Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes

The entire mitochondrial genome was sequenced in a prostriate tick, Ixodes hexagonus, and a metastriate tick, Rhipicephalus sanguineus. Both genomes encode 22 tRNAs, 13 proteins, and two ribosomal RNAs. Prostriate ticks are basal members of Ixodidae and have the same gene order as Limulus polyphemus. In contrast, in R. sanguineus, a block of genes encoding NADH dehydrogenase subunit 1 (ND1), tRNA(Leu)(UUR), tRNA(Leu)(CUN), 16S rDNA, tRNA(Val), 12S rDNA, the control region, and the tRNA(Ile) and tRNA(Gln) have translocated to a position between the tRNA(Glu) and tRNA(Phe) genes. The tRNA(Cys) gene has translocated between the control region and the tRNA(Met) gene, and the tRNA(Leu)(CUN) gene has translocated between the tRNA(Ser)(UCN) gene and the control region. Furthermore, the control region is duplicated, and both copies undergo concerted evolution. Primers that flank these rearrangements confirm that this gene order is conserved in all metastriate ticks examined. Correspondence analysis of amino acid and codon use in the two ticks and in nine other arthropod mitochondrial genomes indicate a strong bias in R. sanguineus towards amino acids encoded by AT-rich codons.      

Mitochondrial sequence variation in Carlos capensis (Neumann), a parasite of seabirds,collected on Torishima Island in Japan

Partial mitochondrial 16S ribosomal ribonucleic acid gene sequences in the ticks Carios capensis collected from black-footed albatross. Diomedea nigripes, colonies on Torishima Island, Japan (30 degrees 28'N, 140 degrees 18'E), were examined. The sequence was compared with those of C. capensis from Hawaii, South Carolina, and Texas. The sequences were all identical in ticks from Torishima and 2 from Hawaii. There were 2-3 transitions between the other Hawaiian and Texas ticks and Torishima specimens. Two transitions were also observed when compared with the ticks from South Carolina. The results suggest the possibility of gene flow between tick populations at each of the 2 geographic sites, which probably was accomplished by tick-infested migratory seabirds at their breeding sites. Sequence comparison analysis indicated that the C. capensis ticks are on the branch with C. marginatus and C. mexicanus ticks and not with Ornithodoros. This supports the revision suggested by Klompen and Oliver (1993).     

Mitochondrial sequence evolution in spiders: intraspecific variation in tRNAs lacking the TPsiC Arm

Analyses of mitochondrial DNA sequences from three species of Habronattus jumping spiders (Chelicerata: Arachnida: Araneae) reveal unusual inferred tRNA secondary structures and gene arrangements, providing new information on tRNA evolution within chelicerate arthropods. Sequences from the protein-coding genes NADH dehydrogenase subunit 1 (ND1), cytochrome oxidase subunit I (COI), and subunit II (COII) were obtained, along with tRNA, tRNA, and large-subunit ribosomal RNA (16S) sequences; these revealed several peculiar features. First, inferred secondary structures of tRNA and, likely, tRNA, lack the TPsiC arm and the variable arm and therefore do not form standard cloverleaf structures. In place of these arms is a 5-6-nt T arm-variable loop (TV) replacement loop such as that originally described from nematode mitochondrial tRNAs. Intraspecific variation occurs in the acceptor stem sequences in both tRNAs. Second, while the proposed secondary structure of the 3' end of 16S is similar to that reported for insects, the sequence at the 5' end is extremely divergent, and the entire gene is truncated about 300 nt with respect to Drosophila yakuba. Third, initiation codons appear to consist of ATY (ATT and ATC) and TTG for ND1 and COII, respectively. Finally, Habronattus shares the same ND1-tRNA-16S gene arrangement as insects and crustaceans, thus illustrating variation in a tRNA gene arrangement previously proposed as a character distinguishing chelicerates from insects and crustaceans.     

