Primate genus Miopithecus: evidence for the existence of species and subspecies of dwarf guenons based on cellular and endogenous viral sequences

Sequence data from the mitochondrial 12S rRNA gene were combined with endogenous retrovirus sequences to study the position of the genus Miopithecus in the primate tree. The mitochondrial sequences indicated that Miopithecus is a true genus distinct from Cercopithecus, although talapoin monkeys are commonly referred to as dwarf guenons. The existence of two species of dwarf guenons, suggested by differences in coat color, pigmentation, and geographic location, was supported by substantial mitochondrial 12S rRNA gene divergence. In line with the informal proposal of J. Kingdon (1997, "The Kingdon Field Guide to African Mammals," Academic Press, London), we use the names Miopithecus talapoin for the southern, darker species and Miopithecus ougouensis for the northern, lighter-colored monkeys. Different 12S rRNA gene haplotypes found in M. ougouensis individuals suggest the possible existence of additional subspecies. Simian endogenous retrovirus (SERV) strain 23. 1 proviruses were introduced in the primate germ-line after the Cercopithecinae split from the Colobinae, estimated at around 9-14 million years ago. SERV sequences were used for timing of divergence events in Cercopithecinae and confirmed the close relationship between the genera Cercopithecus and Miopithecus, which was only weakly supported by the more variable mtDNA sequences in a distance analysis, demonstrating the utility of these pseudogenes in phylogenetic grouping.     

Primate Genus Miopithecus: Evidence for the Existence of Species and Subspecies of Dwarf Guenons Based on Cellular and Endogenous Viral Sequences

Sequence data from the mitochondrial 12S rRNA gene were combined with endogenous retrovirus sequences to study the position of the genus Miopithecus in the primate tree. The mitochondrial sequences indicated that Miopithecus is a true genus distinct from Cercopithecus, although talapoin monkeys are commonly referred to as dwarf guenons. The existence of two species of dwarf guenons, suggested by differences in coat color, pigmentation, and geographic location, was supported by substantial mitochondrial 12S rRNA gene divergence. In line with the informal proposal of J. Kingdon (1997, “The Kingdon Field Guide to African Mammals,” Academic Press, London), we use the names Miopithecus talapoin for the southern, darker species and Miopithecus ougouensis for the northern, lighter-colored monkeys. Different 12S rRNA gene haplotypes found in M. ougouensis individuals suggest the possible existence of additional subspecies. Simian endogenous retrovirus (SERV) strain 23.1 proviruses were introduced in the primate germ-line after the Cercopithecinae split from the Colobinae, estimated at around 9–14 million years ago. SERV sequences were used for timing of divergence events in Cercopithecinae and confirmed the close relationship between the genera Cercopithecus and Miopithecus, which was only weakly supported by the more variable mtDNA sequences in a distance analysis, demonstrating the utility of these pseudogenes in phylogenetic grouping.     

Nuclear counterparts of the cytoplasmic mitochondrial 12S rRNA gene: A problem of ancient DNA and molecular phylogenies

Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results.Correspondence to: A.C. van der Kuyl     

基于涉案兽类12S rRNA基因的NCBI数据库序列可靠性分析

本研究以常用于动物种属鉴定的12S rRNA基因位点为研究对象,利用所测得的17种常见涉案兽类12S rRNA基因部分片段序列及NCBI数据库中下载的该物种DNA序列及其近缘物种DNA序列,构建系统进化树。根据进化树的聚类情况,判断NCBI数据库中的相关基因序列或物种名称的正确性,并对其中错误序列的登陆号进行标记,以防对后续涉案动物的准确鉴定造成影响。分别从17种常见涉案兽类(共26份样本)中提取线粒体DNA,并利用通用引物扩增线粒体DNA上的12S rRNA基因部分片段并进行测序分析。通过NCBI数据库的Blast比对功能,筛选出与本研究物种同源性由高到低的物种,并从NCBI基因数据库中下载此类近缘物种的12S rRNA基因序列共351条,利用MEGA7.0软件构建该物种及其近缘物种系统进化树。通过比对发现NCBI中登录号为KP202279等3个序列所对应物种拉丁名错误。登录号为AY184436等11个序列所对应物种拉丁名可能存在疑问。GenBank中某些物种拉丁名有同种异名现象。因此,NCBI数据库数据可靠性有待进一步验证,只能作为涉案物种鉴定的参考数据之一,可借助构建系统进化树等方法来确认其结果的准确性。     

