mt16S rRNA Research Articles

Morphological and Molecular Characters Differentiate Common Morphotypes of Atlantic Holopelagic Sargassum

Since 2011, massive new strandings of holopelagic Sargassum have been reported on the coasts of the Caribbean, northern Brazil, Guiana, and West Africa, causing severe economic and ecological damage. Three common morphotypes (S. fluitans III, S. natans I, and S. natans VIII) were identified as responsible for these catastrophic events, with dominance shifts between them over time. However, the taxonomic status of these holopelagic Sargassum morphotypes remains unclear. Using an integrative taxonomy framework, combining a morphological study and molecular analyses, this study aimed to clarify their taxonomic status. Morphological analyses of 54 characters revealed no intermediate form between the three morphotypes, with the overall shape, nature of the axis, and size and shape of blades and vesicles being the most discriminating. An analysis of mitochondrial (IGS, cox2, cox3, mt16S rRNA, and nad6) and plastid (rbcL) markers confirmed the genetic divergence among the three morphotypes, with a lower level of divergence between the two S. natans morphotypes. Without additional molecular characterization, these morphotypes cannot be classified as three distinct species. However, due to their distinct morphological characteristics and sympatry within drifting aggregations, a revision of holopelagic species names is proposed, with Sargassum fluitans var. fluitans (for S. fluitans III), Sargassum natans var. natans (for S. natans I), and S. natans var. wingei (for S. natans VIII). This revision provides necessary clarity on the species involved in inundations of the tropical Atlantic.

Molecular Investigation of Mitochondrial RNA19 Role in the Pathogenesis of MELAS Disease.

In mammalian mitochondria, the processing of primary RNA transcripts involves a coordinated series of cleavage and modification events, leading to the formation of processing intermediates and mature mt-RNAs. RNA19 is an unusually stable unprocessed precursor, physiologically polyadenylated, which includes the 16S mt-rRNA, the mt-tRNALeuUUR and the mt-ND1 mRNA. These peculiarities, together with the alteration of its steady-state levels in cellular models with defects in mitochondrial function, make RNA19 a potentially important molecule for the physiological regulation of mitochondrial molecular processes as well as for the pathogenesis of mitochondrial diseases. In this work, we quantitatively and qualitatively examined RNA19 in MELAS trans-mitochondrial cybrids carrying the mtDNA 3243A>G transition and displaying a profound mitochondrial translation defect. Through a combination of isokinetic sucrose gradient and RT-qPCR experiments, we found that RNA19 accumulated and co-sedimented with the mitoribosomal large subunit (mt-LSU) in mutant cells. Intriguingly, exogenous expression of the isolated LARS2 C-terminal domain (Cterm), which was shown to rescue defective translation in MELAS cybrids, decreased the levels of mt-LSU-associated RNA19 by relegating it to the pool of free unbound RNAs. Overall, the data reported here support a regulatory role for RNA19 in mitochondrial physiopathological processes, designating this RNA precursor as a possible molecular target in view of therapeutic strategy development.

A late-stage assembly checkpoint of the human mitochondrial ribosome large subunit

Many cellular processes, including ribosome biogenesis, are regulated through post-transcriptional RNA modifications. Here, a genome-wide analysis of the human mitochondrial transcriptome shows that 2’-O-methylation is limited to residues of the mitoribosomal large subunit (mtLSU) 16S mt-rRNA, introduced by MRM1, MRM2 and MRM3, with the modifications installed by the latter two proteins being interdependent. MRM2 controls mitochondrial respiration by regulating mitoribosome biogenesis. In its absence, mtLSU particles (visualized by cryo-EM at the resolution of 2.6 Å) present disordered RNA domains, partial occupancy of bL36m and bound MALSU1:L0R8F8:mtACP anti-association module, allowing five mtLSU biogenesis intermediates with different intersubunit interface configurations to be placed along the assembly pathway. However, mitoribosome biogenesis does not depend on the methyltransferase activity of MRM2. Disruption of the MRM2 Drosophila melanogaster orthologue leads to mitochondria-related developmental arrest. This work identifies a key checkpoint during mtLSU assembly, essential to maintain mitochondrial homeostasis.

