Revealing less derived nature of cartilaginous fish genomes with their evolutionary time scale inferred with nuclear genes

Abstract

Cartilaginous fishes, divided into Holocephali (chimaeras) and Elasmoblanchii (sharks, rays and skates), occupy a key phylogenetic position among extant vertebrates in reconstructing their evolutionary processes. Their accurate evolutionary time scale is indispensable for better understanding of the relationship between phenotypic and molecular evolution of cartilaginous fishes. However, our current knowledge on the time scale of cartilaginous fish evolution largely relies on estimates using mitochondrial DNA sequences. In this study, making the best use of the still partial, but large-scale sequencing data of cartilaginous fish species, we estimate the divergence times between the major cartilaginous fish lineages employing nuclear genes. By rigorous orthology assessment based on available genomic and transcriptomic sequence resources for cartilaginous fishes, we selected 20 protein-coding genes in the nuclear genome, spanning 2973 amino acid residues. Our analysis based on the Bayesian inference resulted in the mean divergence time of 421 Ma, the late Silurian, for the Holocephali-Elasmobranchii split, and 306 Ma, the late Carboniferous, for the split between sharks and rays/skates. By applying these results and other documented divergence times, we measured the relative evolutionary rate of the Hox A cluster sequences in the cartilaginous fish lineages, which resulted in a lower substitution rate with a factor of at least 2.4 in comparison to tetrapod lineages. The obtained time scale enables mapping phenotypic and molecular changes in a quantitative framework. It is of great interest to corroborate the less derived nature of cartilaginous fish at the molecular level as a genome-wide phenomenon.

Figure 1. Relationship of chondrichthyan species.

Species tree illustrating the relationship of all chondrichthyan species employed in our analyses, either in the divergence time study or evolutionary rate analysis (see text for alternative views of the phylogenetic relationship). Circles indicate the nodes referred to in the divergence time analysis. Widths of triangles are proportional to the numbers of species for individual groups according to Compagno et al. .

Figure 2. Work flow of gene family selection for divergence time estimation within chondrichthyans.

See Methods for details of elasmobranch EST assembly and gene prediction on C. milii genomic genomic contigs. Abbreviations: EST, expressed sequence tags; GSS, genome survey sequence.

Figure 3. Phylogenetic tree ofRAN binding protein 1 genes.

This gene is listed as candidate #9 in Table 1. The tree was reconstructed with the maximum-likelihood (ML) method (see Methods). Bootstrap values were calculated with 100 resamplings. Support values at nodes indicate, in order, probabilities in the ML and the neighbor-joining (NJ) analysis. 119 amino acid sites were included for tree inference (shape parameter for gamma distribution α = 0.38). Note that the topology of this ML tree is not consistent with the generally accepted species phylogeny, but the log-likelihood of the tree topology consistent with the species phylogeny was not significantly lower than that of the ML tree (Table 1). For this reason, this gene was included in the final dataset.

Figure 4. Estimated timetree of vertebrates.

Timetree produced by MCMCTREE in PAML 4.4 implementing the relaxed molecular clock method. A total of 19 time constraints (see Table S2) used for the calculation are shown as arrowheads at the eleven nodes. 2973 amino acid sites were analyzed derived from a total of 20 nuclear genes. Horizontal bars indicate 95% confidence intervals (CI) of the divergence time estimates. All estimates and 95% CIs are listed in Table 1. The marginal densities obtained in TRACER 1.5 are shown in light grey above the bars. Rates given by MCMCTREE are shown above the individual branches.