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.

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.

Molecular phylogeny and biogeography of West Indian toads (Anura: Bufonidae).

Toads of the Bufo peltocephalus Group (Anura: Bufonidae) occur throughout the Greater Antilles (Cuba to the Virgin Islands), a geographic region of relatively high endemicity. Previous morphological and immunological studies suggested that the West Indian toads are a monophyletic lineage derived from Neotropical Bufo but were unable to clarify relationships within the group. We examined the evolutionary relationships and biogeography of this group of frogs by collecting approximately 2 kb of mitochondrial DNA sequence data from eight West Indian species and selected non-West Indian species from the New World and the Old World. Our analyses support the monophyly of native West Indian toads and a New World origin for the group. Relationships among the West Indian species are less certain, but a Cuban lineage is defined in most analyses.

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.

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.

Human and ape molecular clocks and constraints on paleontological hypotheses.

Although the relationships of the living hominoid primates (humans and apes) are well known, the relationships of the fossil species, times of divergence of both living and fossil species, and the biogeographic history of hominoids are not well established. Divergence times of living species, estimated from molecular clocks, have the potential to constrain hypotheses of the relationships of fossil species. In this study, new DNA sequences from nine protein-coding nuclear genes in great apes are added to existing datasets to increase the precision of molecular time estimates bearing on the evolutionary history of apes and humans. The divergence of Old World monkeys and hominoids at the Oligocene-Miocene boundary (approximately 23 million years ago) provides the best primate calibration point and yields a time and 95% confidence interval of 5.4 +/- 1.1 million years ago (36 nuclear genes) for the human-chimpanzee divergence. Older splitting events are estimated as 6.4 +/- 1.5 million years ago (gorilla, 31 genes), 11.3 +/- 1.3 million years ago (orangutan, 33 genes), and 14.9 +/- 2.0 million years ago (gibbon, 27 genes). Based on these molecular constraints, we find that several proposed phylogenies of fossil hominoid taxa are unlikely to be correct.

The early history of modern birds inferred from DNA sequences of nuclear and mitochondrial ribosomal genes.

The traditional view of avian evolution places ratites and tinamous at the base of the phylogenetic tree of modern birds (Neornithes). In contrast, most recent molecular studies suggest that neognathous perching birds (Passeriformes) compose the oldest lineage of modern birds. Here, we report significant molecular support for the traditional view of neognath monophyly based on sequence analyses of nuclear and mitochondrial DNA (4.4 kb) from every modern avian order. Phylogenetic analyses further show that the ducks and gallinaceous birds are each other's closest relatives and together form the basal lineage of neognathous birds. To investigate why other molecular studies sampling fewer orders have reached different conclusions regarding neognath monophyly, we performed jackknife analyses on our mitochondrial data. Those analyses indicated taxon-sampling effects when basal galloanserine birds were included in combination with sparse taxon sampling. Our phylogenetic results suggest that the earliest neornithines were heavy-bodied, ground-dwelling, nonmarine birds. This inference, coupled with a fossil bias toward marine environments, provides a possible explanation for the large gap in the early fossil record of birds.

Phylogenetic relationships of xenodontine snakes inferred from 12S and 16S ribosomal RNA sequences.

The phylogenetic relationships of xenodontine snakes are inferred from sequence analyses of portions of two mitochondrial genes (12S and 16S ribosomal RNA) in 85 species. Although support values for most of the basal nodes are low, the general pattern of cladogenesis observed is congruent with many independent molecular, morphological, and geographical data. The monophyly of xenodontines and the basal position of North American xenodontines in comparison with Neotropical xenodontines are favored, suggesting an Asian-North American origin of xenodontines. West Indian xenodontines (including endemic genera and members of the genus Alsophis) appear to form a monophyletic group belonging to the South American clade. Their mid-Cenozoic origin by dispersal using ocean currents is supported. Within South American mainland xenodontines, the tribes Hydropsini, Pseudoboini, and Xenodontini are monophyletic. Finally, our results suggest that some morphological and ecological traits concerning maxillary dentition, macrohabitat use, and foraging strategy have appeared multiple times during the evolution of xenodontine snakes.

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.

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 multiple origins of Insectivora and for a new order of endemic African insectivore mammals.

The traditional views regarding the mammalian order Insectivora are that the group descended from a single common ancestor and that it is comprised of the following families: Soricidae (shrews), Tenrecidae (tenrecs), Solenodontidae (solenodons), Talpidae (moles), Erinaceidae (hedgehogs and gymnures), and Chrysochloridae (golden moles). Here we present a molecular analysis that includes representatives of all six families of insectivores, as well as 37 other taxa representing marsupials, monotremes, and all but two orders of placental mammals. These data come from complete sequences of the mitochondrial 12S rRNA, tRNA-Valine, and 16S rRNA genes (2.6 kb). A wide range of different methods of phylogenetic analysis groups the tenrecs and golden moles (both endemic to Africa) in an all-African superordinal clade comprised of elephants, sirenians, hyracoids, aardvark, and elephant shrews, to the exclusion of the other four remaining families of insectivores. Statistical analyses reject the idea of a monophyletic Insectivora as well as traditional concepts of the insectivore suborder Soricomorpha. These findings are supported by sequence analyses of several nuclear genes presented here: vWF, A2AB, and alpha-beta hemoglobin. These results require that the order Insectivora be partitioned and that the two African families (golden moles and tenrecs) be placed in a new order. The African superordinal clade now includes six orders of placental mammals.

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.