First record of the bacterial endosymbiont Wolbachia for phytophagous hoverflies from genus Merodon (Diptera: Syrphidae)

Wolbachia is a widespread bacterial endosymbiont among arthropod species. It influences the reproduction of the host species and also mitochondrial DNA diversity. Until now there were only a few studies that detected Wolbachia infections in hoverflies (Diptera: Syrphidae), and this is the first broader study with the aim of examining the incidence of Wolbachia in the hoverfly genus Merodon. The obtained results indicate an infection rate of 96% and the presence of both Wolbachia supergroup A and B, which are characteristic for most of the infected arthropod species. Additionally, the presence of multiple Wolbachia strains in the Merodon aureus group species was detected and the mitochondrial DNA COI‐based relationships of the group are discussed in the light of infection. Finally, we discuss plant‐mediated horizontal transmission of Wolbachia strains among the studied hoverfly species.     

Molecular diversity of populations of the Merodon ruficornis group (Diptera, Syrphidae) on the Balkan Peninsula

Genetic diversity of 11 populations of the Merodon ruficornis group (Diptera, Syrphidae) on the Balkan Peninsula was quantified using allozyme electrophoresis and nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I. A total of 10 haplotypes of mtDNA were found in 22 analysed individuals. One haplotype was shared between M. loewi (from Pindos and Duba?nica Mts) and M. armipes, one confined to M. auripes (haplotype III) and M. armipes (VII) and three to M. trebevicensis (VIII, IX and X), M. ruficornis (I, II and IV) and M. loewi (V, VI and VII). Analysis of the population genetic structure parameters showed great differences among analysed species. Different levels of genetic variability suggested that a variety of biological, environmental and/or historical factors had influenced these hoverfly populations. The unweighted pair group method with arithmetic average dendrogram constructed using allozyme data suggested that M. loewi was the most divergent species in comparison with M. armipes, M. auripes, M. ruficornis and M. trebevicensis. Clear separation of the populations ((((M. armipes + M. auripes) + M. ruficornis) + M. trebevicensis) + M. loewi) was observed. The parsimony analysis of the M. ruficornis group using Merodon avidus as outgroup resulted in three equally parsimonious trees, and the strict consensus presented the following relationships ((((M. loewi + M. armipes) + (M. auripes)) + (M. trebevicensis)) + (M. ruficornis)))). The uncorrected interspecies pairwise sequence divergences ranged from 1.69% (between haplotype III of M. armipes and haplotype V of M. loewi) to 4.94% (between M. ruficornis and M. trebevicensis).     

Oligocene swamp sediments of Lesvos Island,Greece (geochemistry and mineralogy)

Summary The Tertiary Lapsarna swamp sedimentary rocks include thin horizons (organic-rich sediments, up to 22% C_org). Analyses of major elements of carbonates, shaly and siliceous rocks, and a thin, intensively silicified coaly horizon were taken (10 representative samples). Pure carbonates consistalmost completely of calcite, the siliceous sediments of quartz and calcite, and the shales of montmorillonite, feldspar, quartz, dolomite and calcite. The shales contain montmorillonite as the only clay component. The presence of montmorillonite in all shales suggests that the chemistry of the clay-forming solutions lies within the stability field of montmorillonite. Geochemical data indicate that montmorillonite and associated immobile elements (La, Zr, Y, Nb) are the products of hydrolysis of volcanic material in a subtropical region. The intense silification of the coaly horizon is due to a contact with silica-rich solutions that formed during the formation of montmorillonite. Quartz is produced diagenetically by transformation of amorphous opal-A after burial.     

Species identification of aphids (Insecta: Hemiptera: Aphididae) through DNA barcodes

A 658-bp fragment of mitochondrial DNA from the 5' region of the mitochondrial cytochrome c oxidase 1 (COI) gene has been adopted as the standard DNA barcode region for animal life. In this study, we test its effectiveness in the discrimination of over 300 species of aphids from more than 130 genera. Most (96%) species were well differentiated, and sequence variation within species was low, averaging just 0.2%. Despite the complex life cycles and parthenogenetic reproduction of aphids, DNA barcodes are an effective tool for identification.     

