Examining the utility of categorical models and alleviating artifacts in phylogenetic reconstruction of the Squamata (Reptilia).

Reconstruction artifacts are a serious hindrance to the elucidation of phylogenetic relationships and a number of methods have been devised to alleviate them. Previous studies have demonstrated a striking disparity in the evolutionary rates of the mitochondrial (mt) genomes of squamate reptiles (lizards, worm lizards and snakes) and the reconstruction artifacts that may arise from this. Here, to examine basal squamate relationships, we have added the mt genome of the blind skink Dibamus novaeguineae to the mitogenomic dataset and applied different models for resolving the squamate tree. Categorical models were found to be less susceptible to artifacts than were the commonly used noncategorical phylogenetic models GTR and mtREV. The application of different treatments to the data showed that the removal of the fastest evolving sites in snakes improved phylogenetic signal in the dataset. Basal divergences remained, nevertheless, poorly resolved. The proportion of both fast-evolving and conserved sites in the squamate mt genomes relative to sites with intermediate rates of evolution suggests rapid early divergences among squamate taxa and at least partly explains the short internal relative to external branches in the squamate tree. Thus, mt and nuclear trees may never reach full agreement because of the short branches characterizing these divergences.

Mitogenomic analyses of eutherian relationships.

Reasonably correct phylogenies are fundamental to the testing of evolutionary hypotheses. Here, we present phylogenetic findings based on analyses of 67 complete mammalian mitochondrial (mt) genomes. The analyses, irrespective of whether they were performed at the amino acid (aa) level or on nucleotides (nt) of first and second codon positions, placed Erinaceomorpha (hedgehogs and their kin) as the sister group of remaining eutherians. Thus, the analyses separated Erinaceomorpha from other traditional lipotyphlans (e.g., tenrecs, moles, and shrews), making traditional Lipotyphla polyphyletic. Both the aa and nt data sets identified the two order-rich eutherian clades, the Cetferungulata (comprising Pholidota, Carnivora, Perissodactyla, Artiodactyla, and Cetacea) and the African clade (Tenrecomorpha, Macroscelidea, Tubulidentata, Hyracoidea, Proboscidea, and Sirenia). The study corroborated recent findings that have identified a sister-group relationship between Anthropoidea and Dermoptera (flying lemurs), thereby making our own order, Primates, a paraphyletic assembly. Molecular estimates using paleontologically well-established calibration points, placed the origin of most eutherian orders in Cretaceous times, 70-100 million years before present (MYBP). The same estimates place all primate divergences much earlier than traditionally believed. For example, the divergence between Homo and Pan is estimated to have taken place approximately 10 MYBP, a dating consistent with recent findings in primate paleontology.

The complete mitochondrial genome of the hagfish Myxine glutinosa: unique features of the control region.

The complete sequence of the mitochondrial DNA of the hagfish Myxine glutinosa has been determined. The hagfish mtDNA (18,909 bp) is the longest vertebrate mtDNA determined so far. The gene arrangement conforms to the consensus vertebrate type and differs from that of lampreys. The exceptionally long (3628-bp) control region of the hagfish contains the typical conserved elements found in other vertebrate mtDNAs but is characterized by a large number of putative hairpins, which can potentially fold into a highly compact secondary structure that appears to be unique to hagfish. The comparison of the mtDNAs of two M. glutinosa specimens, excluding the control region, shows a 0.6% divergence at the nucleotide level as a sample of intraspecies polymorphism.

The mitochondrial genomes of the iguana (Iguana iguana) and the caiman (Caiman crocodylus): implications for amniote phylogeny.

The complete mitochondrial genomes of two reptiles, the common iguana (Iguana iguana) and the caiman (Caiman crocodylus), were sequenced in order to investigate phylogenetic questions of tetrapod evolution. The addition of the two species allows analysis of reptilian relationships using data sets other than those including only fast-evolving species. The crocodilian mitochondrial genomes seem to have evolved generally at a higher rate than those of other vertebrates. Phylogenetic analyses of 2889 amino-acid sites from 35 mitochondrial genomes supported the bird-crocodile relationship, lending no support to the Haematotherma hypothesis (with birds and mammals representing sister groups). The analyses corroborated the view that turtles are at the base of the bird-crocodile branch. This position of the turtles makes Diapsida paraphyletic. The origin of the squamates was estimated at 294 million years (Myr) ago and that of the turtles at 278 Myr ago. Phylogenetic analysis of mammalian relationships using the additional outgroups corroborated the Marsupionta hypothesis, which joins the monotremes and the marsupials to the exclusion of the eutherians.