Consistent genetic divergence observed among pelagic Sargassum morphotypes in the western North Atlantic

AbstractSargassum natans and Sargassum fluitans are uniquely holopelagic macroalgae, providing open ocean nursery and foraging habitat for commercially and ecologically important species. Recent basin‐wide changes in pelagic Sargassum diversity and distribution have manifested in proliferation of a previously rare morphotype, Sargassum natans VIII, to rival biomass levels of historically dominant S. natans I and S. fluitans III. Precise genetic identification of these morphotypes can improve accuracy and interpretation of ecological studies as well as clarify evolutionary history and population connectivity. For 139 field samples collected from the subtropical and tropical North Atlantic, three mitochondrial genes (cox3, nad6, and mt16S rRNA) were used to examine genetic divergence among the three common pelagic Sargassum morphotypes. These gene sequences successfully differentiated among morphotypes regardless of geographic origin, confirming in situ morphology‐based identifications. Sargassum natans I and S. natans VIII exhibited divergence consistent with that between the S. natans‐complex and S. fluitans III. Phylogenetic analysis of these samples also indicated evolutionary divergence between Sargassum morphologies. The genetic divergence among morphotypes, compared with benthic Sargassum species, suggested that taxonomic reclassification of the three most common pelagic morphotypes may be warranted.

Heterologous Inferential Analysis (HIA) and Other Emerging Concepts: In Understanding Mitochondrial Variation In Pathogenesis: There is no More Low-Hanging Fruit.

Here we summarize our latest efforts to elucidate the role of mtDNA variants affecting the mitochondrial translation machinery, namely variants mapping to the mt-rRNA and mt-tRNA genes. Evidence is accumulating to suggest that the cellular response to interference with mitochondrial translation is different from that occurring as a result ofmutations in genes encodingOXPHOS proteins. As a result, it appears safe to state that a complete view of mitochondrial disease will not be obtained until we understand the effect of mt-rRNA and mt-tRNA variants on mitochondrial protein synthesis. Despite the identification of a large number of potentially pathogenic variants in the mitochondrially encoded rRNA (mt-rRNA) genes, we lack direct methods to firmly establish their pathogenicity. In the absence of such methods, we have devised an indirect approach named heterologous inferential analysis (HIA ) that can be used to make predictions concerning the disruptive potential of a large subset of mt-rRNA variants. We have used HIA to explore the mutational landscape of 12S and 16S mt-rRNA genes. Our HIA studies include a thorough classification of all rare variants reported in the literature as well as others obtained fromstudies performed in collaboration with physicians. HIA has also been used with non-mammalian mt-rRNA genes to elucidate how mitotypes influence the interaction of the individual and the environment. Regarding mt-tRNA variations, rapidly growing evidence shows that the spectrum of mutations causing mitochondrial disease might differ between the different mitochondrial haplogroups seen in human populations.

Human GTPBP5 (MTG2) fuels mitoribosome large subunit maturation by facilitating 16S rRNA methylation.

Biogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome.

Sequence Variations and Molecular Phylogeny of some Red Sea Parrotfishes (Scaridae) Using Mitochondrial Gene Sequences

