Resolution of phylogenetic relationships among recently evolved species as a function of amount of DNA sequence: an empirical study based on woodpeckers (Aves: Picidae)

Synonymous substitutions in the 13 mitochondrial encoded protein genes form a large pool of characters that should approach the ideal for phylogenetic analysis of being independently and identically distributed. Pooling sequences from multiple mitochondrial protein-coding genes should result in statistically more powerful estimates of relationships among species that diverged sufficiently recently that most nucleotide substitutions are synonymous. Cytochrome oxidase I (COI) was sequenced for woodpecker species for which cytochrome b (cyt b) sequences were available. A pairing-design test based on the normal distribution indicated that cyt b evolves more rapidly than COI when all nucleotides are compared but their rates are equal for synonymous substitutions. Nearly all of the phylogenetically informative substitutions among woodpeckers are synonymous. Statistical support for relationships, as measured by bootstrap proportions, increased as the number of nucleotides increased from 1047 (cyt b) to 1512 (COI) to 2559 nucleotides (aggregate data set). Pseudo-bootstrap replicates showed the same trend and increasing the amount of sequence beyond the actual length of 2559 nucleotides to 5120 (2x) resulted in stronger bootstrap support, even though the amount of phylogenetic information was the same. However, the amount of sequence required to resolve an internode depends on the length of the internode and its depth in the phylogeny.     

Different patterns of selection on the nuclear genes IRBP and DMP-1 affect the efficiency but not the outcome of phylogeny estimation for didelphid marsupials

Selection at the protein-level can influence nucleotide substitution patterns for protein-coding genes, which in turn can affect their performance as phylogenetic characters. In this study, we compare two protein-coding nuclear genes that appear to have evolved under markedly different selective constraints and evaluate how selection has shaped their phylogenetic signal. We sequenced 1,100+ bp of exon 6 of the gene encoding dentin matrix protein 1 (DMP1) from most of the currently recognized genera of New World opossums (family: Didelphidae) and compared these data to an existing matrix of sequences from the interphotoreceptor retinoid-binding protein gene (IRBP) and morphological characters. In comparison to IRBP, DMP1 has far fewer sites under strong purifying selection and exhibits a number of sites under positive directional selection. Furthermore, selection on the DMP1 protein appears to conserve short, acidic, serine-rich domains rather than primary amino acid sequence; as a result, DMP1 has significantly different nucleotide substitution patterns from IRBP. Using Bayesian methods, we determined that DMP1 evolves almost 30% faster than IRBP, has 2.5 times more variable sites, has less among-site rate heterogeneity, is skewed toward A and away from CT (IRBP has relatively even base frequencies), and has a significantly lower rate of change between adenine and any other nucleotide. Despite these different nucleotide substitution patterns, estimates of didelphid relationships based on separate phylogenetic analyses of these genes are remarkably congruent whether patterns of nucleotide substitution are explicitly modeled or not. Nonetheless, DMP1 contains more phylogenetically informative characters per unit sequence and resolves more nodes with higher support than does IRBP. Thus, for these two genes, relaxed functional constraints and positive selection appear to improve the efficiency of phylogenetic estimation without compromising its accuracy.     

Alignment and phylogenetic analysis of beta-fibrinogen intron 7 sequences among avian orders reveal conserved regions within the intron

We sequenced beta-fibrinogen intron 7 (beta-fibint 7) from 28 species of birds, representing 18 families in nine orders. Although the antiquity of the avian orders is estimated to be 55 to 90 Myr, and numerous indels have accrued among diverging lineages, the intron sequences were not difficult to align. However, alignment of avian sequences with mammal or snake sequences was difficult, and the residual phylogenetic signal was weak. beta-fibint 7 is an AT-rich intron, and its base composition varies little over the diversity of birds represented by our sample. Alignment of these anciently diverged sequences reveals at least five clusters of conserved nucleotides; at least two clusters appear to be in excess of the minimal set usually associated with intron excision, but their functions are unknown. Two equally most-parsimonious (MP) trees were found when indels were not included in the phylogenetic analysis, and six such trees were found when indels were included. The Neighbor-Joining and maximum-likelihood trees were identical to each other and to one of the MP trees in each MP analysis. Indels, as well as nucleotide substitutions, are phylogenetically informative, and bootstrap support exceeded 90% for 21 of 24 inferred nodes when indels were included in the MP analysis. All traditional orders represented by two or more species appear monophyletic. Relationships among avian orders are strongly supported with the exception of an inferred sister-group relationship between Caprimulgiformes and Columbiformes. A relatively close relationship between Piciformes and Passeriformes is inferred, at odds with earlier DNA-DNA hybridization studies but consistent with traditional classifications. Among Passeriformes, the traditional perspective of a sister-group relationship of suboscines and oscines is supported, as is the subsequent split of the oscines into a lineage representative of the Corvida before the diversification of the Passerida. The four species of owls divide into two strongly supported clades, corresponding to the widely accepted bifurcation of owls into two families, Tytonidae and Strigidae. A sister-group relationship between gallinaceous birds and waterfowl, the Galloanserae, is also strongly supported.     

