Revisiting Jiggins et al. 2001

In a paper published in Nature in 2001, Chris Jiggins, Russell Naisbit, Rebecca Coe and James Mallet showed that divergence in mimicry of colour pattern was responsible for the origin of two Heliconia butterfly species. Using experiments, Jiggins and colleagues showed that differences in mimicry pattenrs led to assortative mating of the sister species and consequently low hybridisation and non-mimetic intermediates that were poorly adapted. Sixteen yearsa after the paper was published, I spoke to Chris Jiggins about his motivation to do this study, memories of experimental work and what we have learnt since about the role of mimicry in speciation.

Citation: Jiggins, C. D., Naisbit, R. E., Coe, R. L., & Mallet, J. (2001). Reproductive isolation caused by colour pattern mimicry. Nature, 411(6835), 302-305.

Date of interview: 17 March 2017 (via Skype)

Hari Sridhar: I want to start by asking you a little bit about your motivation to write this paper. From looking at your publication profile, it looks like you did this work during your PhD. I wanted to ask you what the motivation for this particular paper was, in relation to everything else that you did in your PhD.

Chris Jiggins: Sure. Well, actually, it was a post-doc. I did my PhD on studying a hybrid zone in Ecuador, in the south of Ecuador. And this was a hybrid zone between two taxa that were on the cusp of becoming species. But hybrids formed only about 10% of the population. I guess they were hybridizing but hybrids were less common than you expected at random. So we looked at reproductive isolation, and what were the factors that made these populations distinct, and talked about how they could coexist. And so then, for my post-doc project, we were interested in actually moving to look more at really sympatric species that sort of were more along the speciation process; which could coexist stably in sympatry. So we turned to these two species melpomene and cydno. And so, yeah, we were based in Panama, and we were interested in dissecting the reproductive isolation between them.

In my PhD work, we had shown that the general focus of speciation biology at the time was very much derived from Drosophila. It was very focused on hybrid sterility and hybrid inviability, hybrid breakdown, genetic incompatibilities, which is what everyone was studying, and everyone sort of talked about, as if that was the be all and end all of speciation. And then these taxa we studied in Ecuador actually had no post-mating isolation; just very strong assortative mating and probably selection – ecological selection – against hybrids because of their color patterns. So that sort of really shifted our thinking about speciation. It was like, hang on, actually, no, maybe it’s the behavior and the pre-mating isolation, which is really important in the early stages. So that was the kind of background through which I came to the post-doc.

HS: Did you do your PhD and post-doc from the same university?

CJ: Yeah, that’s right. I actually took a year off after my PhD, worked in a conservation project in Ecuador for about a year. And I was flying back, in New York on my way back to the UK, when I spoke on the phone to Jim Mallet, who’d been my PhD advisor, and he said, I’ve got this post-doc project, do you want to come back and work on this? I jumped at the opportunity because I was keen to get back into science at that point.

HS: Was this partly at the university and partly at STRI [Smithsonian Tropical Research Institute]?

CJ: That’s right. It was funded by a NERC grant to UCL [University College London] in London, but I spent most of my time in STRI.

HS: Stepping back a bit, how did you get interested in butterflies and speciation and decide to study these for your PhD?

CJ: I’d been to Borneo as an undergraduate and just fell in love with the diversity of insects and rainforests, and wanted to study the evolution of biodiversity, I guess. I liked butterflies, but I wasn’t particular a butterfly person at that stage. I just was fascinated by insects. And then I applied for various PhDs and this one with Jim Mallet looked very exciting. So I was very pleased to be offered that opportunity. It was just chance really, and now here I am still working on the same species.

HS: How did the authors of this paper come together and what did each contribute to this work?

CJ: So there was myself, obviously, and Jim Mallet. Jim Mallet was the PI and had the grant. So he was sort of the inspiration for the projects, I suppose. And then there was Russ Naisbit. He was a PhD student who worked with me on the project. And then Rebecca was an undergrad who came out to help us from UCL. Rebecca did a lot of the, actually, sitting in the cage, waving the models and doing the experiments. She actually collected a lot of data.