Targeted DNA fragments were isolated from four scarids species; Scarus collana Ruppell 1835; Scarus frenatus Lacepede 1802; Scarus (Chlorurus) sordidus Forsskal 1775 and Scarus niger Forsskal 1775 using mitochondrial 16S rRNA gene-specific primers. Analysis of obtained partial 16S rRNA gene nucleotide sequences showed a high level of nucleotide identity in the studied regions. This reflects a close genetic relationship and shared ancestry among studied parrotfishes. Nucleotide compositions of partial 16S rRNA gene nucleotide sequence biased towards adenine and similar preference towards thymine, cytocine, and guanine. Also, base constitutions revealed preference towards higher DNA conservations. Phylogenetic analysis displayed patterns of assembly for studied species, and other included related taxa, which reflect their similar genetic makeup and their tendency to have similar niches. The phylogenetic trees revealed two evolutionary lineages splitting Scaridae and Wrasses which assumed that Scaridae should maintain the family status. Obtained data could be beneficial for parootfishes classification, conservation, and their needed environments. Therefore, the acquisition of nucleotide sequences from other parrotfishes using the developed mt16S rRNA gene-specific primers utilized here would contribute in the future to the phylogenetic

TDP-43 stabilises the processing intermediates of mitochondrial transcripts

The 43-kDa trans-activating response region DNA-binding protein 43 (TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS). Despite of accumulating evidence that mitochondrial dysfunction underlies the pathogenesis of TDP-43–related ALS, the roles of wild-type TDP-43 in mitochondria are unknown. Here, we show that the small TDP-43 population present in mitochondria binds directly to a subset of mitochondrial tRNAs and precursor RNA encoded in L-strand mtDNA. Upregulated expression of TDP-43 stabilised the processing intermediates of mitochondrial polycistronic transcripts and their products including the components of electron transport and 16S mt-rRNA, similar to the phenotype observed in cells deficient for mitochondrial RNase P. Conversely, TDP-43 deficiency reduced the population of processing intermediates and impaired mitochondrial function. We propose that TDP-43 has a novel role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts.

Maturation of selected human mitochondrial tRNAs requires deadenylation.

Human mitochondria contain a genome (mtDNA) that encodes essential subunits of the oxidative phosphorylation system. Expression of mtDNA entails multi-step maturation of precursor RNA. In other systems, the RNA life cycle involves surveillance mechanisms, however, the details of RNA quality control have not been extensively characterised in human mitochondria. Using a mitochondrial ribosome profiling and mitochondrial poly(A)-tail RNA sequencing (MPAT-Seq) assay, we identify the poly(A)-specific exoribonuclease PDE12 as a major factor for the quality control of mitochondrial non-coding RNAs. The lack of PDE12 results in a spurious polyadenylation of the 3' ends of the mitochondrial (mt-) rRNA and mt-tRNA. While the aberrant adenylation of 16S mt-rRNA did not affect the integrity of the mitoribosome, spurious poly(A) additions to mt-tRNA led to reduced levels of aminoacylated pool of certain mt-tRNAs and mitoribosome stalling at the corresponding codons. Therefore, our data uncover a new, deadenylation-dependent mtRNA maturation pathway in human mitochondria.

The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNAMet, and mt-tRNAPhe, and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression.

Unequal distribution of 16S mtrRNA at the 2-cell stage regulates cell lineage allocations in mouse embryos

Cell lineage determination during early embryogenesis has profound effects on adult animal development. Pre-patterning of embryos, such as that of Drosophila and Caenorhabditis elegans, is driven by asymmetrically localized maternal or zygotic factors, including mRNA species and RNA binding proteins. However, it is not clear how mammalian early embryogenesis is regulated and what the early cell fate determinants are. Here we show that, in mouse, mitochondrial ribosomal RNAs (mtrRNAs) are differentially distributed between 2-cell sister blastomeres. This distribution pattern is not related to the overall quantity or activity of mitochondria which appears equal between 2-cell sister blastomeres. Like in lower species, 16S mtrRNA is found to localize in the cytoplasm outside of mitochondria in mouse 2-cell embryos. Alterations of 16S mtrRNA levels in one of the 2-cell sister blastomere via microinjection of either sense or anti-sense RNAs drive its progeny into different cell lineages in blastocyst. These results indicate that mtrRNAs are differentially distributed among embryonic cells at the beginning of embryogenesis in mouse and they are functionally involved in the regulation of cell lineage allocations in blastocyst, suggesting an underlying molecular mechanism that regulates pre-implantation embryogenesis in mouse.

