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.     

Identification of Channa species using the partial cytochrome c oxidase subunit I (COI) gene as a DNA barcoding marker

The snakehead fish of the genus Channa are an important food fish in China. However, the molecular identification and phylogeny of this genus is poorly understood. Here, we present the utility of partial sequences of the COI gene for use in DNA barcoding for the identification of Channa individuals, which includes four species: Channa argus, Channa maculata, Channa asiatica, and Channa striata. A total of 19 haplotypes were identified in this study. The interspecific K2P distances were higher than intraspecific distances. The lowest interspecific distance (0.091) was between C. argus and C. maculata while the highest interspecific distance (0.219) was between C. argus and C. striata. No intraspecific–interspecific distance overlaps were observed, and a distinct barcoding gap was found between intraspecific and interspecific distances in each species. Our results showed that the partial COI gene is an effective DNA barcoding marker for identifying Channa species.     

Protein–protein interactions of the cytochrome c oxidase DNA barcoding region

Enzymatic amplification of homologous regions of DNA using ‘universal’ polymerase chain reaction primers has provided insight into insect systematics, phylogeography, molecular evolution and species identification. One of the more commonly amplified and sequenced regions is a short region of the cytochrome c oxidase subunit I gene (COI), commonly called the barcoding region. COI is one of three mitochondrial‐encoded subunits of complex IV (Cox) of the electron transport chain. In addition to the mitochondrial subunits there are nine nuclear‐encoded subunits of the complex in Drosophila. Whereas a number of phylogenetic biases associated with this region have been examined and the quaternary structure of Cox has been modelled, the influence of protein–protein interactions on the observed patterns of evolution in this barcoding region of insects has never been examined critically. Using a well‐resolved independently derived phylogeny of 38 Diptera species, we examined the homogeneity of the substitution processes within the barcoding region. We show that, within Diptera, amino acid residues interacting with nuclear‐encoded subunits of Cox are evolving at elevated rates across the phylogeny. Furthermore, we show that codon position two is biased by protein–protein interactions. In contrast, third codon positions provide a less biased estimate of genetic variation in the region. This study highlights the need to examine the potential for systematic bias in DNA barcoding regions as part of the critical assessment of evidence in systematics and in biodiversity assessments.     

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.     

DNA barcoding of sunn pest adult parasitoids using cytochrome c oxidase subunit I (COI)

In this study, DNA barcoding was used in the identification of potential biological control agents of sunn pest adult parasitoid species, including Eliozeta helluo (F.), Phasia subcoleoptrata (L.), Ectophasia crassipennis (F.) and Elomyia lateralis (Meig). DNA analyses were assessed by sequencing cytochrome c oxidase subunit I (COI) gene. The obtained sequences were analyzed in terms of nucleotide composition, nucleotide pair frequency and haplotype diversity. Genetic divergence among haplotypes was estimated by constructing genetic distance matrix using DNA sequence variations, by Kimura 2-parameter model. Variable sites and average variations of the sequenced 603 base pair long DNA fragment were calculated. All COI barcodes were matched with reference sequences of expected species according to morphological identification. Neighbor-joining tree was drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. The evolutionary history inferred using the UPGMA method indicated two distinct mitochondrial haplotype lineages. The genetic variation between sunn pest adult parasitoids will be useful in sunn pest management, regulatory and environmental applications.     

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.     

Genetic variability in the Arabian oryx (Oryx leucoryx)

An electrophoretic survey of blood markers of the Arabian oryx (Oryx leucoryx) was undertaken in order to ascertain the genetic variability in a sample of 85 individuals, mainly from Saudi Arabian (Taíf) and Jordanian herds. Three out of 18 loci were found to be polymorphic (P = 16.7%) and the mean heterozygosity (H = 0.052) appears to be relatively high with respect to the severe demographic bottlenecks expressed by the species since the 1960s. No genetic differentiation was found between Arabian and Jordanian samples considered. Consequences of these findings for the management of the Taíf herd and for such procedures as pedigree determination are discussed, and an example of this latter application is given for a case of doubtful parentage.     