HS: Could you give us a sense of what the work involved you, both in the field and the experiments.What was your daily routine and who helped you do all the collections and the experiments in field?

CJ: Yeah, so it was mostly myself and Russ. We were based in Gamboa, where there’s now a very large kind of Heliconius research facility. But at the time, we were the first to establish Heliconius research in Gamboa. We built some outdoor cages where we used to do crosses between the species to study the genetic basis of their wing pattern differences and their hybrid strategy. We went out into the field on Pipeline road, which is a very famous bit of rain forest in the Soberania National Park, where we would collect the two species and also did some ecological studies. So we’d go out and collect butterflies from there, bring them into the insectary, and do experiments on all aspects of reproductive isolation. The key to this paper really was the insight that the wing patterns were not only selected for mimicry and for signaling to predators, but were also important in signaling to mates and reproductive isolation.That came about actually because we were out on Pipeline road and one of the old kind of tricks for catching Heliconius is to vigorously wave a red rag and they come down out of the forest canopy to investigate this moving red signal, probably because they feed on red flowers and they are obviously attracted to mates by their colors as well.The attraction to red was a well-known fact. And then one day when we were out, Jim didn’t have a red rag so he said, Well, cydno are black and white, maybe they’ll come to a white rag. So he got his white handkerchief out and waved that and the cydno kind of came sailing down to this white handkerchief. That really got us thinking that maybe the preferences of the two species would be for their different colors.  That was a moment of insight that maybe the different species could have different color preferences. And that made the link between the ecologically selected trait and pre-mating isolation, which was the key insight in this paper. So then we designed some experiments to try and test that by waving little colored models at the male butterflies. We had a sort of barbecue poker that we found in a garage, put some models on the end of a bit of wire, waved them in a cage full of males and watched the attraction.It was a very low tech experiment. And we showed that you could do that both with printed paper models and with real wings. Therefore, you could really, in that way, separate the color from other cues, by demonstrating that they showed a similar response to just a printed paper model.

HS: Who made the model?

CJ: I think I did.

HS: Did you just use paper painted on both sides?

CJ: The first ones we did were with the wings of the butterfly. So we would have butterflies that we’d raised in the lab and we’d remove the wings and just use a piece of tape and attach them actually a little bit further apart than they really were, so that we would have a bit of flexibility, and they would flutter as you wave to the poker. And then we attached those to a little straw, so you could poke the straw onto the piece of wire and swap the patterns over quite readily. That worked pretty well. And then we went on to make models. Inkjet printers were becoming fairly common – color inkjet printers – so we just printed them out. Nowadays, we actually look at the reflectance and try to match colors to the actual butterflies, but in those days we just took a photo and tried and made it look about right to the human vision.We didn’t have the methods to kind of use bird vision models to match the models.

HS: But in the paper you mention that you match reflectance…

CJ: Yeah, it was a post-hoc thing. So we went back and measured the reflectance of the models and of the real wings and showed that they were broadly similar. I mean, nowadays, we actually alter the printing to try and get them as close as we can. But in that case, it was just a matter of printing something out that looked about right with the spectrometer. But it was adequate. We weren’t missing some sort of UV signal or something that was in one and not the other.

HS: Do you still use the same kind of methods for experiments today?

CJ: We did a lot of experiments over the following years where we used very similar methods. We’ve sort of moved on a bit from that now. We’re more interested now in female choice. I think we’ve established pretty clearly that males have strong preferences. But, at the time, we hadn’t looked too much at the females; they’re harder to work with. So we’re trying to focus on female preferences now, which are more driven by chemical signals. The research has moved on a little bit.

HS: Do you still have models that you used in these experiments?

CJ: I have a house in Panama. The barbecue poker with the wire attached on it and some of the models are still in a box in the house.

HS: Were the illustrations of the butterflies in the paper made digitally?

CJ: Yeah, they were digital photos of pinned specimens. You can actually see the labels – I’m looking at it now – you can see the paper labels, which I obviously hadn’t removed.

HS: You spoke about this method of using a red rag to attract the butterflies.Was this something that had been was known for a long time and do you know who discovered that this works?

