Identification of the structural gene for yeast cytochrome c oxidase subunit I on mitochondrial DNA.

Mitochondrial protein synthesis was analyzed in the yeast mit^? mutants of $\text{Saccharomyces}$$\text{cerevisiae}$ which specifically lack cytochrome $\text{c}$ oxidase. [³H]leucine labeled polypeptides synthesized in yeast OXI 3 mutant were analyzed by means of immunoprecipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When compared to control, subunit I was not detectable. This result was substantiated by growing OXI 3 mutant in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Under such conditions SDS-PAGE analysis of [³H]leucine labeled immunoprecipitate shows the absence of subunit I. These data show that the OXI 3 locus contains the structural gene for cytochrome $\text{c}$ oxidase subunit I.     

Species of tetrahymena identical by small subunit rRNA gene sequences are discriminated by mitochondrial cytochrome c oxidase I gene sequences

The mitochondrial cytochrome c oxidase 1 (CO1) genes of two isolates of each of the seven mating types of Tetrahymena thermophila were sequenced and found to differ by < 1% in nucleotide sequence and to be identical by putative protein sequence. As this gene was highly conserved in this species, the CO1 gene sequence was determined for four pairs of Tetrahymena species identical in their small subunit rRNA gene sequences. The following pairs of species showed from 1% to 12% divergence at the nucleotide level, enabling discrimination of all these species: (1) Tetrahymena pyriformis strain T and Tetrahymena setosa strain HZ-1; (2) Tetrahymena canadensis strain UM1215 and Tetrahymena rostrata strain ID-3; (3) Tetrahymena pigmentosa strain UM1285 and Tetrahymena hyperangularis strain EN112; and (4) Tetrahymena tropicalis strain TC-105 and Tetrahymena mobilis. However, because of the synonymous nature of the majority of substitutions, the pairs of species were identical based on the putative protein sequence.     

Identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit I and cytochrome b gene sequences

Abstract.  Primer pairs were designed and protocols developed to selectively amplify segments of vertebrate mitochondrial cytochrome oxidase subunit 1 (COI) and cytochrome b (Cyt b ) mtDNA from the bloodmeals of mosquitoes (Diptera: Culicidae). The protocols use two pairs of nested COI primers and one pair of Cyt b primers to amplify short segments of DNA. Resultant sequences are then compared with sequences in GenBank, using the BLAST function, for putative host identification. Vertebrate DNA was amplified from 88% of our sample of 162 wild-caught, blood-fed mosquitoes from Oregon, U.S.A. and GenBank BLAST searches putatively identified 98% of the amplified sequences, including one amphibian, seven mammalian and 14 avian species. Criteria and caveats for putative identification of bloodmeals are discussed.     

Patterns of evolution of mitochondrial cytochrome c oxidase I and II DNA and implications for DNA barcoding

DNA barcoding has focused increasing attention on the use of specific regions of mitochondrial cytochrome c oxidase I and II genes (COI-COII) to diagnose and delimit species. However, our understanding of patterns of molecular evolution within these genes is limited. Here we examine patterns of nucleotide divergence in COI-COII within species and between species pairs of Lepidoptera and Diptera using a sliding window analysis. We found that: (1) locations of maximum divergence within COI-COII were highly variable among taxa surveyed in this study; (2) there was major overlap in divergence within versus between species, including within individual COI-COII profiles; (3) graphical DNA saturation analysis showed variation in percent nucleotide transitions throughout COI-COII and only limited association with levels of DNA divergence. Ultimately, no single optimally informative 600 bp location was found within the 2.3 kb of COI-COII, and the DNA barcoding region was no better than other regions downstream in COI. Consequently, we recommend that researchers should maximize sequence length to increase the probability of sampling regions of high phylogenetic informativeness, and to minimize stochastic variation in estimating total divergence.     

