The complete mitochondrial genome of Rhynchocypris oxycephalus (Teleostei: Cyprinidae) and its phylogenetic implications

Rhynchocypris oxycephalus (Teleostei: Cyprinidae) is a typical small cold water fish, which is distributed widely and mainly inhabits in East Asia. Here, we sequenced and determined the complete mitochondrial genome of R. oxycephalus and studied its phylogenetic implication. R. oxycephalus mitogenome is 16,609 bp in length (GenBank accession no.: MH885043), and it contains 13 protein‐coding genes (PCGs), two rRNA genes, 22 tRNA genes, and two noncoding regions (the control region and the putative origin of light‐strand replication). 12 PCGs started with ATG, while COI used GTG as the start codon. The secondary structure of tRNA‐Ser (AGN) lacks the dihydrouracil (DHU) arm. The control region is 943bp in length, with a termination‐associated sequence, six conserved sequence blocks (CSB‐1, CSB‐2, CSB‐3, CSB‐D, CSB‐E, CSB‐F), and a repetitive sequence. Phylogenetic analysis was performed with maximum likelihood and Bayesian methods based on the concatenated nucleotide sequence of 13 PCGs and the complete sequence without control region, and the result revealed that the relationship between R. oxycephalus and R. percnurus is closest, while the relationship with R. kumgangensis is farthest. The genus Rhynchocypris is revealed as a polyphyletic group, and R. kumgangensis had distant relationship with other Rhynchocypris species. In addition, COI and ND2 genes are considered as the fittest DNA barcoding gene in genus Rhynchocypris. This work provides additional molecular information for studying R. oxycephalus conservation genetics and evolutionary relationships.     

Vicariance of Pyrocoelia fireflies (Coleoptera: Lampyridae) in the Ryukyu islands,Japan

We performed molecular phylogenetic analyses based on the mitochondrial COI gene (687 bp) and the nuclear 28S rRNA gene (715 bp) and reconstructed phylogenetic trees of the Pyrocoelia fireflies in the Ryukyu Islands and eastern Asia. Age calibration was done using a robust geological constraint: the Okinawa trough and associated straits began to rift at 1.55 Ma, isolating the Ryukyu Islands from the Chinese continent, Japanese islands, Taiwan island and some of the islands from each other. We suggest that the physical isolation of these islands began to generate the allopatric speciation within these islands, so the timing of this isolation was assigned to an appropriate node. The topology is completely concordant among phylogenetic trees reconstructed using MEGA (maximum‐likelihood), raxmlGUI (maximum‐likelihood) and BEAST (Bayesian inference; including combined analysis of COI and 28S rRNA genes). Two lineages are recognized, related to their emergence time; spring to summer, and autumn. In each lineage, vicariance is inferred to have begun at 1.55 Ma from our phylogenetic and geological analyses. In lineage 1, P. oshimana (Amami), P. matsumurai (Okinawa), P. discicollis (W. Japan), P. fumosa (E. Japan) and P. abdominalis (Yaeyama) were differentiated. In lineage 2, P. rufa (Tsushima and Korea), P. miyako (Miyako‐jima), P. atripennis (Ishigaki‐jima) and P. praetexta (Taiwan and HongKong) were differentiated. Pyrocoelia analis (Taiwan and China) emerges throughout the year except for winter, and constitutes another lineage. We suggest that Pyrocoelia fireflies differentiated at 2 Ma to generate these three lineages. The base substitution rate for the COI gene is estimated as 4.48% Myr^–1 and that for the 28S rRNA gene is 0.394%  Myr^–1, and these rates were used in a combined BEAty analysis in BEAST.     

Deep sympatric mtDNA divergence in the autumnal moth (Epirrita autumnata)

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species‐level taxonomy. We found five COI sub‐clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub‐clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.     

Italian Peninsula preserves an evolutionary lineage of the fat dormouse Glis glis L. (Rodentia: Gliridae)

The present study examines the population genetic structure of fifty‐nine specimens of Glis glis (Linneaus, 1766) from thirteen localities in central Europe, sequencing a 400‐bp segment of the mitochondrial cytochrome b (cyt b) gene and a 673‐bp segment of the cytochrome c oxidase subunit I (COI) gene. The consensus tree obtained from Bayesian analysis revealed a robust dichotomy, showing two sister groups: one clade includes samples from a wide geographical area, extending from north‐central Europe to northern Italy (major branch sensu Bilton), and the other comprises samples collected in central and southern Italy and in Sicily (Italian branch). According to the Tajima–Nei model, the two phylogroups were separated by a sequence divergence of 0.8% (cyt b) – 2.6% (COI), showing the COI gene to be more informative than cyt b. On a smaller geographical scale, the Italian clade was further substructured, displaying geographical differentiation along the Peninsula. The gene pool in this area was patchy; whereas populations from Sicily Island demonstrated fixed cyt b and COI haplotypes, assuming processes of isolation and selection. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 11–21.     

