Papers

Reproductive isolation via a switch in Müllerian mimetic wing coloration has become the paradigm for speciation in aposematic Heliconius butterflies. Ecological speciation associated with a shift in mimicry is likely to have contributed to many speciation events in the clade. However, an increasing body of phylogenetic evidence suggests that reproductive isolation in Heliconius can also arise, or at least be maintained, without colour pattern differences. Here, we use molecular data to test for the presence of a cryptic species within Heliconius demeter by testing for clusters of multilocus genotypes and reciprocally monophyletic clades near Tarapoto, northern Peru. Using AFLPs, we identify two genotypic clusters corresponding most closely in morphology to subspecies H. demeter ucayalensis and H. demeter cf. demeter. These subspecies are also reciprocally monophyletic for the mitochondrial genes CoI and CoII and exhibit marked differences in adult and larval morphology, oviposition behaviour and host plant use. Nuclear markers are, however, characterised by abundant polyphyly, which we interpret as retained ancestral polymorphisms. CoI extracted from 12 of the 15 currently recognized subspecies suggest the observed differences are reflected across the range of H. demeter, with a deep phylogenetic split between the southern and northern Amazonian races. We raise H. demeter eratosignis from central Brazil to H. eratosignis based on nomenclatural priority, comprising H. demeter ucayalensis and two other southern Amazonian races. H. demeter cf. demeter along with northern Amazonian and Guianese subspecies remain within H. demeter sensu stricto. Unlike H. eratosignis, H. demeter is characterised by unusual sexual dimorphism and highly divergent host plant use. In light of this, we discuss possible drivers of their speciation. Our study documents yet another discovery of a cryptic, sympatric species in what previously seemed a very well-studied group.

Understanding why species richness peaks along the Andes is a fundamental question in the study of Neotropical biodiversity. Several biogeographic and diversification scenarios have been proposed in the literature, but there is confusion about the processes underlying each scenario, and assessing their relative contribution is not straightforward. Here, we propose to refine these scenarios into a framework which evaluates four evolutionary mechanisms: higher speciation rate in the Andes, lower extinction rates in the Andes, older colonization times and higher colonization rates of the Andes from adjacent areas. We apply this framework to a species-rich subtribe of Neotropical butterflies whose diversity peaks in the Andes, the Godyridina (Nymphalidae: Ithomiini). We generated a time-calibrated phylogeny of the Godyridina and fitted time-dependent diversification models. Using trait-dependent diversification models and ancestral state reconstruction methods we then compared different biogeographic scenarios. We found strong evidence that the rates of colonization into the Andes were higher than the other way round. Those colonizations and the subsequent local diversification at equal rates in the Andes and in non-Andean regions mechanically increased the species richness of Andean regions compared to that of non-Andean regions (‘species-attractor’ hypothesis). We also found support for increasing speciation rates associated with Andean lineages. Our work highlights the importance of the Andean slopes in repeatedly attracting non-Andean lineages, most likely as a result of the diversity of habitats and/or host plants. Applying this analytical framework to other clades will bring important insights into the evolutionary mechanisms underlying the most species-rich biodiversity hotspot on the planet.

The major revolution in Charles Darwin’s ‘On the Origin of Species’ was the proposal that evolutionary change took place by natural selection. The ‘Origin’ was highly influential primarily because of its convincing, logical arguments, but in 1859 Darwin was unable to provide a single empirical case of natural selection. By the late 19th century, two key examples of natural selection became known: mimicry in heliconian butterflies and rapid increases in melanic forms of the peppered moth (Biston betularia) as well as of many other moth species in industrial Britain [1, 2]. Only now, however, are we beginning to catch a glimpse of the genetics underlying these adaptive changes. Remarkably, two independent and different-looking colour pattern switches in Lepidoptera — one in wing colour patterning and one that melanizes all scales over the wings and body — have been mapped to exactly the same gene in Heliconius and Biston [3, 4].

