Argynnis aglaja, the Dark Green Fritillary, at Burcombe, Wilts., on 30 June 2015
In the course of trying to decide whether Henry Walter Bates was confused by mimicry in Heliconius melpomene vs. erato (and he clearly was, at least by the pictures referring to the new names in Cramer’s Pap. Exot.), I have found a puzzle in the types of melpomene herself. Linnaeus described Papilio melpomene.
At least one of the designated types, the male, is clearly an erato (enlarged image, bottom, cited as paralectotype in Butterflies of America (refers to http://linnean-online.org/14407/)), and even the top one (enlarged image), a female (cited as lectotype in BoA (http://linnean-online.org/14406/)) is somewhat dubious. After mature consideration. and a few beers, I believe that the lectotype is indeed a melpomene , but it’s a small specimen that is a bloody good mimic of erato, including reducing some of the whitish scales on the underside so that the red shows through (a VERY erato trait — and females are always best at everything, including mimicry, don’t you find?). But the paralectotype is clearly an erato.
These specimens are now around 240 years old, and were purchased from Linnaeus’ widow in 1783 by Sir James Edward Smith, who founded, and served as first President of, the Linnean Society of London.
Today, we know (more or less) what we mean by Heliconius melpomene (except that we’ve discovered cryptic taxa belonging to H. timareta within it, and except that we’ve discovered that Guiana and Panama melpomene are partially intersterile, but, really, we know this species almost as well as anyone knows any species on the planet). So I suggest that whatever the putative type is (and it has to be putative, because it is not entirely clear which specimen is what in Linnaus’ writing), we should move as a group to support the current usage.
Heliconius erato has had its own spotty history. The Linnean type is clearly a doris! See: http://www.butterfliesofamerica.com/L/heliconius_e_erato_historical.htm
But the H. erato we know has received ICZN name conservation approval after a petition by John R.G. Turner.
Comments welcome! I suggest you vote for top and bottom specimens (of the image above) being erato or melpomene; I’d be interested in what you all think.
This is a time lapse video of coordinated feeding in Heliconius doris caterpillars, eating a leaf of Passiflora ambigua
Filmed in Gamboa with my iPhone
This post is written in response to a journal club blog post by the Ross-Ibarra lab on our recent paper in Genome Research, where we used RAD data to study parallel hybrid zones in Heliconius erato and H. melpomene. First, we’d like to say many thanks for your comments and discussion! Here are responses to a few of the main points:
We compared two methods of analysing the data in our paper – first de novo assembly of the sequences, and second alignment to the reference genome. In response to your criticism that this comparison is a ‘little unfair’ – well we largely agree with this. We started out by using two ‘off-the-shelf’ solutions to this problem, first the program Stacks for de novo assembly, and second a well established pipeline that we use in the lab for analysis of resequence data based on the read-mapper Stampy and base-caller GATK. What we found after this first round of analysis was that, much to our surprise, the read mapping approach worked really well even when the reference genome was quite distant from the study species (i.e. when mapping H. erato reads to the H. melpomene genome). We therefore pursued this approach for the rest of the paper and did not invest more time in the Stacks-based approach. As highlighted in the post, this isn’t a very fair comparison as there is currently no way of adding the paired end data and calling SNPs in Stacks – so basically Stacks is throwing away at least half the data. It should be possible to get around this, but it would require quite a bit of compute time and script-writing (we previously did something similar in an analysis of data for the diamondback moth – Baxter et al., PLoS One 6, e19315). We decided not to do this because the read-mapping approach worked pretty well for both species. However, we did decide to report both analyses throughout the paper, as the results reflect what can be achieved with relatively straightforward application of existing programs. However we fully acknowledge that the de novo assembly approach could be taken much further and recommend that readers consider both approaches in analysis of similar data sets.
