Identification of five species of the Anopheles dirus complex from Thailand, using allele-specific polymerase chain reaction

The Anopheles dirus complex of mosquitoes contains some of the most important vectors of malaria in Southeast Asia. To distinguish five species of the complex that occur in Thailand, a method using the polymerase chain reaction (PCR) was developed. The method utilizes allele-specific amplification to detect fixed differences between the species in the DNA sequence of the ribosomal DNA internal transcribed spacer 2. Primers were designed to amplify fragments of diagnostic length from the DNA of the different species. The method was tested on 179 mosquitoes of the An. dirus complex from many parts of Thailand and shown to be effective. Every specimen was unambiguously identified as species A, B, C, D or F (i.e. An. dirus s.s. species B, C, D or An. nemophilous, respectively) by the PCR method, with confirmation of 58/61 identifications from polytene chromosome characteristics. For the other three specimens (3/44 from Kanchanaburi 5 locality), there was disagreement between the PCR and chromosomal methods of species identification (probably due to errors in the chromosomal identifications). Primers can be combined in a single PCR reaction providing a rapid, sensitive and straightforward method of species identification. Only small quantities of DNA are required, leaving most of the mosquito to be used for other analyses.     

Identification of North Sea molluscs with DNA barcoding

Sequence‐based specimen identification, known as DNA barcoding, is a common method complementing traditional morphology‐based taxonomic assignments. The fundamental resource in DNA barcoding is the availability of a taxonomically reliable sequence database to use as a reference for sequence comparisons. Here, we provide a reference library including 579 sequences of the mitochondrial cytochrome c oxidase subunit I for 113 North Sea mollusc species. We tested the efficacy of this library by simulating a sequence‐based specimen identification scenario using Best Match, Best Close Match (BCM) and All Species Barcode (ASB) criteria with three different threshold values. Each identification result was compared with our prior morphology‐based taxonomic assignments. Our simulation resulted in 87.7% congruent identifications (93.8% when excluding singletons). The highest number of congruent identifications was obtained with BCM and ASB and a 0.05 threshold. We also compared identifications with genetic clustering (Barcode Index Numbers, BINs) computed by the Barcode of Life Datasystem (BOLD). About 68% of our morphological identifications were congruent with BINs created by BOLD. Forty‐nine sequences were clustered in 16 discordant BINs, and these were divided in two classes: sequences from different species clustered in a single BIN and conspecific sequences divided in more BINs. Whereas former incongruences were probably caused by BOLD entries in need of a taxonomic update, the latter incongruences regarded taxa requiring further investigations. These include species with amphi‐Atlantic distribution, whose genetic structure should be evaluated over their entire range to produce a reliable sequence‐based identification system.     

传粉网络构建的定性定量分子研究: 应用与展望

传粉者是重要的生态功能提供者, 在维持稳定的生态系统和高效的农业生产力中发挥着重要作用。因此, 传粉网络的构建和监测工作对评价生态系统平衡和调控农业生产至关重要。该工作的基础就是通过对传粉者及植物的物种鉴定构建其相关性。传统的形态学物种鉴定对分类学专家的专业知识、时间和经验都提出较高的要求。DNA条形码和高通量测序技术(high-throughput sequencing, HTS)的发展及其在传粉网络研究中的应用, 提供了高效、准确鉴定传粉者与植物的方法, 大大提高了传粉网络构建的效率。本文阐述了传粉网络研究相关的研究方法和技术进展, 并提出利用高通量测序技术结合无PCR扩增(PCR-free)的“超级条形码”技术, 有望实现以更高的灵敏度和分辨率对混合物种样品进行定性及相对定量的监测。该方法的有效性已在其他生物多样性研究中得以验证, 在传粉网络研究中虽处于初始阶段, 但应用前景广阔。     

Linking camera‐trap data to taxonomy: Identifying photographs of morphologically similar chipmunks

Remote cameras are a common method for surveying wildlife and recently have been promoted for implementing large‐scale regional biodiversity monitoring programs. The use of camera‐trap data depends on the correct identification of animals captured in the photographs, yet misidentification rates can be high, especially when morphologically similar species co‐occur, and this can lead to faulty inferences and hinder conservation efforts. Correct identification is dependent on diagnosable taxonomic characters, photograph quality, and the experience and training of the observer. However, keys rooted in taxonomy are rarely used for the identification of camera‐trap images and error rates are rarely assessed, even when morphologically similar species are present in the study area. We tested a method for ensuring high identification accuracy using two sympatric and morphologically similar chipmunk (Neotamias) species as a case study. We hypothesized that the identification accuracy would improve with use of the identification key and with observer training, resulting in higher levels of observer confidence and higher levels of agreement among observers. We developed an identification key and tested identification accuracy based on photographs of verified museum specimens. Our results supported predictions for each of these hypotheses. In addition, we validated the method in the field by comparing remote‐camera data with live‐trapping data. We recommend use of these methods to evaluate error rates and to exclude ambiguous records in camera‐trap datasets. We urge that ensuring correct and scientifically defensible species identifications is incumbent on researchers and should be incorporated into the camera‐trap workflow.     