CJ: I don’t know. As far as I know, it’s sort of long-term knowledge. Jim Mallet worked with Larry Gilbert as his PhD advisor, and there was a big group of Heliconius biologists working in Costa Rica in the 1970s and 80s. I guess it was part of the common knowledge among that group. Someone else might be able to tell you who invented it.  I think it’s a long-standing method.

HS: In the Methods, you mention another site in French Guiana.Was that also a site that you visited and collected butterflies?

CJ: That’s right. So we were interested in populations of melpomene that were not sympatric with cydno. Some early mitochondrial sequencing had shown that, actually, this French Guiana population was potentially ancestral to the speciation of melpomene and cydno. So we were sort of interested in that as a possible out-group to the speciation event. That actually turns out not to be true. So when you look at the whole genomes, melpomene is a coherent species. But at the time we were interested in that population because it looked to make the relationships of melpomene and cydno paraphyletic. But actually, it was also a very useful kind of allopatric control. So we had a population of melpomene that didn’t have a history of sympatry with cydno. And there was some evidence in the paper for reinforcement. There was much stronger reproductive isolation between the populations from Panama than there was between cydno and melpomene from French Guiana. That was not very strong evidence, not very convincing, because it’s only one comparison, but certainly consistent with the role for reinforcement in driving the speciation event.

HS: This is just a minor detail. In Figure 1, you cite a personal communication from V. Bull and M.Beltrán for some unpublished sequences from nuclear loci. Could you tell us a little more about who these people were?  

CJ: Well, actually, M. Beltrán is my ex-wife. She went on to do a PhD with Jim. Vanessa Bull was also a PhD student with Jim. They were both working in the lab in Panama doing sequencing of these species, and they had a series of papers, published in the following years where they showed evidence for gene flow between melpomene and cydno.

HS: One final question about the methods. Can you tell us a little more about the insectaries you built for this study?

CJ: They were mostly built with aluminium poles and with the kind of mosquito mesh that people put on their houses in the tropics. The design came from Mauricio Linares, who was our collaborator. A bit of history there – we originally planned to do the project in Colombia. We went to Colombia, worked with Mauricio for five months or so, and then, just because of the political situation and the insecurity we moved to Panama.

HS: So the insectary design was borrowed from him?

CJ: Yeah, actually we bought all these materials in Colombia, and packed and took them on the plane to Panama. So we arrived in Panama, with butterflies, and all of these aluminium poles; maybe we didn’t bring the mesh. But we definitely brought these bundles of aluminium poles with which we constructed the first insectaries.

HS: Do you continue to work in the sites in Panama and French Guiana?

CJ: We’ve been back to French Guiana a few times. We’ve had some recent papers where we’ve again used French Guiana as a sort of an out-group to look at gene flow between melpomene and cydno in a whole genome context. So we’ve carried on with that design. And we almost always have someone from the lab working in Panama. We’ve carried on working in Panama. And I have had a very productive collaborations with Colombia as well, which is still ongoing, including a couple of PhD students that have worked in my labs.

HS: When was the last time you visited Panama?

CJ: Well, I was in Colombia in January this year; Panama, not for a year or so.

HS: Would you say these sites have changed a lot from the time you worked there for this study?

CJ: That’s an interesting question. In Panama, the forest hasn’t changed much, but Panama as a country has changed enormously. There’s been a huge amount of economic development and Panama City has transformed beyond recognition, in terms of high rise buildings and construction. The area of Gamboa has changed in some ways, culturally. I was there over the period when it was still part of the Canal Zone run by the Americans. And then it became part of Panama. So that was a big change. But if you stood there and looked around, there’s not a huge amount of change. The houses look pretty much the same and the forest looks pretty much intact.

HS: What about the facilities in the field stations? Has that changed a lot?

CJ: Yeah, that’s changed enormously, actually. When I was first there, Gamboa was a bit of a forgotten corner of the Smithsonian Institute. We just had little cages in the garden of one of their buildings. Now there’s a huge brand new lab. There are enormous sort of aluminium insectaries. It’s all very, very dynamic with a lot of people working there. So it’s really changed; the research has changed out of recognition, really.