An interesting question is whether these populations are differentiated at loci other than those involved in colour pattern. We suggested that some differentiated SNPs that were not associated with wing pattern traits might represent such ecological differentiation, perhaps through adaptation to altitude. There is a major problem here in that colour pattern and ecology are strongly correlated, with one wing pattern form typically found at higher altitudes in each zone. However, we did have some power to separate such effects as the correlation between colour pattern and altitude in the samples wasn’t perfect (some of the phenotypically “pure” individuals were sampled from closer to the centre of the hybrid zone) – so we did try to test for associations with altitude. We didn’t find evidence for genomic regions uniquely associated with altitude but there were some loci that did show high Fst but not colour pattern associations and that might be due to ecological differences. However, as you correctly pointed out, this could also be due to small sample sizes or colour pattern phenotypes we didn’t measure. Future work will need much larger sample sizes, but in general we were surprised and pleased by how much information could be extracted from these relatively small sample sizes (especially after some of the individuals dropped out due to hidden cryptic species!).
In response to your comment about divergence times, we would completely agree that our data are not well designed to estimate relative divergence times for the two species (mainly because we carried out separate phylogenetic analyses in the two species). Our discussion of co-divergence was mainly focussed on the order of divergence of the co-mimetic subspecies in the phylogeographic tree, which did strike us as rather surprisingly similar in the two species. Not entirely sure how to interpret that though, and as you state this may not tell us much about the history of Mullerian mimicry.
On a similar note, it is indeed surprising how genetically similar these races are to one another, despite their huge differences in appearance. If, populations are compared from further afield, such as Brazil, there is more genetic differentiation across larger distances, but we think this is due to isolation-by-distance rather than being associated with wing patterning. John Turner suggested in 1979 that there were ‘Contrasted modes of evolution in the same genome’ (Turner PNAS 76:1924-1928; it’s a great paper, worth reading!). Everything we have discovered recently supports this assertion – the wing pattern races differ at little apart from a few major wing patterning loci, which change abruptly across narrow hybrid zones. These therefore show very different patterns of evolution as compared to the rest of the genome, which shows a more typical geographic structuring.
Thanks for your observation about the divergent region on Chromosome 13 of Ecuadorian H. melpomene, you may be correct that this could be due to the homologue of Ro in H. melpomene. However, we don’t see any divergence outliers in H. melpomene on the scaffold that has the strongest Ro associations in H. erato, so the idea that we have found the Ro homologue in H. melpomene remains quite tentative at this stage. Again, you are right to highlight the small sample sizes here which will have reduced our power to detect associations in this population.
In summary, the major findings of our paper were that 1) mapping of short-read RAD data to a reference genome even ~15% divergent worked remarkably well 2) RAD data provide a great deal of power to detect large-effect loci in hybrid zones, which is extremely promising for other species where there isn’t a long history of traditional mapping studies as in Heliconius and 3) we found a new cryptic species in our samples! However our sample sizes were not large enough to identify small-effect loci reliably, or to reliably separate highly correlated factors such as wing pattern and altitude, and these interesting questions will only be resolved with larger studies.
Chris and Nicola
How do evolutionary biologists look into the past? How can we know when and where an organism has lived, if it left no fossils (like most butterflies)? How can we tell if genes were exchanged between species?
All these questions are studied by a field of biology called phylogenetics, from the Greek phyle=kind and genesis=origin.
Reconstructing the tree of life
Our understanding of the evolution of butterflies and other forms of life rests on the knowledge of how species are related to each other. These relations are often depicted as a tree, where the tips of branches are species, and connections between them represent ancient ancestors. These relations can be inferred from DNA sequences. Some mutations in DNA are not purged and accumulate with time at steady rate. If we compare the same gene from two butterfly species, we are likely to find some differences: the more variation, the more time must have passed since the two organisms had a common ancestor. For instance, the genomic sequences from humans and chimpanzees differ a lot less than those from humans and macaques, illustrating our closer evolutionary affinity to chimps.
Figure 1. An alignment of the 16S gene from four species of butterflies. The three South American Heliconius are more similar to each other than they are to the African Acraea, reflecting evolutionary history.