尼罗鳄线粒体基因组全序列分析及鳄类系统发生关系的探讨

大多数脊椎动物的线粒体基因组（约16—18kb）的组成是相对较稳定的,但在不同类群中,线粒体基因组在基因结构和基因排列方式等方面均显示了极大的多样性,这种多样性可能反映了真核细胞不同的进化路线（Saccone et al．,1999）。就目前的研究而言,线粒体基因组是惟一一个能够从基因组水平上来分析动物系统发生的分子标记,可以从线粒体基因组序列信息、基因组成及基因排列方式等进行多方位的分子进化研究,因而线粒体基因组全序列将成为动物分子系统发生最有力的证据（Saccone et al．,1999）。     

12S rRNA和Cyt b基因序列测定在獐乳制品鉴定中的应用

采用改良的蛋白酶K消化和酚/氯仿抽提的方法从脂肪含量很高的动物乳制品及胃组织中提取出基因组总DNA,利用特异引物扩增了线粒体12S rRNA基因和Cyt b基因的部分片段,测定了12S rRNA和Cyt b基因的PCR扩增产物序列,使用BLAST搜索软件将其序列与GenBank中的同源序列进行比对,并利用DNAMAN分析软件分析序列同源性。结果表明3件检材均来源于獐Hydropotes inermis。本研究所用方法在野生动物乳制品鉴定中具有较高的应用价值。     

Evaluation of the 16S and 12S rRNA genes as universal markers for the identification of commercial fish species in South Africa

The development of DNA-based methods for the identification of fish species is important for fisheries research and control, as well as for the detection of unintentional or fraudulent species substitutions in the marketplace. The aim of this study was to generate a comprehensive reference database of DNA sequences from the mitochondrial 16S and 12S ribosomal RNA (rRNA) genes for 53 commercial fish species in South Africa and to evaluate the applicability of these genetic markers for the identification of fish at the species level. The DNA extracted from all target species was readily amplified using universal primers targeting both rRNA gene regions. Sequences from the 16S and 12S rRNA genes were submitted to GenBank for the first time for 34% and 53% of the fish species, respectively. Cumulative analysis of the 16S rRNA gene sequences revealed mean conspecific, congeneric and confamilial Kimura two parameter (K2P) distances of 0.03%, 0.70% and 5.10% and the corresponding values at the 12S level were 0.03%, 1.00% and 5.57%. K2P neighbour-joining trees based on both sequence datasets generally clustered species in accordance with their taxonomic classifications. The nucleotide variation in both the 16S and 12S sequences was suitable for identifying the large majority of the examined fish specimens to at least the level of genus, but was found to be less useful for the explicit differentiation of certain congeneric fish species. It is recommended that one or more faster-evolving DNA regions be analysed to confirm the identities of closely-related fish species in South Africa.     

从rRNA基因序列探讨云斑蛛属Cyrtophora的系统发生地位

由于云斑蛛属Cyrtophora蜘蛛所织的网形态上较为特殊,所以该属分类地位长期以来有异议.为从分子水平探讨该属系统发生地位,本文对相关的11种蜘蛛线粒体12S rRNA基因和核18S rRNA基因部分序列进行了测定.基于12S rRNA和18S rRNA基因序列联合数据进行的分子系统发生分析结果表明云斑蛛属属于园蛛科,该结果提示在园蛛总科分类实践中以蛛网形态特征为分类依据时要慎重考虑其可靠性.     