HS: Could we go over the list of people you acknowledge to get a sense of how you knew them and how they helped?

CJ: Sure. Yeah. If I can remember them all.

HS: The first name is C. Paresce.

CJ: Yeah, so she was Camille. She was an undergraduate from UCL who also came out and helped us for a while. Okay, then the next one is Catalina Estrada, who was a fantastic Master’s student from Colombia who worked with me for a long time in Panama and then went on to do a PhD in Texas, and actually now is living in London. Richard Woods was an undergrad. from UCL, who came out with us to Colombia at the beginning. Margarita Beltrán is my ex-wife. And then Leanne O’Donovan. She came out with Rebecca Coe, and they both did some of these experiments, although Leanne went home a lot earlier. I think Rebecca just contributed a lot more to the data collection, so she ended up as an author.

Bernard Hermier was a French amateur collector who lived in Cayenne, in French Guiana. He was a local collector who knew the localities where to go and find the butterflies, and we had a previous paper about where melpomene could be found in French Guiana. The first time, we drove all over the place in Cayenne, and then we went and met him for dinner at a rather fancy French restaurant in Cayenne. Over dinner, on a napkin he sort of sketched out agood bit of local knowledge, the locations where we should park and where we should go, for example just behind this building there’s a flower and then just behind that you’ll find melpomene. And then sure enough, we went out the next day and there they were. F. Jiggins is my brother. I think we had some useful discussions as he is also an evolutionary biologist. Igor Emelianov was a postdoc at UCL and Mathieu Joron is another Heliconius biologist, now working in Montpellier.

Then there’s Eldredge Bermingham. He was my contact. He was later the director of STRI in Panama. He ran the lab, the molecular ecology lab, where we’ve worked. And he was my sort of host in the Smithsonian. Greg Hurst is a professor in Liverpool now who works on male-killing bacteria and incompatibilities. I called him up a few times from Panama to discuss the inheritance of sterility and incompatibility and possible genetic causes of that; so we had some interesting discussions. And then S Upson. I can’t remember his first name. He helped with reflectance measurements.

HS: What is Perkin Elmer?

CJ: That’s an equipment company. They made this reflectance spectrometer we used to compare the colors of the models and the wings.

HS: Do you remember how long, approximately, it took to write the paper, and when and where you did most of the writing?

CJ: I can’t really remember how long, but I know that I was in Panama and Jim was in London. And we obviously had email contact. And I remember we were really going just very intensively to and fro, sometimes several times a day, with discussions of particular paragraphs. Even though we were on different continents, we worked very closely and quite intensively on the writing. I think it was an experience that taught me a great deal about the importance of writing clearly, and sort of the intensiveness of the process of putting together one of these short papers that conveys things clearly. So yeah, I do, quite vividly, remember sitting in my office on the second floor of my house in Gamboa, probably, you know, in rather hot conditions, emailing back and forth with Jim about the text.

HS: Did the other two authors get involved in the writing?

CJ: Russ was there with me in Panama so he helped.He looked at drafts and worked on things. Rebecca, not so much. She was an undergrad. I think she’d gone back to UK at that point. She probably looked at a couple of versions, but she wasn’t particularly involved in writing.But she went on to do a PhD at the University of Edinburgh.

HS: Did this paper have a relatively smooth right to peer review? Was Nature the first place you submitted to?

CJ: Nature was the first place. It’s a little bit far back now to remember, but I think we had a slightly mixed response initially, you know, some positive reviews and some slightly less so and so.We went through a couple of rounds of revision,as far as I recall. I think we were perhaps initially rejected and Jim kind of argued that there was enough positive arguments, as we had a couple of really good reviews; positive reviews.

HS: At the time when the paper was published, do you remember how it was received?  Did it attract a lot of attention?

CJ: I don’t think so.I was sort of less aware really, then. I don’t think we had much press attention. I was out in Panama as well. So, probably, if there was, I wasn’t all that involved. But I don’t remember there being much. I mean, I was excited of course.

HS: This paper has been cited over 450 times. Did you anticipate that it would have this kind of an impact? And do you have a sense of what it mostly gets cited for?

