Phenotypic and genomic dissection of colour pattern variation in a reef fish radiation.

Coral reefs rank among the most diverse species assemblages on Earth. A particularly striking aspect of coral reef communities is the variety of colour patterns displayed by reef fishes. Colour pattern is known to play a central role in the ecology and evolution of reef fishes through, for example, signalling or camouflage. Nevertheless, colour pattern is a complex trait in reef fishes-actually a collection of traits-that is difficult to analyse in a quantitative and standardized way. This is the challenge that we address in this study using the hamlets (Hypoplectrus spp., Serranidae) as a model system. Our approach involves a custom underwater camera system to take orientation- and size-standardized photographs in situ, colour correction, alignment of the fish images with a combination of landmarks and Bézier curves, and principal component analysis on the colour value of each pixel of each aligned fish. This approach identifies the major colour pattern elements that contribute to phenotypic variation in the group. Furthermore, we complement the image analysis with whole-genome sequencing to run a multivariate genome-wide association study for colour pattern variation. This second layer of analysis reveals sharp association peaks along the hamlet genome for each colour pattern element and allows to characterize the phenotypic effect of the single nucleotide polymorphisms that are most strongly associated with colour pattern variation at each association peak. Our results suggest that the diversity of colour patterns displayed by the hamlets is generated by a modular genomic and phenotypic architecture.

Rapid radiation in a highly diverse marine environment.

Rapid diversification is often observed when founding species invade isolated or newly formed habitats that provide ecological opportunity for adaptive radiation. However, most of the Earth's diversity arose in diverse environments where ecological opportunities appear to be more constrained. Here, we present a striking example of a rapid radiation in a highly diverse marine habitat. The hamlets, a group of reef fishes from the wider Caribbean, have radiated into a stunning diversity of color patterns but show low divergence across other ecological axes. Although the hamlet lineage is ∼26 My old, the radiation appears to have occurred within the last 10,000 generations in a burst of diversification that ranks among the fastest in fishes. As such, the hamlets provide a compelling backdrop to uncover the genomic elements associated with phenotypic diversification and an excellent opportunity to build a broader comparative framework for understanding the drivers of adaptive radiation. The analysis of 170 genomes suggests that color pattern diversity is generated by different combinations of alleles at a few large-effect loci. Such a modular genomic architecture of diversification has been documented before in Heliconius butterflies, capuchino finches, and munia finches, three other tropical radiations that took place in highly diverse and complex environments. The hamlet radiation also occurred in a context of high effective population size, which is typical of marine populations. This allows for the accumulation of new variants through mutation and the retention of ancestral genetic variation, both of which appear to be important in this radiation.

Rampant Genome-Wide Admixture across the Heliconius Radiation.

How frequent is gene flow between species? The pattern of evolution is typically portrayed as a phylogenetic tree, yet gene flow between good species may be an important mechanism in diversification, spreading adaptive traits and leading to a complex pattern of phylogenetic incongruence. This process has thus far been studied mainly among a few closely related species, or in geographically restricted areas such as islands, but not on the scale of a continental radiation. Using a genomic representation of 40 out of 47 species in the genus, we demonstrate that admixture has played a role throughout the evolution of the charismatic Neotropical butterflies Heliconius. Modeling of phylogenetic networks based on the exome uncovers up to 13 instances of interspecific gene flow. Admixture is detected among the relatives of Heliconius erato, as well as between the ancient lineages leading to modern clades. Interspecific gene flow played a role throughout the evolution of the genus, although the process has been most frequent in the clade of Heliconius melpomene and relatives. We identify Heliconius hecalesia and relatives as putative hybrids, including new evidence for introgression at the loci controlling the mimetic wing patterns. Models accounting for interspecific gene flow yield a more complete picture of the radiation as a network, which will improve our ability to study trait evolution in a realistic comparative framework.

Developmental plasticity shapes social traits and selection in a facultatively eusocial bee.

Developmental plasticity generates phenotypic variation, but how it contributes to evolutionary change is unclear. Phenotypes of individuals in caste-based (eusocial) societies are particularly sensitive to developmental processes, and the evolutionary origins of eusociality may be rooted in developmental plasticity of ancestral forms. We used an integrative genomics approach to evaluate the relationships among developmental plasticity, molecular evolution, and social behavior in a bee species (Megalopta genalis) that expresses flexible sociality, and thus provides a window into the factors that may have been important at the evolutionary origins of eusociality. We find that differences in social behavior are derived from genes that also regulate sex differentiation and metamorphosis. Positive selection on social traits is influenced by the function of these genes in development. We further identify evidence that social polyphenisms may become encoded in the genome via genetic changes in regulatory regions, specifically in transcription factor binding sites. Taken together, our results provide evidence that developmental plasticity provides the substrate for evolutionary novelty and shapes the selective landscape for molecular evolution in a major evolutionary innovation: Eusociality.