Morphological and genetic differentiation of heteromorphy in Labidocera rotunda (Copepoda, Calanoida, Pontellidae).

The pontellid calanoid Labidocera rotunda Mori, 1929 is relatively widespread in the inshore surface waters of East Asia. In this study, some heteromorphic specimens have been observed with extreme morphological discrepancies in the female, including the genital double-somite, the second urosomite, the caudal rami, and the fifth leg. To evaluate the validity of species assignment of the morphological variants, we analyzed the DNA sequences of two mitochondrial genes, 16S rRNA and cytochrome oxidase subunit I (mtCOI). The specimens were collected in the Yellow Sea, the East China Sea and the East Sea (Sea of Japan) around Korea. They differed by <2.9% for mt16S rRNA and by 3.7%-4.7% for mtCOI, suggesting that they are conspecific despite their considerable morphological differences. In contrast, Labidocera japonica Mori, 1935, a morphologically similar species to L. rotunda, displayed interspecific variation of 6.1%-7.2% for mt16S rRNA and 15.1%-16.7% for mtCOI.

Molecular Phylogeny of Viperidae Family from Different Provinces in Saudi Arabia

Molecular systematic is important in solving the problematic taxonomy of venomous snakes and development of antivenins. The current study aimed to investigate the molecular phylogeny of 4 venomous species in Saudi Arabia using mitochondrial (mt) 16S rRNA gene. DNA extracted from blood and mt16S rRNA gene amplified by PCR. Sequences submitted to gene bank, examined for similarity with other sequences in the data base using BLAST search, aligned using Clustal W method, and phylogenetic tree was constructed. E. coloratus clustered as a separate group along with the isolate from Oman and Yemen with insignificant relation. Cerastes and Bitis arietans groups strongly correlated as sister taxa. The current B. arietans sample did not significantly correlate to the previously published sample from the same province (Taif). Cerastes groups did not significantly correlate with samples from Egypt or Israel. This information might reflect the need for multiple gene markers for better molecular systematic.

Species identification and phylogenetic analysis of genus Nassarius (Nassariidae) based on mitochondrial genes

Genus Nassarius contains many subgenera, such as Zeuxis, Telasco, Niotha, Varicinassa, Plicarcularia, Nassarius s. str. and Reticunassa. On the basis of morphological characteristics of the shell and radula and sequences of mitochondrial cytochrome oxidase subunit I (COI) and 16S rRNA genes, Nassarius specimens collected from the South China Sea were identified and phylogenetically analyzed. Although Nassarius sp. and Nassarius (Varicinassa) variciferus were morphologically different in their shells, few variations were found among their radular teeth and sequences of mtCOI and mt16S RNA genes. Therefore, Nassarius sp. should be classified as N. (Varicinassa) variciferus. Nassarius (Zeuxis) sp. has only a subtle difference from Nassarius (Zeuxis) algidus on the shell, but it shows obvious differences in radular teeth and DNA sequence, indicating that they are two distinct species. Sequence divergence of mtCOI and mt16S RNA genes within Nassarius species was much lower than that between species, suggesting that these two genes are suitable for Nassarius species identification. Phylogenetic analysis (neighbor-joining and maximum parsimony) based on mtCOI and mt16S rRNA genes revealed the presence of two groups in genus Nassarius and a closest relationship between subgenera Zeuxis and Telasco. Species of subgenus Plicarcularia did not form a single clade. The molecular phylogeny was not congruent with the previous morphological phylogeny. The subgeneric divisions of genus Nassarius appear to be uncertain and unreliable.