Evolution of the WANCY region in amniote mitochondrial DNA

In most vertebrate mitochondrial genomes, the site for initiation of light-strand replication, OL, is found within a cluster of five transfer RNA (tRNA) genes (tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), and tRNA(Tyr)). This region and part of the adjacent cytochrome c oxydase subunit I (COI) gene were sequenced for two crocodilian, two turtle, and one snake species and for Sphenodon punctatus; part of the adjacent nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2) gene was also sequenced for the crocodilian and turtle species. All had the typical vertebrate gene order. The turtles and the snake have a lengthy noncoding sequence between the tRNA(Asn) and tRNA(Cys) genes that we assumed to be homologous to the mammalian OL. The crocodilians and Sphenodon lack such a sequence, a condition they share with birds. Most proposed phylogenies for the amniotes require that OL at this position was lost at least twice during their diversification or was evolved independently more than once. Within the five tRNA genes, frequencies of substitutions are much higher in loops than in stems. Many loops vary dramatically in size among the species; in the most extreme case, the D-arm of the Sphenodon tRNA(Cys) is a "D-arm replacement" loop of seven nucleotides. Frequency of transitions in stems is relatively uniform across tRNAs, but frequency of transversions varies greatly. Mismatches in stems are infrequent, and their relative frequency in a specific tRNA is unrelated to the frequency of substitution in the corresponding gene. Several features of mammalian mitochondrial tRNAs are conserved in WANCY tRNAs throughout amniotes. The inferred initiation codon for COI is GTG in crocodilians, turtles, and the snake, a condition they share with fishes, certain amphibians, and birds. TTG appears to be the initiation codon for COI in Sphenodon; if correct, this would be a novel initiation codon for vertebrate mitochondrial DNA. Phylogenetic analyses of the inferred amino acid sequences of ND2 and COI support the sister-group relationship of birds and crocodilians and suggest that mammals are an early derived lineage within the amniotes.      

Novel features of metazoan mtDNA revealed from sequence analysis of three mitochondrial DNA segments of the land snail Albinaria turrita (Gastropoda: Clausiliidae)

The mitochondrial DNA (mtDNA) size of the terrestrial gastropod Albinaria turrita was determined by restriction enzyme mapping and found to be approximately 14.5 kb. Its partial gene content and organization were examined by sequencing three cloned segments representing about one-fourth of the mtDNA molecule. Complete sequences of cytochrome c oxidase subunit II (COII), and ATPase subunit 8 (ATPase8), as well as partial sequences of cytochrome c oxidase subunit I (COI), NADH dehydrogenase subunit 6 (ND6), and the large ribosomal RNA (IrRNA) genes were determined. Nine putative tRNA genes were also identified by their ability to conform to typical mitochondrial tRNA secondary structures. An 82-nt sequence resembles a noncoding region of the bivalve Mytilus edulis, even though it might contain a tenth tRNA gene with an unusual 5-nt overlap with another tRNA gene. The genetic code of Albinaria turrita appears to be the same as that of Drosophila and Mytilus edulis. The structures of COI and COII are conservative, but those of ATPase8 and ND6 are diversified. The sequenced portion of thelrRNA gene (1,079 nt) is characterized by conspicuous deletions in the 5 and 3 ends; this gene represents the smallest coelomate IrRNA gene so far known. Sequence comparisons of the identified genes indicate that there is greater difference between Albinaria and Mytilus than between Albinaria and Drosophila. An evolutionary analysis, based on COII sequences, suggests a possible nonmonophyletic origin of molluskan mtDNA. This is supported also by the absence of the ATPase8 gene in the mtDNA of Mytilus and nematodes, while this gene is present in the mtDNA of Albinaria and Cepaea nemoralis and in all other known coelomate metazoan mtDNAs.     

