DNA Barcoding of Morphologically Characterized Mosquitoes Belonging to the Genus Mansonia from the Atlantic Forest and Brazilian Savanna.

The identification of mosquito species is necessary for determining the entomological components of disease transmission. However, identification can be difficult in species that are morphologically similar. The cytochrome c oxidase subunit I (COI) DNA barcode region is considered a valuable and reliable diagnostic tool for mosquito species recognition, including those that belong to species complexes. Mansonia mosquitoes are found in forests near swampy areas. They are nocturnal and are highly attracted to light. Hematophagous adult females exhibit aggressive biting behavior and can become infected with and transmit pathogens during their feeding, including some epizootic viruses and avian malaria. In Brazil, twelve Mansonia species have been reported. In a recent study from the São Paulo Zoo in Brazil, three morphologically distinct species were collected and identified, namely: Mansonia (Mansonia) indubitans, Ma. (Man.) pseudotitillans and Ma. (Man.) titillans. However, confirmation of these species by molecular identification was unsuccessful due to a lack of COI sequences in the GenBank database. Thus, this research aimed to describe the COI DNA barcode sequences of some morphologically characterized Mansonia (Man.) species from Brazil and to determine their utility in delimiting species collected from the Atlantic Forest and Brazilian Savanna. Accordingly, we provide tools for the genetic identification of species that play a significant role in pathogen transmission in wildlife and potentially humans. We show that the delimitation of Mansonia species via five different approaches based on COI DNA sequences (BI, NJ, ASAP, bPTP and GMYC) yield basically the same groups identified by traditional taxonomy, and we provide the identification of specimens that were previously identified only up to the subgenus level. We also provide COI sequences from two Mansonia species that were not previously available in sequence databases, Ma. wilsoni and Ma. pseudotitillans, and thus contribute to the ongoing global effort to standardize DNA barcoding as a molecular means of species identification.

Notes on the taxonomic status and distribution of some Cylindrotomidae (Diptera, Tipuloidea), with emphasis on Japanese species.

A morphological and molecular study of 17 Cylindrotomidae species revealed that the two subspecies of Cylindrotomadistinctissima, the Nearctic C.americana Osten Sacken, 1865, stat. reval. and the Palearctic C.distinctissima (Meigen, 1818), represent separated lineages and consequently are raised to species level. Cylindrotomajaponica Alexander, 1919, syn. nov. and C.distinctissimaalpestris Peus, 1952, syn. nov. are now known to be junior synonyms of C.distinctissima. Triogmakuwanailimbinervis Alexander, 1953, syn. nov. and T.nimbipennis Alexander, 1941, syn. nov. are now placed into synonymy under Triogmakuwanai (Alexander, 1913). The Japanese Cylindrotomidae are all redescribed and all available literature and distribution data are summarised. Supplementary descriptions and illustrations for male and female terminalia of Cylindrotomanigriventris Loew, 1849, Diogmadmitrii Paramonov, 2005, Liogmanodicornis (Osten Sacken, 1865), Phalacrocerareplicata (Linnaeus, 1758), P.tipulina Osten Sacken, 1865, and Triogmatrisulcata (Schummel, 1829) are provided. The following new distribution records are outlined; Diogmacaudata Takahashi, 1960 from Arkhangelsk Oblast, Russia; D.glabrata (Meigen, 1818) from Belarus, Latvia, and Altai Republic, Amur Oblast, Novgorod Oblast, Magadan Oblast, Samara Oblast, and Kuril Islands (Shikotan I and Paramushir I) in Russia; Liogmaserraticornis Alexander, 1919 from Khabarovsk Krai, Russia; Phalacrocerareplicata from Khabarovsk Krai, Russia; and the presence of Cylindrotomanigriventris in Altai Republic, Russia is confirmed.

A new European species of Ceratophysella (Collembola, Hypogastruridae) revealed by morphological data and DNA barcodes.

A new species, Ceratophysella stachi, from Denmark, Germany, Luxembourg, Norway, Poland, and Ukraine is described based on morphological data and DNA barcodes. It belongs to a small European group of species with type B chaetotaxy and strong tegumentary granulation with distinct fields of coarse granules: C. granulata Stach, 1949, C. lawrencei (Gisin, 1963), C. neomeridionalis (Nosek & Cervek, 1970), C. scotica (Carpenter & Evans, 1899), and C. silvatica Rusek, 1964. It differs from all of them in the chaetotaxy of lateral parts of thoracic terga II-III (setae m6 present and one additional seta outside lateral sensillum m7 present or absent) that is exceptional within the whole C. armata-group. Notes on closely related species C. granulata are also given.