Molecular identification of mosquitoes (Diptera: Culicidae) in southeastern Australia

DNA barcoding is a modern species identification technique that can be used to distinguish morphologically similar species, and is particularly useful when using small amounts of starting material from partial specimens or from immature stages. In order to use DNA barcoding in a surveillance program, a database containing mosquito barcode sequences is required. This study obtained Cytochrome Oxidase I (COI) sequences for 113 morphologically identified specimens, representing 29 species, six tribes and 12 genera; 17 of these species have not been previously barcoded. Three of the 29 species ─ Culex palpalis, Macleaya macmillani, and an unknown species originally identified as Tripteroides atripes ─ were initially misidentified as they are difficult to separate morphologically, highlighting the utility of DNA barcoding. While most species grouped separately (reciprocally monophyletic), the Cx. pipiens subgroup could not be genetically separated using COI. The average conspecific and congeneric p‐distance was 0.8% and 7.6%, respectively. In our study, we also demonstrate the utility of DNA barcoding in distinguishing exotics from endemic mosquitoes by identifying a single intercepted Stegomyia aegypti egg at an international airport. The use of DNA barcoding dramatically reduced the identification time required compared with rearing specimens through to adults, thereby demonstrating the value of this technique in biosecurity surveillance. The DNA barcodes produced by this study have been uploaded to the ‘Mosquitoes of Australia–Victoria’ project on the Barcode of Life Database (BOLD), which will serve as a resource for the Victorian Arbovirus Disease Control Program and other national and international mosquito surveillance programs.     

Recovery of DNA barcodes from blackfly museum specimens (Diptera: Simuliidae) using primer sets that target a variety of sequence lengths

In this study, we evaluated the efficacy of various primers for the purpose of DNA barcoding old, pinned museum specimens of blackflies (Diptera: Simuliidae). We analysed 271 pinned specimens representing two genera and at least 36 species. Due to the age of our material, we targeted overlapping DNA fragments ranging in size from 94 to 407 bp. We were able to recover valid sequences from 215 specimens, of which 18% had 500‐ to 658‐bp barcodes, 36% had 201‐ to 499‐bp barcodes and 46% had 65‐ to 200‐bp barcodes. Our study demonstrates the importance of choosing suitable primers when dealing with older specimens and shows that even very short sequences can be diagnostically informative provided that an appropriate gene region is used. Our study also highlights the lack of knowledge surrounding blackfly taxonomy, and we briefly discuss the need for further phylogenetic studies in this socioeconomically important family of insects.     

Diurnal activity patterns in hoverflies (Diptera, Syrphidae)

Abstract. 1. Time-budgets are constructed from censuses of hoverflies. Larger species spend less time in flight.
2. Most species are active under similar conditions of light intensity, but a shade-tolerant (Melanostoma scalare Fabr.) and a sun-loving species (Metasyrphus corollae Fabr.) were identified.
3. Large species become active (i.e. move, by flying or feeding) at lower temperatures than small ones, except M.scalare, which is tolerant to cool temperatures. This means that, M.scalare apart, the order of species appearing during the day is size-dependent, largest first.
4. Previous claims about the diel periodicity of syrphids can be reconciled by considering the importance of individual thermal balance.
5. Tolerance to low temperatures in small hoverflies is associated with feeding on anemophilous pollen; this may indicate a proline-fuelled flight.     

Utility of GenBank and the Barcode of Life Data Systems (BOLD) for the identification of forensically important Diptera from Belgium and France

Fly larvae living on dead corpses can be used to estimate post-mortem intervals. The identification of these flies is decisive in forensic casework and can be facilitated by using DNA barcodes provided that a representative and comprehensive reference library of DNA barcodes is available.We constructed a local (Belgium and France) reference library of 85 sequences of the COI DNA barcode fragment (mitochondrial cytochrome c oxidase subunit I gene), from 16 fly species of forensic interest (Calliphoridae, Muscidae, Fanniidae). This library was then used to evaluate the ability of two public libraries (GenBank and the Barcode of Life Data Systems – BOLD) to identify specimens from Belgian and French forensic cases. The public libraries indeed allow a correct identification of most specimens. Yet, some of the identifications remain ambiguous and some forensically important fly species are not, or insufficiently, represented in the reference libraries. Several search options offered by GenBank and BOLD can be used to further improve the identifications obtained from both libraries using DNA barcodes.     