Concepts and definitions of species have been debated by generations of biologists and remain controversial. Microbes pose a particular challenge because of their genetic diversity, asexual reproduction, and often promiscuous horizontal gene transfer (HGT). However, microbes also present an opportunity to study and understand speciation because of their rapid evolution, both in nature and in the lab, and small, easily sequenced genomes. Here, we review how microbial population genomics has enabled us to catch speciation ?in the act? and how the results have challenged and enriched our concepts of species, with implications for all domains of life. We describe how recombination (including HGT and introgression) has shaped the genomes of nascent microbial, animal, and plant species and argue for a prominent role of natural selection in initiating and maintaining speciation. We ask how universal is the process of speciation across the tree of life, and what lessons can be drawn from microbes? Comparative genomics showing the extent of HGT in natural populations certainly jeopardizes the relevance of vertical descent (i.e., the species tree) in speciation. Nevertheless, we conclude that species do indeed exist as clusters of genetic and ecological similarity and that speciation is driven primarily by natural selection, regardless of the balance between horizontal and vertical descent.

The Heliconius butterflies are a widely studied adaptive radiation of 46 species spread across Central and South America, several of which are known to hybridize in the wild. Here, we present a substantially improved assembly of the Heliconius melpomene genome, developed using novel methods that should be applicable to improving other genome assemblies produced using short read sequencing. First, we whole-genome-sequenced a pedigree to produce a linkage map incorporating 99% of the genome. Second, we incorporated haplotype scaffolds extensively to produce a more complete haploid version of the draft genome. Third, we incorporated ∼20x coverage of Pacific Biosciences sequencing, and scaffolded the haploid genome using an assembly of this long-read sequence. These improvements result in a genome of 795 scaffolds, 275 Mb in length, with an N50 length of 2.1 Mb, an N50 number of 34, and with 99% of the genome placed, and 84% anchored on chromosomes. We use the new genome assembly to confirm that the Heliconius genome underwent 10 chromosome fusions since the split with its sister genusEueides, over a period of about 6 million yr.

BACKGROUND: 

An important goal in evolutionary biology is to understand the genetic changes underlying novel morphological structures. We investigated the origins of a complex wing pattern found among Amazonian Heliconius butterflies. Genome sequence data from 142 individuals across 17 species identified narrow regions associated with two distinct red colour pattern elements, dennis and ray. We hypothesise that these modules in non-coding sequence represent distinct cis-regulatory loci that control expression of the transcription factor optix, which in turn controls red pattern variation across Heliconius. Phylogenetic analysis of the two elements demonstrated that they have distinct evolutionary histories and that novel adaptive morphological variation was created by shuffling these cis-regulatory modules through recombination between divergent lineages. In addition, recombination of modules into different combinations within species further contributes to diversity. Analysis of the timing of diversification in these two regions supports the hypothesis of introgression moving regulatory modules between species, rather than shared ancestral variation. The dennis phenotype introgressed into Heliconius melpomene at about the same time that ray originated in this group, while ray introgressed back into H. elevatus much more recently. We show that shuffling of existing enhancer elements both within and between species provides a mechanism for rapid diversification and generation of novel morphological combinations during adaptive radiation.

Despite the greatest butterfly diversity on Earth occurring in the Neotropical Andes and Amazonia, there is still keen debate about the origins of this exceptional biota. A densely sampled calibrated phylogeny for a widespread butterfly subtribe, Oleriina (Ithomiini: Nymphalidae) was used to estimate the origin, colonization history and diversification of this species-rich group.

We estimated the spontaneous mutation rate in Heliconius melpomene by genome sequencing of a pair of parents and 30 of their offspring, based on the ratio of number of de novo heterozygotes to the number of callable site-individuals. We detected nine new mutations, each one affecting a single site in a single offspring. This yields an estimated mutation rate of 2.9 x 10(-9) (95% confidence interval, 1.3 x 10(-9)-5.5 x 10(-9)), which is similar to recent estimates in Drosophila melanogaster, the only other insect species in which the mutation rate has been directly estimated. We infer that recent effective population size of H. melpomene is about 2 million, a substantially lower value than its census size, suggesting a role for natural selection reducing diversity. We estimate that H. melpomene diverged from its Mullerian comimic H. erato about 6 Ma, a somewhat later date than estimates based on a local molecular clock.

