Islet hormones from the African bullfrog Pyxicephalus adspersus (Anura:Ranidae): structural characterization and phylogenetic implications

The African bullfrog Pyxicephalus adspersus is generally classified along with frogs of the genus Rana in the subfamily Raninae of the family Ranidae but precise phylogenetic relationships between species are unclear. Pancreatic polypeptide (PP), insulin, and glucagon-like peptide (GLP-1) were isolated from an extract of P. adspersus pancreas and characterized structurally. A comparison of the amino acid sequence of Pyxicephalus PP (APSEPQHPGG(10)QATPEQLAQY(20)YSDLYQYITF(30)ITRPRF++ +. NH(2)) with those of the known amphibian PP molecules in a maximum parsimony analysis generates a single phylogenetic tree in which Pyxicephalus is the sister to the clade comprising the members of the genus Rana. The three orders of living amphibians form discrete clades with the representative of the Gymnophiona appearing as sister to the Caudata-Anura. In contrast, Pyxicephalus insulin (A chain, GIVEQCCHSA(10)CSLYDLENYC(20)N; B-chain, LANQHLCGSH(10)LVEALYMVCG(20)ERGFFYYPKS(30)) and and GLP-1 (HAEGTFTSDM(10)TSYLEEKAAK(20)EFVDWLIKGR(30)PK) resemble more closely the corresponding peptides from the cane toad Bufo marinus than the peptides from any species of Rana. Cladistic analysis based upon the amino acid sequences of insulin produced a polyphyletic assemblage with the Gymnophiona nesting within an unresolved clade containing the non-ranid frogs. The data support the assertion that the amino acid sequence of PP, but not those of the other islet hormones, is of value as a molecular marker for inferring phylogenetic relationships between early tetrapod species.     

Seed plant phylogeny inferred from all three plant genomes: monophyly of extant gymnosperms and origin of Gnetales from conifers

Phylogenetic relationships among the five groups of extant seed plants are presently quite unclear. For example, morphological studies consistently identify the Gnetales as the extant sister group to angiosperms (the so-called "anthophyte" hypothesis), whereas a number of molecular studies recover gymnosperm monophyly, and few agree with the morphology-based placement of Gnetales. To better resolve these and other unsettled issues, we have generated a new molecular data set of mitochondrial small subunit rRNA sequences, and have analyzed these data together with comparable data sets for the nuclear small subunit rRNA gene and the chloroplast rbcL gene. All nuclear analyses strongly ally Gnetales with a monophyletic conifers, whereas all mitochondrial analyses and those chloroplast analyses that take into account saturation of third-codon position transitions actually place Gnetales within conifers, as the sister group to the Pinaceae. Combined analyses of all three genes strongly support this latter relationship, which to our knowledge has never been suggested before. The combined analyses also strongly support monophyly of extant gymnosperms, with cycads identified as the basal-most group of gymnosperms, Ginkgo as the next basal, and all conifers except for Pinaceae as sister to the Gnetales + Pinaceae clade. According to these findings, the Gnetales may be viewed as extremely divergent conifers, and the many morphological similarities between angiosperms and Gnetales (e.g., double fertilization and flower-like reproductive structures) arose independently.     

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.     

Global patterns of diversification in the history of modern amphibians

The fossil record of modern amphibians (frogs, salamanders, and caecilians) provides no evidence for major extinction or radiation episodes throughout most of the Mesozoic and early Tertiary. However, long-term gradual diversification is difficult to reconcile with the sensitivity of present-day amphibian faunas to rapid ecological changes and the incidence of similar environmental perturbations in the past that have been associated with high turnover rates in other land vertebrates. To provide a comprehensive overview of the history of amphibian diversification, we constructed a phylogenetic timetree based on a multigene data set of 3.75 kb for 171 species. Our analyses reveal several episodes of accelerated amphibian diversification, which do not fit models of gradual lineage accumulation. Global turning points in the phylogenetic and ecological diversification occurred after the end-Permian mass extinction and in the late Cretaceous. Fluctuations in amphibian diversification show strong temporal correlation with turnover rates in amniotes and the rise of angiosperm-dominated forests. Approximately 86% of modern frog species and >81% of salamander species descended from only five ancestral lineages that produced major radiations in the late Cretaceous and early Tertiary. This proportionally late accumulation of extant lineage diversity contrasts with the long evolutionary history of amphibians but is in line with the Tertiary increase in fossil abundance toward the present.     