Expression of a family of noncoding mitochondrial RNAs distinguishes normal from cancer cells

We reported the presence in human cells of a noncoding mitochondrial RNA that contains an inverted repeat (IR) of 815 nucleotides (nt) covalently linked to the 5' end of the mitochondrial 16S RNA (16S mtrRNA). The transcript contains a stem-loop structure and is expressed in human proliferating cells but not in resting cells. Here, we demonstrate that, in addition to this transcript, normal human proliferating cells in culture express 2 antisense mitochondrial transcripts. These transcripts also contain stem-loop structures but strikingly they are down-regulated in tumor cell lines and tumor cells present in 17 different tumor types. The differential expression of these transcripts distinguishes normal from tumor cells and might contribute a unique vision on cancer biology and diagnostics.

Expression of a novel non-coding mitochondrial RNA in human proliferating cells

Previously, we reported the presence in mouse cells of a mitochondrial RNA which contains an inverted repeat (IR) of 121 nucleotides (nt) covalently linked to the 5′ end of the mitochondrial 16S RNA (16S mtrRNA). Here, we report the structure of an equivalent transcript of 2374 nt which is over-expressed in human proliferating cells but not in resting cells. The transcript contains a hairpin structure comprising an IR of 815 nt linked to the 5′ end of the 16S mtrRNA and forming a long double-stranded structure or stem and a loop of 40 nt. The stem is resistant to RNase A and can be detected and isolated after digestion with the enzyme. This novel transcript is a non-coding RNA (ncRNA) and several evidences suggest that the transcript is synthesized in mitochondria. The expression of this transcript can be induced in resting lymphocytes stimulated with phytohaemagglutinin (PHA). Moreover, aphidicolin treatment of DU145 cells reversibly blocks proliferation and expression of the transcript. If the drug is removed, the cells re-assume proliferation and over-express the ncmtRNA. These results suggest that the expression of the ncmtRNA correlates with the replicative state of the cell and it may play a role in cell proliferation.

Molecular dysfunction associated with the human mitochondrial 3302A>G mutation in the MTTL1 (mt-tRNALeu(UUR)) gene

The gene encoding mt-tRNALeu(UUR), MT-TL1, is a hotspot for pathogenic mtDNA mutations. Amongst the first to be described was the 3302A>G transition which resulted in a substantial accumulation in patient muscle of RNA19, an unprocessed RNA intermediate including mt-16S rRNA, mt-tRNALeu(UUR) and MTND1. We have now been able to further assess the molecular aetiology associated with 3302A>G in transmitochondrial cybrids. Increased steady-state levels of RNA19 was confirmed, although not to the levels previously reported in muscle. This data was consistent with an increase in RNA19 stability. The mutation resulted in decreased mt-tRNALeu(UUR) levels, but its stability was unchanged, consistent with a defect in RNA19 processing responsible for low tRNA levels. A partial defect in aminoacylation was also identified, potentially caused by an alteration in tRNA structure. These deficiencies lead to a severe defect in respiration in the transmitochondrial cybrids, consistent with the profound mitochondrial disorder originally associated with this mutation.

Phylogeny of Japanese Stag Beetles (Coleoptera: Lucanidae) Inferred from 16S mtrRNA Gene Sequences, with Reference to the Evolution of Sexual Dimorphism of Mandibles

