Phylogenetic and expression analysis of the basic helix-loop-helix transcription factor gene family: genomic approach to cellular differentiation

A phylogenetic analysis of seven different species (human, mouse, rat, worm, fly, yeast, and plant) utilizing all (541) basic helix-loop-helix (bHLH) genes identified, including expressed sequence tags (EST), was performed. A super-tree involving six clades and a structural categorization involving the entire coding sequence was established. A nomenclature was developed based on clade distribution to discuss the functional and ancestral relationships of all the genes. The position/location of specific genes on the phylogenetic tree in relation to known bHLH factors allows for predictions of the potential functions of uncharacterized bHLH factors, including EST's. A genomic analysis using microarrays for four different mouse cell types (i.e. Sertoli, Schwann, thymic, and muscle) was performed and considered all known bHLH family members on the microarray for comparison. Cell-specific groups of bHLH genes helped clarify those bHLH genes potentially involved in cell specific differentiation. This phylogenetic and genomic analysis of the bHLH gene family has revealed unique aspects of the evolution and functional relationships of the different genes in the bHLH gene family.     

Marmot phylogeny revisited: molecular evidence for a diphyletic origin of sociality

We established the phylogeny of 11 species of the genus Marmota based on the entire sequence of the mitochondrial cytochrome b ( cyt-b ) gene (1.1 kb) and a partial sequence of the NADH dehydrogenase subunit 4 ( ND4 ) gene (1.2 kb). In three species ( Marmota caligata , Marmota olympus , and Marmota bobac ) full-sized nuclear pseudogenes of the mitochondrial cyt-b were identified. The mitochondrial cyt-b genes and the three pseudogenes form separate clusters in the maximum parsimony dendrogram. This finding suggests that the pseudogenes originated from a single transfer to the nucleus that may have occurred prior to the radiation of the genus Marmota . Notably, compared with their functional mitochondrial equivalents the pseudogenes show a much lower substitution rate. In the dendrograms deduced from the mitochondrial sequences two distinct clusters become apparent: one cluster consists of the North-west American species, the other contains the Eurasian species together with the North American species Marmota monax . The position of M. monax as a member of the Eurasian clade is in accordance with the evolution of chromosome numbers. The results are of special interest with respect to the evolution of social systems in the genus that vary from solitary species ( M. monax ) to highly social species living in family groups (e.g. Marmota marmota ). The molecular phylogeny suggests a diphyletic origin of high sociality in the genus Marmota .     

Phylogeny of eagles, Old World vultures, and other Accipitridae based on nuclear and mitochondrial DNA

We assessed phylogenetic relationships for birds of prey in the family Accipitridae using molecular sequence from two mitochondrial genes (1047 bases ND2 and 1041 bases cyt-b) and one nuclear intron (1074 bases beta-fibrinogen intron 7). We sampled representatives of all 14 Accipitridae subfamilies, focusing on four subfamilies of eagles (booted eagles, sea eagles, harpy eagles, and snake eagles) and two subfamilies of Old World vultures (Gypaetinae and Aegypiinae) with nearly all known species represented. Multiple well-supported relationships among accipitrids identified with DNA differ from those traditionally recognized based on morphology or life history traits. Monophyly of sea eagles (Haliaeetinae) and booted eagles (Aquilinae) was supported; however, harpy eagles (Harpiinae), snake eagles (Circaetinae), and Old World vultures were found to be non-monophyletic. The Gymnogene (Polyboroides typus) and the Crane Hawk (Geranospiza caerulescens) were not found to be close relatives, presenting an example of convergent evolution for specialized limb morphology enabling predation on cavity nesting species. Investigation of named subspecies within Hieraaetus fasciatus and H. morphnoides revealed significant genetic differentiation or non-monophyly supporting recognition of H. spilogaster and H. weiskei as distinctive species.     