Phylogeny, evolution, and biogeography of Asiatic Salamanders (Hynobiidae)

We sequenced 15 complete mitochondrial genomes and performed comprehensive molecular phylogenetic analyses to study the origin and phylogeny of the Hynobiidae, an ancient lineage of living salamanders. Our phylogenetic analyses show that the Hynobiidae is a clade with well resolved relationships, and our results contrast with a morphology-based phylogenetic hypothesis. These salamanders have low vagility and are limited in their distribution primarily by deserts, mountains, and oceans. Our analysis suggests that the relationships among living hynobiids have been shaped primarily by geography. We show that four-toed species assigned to Batrachuperus do not form a monophyletic group, and those that occur in Afghanistan and Iran are transferred to the resurrected Paradactylodon. Convergent morphological characters in different hynobiid lineages are likely produced by similar environmental selective pressures. Clock-independent molecular dating suggests that hynobiids originated in the Middle Cretaceous [ approximately 110 million years ago (Mya)]. We propose an "out of North China" hypothesis for hynobiid origins and hypothesize an ancestral stream-adapted form. Given the particular distributional patterns and our molecular dating estimates, we hypothesize that: (i) the interior desertification from Mongolia to Western Asia began approximately 50 Mya; (ii) the Tibetan plateau (at least on the eastern fringe) experienced rapid uplift approximately 40 Mya and reached an altitude of at least 2,500 m; and (iii) the Ailao-Red River shear zone underwent the most intense orogenic movement approximately 24 Mya.     

Multiple forms of gonadotropin-releasing hormone in amphibian brains

Several forms of gonadotropin-releasing hormone (GnRH)-like molecules were found in brains of both anurans (frogs) and urodeles (salamanders). The presence of the mammalian-like GnRH molecule was confirmed by HPLC and cross-reactivity studies. Small amounts of salmonid-like GnRH molecules in the brains of frogs (Rana pipiens, Hyla regilla) and salamanders (Taricha granulosa, Ambystoma gracile) were detected by comparing the HPLC chromatographic pattern and immunological reactivity of the brain extracts with native trout and synthetic salmon GnRH. This nonmammalian form of GnRH in the amphibian brain is similar and perhaps identical, at least by indirect evidence, to a form of GnRH reported earlier to be in sympathetic ganglion, retina, chromaffin tissue, and tadpole brain. If two of the amphibian GnRH molecules prove to be mammalian and salmon GnRH, then it is likely that two separate genes in amphibians code for the distinct primary structures of the molecules. The most parsimonious interpretation of the presence of both mammalian- and salmon-like GnRH in anurans and urodeles is that a common phylogenetic ancestor also possessed the two forms of GnRH. Thus the mammalian form of GnRH may well have been present in labyrinthodont amphibians. Independent of evolutionary origin, the functions of the different GnRH molecules in amphibians are unknown.     