Patterns of Y and X chromosome DNA sequence divergence during the Felidae radiation.

The 37 species of modern cats have evolved from approximately eight phylogenetic lineages within the past 10 to 15 million years. The Felidae family has been described with multiple measures of morphologic and molecular evolutionary methods that serve as a framework for tracking gene divergence during brief evolutionary periods. In this report, we compare the mode and tempo of evolution of noncoding sequences of a large intron within Zfy (783 bp) and Zfx (854 bp), homologous genes located on the felid Y and X chromosomes, respectively. Zfy sequence variation evolves at about twice the rate of Zfx, and both gene intron sequences track feline hierarchical topologies accurately. As homoplasies are infrequent in patterns of nucleotide substitution, the Y chromosome sequence displays a remarkable degree of phylogenetic consistency among cat species and provides a highly informative glimpse of divergence of sex chromosome sequences in Felidae.     

Molecular phylogenetic relationships of pond frogs distributed in the Palearctic region inferred from DNA sequences of mitochondrial 12S ribosomal RNA and cytochrome b genes

The evolutionary relationships of pond frogs distributed in the Far East and Europe were investigated by analyses of nucleotide sequences of mitochondrial 12S ribosomal RNA (12S rRNA) and cytochrome b (cyt b) genes. The nucleotide sequences of a 412-bp segment of the 12S rRNA gene and a 534-bp segment of the cyt b gene were determined by the PCR-direct sequencing method using 19 frogs belonging to six species and one subspecies distributed in the Palearctic region. Phylogenetic trees were constructed by the neighbor-joining and maximum-likelihood methods using Rana catesbeiana or Xenopus laevis as an outgroup. The 412-bp segment of the 12S rRNA gene contained 65 variable sites including gap sites, and the 534-bp segment of the cyt b gene contained 160 variable sites. The nucleotide sequence divergences of the 12S rRNA gene were 0.25-4.83% within the Far Eastern frogs, 0.25-6.22% within the European frogs, and 8.74-11.24% between the Far Eastern and the European frogs, whereas those of the cyt b gene were 3.64-14.73% within the Far Eastern frogs, 0.38-14.42% within the European frogs, and 16.53-23.58% between the Far Eastern and the European frogs. Although most nucleotide substitutions were at the third codon position of the cyt b gene and were silent mutations, 4 amino acid replacements occurred within the Far Eastern frogs, 4 within the European frogs, and 11 between the Far Eastern and the European frogs. The phylogenetic trees constructed from the nucleotide sequence divergences showed slightly different topologies for the 12S rRNA and cyt b genes. R. esculenta from Ukraine was closely related to R. lessonae from Luxembourg in both the 12S rRNA and the cyt b gene sequences.     

Molecular phylogeny of avian genus Syrmaticus based on the mitochondrial cytochrome B gene and control region

Mitochondrial DNA cytochrome b (cyt b) and control region (CR) nucleotide sequences were used to study the molecular phylogeny of the genus Syrmaticus. We found that the substitution rates among the three codon positions of cyt b were heterogeneous and the transition-transversion ratio was highly biased. As to CR sequences of the genus, most variable sites were in the peripheral domains. All molecular phylogenetic trees based on the two genes showed that: 1) the Syrmaticus was monophyletic and included five species with the following cladistic relationship: (S. reevesii, (S. soemmerringii, (S. mikado, (S. humiae and S. ellioti)))). Using the TN genetic distance of cyt b, we inferred the divergence time of the five species according to putative molecular clock and found that values were largely in agreement with the geological scenarios. The origin and speciation processes of the studied group were inferred by combining molecular and biogeographical evidences.     