Relationship among coelacanths,lungfishes, and tetrapods: A phylogenetic analysis based on mitochondrial cytochrome oxidase I gene sequences

To clarify the relationship among coelacanths, lungfishes, and tetrapods, the amino acid sequences deduced from the nucleotide sequences of mitochondrial cytochrome oxidase subunit I (COI) genes were compared. The phylogenetic tree of these animals, including the coelacanth Latimeria chalumnae and the lungfish Lepidosiren paradoxa, was inferred by several methods. These analyses consistently indicate a coelacanth/lungfish clade, to which little attention has been paid by previous authors with the exception of some morphologists. Overall evidence of other mitochondrial genes reported previously and the results of this study equally support the coelacanth/lungfish and lungfish/tetrapod clades, ruling out the coelacanth/tetrapod clade.Correspondence to: K. Watanabe 0592     

基于线粒体基因Cytb和COI的蝗科中五亚科分子系统发育关系分析（直翅目: 蝗总科）（英文）

本研究基于Cytb 基因和COI基因的部分序列来推断17种蝗虫之间的系统发育关系。这17种蝗虫均采自国内,代表了蝗科(Acrididae)5个亚科：黑蝗亚科(Melanoplinae)、斑腿蝗亚科(Catantopinae)、刺胸蝗亚科(Cyrtacanthacridinae)、斑翅蝗亚科(Oedipodinae)和大足蝗亚科(Gomphocerinae)。采用联合序列方法进行分析,结果显示：Cytb 和COI联合序列长度为1 998 bp,其中A和T总含量为72.13%,G和C总含量为27.87%。联合序列共包含了889个保守位点,1 109个变异位点,在这些变异位点中有838个简约信息位点。系统发生树采用邻接法（NJ）、最大简约法（MP）和最大似然法（ML）进行构建。使用蜢总科的变色乌蜢Erianthus versicolor 和 Erianthus sp. 两个种作为外群。结果表明：大足蝗亚科和斑腿蝗亚科的单系性没有得到支持。斑翅蝗亚科内部各种聚成一个大支,在本研究中该亚科的单系性得到支持,与前人的研究结论相同。大足蝗亚科、斑腿蝗亚科、刺胸蝗亚科和黑蝗亚科这4科关系非常近,可以考虑将其合并为一个亚科。同时,我们发现基于Cytb和COI基因联合序列推断蝗科内各亚科间的系统发生关系并不十分可靠。     

Molecular phylogeny of parasitic Platyhelminthes based on sequences of partial 28S rDNA D1 and mitochondrial cytochrome c oxidase subunit I

The phylogenic relationships existing among 14 parasitic Platyhelminthes in the Republic of Korea were investigated via the use of the partial 28S ribosomal DNA (rDNA) D1 region and the partial mitochondrial cytochrome c oxidase subunit 1 (mCOI) DNA sequences. The nucleotide sequences were analyzed by length, G + C %, nucleotide differences and gaps in order to determine the analyzed phylogenic relationships. The phylogenic patterns of the 28S rDNA D1 and mCOI regions were closely related within the same class and order as analyzed by the PAUP 4.0 program, with the exception of a few species. These findings indicate that the 28S rDNA gene sequence is more highly conserved than are the mCOI gene sequences. The 28S rDNA gene may prove useful in studies of the systematics and population genetic structures of parasitic Platyhelminthes.     

Comparative study on DNA sequences of ribosomal DNA and cytochrome c oxidase subunit 1 of mitochondrial DNA among five species of gnathostomes

The nucleotide sequences of partial 18S, complete internal transcribed spacer region 1 (ITS1), complete 5.8S, complete ITS2 and partial 28S of ribosomal DNA (rDNA) and cytochrome c oxidase subunit 1 of mitochondrial DNA (MCOI) from five species of gnathostomes (G. spinigerum, G. doloresi, G. nipponicum, G. hispidum and G. binucleatum with the former four species being distributed in Japan and Asia) that cause human gnathostomiasis were compared by direct polymerase chain reaction cycle-sequencing. The nucleotide sequences of each region of the18S (613 bp), 5.8S (158 bp) and 28S (598 bp) rDNA from the five species were almost identical. The ITS1 region was different in length for the five species. The nucleotide sequences of each region of ITS2 and partial MCO1 regions were different among the five species. Therefore, these two regions can be used as genetic markers for identification of worms.     