As a first step in reconstructing the phylogeny of world stag beetles (Coleoptera: Lucanidae), phylogenetic relationships among the major members of Japanese stag beetles were explored by analyzing a sequence of 1030 nucleotides from the mitochondrial 16S ribosomal RNA (16S rRNA) gene. A total of 20 species and three additional subspecies representing 13 genera were examined to provide basic information on the phylogeny of world Lucanidae. The resultant phylogenetic tree indicates that the family Lucanidae is monophyletic, and contains two major lineages: one consists of the genera Platycerus, Aesalus, Ceruchus, and Nicagus, and the other includes Dorcus, Rhaetulus, Prosopocoilus, Aegus, Neolucanus, Prismognathus, Lucanus, Figulus, and Nigidius. Generic members of the latter lineage are further divided into the following four sublineages: i) Figulus and Nigidius; ii) Prismognathus and Lucanus; iii) Aegus and Neolucanus; and iv) Dorcus, Rhaetulus, and Prosopocoilus. These molecular phylogenetic relationships are used as a basis for a preliminary exploration of the evolution of sexual dimorphism in the shape of the mandible. The results of this investigation suggest that strong sexual dimorphism with well-developed mandibles in males evolved independently at least twice, once in the genus Aegus and once in the ancestor of the Lucanus-Prismognathus and Dorcus-Rhaetulus-Prosopocoilus clades. Alternatively, it is possible that sexual dimorphism of mandibles has undergone secondary loss in the genera Figulus and Nigidius.

Phylogenetic Analysis of Mediterranean Mugilids by Allozymes and 16S mt-rRNA Genes Investigation: Are the Mediterranean Species of Liza Monophyletic?

The family Mugilidae (Pisces, Mugiliformes) includes species which are present in all tropical and temperate regions. Six species, Chelon labrosus, Mugil cephalus, Liza aurata, L. ramada, L. saliens, Oedalechilus labeo, are commonly found in the Mediterranean. These species have been widely studied through morphological, biochemical, and molecular markers. However, their phylogenetic relationships, and therefore the assumed monophyly of Liza species, still remain unclear: To further investigate this topic, gene-enzyme systems and sequences of the partial 16S rRNA mitochondrial gene were analyzed in Italian samples of all six Mediterranean species. The phylogenetic reconstructions indicated M. cephalus as being the most divergent species and the existence of a main cluster including all the Mediterranean species of Liza and C. labrosus. The parametric bootstrap approach adopted to test alternative phylogenetic hypotheses indicated that the Mediterranean species of Liza do not form a monophyletic group exclusive of Chelon.

Multiplexed species-specific PCR protocol to discriminate four N. Atlantic Calanus species, with an mtCOI gene tree for ten Calanus species

Accurate species identification is the cornerstone of any ecological study – yet this fundamental step is not always possible for marine zooplankton. Routine species' identification, especially of juvenile andlarval stages, is difficult for Calanus species (Copepoda; Calanoida) in the N. Atlantic Ocean, where two or three species may co-occur. A rapid, simple, and inexpensive molecularly based protocol to identify individual copepods of any life stage has beendeveloped. This protocol will routinely identify four Calanus species in the N. Atlantic and will help to accurately understand the role of each species in coastal and open ocean ecosystems. The DNA sequence of a 633 bp region of the mitochondrial cytochrome oxidase I (mtCOI) was determined for ten Calanus species: C.australis (Brodsky, 1959),C.chilensis (Brodsky, 1959),C.finmarchicus (Gunnerus, 1770),C.glacialis (Jaschnov, 1955),C.helgolandicus (Claus, 1863),C.hyperboreus (Kroyer, 1838),C.marshallae (Frost, 1974),C.pacificus (Brodsky, 1948),C.simillimus (Giesbrecht, 1902), and C.sinicus (Brodsky, 1965). MtCOI sequences were used to design species-specific oligonucleotide primers for C.finmarchicus,C.glacialis,C.helgolandicus, and C.hyperboreus and to optimize a competitive, multiplexed, species-specific PCR (SS-PCR) protocol to discriminate the four species. This corrects and improves a previously published protocol for three Calanus species (Bucklin et al. 1999: Hydrobiologia 401:239–254), unambiguously identifying individual copepods and copepodites from diverse geographic regions of the four species' ranges. In order to further examine the pattern of mtCOI evolution within Calanus (an important consideration for molecular systematic characters), consensus mtCOI sequences were used to reconstruct phylogenetic relationships among the ten species; the mtCOI gene tree agreed with morphological and molecular (based on mt16S rRNA) phylogenies, except that the affiliation of C.sinicus could not be resolved.