Molecular evidence of an African Phiomorpha-South American Caviomorpha clade and support for Hystricognathi based on the complete mitochondrial genome of the cane rat (Thryonomys swinderianus).

The complete mitochondrial genome of an African cane rat, Thryonomys swinderianus (Rodentia, Hystricognathi), was included in a phylogenetic analysis along with 4 rodents, 14 additional eutherians, and 3 noneutherian outgroups. Monophyly of the suborder Hystricognathi, represented by the cane rat and the South American guinea pig, Cavia porcellus, was strongly supported by maximum-parsimony, neighbor-joining, and maximum-likelihood methods. The molecular-based estimate of the divergence time of Old and New World Hystricognathi (approximately 85 million years before present, MYBP) is consistent with an hypothesis of vicariance divergence due to the rifting of the African and South American continents 86-100 MYBP. Monophyly of Rodentia or the superordinal clade Glires (Rodentia and Lagomorpha) were not supported.

The mitochondrial genome of the sperm whale and a new molecular reference for estimating eutherian divergence dates.

Extant cetaceans are systematically divided into two suborders: Mysticeti (baleen whales) and Odontoceti (toothed whales). In this study, we have sequenced the complete mitochondrial (mt) genome of an odontocete, the sperm whale (Physeter macrocephalus), and included it in phylogenetic analyses together with the previously sequenced complete mtDNAs of two mysticetes (the fin and blue whales) and a number of other mammals, including five artiodactyls (the hippopotamus, cow, sheep, alpaca, and pig). The most strongly supported cetartiodactyl relationship was: outgroup,((pig, alpaca), ((cow, sheep),(hippopotamus,(sperm whale,(baleen whales))))). As in previous analyses of complete mtDNAs, the sister-group relationship between the hippopotamus and the whales received strong support, making both Artiodactyla and Suiformes (pigs, peccaries, and hippopotamuses) paraphyletic. In addition, the analyses identified a sister-group relationship between Suina (the pig) and Tylopoda (the alpaca), although this relationship was not strongly supported. The paleontological records of both mysticetes and odontocetes extend into the Oligocene, suggesting that the mysticete and odontocete lineages diverged 32-34 million years before present (MYBP). Use of this divergence date and the complete mtDNAs of the sperm whale and the two baleen whales allowed the establishment of a new molecular reference, O/M-33, for dating other eutherian divergences. There was a general consistency between O/M-33 and the two previously established eutherian references, A/C-60 and E/R-50. Cetacean (whale) origin, i.e., the divergence between the hippopotamus and the cetaceans, was dated to approximately 55 MYBP, while basal artiodactyl divergences were dated to >/=65 MYBP. Molecular estimates of Tertiary eutherian divergences were consistent with the fossil record.

The phylogenetic position of the Talpidae within eutheria based on analysis of complete mitochondrial sequences.

The complete mitochondrial (mt) genome of the mole Talpa europaea was sequenced and included in phylogenetic analyses together with another lipotyphlan (insectivore) species, the hedgehog Erinaceus europaeus, and 22 other eutherian species plus three outgroup taxa (two marsupials and a monotreme). The phylogenetic analyses reconstructed a sister group relationship between the mole and fruit bat Artibeus jamaicensis (order Chiroptera). The Talpa/Artibeus clade constitutes a sister clade of the cetferungulates, a clade including Cetacea, Artiodactyla, Perissodactyla, and Carnivora. A monophyletic relationship between the hedgehog and the mole was significantly rejected by maximum parsimony and maximum likelihood. Consistent with current systematic schemes, analyses of complete cytochrome b genes including the shrew Sorex araneus (family Soricidae) revealed a close relationship between Talpidae and Soricidae. The analyses of complete mtDNAs, along with the findings of other insectivore studies, challenge the maintenance of the order Lipotyphla as a taxonomic unit and support the elevation of the Soricomorpha (with the families Talpidae and Soricidae and possibly also the Solenodontidae and Tenrecidae) to the level of an order, as previously proposed in some morphological studies.

Molecular estimates of primate divergences and new hypotheses for primate dispersal and the origin of modern humans.

