Gavin J. P. Naylor

A DNA Sequence–Based Approach To the Identification of Shark and Ray Species and Its Implications for Global Elasmobranch Diversity and Parasitology

Abstract In an effort to provide a framework for the accurate identification of elasmobranchs, driven in large part by the needs of parasitological studies, a comprehensive survey of DNA sequences derived from the mitochondrial NADH2 gene was conducted for elasmobranchs collected from around the world. Analysis was based on sequences derived from 4283 specimens representing an estimated 574 (of ∼1221) species (305 sharks, 269 batoids), each represented by 1 to 176 specimens, in 157 (of 193 described) elasmobranch genera in 56 (of 57 described) families of elasmobranchs (only Hypnidae was not represented). A total of 1921 (44.9%) of the samples were represented by vouchers and/or images available in an online host specimen database (http://elasmobranchs.tapewormdb.uconn.edu). A representative sequence for each of the 574 species identified in this survey, as well as an additional 11 sequences for problematic complexes, has been deposited in GenBank. Neighbor-joining analysis of the data revealed a substantial amount of previously undocumented genetic diversity in elasmobranchs, suggesting 79 potentially new taxa (38 sharks, 41 batoids). Within-species p-distance variation in NADH2-percent sequence divergence ranged from 0 to 2.12 with a mean of 0.27; within-genus p-distance variation ranged from 0.03 to 27.01, with a mean of 10.16. These values are roughly consistent with estimates from prior studies based on barcode COI sequences for elasmobranchs and fishes. While biogeographic influences have likely shaped the diversification of the entire group, the traces left by older influences tend to be overprinted by newer ones. As a result, the most clearly interpretable influences are those associated with recently diverged taxa. Among closely related elasmobranchs, four regions appear to be of particular importance: (1) the Atlantic Ocean, (2) Arabian Sea, Persian Gulf, and Red Sea, (3) Southeast Asia, and (4) Australia. Each of these regions has a substantial proportion of taxa that are genetically distinct from their closest relatives in other regions. These results suggest that great care should be taken in establishing the identities of elasmobranch hosts in parasitological studies. Furthermore, it is likely that many existing host records require confirmation.

Evidence for an ancient adaptive episode of convergent molecular evolution

Documented cases of convergent molecular evolution due to selection are fairly unusual, and examples to date have involved only a few amino acid positions. However, because convergence mimics shared ancestry and is not accommodated by current phylogenetic methods, it can strongly mislead phylogenetic inference when it does occur. Here, we present a case of extensive convergent molecular evolution between snake and agamid lizard mitochondrial genomes that overcomes an otherwise strong phylogenetic signal. Evidence from morphology, nuclear genes, and most sites in the mitochondrial genome support one phylogenetic tree, but a subset of mostly amino acid-altering substitutions (primarily at the first and second codon positions) across multiple mitochondrial genes strongly supports a radically different phylogeny. The relevant sites generally evolved slowly but converged between ancient lineages of snakes and agamids. We estimate that ≈44 of 113 predicted convergent changes distributed across all 13 mitochondrial protein-coding genes are expected to have arisen from nonneutral causes—a remarkably large number. Combined with strong previous evidence for adaptive evolution in snake mitochondrial proteins, it is likely that much of this convergent evolution was driven by adaptation. These results indicate that nonneutral convergent molecular evolution in mitochondria can occur at a scale and intensity far beyond what has been documented previously, and they highlight the vulnerability of standard phylogenetic methods to the presence of nonneutral convergent sequence evolution.

Structural biology and phylogenetic estimation.

When reconstructing evolutionary trees from DNA sequences, it is often assumed that increasing the amount of sequence will improve the phylogenetic estimate. This is based on the notion that historical ‘signal’ will rise above misleading ‘noise’ as more sequence is gathered. Our analysis of mitochondrial genomes fails to support this assumption, but suggests a way to select objectively for data with maximum ‘signal-to-noise’ potential.

The interface of protein structure, protein biophysics, and molecular evolution

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state‐of‐the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high‐throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction.

Capturing protein-coding genes across highly divergent species

DNA hybridization capture combined with next generation sequencing can be used to determine the sequences of hundreds of target genes across hundreds of individuals in a single experiment. However, the approach has thus far only been successfully applied to capture targets that are highly similar in sequence to the bait molecules. Here we introduce modifications that extend the reach of the method to allow efficient capture of highly divergent homologous target sequences using a single set of baits. These modifications have important implications for comparative biology.