Phylogenetic relationships and altered genome structures among Tetrahymena mitochondrial DNAs.

Tetrahymena thermophila mitochondrial DNA is a linear molecule with two tRNAs, large subunit beta (LSU beta) rRNA (21S rRNA) and LSU alpha rRNA (5.8S-like RNA) encoded near each terminus. The DNA sequence of approximately 550 bp of this region was determined in six species of Tetrahymena. In three species the LSU beta rRNA and tRNA(leu) genes were not present on one end of the DNA, demonstrating a mitochondrial genome organization different from that of T. thermophila. The DNA sequence of the LSU alpha rRNA was used to construct a mitochondrial phylogenetic tree, which was found to be topologically equivalent to a phylogenetic tree based on nuclear small subunit rRNA sequences (Sogin et al. (1986) EMBO J. 5, 3625-3630). The mitochondrial rRNA gene was found to accumulate base-pair substitutions considerably faster than the nuclear rRNA gene, the rate difference being similar to that observed for mammals.     

尖塘鳢属鱼类线粒体12SrRNA基因序列分析

利用PCR技术扩增和测序了线纹尖塘鳢、云斑尖塘鳢和海丰沙塘鳢线粒体12SrRNA基因,结合从GenBank中下载的部分同源序列,共分析了5种鱼类的系统发育关系。在Kimura2-parameter模型构建的邻接树中,原产泰国的云斑尖塘鳢与原产澳州线纹尖塘鳢均为单系类群,二者为亲缘关系最为密切的姐妹群,海丰沙塘鳢与其它群体的亲缘关系较远,支持将尖塘鳢属从塘鳢属中分出的传统分类处理。尖塘鳢属内云斑尖塘鳢和线纹尖塘鳢鱼类种内DNA序列无差异,而种间差异明显,表明线粒体12SrRNA基因可作为塘鳢科鱼类种类鉴定的良好分子标记。     

The rate of mitochondrial 12S rRNA gene evolution is similar in freshwater turtles and marsupials

Assertions that the ``conventional' rate of mitochondrial DNA (mtDNA) evolution is reduced in poikilotherms in general and turtles in particular were tested for side-necked turtles (Pleurodira: Chelidae). Homologous data sets of mitochondrial 12S rRNA gene sequences were used to compare the average divergence between the Australian and South American species for two Gondwanan groups: the chelid turtles and the marsupials. The mean nucleotide divergences between continental groups for both the turtles and the marsupials are remarkably similar. These data suggest that the rate of evolution of mitochondrial 12S rRNA gene is not substantially slower in turtles than in the homeothermic marsupials. Received: 24 February 1997 / Accepted: 30 June 1997     

Utility of nuclear DNA intron markers at lower taxonomic levels: phylogenetic resolution among nine Tragelaphus spp

Phylogenetic relationships among the nine spiral-horn antelope species of the African bovid tribe Tragelaphini are controversial. In particular, mitochondrial DNA sequencing studies are not congruent with previous morphological investigations. To test the utility of nuclear DNA intron markers at lower taxonomic levels and to provide additional data pertinent to tragelaphid evolution, we sequenced four nuclear DNA segments (MGF, PRKCI, SPTBN, and THY) and combined these data with mitochondrial DNA sequences from three genes (cytochrome b, 12S rRNA, and 16S rRNA). Our molecular supermatrix comprised 4682 characters which were analyzed independently and in combination. Parsimony and model based phylogenetic analyses of the combined nuclear DNA data are congruent with those derived from the analysis of mitochondrial gene sequences. The corroboration between nuclear and mtDNA gene trees reject the possibility that genetic processes such as lineage sorting, gene duplication/deletion and hybrid speciation account for the conflict evident in the previously published phylogenies. It suggests rather that the morphological characters used to delimit the Tragelaphid species are subject to convergent evolution. Divergence times among species, calculated using a relaxed Bayesian molecular clock, are consistent with hypotheses proposing that climatic oscillations and their impact on habitats were the major forces driving speciation in the tribe Tragelaphini.     