Multiple independent origins of mitochondrial gene order in birds

Mitochondrial genomes of all vertebrate animals analyzed to date have the same 37 genes, whose arrangement in the circular DNA molecule varies only in the relative position of a few genes. This relative conservation suggests that mitochondrial gene order characters have potential utility as phylogenetic markers for higher-level vertebrate taxa. We report discovery of a mitochondrial gene order that has had multiple independent originations within birds, based on sampling of 137 species representing 13 traditionally recognized orders. This provides evidence of parallel evolution in mitochondrial gene order for animals. Our results indicate operation of physical constraints on mitochondrial gene order changes and support models for gene order change based on replication error. Bird mitochondria have a displaced O_L (origin of light-strand replication site) as do various other Reptilia taxa prone to gene order changes. Our findings point to the need for broad taxonomic sampling in using mitochondrial gene order for phylogenetic analyses. We found, however, that the alternative mitochondrial gene orders distinguish the two primary groups of songbirds (order Passeriformes), oscines and suboscines, in agreement with other molecular as well as morphological data sets. Thus, although mitochondrial gene order characters appear susceptible to some parallel evolution because of mechanistic constraints, they do hold promise for phylogenetic studies.     

Multiple molecular evidences for a living mammalian fossil

Laonastes aenigmamus is an enigmatic rodent first described in 2005. Molecular and morphological data suggested that it is the sole representative of a new mammalian family, the Laonastidae, and a member of the Hystricognathi. However, the validity of this family is controversial because fossil-based phylogenetic analyses suggest that Laonastes is a surviving member of the Diatomyidae, a family considered to have been extinct for 11 million years. According to these data, Laonastes and Diatomyidae are the sister clade of extant Ctenodactylidae (i.e., gundies) and do not belong to the Hystricognathi. To solve the phylogenetic position of Laonastes, we conducted a large-scale molecular phylogeny of rodents. The analysis includes representatives of all major rodent taxonomic groups and was based on 5.5 kb of sequence data from four nuclear and two mitochondrial genes. To further validate the obtained results, a short interspersed element insertion analysis including 11 informative loci was also performed. Our molecular data based on sequence and short interspersed element analyses unambiguously placed Laonastes as a sister clade of gundies. All alternative hypotheses were significantly rejected based on Shimodaira-Hasegawa tests, supporting the idea that Laonastes does not belong to the Hystricognathi. Molecular dating analysis also supports an ancient divergence, approximately 44 Mya ago, between Ctenodactylidae and Laonastes. These combined analyses support the hypothesis that Laonastes is indeed a living fossil. Protection of this surviving species would conserve an ancient mammalian family.     

The evolution of coloration and toxicity in the poison frog family (Dendrobatidae)

The poison frogs (family Dendrobatidae) are terrestrial anuran amphibians displaying a wide range of coloration and toxicity. These frogs generally have been considered to be aposematic, but relatively little research has been carried out to test the predictions of this hypothesis. Here we use a comparative approach to test one prediction of the hypothesis of aposematism: that coloration will evolve in tandem with toxicity. Recently, we developed a phylogenetic hypothesis of the evolutionary relationships among representative species of poison frogs, using sequences from three regions of mitochondrial DNA. In our analysis, we use that DNA-based phylogeny and comparative analysis of independent contrasts to investigate the correlation between coloration and toxicity in the poison frog family (Dendrobatidae). Information on the toxicity of different species was obtained from the literature. Two different measures of the brightness and extent of coloration were used. (i) Twenty-four human observers were asked to rank different photos of each different species in the analysis in terms of contrast to a leaf-littered background. (ii) Color photos of each species were scanned into a computer and a computer program was used to obtain a measure of the contrast of the colors of each species relative to a leaf-littered background. Comparative analyses of the results were carried out with two different models of character evolution: gradual change, with branch lengths proportional to the amount of genetic change, and punctuational change, with all change being associated with speciation events. Comparative analysis using either method or model indicated a significant correlation between the evolution of toxicity and coloration across this family. These results are consistent with the hypothesis that coloration in this group is aposematic.     

Plant mitochondrial nucleic acid sequences as a tool for phylogenetic analysis.

