Guillermo Ortí

Genome 10K: A Proposal to Obtain Whole-Genome Sequence for 10 000 Vertebrate Species

The human genome project has been recently complemented by whole-genome assessment sequence of 32 mammals and 24 nonmammalian vertebrate species suitable for comparative genomic analyses. Here we anticipate a precipitous drop in costs and increase in sequencing efficiency, with concomitant development of improved annotation technology and, therefore, propose to create a collection of tissue and DNA specimens for 10,000 vertebrate species specifically designated for whole-genome sequencing in the very near future. For this purpose, we, the Genome 10K Community of Scientists (G10KCOS), will assemble and allocate a biospecimen collection of some 16,203 representative vertebrate species spanning evolutionary diversity across living mammals, birds, nonavian reptiles, amphibians, and fishes (ca. 60,000 living species). In this proposal, we present precise counts for these 16,203 individual species with specimens presently tagged and stipulated for DNA sequencing by the G10KCOS. DNA sequencing has ushered in a new era of investigation in the biological sciences, allowing us to embark for the first time on a truly comprehensive study of vertebrate evolution, the results of which will touch nearly every aspect of vertebrate biological enquiry.

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.

Population structure and biogeography of migratory freshwater fishes ( Prochilodus : Characiformes) in major South American rivers

Mitochondrial DNA (mtDNA) sequences were used to infer the phylogenetic relationships of Prochilodus species in the Paraná, Amazonas, Orinoco, and Magdalena basins. Sequences of ATPase subunits 6 and 8 (total 840 bp) were obtained for 21 Prochilodus specimens from the four river systems. Using Semaprochilodus as an outgroup, phylogenetic analyses showed that: (i) each river basin contains a monophyletic group of mtDNA lineages; and (ii) the branching order places Magdalena in a basal position with subsequent branching of Orinoco, Amazon and Paraná. The mitochondrial control region was sequenced for 26 P. lineatus (from the Paraná basin) and six other Prochilodus specimens from the Magdalena, Orinoco and Amazon. All 26 control region haplotypes were unique with sequence divergence ranging from 0.3 to 3.6%. The control region phylogeny is well resolved but phylogenetic structure is not associated with geography. For example, mtDNA haplotypes from the upper Paraná (Mogui Guassú) and the upper Bermejo, separated by at least 2600 km, have close genealogical ties. Phylogeographic analyses, including nested clade analysis, suggest high levels of gene flow within this basin.

GLOBAL SURVEY OF MITOCHONDRIAL DNA SEQUENCES IN THE THREESPINE STICKLEBACK: EVIDENCE FOR RECENT MIGRATIONS

Phylogenetic analyses of mitochondrial DNA (mtDNA) sequences were used to assess the matriarchal genetic structure of the threespine stickleback, Gasterosteus aculeatus. A 747 base‐pair (bp) fragment of the cytochrome b was sequenced from 36 individuals collected from 25 localities in Europe, North America, and Japan. Two major divergent clades were revealed: one widespread in Japan but with representatives in some Alaskan and British Columbian lakes and the other common in Europe and North America. A simple diagnostic test using the polymerase chain reaction (PCR) and a restriction enzyme was used to assay additional individuals, confirming the absence of the Japanese clade in the Atlantic basin. Geographic distribution of mtDNA variation suggests (1) a recent origin of the Atlantic populations, and (2) support for previous hypotheses about the existence of Pleistocene refugia for freshwater fishes in Alaska and British Columbia. Silent substitution rates were used to date the colonization of the Atlantic at 90,000 to 260,000 yr before present, which conflicts with earlier dates implied by the fossil record. The recent replacement of Atlantic mitochondrial lineages suggested by our data may be explained by severe reduction or extinction of northern Atlantic populations during the Pleistocene, followed by a recent reinvasion from the Pacific. With a global perspective of the distribution of genetic variation as a framework, meaningful comparisons at a smaller geographical scale will now be possible.

Phylogenetic relationships within the speciose family Characidae (Teleostei: Ostariophysi: Characiformes) based on multilocus analysis and extensive ingroup sampling

BackgroundWith nearly 1,100 species, the fish family Characidae represents more than half of the species of Characiformes, and is a key component of Neotropical freshwater ecosystems. The composition, phylogeny, and classification of Characidae is currently uncertain, despite significant efforts based on analysis of morphological and molecular data. No consensus about the monophyly of this group or its position within the order Characiformes has been reached, challenged by the fact that many key studies to date have non-overlapping taxonomic representation and focus only on subsets of this diversity.ResultsIn the present study we propose a new definition of the family Characidae and a hypothesis of relationships for the Characiformes based on phylogenetic analysis of DNA sequences of two mitochondrial and three nuclear genes (4,680 base pairs). The sequences were obtained from 211 samples representing 166 genera distributed among all 18 recognized families in the order Characiformes, all 14 recognized subfamilies in the Characidae, plus 56 of the genera so far considered incertae sedis in the Characidae. The phylogeny obtained is robust, with most lineages significantly supported by posterior probabilities in Bayesian analysis, and high bootstrap values from maximum likelihood and parsimony analyses.ConclusionA monophyletic assemblage strongly supported in all our phylogenetic analysis is herein defined as the Characidae and includes the characiform species lacking a supraorbital bone and with a derived position of the emergence of the hyoid artery from the anterior ceratohyal. To recognize this and several other monophyletic groups within characiforms we propose changes in the limits of several families to facilitate future studies in the Characiformes and particularly the Characidae. This work presents a new phylogenetic framework for a speciose and morphologically diverse group of freshwater fishes of significant ecological and evolutionary importance across the Neotropics and portions of Africa.

