A molecular phylogeny of reptiles.

The classical phylogeny of living reptiles pairs crocodilians with birds, tuataras with squamates, and places turtles at the base of the tree. New evidence from two nuclear genes, and analyses of mitochondrial DNA and 22 additional nuclear genes, join crocodilians with turtles and place squamates at the base of the tree. Morphological and paleontological evidence for this molecular phylogeny is unclear. Molecular time estimates support a Triassic origin for the major groups of living reptiles.

Molecular evidence for the early colonization of land by fungi and plants.

The colonization of land by eukaryotes probably was facilitated by a partnership (symbiosis) between a photosynthesizing organism (phototroph) and a fungus. However, the time when colonization occurred remains speculative. The first fossil land plants and fungi appeared 480 to 460 million years ago (Ma), whereas molecular clock estimates suggest an earlier colonization of land, about 600 Ma. Our protein sequence analyses indicate that green algae and major lineages of fungi were present 1000 Ma and that land plants appeared by 700 Ma, possibly affecting Earth's atmosphere, climate, and evolution of animals in the Precambrian.

A genomic timescale for the origin of eukaryotes.

BACKGROUND: Genomic sequence analyses have shown that horizontal gene transfer occurred during the origin of eukaryotes as a consequence of symbiosis. However, details of the timing and number of symbiotic events are unclear. A timescale for the early evolution of eukaryotes would help to better understand the relationship between these biological events and changes in Earth's environment, such as the rise in oxygen. We used refined methods of sequence alignment, site selection, and time estimation to address these questions with protein sequences from complete genomes of prokaryotes and eukaryotes. RESULTS: Eukaryotes were found to evolve faster than prokaryotes, with those eukaryotes derived from eubacteria evolving faster than those derived from archaebacteria. We found an early time of divergence (approximately 4 billion years ago, Ga) for archaebacteria and the archaebacterial genes in eukaryotes. Our analyses support at least two horizontal gene transfer events in the origin of eukaryotes, at 2.7 Ga and 1.8 Ga. Time estimates for the origin of cyanobacteria (2.6 Ga) and the divergence of an early-branching eukaryote that lacks mitochondria (Giardia) (2.2 Ga) fall between those two events. CONCLUSIONS: We find support for two symbiotic events in the origin of eukaryotes: one premitochondrial and a later mitochondrial event. The appearance of cyanobacteria immediately prior to the earliest undisputed evidence for the presence of oxygen (2.4-2.2 Ga) suggests that the innovation of oxygenic photosynthesis had a relatively rapid impact on the environment as it set the stage for further evolution of the eukaryotic cell.

Evolutionary history of the enolase gene family.

The enzyme enolase [EC 4.2.1.11] is found in all organisms, with vertebrates exhibiting tissue-specific isozymes encoded by three genes: alpha (alpha), beta (beta), and gamma (gamma) enolase. Limited taxonomic sampling of enolase has obscured the timing of gene duplication events. To help clarify the evolutionary history of the gene family, cDNAs were sequenced from six taxa representing major lineages of vertebrates: Chiloscyllium punctatum (shark), Amia calva (bowfin), Salmo trutta (trout), Latimeria chalumnae (coelacanth), Lepidosiren paradoxa (South American lungfish), and Neoceratodus forsteri (Australian lungfish). Phylogenetic analysis of all enolase and related gene sequences revealed an early gene duplication event prior to the last common ancestor of living organisms. Several distantly related archaebacterial sequences were designated as 'enolase-2', whereas all other enolase sequences were designated 'enolase-1'. Two of the three isozymes of enolase-1, alpha- and beta-enolase, were discovered in actinopterygian, sarcopterygian, and chondrichthian fishes. Phylogenetic analysis of vertebrate enolases revealed that the two gene duplications leading to the three isozymes of enolase-1 occurred subsequent to the divergence of living agnathans, near the Proterozoic/Phanerozoic boundary (approximately 550Mya). Two copies of enolase, designated alpha(1) and alpha(2), were found in the trout and are presumed to be the result of a genome duplication event.