Developing DNA barcodes for species identification in Podophylloideae (Berberidaceae)

Species of Podophyllum, Dysosma, Sinopodophyllum, and Diphylleia, genera from Podophylloideae of Berberidaceae, have long been used in traditional herbal medicine in East Asia and/or North America. Accurate identification of the species of these four genera is crucial to their medicinal uses. In this study, we tested the utility of nine barcodes (matK, rbcL, atpH-atpI, rpl32-trnL^UAG, rps18-clpp, trnL-trnF, trnL-ndhJ, trnS-trnfM, and internal transcribed spacer (ITS)) to discriminate different species of Podophylloideae. Thirty-six individuals representing 12 species of Podophylloideae were collected from different locations in China, Japan, and North America. We assessed the feasibility of amplification and sequencing of all markers, examined the levels of the barcoding gap based on DNA sequence divergence between ranges of intra- and interspecific variation using pairwise distances, and further evaluated successful identifications using each barcode by similarity-based and tree-based methods. Results showed that nine barcodes, except rps18-clpp, have a high level of primer universality and sequencing success. As a single barcode, ITS has the most variable sites, greater intra- and interspecific divergences, and the highest species discrimination rate (83%), followed by matKwhich has moderate variation and also high species discrimination rates. However, these species can also be discriminated by ITS alone, except Dysosma versipellis (Hance) M. Cheng ex T. S. Ying and D. pleiantha (Hance) Woodson. The combination of ITS + matK did not improve species resolution over ITS alone. Thus, we propose that ITS may be used as a sole region for identification of most species in Podophylloideae. The failure of ITS to distinguish D. versipellis and D. pleiantha is likely attributed to incomplete lineage sorting due to recent divergence of the two species.     

北京地区7种常见嗜尸性蝇类的COI基因序列分析及DNA条形码的建立

嗜尸性蝇类在命案死亡时间和现场推断方面有着十分重要的应用, 而DNA 条形编码技术能摆脱对虫卵和幼虫的饲养以及后续物种鉴定方面专业知识的依赖, 有助于实现现场采集蝇类样本的快速鉴定。本研究采集了北京地区7个嗜尸性蝇类优势种共77个个体的样本, 测定了所有个体线粒体DNA 上细胞色素C氧化酶亚基Ⅰ（COI）基因1 120 bp的序列。基于序列的系统发生分析显示, 同一物种不同个体的序列均以高达99%的支持值聚集在一起。序列间的分歧统计表明这些蝇类在物种内的个体分歧不超过1%, 而不同物种间的净分歧均超过7.74%, 最高可达14.85%。滑动窗口分析表明, 在整个序列区段种间差异位点存在较平均的分布。通过测定COI基因的序列, 建立了北京地区7个嗜尸性蝇类优势种的DNA条形码, 据此实现了对这些物种准确、快速、简单的区分和鉴定, 同时也为后续应用于物种鉴定的种属特异性位点之筛选提供了基础数据。     

Using COI barcodes to identify forensically and medically important blowflies

Abstract.  The utility of cytochrome oxidase I (COI) DNA barcodes for the identification of nine species of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae), from Australia, was tested. A 658-bp fragment of the COI gene was sequenced from 56 specimens, representing all nine Chrysomya species and three calliphorid outgroups. Nucleotide sequence divergences were calculated using the Kimura-two-parameter distance model and a neighbour-joining (NJ) analysis was performed to provide a graphic display of the patterns of divergence among the species. All species were resolved as reciprocally monophyletic on the NJ tree. Mean intraspecific and interspecific sequence divergences were 0.097% (range 0–0.612%, standard error [SE] = 0.119%) and 6.499% (range 0.458–9.254%, SE = 1.864%), respectively. In one case, a specimen that was identified morphologically was recovered with its sister species on the NJ tree. The hybrid status of this specimen was established by sequence analysis of the second ribosomal internal transcribed spacer (ITS2). In another instance, this nuclear region was used to verify four cases of specimen misidentification that had been highlighted by the COI analysis. The COI barcode sequence was found to be suitable for the identification of Chrysomya species from the east coast of Australia.     