Complete mitochondrial genome of a carabid beetle,Damaster mirabilissimus mirabilissim (Coleoptera: Carabidae)

In the present study, we report the 16 823‐bp long complete mitochondrial genome (mitogenome) of a carabid beetle, Damaster mirabilissimus mirabilissim (Coleoptera: Carabidae), which is endangered in Korea. The gene arrangement of D. m. mirabilissim mitogenome is identical to the most common type found in insects. The start codon of the D. m. mirabilissim COI gene is a typical ATN codon. On the other hand, the initiation codon for ND1 gene is TTG, instead of ATN. All transfer RNAs (tRNAs) exhibit a stable canonical clover‐leaf structure, except for tRNA^Ser(AGN), the dihydrouridine arm of which forms a simple loop. The 1703‐bp long A+T‐rich region is the second longest among the complete adephagan mitogenome sequences, next to Macrogyrus oblongus belonging to Gyrinoidea. One of the unusual features of the genome is the presence of a tRNA^Leu(UUR)‐like sequence in the A+T‐rich region. This sequence displays the proper anticodon sequence and the potential to form secondary structures, but also harbors many mismatches in the stems.     

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well‐supported clusters. Intraspecific Kimura 2‐parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra‐ and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.     

Molecular identification of cryptic bumblebee species from degraded samples using PCR–RFLP approach

The worldwide decline and local extinctions of bumblebees have raised a need for fast and accurate tools for species identification. Morphological characters are often not sufficient, and molecular methods have been increasingly used for reliable identification of bumblebee species. Molecular methods often require high‐quality DNA which makes them less suitable for analysis of low‐quality or older samples. We modified the PCR–RFLP protocol for an efficient and cost‐effective identification of four bumblebee species in the subgenus Bombus s. str. (B. lucorum, B. terrestris, B. magnus and B. cryptarum). We used a short partial mitochondrial COI fragment (446 bp) and three diagnostic restriction enzymes (Hinf I, Hinc II and Hae III) to identify species from degraded DNA material. This approach allowed us to efficiently determine the correct species from all degraded DNA samples, while only a subset of samples 64.6% (31 of 48) resulted in successful amplification of a longer COI fragment (1064 bp) using the previously described method. This protocol can be applied for conservation and management of bumblebees within this subgenus and is especially useful for fast species identification from degraded samples.     

Genetic polymorphism at the cytochrome oxidase I gene in mediterranean populations of Batrocera Oleae (Diptera: Tephritidae)

The tephritid Bactrocera oleae (Gmelin) is a harmful pest of olive crops that cause important agricultural and economic losses in the Mediterranean area where 90% of the world olive trees are cultivated. The knowledge of the genetic diversity in insect pest species populations is critical for decisions concerning appropriate control management strategies. In the present work, the genetic variability within and among 7 populations ‐five from Spain, one from Italy and one from Tunisia‐, was assessed by sequencing 1151 bp of the COI gene. A total of 21 haplotypes were observed. The intraspecific diversity was high, particularly in the Spanish populations (haplotype and nucleotide diversity 0.84 and 0.00137, respectively). However, the genetic differentiation among the populations was low in the case of Spanish ones (Fst between 0 and 0.041), and higher –and statistically significant– when comparing with the Italian and Tunisian samples. The haplotypes distribution and the PCoA analysis show three clear groups of populations: Spanish, Italian and Tunisian. The results might indicate the length of time elapsed since B. oleae became established in the Mediterranean region, the large effective sizes expected of its populations and the high gene flow among Iberian populations. The information could be relevant for integrated control programmes coordination.     