基于线粒体细胞色素c氧化酶亚基I基因序列的帘蛤科贝类分子系统发育研究

对21种帘蛤科贝类线粒体细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunit I,COI)基因核苷酸序列进行了分析,以探讨这一序列在种质鉴定、分子系统发生研究中的应用价值。测序结果表明,所有物种扩增片段长度均为707 bp(含引物),序列A+T含量(62.4%—67.8%)明显高于G+C含量。物种间共有变异位点379个,其中简约信息位点334个;此区段共编码235个氨基酸,种间共有氨基酸变异位点100个。以COI基因片段序列为标记,用中国蛤蜊(Mactra chinensis)作外群,构建了35种帘蛤科贝类(其中14种贝类COI序列从GenBank下载)的系统发生树,结合拓扑结构分析和序列比对分析,结果表明:支持将短文蛤(Meretrix petechinalis)和丽文蛤(M.lusoria)订为文蛤(M.meretrix)的同物异名的观点,建议将丽文蛤和短文蛤订为文蛤的地理亚种;支持将薄片镜蛤(Dosinia corrugata)和D.angulosa订为2个独立种的观点;认为将波纹巴非蛤(Paphia undulata)和织锦巴非蛤(P.textile)订为2个独立种是合适的。COI基因序列含有丰富的遗传信息,适合作为帘蛤科贝类种群遗传结构和系统发生研究的分子标记。     

Species-specific identification of human hookworms by PCR of the mitochondrial cytochrome oxidase I gene.

Significant differences in the life histories of the human hookworms Ancylostoma duodenale and Necator americanus necessitate their differentiation for epidemiological studies and the design of control programs. Current methods of identification require time-consuming, labor-intensive techniques. A polymerase chain reaction (PCR)-based method that enables rapid species identification is described. The mitochondrial cytochrome oxidase I genes of both species were sequenced, and species-specific primer sets were designed. The primers were used in PCR to amplify 585-bp fragments of the cytochrome oxidase gene from individual hookworm eggs, larvae, and adults. The technique was also able to identify mixed infections containing equal amounts of eggs from each species. The technique is rapid, technically simple, and sensitive and will permit the accurate identification of human hookworms in epidemiological field studies.     

Phylogenetic analysis of six species of pacific abalone (Haliotidae) based on DNA sequences of 16s rRNA and cytochrome c oxidase subunit I mitochondrial genes

Six species of abalones (Haliotidae) are found on the Korean coasts. Identification and characterization of these abalones are usually based on morphologic characters. In this research we compared the partial sequences of the mitochondrial 16S ribosomal RNA and cytochrome c oxidase subunit I genes to identify species using molecular data and to determine their phylogenetic relationships. Sequence alignments and phylogenetic analysis revealed that the 6 species fell into 2 distinct groups which were genetically distant from each other and exhibited little internal phylogenetic resolution. One group included Haliotis discus hannai, H. discus discus, H. madaka, and H. gigantea, while the other group contained H. diversicolor supertexta and H. diversicolor diversicolor. The 16S rRNA sequences were relatively more conserved than to the COI sequences, but both gene sequences provided sufficient phylogenetic information to distinguish among the 6 species of Pacific abalone, and thus could be valuable molecular characters for species identification.     

New DNA barcodes for identification of Korean birds

DNA barcode is a short sequence of standardized genomic region that is specific to a species and therefore, helps in species identification. According to studies of animal species, the 648-bp sequence of the mitochondrial gene encoding cytochrome c oxidase 1 (CO1) is extremely useful for species identification. Several studies of birds have already ascertained the reliability of CO1 barcodes. In this study, we investigated the validity of DNA barcoding in Korean bird species by using additional barcode records. We analyzed the CO1 barcodes of 154 species of Korean birds, and discovered that the average genetic distance between congeneric species was 25 times higher than the average genetic distance within species. Most (98.7 %) bird species possessed a barcode distinct from that of other bird species. However, among the remaining 1.3 %, species had overlapping barcode clusters. Thus, we reemphasize that CO1 barcodes are an effective identification tool for Korean bird species.     