CJ: Yeah, sure. As I alluded to earlier, since sort of the late 90s, when I first got interested in speciation research, it was dominated by people studying Drosophila and studying hybrid sterility and incompatibility. The big shift has been this sort of emphasis on ecology, and on these sorts of mechanisms that link or that allow species to persist and diverge in the face of ongoing gene flow. That’s really the challenge, that species diverge despite the fact that they’re sort of mixing through hybridization.So one of the mechanisms that facilitates that process is coupling, where the same trait affects both reproductive isolation and ecological adaptation; or, effectively, the same trait affecting multiple aspects of species divergence. And that sort of helps to allow these species to persist in the face of gene flow and hybridization. So I think that’s why it’s been cited, because it’s actually a really nice and intuitive and visually appealing example of that phenomenon. And it’s been cited because the whole field has very much shifted towards recognizing this as an important process in speciation. Did I foresee that? No, I don’t think so. I think we thought we were onto something. I think we felt a bit like we were in a minority at the time actually, that wasn’t really the main emphasis of speciation. Although, to be fair, by the time this paper came out, we certainly weren’t the only people saying this. But when my PhD papers were published, then there was much less emphasis on this kind of behavioral and ecological aspects of speciation. So, yeah, it was sort of timely, really, in the sense that it coincided with a point where the field really changed in emphasis.

HS: Did this paper have a big impact on your career?

CJ: Absolutely. I mean, obviously, you can’t say for sure what was important, but soon after this I was awarded a Royal Society University Research Fellowship.That was amazing, very prestigious, and turned out to be eight years of research fellowship funding, which I took to Edinburgh. That defined my career and allowed me the freedom to move in different directions. No question, I think that this paper helped. Whether I’d have got it without this paper, I don’t know that. It came at a critical moment in my career.

HS: What impact did this paper have on your research trajectory, both in the short-term and in the long-term? Did you follow up on this work immediately? Has this been a major area of research since then?

CJ: Yeah, it really has. I have an ERC [European Research Council] grant at the moment, which is working on speciation in melpomene and cydno; exactly the same species. So we’re doing a lot more genomics and a lot more of the genetic side of things than we were then. But it’s really based on the story of what we demonstrated in this paper. So I think it has provided the foundation for my career. What I like doing is kind of looking at one system from multiple angles. And so we are working on chemical ecology and genomics and genome structure and all sorts of different aspects of speciation, but focused on this species group. So this was background, this was important work that built the foundations for what I’m still doing today. So it has been very influential.

HS: It’s been 16 years since this paper was published. Would you say that the main conclusion of this paper still holds true more-or-less? I’d like to read a couple of lines from the paper: You say “In conclusion, pre- and post-mating isolation between H. melpomene and H. cydno has resulted from an adaptive shift in ecology and mimicry, in association with partial hybrid sterility. Subsequently, assortative mating between sympatric populations has become enhanced, possibly owing to reinforcement”. If you were to rewrite those lines today, would you write them the same way?

CJ: I don’t think I’d change anything. We understand more about things now, but I think that still stands.

HS: If you were to redo this study today, what would you do differently?

CJ: I do have one criticism of the methods, actually, which was that we had this design where we put all these males in a cage, and then just kind of waved models at them. That’s sort of a pseudo-replication inherent in that way. You’re not really separating the responses of individual males. We did it properly in subsequent papers and the result holds up. It’s not wrong, but we should have measured the behavior of individual males. And that’s what we’ve done since and the result still holds up. But that’s one specific kind of methodological critique I would make of what we did in those days.

HS: What abou tin terms of the methods – would you take advantage of the technology available today – video recording, new genetic tools etc.?

CJ: What we’re doing now is actually trying to look at the genetic basis of these behaviors. Richard Merrill, who’s just gone on to an independent fellowship, but has been in my lab for a long time, has been doing these kinds of experiments, using genetic markers, genomic markers, to map the quantitative trait loci that control these behaviors. I guess that’s how things have moved on. But the basic behavioral methodology I wouldn’t change. I mean, obviously, there is a potential now for automating the recording of behavioral data. We haven’t really got that working as yet. But that’s something Richard wants to start doing. So that would obviously be an exciting possibility. In terms of the genetics, that’s absolutely, completely transformed since this time. We used to talk about mitochondrial sequences and now we’re doing whole genome sequencing for large numbers of butterflies. That’s changed completely.

