collections in the world museums I have visited have some misidentifications for this reason. The highly accurate shape perception that we vertebrates use to detect the clear cases of Heliconius mimicry is a communication channel unavailable to insects. A mere convergence in “whole body coloration” would not require detailed resemblance in shape of pattern outlines.

2) Mouy ignores a section in our paper where we discuss how mimicry colour patterns are often involved as intraspecific cues in mate choice, and their association with speciation (Mallet & Gilbert, 1995: 176). Heliconius cydno (including its semispecies allies, H. pachinus and H. timareta) differs from its sibling species H. melpomene. The latter is red and black with a yellow hindwing bar, and males in particular tend to be attracted to red rags waved in the forest; H. cydno, on the other hand is white and black often with iridescent blue in the melanic regions, and is attracted instead to white rags. Notice that these signals are non-specific as the coloured rags do not look much like real Heliconius butterflies. We concluded that “while mate choice may evolve during mimetic divergence, it is rather unlikely that evolution of mate choice itself drives divergence” (Mallet & Gilbert, 1995: 176).

We later showed experimentally that colour is indeed involved in the initial courtship approach by males of these two species to females, or to crude coloured paper models (Jiggins et al., 2001). We thought that perhaps most speciation in Heliconius was accompanied by a mimicry switch (Turner, 1976). But colour pattern loci acting as important “speciation genes” is not the norm even in Heliconius. We later discovered that highly mimetic forms of H. timareta (part of the H. cydno superspecies) were sympatric with identically patterned H. melpomene (Giraldo et al., 2008, Mérot et al., 2013). Later genomic work proved that H. timareta gained its colour pattern mimicry via occasional hybridization with the local forms of H. melpomene (Heliconius Genome Consortium, 2012). These cryptic pairs of sympatric forms were previously confused by workers on Heliconius, including myself. Similarly, we recently discovered another cryptic pair of sister species in a different subgroup of Heliconius, H. eratosignis and H. demeter, that also share mimetic patterns, and yet successfully remain distinct in sympatry (Rosser et al., 2019). Thus although intraspecific preferences do sometimes make use of differences in mimetic colour patterns, they are not the primary feature keeping species apart. Instead, pheromone differences between species are almost certainly the most important cue that leads to strong assortative mating among these species (Mérot et al., 2015, Jiggins et al., 2001). Similarly, it is most unlikely that aggregation signalling is the primary function of mimetic colour patterns.

3) As it happens, a certain amount is known about cues that attract Heliconius to gregarious roost mates, and it conflicts with Mouy’s aggregation signalling hypothesis for mimicry and bright colour patterns. As with courtship, roosting aggregation involves an initial generalized approach to a visual cue, followed by a close range “fanning” or hovering behaviour, during which time both males and females appear to exchange odour cues (Mallet, 1980, Mallet, 1986). It is likely, in fact, that courtship behaviour has been coopted in these species to facilitate aggregation behaviour. While colour pattern and contrast patterns are involved in initial attraction, even crude black or white oblong cardboard models can elicit close approach and fanning behaviour if in a setting that is contrasty enough (Mallet, 1980). Furthermore, there are plenty of examples of non-mimics roosting gregariously, in spite of a bias towards co-mimics (Mallet & Gilbert, 1995). Detailed mimicry is clearly not required for attraction of Heliconius to heterospecific roosts.

Is the theory of aposematism and mimicry in need of a revision? (The answer is no!)

In science, no hypothesis is actually proved. Rather, we tend to disprove alternatives, or at least to show why the alternative hypotheses are unlikely. Sometimes, however, prevailing opinion is overturned. For instance, before Copernicus, people generally seem to have believed the Ptolemaic theory that the sun revolved around the earth. The Copernican/Newtonian revolution explained that it was more likely instead that the earth revolved around the sun, with the mechanism for elliptical orbits being gravity. Today no serious scientist believes any alternative, and alternatives now seem so unlikely that one can say that rotation round the sun is an established fact. So is Henri Mouy a new Copernicus of mimicry theory? Or is his aggregation hypothesis just wrong? It seems clear that the latter is the correct interpretation.