Comparative evolution of the mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> gene and nuclear <Emphasis Type="Italic">S7</Emphasis> ribosomal protein gene intron 1 in sinipercid fishes and their relatives

The extant sinipercids are a group of freshwater percoid fishes endemic to East Asia. A recent mitochondrial cytochrome b phylogeny of sinipercids has challenged some aspects for their traditional taxonomy and molecular phylogeny, especially for the monophyly of Sinipercidae. In this study, we analyzed mitochondrial cytochrome b and nuclear encoded S7 ribosomal protein gene intron 1 for 10 sinipercid species and 11 related species to compare the phylogenetic signal and nucleotide substitution properties of these two gene sequences. The length of S7 intron 1 ranged from 461 to 719 bp, but alignment was not difficult, and the indels, the proportion of which in the total nucleotides ranged from 3.76 to 45.83%, were phylogenetically informative. Our results indicate that: (1) the relative rate presented by cyt b is five times that of S7 intron 1; (2) the proportion of phylogenetic information is higher in S7 than in cyt b; (3) S7 intron 1 has more base composition bias, but more uniform nucleotide substitution properties; (4) the overall ratio between transitions and transversions in S7 intron 1 is lower than in cyt b. Maximum parsimony and Bayesian analyses of aligned S7 intron 1 and the combined S7 and cyt b dataset resulted in phylogenies that contained the previously identified genera Siniperca and Coreoperca, whereas the monophyly of Coreoperca cannot be corroborated by separate cyt b analysis. The monophyly of Sinipercidae is not supported in separate and combined dataset analyses, although the alternative hypothesis cannot be significantly rejected based on approximately unbiased tests and Shimodaira–Hasegawa tests. Overall, maximum parsimony analyses result in trees with a lack of phylogenetic resolution in deep nodes, and the signal from S7 intron 1 conflicts the cyt b signal in the combined dataset analyses. The reasons for the poor performance of cyt b to S7 intron 1 in the phylogeny are discussed.     

Type I STS markers are more informative than cytochrome B in phylogenetic reconstruction of the Mustelidae (Mammalia: Carnivora)

We compared the utility of five nuclear gene segments amplified with type I sequence-tagged site (STS) primers versus the complete mitochondrial cytochrome b (cyt b) gene in resolving phylogenetic relationships within the Mustelidae, a large and ecomorphologically diverse family of mammalian carnivores. Maximum parsimony and likelihood analyses of separate and combined data sets were used to address questions regarding the levels of homoplasy, incongruence, and information content within and among loci. All loci showed limited resolution in the separate analyses because of either a low amount of informative variation (nuclear genes) or high levels of homoplasy (cyt b). Individually or combined, the nuclear gene sequences had less homoplasy, retained more signal, and were more decisive, even though cyt b contained more potentially informative variation than all the nuclear sequences combined. We obtained a well-resolved and supported phylogeny when the nuclear sequences were combined. Maximum likelihood and Bayesian phylogenetic analyses of the total combined data (nuclear and mitochondrial DNA sequences) were able to better accommodate the high levels of homoplasy in the cyt b data than was an equally weighted maximum parsimony analysis. Furthermore, partition Bremer support analyses of the total combined tree showed that the relative support of the nuclear and mitochondrial genes differed according to whether or not the homoplasy in the cyt b gene was downweighted. Although the cyt b gene contributed phylogenetic signal for most major groupings, the nuclear gene sequences were more effective in reconstructing the deeper nodes of the combined tree in the equally weighted parsimony analysis, as judged by the variable-length bootstrap method. The total combined data supported the monophyly of the Lutrinae (otters), whereas the Melinae (badgers) and Mustelinae (weasels, martens) were both paraphyletic. The American badger, Taxidea taxus (Taxidiinae), was the most basal taxon. Because hundreds of type I STS primer sets spanning the complete genomes of the human and mouse have been published and thus represent many independently segregating loci, the potential utility of these markers for molecular systematics of mammals and other groups is enormous.     

