Cytochrome Oxidase I Gene Sequences Research Articles

Razor Clams (Class Bivalvia) of Kuala Selangor, Malaysia: Morphology, Genetic Diversity and Heavy Metal Concentration

Razor Clam (Bivalvia) could be easily identified as having two long, narrow shell valves connected by hinges, two siphons and one strong foot. It plays important role in ecological services besides providing food and extra income for the coastal communities. As filter feeder, razor clam is a good indicator for heavy metal contamination in aquatic environments. In Selangor, razor clam is locally known as ‘siput buluh’, yet, there is no proper scientific documentation on this species. This short study was carried out to assess the morphology of razor clams in Selangor for species identification and to sequence the Cytochrome Oxidase I (COI) gene for determination of its genetic diversity. Heavy sea traffics occurred in the sea near to the mudflat where razor clams were abundantly found has raised question whether razor clams were safe to be consumed. Therefore, this study is also designed to determine the level of selected heavy metals concentration in different body parts of razor clams. Based on selected morphometric and meristic data, razor clams of Kuala Selangor obtained during this study were Solen sarawakensis although many individuals displayed darker outer shell colouration and slightly thicker, longer and heavier shells. Based on analyses of putative COI gene sequences of approximately 677bp, there was a strong genetic divergence (11.7%) between S. sarawakensis inhabiting Kuala Selangor and Kuching Bay, Sarawak suggesting that although both razor clams were very similar morphologically, they could actually be two different species. Tissues of razor clams sampled from mudflat area in Kuala Selangor showed high concentration of Fe (146.50 mg/kg) and Mn (94.00 mg/kg). Other heavy metal elements namely Cr (42.50 mg/kg) and Cd (33.00 mg/kg) were also recorded during this study, occurring mainly in the shells. Traces of Hg were also detected, and its concentration did not differ much among the different parts analyzed.

Exploited but Unevaluated: DNA Barcoding Reveals Skates and Stingrays (Chordata, Chondrichthyes) Species Landed in the Indonesian Fish Market

Reliable and precise species identification is important to fisheries management and conservation. However, many rays and skates in Indonesia are currently being exploited and landed into traditional fish market without a proper identification. Therefore, this study was conducted to identify species of skates and stingrays that were landed and traded in three fish markets in Indonesia (Palabuhanratu, Muara Saban, and Lampung) using molecular techniques and to determine the conservation status of the identified species based on IUCN (International Union for Conservation of Nature and Natural Resources) as well as defined by CITES (Convention on International Trade in Endangered Species). The mitochondrial cytochrome oxidase I (COI) gene was amplified by polymerase chain reaction (PCR) using a pair of primer, fish-BCL and fish-BCH. Of 29 tissue samples collected from the study sites, a total of five species were successfully identified: Dipturus chilensis (4), Himantura walga (1), Neotrygon kuhlii (11), Taeniura lymma (9) and Rhinoptera javanica (4). The Neighbor Joining phylogeny of mitochondrial lineages, based on partial COI gene sequences, the ingroup haplotypes were clustered into five main clades representing each species. The identified stingrays were being listed as vulnerable (D. chilensis and R. javanica), near threatened (H. walga and T. lymma), and data deficient (N. kuhlii) by IUCN, with two species (D. chilensis and H. walga) population were indicated decreased. Unfortunately, all of identified species have not been evaluated by CITES regarding their trade status. As a consequences, a valuable effort should be placed to create a scientific network for monitoring programmes not only on a local scale, and to make pressure on governments for adopting molecular techniques as tools for controlling and avoiding misidentification. Keywords: Mitochondrial DNA, Phylogeny, Coral Triangle, Taxonomy, Fisheries

Are microbes fundamentally different than macroorganisms? Convergence and a possible case for neutral phenotypic evolution in testate amoeba (Amoebozoa: Arcellinida).