Phylogenetic relationships of leaf monkeys (Presbytis; Colobinae) based on cytochrome b and 12S rRNA genes

Little is known about the classification and phylogenetic relationships of the leaf monkeys (Presbytis). We analyzed mitochondrial DNA sequences of cytochrome b (Cyt b) and 12S rRNA to determine the phylogenetic relationships of the genus Presbytis. Gene fragments of 388 and 371 bp of Cyt b and 12S rRNA, respectively, were sequenced from samples of Presbytis melalophos (subspecies femoralis, siamensis, robinsoni, and chrysomelas), P. rubicunda and P. hosei. The genus Trachypithecus (Cercopithecidae) was used as an outgroup. The Cyt b NJ and MP phylogeny trees showed P. m. chrysomelas to be the most primitive, followed by P. hosei, whereas 12S rRNA tree topology only indicated that these two species have close relationships with the other members of the genus. In our analysis, chrysomelas, previously classified as a subspecies of P. melalophos, was not included in either the P. m. femoralis clade or the P. m. siamensis clade. Whether or not there should be a separation at the species level remains to be clarified. The tree topologies also showed that P. m. siamensis is paraphyletic with P. m. robinsoni, and P. m. femoralis with P. rubicunda, in two different clades. Cyt b and 12S rRNA are good gene candidates for the study of phylogenetic relationships at the species level. However, the systematic relationships of some subspecies in this genus remain unclear.     

A multi‐locus approach resolves the phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups in Malaysia: evidence for distinct evolutionary lineages

A multi‐locus approach was used to examine the DNA sequences of 10 nominal species of blackfly in the Simulium subgenus Gomphostilbia (Diptera: Simuliidae) in Malaysia. Molecular data were acquired from partial DNA sequences of the mitochondria‐encoded cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA genes, and the nuclear‐encoded 18S rRNA and 28S rRNA genes. No single gene, nor the concatenated gene set, resolved all species or all relationships. However, all morphologically established species were supported by at least one gene. The multi‐locus sequence analysis revealed two distinct evolutionary lineages, conforming to the morphotaxonomically recognized Simulium asakoae and Simulium ceylonicum species groups.     

Molecular structures of mitochondrial-DNA-like sequences in human nuclear DNA.

Two lambda phage clones carrying mitochondrial-DNA-like (mtDNA-like) sequences isolated from a human gene library were named Lm E-1 and Lm C-2, and their DNA structures were characterized. Lm E-1 contains about 0.4 kb DNA homologous to the 5' portion of the mitochondrial 16S ribosomal RNA (rRNA) gene and Lm C-2, a 1.6 kb DNA homologous to the 3' portion of the 12S rRNA gene and to almost all of the 16S rRNA gene. Comparisons of their nucleotide sequences with those of the corresponding regions of the human mtDNA revealed no detectable DNA rearrangement and their homologies to the human mtDNA are 84% and 80%, respectively. There are neither terminal repeats in the nuclear mtDNA-like sequences nor duplications of the nuclear DNAs flanking the mtDNA-like sequences. Evolutionary relationship between these two human nuclear mtDNA-like sequences and the human and bovine mtDNAs is discussed.     