Effect of Leaf Type and Pesticide Exposure on Abundance of Bacterial Taxa in Mosquito Larval Habitats

Lentic freshwater systems including those inhabited by aquatic stages of mosquitoes derive most of their carbon inputs from terrestrial organic matter mainly leaf litter. The leaf litter is colonized by microbial communities that provide the resource base for mosquito larvae. While the microbial biomass associated with different leaf species in container aquatic habitats is well documented, the taxonomic composition of these microbes and their response to common environmental stressors is poorly understood. We used indoor aquatic microcosms to determine the abundances of major taxonomic groups of bacteria in leaf litters from seven plant species and their responses to low concentrations of four pesticides with different modes of action on the target organisms; permethrin, malathion, atrazine and glyphosate. We tested the hypotheses that leaf species support different quantities of major taxonomic groups of bacteria and that exposure to pesticides at environmentally relevant concentrations alters bacterial abundance and community structure in mosquito larval habitats. We found support for both hypotheses suggesting that leaf litter identity and chemical contamination may alter the quality and quantity of mosquito food base (microbial communities) in larval habitats. The effect of pesticides on microbial communities varied significantly among leaf types, suggesting that the impact of pesticides on natural microbial communities may be highly complex and difficult to predict. Collectively, these findings demonstrate the potential for detritus composition within mosquito larval habitats and exposure to pesticides to influence the quality of mosquito larval habitats.     

Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species

Biodiversity studies require species level analyses for the accurate assessment of community structures. However, while specialized taxonomic knowledge is only rarely available for routine identifications, DNA taxonomy and DNA barcoding could provide the taxonomic basis for ecological inferences. In this study, we assessed the community structure of sediment dwelling, morphologically cryptic Chironomus larvae in the Rhine-valley plain/Germany, comparing larval type classification, cytotaxonomy, DNA taxonomy and barcoding. While larval type classification performed poorly, cytotaxonomy and DNA-based methods yielded comparable results: detrended correspondence analysis and permutation analyses indicated that the assemblages are not randomly but competitively structured. However, DNA taxonomy identified an additional species that could not be resolved by the traditional method. We argue that DNA-based identification methods such as DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of the taxonomic assessment in biodiversity and community ecology studies.     

A ranking system for reference libraries of DNA barcodes: application to marine fish species from Portugal

Background

The increasing availability of reference libraries of DNA barcodes (RLDB) offers the opportunity to the screen the level of consistency in DNA barcode data among libraries, in order to detect possible disagreements generated from taxonomic uncertainty or operational shortcomings. We propose a ranking system to attribute a confidence level to species identifications associated with DNA barcode records from a RLDB. Here we apply the proposed ranking system to a newly generated RLDB for marine fish of Portugal.

Methodology/Principal Findings

Specimens (n = 659) representing 102 marine fish species were collected along the continental shelf of Portugal, morphologically identified and archived in a museum collection. Samples were sequenced at the barcode region of the cytochrome oxidase subunit I gene (COI-5P). Resultant DNA barcodes had average intra-specific and inter-specific Kimura-2-parameter distances (0.32% and 8.84%, respectively) within the range usually observed for marine fishes. All specimens were ranked in five different levels (A–E), according to the reliability of the match between their species identification and the respective diagnostic DNA barcodes. Grades A to E were attributed upon submission of individual specimen sequences to BOLD-IDS and inspection of the clustering pattern in the NJ tree generated. Overall, our study resulted in 73.5% of unambiguous species IDs (grade A), 7.8% taxonomically congruent barcode clusters within our dataset, but awaiting external confirmation (grade B), and 18.7% of species identifications with lower levels of reliability (grades C/E).

Conclusion/Significance

We highlight the importance of implementing a system to rank barcode records in RLDB, in order to flag taxa in need of taxonomic revision, or reduce ambiguities of discordant data. With increasing DNA barcode records publicly available, this cross-validation system would provide a metric of relative accuracy of barcodes, while enabling the continuous revision and annotation required in taxonomic work.     

Integrative taxonomy for continental-scale terrestrial insect observations

Although 21(st) century ecology uses unprecedented technology at the largest spatio-temporal scales in history, the data remain reliant on sound taxonomic practices that derive from 18(th) century science. The importance of accurate species identifications has been assessed repeatedly and in instances where inappropriate assignments have been made there have been costly consequences. The National Ecological Observatory Network (NEON) will use a standardized system based upon an integrative taxonomic foundation to conduct observations of the focal terrestrial insect taxa, ground beetles and mosquitoes, at the continental scale for a 30 year monitoring program. The use of molecular data for continental-scale, multi-decadal research conducted by a geographically widely distributed set of researchers has not been evaluated until this point. The current paper addresses the development of a reference library for verifying species identifications at NEON and the key ways in which this resource will enhance a variety of user communities.     

