Project description:Background: Heliconius butterflies are an excellent model system for studies of adaptive convergent and divergent phenotypic traits. Wing colour patterns are used as signals to both predators and potential mates and are inherited in a Mendelian manner. The underlying genetic mechanisms of pattern formation have been studied for many years and shed light on broad issues, such as the repeatability of evolution. In Heliconius melpomene, the yellow hindwing bar is controlled by the HmYb locus and several genes in this region show expression pattern differences across races. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that have key roles in many biological processes, including development. It seems likely that miRNAs could act as downstream regulators of genes involved in wing development, patterning and pigmentation. For this reason we characterised miRNAs in developing butterfly wings and examined differences in their expression between colour pattern races. Results: We sequenced small RNA libraries from two colour pattern races and detected 142 Heliconius miRNAs with homology to others found in miRBase. Several highly abundant miRNAs appeared to be differentially expressed between colour pattern races and this was tested further in different developing pupal wing stages using Northern blots. These revealed that differences in expression were due to developmental stage rather than colour pattern. Assembly of sequenced reads to the HmYb region identified miR-193 and miR-2788; located 2380bp apart in an intergenic region. A search for miRNAs in all available H. melpomene BAC sequences (~2.5Mb) did not reveal any other miRNA genes and no novel miRNAs were predicted. There were several regions where other small RNA sequences assembled to the HmYb region and appeared to be differentially expressed.These might represent other regulatory RNAs. Conclusions: Here we describe the first butterfly miRNAs and characterise their expression in developing wings. Some show differences in expression across developing pupal stages. Two miRNAs were located in the HmYb region. Future work will examine the expression of these miRNAs in different colour pattern races and identify miRNA targets among wing patterning genes.

Project description:Background: Heliconius butterflies are an excellent model system for studies of adaptive convergent and divergent phenotypic traits. Wing colour patterns are used as signals to both predators and potential mates and are inherited in a Mendelian manner. The underlying genetic mechanisms of pattern formation have been studied for many years and shed light on broad issues, such as the repeatability of evolution. In Heliconius melpomene, the yellow hindwing bar is controlled by the HmYb locus and several genes in this region show expression pattern differences across races. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that have key roles in many biological processes, including development. It seems likely that miRNAs could act as downstream regulators of genes involved in wing development, patterning and pigmentation. For this reason we characterised miRNAs in developing butterfly wings and examined differences in their expression between colour pattern races. Results: We sequenced small RNA libraries from two colour pattern races and detected 142 Heliconius miRNAs with homology to others found in miRBase. Several highly abundant miRNAs appeared to be differentially expressed between colour pattern races and this was tested further in different developing pupal wing stages using Northern blots. These revealed that differences in expression were due to developmental stage rather than colour pattern. Assembly of sequenced reads to the HmYb region identified miR-193 and miR-2788; located 2380bp apart in an intergenic region. A search for miRNAs in all available H. melpomene BAC sequences (~2.5Mb) did not reveal any other miRNA genes and no novel miRNAs were predicted. There were several regions where other small RNA sequences assembled to the HmYb region and appeared to be differentially expressed.These might represent other regulatory RNAs. Conclusions: Here we describe the first butterfly miRNAs and characterise their expression in developing wings. Some show differences in expression across developing pupal stages. Two miRNAs were located in the HmYb region. Future work will examine the expression of these miRNAs in different colour pattern races and identify miRNA targets among wing patterning genes. High-throughput sequencing of Heliconius melpomene endogenous small RNAs. Size fractionated small RNA from total RNA extracts of two different Heliconius melpomene races (Heliconius melpomene melpomene and Heliconius melpomene rosina) were isolated from wing tissue using miRVana kit. 100Âµg RNA from 11 individuals of different developmental stages was pooled for each race as follows: 4.1% larval stage <1; 2% larval stage 1-1.75; 2.9% larval stage 2-2.5; 22% larval stage 2.75-3; 19% larval stage > 3; 25% early pupae; 25% mid-melanin pupae. Sequences were ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Solexa/Illumina high throughput pyrosequencing. Please see www.illumina.com for details of the sequencing technology.

Project description:The unpalatable and warning-patterned butterflies Heliconius erato and Heliconius melpomene provide the best studied example of mutualistic Müllerian mimicry, thought-but rarely demonstrated-to promote coevolution. Some of the strongest available evidence for coevolution comes from phylogenetic codivergence, the parallel divergence of ecologically associated lineages. Early evolutionary reconstructions suggested codivergence between mimetic populations of H. erato and H. melpomene, and this was initially hailed as one of the most striking known cases of coevolution. However, subsequent molecular phylogenetic analyses found discrepancies in phylogenetic branching patterns and timing (topological and temporal incongruence) that argued against codivergence. We present the first explicit cophylogenetic test of codivergence between mimetic populations of H. erato and H. melpomene, and re-examine the timing of these radiations. We find statistically significant topological congruence between multilocus coalescent population phylogenies of H. erato and H. melpomene. Cophylogenetic historical reconstructions support repeated codivergence of mimetic populations, from the base of the sampled radiations. Pairwise distance correlation tests, based on our coalescent analyses plus recently published AFLP and wing colour pattern gene data, also suggest that the phylogenies of H. erato and H. melpomene show significant topological congruence. Divergence time estimates, based on a Bayesian coalescent model, suggest that the evolutionary radiations of H. erato and H. melpomene occurred over the same time period, and are compatible with a series of temporally congruent codivergence events. Our results suggest that differences in within-species genetic divergence are the result of a greater overall effective population size for H. erato relative to H. melpomene and do not imply incongruence in the timing of their phylogenetic radiations. Repeated codivergence between Müllerian co-mimics, predicted to exert mutual selection pressures, strongly suggests coevolution. Our results therefore support a history of reciprocal coevolution between Müllerian co-mimics characterised by phylogenetic codivergence and parallel phenotypic change.

Dataset Information

The genetic basis of structural colour variation in mimetic Heliconius butterflies

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