A PCR-based method for diet analysis in freshwater organisms using 18S rDNA barcoding on faeces

The development of DNA barcoding from faeces represents a promising method for animal diet analysis. However, current studies mainly rely on prior knowledge of prey diversity for a specific predator rather than on a range of its potential prey species. Considering that the feeding behaviour of teleosts may evolve with their environment, it could prove difficult to establish an exhaustive listing of their prey. In this article, we extend the DNA barcoding approach to diet analysis to allow the inclusion of a wide taxonomic range of potential prey items. Thirty-four ecological clade-specific primer sets were designed to cover a large proportion of prey species found in European river ecosystems. Selected primers sets were tested on isolated animal, algal or plant tissues and thereafter on fish faeces using nested PCR to increase DNA detection sensitivity. The PCR products were sequenced and analysed to confirm the identity of the taxa and to validate the method. The methodology developed here was applied to a diet analysis of three freshwater cyprinid species that are assumed to have similar feeding behaviour [Chondrostoma toxostoma toxostoma (Vallot 1837), Chondrostoma nasus nasus (Linnaeus, 1758) and Barbus barbus, (Linneaus 1758)]. These three species were sampled in four different hydrographic basins. Principal Component Analysis based on prey proportions identified distinct perilithon grazer and benthophagous behaviours. Furthermore, our results were consistent with the available literature on feeding behaviour in these fish. The simplicity of the PCR-based method and its potential generalization to other freshwater organisms may open new perspectives in food web ecology.     

Discovering hidden biodiversity: the use of complementary monitoring of fish diet based on DNA barcoding in freshwater ecosystems

Ecological monitoring contributes to the understanding of complex ecosystem functions. The diets of fish reflect the surrounding environment and habitats and may, therefore, act as useful integrating indicators of environmental status. It is, however, often difficult to visually identify items in gut contents to species level due to digestion of soft‐bodied prey beyond visual recognition, but new tools rendering this possible are now becoming available. We used a molecular approach to determine the species identities of consumed diet items of an introduced generalist feeder, brown trout (Salmo trutta), in 10 Tasmanian lakes and compared the results with those obtained from visual quantification of stomach contents. We obtained 44 unique taxa (OTUs) belonging to five phyla, including seven classes, using the barcode of life approach from cytochrome oxidase I (COI). Compared with visual quantification, DNA analysis showed greater accuracy, yielding a 1.4‐fold higher number of OTUs. Rarefaction curve analysis showed saturation of visually inspected taxa, while the curves from the DNA barcode did not saturate. The OTUs with the highest proportions of haplotypes were the families of terrestrial insects Formicidae, Chrysomelidae, and Torbidae and the freshwater Chironomidae. Haplotype occurrence per lake was negatively correlated with lake depth and transparency. Nearly all haplotypes were only found in one fish gut from a single lake. Our results indicate that DNA barcoding of fish diets is a useful and complementary method for discovering hidden biodiversity.     

Taxon-specific PCR for DNA barcoding arthropod prey in bat faeces

The application of DNA barcoding to dietary studies allows prey taxa to be identified in the absence of morphological evidence and permits a greater resolution of prey identity than is possible through direct examination of faecal material. For insectivorous bats, which typically eat a great diversity of prey and which chew and digest their prey thoroughly, DNA-based approaches to diet analysis may provide the only means of assessing the range and diversity of prey within faeces. Here, we investigated the effectiveness of DNA barcoding in determining the diets of bat species that specialize in eating different taxa of arthropod prey. We designed and tested a novel taxon-specific primer set and examined the performance of short barcode sequences in resolving prey species. We recovered prey DNA from all faecal samples and subsequent cloning and sequencing of PCR products, followed by a comparison of sequences to a reference database, provided species-level identifications for 149/207 (72%) clones. We detected a phylogenetically broad range of prey while completely avoiding detection of nontarget groups. In total, 37 unique prey taxa were identified from 15 faecal samples. A comparison of DNA data with parallel morphological analyses revealed a close correlation between the two methods. However, the sensitivity and taxonomic resolution of the DNA method were far superior. The methodology developed here provides new opportunities for the study of bat diets and will be of great benefit to the conservation of these ecologically important predators.     