This study reveals extensive phenotypic convergence based on the non-monophyly of genera and morphospecies of testate (shelled) amoebae. Using two independent markers, small subunit ribosomal DNA (ssu-rDNA) and mitochondrial cytochrome oxidase I (COI), we demonstrate discordance between morphology and molecules for ‘core Nebela’ species (Arcellinida; Amoebozoa). Prior work using just a single locus, ssu-rDNA, also supported the non-monophyly of the genera Hyalosphenia and Nebela as well as for several morphospecies within these genera. Here, we obtained COI gene sequences of 59 specimens from seven morphospecies and ssu-rDNA gene sequences of 50 specimens from six morphospecies of hyalosphenids. Our analyses corroborate the prior ssu-rDNA findings of morphological convergence in test (shell) morphologies, as COI and ssu-rDNA phylogenies are concordant. Further, the monophyly of morphospecies is rejected using approximately unbiased tests. Given that testate amoebae are used as bioindicators in both palaeoecological and contemporary studies of threatened ecosystems such as bogs and fens, understanding the discordance between morphology and genetics in the hyalosphenids is essential for interpretation of indicator species. Further, while convergence is normally considered the result of natural selection, it is possible that neutrality underlies phenotypic evolution in these microorganisms.

Molecular Identification of Forensically Important Indian Species of Flesh Flies (Diptera: Sarcophagidae) by Using COI Gene of Mitochondrial DNA

Identification of the fauna collected from and around the corpse is an absolutely vital prerequisite for the estimation of post mortem interval(PMI) in the field of forensic entomology. Morphological identification of flesh flies (Diptera; Sarcophagidae) can be perplexing due similar attributes of the species. So, to simplify the identification method, amplification of cytochrome oxidase I (COI) gene is often tried. We illustrate the use of 450 bp fragment of COI gene for differentiation of ten forensically significant species of flesh flies in India. The outcome displays the robustness of COI gene as a diagnostic marker, since its nucleotide variability endows reliable distinction to be drawn between species. Four new COI gene sequences have been added to GenBank which may be of interest for correct species identification for future workers.

DNA Barcoding and Elucidation of Cryptic Diversity in Thrips (Thysanoptera)

Accurate and timely identification of invasive insect pests underpins most biological endeavors ranging from biodiversity estimation to insect pest management. In this regard, identification of thrips, an invasive insect pest is important and challenging due to their complex life cycles, parthenogenetic mode of reproduction, sex and color morphs. In the recent years, DNA barcoding employing 5′ region of the mitochondrial Cytochrome Oxidase I (CO-I) gene has become a popular tool for species identification. In this study, we employed CO-I gene sequences for discriminating 151 species of thrips for the first time. Analyses of the intraspecific and intrageneric distances of the CO-I sequences ranged from 0.0 to 7.91% and 8.65% to 31.15% respectively. This study has revealed the existence of cryptic species in Thrips hawaiiensis (Morgan) (Thysanoptera: Thripidae) and Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) for the first time, along with previously reported cryptic species such as Thrips palmi Karny (Thysanoptera: Thripidae), T. tabaci Lindeman, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), Scirtothrips dorsalis Hood. We are proposing, the feasibility of hosting an independent integrated taxonomy library for thrips and indicate that it can serve as an effective system for species identification, this approach could potentially play a key role in formulating effective insect pest management strategies.

Homology difference analysis of invasive mealybug species Phenacoccus solenopsis Tinsley in Southern China with COI gene sequence variability.

The mealybug species Phenacoccus solenopsis (P. solenopsis) has caused much agricultural damage since its recent invasion in China. However, the source of this invasion remains unclear. This study uses molecular methods to clarify the relationships among different population of P. solenopsis from China, USA, Pakistan, India, and Vietnam to determine the geographic origin of the introduction of this species into China. P. solenopsis samples were collected from 25 different locations in three provinces of Southern China. Samples from the USA, Pakistan, and Vietnam were also obtained. Parts of the mitochondrial genes for cytochrome oxidase I (COI) were sequenced for each sample. Homologous DNA sequences of the samples from the USA and India were downloaded from Gen Bank. Two haplotypes were found in China. The first was from most samples from the Guangdong, Guangxi, and Hainan populations in the China and Pakistan groups, and the second from a few samples from the Guangdong, Guangxi, Hainan populations in the China, Pakistan, India, and Vietnam groups. As shown in the maximum likelihood of trees constructed using the COI sequences, these samples belonged to two clades. Phylogenetic analysis suggested that most P. solenopsis mealybugs in Southern China are probably closely related to populations in Pakistan. The variation, relationship, expansion, and probable geographic origin of P. solenopsis mealybugs in Southern China are also discussed.