High molecular and phenotypic diversity in the Merodon avidus complex (Diptera,Syrphidae): cryptic speciation in a diverse insect taxon

This paper examines molecular and phenotypic variability in the widely spread European hoverfly species complex Merodon avidus. Herein, based on the mitochondrial DNA (mtDNA) sequences of the cytochrome c oxidase subunit I (COI) and morphometric wing parameters, M. avidus is shown to comprise a complex of cryptic species, and one variety is redefined as a valid species: M. bicolor Gil Collado, 1930 (as var. of spinipes). The species M. bicolor, M. avidus A, and M. avidus B were clearly delimited based on their wing size. A total of 29 M. avidus and M. bicolor individuals presented 20 mtDNA haplotypes, four of which were shared by M. avidus A and M. avidus B, three were confined to M. bicolor, seven to M. avidus A, and six to M. avidus B. Sequence divergences between lineages occurring in the Balkan and in Spain ranged from 4.93 to 6.0 (uncorrected p in %) whereas divergences between M. avidus A and M. avidus B were 0.26 to 1.56. Divergence among morphologically identified individuals of M. avidus A and M. avidus B species ranged from 0.13 to 1.58, and from 0.13 to 0.52, respectively. The phenotypic substructuring and observed genetic uniqueness of populations in spatially and temporally fragmented M. avidus taxa were used to identify genetic units. The early split of two allopatric lineages, Spanish M. bicolor and Balkan M. avidus, was followed by diversification in each lineage. Present‐day morphological uniformity masks much of the genetic complexity of lineages within the M. avidus complex. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 819–833.     

Assessing the diversity of New Zealand freshwater harpacticoid copepods (Crustacea: Copepoda) using mitochondrial DNA (COI) barcodes

Taxonomic and ecological studies of freshwater harpacticoid copepods are limited globally by the ability to easily and accurately identify specimens. Here, we test the use of the mitochondrial cytochrome c oxidase subunit I (COI) gene locus as a tool for assessing the diversity of freshwater Harpacticoida. We obtained sequences from New Zealand harpacticoid copepods, representing two families, five genera and nine species, including the non-indigenous Elaphoidella sewelli. All species were delineated by the COI gene. However, high intraspecific diversity was evident among populations of Elaphoidella bidens (>12%), and between North and South Island populations of Bryocamptus pygmaeus (>18%), potentially indicating the presence of morphologically cryptic taxa. We suggest that mitochondrial DNA (COI) sequences can provide a useful tool for the routine identification of freshwater harpacticoid copepods. Applications of these data will include assessing species diversity and biogeography as well as assisting with the detection of non-indigenous species.     

Cryptic speciation in the Merodon luteomaculatus complex (Diptera: Syrphidae) from the eastern Mediterranean

The Merodon aureus group is characterized by high endemism and the presence of morphologically cryptic species. Within one of its subgroups, M. bessarabicus, seven species and four more species complexes have been described to date. One of these complexes, the M. luteomaculatus, comprises new taxa that are the subject of the present study. Its members have allopatric ranges restricted to the Balkan Peninsula and Aegean islands. This complex exhibits morphological variability that could not be characterized using a traditional morphological approach. Thus, we used integrative taxonomy with independent character sets (molecular, geometric morphometric, distributional) to delimit species boundaries. Data on three molecular markers (COI, 28S rRNA, and ISSR) and geometric morphometry of the wing and male genitalia, together with distributional data, enabled recognition of six cryptic species within the complex: M. andriotes sp. n., M. euri sp. n., M. erymanthius sp. n., M. luteomaculatus sp. n., M. naxius sp. n., and M. peloponnesius sp. n. We discuss the possible influence of Aegean paleogeographical history on the speciation of this complex.     

Species identification of North American guinea worms (Nematoda: Dracunculus) with DNA barcoding

Dracunculus insignis is a nematode parasite that infects the subcutaneous tissues of mammals such as raccoon (Procyon lotor), mink (Neovison vison) and fisher (Martes pennanti). D. lutrae, a morphologically similar species, has only been recovered from the otter (Lontra canadensis). Species identification of these two North American guinea worms has only been achieved by morphology of males and host identity. As a result, where only female specimens are present, accurate identifications are not possible. To date, specimens recovered from otter have been assumed to be D. lutrae, while those from all other hosts are assumed to be D. insignis. This study uses DNA barcoding to differentiate between these two North American dracunculoids. Our results show that D. insignis is a 'true' generalist, showing little sequence divergence regardless of host association, although our studies did validate its occurrence in a new host - the otter. Interestingly, specimens of the host specialist, D. lutrae, showed some sequence divergence, although it was low. The finding of D. insignis in otter substantiates the need to supplement morphology-based methods in providing species identifications for certain dracunculoids.     

Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application

Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage to horticulture industries. Immature stages of these two fruit fly species have been intercepted in New Zealand a number of times. Identification to species was not possible using morphological characters; thus, it is important to develop an assay for their species‐level identification. Here, the real‐time PCR assays for rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity by amplifying the two target species successfully, with no cross‐reactions observed in the tested tephritid species. The in silico test of the primer and probe binding sites of the two assays also demonstrated the assays’ specificity by no mismatches present in the binding regions of the target species, but 1–4 mismatches in the binding regions of the non‐target fruit fly species. The thresholds of detection for the two assays are as low as 10 copies/µl of the target DNA, indicating that the assays have a very high sensitivity. The application of the real‐time PCR assays has greatly assisted in routine pest identifications at the New Zealand border and surveillance programme. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations worldwide.     

Saproxylic hoverflies benefit by modern forest management (Diptera: Syrphidae)

Saproxylic organisms are regarded as threatened in many European countries. Nevertheless, there have been some signals indicating a recent increase of saproxylic hoverflies in the Netherlands. This paper examines the change in occurrence of saproxylic hoverflies compared to other ecological groups of the same family. The trend analysis is based on the database of an extensive recording programme carried out in the Netherlands. The results show an increase in occupied grid cells for a majority of saproxylic species in relation to other ecological groups. This positive trend occurs among all five ecological subcategories of saproxylic hoverflies recognized in this paper. It is suggested that this increase could be attributed to the changes in the Dutch forests during the past 50 years. Important aspects of these changes are: the increase in forest area (including an increase in area of forest with large and very large trees), the increase in age of forests and the changes in forest management in favour of a more natural approach towards dead and ill trees. In the discussion the possible influence of climate change and the method of trend calculation are discussed.     

DNA条形码技术在青海海东地区小型兽类鉴定中的应用

为弥补传统形态分类方法的不足,探究应用DNA条形码技术进行分子生物学鉴定的可行性,本研究用DNA条形码技术检测了青海省海东地区3目6科14属18种110只小型兽类的COI基因部分序列。分析所测COI基因序列可知:种内遗传距离≤3%,种间遗传距离5-10%,属间遗传距离12-19%,种间遗传距离显著大于种内遗传距离。NJ树显示同种个体聚为有很高支持度的单一分支。有6个个体(4只黄胸鼠、2只小家鼠)在现场鉴定中被误定为其他种类。研究结果表明使用条形码技术能纠正形态学鉴定中的错误,也说明动物线粒体COI基因是一个有效的DNA条形码标准基因。     

DNA barcodes as a tool in biodiversity research: testing pre-existing taxonomic hypotheses in Delphic Apollo butterflies (Lepidoptera,Papilionidae)

Numerous studies have demonstrated that DNA barcoding is an effective tool for detecting DNA clusters, which can be viewed as operational taxonomic units (OTUs), useful for biodiversity research. Frequently, the OTUs in these studies remained unnamed, not connected with pre-existing taxonomic hypotheses, and thus did not really contribute to feasible estimation of species number and adjustment of species boundaries. For the majority of organisms, taxonomy is very complicated with numerous, often contradictory interpretations of the same characters, which may result in several competing checklists using different specific and subspecific names to describe the same sets of populations. The highly species-rich genus Parnassius (Lepidoptera: Papilionidae) is but one example, such as several mutually exclusive taxonomic systems have been suggested to describe the phenotypic diversity found among its populations. Here we provide an explicit flow chart describing how the DNA barcodes can be combined with the existing knowledge of morphology-based taxonomy and geography (sympatry versus allopatry) of the studied populations in order to support, reject or modify the pre-existing taxonomic hypotheses. We then apply this flow chart to reorganize the taxa within the Parnassius delphius species group, solving long-standing taxonomic problems.