Divergence time estimates for the early history of animal phyla and the origin of plants, animals and fungi.

In the past, molecular clocks have been used to estimate divergence times among animal phyla, but those time estimates have varied widely (1200-670 million years ago, Ma). In order to obtain time estimates that are more robust, we have analysed a larger number of genes for divergences among three well-represented animal phyla, and among plants, animals and fungi. The time estimate for the chordate-arthropod divergence, using 50 genes, is 993 +/- 46 Ma. Nematodes were found to have diverged from the lineage leading to arthropods and chordates at 1177 +/- 79 Ma. Phylogenetic analyses also show that a basal position of nematodes has strong support (p > 99%) and is not the result of rate biases. The three-way split (relationships unresolved) of plants, animals and fungi was estimated at 1576 +/- 88 Ma. By inference, the basal animal phyla (Porifera, Cnidaria, Ctenophora) diverged between about 1200-1500 Ma. This suggests that at least six animal phyla originated deep in the Precambrian, more than 400 million years earlier than their first appearance in the fossil record.

Convergence and divergence in the evolution of aquatic birds.

Aquatic birds exceed other terrestrial vertebrates in the diversity of their adaptations to aquatic niches. For many species this has created difficulty in understanding their evolutionary origin and, in particular, for the flamingos, hamerkop, shoebill and pelecaniforms. Here, new evidence from nuclear and mitochondrial DNA sequences and DNA-DNA hybridization data indicates extensive morphological convergence and divergence in aquatic birds. Among the unexpected findings is a grouping of flamingos and grebes, species which otherwise show no resemblance. These results suggest that the traditional characters used to unite certain aquatic groups, such as totipalmate feet, foot-propelled diving and long legs, evolved more than once and that organismal change in aquatic birds has proceeded at a faster pace than previously recognized.

Molecular evidence for the origin of birds.

The major groups of amniote vertebrates appeared during a relatively short time span at the end of the Paleozoic Era, a fact that has caused difficulty in estimating their relationships. The fossil record suggests that crocodilians are the closest living relatives of birds. However, morphological characters and molecular sequence data from living amniotes have repeatedly challenged this hypothesis by indicating a bird-mammal relationship. DNA sequences from four slow-evolving genes (mitochondrial 12S and 16S rRNA, tRNAVal, and nuclear alpha-enolase) now provide strong statistical support for a bird-crocodilian relationship.

Monophyly of the order Rodentia inferred from mitochondrial DNA sequences of the genes for 12S rRNA, 16S rRNA, and tRNA-valine.

A recent analysis of amino acid sequence data (Graur et al.) suggested that the mammalian order Rodentia is polyphyletic, in contrast to most morphological data, which support rodent monophyly. At issue is whether the hystricognath rodents, such as the guinea pig, represent an independent evolutionary lineage within mammals, separate from the sciurognath rodents. To resolve this problem, we sequenced a region (2,645 bp) of the mitochondrial genome of the guinea pig containing the complete 12S ribosomal RNA, 16S ribosomal RNA, and transfer RNA(VAL) genes for comparison with the available sciurognath and other mammalian sequences. Several methods of analysis and statistical tests of the data all show strong support for rodent monophyly (91%-98% bootstrap probability, or BP). Calibration with the mammalian fossil record suggests a Cretaceous date (107 mya) for the divergence of sciurognaths and hystricognaths. An older date (38 mya) for the controversial Mus-Rattus divergence also is supported by these data. Our neighbor-joining analyses of all available sequence data (25 genes) confirm that some individual genes support rodent polyphyly but that tandem analysis of all data does not. We propose that the conflicting results are due to several compounding factors. The unique biochemical properties of some hystricognath metabolic proteins, largely responsible for generating this controversy, may have a single explanation: a cascade effect resulting from inactivation of the zinc-binding abilities of insulin. After excluding six genes possibly affected by insulin inactivation, analyses of all available sequence data (7,117 nucleotide sites, 3,099 amino acid sites) resulted in strong support for rodent monophyly (94% BP for DNA sequences, 90% for protein sequences), which lends support to the insulin-cascade hypothesis.