The Mitochondrial Genomes of Nuttalliella namaqua (Ixodoidea: Nuttalliellidae) and Argas africolumbae (Ixodoidae: Argasidae): Estimation of Divergence Dates for the Major Tick Lineages and Reconstruction of Ancestral Blood-Feeding Characters

Ixodida are composed of hard (Ixodidae), soft (Argasidae) and the monotypic Nuttalliellidae (Nuttalliella namaqua) tick families. Nuclear 18S rRNA analysis suggested that N. namaqua was the closest extant relative to the last common ancestral tick lineage. The mitochondrial genomes of N. namaqua and Argas africolumbae were determined using next generation sequencing and de novo assembly to investigate this further. The latter was included since previous estimates on the divergence times of argasids lacked data for this major genus. Mitochondrial gene order for both was identical to that of the Argasidae and Prostriata. Bayesian analysis of the COI, Cytb, ND1, ND2 and ND4 genes confirmed the monophyly of ticks, the basal position of N. namaqua to the other tick families and the accepted systematic relationships of the other tick genera. Molecular clock estimates were derived for the divergence of the major tick lineages and supported previous estimates on the origins of ticks in the Carboniferous. N. namaqua larvae fed successfully on lizards and mice in a prolonged manner similar to many argasids and all ixodids. Excess blood meal-derived water was secreted via the salivary glands, similar to ixodids. We propose that this prolonged larval feeding style eventually gave rise to the long feeding periods that typify the single larval, nymphal and adult stages of ixodid ticks and the associated secretion of water via the salivary glands. Ancestral reconstruction of characters involved in blood-feeding indicates that most of the characteristics unique to either hard or soft tick families were present in the ancestral tick lineage.     

Nucleotide sequence of nine protein-coding genes and 22 tRNAs in the mitochondrial DNA of the sea starPisaster ochraceus

Summary We have cloned and sequenced over 9 kb of the mitochondrial genome from the sea starPisaster ochraceus. Within a continuous 8.0-kb fragment are located the genes for NADH dehydrogenase subunits 1, 2, 3, and 4L (ND1, ND2, ND3, and ND4L), cytochrome oxidase subunits I, II, and III (COI, COII, and COIII), and adenosine triphosphatase subunits 6 and 8 (ATPase 6 and ATPase 8). This large fragment also contains a cluster of 13 tRNA genes between ND1 and COI as well as the genes for isoleucine tRNA between ND1 and ND2, arginine tRNA between COI and ND4L, lysine tRNA between COII and ATPase 8, and the serine (UCN) tRNA between COIII and ND3. The genes for the other five tRNAs lie outside this fragment. The gene for phenylalanine tRNA is located between cytochrome b and the 12S ribosomal genes. The genes for tRNA^glu and tRNA^thr are 3 to the 12S ribosomal gene. The tRNAs for histidine and serine (AGN) are adjacent to each other and lie between ND4 and ND5. These data confirm the novel gene order in mitochondrial DNA (mtDNA) of sea stars and delineate additional distinctions between the sea star and other mtDNA molecules.     

Combined molecular and morphological phylogenetic analyses of the New Zealand wolf spider genus Anoteropsis (Araneae: Lycosidae)

Datasets from the mitochondrial gene regions NADH dehydrogenase subunit I (ND1) and cytochrome c oxidase subunit I (COI) of the 20 species in the New Zealand wolf spider (Lycosidae) genus Anoteropsis were generated. Sequence data were phylogenetically analysed using parsimony and maximum likelihood analyses. The phylogenies generated from the ND1 and COI sequence data and a previously generated morphological dataset were significantly congruent (p<0.001). Sequence data were combined with morphological data and phylogenetically analysed using parsimony. The ND1 region sequenced included part of tRNA(Leu(CUN)), which appears to have an unstable amino-acyl arm and no TpsiC arm in lycosids. Analyses supported the existence of five species groups within Anoteropsis and the monophyly of species represented by multiple samples. A radiation of Anoteropsis species within the last five million years is inferred from the ND1 and COI likelihood phylograms, habitat and geological data, which also indicates that Anoteropsis arrived in New Zealand some time after it separated from Gondwana.     

