Chenhong Li

The Tree of Life and a New Classification of Bony Fishes

The tree of life of fishes is in a state of flux because we still lack a comprehensive phylogeny that includes all major groups. The situation is most critical for a large clade of spiny-finned fishes, traditionally referred to as percomorphs, whose uncertain relationships have plagued ichthyologists for over a century. Most of what we know about the higher-level relationships among fish lineages has been based on morphology, but rapid influx of molecular studies is changing many established systematic concepts. We report a comprehensive molecular phylogeny for bony fishes that includes representatives of all major lineages. DNA sequence data for 21 molecular markers (one mitochondrial and 20 nuclear genes) were collected for 1410 bony fish taxa, plus four tetrapod species and two chondrichthyan outgroups (total 1416 terminals). Bony fish diversity is represented by 1093 genera, 369 families, and all traditionally recognized orders. The maximum likelihood tree provides unprecedented resolution and high bootstrap support for most backbone nodes, defining for the first time a global phylogeny of fishes. The general structure of the tree is in agreement with expectations from previous morphological and molecular studies, but significant new clades arise. Most interestingly, the high degree of uncertainty among percomorphs is now resolved into nine well-supported supraordinal groups. The order Perciformes, considered by many a polyphyletic taxonomic waste basket, is defined for the first time as a monophyletic group in the global phylogeny. A new classification that reflects our phylogenetic hypothesis is proposed to facilitate communication about the newly found structure of the tree of life of fishes. Finally, the molecular phylogeny is calibrated using 60 fossil constraints to produce a comprehensive time tree. The new time-calibrated phylogeny will provide the basis for and stimulate new comparative studies to better understand the evolution of the amazing diversity of fishes.

A practical approach to phylogenomics: the phylogeny of ray-finned fish (Actinopterygii) as a case study

BackgroundMolecular systematics occupies one of the central stages in biology in the genomic era, ushered in by unprecedented progress in DNA technology. The inference of organismal phylogeny is now based on many independent genetic loci, a widely accepted approach to assemble the tree of life. Surprisingly, this approach is hindered by lack of appropriate nuclear gene markers for many taxonomic groups especially at high taxonomic level, partially due to the lack of tools for efficiently developing new phylogenetic makers. We report here a genome-comparison strategy to identifying nuclear gene markers for phylogenetic inference and apply it to the ray-finned fishes – the largest vertebrate clade in need of phylogenetic resolution.ResultsA total of 154 candidate molecular markers – relatively well conserved, putatively single-copy gene fragments with long, uninterrupted exons – were obtained by comparing whole genome sequences of two model organisms, Danio rerio and Takifugu rubripes. Experimental tests of 15 of these (randomly picked) markers on 36 taxa (representing two-thirds of the ray-finned fish orders) demonstrate the feasibility of amplifying by PCR and directly sequencing most of these candidates from whole genomic DNA in a vast diversity of fish species. Preliminary phylogenetic analyses of sequence data obtained for 14 taxa and 10 markers (total of 7,872 bp for each species) are encouraging, suggesting that the markers obtained will make significant contributions to future fish phylogenetic studies.ConclusionWe present a practical approach that systematically compares whole genome sequences to identify single-copy nuclear gene markers for inferring phylogeny. Our method is an improvement over traditional approaches (e.g., manually picking genes for testing) because it uses genomic information and automates the process to identify large numbers of candidate makers. This approach is shown here to be successful for fishes, but also could be applied to other groups of organisms for which two or more complete genome sequences exist, which has important implications for assembling the tree of life.

Multi-locus phylogenetic analysis reveals the pattern and tempo of bony fish evolution

Over half of all vertebrates are “fishes”, which exhibit enormous diversity in morphology, physiology, behavior, reproductive biology, and ecology. Investigation of fundamental areas of vertebrate biology depend critically on a robust phylogeny of fishes, yet evolutionary relationships among the major actinopterygian and sarcopterygian lineages have not been conclusively resolved. Although a consensus phylogeny of teleosts has been emerging recently, it has been based on analyses of various subsets of actinopterygian taxa, but not on a full sample of all bony fishes. Here we conducted a comprehensive phylogenetic study on a broad taxonomic sample of 61 actinopterygian and sarcopterygian lineages (with a chondrichthyan outgroup) using a molecular data set of 21 independent loci. These data yielded a resolved phylogenetic hypothesis for extant Osteichthyes, including 1) reciprocally monophyletic Sarcopterygii and Actinopterygii, as currently understood, with polypteriforms as the first diverging lineage within Actinopterygii; 2) a monophyletic group containing gars and bowfin (= Holostei) as sister group to teleosts; and 3) the earliest diverging lineage among teleosts being Elopomorpha, rather than Osteoglossomorpha. Relaxed-clock dating analysis employing a set of 24 newly applied fossil calibrations reveals divergence times that are more consistent with paleontological estimates than previous studies. Establishing a new phylogenetic pattern with accurate divergence dates for bony fishes illustrates several areas where the fossil record is incomplete and provides critical new insights on diversification of this important vertebrate group.