One hundred and sixty years ago, the evolutionary hypothesis of mimicry was born (Bates, 1862): predators avoid attacking prey that are unprofitable and learn to recognize their colour patterns and avoid them; other either palatable or unpalatable species benefit from reduced predation pressure by having copy-cat  colour patterns. Soon after, the hypothesis of warning colour was proposed to explain bright colour patterns in unpalatable insects (Wallace, 1867): individuals displaying these advertising colour patterns were recognized by predators, after conditioning by previous encounters, and avoided as prey. This evolutionary theory of warning coloration as a signal was conceptualized as “aposematism” by a classically educated Oxford University entomologist who enjoyed coining neologisms based on Greek roots (Poulton, 1890) – “sympatry” was another term he coined. Although there are some who would question minor details of mimicry and warning coloration, almost all of today’s work on the phenomena of mimicry and aposematism accepts these ideas as being largely correct. The bright aposematic colour patterns of skunks and wasps are so clearly warnings that nobody doubts it; the examples of wasp mimicry by flies and Lepidoptera have nothing to do with aggregations, and show that these signals are indeed learnt by potential predators, just as humans learn to avoid wasps after being stung. These same hypotheses are supported in most evolution textbooks (Futuyma & Kirkpatrick, 2017, Ruxton et al., 2018). A rejection of these ideas would be a major upset in evolutionary biology.

Evolutionary theories of aposematism and mimicry were initially based on comparative evidence and theory. These ideas were later backed up with extensive observations of the behaviour of birds and their interactions with insects; Poulton was especially active in the early part of the 20^th Century in gathering and publishing observations from around the world (see above). In 1958 the first carefully controlled behavioural experiments on mimicry were carried out (Brower, 1958), and soon thereafter with heliconiine butterflies (Brower et al., 1963). Subsequently experiments in field and laboratory have carefully documented predator perception and learning, prey behaviour, and the strength of natural selection, as well as other evidence on the function of aposematism, mimicry and aggregations of potential prey on predators (Mallet & Joron, 1999, Jiggins, 2017, Ruxton et al., 2018). Heliconius remains an extraordinarily useful model genus for understanding the evolution of mimicry and aposematism, and there really is no doubt that that aposematism and mimicry are very strongly supported as the primary explanation for bright colours. In contrast, although colour patterns are sometimes exploited in a general way by Heliconius as intraspecific cues, this is clearly only a secondary re-use rather than the primary function for the bright colours and mimicry.

Conclusion

I am somewhat astonished that Henri Mouy, an “independent researcher,” who provides no observations of his own on butterflies in the wild, seems to have persuaded the editors and reviewers of an academic journal that his hypothesis might overturn consensus, a century and a half old, on warning colour and Müllerian mimicry.

Acknowledgments

I am very grateful to Graeme Ruxton, an Editor at Evolutionary Ecology, for considering this piece for publication in Evolutionary Ecology as a rebuttal. However, I decided that it would be better as a blog post. I am also grateful to Henri Mouy for his correspondence on the issue, and to my lab group (Neil Rosser, Fernando Seixas, Yuttapong Thawornwattana, and Sarah Dendy) for advice.

References

Bates, H. W. 1862. Contributions to an insect fauna of the Amazon valley. Lepidoptera: Heliconidae. Transactions of the Linnean Society of London 23: 495-566.

Bocak, L. & Yagi, T. 2010. Evolution of mimicry patterns in Metriorrhynchus (Coleoptera: Lycidae): the history of dispersal and speciation in south east Asia. Evolution 64: 39-52.

Brower, J. V. Z. 1958. Experimental studies of mimicry in some North American butterflies. Part I. The monarch, Danaus plexippus, and viceroy, Limenitis archippus archippus. Evolution 12: 32-47.

Brower, L. P., Brower, J. V. Z. & Collins, C. T. 1963. Experimental studies of mimicry. 7. Relative palatability and Müllerian mimicry among Neotropical butterflies of the subfamily Heliconiinae. Zoologica, New York 48: 65-84.

Finkbeiner, S. D., Briscoe, A. D. & Reed, R. D. 2012. The benefit of being a social butterfly: communal roosting deters predation. Proceedings of the Royal Society B: Biological Sciences 279: 2769-2776.

Finkbeiner, S. D., Briscoe, A. D. & Reed, R. D. 2014. Warning signals are seductive: relative contributions of color and pattern to predator avoidance and mate attraction in Heliconius butterflies. Evolution 68: 3410-3420.

Futuyma, D. & Kirkpatrick, M. 2017. Evolution. Fourth Edition.Sinauer, Sunderland, Mass.

Giraldo, N., Salazar, C., Jiggins, C. D., Bermingham, E. & Linares, M. 2008. Two sisters in the same dress: Heliconius cryptic species. BMC Evolutionary Biology 8: 324.