Phylogeny of xantusiid lizards: concern for data and analysis.

Phylogenetic analyses of new DNA sequence data from the mitochondrial 16S rRNA gene in xantusiid lizards support the intergeneric relationships obtained previously (S.B. Hedges, R.L. Bezy, and L.B. Maxson, 1991, Mol. Biol. Evol. 8:767-780) with data from the 12S rRNA and cytochrome b (cyt b) genes. The total data set now includes 1028 alignable sites, 471 of which are variable and informative for the distance analyses and 281 of which are informative for the parsimony analyses. Crother and Presch (1993), Mol. Phylogenet. Evol. 1:289-294) claim that their reanalyses of our 12S rRNA and cyt b sequence data do not support a robust phylogeny for xantusiid lizards. However, that conclusion is not supported by their own analyses of the combined data from those two genes, which result in the same phylogenetic tree of xantusiid genera that we obtained in the original study with the same method (maximum parsimony). This result was unchanged when Crother and Presch eliminated sites containing insertions/deletions and ambiguities, and when transversions were weighted. The less robust result for the separate cyt b analyses, probably due to the smaller size of the data set, was already noted (Hedges et al., 1991). We believe that the best estimate of relationships, in this case, is obtained by combining the sequence data from these tightly linked mitochondrial genes. We also refute the criticisms by Crother and Presch of the neighbor-joining method. To correct for a higher rate of transitions in mitochondrial sequence data, they weight transversions more heavily (5x) than transitions. We present theoretical criticisms of this weighting method and advocate the use of scaled corrections available with distance methods. Crother and Presch also claim that a robust phylogeny of xantusiid lizards is not obtained when some morphological data (13 informative characters) are combined with the molecular data in a single analysis. However, there are serious problems with their morphological data and methods of analysis. We reevaluate the three pivotal morphological characters in their alternative phylogeny for xantusiid genera and demonstrate that none of the three provides unambiguous support for their alternative arrangement. Crother and Presch implement a new approach whereby the entire morphological data set is weighted equally to the molecular data set in a combined analysis, thus resulting in a very inflated weight assigned to each character in the small morphological data set. Using this rationale, each of the 13 informative morphological characters would receive a greater than million-fold weight if combined with the sequence data for the entire genome of these lizards.(ABSTRACT TRUNCATED AT 400 WORDS)

Origin of West Indian populations of the geographically widespread boa Corallus enydris inferred from mitochondrial DNA sequences.

Corallus enydris (Serpentes: Boidae: Boinae) is an arboreal snake with an extremely wide mainland distribution from southern Costa Rica to southeastern Brazil and is one of two boine species that has invaded the Lesser Antilles (Grenada Bank and St. Vincent). Mitochondrial DNA sequences of samples from seven geographically disparate localities provided evidence of phylogenetic relationships. The monophyly of C. enydris is corroborated and a major dichotomy between northern samples (Panama and Trinidad) and southern samples (Guyana, Perú, southeastern Brazil) was found and corresponds to the two currently recognized subspecies. Unexpectedly, the two samples from the West Indies (southern Lesser Antilles) cluster with the southern rather than the geographically closer northern samples (e.g., Trinidad). The results imply a fairly recent Guianan-Amazonian origin of West Indian populations.

Molecules vs. morphology in avian evolution: the case of the "pelecaniform" birds.

