The relative roles of regulatory and protein evolution in the origin and loss of convergent phenotypic traits is a core question in evolutionary biology. Here we combine phylogenomic, epigenomic and developmental data to show that convergent evolution of regulatory regions, but not protein-coding genes, is associated with flightlessness in palaeognathous birds, a classic example of a convergent phenotype. Eleven new genomes, including a draft genome from an extinct moa, resolve palaeognath phylogeny and show that the incidence of independent, convergent accelerations among 284,000 conserved non-exonic elements is significantly more frequent in ratites than other bird lineages. Ratite-specific acceleration of conserved regions and measures of open chromatin across eight tissues in the developing chick identify candidate regulatory regions that may have modified or lost function in ratites. Enhancer activity assays conducted in the early developing chicken forelimb confirm that volant versions of a conserved element in the first intron of the TEAD1 gene display conserved enhancer activity, whereas an accelerated flightless version fails to drive reporter gene expression. Our results show that convergent molecular changes associated with loss of flight are largely regulatory in nature.

The hoatzin (Opisthocomus hoazin) is a bizarre, long-tailed, crested bird that inhabits the riparian lowlands of South America. Among its peculiar attributes are (1) microbial foregut fermentation to convert plant cellulose in consumed foliage into simple sugars, (2) a highly modified skeleton to accommodate its large crop, and (3) in the young of this species, wing claws at the wrist joint which are used to climb among the branches of the nest tree. Consequently, the taxonomic position of this unusual bird has perplexed systematists since its description over 200 years ago. Traditionally classified among the fowl-like birds (Galliformes), recent studies have favored its placement with the cuckoos (Cuculiformes: Cuculidae). To help resolve this systematic uncertainty, we sequenced six mitochondrial genes (cytochrome oxidase I, II, and III, ATPase 8, ATPase 6, and cytochrome b) and one nuclear gene (c-mos), totaling 5,487 base pairs. With this large data set and an appropriate range of outgroup taxa, we demonstrate that the hoatzin should not be classified among the cuckoos or Galliformes. Instead, our analyses indicate that the hoatzin is most closely related to the turacos (Musophagiformes: Musophagidae), a small family of arboreal, frugivorous birds inhabiting sub-Saharan Africa. This phylogenetic relationship is also supported by osteological behavioral, and fossil evidence.

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