Hamilton, W. D. 1971. Geometry for the selfish herd. Journal of Theoretical Biology 31: 295-311.

Heliconius Genome Consortium 2012. Butterfly genome reveals promiscuous exchange of mimicry adaptations among species. Nature (London) 487: 94-98.

Jiggins, C. D. 2017. The Ecology and Evolution of Heliconius Butterflies.Oxford University Press, Oxford.

Jiggins, C. D., Naisbit, R. E., Coe, R. L. & Mallet, J. 2001. Reproductive isolation caused by colour pattern mimicry. Nature (London) 411: 302-305.

Jones, F. M. 1930. The sleeping heliconias of Florida. Natural History 30: 635-644.

Leimar, O., Enquist, M. & Sillén-Tullberg, B. 1986. Evolutionary stability of aposematic coloration and prey unprofitability: a theoretical analysis. American Naturalist 128: 469-490.

Mallet, J. 1980. A laboratory study of roosting in the gregarious butterfly Heliconius melpomene. UCL Discovery.

Mallet, J. 1986. Gregarious roosting and home range in Heliconius butterflies. National Geographic Research 2: 198-215.

Mallet, J. 2001. Causes and consequences of a lack of coevolution in Müllerian mimicry. Evolutionary Ecology 13: 777-806.

Mallet, J. 2010. Shift happens! Evolution of diversity in warning colour and mimicry. Ecological Entomology 35: 90-104.

Mallet, J. & Gilbert, L. E. 1995. Why are there so many mimicry rings? Correlations between habitat, behaviour and mimicry in Heliconius butterflies. Biological Journal of the Linnean Society 55: 159-180.

Mallet, J. & Joron, M. 1999. The evolution of diversity in warning colour and mimicry: polymorphisms, shifting balance, and speciation. Annual Review of Ecology and Systematics 30: 201-233.

Mallet, J. & Singer, M. C. 1987. Individual selection, kin selection, and the shifting balance in the evolution of warning colours: the evidence from butterflies. Biological Journal of the Linnean Society 32: 337-350.

Mérot, C., Frérot, B., Leppik, E. & Joron, M. 2015. Beyond magic traits: multimodal mating cues in Heliconius butterflies. Evolution 69: 2891-2904.

Mérot, C., Mavárez, J., Evin, A., Dasmahapatra, K. K., Mallet, J., Lamas, G. & Joron, M. 2013. Genetic differentiation without mimicry shift in a pair of hybridizing Heliconius species (Lepidoptera: Nymphalidae). Biological Journal of the Linnean Society 109: 830-847.

Mouy, H. 2022. Colours as aggregation signals in Lepidoptera: Are Heliconius Müllerian mimics? Evolutionary Ecology 36: 341–353.

Müller, F. 1879. Ituna and Thyridia; a remarkable case of mimicry in butterflies. Proceedings of the Entomological Society of London 1879: xx-xxix.

Poulton, E. B. 1890. The Colours of Animals.Trübner & Co Ltd, London.

Poulton, E. B. 1931a. Acraeine butterflies congregating in a small area for the night’s rest, observed by Dr. Hale Carpenter in Portuguese East Africa. Proceedings of the Entomological Society of London 6: 71-.

Poulton, E. B. 1931b. The gregarious sleeping habits of a heliconian and an ithomiine butterfly in Trinidad, observed by P. Lechmere Guppy. Proceedings of the Entomological Society of London 6: 68-69.

Poulton, E. B. 1931c. The gregarious sleeping habits of Heliconius charitonia, L. Proceedings of the Entomological Society of London 6: 4-10.

Poulton, E. B. 1933. The gregarious resting habits of danaine butterflies in Australia; also of heliconiine and ithomiine butterflies in tropical America. Proceedings of the Royal Entomological Society of London 7: 64-67.

Rosser, N., Freitas, A. V. L., Huertas, B., Joron, M., Lamas, G., Mérot, C., Simpson, F., Willmott, K. R., Mallet, J. & Dasmahapatra, K. K. 2019. Cryptic speciation associated with geographic and ecological divergence in two Amazonian Heliconius butterflies. Zoological Journal of the Linnean Society 186: 233-249.

Ruxton, G. D., Allen, W. L., Sherratt, T. N. & Speed, M. 2018. Avoiding Attack: The Evolutionary Ecology of Crypsis, Aposematism, and Mimicry. Second Edition.Oxford University Press, Oxford.