Northern fowl mite (Ornithonyssus sylviarum) in Sweden

Haematophagous mites were collected from the vent region and plumage of chickens in six hobby flocks of ornamental breeds in Sweden, one of which included turkeys. Soiled vent skin and feathers, dermatitis, hyperkeratosis, skin necroses and ulcers were observed in 12 necropsied birds from two of the flocks. The mites were identified as the northern fowl mite Ornithonyssus sylviarum (Mesostigmata: Macronyssidae). This was supported by sequence analysis of a 642‐bp region in the mitochondrial cytochrome oxidase subunit 1 (COI) gene (COI) in mites collected from five flocks, which showed 97–99% sequence similarity to O. sylviarum by blast analysis. Pairwise sequence comparisons revealed nucleotide variations in the range of 0–2.8%, whereas amino acid sequences were highly conserved. This paper represents one of very few records of O. sylviarum in European poultry, and is the first to report COI sequence data for O. sylviarum from poultry in Europe.     

Identification of a short interspersed repetitive element insertion polymorphism in the porcine MX1 promoter associated with resistance to porcine reproductive and respiratory syndrome virus infection

The myxovirus resistance (Mx) proteins belong to the dynamin superfamily and are important for innate host defence against RNA viruses. In this study, we demonstrate that positive elements are present in the two promoter regions of ?2713 to ?2565 and ?688 to ?431 in the porcine MX1 gene. Sequencing and alignment of the amplified porcine MX1 gene promoter region identified a short interspersed repetitive element (SINE) insertion of 275 bp at site ?547. At this site, allele B (an insertion of 275 bp) is dominant in Chinese indigenous pig breeds but has a workable minor allele frequency in western lean‐type pig breeds. Luciferase activity was compared between promoters with and without the insertion of the 275‐bp fragment in transiently transfected MARC‐145 cells. The insertion of the 275‐bp fragment increased the luciferase activity significantly (P < 0.05) both prior to and post‐porcine reproductive and respiratory syndrome (PRRS) virus inoculation. These results suggest that the SINE insertion polymorphism at site ?547 of the MX1 gene promoter region is a potential DNA marker for PRRS resistance in pigs.     

DNA barcoding of Bradysia (Diptera: Sciaridae) for detection of the immature stages on agricultural crops

We investigated the usefulness of mitochondrial cytochrome c oxidase (COI) DNA barcoding of the genus Bradysia for the detection of immature stages and cryptic species complex. Although the larvae of some species in this genus are agricultural pests, immature stages are rarely identified due to the lack of key morphological characteristics. We constructed partial sequences of the COI gene for 25 species of Bradysia as a first step towards a DNA barcode. Using these data, Bradysia impatiens, B. procera and B. peraffinis were identified from larval specimens collected, respectively, from paprika, ginseng and oak sawdust beds used for cultivating shiitake. Our findings reveal a complex of three species within the B. tilicola group. These species were all identified as important pest B. ocellaris based on the morphology of male genital structures; however, the interspecific genetic divergence of the COI region was significantly greater (16.1–19.4%) than the intraspecific variation in each species. Therefore, B. ocellaris may consist of at least three species. The results demonstrate that COI DNA barcodes are useful for Bradysia species identification.     

Development and characterization of EST‐SSR markers in an East Asian temperate plant genus Diabelia (Caprifoliaceae)

A set of expressed sequence tag (EST) simple sequence repeat (SSR) markers were developed and characterized using next‐generation sequencing technology for the genus Diabelia (Caprifoliaceae). De novo assembly of RNA‐seq reads resulted in 58 669 contigs with the N50 length of 1211 bp. A total of 2746 contigs were identified to harbor SSR motifs, of which 48 primer pairs were designed and 11 were shown to be polymorphic across three morphospecies of Diabelia. When evaluated with 30 individuals, the number of alleles per locus ranged from 2 to 11 and the expected heterozygosity varied from 0.399 to 0.873, respectively. Distance‐based clustering indicated that the EST‐SSR markers can provide sufficient power to distinguish the three species (or populations). These markers will be useful for evaluating the range‐wide genetic diversity of each species and examining genetic divergence and gene flow between the three species.     