The origin of the Tibetan Mastiff and species identification of Canis based on mitochondrial cytochrome c oxidase subunit I (COI) gene and COI barcoding

DNA barcoding is an effective technique to identify species and analyze phylogenesis and evolution. However, research on and application of DNA barcoding in Canis have not been carried out. In this study, we analyzed two species of Canis, Canis lupus (n = 115) and Canis latrans (n = 4), using the cytochrome c oxidase subunit I (COI) gene (1545 bp) and COI barcoding (648 bp DNA sequence of the COI gene). The results showed that the COI gene, as the moderate variant sequence, applied to the analysis of the phylogenesis of Canis members, and COI barcoding applied to species identification of Canis members. Phylogenetic trees and networks showed that domestic dogs had four maternal origins (A to D) and that the Tibetan Mastiff originated from Clade A; this result supports the theory of an East Asian origin of domestic dogs. Clustering analysis and networking revealed the presence of a closer relative between the Tibetan Mastiff and the Old English sheepdog, Newfoundland, Rottweiler and Saint Bernard, which confirms that many well-known large breed dogs in the world, such as the Old English sheepdog, may have the same blood lineage as that of the Tibetan Mastiff.     

Evolution of the mitochondrial DNA cytochrome b gene in Tetraonidae birds

Mitochondrial fragments containing the cytochrome b gene (1020 bp in size) of four bird species belonging to four genera of the family Tetraonidae (Tetrao parvirostris, Bonasa umbellus, Lagopus lagopus scoticus, and Falcipennis falcipennis) were directly sequenced. Of the 1020 nucleotide positions, 186 were variable and uniformly distributed over the gene and only 46 were parsimony informative. Most substitutions were synonymous. Replacement substitutions were detected for 15 out of 340 amino acid sites; only four replacements were parsimony informative. The greatest codon bias was found for leucine and serine. The C-T transitions and the G-C transversions were, respectively, the most common (60.7%) and the most rare (5.9%). The mutation frequencies were high at the third codon position (85.2%) and relatively low at the first and the second position. At the third codon position of the species examined, the guanine content was the lowest (3.3%) and the cytosine content was the highest (44.5%). Based on the cytochrome b gene sequences, phylogenetic relationships in the order Galliformes are inferred.     

Phylogeny of the genus Chironomus (Diptera) inferred from DNA sequences of mitochondrial cytochrome b and cytochrome oxidase I

Two mitochondrial genes, Cytochrome b (Cytb) and Cytochrome c oxidase subunit I (COI), have been used as phylogenetic markers in Chironomids. The nucleotide sequences of 685 bp from Cytb and 596 bp from COI have been determined for 36 Chironomus species from the Palearctic, or Holarctic, and Australasia. The concatenated sequence of 1281 bp from both genes was used to investigate the phylogenetic relationships among these species. The nucleotide sequence alignments were used for construction of phylogenetic trees based on maximum-parsimony and neighbor-joining methods. Both techniques produced similar phylogenies. Monophyly of the genus Chironomus is supported by a bootstrap value of 100% at the basal branch. Six clusters of species have been revealed with high bootstrap values supporting both monophyly of each cluster and the validity of the branching order within each cluster. Four species, C. circumdatus, C. nepeanensis, C. dorsalis, and C. crassiforceps, cannot be placed into any cluster. Cytological phylogenies were constructed using the same set of species, except for C. biwaprimus. These trees showed many similarities to that obtained from the mitochondrial (mt) sequence analysis, but also a number of significant differences. When compared with the tree constructed from the sequence of 23 species available for one of the globin genes, globin 2b (gb2b), there was better support for the mt tree than for the cytological trees. An intron, which varies in its occurrence and position in gb2b, was also investigated and the distribution of the introns supports the phylogenetic history of the genus Chironomus obtained with mt data. The differences observed in the cytological trees seem to be attributable more to the retention of the same chromosome banding sequence across several species, rather than convergent evolutionary events. An important question is the determination of the position of the subgenus Camptochironomus in relation to the representatives of the nominal subgenus Chironomus, since it has been suggested that this is a separate genus. The Camptochironomus species are internal to the trees and have arisen more recently than some of the species of the subgenus Chironomus, indicating that they are not sufficiently differentiated to be considered more than a subgenus.     