Salcedo, C. 2011. Evidence of predation and disturbance events at Heliconius (Insecta: Lepidoptera: Nymphalidae) nocturnal aggregations in Panama and Costa Rica. Journal of Natural History 45: 1715-1721.

Turner, J. R. G. 1976. Adaptive radiation and convergence in subdivisions of the butterfly genus Heliconius (Lepidoptera: Nymphalidae). Zoological Journal of the Linnean Society 58: 297-308.

Wallace, A. R. 1867. Mimicry and other protective resemblances among animals. Westminster and Foreign Quarterly Review 32: 1-43. [Note added 20 Jan 2024: The warning colour (aposematism) hypothesis is not well developed in this paper. I think the Wallace reference I was thinking of is: Wallace, A.R. 1867. Letter 5416 – Wallace, A. R. to Darwin, C. R., 24 Feb 1867, Reply to Darwin about brightly coloured catterpillars (sic), 8 pp. Darwin Correspondence Project. Cambridge, University of Cambridge. https://www.darwinproject.ac.uk/letter/DCP-LETT-5416.xml.]

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[1] Cambridge, MA, USA

Henri Mouy has recently answered my criticism. See: https://www.heliconius.org/2024/heliconius-are-neither-aposematic-nor-mullerian-mimics-henri-mouy-on-mallet-2024-on-mouy-2022/

A fascinating recent paper by Jennifer Hoyal Cuthill et al. (2019) published in Science Advances uses a “deep convolutional neural network” to classify photos of upper and underside of 1234 butterfly specimens, all of them named subspecies of Heliconius erato and H. melpomene, into phenotypic clusters. The researchers worked with Blanca Huertas and Robyn Crowther to photograph specimens from the London Natural History Museum, that has one of the most complete collections of Heliconius worldwide. They compare the “phylogenetic analyses” based on phenotypic classification with those of genes known or thought to be close to sites of colour pattern switch loci, and those of “neutral” genes selected from around the genome. They conclude that the species have converged within each geographic region, due to Müllerian mimicry. They also argue that the results prove that convergence is due to mutual coevolution.

This topic is of particular interest to me because (a) it concerns my favourite organisms, (b) the coevolution conclusion rebuts a paper I wrote arguing that there is little convincing evidence for coevolution in Müllerian mimicry (Mallet 2001). I’ve communicated with Jennifer on the paper, and discussed the difficulty of classifying hybrids between races, and helped to correct her classifications with some of the obvious inter-subspecific hybrids I found in a sample of their photos from the Natural History Museum’s collections of each subspecies. Blanca Huertas also was my PhD student at UCL, so I am somewhat conflicted about this paper.

Firstly how does the algorithm perform? I don’t fully understand how the deep convolutional neural network works, and in fact maybe nobody really does, but the algorithm itself apparently does! It clearly classifies some mimics together. For instance H. erato phyllis is reasonably near H. melpomene nanna and H. melpomene burchelli in Fig. 1A, which is expected because they are usually classified as co-mimics. I assume that the Euclidean “phenotypic distance” referred to in this figure is measured in 64-dimensional space using the algorithm’s classification of each specimen.  But H. erato venustus (https://butterfliesofamerica.com/L/heliconius_erato_venustus.htm) is classified by the algorithm a long way from its common co-mimic in Bolivia H. melpomene penelope (see: http://media.api.aucklandmuseum.com/id/media/v/576002?rendering=original.jpg for a series of the pure subspecies). For some reason penelope is not called a mimic according to the colour coding in Fig. 1, for reasons I don’t understand. And penelope (no. 28) and four other numbered subspecies are completely missed from Table 1, for reasons that are not explained. I am wondering if the Natural History Museum just happens not to have a good collection of pure penelope, and that’s why we have this apparent screw up. It was mentioned in the methods that there was only one “pure” penelope, and maybe it happened to be one of the obvious hybrids in the type series in the Natural History Museum (https://butterfliesofamerica.com/L/t/Heliconius_melpomene_penelope_a.htm). Also, Heliconius melpomene vulcanus is mis-classified to be phenotypically close to H. m. melpomene and H. erato hydara both of which lack a yellow hindwing bar that vulcanus has on the underside, and penelope which is rayed. The authors say that this is because the blue iridescence of H. melpomene vulcanus is reduced compared to the true comimic from Western Colombia, H. erato venus (p. 3). This is true, although this may also be due to the use of photographs taken in artificial light rather than under natural sunlit conditions. Some Heliconius colours, particularly iridescence, are particularly hard to render photographically.