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.

Addressing Gene Tree Discordance and Non-Stationarity to Resolve a Multi-Locus Phylogeny of the Flatfishes (Teleostei: Pleuronectiformes)

Non-homogeneous processes and, in particular, base compositional non-stationarity have long been recognized as a critical source of systematic error. But only a small fraction of current molecular systematic studies methodically examine and effectively account for the potentially confounding effect of non-stationarity. The problem is especially overlooked in multi-locus or phylogenomic scale analyses, in part because no efficient tools exist to accommodate base composition heterogeneity in large data sets. We present a detailed analysis of a data set with 20 genes and 214 taxa to study the phylogeny of flatfishes (Pleuronectiformes) and their position among percomorphs. Most genes vary significantly in base composition among taxa and fail to resolve flatfish monophyly and other emblematic groups, suggesting that non-stationarity may be causing systematic error. We show a strong association between base compositional bias and topological discordance among individual gene partitions and their inferred trees. Phylogenetic methods applying non-homogeneous models to accommodate non-stationarity have relatively minor effect to reduce gene tree discordance, suggesting that available computer programs applying these methods do not scale up efficiently to the data set of modest size analysed in this study. By comparing phylogenetic trees obtained with species tree (STAR) and concatenation approaches, we show that gene tree discordance in our data set is most likely due to base compositional biases than to incomplete lineage sorting. Multi-locus analyses suggest that the combined phylogenetic signal from all loci in a concatenated data set overcomes systematic biases induced by non-stationarity at each partition. Finally, relationships among flatfishes and their relatives are discussed in the light of these results. We find support for the monophyly of flatfishes and confirm findings from previous molecular phylogenetic studies suggesting their close affinity with several carangimorph groups (i.e., jack and allies, barracuda, archerfish, billfish and swordfish, threadfin, moonfish, beach salmon, and snook and barramundi).

Exon-primed intron-crossing (EPIC) markers for non-model teleost fishes

BackgroundExon-primed intron-crossing (EPIC) markers have three advantages over anonymous genomic sequences in studying evolution of natural populations. First, the universal primers designed in exon regions can be applied across a broad taxonomic range. Second, the homology of EPIC-amplified sequences can be easily determined by comparing either their exon or intron portion depending on the genetic distance between the taxa. Third, having both the exon and intron fragments could help in examining genetic variation at the intraspecific and interspecific level simultaneously, particularly helpful when studying species complex. However, the paucity of EPIC markers has hindered multilocus studies using nuclear gene sequences, particularly in teleost fishes.ResultsWe introduce a bioinformatics pipeline for developing EPIC markers by comparing the whole genome sequences between two or more species. By applying this approach on five teleost fishes whose genomes were available in the Ensembl database http://www.ensembl.org, we identified 210 EPIC markers that have single-copy and conserved exon regions with identity greater than 85% among the five teleost fishes. We tested 12 randomly chosen EPIC markers in nine teleost species having a wide phylogenetic range. The success rate of amplifying and sequencing those markers varied from 44% to 100% in different species. We analyzed the exon sequences of the 12 EPIC markers from 13 teleosts. The resulting phylogeny contains many traditionally well-supported clades, indicating the usefulness of the exon portion of EPIC markers in reconstructing species phylogeny, in addition to the value of the intron portion of EPIC markers in interrogating the population history.ConclusionsThis study illustrated an effective approach to develop EPIC markers in a taxonomic group, where two or more genome sequences are available. The markers identified could be amplified across a broad taxonomic range of teleost fishes. The phylogenetic utility of individual markers varied according to intron size and amplifiability. The bioinformatics pipelines developed are readily adapted to other taxonomic groups.

The phylogenetic placement of sinipercid fishes (“Perciformes”) revealed by 11 nuclear loci

Sinipercids are freshwater fishes from East Asia assigned to the genera Siniperca (8-9 species) and Coreoperca (3 species). Although supported as a monophyletic group, the phylogenetic placement of sinipercids among percomorph fishes and the identification of their closest relatives have perplexed generations of taxonomists. Here, we collect and analyze new molecular evidence from 11 nuclear genes - six of them novel markers developed for this study - sampled from seven sinipercid species and several closely-related species that have been proposed as their putative sister taxa in the past. The concatenated alignment of the 11 loci included 10,560bp that were split into 18 blocks for a partitioned analysis using Bayesian and Maximum Likelihood methods. Both approaches resulted in nearly identical topologies, in which the monophyly of the sinipercids was strongly supported (Bootstrap Support=99; Posterior Probability=1.0) with Centrarchidae as their sister taxon (BS=87; PP=1.0). Percichthyidae formed a sister group to the sinipercid-centrarchid clade (BS=100; PP=1.0). Tests for alternative hypotheses of relationships significantly rejected the placement of sinipercids within the Serranidae, Moronidae, or Centropomidae, or as sister taxon to Lateolabrax japonicus, Perca flavescens, or Pristigenys alta (SH test, p<0.001,). The contribution of individual gene genealogies to support this result was explored further using a multispecies coalescent approach (BEST) to infer the species phylogeny. The sister-group relationship with Centrarchidae also is supported by this approach but relationships among species within the genus Siniperca showed a different pattern than the results obtained with the concatenated data set. Our study supports the designation of a distinct family, Sinipercidae, for this group of fishes (Siniperca and Coreoperca).