Molecular diet analysis of neotropical bats based on fecal DNA metabarcoding

Bat communities in the Neotropics are some of the most speciose assemblages of mammals on Earth, with regions supporting more than 100 sympatric species with diverse feeding ecologies. Because bats are small, nocturnal, and volant, it is difficult to directly observe their feeding habits, which has resulted in their classification into broadly defined dietary guilds (e.g., insectivores, carnivores, and frugivores). Apart from these broad guilds, we lack detailed dietary information for many species and therefore have only a limited understanding of interaction networks linking bats and their diet items. In this study, we used DNA metabarcoding of plants, arthropods, and vertebrates to investigate the diets of 25 bat species from the tropical dry forests of Lamanai, Belize. Our results report some of the first detection of diet items for the focal bat taxa, adding rich and novel natural history information to the field of bat ecology. This study represents a comprehensive first effort to apply DNA metabarcoding to bat diets at Lamanai and provides a useful methodological framework for future studies testing hypotheses about coexistence and niche differentiation in the context of modern high‐throughput molecular data.     

Molecular identification of fine roots of trees from the Alps: reliable and fast DNA extraction and PCR-RFLP analyses of plastid DNA

Fine roots of trees are intensively used as indicators to assess soil alterations, e.g. those owing to atmospheric inputs of acidifying substances, but their identification to species with morphological criteria is difficult. In this study, we established molecular techniques in order to identify fine roots of the 30 most common tree species of the Alps. We developed a protocol for efficient isolation of DNA from fine roots with extraction of DNA in the presence of polyvinylpyrrolidone (PVP) and polyvinylpolypyrrolidone (PVPP). The trnL (UAA) intron of plastid DNA was used as a marker for fine root identification. We amplified and sequenced this intron with plant universal primers. The size of the sequences ranged from 444 to 672 bp. A synoptic key for species identification was designed on the basis of restriction fragment patterns predicted from sequence data. Using the restriction enzyme TaqI as key enzyme, and where necessary HinfI, RsaI and CfoI, 16 taxa, including Picea abies, Larix decidua, Abies alba, and Fagus sylvatica, the dominant tree species of the Alpine region could be identified by agarose gel electrophoresis of restriction fragments. Fourteen taxa could be identified to the genus level, among them Quercus, Salix and Populus species. In a field study, conducted in a 20 x 30 m plot of a mixed forest with five tree species, fine roots of 43 out of 46 samples were identified and their distributions were mapped. These results demonstrate the utility of our DNA extraction method and of the trnL intron for the identification of fine tree roots.     

VIP Barcoding: composition vector‐based software for rapid species identification based on DNA barcoding

Species identification based on short sequences of DNA markers, that is, DNA barcoding, has emerged as an integral part of modern taxonomy. However, software for the analysis of large and multilocus barcoding data sets is scarce. The Basic Local Alignment Search Tool (BLAST) is currently the fastest tool capable of handling large databases (e.g. >5000 sequences), but its accuracy is a concern and has been criticized for its local optimization. However, current more accurate software requires sequence alignment or complex calculations, which are time‐consuming when dealing with large data sets during data preprocessing or during the search stage. Therefore, it is imperative to develop a practical program for both accurate and scalable species identification for DNA barcoding. In this context, we present VIP Barcoding: a user‐friendly software in graphical user interface for rapid DNA barcoding. It adopts a hybrid, two‐stage algorithm. First, an alignment‐free composition vector (CV) method is utilized to reduce searching space by screening a reference database. The alignment‐based K2P distance nearest‐neighbour method is then employed to analyse the smaller data set generated in the first stage. In comparison with other software, we demonstrate that VIP Barcoding has (i) higher accuracy than Blastn and several alignment‐free methods and (ii) higher scalability than alignment‐based distance methods and character‐based methods. These results suggest that this platform is able to deal with both large‐scale and multilocus barcoding data with accuracy and can contribute to DNA barcoding for modern taxonomy. VIP Barcoding is free and available at http://msl.sls.cuhk.edu.hk/vipbarcoding/ .     