Table 1 itself also shows that penelope groups fairly closely with H. melpomene burchelli and nanna, but that H. erato phyllis is not classified together with these two, but instead with Heliconius erato petiverana from Mexico. In sum, the algorithm, after being trained on 1500 images, does a reasonably good job of classifying the remaining 968 images to subspecies with 86% accuracy. Interestingly, some of the principal component axes (3 and 4) are apparently also able to distinguish H. erato and H. melpomene (Fig. 2F), suggesting that some useful phylogenetic information, as well as mimicry, is detected by the algorithm.

Overall, I’m reasonably impressed with the algorithm’s performance, although it seems only to reach a not-so good taxonomists’ level thus far: a good human taxonomist will still beat it. Deep Convolutional Neural Network (ButterflyNet) is not yet the Deep Blue of taxonomy! But this is the first time I believe that such an extensive classification of this nature has been done by machine learning, and so this represents a considerable achievement for this paper and these authors.

Next, let’s consider the evidence for the authors’ argument for coevolution in Müllerian mimicry. Readers will probably be aware that Müllerian mimicry occurs between species that are both generally rejected by predators, and it is classified as a mutualism because both partners in the mimicry ring benefit. Mimicry evolves because each species benefits from the association due to mortality during predator learning; each species is sharing the costs of educating predators. On the other hand, Batesian mimicry is the case where an edible species evolves to mimic a poisonous species. In this case, the relation is parasitic, because a pleasant experience with an edible mimic may induce a predator to attack the model. The alternative is one-sided “advergent” mimicry (Brower & Brower 1972), whereby a mimic converges to an unpalatable model, but the model does not respond by evolving towards the mimic. Advergent mimicry is expected in parasitic Batesian mimicry, while Müllerian mimicry is a mutualism, and most people imagine that all mutualisms involve coevolution.

First we need some history. Müllerian mimicry has two special features.

First, Müller’s paper was indeed “evolution’s oldest mathematical model” (Müller 1879). Müller was a maths teacher at the college at Desterro, Brazil as well as a correspondent of Darwin and a major naturalist who documented the local fauna and flora. Müller suggested that predators probably learned to avoid unplatable species after killing a certain number of these per year. If two unpalatable prey species shared the same colour pattern so that they were indistinguishable to the predators, then they would each lose fewer individuals killed during the process of learning, making this a mutualism. We expect the rarer species to gain most of the association. Müller showed that each species gains a proportional advantage by becoming an indistinguishable mimic given by the inverse square of the relative abundance. For example, if a species has a tenth of the abundance of its comimic, it gains 100x the fitness benefit from the mimicry. The same will apply to the degree of unpalatability; measured as the reduction in the number killed, unpalatability and abundance combine to reduce predation in the same way (Mallet 2001). Thus, the benefits of Müllerian mimicry will typically be very one-sided unless abundance and unpalatability are closely balanced.

Second, there’s another wrinkle. John R.G. Turner (1977, 1988), citing a 1927 paper by A.J. Nicholson, pointed out that an abundant unpalatable species would typically not gain by mimicking a rarer unpalatable species, because local predators will be better trained by the most abundant (and/or most unpalatable) species. Therefore, a common or very unpalatable mimic has very little interest in converging on a rarer or less unpalatable species, even though, once it did occur, it would ultimately be beneficial for both species (albeit much reduced by the Müller inverse square law for the common species). Turner argued that most Müllerian mimicry would occur with rarer species approaching commoner species and gaining the evolutionary benefits of mimicry. In his “two-step” model, however, Turner did also propose a subsequent truly coevolutionary mechanism for Müllerian mimicry. Once the proto-mimic species overlapped the model species due to advergence in the first step of mimicry, and became somewhat indistinguishable in the perceptual consideration of the predators, then the second step, mutual convergence, became possible (Turner 1977, 1984, 1988).

Thus these two considerations argue that coevolution will occur perhaps rarely between Müllerian mimics, unless some lucky combination of density fluctuations or palatability details are met, at least until after the two species become so close as to be within a tight domain of protection by the most common species.