Use of mitogenomic information in teleostean molecular phylogenetics: a tree-based exploration under the maximum-parsimony optimality criterion

We explored the phylogenetic utility and limits of the individual and concatenated mitochondrial genes for reconstructing the higher-level relationships of teleosts, using the complete (or nearly complete) mitochondrial DNA sequences of eight teleosts (including three newly determined sequences), whose relative phylogenetic positions were noncontroversial. Maximum-parsimony analyses of the nucleotide and amino acid sequences of 13 protein-coding genes from the above eight teleosts, plus two outgroups (bichir and shark), indicated that all of the individual protein-coding genes, with the exception of ND5, failed to recover the expected phylogeny, although unambiguously aligned sequences from 22 concatenated transfer RNA (tRNA) genes (stem regions only) recovered the expected phylogeny successfully with moderate statistical support. The phylogenetic performance of the 13 protein-coding genes in recovering the expected phylogeny was roughly classified into five groups, viz. very good (ND5, ND4, COIII, COI), good (COII, cyt b), medium (ND3, ND2), poor (ND1, ATPase 6), and very poor (ND4L, ND6, ATPase 8). Although the universality of this observation was unclear, analysis of successive concatenation of the 13 protein-coding genes in the same ranking order revealed that the combined data sets comprising nucleotide sequences from the several top-ranked protein-coding genes (no 3rd codon positions) plus the 22 concatenated tRNA genes (stem regions only) best recovered the expected phylogeny, with all internal branches being supported by bootstrap values >90%. We conclude that judicious choice of mitochondrial genes and appropriate data weighting, in conjunction with purposeful taxonomic sampling, are prerequisites for resolving higher-level relationships in teleosts under the maximum-parsimony optimality criterion.     

Molecular phylogenetics and historical biogeography among salamandrids of the "true" salamander clade: rapid branching of numerous highly divergent lineages in Mertensiella luschani associated with the rise of Anatolia

Phylogenetic relationships among salamandrids of the "true" salamander clade are investigated using 2019 aligned base positions (713 parsimony informative) of 20 mitochondrial DNA sequences from the genes encoding ND1 (subunit one of NADH dehydrogenase), tRNA(Ile), tRNA(Gln), tRNA(Met), ND2, tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), tRNA(Tyr), and COI (subunit I of cytochrome c oxidase), plus the origin for light-strand replication (O(L)) between the tRNA(Asn) and the tRNA(Cys) genes. Parsimony analysis produces a robust phylogenetic estimate for the relationships of the major groups of "true" salamanders. Strong support is provided for the sister taxon relationship of Chioglossa and Mertensiella caucasica and for the placement of Salamandra and Mertensiella luschani as sister taxa. These relationships suggest two vicariant events between Europe and Anatolia caused by the formation of seaways in the Mediterranean Basin. Molecular divergence indicates an Early Miocene separation of Chioglossa and M. caucasica and a Late Miocene separation of Salamandra and M. luschani. The traditional phylogenetic hypothesis of a monophyletic Mertensiella is statistically rejected, indicating that southwestern and northeastern Anatolian populations have separate historical biogeographic origins. Therefore, we recommend placement of M. luschani in the genus Salamandra. Within M. luschani, six highly divergent lineages showing 7.6 to 10.1% pairwise sequence divergence are identified. Tests using four-taxon subsamples suggest that these lineages diverged nearly simultaneously in the Late Miocene, approximately 6 to 8 million years ago, when extensive uplifting of Anatolia occurred in response to the Arabian collision.     

Mitochondrial DNA sequence-based phylogenetic relationship among flesh flies of the genus <Emphasis Type="Italic">Sarcophaga</Emphasis> (Sarcophagidae: Diptera)

The phylogenetic relationships among flesh flies of the family Sarcophagidae has been based mainly on the morphology of male genitalia. However, the male genitalic character-based relationships are far from satisfactory. Therefore, in the present study mitochondrial DNA has been used as marker to unravel genetic relatedness and to construct phylogeny among five sympatric species of the genus Sarcophaga. Two mitochondrial genes viz., cytochrome oxidase subunit 1 (COI) and NAD dehydrogenase subunit 5 (ND5) were sequenced and genetic distance values were calculated on the basis of sequence differences in both the mitochondrial genes. The data revealed very few genetic difference among the five species for the COI and ND5 gene sequences.     