To evaluate the potential of mitochondrial nucleic acid sequences as a phylogenetic tool, we have analyzed cytochrome oxidase subunit III (coxIII) coding sequences in representatives of the major groups of land plants. The phylogenetic tree derived from these mitochondrial sequences confirms the monophyletic origin of land plant mitochondria with the general order and descent of land plants deduced by other molecular, physiological, and morphological traits. The mitochondrial sequences strongly suggest a close phylogenetic relationship between Bryophyta and Lycopodiatae, whereas Psilophytatae cluster with the other vascular plants. In addition to the high sequence similarity, both Hepaticophytina and Lycopodiatae contain a related intron in the coxIII gene that, to our knowledge, is not found in any other plant species. The slowly evolving mitochondrial sequences of plants are shown to provide a useful phylogenetic tool to evaluate distant evolutionary relationships within this kingdom.     

Morphometrics and hominoid phylogeny: Support for a chimpanzee-human clade and differentiation among great ape subspecies

Taxonomic and phylogenetic analyses of great apes and humans have identified two potential areas of conflict between molecular and morphological data: phylogenetic relationships among living species and differentiation of great ape subspecies. Here we address these problems by using morphometric data. Three-dimensional landmark data from the hominoid temporal bone effectively quantify the shape of a complex element of the skull. Phylogenetic analysis using distance-based methods corroborates the molecular consensus on African ape and human phylogeny, strongly supporting a Pan-Homo clade. Phenetic differentiation of great ape subspecies is pronounced, as suggested previously by mitochondrial DNA and some morphological studies. These results show that the hominoid temporal bone contains a strong phylogenetic signal and reveal the potential for geometric morphometric analysis to shed light on phylogenetic relationships.     

Molecular phylogeny of trematodes in Family Heterophyidae based on mitochondrial cytochrome c oxidase subunit I (mCOI)

ObjectiveTo analyze a phylogenetic tree for understanding the molecular systematic of trematode in Family Heterophyidae, which are highly distributed in Thailand.MethodsBased on thirteen sequences of mitochondrial cytochrome c oxidase subunit I (mCOI) gene from six genera of heterophyid trematodes, viz. Haplorchis, Stellantchasmus, Centrocestus, Metagonimus, Pygidopsis, and Haplorchoides were aligned automatically using the Clustal × 2.0 program. A phylogenetic tree was constructed by maximum likeinghood (ML) and neighbor-joining (NJ) methods, with 1 000 bootstrap using the 5.0 program.ResultsThe phylogenetic relationship from both methods was similar and separated into three groups consisting of Haplorchoides pumilio group, Haplorchoides taichui group and another heterophyid genera.ConclusionsThe sequence data of mtCOI can be used to investigate the phylogenetic relationships of trematodes at the genus level. Each clade of different genera of heterophyid trematodes can be separated into sister groups that correlated with the morphological characteristic, kind of secondary intermediate host and geographic distribution.     

Biodiversity of Costa Rican salamanders: implications of high levels of genetic differentiation and phylogeographic structure for species formation

Although salamanders are characteristic amphibians in Holarctic temperate habitats, in tropical regions they have diversified evolutionarily only in tropical America. An adaptive radiation centered in Middle America occurred late in the history of a single clade, the supergenus Bolitoglossa (Plethodontidae), and large numbers of species now occur in diverse habitats. Sublineages within this clade decrease in number from the northern to southern parts of Middle America, and in Costa Rica, there are but three. Despite this phylogenetic constraint, Costa Rica has many species; the number of salamander species on one local elevational transect in the Cordillera de Talamanca may be the largest for any such transect in the world. Extraordinary variation in sequences of the mitochondrial gene cytochrome b within a clade of the genus Bolitoglossa in Costa Rica reveals strong phylogeographic structure within a single species, Bolitoglossa pesrubra. Allozymic variation in 19 proteins reveals a pattern largely concordant with the mitochondrial DNA phylogeography. More species exist than are currently recognized. Diversification occurs in restricted geographic areas and involves sharp geographic and elevational differentiation and zonation. In their degree of genetic differentiation at a local scale, these species of the deep tropics exceed the known variation of extratropical salamanders, which also differ in being less restricted in elevational range. Salamanders display "tropicality" in that although speciose, they are usually local in distribution and rare. They display strong ecological and physiological differentiation that may contribute importantly to morphological divergence and species formation.     