Mullerian mimicry among Neotropical Heliconiini butterflies is an excellent example of natural selection, associated with the diversification of a large continental-scale radiation. Some of the processes driving the evolution of mimicry rings are likely to generate incongruent phylogenetic signals across the assemblage, and thus pose a challenge for systematics. We use a data set of 22 mitochondrial and nuclear markers from 92% of species in the tribe, obtained by Sanger sequencing and de novo assembly of short read data, to re-examine the phylogeny of Heliconiini with both supermatrix and multispecies coalescent approaches, characterize the patterns of conflicting signal, and compare the performance of various methodological approaches to reflect the heterogeneity across the data. Despite the large extent of reticulate signal and strong conflict between markers, nearly identical topologies are consistently recovered by most of the analyses, although the supermatrix approach failed to reflect the underlying variation in the history of individual loci. However, the supermatrix represents a useful approximation where multiple rare species represented by short sequences can be incorporated easily. The first comprehensive, time-calibrated phylogeny of this group is used to test the hypotheses of a diversification rate increase driven by the dramatic environmental changes in the Neotropics over the past 23 myr, or changes caused by diversity-dependent effects on the rate of diversification. We find that the rate of diversification has increased on the branch leading to the presently most species-rich genus Heliconius, but the change occurred gradually and cannot be unequivocally attributed to a specific environmental driver. Our study provides comprehensive comparison of philosophically distinct species tree reconstruction methods and provides insights into the diversification of an important insect radiation in the most biodiverse region of the planet.

For over 100 years, it has been known that polymorphic mimicry is often switched by simple mendelian factors, yet the physical nature of these loci had escaped characterization. Now, the genome sequences of two swallowtail butterfly (Papilio) species have enabled the precise identification of a locus underlying mimicry, adding to unprecedented recent discoveries in mimicry genetics.

Ranitomeya poison frogs in the Peruvian Amazon are a rare example of Mullerian mimicry in vertebrates. These frogs also prefer to court same-coloured mimics. This suggests that divergence in mimicry plays a role in reproductive isolation.

Multilocus clines between Mullerian mimetic races of Heliconius butterflies provide a classic example of the maintenance of hybrid zones and their importance in speciation. Concordant hybrid zones in the mimics Heliconius erato and H. melpomene in northern Peru were carefully documented in the 1980s, and this prior work now permits a historical analysis of the movement or stasis of the zones. Previous work predicted that these zones might be moving toward the Andes due to selective asymmetry. Extensive deforestation and climate change might also be expected to affect the positions and widths of the hybrid zones. We show that the positions and shapes of these hybrid zones have instead remained remarkably stable between 1985 and 2012. The stability of this interaction strongly implicates continued selection, rather than neutral mixing following secondary contact. The stability of cline widths and strong linkage disequilibria (gametic correlation coefficients Rmax = 0.35-0.56 among unlinked loci) over 25 years suggest that mimetic selection pressures on each color pattern locus have remained approximately constant (s approximately 0.13-0.40 per locus in both species). Exceptionally high levels of precipitation at the edge of the easternmost Andes may act as a population density trough for butterflies, trapping the hybrid zones at the foot of the mountains, and preventing movement. As such, our results falsify one prediction of the Pleistocene Refugium theory: That the ranges of divergent species or subspecies should be centered on regions characterized by maxima of rainfall, with hybrid zones falling in more arid regions between them.

Butterflies in the genus Heliconius have undergone rapid adaptive radiation for warning patterns and mimicry, andare excellent models to study the mechanisms underlying diversification. In Heliconius, mimicry rings typicallyinvolve distantly related species, whereas closely related species often join different mimicry rings. Genetic andbehavioural studies have shown how reproductive isolation in many pairs of Heliconius taxa is largely mediatedby natural and sexual selection on wing colour patterns. However, recent studies have uncovered new cases inwhich pairs of closely related species are near-perfect mimics of each other. Here, we provide morphometric andgenetic evidence for the coexistence of two closely related, hybridizing co-mimetic species on the eastern slopes ofthe Andes, H. melpomene amaryllis and H. timareta ssp. nov., which is described here as H. timareta thelxinoe.A joint analysis of multilocus genotyping and geometric morphometrics of wing shape shows a high level ofdifferentiation between the two species, with only limited gene flow and mixing. Some degree of genetic mixing canbe detected, but putative hybrids were rare, only one of 175 specimens being a clear hybrid. In contrast, we foundphenotypic differentiation between populations of H. timareta thelxinoe, possibly indicative of strong selection forlocal mimicry in different communities. In this pair of species, the absence of breakdown of genetic isolation despitenear-identical wing patterns implies that factors other than wing patterns keep the two taxa apart, such aschemical or behavioural signals, or ecological adaptation along a strong altitudinal gradient. © 2013 The LinneanSociety of London, Biological Journal of the Linnean Society, 2013