Vicariant Patterns of Fragmentation among Gekkonid Lizards of the Genus Teratoscincus Produced by the Indian Collision: A Molecular Phylogenetic Perspective and an Area Cladogram for Central Asia

A well-supported phylogenetic hypothesis is presented for gekkonid lizards of the genus Teratoscincus. Phylogenetic relationships of four of the five species are investigated using 1733 aligned bases of mitochondrial DNA sequence from the genes encoding ND1 (subunit one of NADH dehydrogenase), tRNA(Ile), tRNA(Gln), tRNA(Met), ND2, tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), tRNA(Tyr), and COI (subunit I of cytochrome c oxidase). A single most parsimonious tree depicts T. przewalskii and T. roborowskii as a monophyletic group, with T. scincus as their sister taxon and T. microlepis as the sister taxon to the clade containing the first three species. The aligned sequences contain 341 phylogenetically informative characters. Each node is supported by a bootstrap value of 100% and the shortest suboptimal tree requires 29 additional steps. Allozymic variation is presented for proteins encoded by 19 loci but these data are largely uninformative phylogenetically. Teratoscincus species occur on tectonic plates of Gondwanan origin that were compressed by the impinging Indian Subcontinent, resulting in massive montane uplifting along plate boundaries. Taxa occurring in China (Tarim Block) form a monophyletic group showing vicariant separation from taxa in former Soviet Central Asia and northern Afghanistan (Farah Block); alternative biogeographic hypotheses are statistically rejected. This vicariant event involved the rise of the Tien Shan-Pamir and is well dated to 10 million years before present. Using this date for separation of taxa occurring on opposite sides of the Tien Shan-Pamir, an evolutionary rate of 0.57% divergence per lineage per million years is calculated. This rate is similar to estimates derived from fish, bufonid frogs, and agamid lizards for the same region of the mitochondrial genome ( approximately 0.65% divergence per lineage per million years). Evolutionary divergence of the mitochondrial genome has a surprisingly stable rate across vertebrates.     

Phylogenetic and phylogeographic relationships in Ixodes holocyclus and Ixodes cornuatus (Acari: Ixodidae) inferred from COX1 and ITS2 sequences

We inferred the phylogenetic and phylogeographic relationships in ticks, which were identified morphologically as Ixodes holocyclus and Ixodes cornuatus, from mitochondrial cytochrome oxidase subunit 1 (COX1) and nuclear internal transcribed spacer 2 (ITS2) sequences. We obtained COX1 (640bp) and ITS2 (527-568bp) sequences from 429 ticks from 49 localities in Tasmania, Victoria, New South Wales and Queensland, Australia. Our analyses show that there are two species of Ixodes in eastern Australia that cause paralysis in dogs and other mammals: I. holocyclus and I. cornuatus. Further, we conclude that the morphological criteria used to differentiate female I. holocyclus and I. cornuatus are equivocal but I. holocyclus can be distinguished from I. cornuatus using COX1 and/or ITS2 sequences. Intraspecific genetic variation in I. holocyclus and I. cornuatus was less than 0.86% and 0.19% for COX1 and ITS2, respectively. Ixodes holocyclus could be genetically distinguished between different geographic ranges. There were no significant genetic differences between I.cornuatus from Tasmania and mainland Australia, but there are some COX1 haplotypes of I. cornuatus from the mainland that were not detected in Tasmanian and vice versa.     

Taxonomic status of two South American sympatric squat lobsters, Munida gregaria and Munida subrugosa (Crustacea: Decapoda: Galatheidae), challenged by DNA sequence information

We investigated the taxonomic status of two sympatric morphospecies of squat lobsters from southern South America (Beagle Channel, Strait of Magellan, and Burdwood Bank), Munida gregaria and Munida subrugosa , by DNA sequence analysis of three mitochondrial (mt)DNA gene fragments [416 bp of 16S rDNA(165), 566 bp of cytochrome c oxidase subunit I(COI) and 418 bp of NADH dehydrogenase subunit 1 (ND1)]; and the nuclear rDNA internal transcribed spacer (ITS) 1 (883–952 bp). We obtained a total of 79 sequences from 32 individuals. The 16S sequences of all M. gregaria and M. subrugosa were invariant and identical, whereas COI and ND1 showed 12 and 15 variable sites, respectively. These polymorphisms were shared between morphospecies. Interspecific Tamura–Nei distances for COI and ND1 sequences were 0.0024 and 0.0032, respectively, and were not significantly different from intraspecific distances (Kruskal–Wallis tests: P  = 0.58 and P  = 0.69, for COI and ND1, respectively). Similar to the results obtained from the mtDNA sequences, no relationship was found between the ITS1 maximum parsimony tree topology and the morphologic classification of specimens in M. gregaria and M. subrugosa . We conclude that M. gregaria and M. subrugosa from southern South America may either represent a case of a dimorphic species, or a case of incomplete lineage sorting. The fact that these two morphospecies did not show fixed differences over a total of 1947 bp analysed reinforces the hypothesis of a single dimorphic species.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 421–434.     