DNA barcoding in medicinal plants: Testing the potential of a proposed barcoding marker for identification of Uncaria species from China

DNA barcoding, an increasingly popular mean of species identification, has been widely used for global species identification despite a consensus not being reached regarding which DNA sequences can be used as the best plant barcodes. In this study, we tested the feasibility of five candidate DNA barcodes (nrITS, nrITS2, matk, rbcL and trnH-psbA) for identifying Uncaria species. We collected a total of 54 specimens of 10 Uncaria species across its distributional range. BLAST, barcoding gaps, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capability of the candidate DNA barcodes. The results showed that the ITS2 is most suitable as a candidate DNA barcode for identification of medicinal plants of the genus Uncaria.     

Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes

This report describes a set of 21 polymerase chain reaction primers and amplification conditions developed to barcode practically any teleost fish species according to their mitochondrial cytochrome b and nuclear rhodopsin gene sequences. The method was successfully tested in more than 200 marine fish species comprising the main Actinopterygii family groups. When used in phylogenetic analyses, its combination of two genes with different evolutionary rates serves to identify fish at the species level. We provide a flow diagram indicating our validated polymerase chain reaction amplification conditions for barcoding and species identification applications as well as population structure or haplotyping analyses, adaptable to high‐throughput analyses.     

植物DNA条形码研究领域文献计量学及可视化分析

为全面了解植物DNA条形码研究领域的发展和最新动态,探讨中国DNA条形码发展的状态和前景,该文利用Web of Science数据库对该研究领域进行文献计量学统计,并对引用频次、研究热点和研究前沿进行了可视化分析。结果表明:(1)中国、美国、加拿大学者在该领域文献贡献率最大,中国研究机构发文量领先,但美国、加拿大科研机构论文质量较高,影响力较大。(2) 2009年是该领域研究的高峰期,该研究领域的前沿和研究热点主要集中在物种的识别和生物多样性应用、DNA条形码候选序列筛选和鉴定技术的规范化。(3)中国学者在植物DNA条形码领域研究具有领军作用和很高的影响力,国家提倡中药产业的发展也推动了我国DNA条形码蓬勃发展,但论文的质量和影响力与美国、英国、加拿大等发达国家研究还有一定差距,应加大与发达国家科研机构合作,提高研究能力,DNA条形码技术在植物的鉴定、分类和生物多样性的保护起到非常重要的作用。这表明建立一个更全面、通用的全球植物DNA条码库以及开发新的标记并采用新的测序技术是植物DNA条形码研究的未来前景。     

Molecular systematics and biogeography of Crawfurdia, Metagentiana and Tripterospermum (Gentianaceae) based on nuclear ribosomal and plastid DNA sequences

BACKGROUND AND AIMS: The systematic position of the genus Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae. The aim of this paper is to examine the systematic position of Crawfurdia, Metagentiana and Tripterospermum and to clarify their phylogenetic affinities more clearly using ITS and trnL intron sequences. METHODS: Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the plastid DNA trnL (UAA) intron were analysed phylogenetically. Ten of fourteen Metagentiana species were sampled, together with 40 species of other genera in the subtribe Gentianinae. KEY RESULTS: The data support several previously published conclusions relating to the separation of Metagentiana from Gentiana and its closer relationships to Crawfurdia and Tripterospermum based on studies of gross morphology, floral anatomy, chromosomes, palynology, embryology and previous molecular data. The molecular clock hypothesis for the tested sequences in subtribe Gentianinae was not supported by the data (P < 0.05), so the clock-independent non-parametric rate smoothing method was used to estimate divergence time. This indicates that the separation of Crawfurdia, Metagentiana and Tripterospermum from Gentiana occurred about 11.4-21.4 Mya (million years ago), and the current species of these three genera diverged at times ranging from 0.4 to 6.2 Mya. CONCLUSIONS: The molecular analyses revealed that Crawfurdia, Metagentiana and Tripterospermum do not merit status as three separate genera, because sampled species of Crawfurdia and Tripterospermum are embedded within Metagentiana. The speciation and rapid radiation of these three genera is likely to have occurred in western China as a result of upthrust of the Himalayas during the late Miocene and the Pleistocene.     