Comparison of evolutionary rates in the mitochondrial DNA cytochrome b gene and control region and their implications for phylogeny of the Cobitoidea (Teleostei: Cypriniformes)

It is widely accepted that mitochondrial DNA (mtDNA) control region evolves faster than protein encoding genes with few exceptions. In the present study, we sequenced the mitochondrial cytochrome b gene (cyt b) and control region (CR) and compared their rates in 93 specimens representing 67 species of loaches and some related taxa in the Cobitoidea (Order Cypriniformes). The results showed that sequence divergences of the CR were broadly higher than those of the cyt b (about 1.83 times). However, in considering only closely related species, CR sequence evolution was slower than that of cyt b gene (ratio of CR/cyt b is 0.78), a pattern that is found to be very common in Cypriniformes. Combined data of the cyt b and CR were used to estimate the phylogenetic relationship of the Cobitoidea by maximum parsimony, neighbor-joining, and Bayesian methods. With Cyprinus carpio and Danio rerio as outgroups, three analyses identified the same four lineages representing four subfamilies of loaches, with Botiinae on the basal-most clade. The phylogenetic relationship of the Cobitoidea was ((Catostomidae+Gyrinocheilidae)+(Botiinae+(Balitorinae+(Cobitinae+Nemacheilinae)))), which indicated that Sawada's Cobitidae (including Cobitinae and Botiinae) was not monophyletic. Our molecular phylogenetic analyses are in very close agreement with the phylogenetic results based on the morphological data proposed by Nalbant and Bianco, wherein these four subfamilies were elevated to the family level as Botiidae, Balitoridae, Cobitidae, and Nemacheilidae.     

蜂猴线粒体细胞色素b基因变异特点及系统发育分析

测定了蜂猴属（Nycticebus)1个蜂猴（N.coucang)和2个矮蜂猴（N,pygmaseus)个体的线粒体细胞色素b(cyt-b)基因全序列,比较现有的司猴科其他种序列,分析了核苷酸序列差异和碱基替换特点,以指猴为外群重建了系统发育树,结果表明,在所研究的个体中,2个蜂猴物种碱基组成具有哺乳动物的共同特点,它们之间转换比（特别是密码子第3位）是颠换比的6倍多,大于其他种间比较;低的Ka/Ks值（＜0．1）,说明懒猴科cyt-b基因的异义突位点受到强的选择压力作用。由cyt-b基因构建的系统发育树符合懒猴科化石记录和形态学分类观点,根据化石记录和与分化时间有一定线性关系的第3位颠换和同义突变速率,估算蜂猴与倭蜂猴种间,蜂猴与蜂属间可能的分化时间分别为300和600万年。     

Novel primers for complete mitochondrial cytochrome b gene sequencing in mammals

Sequence-based species identification relies on the extent and integrity of sequence data available in online databases such as GenBank. When identifying species from a sample of unknown origin, partial DNA sequences obtained from the sample are aligned against existing sequences in databases. When the sequence from the matching species is not present in the database, high-scoring alignments with closely related sequences might produce unreliable results on species identity. For species identification in mammals, the cytochrome b (cyt b) gene has been identified to be highly informative; thus, large amounts of reference sequence data from the cyt b gene are much needed. To enhance availability of cyt b gene sequence data on a large number of mammalian species in GenBank and other such publicly accessible online databases, we identified a primer pair for complete cyt b gene sequencing in mammals. Using this primer pair, we successfully PCR amplified and sequenced the complete cyt b gene from 40 of 44 mammalian species representing 10 orders of mammals. We submitted 40 complete, correctly annotated, cyt b protein coding sequences to GenBank. To our knowledge, this is the first single primer pair to amplify the complete cyt b gene in a broad range of mammalian species. This primer pair can be used for the addition of new cyt b gene sequences and to enhance data available on species represented in GenBank. The availability of novel and complete gene sequences as high-quality reference data can improve the reliability of sequence-based species identification.     