The main features of the craniate mitochondrial DNA between the ND1 and the COI genes were established in the common ancestor with the lancelet

We have cloned the mitochondrial DNA fragment extending from tRNA-Leu to the cytochrome oxidase subunit 1 (COI) genes of Branchiostoma lanceolatum, Myxine glutinosa, Lampetra fluviatilis, and Scyliorhinus caniculus and have determined their respective gene sequences and organization. In all four species, this region contains the ND1 and ND2 genes and the genes coding eight tRNAs, namely, tRNA-Ile, -Gln, -Met, - Trp, -Ala, -Asn, -Cys, and -Tyr. The gene order is the same in the hagfish, lamprey and dogfish. In the lancelet, the location of the tRNA genes is slightly different. The mitochondrial code of Myxine, Lampetra, and Scyliorhinus is identical to that of vertebrates. The code used by the lancelet is the same with the exception of AGA (a stop codon in vertebrates), which codes for glycine in the lancelet. From the comparison of the four maps with already published ones for other species, we propose that the main features of the craniate mtDNA between the ND1 and COI genes were established in the common ancestor to cephalochordates and vertebrates more than 400 MYA. The origin of replication of the light-strand (Ori-L), usually located between the tRNA-Asn and tRNA-Cys genes in vertebrates, was not found in the lancelet, hagfish, or lamprey (Lampetra). In contrast, it was found in the dogfish. Thus the position of Ori-L was established for the first time in the common ancestor to the Chondrichthyes and Osteichthyes and remained present in all later-emerging vertebrates.      

The mitochondrial genome of the sea anemone Metridium senile (Cnidaria): introns,a paucity of tRNA genes,and a near-standard genetic code.

The circular, 17,443 nucleotide-pair mitochondrial (mt) DNA molecule of the sea anemone, Metridium senile (class Anthozoa, phylum Cnidaria) is presented. This molecule contains genes for 13 energy pathway proteins and two ribosomal (r) RNAs but, relative to other metazoan mtDNAs, has two unique features: only two transfer RNAs (tRNA(f-Met) and tRNA(Trp)) are encoded, and the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes each include a group I intron. The COI intron encodes a putative homing endonuclease, and the ND5 intron contains the molecule''s ND1 and ND3 genes. Most of the unusual characteristics of other metazoan mtDNAs are not found in M. senile mtDNA: unorthodox translation initiation codons and partial translation termination codons are absent, the use of TGA to specify tryptophan is the only genetic code modification, and both encoded tRNAs have primary and secondary structures closely resembling those of standard tRNAs. Also, with regard to size and secondary structure potential, the mt-s-rRNA and mt-1-rRNA have the least deviation from Escherichia coli 16S and 23S rRNAs of all known metazoan mt-rRNAs. These observations indicate that most of the genetic variations previously reported in metazoan mtDNAs developed after Cnidaria diverged from the common ancestral line of all other Metazoa.     