What is the evidence for coevolution among Heliconius co-mimics in nature? My own PhD supervisor, Larry Gilbert, had suggested that it was clear that normally, H. melpomene adverged to H. erato, but he argued that some evidence suggested that the reverse could also be true. In Central America, H. melpomene rosina has a broad yellow hindwing band. In contrast, H. erato petiverana from Mexico, and H. erato forms southward to northeastern Costa Rica also have narrow yellow hindwing bands. Heliconius melpomene is a southern species in Central America, and enters Nicaragua but is not known from further north. Gilbert suggested that although the prevailing direction of mimicry was melpomene -> erato, nevertheless in the case of the hindwing bars, the broadening of the yellow hindwing bar to the south of the H. erato distribution is due to convergent mimicry with the broader bands of H. melpomene (Gilbert 1983). I disputed that. Heliconius erato may have diverged in northern populations to produce narrow yellow bars, but since it did not occur there, H. melpomene would not have been able to follow this trend in the north. In contrast, the main mimicry force on H. melpomene in the south would be to adopt the broader hindwing bars of the local H. erato. Thus the apparent spatial coincidence of H. melpomene rosina and H. erato forms with broad yellow hindwing bars is readily explained by mimicry, but not necessarily by means of coevolution and mutual convergence.

In my own paper I concluded that the null hypothesis for coevolution had not been effectively rejected, and that most indications suggested H. melpomene was largely an advergent mimic of H. erato rather than the other way round. This was evidenced by the generally greater abundance, more widespread geographic distribution, and neater colour patterns of the latter – H. melpomene, although often an extremely accurate mimic, tends to have fuzzier or more variable colour patterns and, together with close sister species H. cydno and H. timareta, mimics a variety of other species or subspecies in the Heliconius erato-sara group of species.

In the new paper, Hoyal Cuthill et al. (2019) follow on from previous attempts (Hoyal Cuthill & Charleston 2012, 2015) to prove that coevolution did in fact occur between H. erato and H. melpomene. Some of their arguments are: (1) based on molecular dating, the subspecies of the two species are roughly the same age, as would be expected if they had co-diverged in different geographic localities, (2) that genealogical topologies of colour pattern genes are more similar to those of co-mimics than are genealogies based on a set of comparator genes more distantly linked to colour pattern genes, and (3) arguments similar to those of Gilbert (1983) about divergence of races of H. erato in the absence of H. melpomene. For instance, Hoyal Cuthill et al. (2019) argue that the narrow red forewing band of W. Ecuador H. erato cyrbia (see figure) is an example of advergence towards narrow-banded H. melpomene cythera (rather than the other way round), apparently because the adjacent Colombian H. melpomene vulcanus also displays a somewhat narrower forewing band than H. erato venus in this western part of South America.

However, for (3) most colour pattern features of H. melpomene cythera indicate fuzziness and variability, as expected if melpomene is approximating the tidy and extraordinarily striking pattern of H. e. cyrbia, rather than being mimicked. The already noted dullness of the blue iridescence of melpomene vulcanus compared with erato venus is cranked up to even greater pitch with melpomene cythera versus erato cyrbia. This suggests that H. melpomene evolutionarily attempts to, but finds it so far impossible follow the extreme flashiness of H. erato cyrbia, and here I mean this literally, if you’ve ever seen cyrbia flying in the sunlight.

I’m not convinced by (2) either, since although Hoyal Cuthill et al. picked “colour pattern genes”, these genes were not at the precise location of colour pattern loci, which are known to be largely in gene desert non-coding regions nearby; they mostly appear to be cis-regulatory elements that control the effector genes. Phylogenetic topologies based on these genes are also similar to topologies based on unlinked loci. I remain also unconvinced by (1), in part because it is circumstantial evidence (same age does not equal coevolution), and in part because we don’t really know how to age subspecies via molecular genetic data given the abundant gene flow between subspecies at their hybrid zones (Nadeau et al. 2012).

The discussion ends with a hypothetical model of how some colour pattern traits might recombine across mutualists so that novel traits can evolve in both species as a result of mutual coevolution and convergence. I agree that this would be true if coevolution was the case, but first I’d need to be convinced that coevolution did occur. For instance, a novel trait for Heliconius erato/melpomene mimicry in H. e. cyrbia is the white hindwing fringe checked with black rays, which does not occur in any other race of this species. Heliconius melpomene also has a version of this novel trait in subspecies cythera, but just knowing that does not make it clear which species first invented it or whether it represents a combination due to mutual convergence.

Overall, this is a fascinating and complicated paper. It achieves for the first time a machine learning classification of the mimicry patterns in Heliconius erato and H. melpomene. But I believe it does not yet prove the case for coevolution between these two iconic co-mimics. 