Effective mosquito and arbovirus surveillance using metabarcoding

Effective vector and arbovirus surveillance requires timely and accurate screening techniques that can be easily upscaled. Next‐generation sequencing (NGS) is a high‐throughput technology that has the potential to modernize vector surveillance. When combined with DNA barcoding, it is termed ‘metabarcoding.’ The aim of our study was to establish a metabarcoding protocol to characterize pools of mosquitoes and screen them for virus. Pools contained 100 morphologically identified individuals, including one Ross River virus (RRV) infected mosquito, with three species present at different proportions: 1, 5, 94%. Nucleic acid extracted from both crude homogenate and supernatant was used to amplify a 269‐bp section of the mitochondrial cytochrome c oxidase subunit I (COI) locus. Additionally, a 67‐bp region of the RRV E2 gene was amplified from synthesized cDNA to screen for RRV. Amplicon sequencing was performed using an Illumina MiSeq, and bioinformatic analysis was performed using a DNA barcode database of Victorian mosquitoes. Metabarcoding successfully detected all mosquito species and RRV in every positive sample tested. The limits of species detection were also examined by screening a pool of 1000 individuals, successfully identifying the species and RRV from a single mosquito. The primers used for amplification, number of PCR cycles and total number of individuals present all have effects on the quantification of species in mixed bulk samples. Based on the results, a number of recommendations for future metabarcoding studies are presented. Overall, metabarcoding shows great promise for providing a new alternative approach to screening large insect surveillance trap catches.     

CHARACTERIZATION OF DIATOM (BACILLARIOPHYCEAE) NITRATE REDUCTASE GENES AND THEIR DETECTION IN MARINE PHYTOPLANKTON COMMUNITIES1

The complete assimilatory nitrate reductase (NR) gene from the pennate diatom Phaeodactylum triconutum Bohlin was sequenced from cDNA and compared with NR sequences from fungi, green algae, vascular plants, and the recently sequenced genome of the centric diatom Thalassiosira pseudonana Hasle and Heimdal CCMP1335. In all the major eukaryotic nitrate reductase (Euk‐NR) functional domains, diatom NR gene sequences are generally 50%–60% identical to plant and alga sequences at the amino acid level. In the less conserved N‐terminal, hinge 1, and hinge 2 regions, homology to other NR sequences is weak, generally<30%. Two PCR primer sets capable of amplifying Euk‐NR from plants, algae, and diatoms were designed. One primer set was used to amplify a 750‐base pair (bp) NR fragment from the cDNA of five additional diatom strains. The PCR amplicon spans part of the well‐conserved dimer interface region, the more variable hinge 1 region, and part of the conserved cytochrome b heme binding region. The second primer set, targeted to the dimer region, was used to amplify an approximately 400‐bp fragment of the NR gene from DNA samples collected in Monterey Bay, California and in central New Jersey inner continental shelf (LEO‐15 site) waters. Only diatom‐like NR sequences were recovered from Monterey Bay samples, whereas LEO‐15 samples yielded NR sequences from a range of photosynthetic eukaryotes. The prospect of using DNA‐ and RNA‐based methods to target the NR genes of diatoms specifically is a promising approach for future physiological and ecological experiments.     

Tissue expression and bioinformatics analysis of the vitellogenin gene of Asian arowana (Scleropages formosus)

The RACE technique was used to clone the full‐length vitellogenin (VTG) cDNA sequence of Asian arowana (Scleropages formosus). The full‐length sequence was 5,550 bp with an open reading frame of 5,238 bp, encoding 1,745 amino acids, and 5′ and 3′ UTRs (untranslated regions) of 45 bp and 267 bp, respectively. Phylogenetic analysis showed that S. formosus and silver arowana (Osteoglossum bicirrhosum) share a close evolutionary relationship (bootstrap 100%). The quantitative real‐time PCR results showed that vtg expression was significantly higher in liver and gonads of male and female fish compared with its expression in the other tissues tested (p < 0.01). The relative expression levels of vtg in liver, gland, kidney, heart, head kidney, and brain of female fish were significantly higher than in the corresponding tissues of male fish (p < 0.05).     

Molecular characterization of a new badnavirus associated with streak symptoms on enset (Ensete ventricosum,Musaceae)

Electron microscopy of leaf samples displaying streak symptoms from enset (Ensete ventricosum), a banana‐like plant widely cultivated in Ethiopia, showed the presence of bacilliform shaped virions as known for badnaviruses. DNA extracts subjected to rolling circle amplification (RCA), polymerase chain reaction (PCR) and cloning and sequence analysis revealed that the virus has a circular double‐stranded DNA genome of 7,163 nucleotides encoding predicted proteins of 21.5 kDa, 14.5 kDa and 202.5 kDa, a genome organization known for badnaviruses. The virus is phylogenetically most closely related to Sugarcane bacilliform Guadeloupe D virus with a nucleotide sequence identity of 77.2% at the conserved RT/RNase‐H region and 73.6% for the whole genome. Following the current species demarcation criteria, the virus should be considered as an isolate of a new species in the genus Badnavirus for which the name Enset leaf streak virus (ELSV) is suggested. Leaf samples from enset and banana were screened using virus‐specific primers, and ELSV was detected in six of 40 enset but not found in any of 61 banana samples. On the other hand, Banana streak OL virus (BSOLV) was detected from the majority (60%) of symptomatic banana samples but not from enset samples. This paper reports the first full‐genome sequence of a putative new badnavirus species infecting plants in the genus Ensete. In addition, this is the first report of the occurrence of BSOLV in Ethiopia.     