Morphological and molecular systematic review of Marphysa Quatrefages, 1865 (Annelida: Eunicidae) species from South Africa.

A vast polychaete fauna is hidden behind complexes of cryptic and pseudo-cryptic species, which has greatly hindered our understanding of species diversity in several regions worldwide. Among the eunicids, Marphysa sanguinea Montagu, 1813 is a typical example, recorded in three oceans and with various species considered its junior synonyms. In South Africa, specimens previously misidentified as M. sanguinea are now known as Marphysa elityeni Lewis & Karageorgopoulos, 2008. Of the six Marphysa Quatrefages, 1865a species recorded from the same region, three have their distributions restricted to South Africa while the others are considered to have worldwide distributions. Here, we evaluated the taxonomic status of the indigenous M. elityeni and investigated the presence of the widespread species Marphysa macintoshi Crossland, 1903 and Marphysa depressa Schmarda, 1861 in South Africa using morphological and molecular data. Our results reveal that M. elityeni is a junior synonym of Marphysa haemasoma, a species previously described from South Africa which is herein reinstated as a valid species. Both M. macintoshi and M. depressa are not present in South Africa and their status as being distributed worldwide deserves further investigation. Marphysa durbanensis Day, 1934 and the new species described here, M. sherlockae n. sp., had been misidentified as M. macintoshi and M. depressa respectively. Thus, the number of Marphysa species with distributions restricted to South Africa increased from three to five. This study reiterates the importance of implementing an integrated taxonomic framework to unravel local biodiversity.

Facultative amphidromy and pelagic larval duration plasticity of Rhinogobius formosanus (Teleostei, Gobioidei).

Rhinogobius formosanus Oshima, 1919 has long been considered an amphidromous goby. However, a landlocked population recently found in the Jingualiao Creek upstream of the Feitsui Reservoir in Taipei suggests that R. formosanus may complete its life in the river. This study aims to verify the habitat use of the landlocked population of R. formosanus collected from the Feitsui Reservoir and an amphidromous population collected in Malian Creek using otolith Sr:Ca ratio analysis. The hypothesis that early life history varies between the landlocked and migratory gobies was also tested. Genetic analyses show that the Feitsui Reservoir and Malian Creek populations are not genetically different. Rhinogobius formosanus from Malian Creek showed high-to-low otolith Sr:Ca ratios suggesting that these specimens spent a planktonic larval stage in the sea followed by a freshwater life at later stages. In contrast, R. formosanus from the Feitsui Reservoir showed constant lower otolith Sr:Ca ratios, implying a landlocked life history of fish in the creek upstream of the reservoir. In addition, the analysis of growth increments showed a longer pelagic larval duration for the fish in the Malian Creek (58.8 days) than those in the Feitsui Reservoir (38.8). Variation of pelagic larval duration in two genetically homogenous populations implies acclimatization to the reservoir by the landlocked gobies. This study shows that R. formosanus, like some other congeners, is capable of adapting to a freshwater landlocked environment in its early developmental stage and supports the hypothesis that landlocked populations may have a shorter pelagic larval duration.

DNA Barcoding: A Reliable Method for the Identification of Thrips Species (Thysanoptera, Thripidae) Collected on Sticky Traps in Onion Fields.

Several thrips species (Insecta, Thysanoptera) are globally known as important crop pests and vectors of viral diseases, but their identification is difficult because of their small body size and inconspicuous morphological differences. Sequencing variation in the mitochondrial cytochrome c oxidase I (COI) region has been proven to be useful for the identification of species of many groups of insect pests. Here, DNA barcoding has been used to identify thrips species collected with the use of sticky traps placed in an open onion field. A total of 238 thrips specimens were analyzed, 151 of which could be identified to species and 27 to genera belonging to the family Thripidae. Fifty-one specimens could not be assigned to any genus, with the closest BLAST match in the GenBank queries being below 98%, whilst six specimens were not recognized as Thysanoptera. The results indicate that, although there are a few pest thrips species not yet barcoded, most of the species that may cause damage to crops in Europe are represented in GenBank and other databases, enabling correct identification. Additionally, DNA barcoding can be considered a valuable alternative to the classic morphology method for identification of major thrips species.

Pamphilius japonicus feeds on Cornus: the first Old World record of Pamphiliidae (Hymenoptera) associated with Cornaceae.