Genetic diversity and phylogenetic relationship among the western and the Asian honey bees based on two mitochondrial gene segments (COI and ND5)

The Asian honey bee species i.e., Apis cerana (the eastern honey bee), A. dorsata (the giant honey bee), and the western or European honey bee (A. mellifera) collected from Pakistan were studied using partial sequences from two mitochondrial genes (i) the Cytochrome c oxidase I (COI) and (ii) the mitochondrially encoded NADH dehydrogenase 5 (ND5) and then compared with other honey bees sequences (already submitted from different countries around the globe) obtained after the national center for biotechnology information (NCBI). DNA sequences were analyzed employing molecular evolutionary genetics analysis and Kimura 2-parameter model, neighbor-joining method was applied to investigate phylogenetic relationships, and DNA sequence polymorphism was applied to measure the genetic diversity within the genus Apis. The phylogenetic analyses yielded consistent results. Based on COI gene fragment in two Asian and European honey bee species from Pakistan and from other countries showed considerable genetic diversity levels and deviation among the species. While in contrast the phylogenetic analyses based on ND5 gene fragment in Asian and European honey bee species from Pakistan and other countries showed comparatively higher genetic diversity indices and variations than the COI gene. So, in the genus Apis, the mitochondrial ND5 region has shown the possibility to answer the interactions among species. A further detailed work (by linking the analysis of other genomic and mitochondrial genes) is required for good quality solution to establish the concise genetic diversity and interaction among the Apis species. The objective of this study was to explore the extent of genetic differences and phylogenetic links among the three kinds of honey bee species from Pakistan and comparing them with other bee species around the globe.     

Phylogenetic analysis of Oryza species, based on simple sequence repeats and their flanking nucleotide sequences from the mitochondrial and chloroplast genomes

Simple sequence repeats (SSR) and their flanking regions in the mitochondrial and chloroplast genomes were sequenced in order to reveal DNA sequence variation. This information was used to gain new insights into phylogenetic relationships among species in the genus Oryza. Seven mitochondrial and five chloroplast SSR loci equal to or longer than ten mononucleotide repeats were chosen from known rice mitochondrial and chloroplast genome sequences. A total of 50 accessions of Oryza that represented six different diploid genomes and three different allopolyploid genomes of Oryza species were analyzed. Many base substitutions and deletions/insertions were identified in the SSR loci as well as their flanking regions. Of mononucleotide SSR, G (or C) repeats were more variable than A (or T) repeats. Results obtained by chloroplast and mitochondrial SSR analyses showed similar phylogenetic relationships among species, although chloroplast SSR were more informative because of their higher sequence diversity. The CC genome is suggested to be the maternal parent for the two BBCC genome species (O. punctata and O. minuta) and the CCDD species O. latifolia, based on the high level of sequence conservation between the diploid CC genome species and these allotetraploid species. This is the first report of phylogenetic analysis among plant species, based on mitochondrial and chloroplast SSR and their flanking sequences.     

Utility and evolution of cytochrome b in insects

Cytochrome b (cyt-b) is widely used in molecular phylogenetic studies of vertebrate, but not invertebrate, taxa. To determine whether this situation is an historical accident or reflects the utility of cyt-b, we compared the abilities of cyt-b, COI, and one nuclear ribosomal gene region (D1 of 28S) to recover intergeneric relationships within the tiger moth tribes Ctenuchini and Euchromiini. Additionally, we compared the rate of sequence and amino acid evolution of cyt-b across insects. Cytochrome b had the same level of sequence variation and A/T bias as COI, but was less useful for recovering intergeneric relationships. The total evidence tree casts doubt on the traditional taxonomy of the group. For the class Insecta, we found that functional conservation of amino acids occurs for the same regions as those found in vertebrates with the exception of Mallophaga (lice). Lice have an accelerated rate of nonsynonymous substitutions. Accelerated rate of cyt-b nucleotide and amino acid evolution in Apidae (bees) may be correlated with increased metabolic rates associated with facultative endothermy (= heterothermy).     

The complete mitochondrial genome of the Korean soft-shelled turtle Pelodiscus sinensis (Testudines, Trionychidae).

