Unlike microevolutionary processes, little is known about the genetic basis of macroevolutionary processes. One of these magnificent examples is the transition from non-avian dinosaurs to birds that has created numerous evolutionary innovations such as self-powered flight and its associated wings with flight feathers. By analysing 48 bird genomes, we identified millions of avian-specific highly conserved elements (ASHCEs) that predominantly (>99%) reside in non-coding regions. Many ASHCEs show differential histone modifications that may participate in regulation of limb development. Comparative embryonic gene expression analyses across tetrapod species suggest ASHCE-associated genes have unique roles in developing avian limbs. In particular, we demonstrate how the ASHCE driven avian-specific expression of gene Sim1 driven by ASHCE may be associated with the evolution and development of flight feathers. Together, these findings demonstrate regulatory roles of ASHCEs in the creation of avian-specific traits, and further highlight the importance of cis-regulatory rewiring during macroevolutionary changes.

IntroductionDespite the great diversity in digit morphology reflecting the adaptation of tetrapods to their lifestyle, the number of digits in extant tetrapod species is conservatively stabilized at five or less, which is known as the pentadactyl constraint.ResultsWe found that an anuran amphibian species, Xenopus tropicalis (western clawed frog), has a clawed protrusion anteroventral to digit I on the foot. To identify the nature of the anterior-most clawed protrusion, we examined its morphology, tissue composition, development, and gene expression. We demonstrated that the protrusion in the X. tropicalis hindlimb is the sixth digit, as is evident from anatomical features, development, and molecular marker expression.ConclusionIdentification of the sixth digit in the X. tropicalis hindlimb strongly suggests that the prehallux in other Xenopus species with similar morphology and at the same position as the sixth digit is also a vestigial digit. We propose here that the prehallux seen in various species of amphibians generally represents a rudimentary sixth digit.Electronic supplementary materialThe online version of this article (doi:10.1186/s40851-015-0019-y) contains supplementary material, which is available to authorized users.

The fibula, a zeugopod bone in the hindlimb, exhibits various morphologies in tetrapod species. The fibula in some species has a similar length with the other zeugopod element, the tibia, while other species have obvious differences in the sizes of the two elements. In the avian hindlimb, for example, the fibula is extremely short, thin, and truncated. Basic morphology of the fibula is established during development, and cartilage primordium of the bone emerges in a certain region defined by a distinct combination of expression of Hox genes (Hox code). In order to elucidate how the different morphologies are produced from a region that is defined as the fixed Hox code, we examined spatial and temporal patterns of Hoxd11/Hoxd12 expression in the developing limb bud, which defines the region from which the fibula emerges, in comparison with the sites of precartilaginous mesenchymal condensations representing regions for cartilage formation among chick, mouse, and gecko embryos. We found that in the chick hindlimb, expression of Hoxd11/Hoxd12 decreased and disappeared from the presumptive zeugopod region before cartilage formation. This heterochronically early decline of expression of Hox genes is strongly correlated with the peculiar trait of the fibula in the avian hindlimb, since in the other species examined, expression of those genes continued after the onset of cartilage formation. This is morphological phenotype-related because the early disappearance was not seen in the chick forelimb. Our results suggest that temporal change of the Hox code governs diversification in morphology of homologous structures among related species.

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The divergent evolution of niche-related traits can facilitate adaptive radiation, yet identification of the genetic or molecular mechanisms underlying such trait changes remains a major challenge in evolutionary biology. Conducting a detailed morphological comparison along growth trajectories is a powerful method for observing the formation of differences in niche-related traits. Here, we focused on hindlimb length of Anolis lizards, differences in which are related to adaptation for use of different microhabitats. We measured the length of hindlimb skeletons in different ecomorphs of anole lizards (A. sagrei, a trunk-ground ecomorph with long hindlimbs, and A. angusticeps, a twig ecomorph with short hindlimbs) from early embryonic stages to adulthood, to determine which hindlimb elements mainly differentiate the species and the timing of the formation of these differences. With respect to the digit, differences between the species mainly occurred during the embryonic stages of interdigit reduction, when the cartilage of the distal phalanges was simultaneously forming. In addition, we compared the relative length of developing autopods in early embryonic stages using whole-mount in situ hybridization before the formation of the cartilaginous bones, and the results showed that the relative growth rate of the Hoxa11-negative distal region in A. sagrei was greater than that in A. angusticeps. Our results show that there are several important developmental stages for hindlimb length differentiation between A. angusticeps and A. sagrei, depending on which hindlimb element is considered. In particular, the species differences were largely due to variations in digit length, which arose at early embryonic stages.