References

Brower, L.P., & Brower, J.V.Z. 1972. Parallelism, convergence, divergence, and the new concept of advergence in the evolution of mimicry. Transactions of the Connecticut Academy of Arts and Sciences 44:57-67

Gilbert, L.E. 1983. Coevolution and mimicry, Pages 263-281 in D.J. Futuyma, ed. Sunderland, Mass., Sinauer Associates

Hoyal Cuthill, J., & Charleston, M. 2012. Phylogenetic codivergence supports coevolution of mimetic Heliconius butterflies. PLoS One 7:e36464

Hoyal Cuthill, J.F., & Charleston, M. 2015. Wing patterning genes and coevolution of Müllerian mimicry in Heliconius butterflies: support from phylogeography, cophylogeny, and divergence times. Evolution 69:3082-3096

Hoyal Cuthill, J.F., Guttenberg, N., Ledger, S., Crowther, R., & Huertas, B. 2019. Deep learning on butterfly phenotypes tests evolution’s oldest mathematical model. Science Advances 5:eaaw4967. https://advances.sciencemag.org/content/advances/5/8/eaaw4967.full.pdf

Mallet, J. 2001. Causes and consequences of a lack of coevolution in Müllerian mimicry. Evolutionary Ecology 13:777-806

Müller, F. 1879. Ituna and Thyridia; a remarkable case of mimicry in butterflies. Proceedings of the Entomological Society of London 1879:xx-xxix

Nadeau, N.J., Whibley, A.C., Jones, R.T., Davey, J.W., Dasmahapatra, K.K., Baxter, S.W., Quail, M.A., ffrench-Constant, R.H., Blaxter, M., Mallet, J., & Jiggins, C. 2012. Genomic islands of divergence in hybridizing Heliconius butterflies identified by large-scale targeted sequencing. Philosophical Transactions of the Royal Society B: Biological Sciences 367:343-353

This month, we published two papers in PloS Biology on the genetic architecture of speciation in Heliconius butterflies, among the best-known study systems in speciation research. Both papers represent the culmination of many years of work, but at first glance they appear to come to contradictory conclusions. In this piece we discuss why this is the case, and show how it relates to a broader debate about the importance of major loci versus polygenic effects in evolution.

In fact, from the start our two papers approach the same system from different directions. In the first paper, by Merrill et al., we focus on the fact that two closely-related species that co-occur in Panama (Heliconius melpomeneand H. cydno) have evolved strong assortative mating behaviour: they really do not like to hybridize, and much prefer to mate with their own kind. This paper asked about the gene(s) underlying divergence in these preference behaviours, which would inform us about a key step in the evolution of distinct species from a common ancestor.

In contrast, in the second paper by Martin et al., we start from the knowledge that these two species are capable of producing fertile male hybrids, and that their genomes carry the signatures of extensive genetic mixing, or ‘admixture’. While hybridisation is rare (on a per-individual basis) very occasional inter-species mating is enough to scramble the genomes of two species, if it occurs over millions of generations. The second paper therefore addresses how natural selection is acting against the hybrids and their offspring, which carry different combinations of genes from both species. This selection acts to keep the species distinct despite the potential to become genetically mixed.

The main finding of the Merrill et al paper is that just three genetic loci control a large proportion of the differences in mating preference between the species. Remarkably, one of these loci controls both the colour of the butterfly wings andpreference for that colour (though this does not necessarily imply that the same genes are involved). The discovery that changes in a complex behaviour can arise through changes at just a few loci seems surprising, and has important implications for speciation. A new species can arise much more readily if natural selection can act on a few genes of large effect, rather than many loci of small effect. This genetic system that we have discovered should therefore make speciation easier.

By contrast, Martin et al show that the landscape of admixture between these species is consistent with a highly ‘polygenic’ barrier, in which the species boundary is maintained by natural selection acting at multitudes of ‘barrier loci’ across the genome, suggesting a minor contribution of any given locus. This joins a number of studies showing similar trends in both plants and animals. So how do we reconcile these findings? Is speciation more dependent on a few major effect loci that prevent hybridisation, or many small effect loci that lead to the elimination of hybrids?

We think there is less of a paradox here than first appears, and that the papers are more complementary than contradictory. This is because the papers not only consider different components of speciation (pre-mating versus post-mating isolation), but also use different strategies (trait-focused versus genome-focused approaches).