中国地鼠线粒体Cyt b基因测序及其分子进化

目的测定中国地鼠线粒体DNA细胞色素b基因部分序列,分析其分子系统进化关系。方法提取中国地鼠肝脏的总基因组DNA。设计合成特异引物进行PCR扩增,经检测进行测序。用Blast与GenBank中啮齿类其他常用实验动物的物种细胞色素b基因进行同源序列比较,分析其碱基组成及变异情况,并用邻接法、最大简约法、最小进化法构建了分子系统树,在分子水平上探讨中国地鼠和常用啮齿类实验动物的进化关系。结果获得了中国地鼠线粒体Cytb基因的部分序列,共936bp。结论中国地鼠和金黄地鼠的亲缘关系最近,与小鼠、大鼠存在的差异相对大,与豚鼠的亲缘关系最远,与传统的分类地位基本吻合。     

A molecular timescale for galliform birds accounting for uncertainty in time estimates and heterogeneity of rates of DNA substitutions across lineages and sites

A recent molecular timescale for major lineages of the Galliformes indicated that Megapodiidae and possibly Cracidae, originated in the Cretaceous, while the remaining families originated in the Tertiary. This timescale was based on clock-like evolution in genetic and taxonomic partitions of mitochondrial ND2 and cyt b DNA sequences, and assumed that ordinal diversification of Galloanserae around 90 million years ago and imposed, whenever appropriate, minimum age constraints based on the fossil record. This approach is not ideal, as it did not account for uncertainty in estimating branch lengths and time, including the calibration time, and heterogeneity in the rate of DNA substitution among sites and in different lineages. Furthermore all the information available in the DNA sequences was not included, and may have been affected by stochastic error in individual gene partitions. Here, we present a follow-up analysis by estimating divergence times using a Bayesian framework that accounts for these possible sources of uncertainty. Our results based on combined and separate analyses of mitochondrial DNA sequences comprised of 1756 sites of 12S rDNA, ND2 and cyt b indicated that (1) Megapodiidae and Cracidae, and likely Odontophoridae, originated in the Cretaceous; (2) estimates based on concatenated genes are less affected by stochastic error among sites and less influenced by the phylogenetic signals of individual gene partitions, which are unequally distributed along the phylogenetic tree; and (3) the use of only an external molecular calibration results in lower estimation of most ingroup node ages. We also point out that galliform fossils may not be as useful for point calibrations as was previously suggested, but instead may be better employed as priors for the estimation of node ages under a Bayesian approach.     

A mitogenome of the Chevrier's field mouse (Apodemus chevrieri) and genetic variations inferred from the cytochrome b gene

The Chevrier's field mouse (Apodemus chevrieri) is an endemic species to China and is an important pest in agriculture and human diseases. In this study, the complete mitochondrial genome of this species was sequenced and its size was 16,298 bases (accession no.: HQ896683). The mitogenome structure was similar compared with other reported rodent mitochondrial genomes and includes 13 protein-coding genes, 2 rRNA genes (12S rRNA and 16S rRNA), 22 tRNA genes, and 1 control region. This was the first complete mitogenome sequenced in genus Apodemus. The phylogenetic analyses based on the sequences of 12 heavy-strand protein-coding genes demonstrated that A. chevrieri clustered together with genus Mus. Additionally, extremely high haplotype and nucleotide diversities (h=0.978, π=2.6%) were observed based on 44 mitochondrial cytochrome b (cyt b) gene sequences. This suggests adaptive divergence of this species to a variety of living habitats and potential refuges in the eastern margin of the Hengduan Mountains during the Quaternary ice ages. No population expansions or genetic bottlenecks were observed in demographic analyses. The phylogenetic analysis of cyt b sequences and haplotypes revealed a genetic differentiation between north and south populations. The divergence between north clade and south clade occurred probably in the middle Pleistocene 1.1815 million years ago (Mya) (95% highest posterior density 2.3189-0.2737 Mya), which was congruent with the periods of the most tense uplift events in the Tibetan Plateau.     