The cytochrome b gene as a phylogenetic marker: the limits of resolution for analyzing relationships among cichlid fishes.

The mitochondrial cytochrome b (cyt-b) gene is widely used in systematic studies to resolve divergences at many taxonomic levels. The present study focuses mainly on the utility of cyt-b as a molecular marker for inferring phylogenetic relationship at various levels within the fish family Cichlidae. A total of 78 taxa were used in the present analysis, representing all the major groups in the family Cichlidae (72 taxa) and other families from the suborders Labroidei and Percoidei. Gene trees obtained from cyt-b are compared to a published total evidence tree derived from previous studies. Minimum evolution trees based on cyt-b data resulted in topologies congruent with all previous analyses. Parsimony analyses downweighting transitions relative to transversions (ts1:tv4) or excluding transitions at third codon positions resulted in more robust bootstrap support for recognized clades than unweighted parsimony. Relative rate tests detected significantly long branches for some taxa (LB taxa) which were composed mainly by dwarf Neotropical cichlids. An improvement of the phylogenetic signal, as shown by the four-cluster likelihood mapping analysis, and higher bootstrap values were obtained by excluding LB taxa. Despite some limitations of cyt-b as a phylogenetic marker, this gene either alone or in combination with other data sets yields a tree that is in agreement with the well-established phylogeny of cichlid fish.

Evolution of pelvic reduction in threespine stickleback fish : a test of competing hypotheses

Reimchen hypothesized that pelvic reduction in threespine stickleback is favored by an absence of piscivorous fishes and the resulting increase in predation by insects, but Giles hypothesized that the predation regime is unimportant and that a low dissolved calcium concentration favors evolution of pelvic reduction. Substantial pelvic reduction in threespine stickleback sampled from 179 lakes around Cook Inlet, Alaska is strongly associated both with an absence of predatory fishes and a low calcium concentration. However, the association of pelvic reduction with low calcium concentration appears to be contingent on the absence of predatory fishes. These results emphasize the importance of interactions between seemingly unrelated environmental variables for selection of a single trait. However, these results also conflict with some observations elsewhere and do not rule out the possibility that other environmental factors are important for selection for pelvic reduction in threespine stickleback.

Total evidence : molecules, morphology, and the phylogenetics of cichlid fishes

We present a most comprehensive phylogenetic analysis of the family Cichlidae. New data analyzed include mitochondrial 16S rRNA sequences and two nuclear loci (Tmo-M27 and Tmo-4C4) for a large taxonomic sampling with emphasis on South American species. We also incorporate a published morphological data set for a total evidence analysis. Character congruence among mitochondrial (74 taxa) and nuclear data (50 taxa) was high. However, partition-homogeneity tests suggest significant heterogeneity among molecular and morphological data. In agreement with results obtained from molecular data alone, total evidence analysis (1,460 characters for 34 taxa) supports a robust phylogenetic hypothesis for the family Cichlidae that is congruent with drift-vicariance events associated with the fragmentation of Gondwana. Our analyses confirm the placement of Malagasy/Indian cichlids as the most basal lineages, with a sister-group relationship to the monophyletic African and Neotropical clades. Total evidence suggests that the controversial African genus Heterochromis is at the base of the African radiation. Among more than 50 Neotropical genera analyzed, Retroculus is identified as the basal taxon, with successive branching of Cichla, Astronotus, geophagines (including crenicichlines) + chaetobranchines, and cichlasomines + heroines. Relative rate tests applied to mitochondrial DNA suggest significantly higher rates of genetic variation in Neotropical than in African taxa, and both mitochondrial and nuclear sequences show that rate heterogeneity among Neotropical lineages is confined to the geophagine cichlids.

Mitochondrial DNA Phylogeny of the Family Cichlidae: Monophyly and Fast Molecular Evolution of the Neotropical Assemblage

Abstract. A mitochondrial DNA (mtDNA) phylogeny of cichlid fish is presented for the most taxonomically inclusive data set compiled to date (64 taxa). 16S rDNA data establish with confidence relationships among major lineages of cichlids, with a general pattern congruent with previous morphological studies and less inclusive molecular phylogenies based on nuclear genes. Cichlids from Madagascar and India are the most basal groups of the family Cichlidae and sister to African–Neotropical cichlids. The cichlid phylogeny suggests drift-vicariance events, consistent with the fragmentation of Gondwana, to explain current biogeographic distributions. Important phylogenetic findings include the placement of the controversial genus Heterochromis basal among African cichlids, the South American genus Retroculus as the most basal taxon of the Neotropical cichlid assemblage, and the close relationship of the Neotropical genera Cichla with Astronotus rather than with the crenicichlines. Based on a large number of South American genera, the Neotropical cichlids are defined as a monophyletic assemblage and shown to harbor significantly higher levels of genetic variation than their African counterparts. Relative rate tests suggest that Neotropical cichlids have experienced accelerated rates of molecular evolution. But these high evolutionary rates were significantly higher among geophagine cichlids.