First Report of a Croton yellow vein mosaic virus (CYVMV) Associated with Tomato Leaf Curl Disease in India

Leaf curl disease symptoms were observed in tomato crop grown in a tomato field at Matera district of Bahraich, India, in March 2013 with an 85% disease incidence. The infected plants exhibited leaf curl symptoms accompanied with puckering, vein swelling and stunting of the whole plant. PCR carried out with begomovirus coat protein gene and DNA beta‐specific primer sets resulted in positive amplification of ~775 bp and 1.35 kbp, respectively, with all symptom‐bearing plant samples. BLASTn and phylogenetic analyses of CP gene sequences showed highest and close relationship with Croton yellow vein mosaic virus (CYVMV) isolates, while the phylogenetic study of betasatellite sequence showed distinct relationships with other begomovirus associated betasatellites reported from India and abroad. This is a first report of a CYVMV associated with tomato leaf curl disease in India.     

Algal Parasite Herpodiscus durvillaeae (Phaeophyceae: Sphacelariales) Inferred to have Traversed the Pacific Ocean with its Buoyant Host

The parasitic phaeophycean endophyte Herpodiscus durvillaeae (Lindauer) G. R. South has previously only been recorded from New Zealand, in association with a single host species, Durvillaea antarctica (Chamisso) Hariot (southern bull‐kelp). Here we use DNA sequence data from plastid and nuclear markers (chloroplast rbcL, ribosomal LSU, and a nuclear pseudogene copy of COI) to test for the presence of H. durvillaeae beyond the New Zealand region, and on host species other than D. antarctica. Analyses of samples from the Falkland Islands confirm the first record of H. durvillaeae from the Atlantic Ocean. We report that Falkland Islands H. durvillaeae are genetically indistinguishable from samples of this species from New Zealand's sub‐Antarctic Campbell Island, suggesting recent dispersal of the parasite across the Pacific Ocean, presumably by rafting with its buoyant macroalgal host. We also here record H. durvillaeae from New Zealand endemics Durvillaea poha Fraser et al. and D. willana Lindauer.     

The first complete mitochondrial genome sequence of Nanorana parkeri and Nanorana ventripunctata (Amphibia: Anura: Dicroglossidae), with related phylogenetic analyses

Members of the Nanorana genus (family Dicroglossidae) are often referred to as excellent model species with which to study amphibian adaptations to extreme environments and also as excellent keystone taxa for providing insights into the evolution of the Dicroglossidae. However, a complete mitochondrial genome is currently only available for Nanorana pleskei. Thus, we analyzed the complete mitochondrial genomes of Nanorana parkeri and Nanorana ventripunctata to investigate their evolutionary relationships within Nanorana and their phylogenetic position in the family Dicroglossidae. Our results showed that the genomes of N. parkeri (17,837 bp) and N. ventripunctata (18,373 bp) encode 13 protein‐coding genes (PCGs), two ribosomal RNA genes, 23 transfer RNA (tRNA) genes, and a noncoding control region. Overall sequences and genome structure of the two species showed high degree of similarity with N. pleskei, although the motif structures and repeat sequences of the putative control region showed clear differences among these three Nanorana species. In addition, a tandem repeat of the tRNA‐Met gene was found located between the tRNA‐Gln and ND2 genes. On both the 5′ and 3′‐sides, the control region possessed distinct repeat regions; however, the CSB‐2 motif was not found in N. pleskei. Based on the nucleotide sequences of 13 PCGs, our phylogenetic analyses, using Bayesian inference and maximum‐likelihood methods, illustrate the taxonomic status of Nanorana with robust support showing that N. ventripunctata and N. pleskei are more closely related than they are to N. parkeri. In conclusion, our analyses provide a more robust and reliable perspective on the evolutionary history of Dicroglossidae than earlier analyses, which used only a single species (N. pleskei).