Among-site rate variation and phylogenetic analysis of 12S rRNA in sigmodontine rodents

We analyze sequences from two mitochondrial genes, cytochrome b (cyt b) and 12S rRNA (12S), for a group of sigmodontine rodents among which phylogenetic relationships are well understood based on concordance of morphological, chromosomal, allozyme, and other DNA data sets. Because these two genes are physically linked on the nonrecombining mitochondrial genome, they necessarily share the same history. Phylogenetic analysis of the cyt b gene recovers the well-corroborated relationships, generally with strong support. None of the methods that we employed, including variously weighted parsimony, neighbor joining on both single-rate and gamma-corrected distances, and maximum likelihood, were able to recover these relationships for the 12S gene. Parsimony analyses of the 12S data resulted in a relatively strongly supported placement of Peromyscus eremicus that conflicts with that suggested by cyt b and all other data. There is extreme among-site rate variation in the 12S sequences and moderate levels in the cyt b sequences. This highly skewed distribution of rates in the 12S gene makes phylogenetic analyses of these sequences particularly susceptible to the misleading effects of nonindependence and other nonrandom noise, suggesting that phylogenetic analyses of data sets that contain a great deal of among-site rate variation be interpreted with caution.      

剑尾鱼线粒体细胞色素b基因的序列分析

目的　克隆和测定剑尾鱼 (Xiphophorushelleri)线粒体细胞色素b基因 (cytb)的全序列。方法　提取剑尾鱼肝脏的总DNA。设计合成特异引物进行PCR扩增。扩增产物经琼脂糖电泳检测、纯化后克隆到pGEM Teasyvectorsystem中的T载体上 ,筛选转化子 ,提取质粒 ,酶切鉴定。挑取重组质粒pGEM T xhcytb 11进行序列测定。结果　获得了剑尾鱼线粒体cytb基因的全序列 ,共 114 0bp。结论　用BLAST与GenBank中的线粒体DNA序列进行比较 ,显示剑尾鱼与其他鱼类的cytb基因具有较高的同源性 ;根据剑尾鱼与其他 13种鱼的cytb基因序列同源性所建立的进化树 ,与传统的分类地位基本吻合     

六种灵猫科动物的分子系统关系研究

对六种灵猫科物种线粒体12 S rRNA基因及其中四种的Cytb基因部分序列进行了测定,并从Gen-Bank获得斑林狸(Prionodon pardicolor)、熊狸(Arctictis binturong)的Cytb基因同源序列。两基因整合序列比对后长755 bp,12 S rRNA基因序列中有70个变异位点,31个简约信息位点,在Cytb基因序列中,共有120个位点呈现变异,60个简约信息位点,Cytb基因的碱基变异百分比高于12 S rRNA基因的碱基变异百分比。使用邻接法(NJ)、最大似然法(ML)重建的分子系统树显示:斑林狸从灵猫亚科中分离出来,支持灵猫亚科的多系起源,而且斑林狸可能是中国起源最早且最特化的灵猫科动物。另外,同属于灵猫亚科的大灵猫(Viverra zibe-tha)、小灵猫(Viverricula indica)聚为一支,同属于棕榈狸亚科的果子狸(Viverricula indica)、熊狸聚为姐妹群,这些与传统形态学分类观点一致。     

Pentatricopeptide repeat proteins constrain genome evolution in chloroplasts

Higher plants encode hundreds of pentatricopeptide repeat proteins (PPRs) that are involved in several types of RNA processing reactions. Most PPR genes are predicted to be targeted to chloroplasts or mitochondria, and many are known to affect organellar gene expression. In some cases, RNA binding has been directly demonstrated, and the sequences of the cis-elements are known. In this work, we demonstrate that RNA cis-elements recognized by PPRs are constrained in chloroplast genome evolution. Cis-elements for two PPR genes and several RNA editing sites were analyzed for sequence changes by pairwise nucleotide substitution frequency, pairwise indel frequency, and maximum likelihood (ML) phylogenetic distances. All three of these analyses demonstrated that sequences within the cis-element are highly conserved compared with surrounding sequences. In addition, we have compared sequences around chloroplast editing sites and homologous sequences in species that lack an editing site due to the presence of a genomic T. Cis-elements for RNA editing sites are highly conserved in angiosperms; by contrast, comparable sequences around a genomically encoded T exhibit higher rates of nucleotide substitution, higher frequencies of indels, and greater ML distances. The loss in requirement for editing to create the ndhD start codon has resulted in the conversion of the PPR gene responsible for editing that site to a pseudogene. We show that organellar dependence on nuclear-encoded PPR proteins for gene expression has constrained the evolution of cis-elements that are required at the level of RNA processing. Thus, the expansion of the PPR gene family in plants has had a dramatic effect on the evolution of plant organelle genomes.