Phylogenetic relationships among megabats, microbats, and primates.

We present 744 nucleotide base positions from the mitochondrial 12S rRNA gene and 236 base positions from the mitochondrial cytochrome oxidase subunit I gene for a microbat, Brachyphylla cavernarum, and a megabat, Pteropus capestratus, in phylogenetic analyses with homologous DNA sequences from Homo sapiens, Mus musculus (house mouse), and Gallus gallus (chicken). We use information on evolutionary rate differences for different types of sequence change to establish phylogenetic character weights, and we consider alternative rRNA alignment strategies in finding that this mtDNA data set clearly supports bat monophyly. This result is found despite variations in outgroup used, gap coding scheme, and order of input for DNA sequences in multiple alignment bouts. These findings are congruent with morphological characters including details of wing structure as well as cladistic analyses of amino acid sequences for three globin genes and indicate that neurological similarities between megabats and primates are due to either retention of primitive characters or to convergent evolution rather than to inheritance from a common ancestor. This finding also indicates a single origin for flight among mammals.     

Mammalian mitogenomic relationships and the root of the eutherian tree

The strict orthology of mitochondrial (mt) coding sequences has promoted their use in phylogenetic analyses at different levels. Here we present the results of a mitogenomic study (i.e., analysis based on the set of protein-coding genes from complete mt genomes) of 60 mammalian species. This number includes 11 new mt genomes. The sampling comprises all but one of the traditional eutherian orders. The previously unrepresented order Dermoptera (flying lemurs) fell within Primates as the sister group of Anthropoidea, making Primates paraphyletic. This relationship was strongly supported. Lipotyphla ("insectivores") split into three distinct lineages: Erinaceomorpha, Tenrecomorpha, and Soricomorpha. Erinaceomorpha was the basal eutherian lineage. Sirenia (dugong) and Macroscelidea (elephant shrew) fell within the African clade. Pholidota (pangolin) joined the Cetferungulata as the sister group of Carnivora. The analyses identified monophyletic Pinnipedia with Otariidae (sea lions, fur seals) and Odobenidae (walruses) as sister groups to the exclusion of Phocidae (true seals).     

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 α-β 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.     

Evolutionary relationships of the coelacanth, lungfishes, and tetrapods based on the 28S ribosomal RNA gene.

The origin of land vertebrates was one of the major transitions in the history of vertebrates. Yet, despite many studies that are based on either morphology or molecules, the phylogenetic relationships among tetrapods and the other two living groups of lobe-finned fishes, the coelacanth and the lungfishes, are still unresolved and debated. Knowledge of the relationships among these lineages, which originated back in the Devonian, has profound implications for the reconstruction of the evolutionary scenario of the conquest of land. We collected the largest molecular data set on this issue so far, about 3,500 base pairs from seven species of the large 28S nuclear ribosomal gene. All phylogenetic analyses (maximum parsimony, neighbor-joining, and maximum likelihood) point toward the hypothesis that lungfishes and coelacanths form a monophyletic group and are equally closely related to land vertebrates. This evolutionary hypothesis complicates the identification of morphological or physiological preadaptations that might have permitted the common ancestor of tetrapods to colonize land. This is because the reconstruction of its ancestral conditions would be hindered by the difficulty to separate uniquely derived characters from shared derived characters in the coelacanth/lungfish and tetrapod lineages. This molecular phylogeny aids in the reconstruction of morphological evolutionary steps by providing a framework; however, only paleontological evidence can determine the sequence of morphological acquisitions that allowed lobe-finned fishes to colonize land.     