A method for reconstructing three-dimensional positions of swarming mosquitoes

We developed a new field method for reconstructing the three-dimensional positions of swarming mosquitoes. This method overcame certain inherent difficulties accompanied by conventional stereoscopic methods and is applicable to three-dimensional measurements of other insect species. Firstly, we constructed a probabilistic model for stereoscopy; if mosquitoes and six reference points with known coordinates were photographed simultaneously from two or more perspectives, then from the positions of images of mosquitoes and the reference points on the photographs, 1) the position of each camera with respect to the reference points is estimated; 2) stereo images which correspond to an identical real mosquito are matched; and 3) the spatial positions of the mosquitoes are determined. We automated the processes 1), 2) and 3), developing computer programs based on our model. We then constructed a portable system for three-dimensional measurements of swarming mosquitoes in the field. Initial data that illustrate the application of our method to studying mosquito swarming were presented.     

DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics

DNA barcoding aims to provide an efficient method for species-level identifications and, as such, will contribute powerfully to taxonomic and biodiversity research. As the number of DNA barcode sequences accumulates, however, these data will also provide a unique 'horizontal' genomics perspective with broad implications. For example, here we compare the goals and methods of DNA barcoding with those of molecular phylogenetics and population genetics, and suggest that DNA barcoding can complement current research in these areas by providing background information that will be helpful in the selection of taxa for further analyses.     

ABUNDANCE OF BLUE AND HUMPBACK WHALES IN THE EASTERN NORTH PACIFIC ESTIMATED BY CAPTURE-RECAPTURE AND LINE-TRANSECT METHODS

We estimated humpback and blue whale abundance from 1991 to 1997 off the west coast of the U. S. and Mexico comparing capture-recapture models based on photographically identified animals and line-transect methods from ship-based surveys. During photo-identification research we obtained 4,212 identifications of 824 humpback whales and 2,403 identifications of 908 blue whales primarily through non-systematic small-boat surveys along the coast of California, Oregon, and Washington. Line-transect surveys from NOAA ships in 1991, 1993, and 1996 covered approximately 39,000 km along the coast of Baja California, California, Oregon, and Washington out to 555 km from shore. The nearshore and clumped distribution of humpback whales allowed photographic identification from small boats to cost-effectively sample a substantial portion of the population, but made it difficult to obtain effective samples in the line-transect surveys covering broad areas. The humpback capture-recapture estimates indicated humpback whale abundance increased over the six years (from 569 to 837). The broader more offshore distribution of blue whales made it harder to obtain a representative sample of identification photographs, but was well suited to the line-transect estimates. The line-transect estimates, after correction for missed animals, indicated approximately 3,000 blue whales (CV = 0.14). Capture-recapture estimates of blue whales were lower than this: approximately 2,000 when using photographs obtained from the line-transect surveys as one of the samples. Comparison of the results from the two methods provides validation, as well as insight into potential biases associated with each method.     

Independent similarity in the morphological evolution of brachiopods

The close similarity of the shell exterior of articulate brachiopods from different orders, which must be taken into account in taxonomic identifications and phylogenetic reconstructions, is analyzed. A possible mechanism of the appearance of such brachiopods in connection with the morphogenetic generality of the structurally similar organisms is evaluated.     

The identification of fish eggs of two species,the ovate sole Solea ovata and black porgy Acanthopagrus schlegelii

Fish eggs of the ovate sole Solea ovata and black porgy Acanthopagrus schlegelii were identified through DNA barcoding of cytochrome c oxidase subunit I (COX1). Visual taxonomic features were achieved, and photographs of the eggs of both species at different developmental stages were reported for the first time. In addition, the dissolution of oil globules caused by ethanol as egg fixatives was observed. This result showed the importance of using formalin as egg fixatives in the case of morphometric analysis and the necessity of combining molecular and visual taxonomic method for morphological study.     