Most speciation events probably involve both pre-mating and post-mating isolating mechanisms: those that limit the production of hybrids as well as those that reduce the survival and reproductive output of any hybrids that are produced. It is perhaps unsurprising that by dissecting these different levels at which isolating mechanisms act, we find different genetic architectures.

The two studies also used entirely different methodologies, designed to detect different things. The approach of Merrill et al., called QTL mapping, is classed as ‘forward genetics’, which starts with a trait of interest and identifies the loci that are associated with that trait. While there almost certainly are some smaller-effect loci that also contribute to the mate preference differences between the species, the QTL mapping approach was only ever likely to find the large-effect ones. Without accurately scoring the phenotypes of an extremely large number of individuals, which is normally very difficult to achieve in behavioural studies, quantitative genetic analyses are unlikely to detect any but the largest effect loci.

Martin et al. used a ‘reverse-genetics’ approach, which starts by identifying signatures of selection in the genome, naive to the trait that is being selected. We searched for variation in the extent of admixture across the genome, as a proxy for the strength of selection against individuals carrying foreign DNA at any genomic locus (i.e. the ‘species barrier’). We found that the species barrier strength is strongly predicted by the recombination rate – the rate at which chromosomes are shuffled in the production of gametes each generation. Increased recombination reduces the extent to which selection acting at a given locus will affect neighbouring parts of the genome. The observed correlation between recombination and admixture can only be explained if manybarrier loci are sprinkled throughout the genome – apparently in contradiction to the fewloci of large effect detected by Merril et al.,.

However, this genome-wide pattern does not preclude the existence of a few barrier loci with a disproportionately large effect, and indeed some genomic regions are particularly resistant to admixture, including the ‘Z’ sex chromosome.  This chromosome is known to harbour loci that contribute to hybrid sterility between the species. We know that natural selection also acts strongly against hybrids with mixed wing patterns, because the hybrid pattern is not recognised by predators as a warning that these butterflies are distasteful and should be avoided. But we also know that there are many other ecological differences between these species, including habitat and host plant preferences, so it is unsurprising that very many loci experience some selection (albeit weak in many cases) against foreign DNA.

One striking finding is that the large effect behaviour loci identified by Merrill et al. did not stand out as particularly strong localised species barriers in the genome scan. This may partly be explained by resolution: QTL mapping only provides an approximate location for the functional locus, so it is difficult to be sure where precisely to expect to see reduced admixture. But another factor is that these loci, although important for speciation, might not act as localised barrier loci. A locus that affects hybrid fitness will tend to show reduced admixture, but that is not necessarily the case for a locus that affects whether hybrids are produced in the first place. An assortative mating locus that prevents production of F1 hybrids could be important for speciation but its impact on the pattern of admixture will depend on what happens in later generation hybrids. 

Finally, one mechanism by which assortative mating may arise is termed ‘reinforcement’, where selection favours traits that reduce the chance of producing unfit (and costly) hybrids. This relies on an association between the genes under selection for hybrid fitness and those that promote assortative mating (like behavioural preferences). In other words, alleles underlying divergent mating behaviours may not themselves be under direct selection, but may rely on finding themselves in butterflies with the matching species specific traits. Whether we expect genome scans to detect loci under this type of indirect selection remains unclear. This is an area that could provide a focus for future theoretical studies.

In summary, we have found evidence both for a highly polygenic architecture, but also loci with major effects on species boundaries. An appealing story would be that early stages of speciation are characterised by ecological selection acting on a few loci of major effect, but that later stages involve accumulation of differences at many traits that are more polygenic – however we cannot distinguish the time course of speciation from these studies that represent a snapshot of a single species pair.  There are also implications here for future speciation studies. The genome scan approach will not necessarily find loci with a large effect on species barriers, even where they exist. Even if no major ‘islands’ of reduced admixture are found, this does not preclude large-effect loci influencing specific traits. As is so often the case in biology, the reality does not fit any single narrative.

This post was written by Chris Jiggins, Simon Martin and Richard Merrill

Kelsey Byers (@plantpollinator) has posted a very useful list of talks and posters at Evolution 2018 Montpellier on Heliconius and related topics such as mimicry and Ithomiini.

See: https://twitter.com/eratosignis/status/1029394638539509760

and:

https://docs.google.com/document/d/1WC8P5ai7k34g24S0XxkZ3pnkZm-DF-WLEKwSJvBUmgI

In front of Museum of Comparative Zoology, Cambridge, Mass.