Sequence and Conservation of a rRNA and tRNAVal Mitochondrial Gene Fragment from Penaeus californiensis and Comparison with Penaeus vannamei and Penaeus stylirostris

Penaeus californiensis is an important species for shrimp fisheries in the Pacific Ocean and has recently been described as a potential cultured species, mainly through the winter season in subtropical regions. A fragment of the mitochondrial 12S rRNA–tRNAVal–16S rRNA genes from P. californiensis was sequenced and compared with the corresponding regions from Penaeus vannamei and Penaeus stylirostris. Purified mitochondrial DNA was used for polymerase chain reaction amplification with primers for 12S and 16S rRNA genes. A 1379 ± 1-bp fragment was obtained, including 90% 16S rRNA, tRNAVal, and a portion of 12S rRNA, cloned, and sequenced. Genetic distances were calculated according to the Kimura 2-parameter distance model, and maximum-likelihood analysis was applied with 1000 bootstrap replications. Sequence identity of P. californiensis with both P. vannamei and P. stylirostris was 0.88, while for P. vannamei and P. stylirostris the identity was 0.92. Maximum-likelihood analysis grouped P. vannamei and P. stylirostris separately from P. californiensis.     

Molecular discrimination of phytoseiids associated with the red palm mite Raoiella indica (Acari: Tenuipalpidae) from Mauritius and South Florida

Phytoseiid populations imported from Mauritius for evaluation for a classical biological control program in Florida, USA, were morphologically identified as Amblyseius largoensis Muma, a species associated with the red palm mite in south Florida and the Caribbean. Bayesian analysis and sequence divergences of the mitochondrial 12S rRNA and nuclear Elongation factor--I alpha (EF-Iα) genes and Neighbor-Joining analysis of High-fidelity-RAPD-PCR markers were used to discriminate between the south Florida and Mauritius populations. High-fidelity-RAPD-PCR markers in addition to Bayesian and sequence divergence analyses of the 12S rRNA sequences suggest that the Mauritius and south Florida populations are genetically different but whether these are species or population differences is unknown. The degenerate EF-Iα primers used to survey the phytoseiids amplified two different elongation factor sequences with distinct amino acid translations, the putative EF-Iα and an unknown elongation factor. Variability within the 12S gene was used to develop population-specific primers for identifying the Mauritius phytoseiids in the event they are released in south Florida.     

Echinococcus granulosus strain differentiation in Iran based on sequence heterogeneity in the mitochondrial 12S rRNA gene

Parasite strain characterization is essential for the establishment of a prevention and control strategy in any endemic area. The aim of this study was to characterize different Echinococcus granulosus isolates from Iran by using DNA sequences of the mitochondrial 12S rRNA gene. Thirty livers and lungs of cattle, sheep and goats naturally infected with E. granulosus were collected from abattoirs in northern and western Iran between June and October 2007. These samples yielded 18 fertile cysts which we used for the genetic work. We designed and tested two new primer pairs which specifically amplify portions of the mitochondrial 12S rRNA gene of the two strains (G1 and G6) of E. granulosus known to occur in Iran. One primer pair amplified a fragment of 259 base pairs (bp) from only the G1 strain. The second pair amplified a fragment of 676 bp from the G6 strain. The G1 genotype was identified in all fertile cyst samples, in agreement with previous studies in Iran. Ten of our samples and a single reference sample of the G6 strain were sequenced and compared with the G1 and G6 sequences deposited in GenBank.     

Nucleotide sequence and evolution of the 18S ribosomal RNA gene in maize mitochondria.

The nucleotide sequence of the gene coding for the 18S ribosomal RNA of maize mitochondria has been determined and a model for the secondary structure is proposed. Dot matrix analysis has been used to compare the extent and distribution of sequence similarities of the entire maize mitochondrial 18S rRNA sequence with that of 15 other small subunit rRNA sequences. The mitochondrial gene shows great similarity to the eubacterial sequences and to the maize chloroplast, and less similarity to mitochondrial rRNA genes in animals and fungi. We propose that this similarity is due to a slow rate of nucleotide divergence in plant mtDNA compared to the mtDNA of animals. Sequence comparisons indicate that the evolution of the maize mitochondrial 18S, chloroplast 16S and nuclear 17S ribosomal genes have been essentially independent, in spite of evidence for DNA transfer between organelles and the nucleus.