We identified newly discovered pamphiliid larvae feeding on Cornus in Tochigi and Ibaraki Prefectures, Honshu, Japan, with Pamphilius japonicus Shinohara, 1985, by molecular methods using mitochondrial cytochrome oxidase subunit 1 (COI) gene sequences. The host plant of P. japonicus was unknown. This is the first record of the Cornaceae as a host plant of the Pamphiliidae in the Old World. The larvae were solitary leaf-rollers on Cornus and discovered mainly on small young trees in shadowy forests.

DNA barcodes and morphology revealed three species masquerading in Xiphydria camelus of authors (Hymenoptera, Xiphydriidae) in Northeast Asia: X. eborata sp. rev. and X. albopicta sp. nov.

Taxonomic status of the widely spread xiphydriid woodwasp Xiphydria camelus (Linné, 1758) was revised by examining morphology of 964 specimens and by molecular analysis using COI barcode sequences. Both morphological and molecular approaches indicated existence of three separate species masquerading in X. camelus of authors in East Asia. The three species were finally determined as X. camelus, X. eborata Konow, 1899, and a new species, X. albopicta. Xiphydria eborata is revived from synonymy with X. camelus and X. albopicta is described as a new species from Russia (Primorskij Kraj), China (Heilongjiang) and Japan (Hokkaido). Collection records are given for all the specimens examined.

Review of the Merodon albifasciatus Macquart species complex (Diptera: Syrphidae): the nomenclatural type located and its provenance discussed.

The Palaearctic Merodon geniculatus species group (Diptera, Syrphidae: Merodontini) currently comprises six Western Mediterranean species and four species with Eastern Mediterranean distribution. One of this group species, Merodon albifasciatus Macquart, 1842, is the only Merodon species listed from the Oriental Region. We discovered the lost holotype, which fit morphologically with European Merodon females of the M. albifasciatus species complex confirmed here and that occurs in the Palaearctic region. An integrative approach to the taxonomy of M. albifasciatus based on morphological and molecular evidences revealed the existence of two additional closely-related species in the Eastern Mediterranean area, M. luteofasciatus Vujic, Radenkovic Ståhls sp. nov. and M. neofasciatus Ståhls Vujic sp. nov.. The females of these three species can only be separated by molecular and distributional data. With the aim of stabilising species concepts within the complex, the taxon distributed in mainland Greece, including the Peloponnese was associated to the old female holotype of M. albifasciatus. The identity of Merodon fractipes Paramonov, described from Rhodes Island (Greece) as subspecies of M. geniculatus Strobl in Czerny Strobl, could not be resolved as the type specimen is lost. Thus the name M. fractipes is suppressed as nomen dubium. We discuss the distribution of the species of M. albifasciatus complex, identify host plants of M. neofasciatus, and provide an identification key to males of Eastern Mediterranean species of Merodon geniculatus group.

Lectotype designation and distribution updates on the freshwater shrimp species Atyaephyra stankoi Karaman 1972.

Atyaephyra stankoi's Karaman 1972 syntypic material has been assumed to be lost. However, M. Karaman's type material has been found within the zoological collections of the University of Novi Sad, Serbia and thus a lectotype is herein designated, while placing aside the designated neotype. An updated distribution of A. stankoi in Greece and F.Y.R.O.M. is provided based on field collections and literature data, while support is provided by COI data. Furthermore, new records in Peloponnesus and Attica expand the known distribution of species in Greece. Finally some preliminary insights are given on the species zoogeography, while there seems to be a correspondence between genetic haplotype groups and geographical location.

Bird droppings on chestnut leaves or sawfly larvae: DNA barcodes verify the occurrence of the archaic Megaxyela togashii (Hymenoptera, Xyelidae) in Hokkaido, Japan.

We made a molecular phylogenetic analysis using mitochondrial cytochrome oxidase subunit 1 (COI) gene sequences for ten unidentified Megaxyela larvae from Hokkaido, Honshu and Shikoku, Japan, and 15 identified adults of four Megaxyela, one Macroxyela and three Xyela species. It revealed that all larvae belonged to M. togashii Shinohara, 1992, which showed rather large intraspecific genetic variability even among the individuals from the same population. This is the first distribution record of M. togashii from Hokkaido. Megaxyela togashii is a univoltine species with a very short larval feeding period, only nine days in one rearing experiment from egg to larval maturation. The larva is a solitary, external leaf-feeder on Juglans ailanthifolia, resting curled around the central leaf vein at the apex of a leaflet, and may resemble the excrement of birds. The prepupa overwinters in an earthen cell whose wall is made only of soil, neither parchment-like nor containing fiber. The mature larva is described and several life traits are discussed.