The concept of recent hominoid divergences has been a mainstay in molecular primatology since the 1970's. However, the ages allocated to the calibration points used to establish these divergence times and the estimates resulting from their application, notably the commonly accepted divergence between Pan (chimpanzees) and Homo 5 million years before present (MYBP), are now palaeontologically refutable. Here we estimate the ages of various primate divergences using three references with a more detailed fossil record than any of the traditional primate calibration points. Our findings suggest that the latter yield datings that are too recent by a factor of about two. For example, our estimates place the divergence between Pan and Homo 10.5-13 MYBP. The revised estimates of primate divergence times suggest a new hypothesis for primate evolution and dispersal: that the divergence between strepsirhines (lorises, lemurs) and anthropoids was contemporary with the break-up of Southern continents about 90 MYBP, with strepsirhines becoming isolated on Madagascar and later dispersing to Africa (and Asia) and anthropoids evolving in South America and subsequently colonizing Africa (and Asia), or possibly North America. In addition we present a new hypothesis, which accommodates the strikingly similar coalescence times for human mitochondrial DNA and the Y-chromosome. This hypothesis posits a common mitochondrial and Y-chromosome bottleneck about 400,000 years ago, associated with the origination of the human 2n = 46 karyotype, obstructing genetic exchange with the 2n = 48 Homo contemporaries.

Molecular studies suggest that cartilaginous fishes have a terminal position in the piscine tree.

The Chondrichthyes (cartilaginous fishes) are commonly accepted as being sister group to the other extant Gnathostomata (jawed vertebrates). To clarify gnathostome relationships and to aid in resolving and dating the major piscine divergences, we have sequenced the complete mtDNA of the starry skate and have included it in phylogenetic analysis along with three squalomorph chondrichthyans-the common dogfish, the spiny dogfish, and the star spotted dogfish-and a number of bony fishes and amniotes. The direction of evolution within the gnathostome tree was established by rooting it with the most closely related non-gnathostome outgroup, the sea lamprey, as well as with some more distantly related taxa. The analyses placed the chondrichthyans in a terminal position in the piscine tree. These findings, which also suggest that the origin of the amniote lineage is older than the age of the oldest extant bony fishes (the lungfishes), challenge the evolutionary direction of several morphological characters that have been used in reconstructing gnathostome relationships. Applying as a calibration point the age of the oldest lungfish fossils, 400 million years, the molecular estimate placed the squalomorph/batomorph divergence at approximately 190 million years before present. This dating is consistent with the occurrence of the earliest batomorph (skates and rays) fossils in the paleontological record. The split between gnathostome fishes and the amniote lineage was dated at approximately 420 million years before present.

The mitochondrial DNA molecule of the aardvark, Orycteropus afer, and the position of the Tubulidentata in the eutherian tree.

An outstanding problem in mammal phylogeny is the relationship of the aardvark (Orycteropus afer), the only living species of the order Tubulidentata, to the extant eutherian lineages. In order to examine this problem the complete mitochondrial DNA (mtDNA) molecule of the aardvark was sequenced and analysed. The aardvark tRNA-Ser (UCN) differs from that of other mammalian mtDNAs reported and appears to have reversed to the ancestral secondary structure of non-mammalian vertebrates and mitochondrial tRNAs in general. Phylogenetic analysis of 12 concatenated protein-coding genes (3325 amino acids) included the aardvark and 15 additional eutherians, two marsupials and a monotreme. The most strongly supported tree identified the aardvark as a sister group of a clade including the armadillo (Xenarthra) and the Cetferungulata (carnivores, perissodactyls, artiodactyls and cetaceans). By applying three molecular calibration points the divergence between the aardvark and armadillo-cetferungulates was estimated at ca. 90 million years before present.

Phylogenetic studies of complete mitochondrial DNA molecules place cartilaginous fishes within the tree of bony fishes.

It is commonly acknowledged that cartilaginous fishes, Chondrichthyes, have a basal position among the Gnathostomata (jawed vertebrates). In order to explore this relationship we have sequenced the complete mitochondrial genome of the spiny dogfish, Squalus acanthias, and included it in a phylogenetic analysis together with a number of bony fishes and amniotes. The phylogenetic reconstructions placed the dogfish among the bony fishes. Thus, and contrary to the common view, the analyses have shown that the position of the sharks is not basal among the gnathostomes. The presently recognized phylogenetic position of the dogfish was identified irrespective of the outgroup used, echinoderms or agnathan fishes. The lungfish was the most basal gnathostome fish, while the teleosteans had an apical position in the piscine tree. A basal position of the dogfish among the gnathostomes was statistically rejected, but the phylogenetic relationship among the coelacanth, spiny dogfish, and teleosts was not conclusively resolved. The findings challenge the current theory that sharks and other chondrichthyans, if monophyletic, are the sister group to all other extant gnathostomes. The results open to question the status of several morphological characters commonly used in piscine phylogenetic reconstruction, most notably the presence versus absence of endochondral bone in the endoskeleton, the macromeric versus micromeric structure of the exoskeleton, and the presence/absence of swimbladder and/or lung. The study also confirmed recent findings demonstrating that the origin of the amniotes is deeper than the diversification of extant bony fishes.