We isolated Korean soft-shelled turtle, Pelodiscus sinensis, mitochondrial DNA by long-polymerase chain reaction (long-PCR) with conserved primers and sequenced this mitochondrial genome (mitogenome) with primer walking using flanking sequences. The P. sinensis mitochondrial DNA has 17,042 bp and its structural organization is conserved compared to those of other reptiles and mammals. To unveil the phylogenetic relationship of the turtles, we used the NJ, MP, and ML analysis methods after inferring those sequences from the mitochondrial 16S rRNA gene. We also compared two P. sinensis variants from Korea and China using the mitochondrial genome. In this study, we report the basic characteristics of the P. sinensis mitochondrial genome, including structural organization and base composition of the rRNAs, tRNAs and protein-coding genes, as well as characteristics of tRNAs. These features are applicable for the study of phylogenetic relationships in turtles.     

Phylogeography of two European Reticulitermes (Isoptera) species: the Iberian refugium

Chemical, i.e. cuticular hydrocarbons, and molecular data were used to probe the phylogeography of Reticulitermes termites collected from various parts of France, Spain and Portugal. Phylogenetic relationships were inferred from sequences of the internal transcribed spacer (ITS2) of nuclear ribosomal RNA genes as well as from two partial mitochondrial DNA segments, the cytochrome oxidase II gene and a sequence combining the tRNA-Leu gene and fragments of the NADH dehydrogenase I and ribosomal 16S genes. Two species, namely, R. grassei and R. banyulensis, were identified based on an analysis of cuticular hydrocarbons and the identification was confirmed by ITS2 haplotyping. However, phylogeny based on the analysis of mitochondrial DNA was not completely in agreement with the conclusions drawn from the chemical and nuclear data. An analysis of 56 R. grassei colonies revealed intraspecific differentiation into two major lineages with distinct geographical ranges. Whereas analysis of cuticular hydrocarbons showed that R. banyulensis was chemically distinct from R. grassei, analysis of mitochondrial DNA showed its close kinship with the R. grassei lineage occurring in southern Spain. This kinship could be explained by their evolution from a common polymorphic ancestor species in this ice age refugium.     

Geographic variation and diversity of the cytochrome b gene in Japanese wild populations of medaka, Oryzias latipes

We conducted a polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome b gene to elucidate the detailed genetic population structure of Japanese wild populations of medaka, Oryzias latipes. The analysis of 1,225 specimens collected from 303 sites identified 67 mitotypes. Subsequently we determined the nucleotide sequences of the complete cytochrome b gene (1141-bp) to clarify the phylogenetic relationships among mitotypes. The phylogenetic tree based on nucleotide sequences indicated three major clades (A, B and C) that differed by 11.3-11.8%, corresponding to three clusters previously identified by RFLP analysis of entire mitochondrial DNAs. The geographic distribution of mitotypes in clades A and B was fully concordant with the Northern and Southern Populations defined by allozymes. Clade A could be subdivided into three subclades and clade B into eleven, with sequence divergences among subclades of 1.3-5.8%. Each distribution of mitotypes in subclades roughly corresponded to that of mtDNA haplotypes in subclusters previously identified. Mitotypes in clade C were found only in the Kanto district. The phylogenetic relationships and the estimated divergence times suggest that three Japanese clades originated from a common ancestor and were separated during the Pliocene, and that the regional differentiation of subclades was closely connected with the geological history of the Quaternary. This study has also demonstrated the possibility of artificial disturbance of natural distribution especially in the Kanto district and the superior efficacy of PCR-RFLP analysis as a simple method for detecting genetic variation and artificial gene flow of medaka.     