Monophyly and interrelationships of Snook and Barramundi (Centropomidae sensu Greenwood) and five new markers for fish phylogenetics

Centropomidae as defined by Greenwood (1976) is composed of three genera: Centropomus, Lates, and Psammoperca. But composition and monophyly of this family have been challenged in subsequent morphological studies. In some classifications, Ambassis, Siniperca and Glaucosoma were added to the Centropomidae. In other studies, Lates+Psammoperca were excluded, restricting the family to Centropomus. Recent analyses of DNA sequences did not solve the controversy, mainly due to limited taxonomic or character sampling. The present study is based on DNA sequence data from thirteen genes (one mitochondrial and twelve nuclear markers) for 57 taxa, representative of all relevant species. Five of the nuclear markers are new for fish phylogenetic studies. The monophyly of Centropomidae sensu Greenwood was supported by both maximum likelihood and Bayesian analyses of a concatenated data set (12,888 bp aligned). No support was found for previous morphological hypotheses suggesting that ambassids are closely allied to the Centropomidae. Putative affinities between centropomids and Glaucosoma, Niphon, or Siniperca were strongly rejected by topology tests. In agreement with previous molecular hypotheses, our results place Centropomidae within a group of fishes that includes carangoids (e.g., jacks, remoras, dolphinfish, roosterfish, and cobia), flatfishes, barracudas, archerfishes, billfishes, moonfish, and threadfins. The phylogeny for the extant Centropomidae is ((Lates, Psammoperca), Centropomus).

EvolMarkers: a database for mining exon and intron markers for evolution, ecology and conservation studies

Recent innovations in next‐generation sequencing have lowered the cost of genome projects. Nevertheless, sequencing entire genomes for all representatives in a study remains expensive and unnecessary for most studies in ecology, evolution and conservation. It is still more cost‐effective and efficient to target and sequence single‐copy nuclear gene markers for such studies. Many tools have been developed for identifying nuclear markers, but most of these have focused on particular taxonomic groups. We have built a searchable database, EvolMarkers, for developing single‐copy coding sequence (CDS) and exon‐primed‐intron‐crossing (EPIC) markers that is designed to work across a broad range of phylogenetic divergences. The database is made up of single‐copy CDS derived from BLAST searches of a variety of metazoan genomes. Users can search the database for different types of markers (CDS or EPIC) that are common to different sets of input species with different divergence characteristics. EvolMarkers can be applied to any taxonomic group for which genome data are available for two or more species. We included 82 genomes in the first version of EvolMarkers and have found the methods to be effective across Placozoa, Cnidaria, Arthropod, Nematoda, Annelida, Mollusca, Echinodermata, Hemichordata, Chordata and plants. We demonstrate the effectiveness of searching for CDS markers within annelids and show how to find potentially useful intronic markers within the lizard Anolis.

DNA capture reveals transoceanic gene flow in endangered river sharks

Significance The river sharks of the genus Glyphis, widely feared as man-eaters throughout India, remain very poorly known to science. The group constitutes five described species, all of which are considered highly endangered and restricted to freshwater systems in Australasia and Southeast Asia. DNA sequence data derived from 19th-century dried museum material augmented with contemporary samples indicates that only three of the five currently described species are valid; that there is a genetically distinct, but as-yet-undescribed, species recorded in Bangladesh and Sarawak in Malaysian Borneo; and that these iconic and mysterious sharks are not restricted to freshwater at all but rather appear to be adapted to both marine and freshwater habitats. For over a hundred years, the “river sharks” of the genus Glyphis were only known from the type specimens of species that had been collected in the 19th century. They were widely considered extinct until populations of Glyphis-like sharks were rediscovered in remote regions of Borneo and Northern Australia at the end of the 20th century. However, the genetic affinities between the newly discovered Glyphis-like populations and the poorly preserved, original museum-type specimens have never been established. Here, we present the first (to our knowledge) fully resolved, complete phylogeny of Glyphis that includes both archival-type specimens and modern material. We used a sensitive DNA hybridization capture method to obtain complete mitochondrial genomes from all of our samples and show that three of the five described river shark species are probably conspecific and widely distributed in Southeast Asia. Furthermore we show that there has been recent gene flow between locations that are separated by large oceanic expanses. Our data strongly suggest marine dispersal in these species, overturning the widely held notion that river sharks are restricted to freshwater. It seems that species in the genus Glyphis are euryhaline with an ecology similar to the bull shark, in which adult individuals live in the ocean while the young grow up in river habitats with reduced predation pressure. Finally, we discovered a previously unidentified species within the genus Glyphis that is deeply divergent from all other lineages, underscoring the current lack of knowledge about the biodiversity and ecology of these mysterious sharks.