Chloroplast DNA phylogeography of the argan tree of Morocco

Polymorphisms in the chloroplast genome of the argan tree (Sapotaceae), an endemic species of south-western Morocco, have been detected by restriction site studies of PCR-amplified fragments. A total of 12 chloroplast DNA (cpDNA) and two mitochondrial DNA (mtDNA) fragments were amplified and digested with a single restriction enzyme ( Hin fI). Polymorphisms were identified in six of the cpDNA fragments, whereas no mtDNA polymorphisms were detected in a survey of 95 individuals from 19 populations encompassing most of the natural range of the species. The cpDNA polymorphisms allowed the identification of 11 haplotypes. Two lineages, one in the south-east and the other in the north-west, divide the range of the argan tree into two distinct areas. The level of genetic differentiation measured at the haplotype level ( G _STc= 0.60) (i.e. with unordered haplotypes) was smaller than when phylogenetic relationships were taken into account ( N _STc= 0.71–0.74) (ordered haplotypes), indicating that population history must be considered in the study of the geographical distribution of cpDNA lineages in this species. If contrasted with the level of nuclear genetic differentiation measured in a previous study with isozymes ( G _STn= 0.25), the results indicate a relatively high level of gene flow by seeds, or conversely a relatively low level of gene flow by pollen, as compared with other tree species. Goats and camels could have played an important role in disseminating the fruits of this tree.     

DNA metabarcoding multiplexing and validation of data accuracy for diet assessment: application to omnivorous diet

Ecological understanding of the role of consumer–resource interactions in natural food webs is limited by the difficulty of accurately and efficiently determining the complex variety of food types animals have eaten in the field. We developed a method based on DNA metabarcoding multiplexing and next‐generation sequencing to uncover different taxonomic groups of organisms from complex diet samples. We validated this approach on 91 faeces of a large omnivorous mammal, the brown bear, using DNA metabarcoding markers targeting the plant, vertebrate and invertebrate components of the diet. We included internal controls in the experiments and performed PCR replication for accuracy validation in postsequencing data analysis. Using our multiplexing strategy, we significantly simplified the experimental procedure and accurately and concurrently identified different prey DNA corresponding to the targeted taxonomic groups, with ≥60% of taxa of all diet components identified to genus/species level. The systematic application of internal controls and replication was a useful and simple way to evaluate the performance of our experimental procedure, standardize the selection of sequence filtering parameters for each marker data and validate the accuracy of the results. Our general approach can be adapted to the analysis of dietary samples of various predator species in different ecosystems, for a number of conservation and ecological applications entailing large‐scale population level diet assessment through cost‐effective screening of multiple DNA metabarcodes, and the detection of fine dietary variation among samples or individuals and of rare food items.     

Discriminating plants using the DNA barcode rbcLb: an appraisal based on a large data set

The ideal DNA barcode for plants remains to be discovered, and the candidate barcode rbcL has been met with considerable skepticism since its proposal. In fact, the variability within this gene has never been fully explored across all plant groups from algae to flowering plants, and its performance as a barcode has not been adequately tested. By analysing all of the rbcL sequences currently available in GenBank, we attempted to determine how well a region of rbcL performs as a barcode in species discrimination. We found that the rbcLb region was more variable than the frequently used rbcLa region. Both universal and plant group‐specific primers were designed to amplify rbcLb, and the performance of rbcLa and rbcLb was tested in several ways. Using blast , both regions successfully identified all families and nearly all genera; however, the successful species identification rates varied significantly among plant groups, ranging from 24.58% to 85.50% for rbcLa and from 36.67% to 90.89% for rbcLb. Successful species discrimination ranged from 5.19% to 96.33% for rbcLa and from 22.09% to 98.43% for rbcLb in species‐rich families, and from 0 to 88.73% for rbcLa and from 2.04% to 100% for rbcLb in species‐rich genera. Both regions performed better for lower plants than for higher plants, although rbcLb performed significantly better than rbcLa overall, particularly for angiosperms. Considering the applicability across plants, easy and unambiguous alignment, high primer universality, high sequence quality and high species discrimination power for lower plants, we suggest rbcLb as a universal plant barcode.     