Genotypic differentiation of twelve Clostridium species by polymorphism analysis of the triosephosphate isomerase (tpi) gene

Housekeeping genes encoding metabolic enzymes may provide alternative markers to 16S ribosomal DNA (rDNA) for genotypic and phylogenetic characterization of bacterial species. We have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay, targeting the triosephosphate isomerase (tpi) gene, which allows the differentiation of twelve pathogenic Clostridium species. Degenerate primers constructed from alignments of tpi sequences of various gram-positive bacteria allowed the amplification of a 501 bp target region in the twelve Clostridium type strains. A phylogenetic tree constructed from the nucleotidic sequences of these tpi amplicons was well correlated with that inferred from analysis of 16S rDNA gene sequences. The analysis of tpi sequences revealed restriction sites of enzyme AluI that could be species-specific. Indeed, AluI digestion of amplicons from the twelve type strains provided distinct restriction patterns. A total of 127 strains (three to sixteen strains for each species) was further analyzed by PCR-RFLP of the tpi gene, and confirmed that each species could be characterized by one to three restriction types (RTs). The differences between RTs within species could be explained by point mutations in AluI restriction sites of the tpi sequences. PCR-restriction analysis of the tpi gene offers an accurate tool for species identification within the genus Clostridium, and provides an alternative marker to 16S rDNA for phylogenetic analyses.     

Molecular systematics of pikas (genus Ochotona) inferred from mitochondrial DNA sequences

The phylogenetic relationships among worldwide species of genus Ochotona were investigated by sequencing mitochondrial cytochrome b and ND4 genes. Parsimony and neighbor-joining analyses of the sequence data yielded congruent results that strongly indicated three major clusters: the shrub-steppe group, the northern group, and the mountain group. The subgeneric classification of Ochotona species needs to be revised because each of the two subgenera in the present classification contains species from the mountain group. To solve this taxonomic problem so that each taxon is monophyletic, i.e. , represents a natural clade, Ochotona could be divided into three subgenera, one for the shrub-steppe species, a second for the northern species, and a third for the mountain species. The inferred tree suggests that the differentiation of this genus in the Palearctic Region was closely related to the gradual uplifting of the Tibet (Qinghai-Xizang) Plateau, as hypothesized previously, and that vicariance might have played a major role in the differentiation of this genus on the Plateau. On the other hand, the North American species, O. princeps, is most likely a dispersal event, which might have happened during the Pliocene through the opening of the Bering Strait. The phylogenetic relationships within the shrub-steppe group are worth noting in that instead of a monophyletic shrub-dwelling group, shrub dwellers and steppe dwellers are intermingled with each other. Moreover, the sequence divergence within the sister taxa of one steppe dweller and one shrub dweller is very low. These findings support the hypothesis that pikas have entered the steppe environment several times and that morphological similarities within steppe dwellers were due to convergent evolution.     

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.     

基于16S rRNA和Cyt b基因序列的鰶亚科系统发育研究

对鰶亚科4属5种鱼类的线粒体基因组16S rRNA和Cyt b基因片段序列进行序列比较和系统发育关系分析。结果显示:5种鰶亚科鱼类的16S rRNA和Cy tb基因片段同源序列长度分别为525 bp和402 bp,序列联合后的序列总长度为927 bp,其中多态位点178个,简约信息位点123个。选取太平洋鲱Clupea pallasii和大西洋鲱C.harengus为外类群,采用邻接法(NJ)、最大简约法(MP)、最大似然法(ML)和贝叶斯法(BI)分别对2个基因片段序列进行了聚类分析,并联合2个基因片段利用邻接法、最大简约法和贝叶斯法进行分析。系统发育分析显示:斑鰶Konosirus punctatus与花鰶Clupanodon thriss亲缘关系最近,分布于美洲大陆的真鰶属Dorosoma鱼类与印度洋、太平洋分布的斑鰶属、花鰶属和海鰶属Nematalosa鱼类亲缘关系较远。     

Species boundaries and phylogenetic relationships in the critically endangered Asian box turtle genus Cuora

