Revealing Less Derived Nature of Cartilaginous Fish Genomes with Their Evolutionary Time Scale Inferred with Nuclear Genes
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Revealing Less Derived Nature of Cartilaginous Fish Genomes with Their Evolutionary Time Scale Inferred with Nuclear Genes Adina J. Renz, Axel Meyer, Shigehiro Kuraku*¤ Chair in Zoology and Evolutionary Biology, Department of Biology, University of Konstanz, Konstanz, Germany 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. Citation: Renz AJ, Meyer A, Kuraku S (2013) Revealing Less Derived Nature of Cartilaginous Fish Genomes with Their Evolutionary Time Scale Inferred with Nuclear Genes. PLoS ONE 8(6): e66400. doi:10.1371/journal.pone.0066400 Editor: Hector Escriva, Laboratoire Arago, France Received January 16, 2013; Accepted May 6, 2013; Published June 25, 2013 Copyright: ß 2013 Renz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Young Scholar Fund of the University of Konstanz and the research grant (KU2669/1-1) from the Deutsche Forschungsgemeinschaft (DFG) to SK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ¤ Current address: Genome Resource and Analysis Unit, RIKEN Center for Developmental Biology, Kobe, Japan Introduction monophylies of each of these two groups (Figure 1) have been supported by several molecular phylogenetic studies employing Chondrichthyans (cartilaginous fishes) occupy a key phyloge- genes in mtDNA [9,14–17] and the nucleus-encoded recombination netic position among extant vertebrates as one of the early- activating gene 1 (RAG1) [18]. Based on this phylogenetic relation- branching lineages [1–3]. Their fossils are considered to be well ship, the monophyletic group containing all sharks is designated preserved, largely because of the abundant deposits of dental ‘Selachimorpha’ (Figure 1). Recently, diverse extinct shark species, material [4]. Living chondrichthyans are divided into two assigned to the {Synechodontiformes, were suggested to form a subclasses, Holocephali (chimaeras) and Elasmobranchii (sharks, monophyletic group, which is sister to all living sharks [19]. This rays and skates) (Figure 1). Based on the fossil records, Holocephali assignment of Synechodontiformes into a monophyletic group and Elasmobranchii are estimated to have diverged in the which originated at the basal position of living sharks shifts the lowermost Devonian 410 million years before present (Ma) [5]. previously assumed minimum constraint of 190 Ma as the origin of The monophyly of each of the two groups was strongly supported modern sharks [20] back to the Late Permian around 250 Ma by morphological analyses [4,6,7], and has been reinforced by [15,19]. recent studies using several genes in the mitochondrial DNA Some chondrichthyan species have been subjected to various (mtDNA) [8,9]. More controversially discussed is the relationship molecular phylogenetic analyses, and they suggested a decreased between batoids (rays and skates) and sharks inside Elasmobran- rate of molecular evolution in the chondrichthyan lineages [21– chii. Earliest morphological studies supported the basal dichotomy 25]. Martin et al. calculated the molecular evolutionary rate using between sharks and batoids [10,11] (Figure 1). However, two mitochondrial genes, cytochrome b (cytb) and cytochrome oxidase I subsequent extensive analyses of morphological variation suggest- (COI), of 13 shark species and showed their slower rate compared ed that batoids were descendants of one derived shark lineage, to the mammalian lineage [21]. Later, Martin et al. reinforced that which is called the ‘‘Hypnosqualea hypothesis’’ [12,13]. More also in nuclear-encoded genes, namely dlx, heat shock protein (HSP) recently, the basal dichotomy between batoids and sharks and 70 and recombination activating gene (RAG) 1, the rates of molecular PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e66400 Time Scale of Cartilaginous Fish Evolution a total of 203 candidate genes were obtained. After investigation of the corresponding alignment of each gene, 122 candidate genes were excluded from further analyses, because they yielded spurious blast hits corresponding to sequences expanded into abundant copies (.500) in the chimaera genome. These 122 discarded candidates included genes whose mammalian orthologs are known to be involved in pathogen recognition and signal transduction [e.g. the nod-like receptor (NLR) family, the tripartite motif (TRIM) family and proteins containing WD repeats or FYVE zinc-finger domains] or are predicted hypothetical genes. The remaining 81 candidate genes were further scrutinized regarding their phylogenetic properties, namely their orthologies within individual datasets (see Methods). Here only three candidates (#1, 13 and 18 in Table 1) yielded the maximum- likelihood (ML) tree compatible with the generally accepted species phylogeny. All other candidate genes whose phylogenetic relationship of included species differed from the generally accepted species phylogeny [1] were further analyzed with the statistical framework of the ML method. That is, we calculated the Figure 1. Relationship of chondrichthyan species. Species tree log-likelihood (logL) of the obtained ML tree and that of tree illustrating the relationship of all chondrichthyan species employed in topology in accordance with the generally accepted species our analyses, either in the divergence time study or evolutionary rate phylogeny. The example of the candidate #9, RAN binding protein analysis (see text for alternative views of the phylogenetic relationship). Circles indicate the nodes referred to in the divergence time analysis. (RANBP) 1 gene, is illustrated in Figure 3. In this ML tree, teleost Widths of triangles are proportional to the numbers of species for fishes, instead of Xenopus laevis, are a sister group of amniotes, individual groups according to Compagno et al. [69]. which does not agree with the generally accepted phylogenetic doi:10.1371/journal.pone.0066400.g001 relationship [1]. However, the likelihoods of this ML tree (Figure 3) and the generally accepted species phylogeny did not significantly evolution for sharks are an order of magnitude slower than those differ (DlogL = 1.2362.35). for mammals [24]. After this assessment, 20 candidate genes remained in the final For the ‘‘Timetree of Life’’ [26], Heinicke et al. performed a dataset with each of them containing from 98 [candidate #15: M- family-level divergence time analysis for the entire Chondrichthyes phase phosphoprotein (MPP) 10] to 381 [candidate #1: phosphoglycerate [20]. They employed nucleotide sequences of the RAG1 gene and kinase (PGK) 1] amino acids in the gene-by-gene alignments mitochondrial 12S and 16S ribosomal RNA (rRNA) genes (Table 1). The total number of amino acids in the final previously sequenced [14,18,27]. Especially among the major concatenated dataset was 2973. chondrichthyan lineages, the analysis employing the RAG1 gene resulted in divergence time estimates of 100 million years older Divergence time estimation employing nuclear protein- than the estimate with 12S and 16S rRNA genes [20]. Most coding genes recently, Inoue et al. inferred divergence times for fourteen Holocephali-Elasmobranchii split. With the concatenated divergence points at the family level of chondrichthyans, using dataset, we first inferred the divergence time between Holocephali whole mtDNA sequences [15]. Summarizing these progresses, and Elasmobranchii (Figure 1). We employed fossil-based time further analyses based on the exhaustive use of latest palaeonto- constraints, which had two options for