Figure 2. The phylogenetic tree of Heliconius and relatives. Using the molecular clock helps us to figure out how old species are, even without fossils. The blue bars indicate statistical error of these estimates.
The molecular clock
Intriguingly, mutations arise at an approximately constant rate. This phenomenon, known as the molecular clock, helps us to infer how long ago two species had a common ancestor and thus how old they are – very useful for invertebrate animals that rarely fossilise! The gene 16S in the example above is found in all insects and accumulates changes at the rate of about 1 DNA base (1 position in sequence) out of a hundred every million years. Finding 20 differences per 100 bases between Heliconius melpomene and Heliconius erato suggests that the two species diverged approximately 10 Million years ago: 10 independent mutations on each branch, one mutation per Million years. Of course, the clock is not perfect and the rate varies between lineages of life – advanced statistical techniques and sequencing large amounts of DNA are needed to get precise estimates.
Figure 3. The molecular clock – the time that passed between past events (like speciation) and today corresponds to the amount of changes accumulated in DNA (red ticks). We find that H. melpomene and H. cydno are more closely related to each other than to H. erato, and that they split about 3 million years ago .
Genes and hybridisation
Heliconius butterflies of different species occasionally exchange adaptive genes, just like modern humans and Neanderthals did. But how can we actually know this without travelling back in time?
If two species do not mix and we compare their genome sequences like in Figure 1, every part of their genome should have the same history. But if some genes were transferred between species, we can expect an unusual pattern of DNA sequences. For example, the warning pattern gene in the Heliconius elevatus genome is unexpectedly closely related to the gene from H. melpomene, suggesting that the sequences were transmitted from H. melpomene to H. elevatus. Such transfer is possible through rare hybridisation, and it is enhanced if the shared genes confer an adaptive advantage of anti-predator warning.
Other applications of phylogenetics
The only illustration in Charles Darwin’s “Origin of Species” is in fact a tree of relations between hypothetical organisms. The modern ability to figure out the relations between DNA sequences is immensely useful in other areas of research. For example, we can use phylogenetic trees to establish where the pandemics of flu and other diseases originate, to show that pathogenic strains of bacteria share the genes for antibiotic resistance, or – in a very famous criminal case – when and where someone got infected with HIV! The same reasoning has also helped us understand that all human populations originated in Africa and how we spread across the globe. If only Darwin new how big the trees will grow!
Krzysztof M. Kozak was raised in Poland and studied in the USA. A bird-watchig trip to Costa Rica and a failed organic synthesis exam convinced him to become an evolutionary biologist rather than a biochemist. Seven years and several trips to the tropics later, he is a happy graduate student at Cambridge, always looking for more ways to combine fieldwork, genetics and computation to understand the tremendous diversity of life. #KrzysztofMKozak
One of the things we have been asked several times at the Royal Society Summer Science Exhibit is whether butterflies always have symmetrical wing patterns. This is almost always the case, because the wing patterns are hard-wired in the genome, and all cells on both wings have the same genetic code. However there are rare exceptions, where a single egg gets fertilised by two sperm, or a single individual developed from two genetically distinct cell lineages. These rare specimens are known as gynandromorphs, and can tell us something about wing development. Here, Martin Thompson describes a few specimens of gynandromorphs in the genus Papilio.
This is a sex mosaic (‘mosaic gynandromorph’) Papilio dardanus. The left wing is mostly the pattern of the female form cenea, but the right wing shows lots of yellow patches of male pigment. There is even a segment at the bottom corner with male patterning and even a little bit of a tail.
It is thought that this kind of pattern arises from the loss of one of the sex chromosomes from one of the two daughters of a cell division, or possibly when an the egg contains an incorrect number of sex chromosomes.
In butterflies and moths, the sex determination system is different to humans – males have two identical sex chromosomes (ZZ rather than XY) and it is the females with two different sex chromosomes (ZW contrasting with XX in humans). If this loss happens very early in development whilst the embryo is only a few cells, the result can be a striking bilateral gynandromorphy, with one half of the body male and one half female:
However, if the error occurs later in development, the result will be a mosaic as in the first picture. Isn’t biology cool?!