Body plan convergence in the evolution of skates and rays (Chondrichthyes: Batoidea)

Skates, rays and allies (Batoidea) comprise more than half of the species diversity and much of the morphological disparity among chondrichthyan fishes, the sister group to all other jawed vertebrates. While batoids are morphologically well characterized and have an excellent fossil record, there is currently no consensus on the interrelationships of family-level taxa. Here we construct a resolved, robust and time-calibrated batoid phylogeny using mitochondrial genomes, nuclear genes, and fossils, sampling densely across taxa. Data partitioning schemes, biases in the sequence data, and the relative informativeness of each fossil are explored. The molecular phylogeny is largely congruent with morphology crownward in the tree, but the branching orders of major batoid groups are mostly novel. Body plan convergence appears to be widespread in batoids. A depressed, rounded pectoral disk supported to the snout tip by fin radials, common to skates and stingrays, is indicated to have been derived independently by each group, while the long, spiny rostrum of sawfishes similarly appears to be convergent with that of sawsharks, which are not batoids. The major extant batoid lineages are inferred to have arisen relatively rapidly from the Late Triassic into the Jurassic, with long stems followed by subsequent radiations in each group around the Cretaceous/Tertiary boundary. The fossil record indicates that batoids were affected with disproportionate severity by the end-Cretaceous extinction event.

Choosing the best genes for the job: the case for stationary genes in genome-scale phylogenetics.

The advent of genomics has fueled optimism for improvement in the reliability and accuracy of phylogenetic trees. An implicit assumption is that there will be an inexorable improvement in phylogenetic accuracy as the number of genes used increases, and that this approach is necessary because there are no identifiable parameters that predict the phylogenetic performance of genes (Gee, 2003; Rokas et al. 2003). These issues were explored in the recent article by Rokas et al. who investigated the phylogenetic signal in a sample of 106 protein-encoding genes selected from the genomes of 8 species of yeast. Rokas et al. (2003) analyzed these genes separately, and in combination, showing that individual genes sometimes support conflicting topologies. Although considerable character incongruence existed in the combined data set, simultaneous analysis of all genes resulted in one tree with 100% bootstrap proportions (BP) at all nodes. This “species tree” was taken to represent the true phylogeny (Fig. 1a topology). The authors then carried out a series of analyses with randomly concatenated data sets of varying size to determine the minimum amount of data required to establish confidence in the species tree at a given level of statistical significance. They concluded that a minimum of 20 randomly concatenated genes was required to infer relationships confidently and that “It is only through the analyses of larger amounts of sequence data that confidence in the proposed phylogenetic reconstruction can be obtained” and further “that analyses based on a single or a small number of genes provide insufficient evidence for establishing or refuting phylogenetic relationships.” They also expressed the opinion that the result for these yeast species was likely to be typical for molecular phylogenetic studies: “. . . we believe that this group is a representative model for key issues that researchers in phylogenetics are confronting,” with the clear implication that the majority of current molecular phylogenies must be considered unreliable. Another important conclusion was that there are no predictors of phylogenetic performance of genes: “there were no identifiable parameters that could systematically account for or predict the performance of single genes.” Similarly, Gee (2003), in discussing the Rokas et al. (2003) paper states, “there are no identifiable parameters that can predict the performance of genes in any systematic way.” Finally, they noted that bootstrap values were lower and variance higher for contiguous gene sequences than for randomly sampled orthologous nucleotides and took this as evidence of the misleading signal in individual genes resulting from the nonindependence of nucleotides within genes. These conclusions, if true, are sobering for those attempting to infer relationships using DNA sequences with limited time and budgets. Herein, we demonstrate that these conclusions require substantial revision. First we show that many genes in the yeast data set published by Rokas et al. (2003) have nucleotide frequencies that have shifted markedly among taxa at third positions of codons. These nucleotide sequences deviate significantly from the stationary condition (see also Phillips et al., 2004). Second, we illustrate through a series of analyses that the stationary gene partition is superior to the nonstationary partition, recovering the underlying phylogeny with many fewer genes. Finally, we show that the conclusion of Rokas et al. regarding the superiority of random sampling of orthologous nucleotides relative to contiguous sequences for phylogenetic analysis is largely an artifact of different bootstrap treatments for these two sampling schemes. Rokas et al. (2003) used several criteria for sampling and retaining genes from seven species of Saccharomyces yeasts, and one outgroup species, Candida albicans (Fig. 1). Genes were spaced at approximately 40-kilobase intervals. Only protein-encoding genes with identifiable and generally alignable homologs in all eight