Genomic architecture and introgression shape a butterfly radiation.

We used 20 de novo genome assemblies to probe the speciation history and architecture of gene flow in rapidly radiating Heliconius butterflies. Our tests to distinguish incomplete lineage sorting from introgression indicate that gene flow has obscured several ancient phylogenetic relationships in this group over large swathes of the genome. Introgressed loci are underrepresented in low-recombination and gene-rich regions, consistent with the purging of foreign alleles more tightly linked to incompatibility loci. Here, we identify a hitherto unknown inversion that traps a color pattern switch locus. We infer that this inversion was transferred between lineages by introgression and is convergent with a similar rearrangement in another part of the genus. These multiple de novo genome sequences enable improved understanding of the importance of introgression and selective processes in adaptive radiation.

Comparative Transcriptomics Provides Insights into Reticulate and Adaptive Evolution of a Butterfly Radiation.

Butterfly eyes are complex organs that are composed of a diversity of proteins and they play a central role in visual signaling and ultimately, speciation, and adaptation. Here, we utilized the whole eye transcriptome to obtain a more holistic view of the evolution of the butterfly eye while accounting for speciation events that co-occur with ancient hybridization. We sequenced and assembled transcriptomes from adult female eyes of eight species representing all major clades of the Heliconius genus and an additional outgroup species, Dryas iulia. We identified 4,042 orthologous genes shared across all transcriptome data sets and constructed a transcriptome-wide phylogeny, which revealed topological discordance with the mitochondrial phylogenetic tree in the Heliconius pupal mating clade. We then estimated introgression among lineages using additional genome data and found evidence for ancient hybridization leading to the common ancestor of Heliconius hortense and Heliconius clysonymus. We estimated the Ka/Ks ratio for each orthologous cluster and performed further tests to demonstrate genes showing evidence of adaptive protein evolution. Furthermore, we characterized patterns of expression for a subset of these positively selected orthologs using qRT-PCR. Taken together, we identified candidate eye genes that show signatures of adaptive molecular evolution and provide evidence of their expression divergence between species, tissues, and sexes. Our results demonstrate: 1) greater evolutionary changes in younger Heliconius lineages, that is, more positively selected genes in the cydno-melpomene-hecale group as opposed to the sara-hortense-erato group, and 2) suggest an ancient hybridization leading to speciation among Heliconius pupal-mating species.

The genomics of an adaptive radiation: insights across the Heliconius speciation continuum.

Fueled by new technologies that allow rapid and inexpensive assessment of fine scale individual genomic variation, researchers are making transformational discoveries at the interface between genomes and biological complexity. Here we review genomic research in Heliconius butterflies - a radiation characterized by extraordinary phenotypic diversity in warningly colored wing patterns and composed of a continuum of taxa across the stages of speciation. These characteristics, coupled with a 50-year legacy of ecological and behavioral research, offer exceptional prospects for genomic studies into the nature of adaptive differences and the formation of new species. Research in Heliconius provides clear connections between genotype, phenotype, and fitness of wing color patterns shown to underlie adaptation and speciation. This research is challenging our perceptions about how speciation occurs in the presence of gene flow and the role of hybridization in generating adaptive novelty. With the release of the first Heliconius genome assembly, emerging genomic studies are painting a dynamic picture of the evolving species boundary. As the field of speciation genomics moves beyond describing patterns, towards a more integrated understanding of the process of speciation, groups such as Heliconius, where there is a clear speciation continuum and the traits underlying adaptation and speciation are known, will provide a roadmap for identifying variation crucial in the origins of biodiversity.

Polyphyly and gene flow between non-sibling Heliconius species.

BACKGROUND: The view that gene flow between related animal species is rare and evolutionarily unimportant largely antedates sensitive molecular techniques. Here we use DNA sequencing to investigate a pair of morphologically and ecologically divergent, non-sibling butterfly species, Heliconius cydno and H. melpomene (Lepidoptera: Nymphalidae), whose distributions overlap in Central and Northwestern South America. RESULTS: In these taxa, we sequenced 30-45 haplotypes per locus of a mitochondrial region containing the genes for cytochrome oxidase subunits I and II (CoI/CoII), and intron-spanning fragments of three unlinked nuclear loci: triose-phosphate isomerase (Tpi), mannose-6-phosphate isomerase (Mpi) and cubitus interruptus (Ci) genes. A fifth gene, dopa decarboxylase (Ddc) produced sequence data likely to be from different duplicate loci in some of the taxa, and so was excluded. Mitochondrial and Tpi genealogies are consistent with reciprocal monophyly, whereas sympatric populations of the species in Panama share identical or similar Mpi and Ci haplotypes, giving rise to genealogical polyphyly at the species level despite evidence for rapid sequence divergence at these genes between geographic races of H. melpomene. CONCLUSION: Recent transfer of Mpi haplotypes between species is strongly supported, but there is no evidence for introgression at the other three loci. Our results demonstrate that the boundaries between animal species can remain selectively porous to gene flow long after speciation, and that introgression, even between non-sibling species, can be an important factor in animal evolution. Interspecific gene flow is demonstrated here for the first time in Heliconius and may provide a route for the transfer of switch-gene adaptations for Müllerian mimicry. The results also forcefully demonstrate how reliance on a single locus may give an erroneous picture of the overall genealogical history of speciation and gene flow.