Mosaics such as these can teach us about how wings are formed and patterned when the butterfly is still a pupae. By looking at a large number of mosaic butterflies, scientists have found that there are several ‘compartments’ to the wing: that there are boundaries which cells never cross during development. These boundaries are usually invisible, but in mosaic gynandromorphy wings, each differently-coloured patch arises as a handful of cells. When we see lots of mosaic patches with similar, sharply-defined and straight boundaries, this tells us about the position of the wing compartments.
Notice that the region where black meets yellow on the forewings is a straight and clear line. This marks a compartment boundary within the wing.
Every living animal and plant starts its life as a single cell. The cell then divides many times, and the end result is a fully functioning organism, like a rose bush or a horsefly or you or Brian Blessed. A human is made of quadrillions of cells, but they all come from that one initial fertilized egg. All (or at least most) of the information that makes you you can be compacted town to one nucleus of one single cell, and then read out in such a way that when that one cell starts to divide quadrillions of times, the new cells congeal into an all-signing, all-dancing organism-y thing. I think this is pretty awesome, and thinking about these problems has led me to working on a PhD studying Heliconius butterflies. This post is a personal perspective on this problem and how I came to study butterflies.
Joe Hanly….or Henry Walter Bates?
The processes that make an animal from a single egg are controlled in such a way that they will always make a pretty accurate version of that animal. This process is really repeatable. Hold your hands together. They’re pretty much exactly the same size and shape, correct? In fact, I’d hazard a guess that your hands are more or less the same shape as the hands of all the other humans you know, too. All of the hands of all humans, including your own two hands, were put together completely independently of each other, yet in most cases they are pretty much perfectly formed (apologies to readers with polysyndactly. And to amputees).
How does that work? How does a hand come to be a hand, and what are the physical mechanisms that can build hands so perfectly and repeatably? How about feet, how do they happen – I guess in a similar way to hands, but with some differences? How does one make a face? I be that’s probably pretty different from the way you make a hand, right? Lungs? Wings? Shells? Roots? I’ve always wondered about how all this stuff gets put together from one egg. I always enjoyed learning about science at school, and studied Biology, Chemistry, Maths and History for my A-levels, and so when I was 17 and I actually had to start deciding what I wanted to do with my life, I decided to go and do a course in Developmental Biology at the University of Manchester.
While I was there, I actually learnt about how hands and faces and lungs and other bits develop, and why we think the process is so symmetrically perfect and repeatable. I’m not going to talk about that here (if you’re interested in these things, then I’ve put some useful links below), but while I was learning about this, I started realise there were even more questions I hadn’t really even considered before.
Firstly, I got really interested in gene regulation. For living things to function, they need to make proteins from genes. While some of these proteins need to be present in all places at all times (for example, the genes important for respiration) others need to be turned on or off in specific places and at specific times, for example, the proteins that make melanin pigments, or the proteins that form the structure of bone. If the proteins that make bones were present everywhere throughout your body, you’d be in trouble. It turns out that while we know quite a bit about the process of turning genes on and off, there is still a lot to be found.
I also got really interested in the way that gene regulation can relate to evolution. Over time, the physical form that a species develops will change in response to selection. Sometimes, these changes might involve the evolution of entirely new proteins or changes to existing proteins, but it turns out that the simplest and most common change that leads to differences in morphology actually affect the ways that genes are turned on and off.
When I finished my degree I decided that I wanted to continue studying science, so I applied for funding for a PhD from the Wellcome Trust, and came to study at the University of Cambridge. I’m working on Heliconius because it turns out to be a good system for investigating how differences in gene regulation can evolve, as we have one species with many different pattern forms, and we can use these patterns to gain an understanding of how the genetics which control phenotypic differences work. I’ll talk more about this in another post, though.
This year the annual Heliconius 2014 meeting will be hosted by the University of Puerto Rico, Rio Piedras campus from June 4th to June 6th. The meeting will be immediately prior to the SMBE meeting in San Juan, Puerto Rico (http://www.smbe.org/).
