Rapid evolution of Beta-keratin genes contribute to phenotypic differences that distinguish turtles and birds from other reptiles

Abstract

Sequencing of vertebrate genomes permits changes in distinct protein families, including gene gains and losses, to be ascribed to lineage-specific phenotypes. A prominent example of this is the large-scale duplication of beta-keratin genes in the ancestors of birds, which was crucial to the subsequent evolution of their beaks, claws, and feathers. Evidence suggests that the shell of Pseudomys nelsoni contains at least 16 beta-keratins proteins, but it is unknown whether this is a complete set and whether their corresponding genes are orthologous to avian beak, claw, or feather beta-keratin genes. To address these issues and to better understand the evolution of the turtle shell at a molecular level, we surveyed the diversity of beta-keratin genes from the genome assemblies of three turtles, Chrysemys picta, Pelodiscus sinensis, and Chelonia mydas, which together represent over 160 Myr of chelonian evolution. For these three turtles, we found 200 beta-keratins, which indicate that, as for birds, a large expansion of beta-keratin genes in turtles occurred concomitantly with the evolution of a unique phenotype, namely, their plastron and carapace. Phylogenetic reconstruction of beta-keratin gene evolution suggests that separate waves of gene duplication within a single genomic location gave rise to scales, claws, and feathers in birds, and independently the scutes of the shell in turtles.

Keywords: Sauropsids; beta-keratins; gene duplication; turtle shell.

Fig. 1.—

Synteny conservation for the beta-keratin cluster on the bird microchromosome 25 across reptiles. Data from Greenwold and Sawyer (2010) and Dalla Valle et al. (2009) were used to identify beta-keratins involved in the formation of the claws (violet), feathers (blue), and scales (green), and those that are turtle specific (red) and ancestral (dark green). Orthologous genes that are not beta-keratins are depicted in black. Protein coding genes without orthologous relationship are in gray. Anolis carolinensis genes displayed in brown are missing from figure 2 due to poor alignments.

Fig. 2.—

Identification of turtle beta-keratins potentially associated with the shell formation. Phylogenetic tree of the beta-keratins in Chrysemys picta (red), Anolis carolinensis (yellow), Crocodylus niloticus (green, three proteins), Gallus gallus (black), and Taeniopygia guttata (blue). The 16 translated cDNA sequences expressed in the skin from shell, soft skin, claws, and digit-tip of Pseudemys nelsoni are represented by black stars; the cDNA sequence with tissue specificity to claws and digit tip is labeled with a purple star. The red-shaded area highlights the putative “shell” beta-keratin clade in C. picta. The chromosomes and scaffolds represented in figure 1 are displayed above the tree. The association between beta-keratins and tissues was established according to phylogenetic affinity with ESTs (Dalla Valle et al. 2009; Greenwold and Sawyer 2010; bird scales: light green, nonshell: dark green, shell: dark red, bird claws: maroon, and feathers: light blue).

Fig. 3.—

Phylogenetic trees of beta-keratins from all species and dating using BEAST. The datings (in Myr), which correspond to 95% confidence intervals, are in square brackets. The turtle-specific clade with 16 out of 17 beta-keratins from Pseudemys nelsoni is labeled as putative shell clade. The bird-specific clade has been labeled in the same way as in figure 2. The clade annotated as ancestral corresponds to the clade with turtle, zebra finch beta-keratin genes. A higher resolution and unedited version of this tree can be found as supplementary figure S3, Supplementary Material online.

Fig. 4.—

Evolutionary model of the beta-keratin genes in the Sauropsids. Evolutionary scenario for the diversification of the beta-keratin clusters on the common ancestor of the chicken microchromosome 25 and Chrysemys picta Group42 scaffold. Dates on the turtle's lineages were estimated using BEAST (fig. 3), and dates for the bird lineage were estimated in Greenwold and Sawyer (2011). “Turtle-specific” and “ancestral” annotations are based on the phylogenetic affinity.