Molecular timing of primate divergences as estimated by two nonprimate calibration points.

The complete mitochondrial DNA (mtDNA) molecule of the hamadryas baboon, Papio hamadryas, was sequenced and included in a molecular analysis of 24 complete mammalian mtDNAs. The particular aim of the study was to time the divergence between Cercopithecoidea and Hominoidea. That divergence, set at 30 million years before present (MYBP) was a fundamental reference for the original proposal of recent hominoid divergences, according to which the split among gorilla, chimpanzee, and Homo took place 5 MYBP. In the present study the validity of the postulated 30 MYBP dating of the Cercopithecoidea/Hominoidea divergence was examined by applying two independent nonprimate molecular references, the divergence between artiodactyls and cetaceans set at 60 MYBP and that between Equidae and Rhinocerotidae set at 50 MYBP. After calibration for differences in evolutionary rates, application of the two references suggested that the Cercopithecoidea/Hominoidea divergence took place >50 MYBP. Consistent with the marked shift in the dating of the Cercopithecoidea/Hominoidea split, all hominoid divergences receive a much earlier dating. Thus the estimated date of the divergence between Pan (chimpanzee) and Homo is 10-13 MYBP and that between Gorilla and the Pan/Homo linage approximately 17 MYBP. The same datings were obtained in an analysis of clocklike evolving genes. The findings show that recalculation is necessary of all molecular datings based directly or indirectly on a Cercopithecoidea/Hominoidea split 30 MYBP.

The complete mitochondrial DNA sequence of the pig (Sus scrofa).

The complete mitochondrial genome sequence of the pig, Sus scrofa, was determined. The length of the sequence presented is 16,679 nucleotides. This figure is not absolute, however, due to pronounced heteroplasmy caused by variable numbers of the motif GTACACGTGC in the control region of different molecules. A phylogenetic study was performed on the concatenated amino acid and nucleotide sequences of 12 protein-coding genes of the mitochondrial genome. The analysis identified the pig (Suiformes) as a sister group of a cow/whale clade, making Artiodactyla paraphyletic. The split between pig and cow/whale was molecularly dated at 65 million years before present.

The complete mitochondrial DNA sequence of the rabbit, Oryctolagus cuniculus.

The nucleotide sequence of the complete mitochondrial DNA (mtDNA) molecule of the rabbit (Oryctolagus cuniculus, order Lagomorpha) was determined. The length of the molecule is 17,245 nt, but the length is not absolute due to the presence of different numbers of repeated motifs in the control region. The organization and gene contents of the mtDNA of the rabbit conform to those of other eutherian species. The putative secondary structures of the tRNAs of the rabbit have been described. These structures as well as the structure of the L-strand origin of replication comply with those characteristic for eutherians in general. The compositional differences between the two mtDNA strands have also been detailed.

The complete mitochondrial genome of Rhea americana and early avian divergences.

The complete mitochondrial DNA, mtDNA, molecule of the greater rhea, Rhea americana, was sequenced. The size of the molecule is 16,710 nucleotides. The organization of the molecule conforms with that described for the chicken and the ostrich. It has been shown previously that relative to other vertebrates the NADH3 gene of the ostrich has an insertion of one nucleotide in position 174 of the gene. The same observation was made in the rhea and in the newly sequenced NADH3 gene of the emu, Dromaius novaehollandiae. Comparison with the NADH3 gene of the chicken and the rook suggests that the inserted nucleotide may be deleted by RNA editing. The divergence between the two struthioniform species, the ostrich and the rhea, was molecularly dated at approximately 51 million years before present, MYBP. This dating is more recent than commonly acknowledged. Phylogenetic analysis of the complete cytochrome b genes of seven avian orders placed the Passeriformes basal in the avian tree with the Struthioniformes among the remaining Neognathae. These findings challenge the commonly accepted notion that the most basal avian divergence is that between the Palaeognathae and Neognathae.

The mitochondrial DNA molecule of the hagfish (Myxine glutinosa) and vertebrate phylogeny.