You can find a brief description of the Heliconius 2014 meeting at the following webpage: http://heliconius.wix.com/heliconiusmeeting
Please register using the form found on the website (http://heliconius.wix.com/heliconiusmeeting).
Breakfast and lunch will be provided for the three days of the meeting and special rate at the UPR housing ($50 per night) will be offered by request (see form on the webpage) to the participants. Possibility to extend the stay at the University housing will also be offered to the participants if requested (especially for the ones that will present at the SMBE meeting).
Gerardo Lamas has issued an online update to his bibliography of neotropical butterflies. Here’s the link:
According to this page, Carlos Pena is currently working on bullying the text file into a database format:
Best wishes to you all
Last week at JournalPub we covered John Turner’s 1979 paper on contrasted modes of evolution:
This was one of the first papers to show that evolution can happen at different rates in different parts of the genome. It seemed strange to us, spoiled as we are by whole genome sequences, that this was considered a significant finding as late as 1979, but it’s difficult to reconstruct the context. We’d be interested to hear from anyone who was around at the time.
Mayr, for example, had argued for ‘genetic revolutions’ following the isolation of a founder population:
Isolating a few individuals (the “founders”) from a variable population which is situated in the midst of the stream of genes which flows ceaselessly through every widespread species will produce a sudden change of the genetic environment of most loci. This change, in fact, is the most drastic genetic change (except for polyploidy and hybridization) which may occur in a natural population, since it may affect all loci at once. Indeed, it may have the character of a veritable “genetic revolution”. Furthermore, this “genetic revolution”, released by the isolation of the founder population, may well have the character of a chain reaction. Changes in any locus will in turn affect the selective values at many other loci, until finally the system has reached a new state of equilibrium.
Heliconius species are usually more like the variable populations Mayr describes than the founder populations. However, single species can have many different wing patterns. By 1979, it was well known that these wing patterns were controlled by a handful of genetic loci, through a long series of genetic crosses showing the perfect segregation of loci with different wing pattern elements: red rays, yellow bands and so on (summarised in Sheppard 1985). Species such as Heliconius erato have races spread across South America with very different patterns, and yet these races can interbreed.
How much of the genome was responsible for these wing pattern differences? The method of choice for answering this question in the 1970s was allozyme electrophoresis, as DNA sequencing was still in its infancy. Proteins with variations in amino acid composition carry different electrical charges. Varying proteins could therefore be separated by running them on a gel. By 1979, a large library of protein variations had been reported in a wide variety of species (documented in detail in Chapter 3 of Lewontin’s The Genetic Basis of Evolutionary Change, available as full free PDFs).
Turner et al were the first authors to test allozyme variation in Heliconius. They took a set of 17 enzymes and tested them for variations in eight species of Heliconius, including multiple races of many species. They showed that none of the enzymes segregated with colour pattern.
For example, wing patterns in Heliconius erato were known to be controlled by between one and seven genetic loci, but the sampled populations of erato were found to be between 93 and 99% identical based on the selected allozymes. This was very similar to the genetic identity seen in Heliconius sara, a species with very few races and only minor wing pattern differences.
The enzymes chosen were the ones that were available, with no intentional bias towards genes that may or may not have been involved in wing patterning. From our point of view, it was unsurprising that such enzymes would not necessarily be involved in wing patterning. But perhaps this was surprising in 1979; perhaps it was expected that loci unrelated to colour patterning would be ‘carried along’ with sharp variation in leading colour pattern loci, following lines of thought like Mayr’s. After all, it is not so different from the ‘islands of speciation’ arguments we are having today.
It may be that we are now used to the idea that only small regions of a few chromosomes are responsible for the variation in colour patterns, with the rest of the genome being mostly very similar and freely flowing between Heliconius races. But we are definitely still struggling with the idea that different parts of the genome can evolve at different rates, under different contraints. And with that, I have to get back to thinking about ABBA-BABA windows…