HS: I want to read some lines from the paper where you talk about lacunae in understanding at that point and what could be done in the future. You say: H. melpomene males sympatric with H. cydno discriminated more strongly than H. melpomene allopatric to H. cydno. This pattern is expected if mate preference has been `reinforced’ to prevent the production of unfit hybrid offspring in sympatry, although the evidence would be strengthened if replicated with other allopatric and sympatric populations”. Has this happened, either through your work or through the work of others?

CJ: Well, not exactly in these species, but Marcus Kronfrost has done some work on cydno and melpomene and pachinus in Costa Rica now and showed populations within pachinus, which is another species in the group, show a similar pattern, as do, I think, melpomene. So it has been replicated in other populations. I didn’t really follow up on that. And the reason was that I sort of felt that even if we could go and collect more populations, they wouldn’t really be truly independent, because they’re all the same species – genetically related. So it wouldn’t really be quite the replication of the result that actually we’d sort of implied by that sentence that you just read. So I didn’t follow up on it, but it has been shown in other populations by other researchers. There’s a fairly consistent pattern, but there is a just an inherent kind of problem of replication. I mean, it would be nice to look at many more other species pairs, but that would be a lot of work.

HS: And then in another place, you say “pleiotropy between mate choice and disruptive selection is an important feature of speciation theory, because it can trigger rapid speciation with a high probability, but only a few other examples are known”. And then, right at the end of the paper, you say, “this [meaning your own study] and other recent examples suggests that ecological adaptation can result in associative mating as a byproduct and maybe an important and largely overlooked cause of speciation”. In the 15 years since this study, have there been other examples, other studies, which have found these characteristics in other cases of speciation?

CJ: It’s actually quite nice to go back and read that, because yeah, absolutely. That has been the case. I think there are lots of examples now and it’s something that people are excited about looking for. There’s a paper on Darwin’s finches where they show that the size of the beak, which is obviously adapted for feeding on different seeds, is also affecting song, which is a pre-mating isolation. There are papers in sticklebacks showing body size is involved in ecological adaptation and in mate choice. There are experiments in Drosophila that are showing cuticular hydrocarbons involved in adaptation to different desiccation regimes, but they’re also involved in mating and reproductive isolation. So lots of and lots of examples now.

HS: In the 15 years since this paper was published, have you ever read the paper again?

CJ: As I just alluded to, not very recently. I’ve certainly read it again, but not particularly recently.

HS: In what context did you read it after it was published?

CJ: Well, I suppose for several years after it was published, we were going back and continuing to work on these problems. And so we were kind of developing the methods, and so I went back and looked at how did we do it before, and how could we improve on that.But I mean the story is quite simple, isn’t it? So it’s not like you need to go back and look at the overall story.

HS: Would you count this as one of your favorites among the papers you have published?

CJ: Yeah, I think so. You asked me about how it was influential in my career. So I think it does stand out as one that, particularly in terms of my ability to secure funding and fellowships and things, was particularly influential.And I was very proud of it at the time and still am proud of it.

HS: What would you say to a student who is about to read this paper today? Would you guide his or her reading in some way? Would you point them to other papers they should read along with this? Would you add any caveats?

CJ: What would I say? I’m not sure I’d want to point them in any particular direction reading this paper. I think most of what we said still stands. There’s a nice review by Maria Servedio on magic traits. She discusses this as perhaps one of the best examples of a magic trait where we understand the role of the trait in post- and pre-mating isolation. And this paper, in combination with one much more recently by Richard Merrill in Proceedings of the Royal Society, where he did selection experiments in the wild with model butterflies and showed selection against the F1 hybrids – that showed that this same color pattern trait was also under post-mating selection – these two studies two combined make an excellent example of what’s now called a magic trait, though I’m not particularly keen on that term, but it’s kind of stuck in the literature. So I guess with that review by Maria, it’s quite a nice story.

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