Turtles are currently the most endangered major clade of vertebrates on earth, and Asian box turtles (Cuora) are in catastrophic decline. Effective management of this diverse turtle clade has been hampered by human-mediated, and perhaps natural hybridization, resulting in discordance between mitochondrial and nuclear markers and confusion regarding species boundaries and phylogenetic relationships among hypothesized species of Cuora. Here, we present analyses of mitochondrial and nuclear DNA data for all 12 currently hypothesized species to resolve both species boundaries and phylogenetic relationships. Our 15-gene, 40-individual nuclear data set was frequently in conflict with our mitochondrial data set; based on its general concordance with published morphological analyses and the strength of 15 independent estimates of evolutionary history, we interpret the nuclear data as representing the most reliable estimate of species boundaries and phylogeny of Cuora. Our results strongly reiterate the necessity of using multiple nuclear markers for phylogeny and species delimitation in these animals, including any form of DNA "barcoding", and point to Cuora as an important case study where reliance on mitochondrial DNA can lead to incorrect species identification.     

A phylogeny of the Caniformia (order Carnivora) based on 12 complete protein-coding mitochondrial genes

Evolutionary relationships of the order Carnivora have been extensively studied. However, phylogenetic studies based on different types of data, species samples, and methods of analysis provide contradictory results. Consequently, phylogenetic relationships of Carnivora remain contentious. Here, the sequence of 12 mitochondrial genes (10,842 nucleotides) from a total of 38 carnivore species was used to investigate the phylogeny of the caniform (dog-like) carnivores. An analysis using maximum parsimony, maximum likelihood, and Bayesian approaches provided a unique and well-supported solution to most contentious relationships within Caniformia. The clade Arctoidea was shown to consist of three major monophyletic groups: Pinnipedia, Ursidae, and Musteloidea. Within Pinnipedia, the families Otariidae and Odobenidae formed a clade, sister to Phocidae. Within Musteloidea, there was a sister relationship between true mustelids (i.e., excluding the skunks) and procyonids, and between ailurids and mephitids (skunks). Despite a high level of confidence obtained at most nodes, uncertainty remained about the relative position of the three major arctoid clades.     

Multiple data sets, high homoplasy, and the phylogeny of softshell turtles (Testudines: Trionychidae)

We present a phylogenetic hypothesis and novel, rank-free classification for all extant species of softshell turtles (Testudines:Trionychidae). Our data set included DNA sequence data from two mitochondrial protein-coding genes and a approximately 1-kb nuclear intron for 23 of 26 recognized species, and 59 previously published morphological characters for a complimentary set of 24 species. The combined data set provided complete taxonomic coverage for this globally distributed clade of turtles, with incomplete data for a few taxa. Although our taxonomic sampling is complete, most of the modern taxa are representatives of old and very divergent lineages. Thus, due to biological realities, our sampling consists of one or a few representatives of several ancient lineages across a relatively deep phylogenetic tree. Our analyses of the combined data set converge on a set of well-supported relationships, which is in accord with many aspects of traditional softshell systematics including the monophyly of the Cyclanorbinae and Trionychinae. However, our results conflict with other aspects of current taxonomy and indicate that most of the currently recognized tribes are not monophyletic. We use this strong estimate of the phylogeny of softshell turtles for two purposes: (1) as the basis for a novel rank-free classification, and (2) to retrospectively examine strategies for analyzing highly homoplasious mtDNA data in deep phylogenetic problems where increased taxon sampling is not an option. Weeded and weighted parsimony, and model-based techniques, generally improved the phylogenetic performance of highly homoplasious mtDNA sequences, but no single strategy completely mitigated the problems of associated with these highly homoplasious data. Many deep nodes in the softshell turtle phylogeny were confidently recovered only after the addition of largely nonhomoplasious data from the nuclear intron.     

Mitochondrial DNA sequence-based phylogenetic relationship among flesh flies of the genus <Emphasis Type="Italic">Sarcophaga</Emphasis> (Sarcophagidae: Diptera)

The phylogenetic relationships among flesh flies of the family Sarcophagidae has been based mainly on the morphology of male genitalia. However, the male genitalic character-based relationships are far from satisfactory. Therefore, in the present study mitochondrial DNA has been used as marker to unravel genetic relatedness and to construct phylogeny among five sympatric species of the genus Sarcophaga. Two mitochondrial genes viz., cytochrome oxidase subunit 1 (COI) and NAD dehydrogenase subunit 5 (ND5) were sequenced and genetic distance values were calculated on the basis of sequence differences in both the mitochondrial genes. The data revealed very few genetic difference among the five species for the COI and ND5 gene sequences.     