Molecular evolution of dengue 2 virus in Puerto Rico: positive selection in the viral envelope accompanies clade reintroduction.

Dengue virus is a circumtropical, mosquito-borne flavivirus that infects 50-100 million people each year and is expanding in both range and prevalence. Of the four co-circulating viral serotypes (DENV-1 to DENV-4) that cause mild to severe febrile disease, DENV-2 has been implicated in the onset of dengue haemorrhagic fever (DHF) in the Americas in the early 1980s. To identify patterns of genetic change since DENV-2's reintroduction into the region, molecular evolution in DENV-2 from Puerto Rico (PR) and surrounding countries was examined over a 20 year period of fluctuating disease incidence. Structural genes (over 20 % of the viral genome), which affect viral packaging, host-cell entry and immune response, were sequenced for 91 DENV-2 isolates derived from both low- and high-prevalence years. Phylogenetic analyses indicated that DENV-2 outbreaks in PR have been caused by viruses assigned to subtype IIIb, originally from Asia. Variation amongst DENV-2 viruses in PR has since largely arisen in situ, except for a lineage-replacement event in 1994 that appears to have non-PR New World origins. Although most structural genes have remained relatively conserved since the 1980s, strong evidence was found for positive selection acting on a number of amino acid sites in the envelope gene, which have also been important in defining phylogenetic structure. Some of these changes are exhibited by the multiple lineages present in 1994, during the largest Puerto Rican outbreak of dengue, suggesting that they may have altered disease dynamics, although their functional significance will require further investigation.

Molecular evolution and phylogeny of dengue type 4 virus in the Caribbean.

We sequenced the E gene and adjacent prM/M and NS1 junctions (1940 bp) of 48 Dengue-4 (DEN-4) isolates collected between 1981 and 1999 from 8 Caribbean islands and from 7 South and Central American countries. Phylogenetic analysis confirms a single introduction in the early 1980s and a high degree of gene flow resulting in a pattern of evolution defined more by time period than geographic origin, especially within the Caribbean basin. A modern Caribbean clade consisting of four distinct lineages has arisen, comprised of isolates from Caribbean islands and nearby regions of South America. This clade is defined by three amino acid substitutions in the E (aa 163 and 351) and NS1 (aa 52) proteins. These findings highlight the importance of migration and gene flow in dengue viral change and suggest that efforts to understand disease dynamics in the Caribbean basin need to focus at regional, rather than local scales.

Phylogenetic discordance at the species boundary: comparative gene genealogies among rapidly radiating Heliconius butterflies.

Recent adaptive radiations provide excellent model systems for understanding speciation, but rapid diversification can cause problems for phylogenetic inference. Here we use gene genealogies to investigate the phylogeny of recent speciation in the heliconiine butterflies. We sequenced three gene regions, intron 3 ( approximately 550 bp) of sex-linked triose-phosphate isomerase (Tpi), intron 3 ( approximately 450 bp) of autosomal mannose-phosphate isomerase (Mpi), and 1,603 bp of mitochondrial cytochrome oxidase subunits I and II (COI and COII), for 37 individuals from 25 species of Heliconius and related genera. The nuclear intron sequences evolved at rates similar to those of mitochondrial coding sequences, but the phylogenetic utility of introns was restricted to closely related geographic populations and species due to high levels of indel variation. For two sister species pairs, Heliconius erato-Heliconius himera and Heliconius melpomene-Heliconius cydno, there was highly significant discordance between the three genes. At mtDNA and Tpi, the hypotheses of reciprocal monophyly and paraphyly of at least one species with respect to its sister could not be distinguished. In contrast alleles sampled from the third locus, Mpi, showed polyphyletic relationships between both species pairs. In all cases, recent coalescence of mtDNA lineages within species suggests that polyphyly of nuclear genes is not unexpected. In addition, very similar alleles were shared between melpomene and cydno, implying recent gene flow. Our finding of discordant genealogies between genes is consistent with models of adaptive speciation with ongoing gene flow and highlights the need for multiple locus comparisons to resolve phylogeny among closely related species.