Morphobiometrical and molecular study of two populations of Demodex folliculorum from humans

A morphobiometrical and molecular study of two populations of Demodex folliculorum from humans isolated from different habitats, skin and eyelashes follicles, were carried out. Morphological and biometrical studies revealed two closely related populations with any distinctive characteristics. For molecular study, a 436-bp region of the 16S rDNA gene and a 453-bp region of the cytochrome oxidase I (COI) gene from individual mites of each population considered were sequenced. Intraindividual and interindividual sequence variation was studied in both populations. Our data show that 16S rDNA is not a useful marker to discriminate between populations; however, COI gene sequences can help to identify the two populations considered, which are morphologically very close and difficult to separate by classic methods. These results are in agreement with the morphological and biometrical differences detected between D. folliculorum from eyelashes and human skin. This study appeals for the revision of the taxonomic status of the D. folliculorum populations, as well as for the species included within genus Demodex.

A new mtDNA COI gene lineage closely related to Anopheles janconnae of the Albitarsis complex in the Caribbean region of Colombia

An understanding of the taxonomic status and vector distribution of anophelines is crucial in controlling malaria. Previous phylogenetic analyses have supported the description of six species of the Neotropical malaria vector Anopheles (Nyssorhynchus) albitarsis s.l. (Diptera: Culicidae): An. albitarsis, Anopheles deaneorum, Anopheles marajoara, Anopheles oryzalimnetes, Anopheles janconnae and An. albitarsis F. To evaluate the taxonomic status of An. albitarsis s.l. mosquitoes collected in various localities in the Colombian Caribbean region, specimens were analyzed using the complete mitochondrial DNA cytochrome oxidase I (COI) gene, the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) region and partial nuclear DNA white gene sequences. Phylogenetic analyses of the COI gene sequences detected a new lineage closely related to An. janconnae in the Caribbean region of Colombia and determined its position relative to the other members of the complex. However, the ITS2 and white gene sequences lacked sufficient resolution to support a new lineage closely related to An. janconnae or the An. janconnae clade. The possible involvement of this new lineage in malaria transmission in Colombia remains unknown, but its phylogenetic closeness to An. janconnae, which has been implicated in local malaria transmission in Brazil, is intriguing.

DNA barcoding of hermit crabs of genus Clibanarius Dana, 1852 (Anomura: Diogenidae) in the Ryukyu Islands, southwestern Japan

The diogenid hermit crab genus Clibanarius Dana, 1852 is a group of decapod crustaceans commonly found in tropical or subtropical shallow waters. The species of the genus are usually characterized by distinct color patterns in life, but the identification of colorless specimens preserved in ethanol is sometimes very difficult. DNA barcoding can facilitate species identification and recognition. In this study, we determined partial sequences of the cytochrome oxidase I (COI) gene of 11 Clibanarius species commonly found in the intertidal zones of the Ryukyu Archipelago, southwestern Japan. The sequences can be used as nametags to distinguish each of the 11 species, as the data differ for each species. The COI gene sequences of Clibanarius show an AT bias, especially in the third codon position (A+T = 76.2%), as has been reported for some other anomuran species. Phylogenetic analyses based on the sequences also offer some suggestions on the interspecific relationships of Clibanarius.

Phylogenetic relationships of Andean-Ecuadorian populations of Anastrepha fraterculus (Wiedemann 1830) (Diptera: Tephritidae) inferred from COI and COII gene sequences

Phylogenetic relationships among Andean-Ecuadorian and other Neotropical populations of Anastrepha fraterculus and related species have been studied using two regions of mtDNA : 405 base pairs within Cytochrome Oxidase I ( COI) and 224 base pairs within Cytochrome Oxidase II (COII). Phylogenetic relationships were inferred using Maximun Parsimony (MP) method and haplotype networks. Andean-Ecuadorian populations of A. fraterculus are monomorphic at the COI locus and fall within a clade of South-American lowland populations of A. fraterculus. They appear to be unrelated with populations of northern Andes of Colombia and Venezuela also assigned to A. fraterculus, meaning that this species, as currently circumscribed, is not monophyletic and is composed of different biological entities that are little differentiated morphologically. At the COII locus, Andean-Ecuadorian populations of A. fraterculus show a major haplotype with a few variants, and form a clade with the lowland populations of southern Brazil an Argentina, but are clearly differentiated from them. Andean-Ecuadorian populations of Anastrepha fraterculus appear to be homogeneous with respect to their mitochondrial genome and thus their identity as members of a single gene pool is confirmed by these results.