CHAPTER 13 – Interrelationships of Lamniform Sharks: Testing Phylogenetic Hypotheses with Sequence Data

This chapter reviews the interrelationships of Lamniform sharks species highlighting the test results of phylogenetic hypotheses with sequence data. In the past decade, much has been made of the power of molecular sequences for inferring evolutionary history. Some advantages seen for DNA sequence data are also highlighted, It critically evaluates phylogenetic inferences derived from molecular data for these species. It provides information about the history of the group derived from the fossil record of these sharks and phylogenies. Results and discussions are detailed for the combinability, nucleotide composition for variable sites, transitions and transversions for each codon, and the phylogenetic analysis tree resulting from the analysis in which all sites are weighed equally. In order to expose any latent phylogenetic bias that might be caused by structured deviations from base compositional stationarity, data were subjected to two types of analysis designed to minimize such influences. It contrasts inferred amount of molecular change (i.e., branch length) and first appearance estimates derived from the fossil record for the new hypothesis. Results are plotted corresponding to the new hypothesis along with the implications of the new hypothesis.

Are the Fossil Data Really at Odds with the Molecular Data/ Morphological Evidence for Cetartiodactyla Phylogeny Reexamined

The phylogenetic position of Cetacea within the mammalian tree has long been a subject of debate. The traditional paleontological view is that an extinct order of mammals, the Mesonychia, is the sister taxon to Cetacea (e.g., Van Valen, 1966; Prothero et al., 1988). This view has recently been supported by morphological studies that examined both fossil and extant material (Geisler and Luo, 1998; O’Leary and Geisler, 1999). The molecular evidence, by contrast, supports a phylogenetic hypothesis in which Cetacea is nested deeply within the Artiodactyla, implying that Artiodactyla is paraphyletic with respect to Cetacea (Sarich, 1985; Milinkovitch et al., 1993; Gatesy et al., 1999, and references therein). Furthermore, several molecular studies have suggested that hippopotamids are the sister taxon to Cetacea (e.g., Irwin and Arnason, 1994; Gatesy et al., 1996; Gatesy, 1997, 1998; Montgelard et al., 1997; Nikaido et al., 1999). Although the “return to water” aspect of this phylogenetic hypothesis has a certain intuitive appeal, it has met with resistance from those who work primarily with morphology (e.g., Geisler and Luo, 1998; O’Leary andGeisler, 1999; O’Leary, 1999). Despite the resurgent interest in the problem, no consensus reconciling the different signals has yet been reached. Obviously, a serious limitation of molecular data is that this information cannot be gathered from the fossil remains of extinct taxa. In contrast, fossils can play an important role in recovering phylogeny inmorphological studies, often providing information about character states of stem lineages that POPPER, K. 1992. Realism and the aim of science. Routledge, London. SIDDALL, M. E., AND M. F. WHITING . 1999. Long branch abstractions. Cladistics 15:9–24.

THE PHYLOGENETIC RELATIONSHIPS AMONG REQUIEM AND HAMMERHEAD SHARKS: INFERRING PHYLOGENY WHEN THOUSANDS OF EQUALLY MOST PARSIMONIOUS TREES RESULT

Abstract— Protein variation among 37 species of carcharhiniform sharks was examined at 17 presumed loci. Evolutionary trees were inferred from these data using both cladistic character and a distance Wagner analysis. Initial cladistic character analysis resulted in more than 30 000 equally parsimonious tree arrangements. Randomization tests designed to evaluate the phylogenetic information content of the data suggest the data are highly significantly different from random in spite of the large number of parsimonious trees produced. Different starting seed trees were found to influence the kind of tree topologies discovered by the heuristic branch swapping algorithm used. The trees generated during the early phases of branch swapping on a single seed tree were found to be topologically similar to those generated throughout the course of branch swapping. Successive weighting increased the frequency and the consistency with which certain clades were found during the course of branch swapping, causing the semi‐strict consensus to be more resolved. Successive weighting also appeared resilient to the bias associated with the choice of initial seed tree causing analyses seeded with different trees to converge on identical final character weights and the same semi‐strict consensus tree.