Identification of water buffalo horn and its adulterants using COI barcode / 药学学报

Bubali cornu (water buffalo horn) has been used as the substitute for Cornu rhinoceri asiatici (rhino horn) in clinical applications, and is the essential ingredient of Angong Niuhuang Wan. In recent years, there are a number of adulterants on the commercial herbal medicine markets. An efficient tool is required for species identification. In this study, 155 Bubali cornu samples have been taken from original animals and collected from commercial herbal medicine markets. 153 COI sequences have been successfully obtained from 155 samples through DNA extraction, PCR amplification, bidirectional sequencing and assembly. 93 COI sequences have been added to the DNA barcoding database of traditional Chinese animal medicine after validation using DNA barcoding GAP and tree-based methods. The species identification of the 62 commercial Bubali cornu medicines has been accomplished on the DNA barcoding system for identifying herbal medicine using the updated animal medicine database (www.tcmbarcode.cn). Except two samples failed to obtain COI sequences, 54.8% of the commercial Bubali cornu medicines were water buffalo horns and 29% were yak horns. Our results showed that yak horn was the major adulterant of Bubali cornu and the DNA barcoding method may accurately discriminate Bubali cornu and their adulterants. Therefore, we recommend that supervision on the herbal medicine markets should be strengthened with this new method to warren the effectiveness of herbal medicines.

Molecular Identification of Metacordyceps Liangshanensis, Its Adulterants and Its Relative Species Based on DNA Barcode / 世界科学技术-中医药现代化

This study was aimed to identify and distinguish Metacordyceps liangshanensis recorded by the Sichuan Province Traditional Chinese Medicine Standard from its adulterants and its relative species by combining ITS and COI barcode sequences in order to study the feasibility of this new method. After extracting DNA of 28 species of Cordyceps samples, DNA were amplified and sequenced. And then, ITS and COI sequences were received. Codon-Code Aligner V3.7.1 and Mega 5.0 were used to analyze the variable site and construct the N-J tree. The results showed that the minimum ITS inter-specific K-2P distance was relatively higher than the maximum intra-specific K-2P distance. The inter-specific sequence divergence between M. liangshanensis and its adulterants exhibited high while intra-specific sequence divergence exhibited low. And COI one was the same case. N-J tree of both ITS and COI indicated that same genus belonged together and each species belonged to relatively independent branch. It was concluded that based on the ITS and COI gene, the technology of DNA barcode can be an excellent identification of M. liangshanensis, its adulterants and its relative species. It provided technical support for the further research on species molecular identification and phylogenetics of Cordyceps .

Evolutionary history and phylogenetic relationship between Auxis thazard and Auxis rochei inferred from COI sequences of mtDNA.

Tunas of the genus Auxis are cosmopolitan species and the smallest members of the tribe Thunnini, the true tunas. In the present study, COI sequences of mtDNA were employed to examine the evolutionary history and phylogenetic relationship between A. thazard and A. rochei. A total of 29 COI sequences were retrieved from NCBI. Historic demographic analyses of sequence data showed that A. thazard has undergone sudden population expansion in the past while population size of A. rochei has been remain constant for long period. Non-significant value of Tajimas's D (P = 0.22400) and Fu's FS (P = 0.21400) test fail to reject the null hypothesis of neutral evolution for A. rochei. Phylogenetic analyses of nucleotide sequences demonstrated separate clusters for both species and are strongly supported by 98% bootstrap value. The results of the present study suggest the recent founding of A. thazard in world ocean while A. rochei represents the ancestral species.

Application of COI-based DNA Barcoding for Identifying Animal Medical Materials in the Chinese Pharmacopoeia / 世界科学技术-中医药现代化

Medicinal animals are important part of Traditional Chinese medicine resources in China. Cytochrome c oxidase subunit I (COI) was selected as the standard DNA barcoding sequence for animal medical materials. In this study, the 51 animal species from 45 animal medical materials in the Chinese Pharmacopoeia were selected and the intra-specific variation and the inter-specific divergence, the barcoding gap, the identification efficiency of their COI sequences were analyzed. The results showed that the inter-specific divergence is higher than intra-specific distance. The barcoding gap existed between inter-specific sequence divergence and intra-specific dis-tance. The identification efficiencies were 100% both at the genus and species level except the Arthropoda. The cluster dendrogram exhibited that different species distinguished from others. Therefore, COI sequence as a bar-code is suitable to identify the species of animal medical materials in Chinese Pharmacopoeia.