Pegasoferae, an unexpected mammalian clade revealed by tracking ancient retroposon insertions

Despite the recent large-scale efforts dedicated to comprehensive phylogenetic analyses using mitochondrial and nuclear DNA sequences, several relationships among mammalian orders remain controversial. Here, we present an extensive application of retroposon (L1) insertion analysis to the phylogenetic relationships among almost all mammalian orders. In addition to demonstrating the validity of Glires, Euarchontoglires, Laurasiatheria, and Boreoeutheria, we demonstrate an interordinal clade that links Chiroptera, Carnivora, and Perissodactyla within Laurasiatheria. Re-examination of a large DNA sequence data set yielded results consistent with our conclusion. We propose a superordinal name "Pegasoferae" for this clade of Chiroptera + Perissodactyla + Carnivora + Pholidota. The presence of a single incongruent L1 locus generates a tree in which the group of Carnivora + Perissodactyla associates with Cetartiodactyla but not with Chiroptera. This result suggests that incomplete lineage sorting of an ancestral dimorphism occurred with regard to the presence or absence of retroposon alleles in a common ancestor of Scrotifera (Pegasoferae + Cetartiodactyla), which was followed by rapid divergence into the extant orders over an evolutionarily short period. Accordingly, Euungulata (Cetartiodactyla + Perissodactyla) and Fereuungulata (Carnivora + Pholidota + Perissodactyla + Cetartiodactyla) cannot be validated as natural groups. The interordinal mammalian relationships presented here provide a cornerstone for future studies in the reconstruction of mammalian classifications, including extinct species, on evolution of large genomic sequences and structure, and in developmental analysis of morphological diversification.     

Multiple, recurring origins of aposematism and diet specialization in poison frogs

Aposematism is the association, in a prey organism, of the presence of a warning signal with unprofitability to predators. The origin of aposematism is puzzling, because of its predicted low probability of establishment in a population due to the prey's increased conspicuousness. Aposematism is a widespread trait in invertebrate taxa, but, in vertebrates, it is mostly evident in amphibians, reptiles, and fishes. Poison frogs (Dendrobatidae) are one of the most well known examples of the co-occurrence of warning coloration and toxicity. This monophyletic group of mostly diurnal leaf-litter Neotropical anurans has both toxic/colorful and palatable/cryptic species. Previous studies suggested a single origin of toxicity and warning coloration, dividing the family in two discrete groups of primitively cryptic and more derived aposematic frogs. Recent molecular phylogenetic analyses using mostly aposematic taxa supported this conclusion and proposed a single tandem origin of toxicity and conspicuous warning coloration. By using expanded taxon and character sampling, we reexamined the phylogenetic correlation between the origins of toxicity and warning coloration. At least four or five independent origins of aposematism have occurred within poison frogs; by using simulations, we rejected hypotheses of one, two, or three origins of aposematism (P < 0.002). We also found that diet specialization is linked with the evolution of aposematism. Specialization on prey, such as ants and termites, may have evolved independently at least two times.     

On the evolutionary origin of the plant mitochondrion and its genome

Higher plants occupy very different positions in the mitochondrial and nuclear lineages of global phylogenetic trees based on conserved regions of small subunit (SSU) and large subunit (LSU) rRNA sequences. In the nuclear subtree, plants branch off late, at a position reflecting a massive radiation of the major multicellular (and some unicellular) groups; in the mitochondrial subtree, in contrast, plants branch off early, near the point of connection between the mitochondrial and eubacterial lineages. Moreover, in the nuclear lineage, plants branch together with the unicellular green alga Chlamydomonas reinhardtii, whereas in the mitochondrial lineage (in both SSU and LSU trees), metaphytes and chlorophyte branch separately. Statistical evaluation indicates that the anomalous branching position of higher plants in the mitochondrial lineage is not a treeing artifact attributable to the relatively rapid rate of sequence divergence of non-plant mitochondrial rRNA sequences. In considering alternative biological explanations for these results, we are led to propose that the rRNA genes in plant mitochondria may be of more recent evolutionary origin than the rRNA genes in other mitochondria. This proposal has implications for monophyletic vs. polyphyletic scenarios of mitochondrial origin and is consistent with other evidence indicating that plant mtDNA is an evolutionary mosaic.