COI (cytochrome oxidase-I) sequence based studies of Carangid fishes from Kakinada coast, India

Mitochondrial DNA, cytochrome oxidase-1 gene sequences were analyzed for species identification and phylogenetic relationship among the very high food value and commercially important Indian carangid fish species. Sequence analysis of COI gene very clearly indicated that all the 28 fish species fell into five distinct groups, which are genetically distant from each other and exhibited identical phylogenetic reservation. All the COI gene sequences from 28 fishes provide sufficient phylogenetic information and evolutionary relationship to distinguish the carangid species unambiguously. This study proves the utility of mtDNA COI gene sequence based approach in identifying fish species at a faster pace.

Lessons from leeches: a call for DNA barcoding in the lab

Many evolution of development labs study organisms that must be periodically collected from the wild. Whenever this is the case, there is the risk that different field collections will recover genetically different strains or cryptic species. Ignoring this potential for genetic variation may introduce an uncontrolled source of experimental variability, leading to confusion or misinterpretation of the results. Leeches in the genus Helobdella have been a workhorse of annelid developmental biology for 30 years. Nearly all early Helobdella research was based on a single isolate, but in recent years isolates from multiple field collections and multiple sites across the country have been used. To assess the genetic distinctness of different isolates, we obtained specimens from most Helobdella laboratory cultures currently or recently in use and from some of their source field sites. From these samples, we sequenced part of the mitochondrial gene cytochrome oxidase I (COI). Sequence divergences and phylogenetic analyses reveal that, collectively, the Helobdella development community has worked on five distinct species from two major clades. Morphologically similar isolates that were thought to represent the same species (H. robusta) actually represent three species, two of which coexist at the same locality. Another isolate represents part of a species complex (the "H. triserialis" complex), and yet another is an invasive species (H. europaea). We caution researchers similarly working on multiple wild-collected isolates to preserve voucher specimens and to obtain from these a molecular "barcode," such as a COI gene sequence, to reveal genetic variation in animals used for research.

Mitochondrial DNA Haplotype Variation in the Parasitic Cirripede Sacculina Carcini Observed in the Cytochrome Oxidase Gene (COI)

Nucleotide sequence data of the mitochondrial cytochrome oxidase I (COI) gene, were compared among 17 specimens of Sacculina carcini parasitising three portunid hosts as a preliminary assessment of haplotype structure in a poorly understood species for which there are few morphological characters on which to base taxonomic analysis. S. carcini parasitising Carcinus maenas (from Sweden, England and Denmark) were compared with S. carcini parasitising Liocarcinus marmoreus (from Ireland) and Liocarcinus holsatus (from Wales). Specimens of three congeneric sacculinid species and one confamilial species were included in the comparison as outgroups. The comparison confirmed that specimens of S. carcini from different hosts and different regions are all the same species. The data also suggested geographically consistent sequence differences among sampled sites, high levels of similarity within sites and very large differences between species, all of which suggests that analysis of the COI gene sequence could be a useful method for resolving population genetics and taxonomy of rhizocephalans.

Mitochondrial DNA sequences of triatomines (Hemiptera: Reduviidae): phylogenetic relationships.

The phylogenetic relationships among 18 species of Triatominae were inferred based on mitochondrial DNA (mtDNA) sequences. The species of Triatoma included 11 belonging to the infestans complex [T. infestans (Klug), T. guasayana Wygodzinsky & Abalos, T. sordida (Stål), T. platensis Neiva, T. brasiliensis Neiva, T. rubrovaria (Blanchard), T. vitticeps (Stål), T. delpontei Romaña & Abalos, T. maculata (Erichson), T. patagonica Del Ponte, and T. matogrossensis Leite & Barbosa] and four others of the same genus but of different complexes [T. circummaculata (Stål), T. protracta (Uhler), T. dimidiata (Latreille), and T. mazzottii Usinger]. As possible outgroups we used Mepraia spinolai Mazza, Panstrongylus megistus (Burmeister), and Rhodnius prolixus Stål. We analyzed mtDNA fragments of the 12S and 16S ribosomal RNA genes from each of the 18 species, as well as of the cytochrome oxidase I (COI) gene from nine. The 12S, 16S, and COI gene sequences were analyzed individually and combined. All of the phylogenetic analyses unambiguously supported two clusters: one including T. infestans, T. platensis, and T. delpontei, and the other T. sordida and T. mutagrossensis. Inclusion of T. circummaculata into the infestans complex was confirmed, although this is in disagreement with the morphological classification. On the other hand, our analyses showed that T. dimidiata is closely related to a phylosoma complex species, T. mazzottii. This is consistent with the tentative classification previously made based on morphological characters. The issue of the monophyly of the genus Triatoma remains unresolved.