The traditional avian Order Pelecaniformes is composed of birds with all four toes connected by a web. This "totipalmate" condition is found in ca. 66 living species: 8 pelicans (Pelecanus), 9 boobies and gannets (Sula, Papasula, Morus), ca. 37 cormorants (Phalacrocorax), 4 anhingas or darters (Anhinga), 5 frigatebirds (Fregata), and 3 tropicbirds (Phaethon). Several additional characters are shared by these genera, and their monophyly has been assumed since the beginning of modern zoological nomenclature. Most ornithologists classify these genera as an order, although tropicbirds have been viewed as related to terns, and frigatebirds as relatives of the petrels and albatrosses. DNA.DNA hybridization data indicated that the pelicans are most closely related to the Shoebill (Balaeniceps rex), a stork-like bird that lives in the swamps of central Africa; the boobies, gannets, cormorants, and anhingas form a closely related cluster; the tropicbirds are not closely related to the other taxa; and the frigatebirds are closest to the penguins, loons, petrels, shearwaters, and albatrosses (Procellarioidea). Most of these results are corroborated by DNA sequences of the 12S and 16S rRNA mitochondrial genes, and they provide another example of incongruence between classifications derived from morphological versus genetic traits.

Molecular evidence for the early history of living amphibians.

The evolutionary relationships of the three orders of living amphibians (lissamphibians) has been difficult to resolve, partly because of their specialized morphologies. Traditionally, frogs and salamanders are considered to be closest relatives, and all three orders are thought to have arisen in the Paleozoic (>250 myr). Here, we present evidence from the DNA sequences of four mitochondrial genes (2.7 kilobases) that challenges the conventional hypothesis and supports a salamander-caecilian relationship. This, in light of the fossil record and distribution of the families, suggests a more recent (Mesozoic) origin for salamanders and caecilians directly linked to the initial breakup of the supercontinent Pangaea. We propose that this single geologic event isolated salamanders and archaeobatrachian frogs on the northern continents (Laurasia) and the caecilians and neobatrachian frogs on the southern continents (Gondwana). Among the neobatrachian frog families, molecular evidence supports a South American clade and an African clade, inferred here to be the result of mid-Cretaceous vicariance.

A molecular timescale for vertebrate evolution.

A timescale is necessary for estimating rates of molecular and morphological change in organisms and for interpreting patterns of macroevolution and biogeography. Traditionally, these times have been obtained from the fossil record, where the earliest representatives of two lineages establish a minimum time of divergence of these lineages. The clock-like accumulation of sequence differences in some genes provides an alternative method by which the mean divergence time can be estimated. Estimates from single genes may have large statistical errors, but multiple genes can be studied to obtain a more reliable estimate of divergence time. However, until recently, the number of genes available for estimation of divergence time has been limited. Here we present divergence-time estimates for mammalian orders and major lineages of vertebrates, from an analysis of 658 nuclear genes. The molecular times agree with most early (Palaeozoic) and late (Cenozoic) fossil-based times, but indicate major gaps in the Mesozoic fossil record. At least five lineages of placental mammals arose more than 100 million years ago, and most of the modern orders seem to have diversified before the Cretaceous/Tertiary extinction of the dinosaurs.

Calibration of avian molecular clocks.

Molecular clocks can be calibrated using fossils within the group under study (internal calibration) or outside of the group (external calibration). Both types of calibration have their advantages and disadvantages. An internal calibration may reduce extrapolation error but may not be from the best fossil record, raising the issue of nonindependence. An external calibration may be more independent but also may have a greater extrapolation error. Here, we used the advantages of both methods by applying a sequential calibration to avian molecular clocks. We estimated a basal divergence within birds, the split between fowl (Galliformes) and ducks (Anseriformes), to be 89.8 +/- 6.97 MYA using an external calibration and 12 rate-constant nuclear genes. In turn, this time estimate was used as an internal calibration for three species-rich avian molecular data sets: mtDNA, DNA-DNA hybridization, and transferrin immunological distances. The resulting time estimates indicate that many major clades of modern birds had their origins within the Cretaceous. This supports earlier studies that identified large gaps in the avian fossil record and suggests that modern birds may have coexisted with other avian lineages for an extended period during the Cretaceous. The new time estimates are concordant with a continental breakup model for the origin of ratites.