Modeling nucleotide evolution at the mesoscale: the phylogeny of the neotropical pitvipers of the Porthidium group (viperidae: crotalinae)

We analyzed the phylogeny of the Neotropical pitvipers within the Porthidium group (including intra-specific through inter-generic relationships) using 1.4 kb of DNA sequences from two mitochondrial protein-coding genes (ND4 and cyt-b). We investigated how Bayesian Markov chain Monte-Carlo (MCMC) phylogenetic hypotheses based on this 'mesoscale' dataset were affected by analysis under various complex models of nucleotide evolution that partition models across the dataset. We develop an approach, employing three statistics (Akaike weights, Bayes factors, and relative Bayes factors), for examining the performance of complex models in order to identify the best-fit model for data analysis. Our results suggest that: (1) model choice may have important practical effects on phylogenetic conclusions even for mesoscale datasets, (2) the use of a complex partitioned model did not produce widespread increases or decreases in nodal posterior probability support, and (3) most differences in resolution resulting from model choice were concentrated at deeper nodes. Our phylogenetic estimates of relationships among members of the Porthidium group (genera: Atropoides, Cerrophidion, and Porthidium) resolve the monophyly of the three genera. Bayesian MCMC results suggest that Cerrophidion and Porthidium form a clade that is the sister taxon to Atropoides. In addition to resolving the intra-specific relationships among a majority of Porthidium group taxa, our results highlight phylogeographic patterns across Middle and South America and suggest that each of the three genera may harbor undescribed species diversity.     

Nuclear gene sequences resolve species phylogeny and mitochondrial introgression in Leptocarabus beetles showing trans-species polymorphisms

We studied the phylogenetic relationships among Japanese Leptocarabus ground beetles, which show extensive trans-species polymorphisms in mitochondrial gene genealogies. Simultaneous analysis of combined nuclear data with partial sequences from the long-wavelength rhodopsin, wingless, phosphoenolpyruvate carboxykinase, and 28S rRNA genes resolved the relationships among the five species, although separate analyses of these genes provided topologies with low resolution. For both the nuclear gene tree resulting from the combined data from four genes and a mitochondrial cytochrome oxidase subunit I (COI) gene tree, we applied a Bayesian divergence time estimation using a common calibration method to identify mitochondrial introgression events that occurred after speciation. Three mitochondrial lineages shared by two or three species were likely subject to introgression due to interspecific hybridization because the coalescent times for these lineages were much shorter than the corresponding speciation times estimated from nuclear gene sequences. We demonstrated that when species phylogeny is fully resolved with nuclear gene sequence data, comparative analysis of nuclear and mitochondrial gene trees can be used to infer introgressive hybridization events that might cause trans-species polymorphisms in mitochondrial gene trees.     

Molecular identification and phylogenetic relationships of seven Indian Sciaenids (Pisces: Perciformes,Sciaenidae) based on 16S rRNA and cytochrome c oxidase subunit I mitochondrial genes

The partial sequences of 16S rRNA and cytochrome c oxidase subunit I (COI) mitochondrial genes were analyzed for species identification and phylogenetic relationships among the commercially important Indian sciaenids (Otolithes cuvieri, Otolithes ruber, Johnius dussumieri, Johnius elongatus, Johnieops vogleri, Otolithoides biauritus and Protonibea diacanthus). Sequence analysis of both genes revealed that the seven species fell into three distinct groups, which were genetically distant from each other and exhibited identical phylogenetic resolution. Partial sequences of both the genes provided sufficient phylogenetic information to distinguish the seven sciaenids indicating the usefulness of mtDNA-based approach in species identification.