Genetic divergence between the South Korean and Mongolian populations of the dung beetle,Gymnopleurus mopsus (Coleoptera: Scarabaeidae) based on mitochondrial cytochrome c oxidase subunit I (COI) gene sequences

The locally extinct dung beetle, Gymnopleurus mopsus Pallas, 1781 (Coleoptera: Scarabaeidae), has not been found in South Korea since the 1970s. This research was conducted to understand the genetic divergence between the South Korean and Mongolian populations of G. mopsus as a part of its reintroduction program in South Korea. The genetic distance and diversity were determined using the mitochondrial cytochrome c oxidase subunit I (COI) gene sequence (658 bp ) corresponding to the DNA barcode region. The mitochondrial COI gene sequences of 64 individuals of G. mopsus collected in South Korea (7 individuals) and Mongolia (57 individuals) showed a relatively high level of genetic diversity (nucleotide diversity, 0.0078 ± 0.0007; Haplotype diversity, 0.965 ± 0.017). The genetic distances between the South Korean and Mongolian populations lay within the intraspecific level and the phylogenetic reconstruction using the neighbor‐joining (NJ) method showed that all individuals belonged to a single clade. This result indicates that the current Mongolian population of G. mopsus is a good candidate source population to restore the locally extinct population of the species in South Korea.     

Mammalian mitochondrial DNA evolution: A comparison of the cytochrome b and cytochrome c oxidase II genes

The evolution of two mitochondrial genes, cytochrome b and cytochrome c oxidase subunit II, was examined in several eutherian mammal orders, with special emphasis on the orders Artiodactyla and Rodentia. When analyzed using both maximum parsimony, with either equal or unequal character weighting, and neighbor joining, neither gene performed with a high degree of consistency in terms of the phylogenetic hypotheses supported. The phylogenetic inconsistencies observed for both these genes may be the result of several factors including differences in the rate of nucleotide substitution among particular lineages (especially between orders), base composition bias, transition/transversion bias, differences in codon usage, and different constraints and levels of homoplasy associated with first, second, and third codon positions. We discuss the implications of these findings for the molecular systematics of mammals, especially as they relate to recent hypotheses concerning the polyphyly of the order Rodentia, relationships among the Artiodactyla, and various interordinal relationships.Correspondence to: R.L. Honeycutt     

Papilio phylogeny based on mitochondrial cytochrome oxidase I and II genes

Butterflies of the genus Papilio have served as the basis for numerous studies in insect physiology, genetics, and ecology. However, phylogenetic work on relationships among major lineages in the genus has been limited and inconclusive. We have sequenced 2.3 kb of DNA from the mitochondrial cytochrome oxidase I and II genes (COI and COII) for 23 Papilio taxa and two outgroups, Pachliopta neptunus and Eurytides marcellus, in order to assess the potential of these genes for use in Papilio phylogenetics and to examine patterns of gene evolution across a broad taxonomic range. Nucleotide and amino acid variation is distributed heterogeneously, both within and between genes. Structural features of the proteins are not always reliable predictors of variation. In a combined analysis, these sequences support a nearly fully resolved topology within subgenera and species groups, though higher level relationships among species groups require additional study. The most noteworthy findings are that neither Papilio alexanor nor P. xuthus belongs in the machaon group and that the subgenus Pterourus is paraphyletic with respect to the subgenus Pyrrhosticta. We leave relationships among members of the phorcas species group as a trichotomy. These two protein coding genes, particularly COI, show excellent performance in resolving relationships at the level of species and species groups among Papilionidae. We strongly endorse a similar approach for future studies aimed at these levels.