The vertebrates are traditionally classified into two distinct groups, Agnatha (jawless vertebrates) and Gnathostomata (jawed vertebrates). Extant agnathans are represented by hagfishes (Myxiniformes) and lampreys (Petromyzontiformes), frequently grouped together within the Cyclostomata. Whereas the recognition of the Gnathostomata as a clade is commonly acknowledged, a consensus has not been reached regarding whether or not Cyclostomata represents a clade. In the present study we have used newly established sequences of the protein-coding genes of the mitochondrial DNA molecule of the hagfish to explore agnathan and gnathostome relationships. The phylogenetic analysis of Pisces, using echinoderms as outgroup, placed the hagfish as a sister group of Vertebrata sensu stricto, i.e., the lamprey and the gnathostomes. The phylogenetic analysis of the Gnathostomata identified a basal divergence between gnathostome fishes and a branch leading to birds and mammals, i.e., between "Anamnia" and Amniota. The lungfish has a basal position among gnathostome fishes with the teleosts as the most recently evolving lineage. The findings portray a hitherto unrecognized polarity in the evolution of bony fishes. The presently established relationships are incompatible with previous molecular studies.

Analyses of mitochondrial genomes strongly support a hippopotamus-whale clade.

Although the sister-group relationship between Cetacea and Artiodactyla is widely accepted, the actual artiodactyl group which is closest to Cetacea has not been conclusively identified. In the present study, we have sequenced the complete mitochondrial genome of the hippopotamus, Hippopotamus amphibius, and included it in phylogenetic analyses together with 15 other placental mammals. These analyses separated the hippopotamus from the other suiform included, the pig, and identified the hippopotamus as the artiodactyl sister group of the cetaceans, thereby making both. Artiodactyla and the suborder. Suiformes paraphyletic. The divergence between the hippopotamid and cetacean lineages was calculated using this molecular data and was estimated at ca. 54 Ma BP.

The complete mitochondrial genome of Alligator mississippiensis and the separation between recent archosauria (birds and crocodiles).

The complete mitochondrial genome of the alligator, Alligator mississippiensis, was sequenced. The size of the molecule is 16,642 nucleotides. Previously reported rearrangements of tRNAs in crocodile mitochondrial genomes were confirmed and, relative to mammals, no other deviations of gene order were observed. The analysis of protein-coding genes of the alligator showed an evolutionary rate that is roughly the same as in mammals. Thus, the evolutionary rate in the alligator is faster than that in birds as well as that in cold-blooded vertebrates. This contradicts hypotheses of constant body temperatures or high metabolic rate being correlated with elevated molecular evolutionary rates. It is commonly acknowledged that birds are the closest living relatives to crocodiles. Birds and crocodiles represent the only archosaurian survivors of the mass extinction at the Cretaceous/Tertiary boundary. On the basis of mitochondrial protein-coding genes, the Haemothermia hypothesis, which defines birds and mammals as sister groups and thus challenges the traditional view, could be rejected. Maximum-likelihood branch length data of amino acid sequences suggest that the divergence between the avian and crocodilian lineages took place at approximately equal to 254 MYA.

The mtDNA sequence of the ostrich and the divergence between paleognathous and neognathous birds.

The complete mitochondrial DNA (mtDNA) molecule of the ostrich, Struthio camelus, was sequenced. The size of the molecule is 16,591 nucleotides. Since the ostrich represents the paleognathous birds, comparison with the mtDNA of the neognathous chicken, the only avian species reported so far in databases, made it possible to identify common and, probably, general avian mtDNA characteristics. Relative to other vertebrates, the avian NADH6 and tRNA-Glu genes are positioned upstream of the control region rather than the cytochrome b gene. The NADH3 gene of the ostrich is terminated by a stop codon at position 207. Thus, the gene is about 140 nucleotides shorter than in other vertebrates. The sequence for L-strand origin of replication is missing in both birds, and four transfer RNA genes of the two avian mtDNAs deviate from common characteristics of tRNAs of vertebrate mtDNAs by having an adenine (and not a thymidine) at position 8. Due to the absence of suitable fossils, most paleontological datings of avian divergences are conjectural. Molecular dating of the divergence between the ostrich and the chicken indicates that these two avian lineages separated 80-90 MYA. Phylogenetic analysis of complete cytochrome b genes of six avian orders showed that Passeriformes represent the earliest divergence among recent birds, contradicting the commonly accepted notion of a basal position of the Palaeognathae among recent birds.