From GenBank to GBIF: Phylogeny-Based Predictive Niche Modeling Tests Accuracy of Taxonomic Identifications in Large Occurrence Data Repositories

Accuracy of taxonomic identifications is crucial to data quality in online repositories of species occurrence data, such as the Global Biodiversity Information Facility (GBIF), which have accumulated several hundred million records over the past 15 years. These data serve as basis for large scale analyses of macroecological and biogeographic patterns and to document environmental changes over time. However, taxonomic identifications are often unreliable, especially for non-vascular plants and fungi including lichens, which may lack critical revisions of voucher specimens. Due to the scale of the problem, restudy of millions of collections is unrealistic and other strategies are needed. Here we propose to use verified, georeferenced occurrence data of a given species to apply predictive niche modeling that can then be used to evaluate unverified occurrences of that species. Selecting the charismatic lichen fungus, Usnea longissima, as a case study, we used georeferenced occurrence records based on sequenced specimens to model its predicted niche. Our results suggest that the target species is largely restricted to a narrow range of boreal and temperate forest in the Northern Hemisphere and that occurrence records in GBIF from tropical regions and the Southern Hemisphere do not represent this taxon, a prediction tested by comparison with taxonomic revisions of Usnea for these regions. As a novel approach, we employed Principal Component Analysis on the environmental grid data used for predictive modeling to visualize potential ecogeographical barriers for the target species; we found that tropical regions conform a strong barrier, explaining why potential niches in the Southern Hemisphere were not colonized by Usnea longissima and instead by morphologically similar species. This approach is an example of how data from two of the most important biodiversity repositories, GenBank and GBIF, can be effectively combined to remotely address the problem of inaccuracy of taxonomic identifications in occurrence data repositories and to provide a filtering mechanism which can considerably reduce the number of voucher specimens that need critical revision, in this case from 4,672 to about 100.     

Electrophoretic differences in the isoenzymes of two mosquito gregarines, Ascogregarina barretti and A. geniculati

Cross-infection experiments demonstrated that Ascogregarina barretti, from Aedes triseriatus, completes its life cycle in Aedes geniculatus. Parasite numbers were comparable to infection with Ascogregarina geniculati, making the separation of these parasites by host preference difficult. However, electrophoresis readily distinguished isoenzymes from the two morphologically similar gregarine species. Different migration rates were obtained for isocitrate dehydrogenase 1 and 2, lactate dehydrogenase, and malate dehydrogenase. The migration rates were also different for parasite and host isoenzymes. When a single, heavily infected gut was subjected to electrophoresis the isocitrate dehydrogenase bands of each were clearly distinguishable on the same electrophoretic track. Electrophoresis appears to be a reliable method for resolving taxonomic complications of mosquito gregarines, a group often with wide host specificities and variable taxonomic characters.     

16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts

Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the gamma-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito.     

BOLD’s role in barcode data management and analysis: a response

DNA barcoding is a very effective tool for the identification of specimens when a carefully validated and taxonomically comprehensive library of reference DNA barcodes is available. Libraries meeting this criterion are now available for some taxonomic groups in some geographic regions, provoking their use as a tool for the identification of samples that would otherwise remain as unknowns. In this article, we emphasize the need for caution in the interpretation of identifications based on a reference library with entries that have seen limited validation. We also emphasize the need for the deposition of sequence records for ‘unknowns’ so that presumptive identifications can be tested by other researchers and updated as the barcode reference library gains increased coverage and validation.     

Taxonomic and phylogenetic analysis of the Oomycota mosquito larvae pathogen Crypticola clavulifera

Beside the taxonomic features displayed by Crypticola clavulifera in culture and unpublished data on its phylogeny, little is known about the main phylogenetic features of this unusual mosquito pathogen. We have PCR amplified the 18S SSU rDNA, ITS, and the partial coding regions of COXII and COXI to study the phylogenetic features of this pathogen within the tree of life. Our phylogenetic data showed C. clavulifera clustered among homologous DNA sequences of the Oomycota class Saprolegniomycetes. Our study support previous taxonomic and unpublished molecular analysis, placing this unusual mosquito larvae pathogen as part of the earliest diverging cluster within the Saprolegniomycetes.     

Assessing macroinvertebrate biodiversity in freshwater ecosystems: advances and challenges in DNA-based approaches

Assessing the biodiversity of macroinvertebrate fauna in freshwater ecosystems is an essential component of both basic ecological inquiry and applied ecological assessments. Aspects of taxonomic diversity and composition in freshwater communities are widely used to quantify water quality and measure the efficacy of remediation and restoration efforts. The accuracy and precision of biodiversity assessments based on standard morphological identifications are often limited by taxonomic resolution and sample size. Morphologically based identifications are laborious and costly, significantly constraining the sample sizes that can be processed. We suggest that the development of an assay platform based on DNA signatures will increase the precision and ease of quantifying biodiversity in freshwater ecosystems. Advances in this area will be particularly relevant for benthic and planktonic invertebrates, which are often monitored by regulatory agencies. Adopting a genetic assessment platform will alleviate some of the current limitations to biodiversity assessment strategies. We discuss the benefits and challenges associated with DNA-based assessments and the methods that are currently available. As recent advances in microarray and next-generation sequencing technologies will facilitate a transition to DNA-based assessment approaches, future research efforts should focus on methods for data collection, assay platform development, establishing linkages between DNA signatures and well-resolved taxonomies, and bioinformatics.