Robertsonian chromosome polymorphism in the Arabian oryx (Oryx leucoryx)

A Robertsonian translocation was found in a herd of Arabian oryx (Oryx leucoryx). The translocated chromosome, when analyzed by G-banding, seemed to involve the fusion of chromosomes 17 and 19. The results of C-banding suggested that the fused chromosome is dicentric. The translocation was traced back through two generations and occurred in a total of 8 of 62 animals in the herd.     

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.     

Molecular characterization of Echinococcus granulosus from Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene

Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosus worldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.     

The G- and C-banding karyotype of the Arabian oryx (Oryx leucoryx)

The chromosomes of the Arabian oryx (Oryx leucoryx) were investigated using GTG and CBG banding technique. Their banding patterns were compared to those of goat and cattle.     

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     

Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3'-end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa3-type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.     

Limitations of mitochondrial gene barcoding in Octocorallia

The widespread assumption that COI and other mitochondrial genes will be ineffective DNA barcodes for anthozoan cnidarians has not been well tested for most anthozoans other than scleractinian corals. Here we examine the limitations of mitochondrial gene barcoding in the sub-class Octocorallia, a large, diverse, and ecologically important group of anthozoans. Pairwise genetic distance values (uncorrected p) were compared for three candidate barcoding regions: the Folmer region of COI; a fragment of the octocoral-specific mitochondrial protein-coding gene, msh1; and an extended barcode of msh1 plus COI with a short, adjacent intergenic region (igr1). Intraspecific variation was <0.5%, with most species exhibiting no variation in any of the three gene regions. Interspecific divergence was also low: 18.5% of congeneric morphospecies shared identical COI barcodes, and there was no discernible barcoding gap between intra- and interspecific p values. In a case study to assess regional octocoral biodiversity, COI and msh1 barcodes each identified 70% of morphospecies. In a second case study, a nucleotide character-based analysis correctly identified 70% of species in the temperate genus Alcyonium. Although interspecific genetic distances were 2× greater for msh1 than COI, each marker identified similar numbers of species in the two case studies, and the extended COI + igr1 + msh1 barcode more effectively discriminated sister taxa in Alcyonium. Although far from perfect for species identification, a COI + igr1 + msh1 barcode nonetheless represents a valuable addition to the depauperate set of characters available for octocoral taxonomy.     

PCR-RFLP analysis of the cytochrome b gene in horse mitochondrial DNA

The mitochondrial DNA sequence of cytochrome b gene in a Thoroughbred horse was determined. By comparing DNA sequences between the Thoroughbred and published sequence data (two horses and one Grevyi zebra), polymerase chain reaction (PCR) primers were designed for amplification of a 590 bp DNA fragment in the cytochrome b gene, and PCR-restriction fragment length polymorphism (RFLP) analysis was studied in 140 horses of six breeds using three restriction enzymes ( AciI, BamHI, RsaI ). Two morphs were found using each of the three enzymes. By combining three enzymes morphs, the 140 horses examined were classified into four types. Type 2 was most frequent in all breeds.     

Chicken mitochondrial cytochrome C oxidase subunit II: comparative analysis among the vertebrates

The chicken cytochrome c oxidase subunit II (COII) was cloned and sequenced. A comparison of the deduced chicken COII sequence with 4 other vertebrate counterparts revealed 64-66% amino acid sequence homology and 68-70% nucleotide sequence homology. Four peptide segments each of nine amino acids long are highly conserved across the 5 species. A redox-center was formed by three of these highly conserved domains, which include two invariant Cys and two invariant His residues for copper ion coordination, three strictly conserved Glu or Asp residues for cytochrome c binding, and highly conserved aromatic acid residues for electron transfer.