The taxonomic status of Digramma (Pseudophyllidea: Ligulidae) inferred from DNA sequences

The traditional classification of the ligulid tapeworms into 2 genera, Ligula Bloch, 1782 and Digramma Cholodkovsky, 1914, remains controversial. Molecular data of sequences for the 5' end of the nuclear 28S ribosomal ribonucleic acid (rRNA) gene, the mitochondrial cytochrome c oxidase subunit I (COI) gene, and the nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) gene, as well as the first internal transcribed spacer (ITS1) of the nuclear ribosomal deoxyribonucleic acid (DNA), were used to characterize Digramma and to investigate its relationship with Ligula. Digramma spp. exhibited identical sequences with Ligula intestinalis both in the 28S rRNA and the COI gene and differed from L. intestinalis by 0.7% in the ITS1 region and 7.4% in the ND1 gene, respectively. A high degree of genetic conservation within 28S ribosomal DNA, COI, ITS1, and even ND1 genes, was found in Ligula and Digramma. The low genetic divergence in the 4 genes between Ligula and Digramma indicates that Digramma is probably not an independent genus. Therefore, it is proposed that Ligula and Digramma should be considered as 2 species within the genus Ligula and the tapeworms of Digramma collected from diverse localities in China belong to the same species. The present study also suggests that ITS1 and ND1 sequences can act as useful genetic markers to distinguish Ligula and Digramma.     

Phylogenetic identification of Sparganum proliferum as a pseudophyllidean cestode by the sequence analyses on mitochondrial COI and nuclear sdhB genes.

Sparganum proliferum is a larval cestode for which the adult stage is unknown. It is characterized by the continuous branching and budding when parasitized to humans, and causes fatal human sparganosis. However, the biological features of S. proliferum, including its taxonomic status, still remain obscure. Our previous investigation suggested that S. proliferum might be phylogenetically distinct from Spirometra erinaceieuropaei, by the analysis on mitochondrial NADH dehydrogenase subunit 3 (ND3) gene. However, mitochondrial DNA sequence in Platyhelminth is known to have heteroplasmy within a species. Therefore, in the present study, we have investigated the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene and the partial nucleotide sequences of nuclear coded succinate dehydrogenase iron-sulfur protein subunit gene (sdhB). The results clearly demonstrated that S. proliferum is a distinct species from S. erinaceieuropaei, and that S. proliferum belongs to the order Pseudophyllidea.     

Ticks feeding on northern pocket gophers (Thomomys talpoides) in central Saskatchewan and the unexpected detection of Ixodes scapularis larvae

Morphological examination of ticks feeding on northern pocket gophers, Thomomys talpoides, near Clavet (Saskatchewan, Canada) revealed the presence of two genera, Ixodes and Dermacentor. All adult ticks collected were identified as I. kingi. Single strand conformation polymorphism (SSCP) analyses and DNA sequencing of the mitochondrial 16S rRNA gene confirmed the species identity of most Ixodes immatures as I. kingi (two nymphs and 82 larvae), and the Dermacentor immatures as D. variabilis (one nymph and one larva) and D. andersoni (three larvae). Six Ixodes larvae feeding on three T. talpoides individuals were identified as four different 16S haplotypes of I. scapularis, which was unexpected because there are no known established populations of this species in Saskatchewan. However, flagging for questing ticks and further examination of the ticks feeding on T. talpoides in two subsequent years failed to detect the presence of I. scapularis near Clavet, suggesting that there is no established population of I. scapularis in this area. Nonetheless, since I. scapularis is a vector of pathogenic agents, passive and active surveillance needs to be conducted in Saskatchewan on an ongoing basis to determine if this tick species and its associated pathogens become established within the province.     

Buffalo species identification and delineation using genetic barcoding markers

Enrichment of barcode databases with mitochondrial cytochrome c oxidase subunit I (COI) barcode sequences in different animal taxa has become important for identification of animal source in food samples to prevent commercial fraud. In this study, COI barcode sequence in seventy one river buffalo samples were determined, analyzed and deposited in Genbank barcode database and barcode of life database (BOLD) to contribute for construction of public reference library for COI barcode sequence in river buffalo. Moreover COI barcode sequence was used to identify the closely related buffalo groups: river buffalo, swamp buffalo, lowland anoa and African buffalo. Results indicated the success of the COI barcode in the identification of each of the tested groups. Whereas a suggested sequence of other mitochondrial segment representing two successive transfer RNA (tRNA) genes; tRNA-Threonine (MT-TT) and tRNA-Proline (MT-TP) was failed to be used as a barcode marker for differentiation between the tested buffalo groups.