Application of DNA barcoding in Roscoea (Zingiberaceae) and a primary discussion on taxonomic status of Roscoea cautleoides var. pubescens

DNA barcoding is a useful tool to define operational taxonomic units based on standardized DNA regions. In this study, three chloroplast markers (rbcL, trnH-psbA and matK) and one nrDNA marker (ITS) were tested for species identification in Roscoea. The ITS and trnH-psbA regions showed high success rate of PCR amplification and bidirectional sequencing, as well as perfect discriminatory ability. On the contrary, rbcL possessed no genetic variation and matK was relatively difficult in PCR amplification and DNA sequencing. Combination of multiple markers greatly improved identification ability of DNA barcoding. ITS + trnH-psbA could effectively discriminate 90% species based on method of the Neighbor-Joining (NJ) tree. Awful PCR amplification and DNA sequencing of matK restricted its efficiency, although it showed rich genetic variability in Roscoea. Moreover, it might be more appropriate to treat Roscoea cautleoides var. pubescens as an independent species based on molecular data, namely R. pubescens Z. Y. Zhu.     

动物食性分析在生态学中的应用研究进展——基于DNA宏条形码技术

动物食性分析是动物营养生态学的重要研究手段,可用于解析动物与环境因素的关联性、捕食者与猎物之间的关系,以及动物物种多样性等科学问题。近年来,基于新一代测序技术的DNA宏条形码技术被广泛应用到生态学多个研究领域,极大地促进了生命科学交叉学科的发展。其中,DNA宏条形码技术在动物食性分析中具有高分辨、高效率、低样本量等优势,具有重要的应用前景。综述了基于DNA宏条形码技术的动物食性分析在生态学中的应用研究进展,并进一步总结了DNA宏条形码技术原理和食性分析方法,着重探讨了基于DNA宏条形码技术的动物食性分析在珍稀濒危动物保护、生物多样性监测、农业害虫防治等生态学研究领域中的应用,并对DNA宏条形码技术在动物食性分析中存在的问题及应用前景进行小结与展望。     

An integrative approach to species discovery in odonates: from character‐based DNA barcoding to ecology

Modern taxonomy requires an analytical approach incorporating all lines of evidence into decision‐making. Such an approach can enhance both species identification and species discovery. The character‐based DNA barcode method provides a molecular data set that can be incorporated into classical taxonomic data such that the discovery of new species can be made in an analytical framework that includes multiple sources of data. We here illustrate such a corroborative framework in a dragonfly model system that permits the discovery of two new, but visually cryptic species. In the African dragonfly genus Trithemis three distinct genetic clusters can be detected which could not be identified by using classical taxonomic characters. In order to test the hypothesis of two new species, DNA‐barcodes from different sequence markers (ND1 and COI) were combined with morphological, ecological and biogeographic data sets. Phylogenetic analyses and incorporation of all data sets into a scheme called taxonomic circle highly supports the hypothesis of two new species. Our case study suggests an analytical approach to modern taxonomy that integrates data sets from different disciplines, thereby increasing the ease and reliability of both species discovery and species assignment.     

New insights on diet variability revealed by DNA barcoding and high-throughput pyrosequencing: chamois diet in autumn as a case study

Characterizing the diet of large herbivores and the determinants of its variation remains a difficult task in wild species. DNA-based techniques have the potential to complement traditional time-consuming methods based on the microhistology of plant cuticle fragments in fecal or rumen samples. Recently, it has been shown that a short chloroplast DNA fragment, the P6 loop of the trnL (UAA) intron, can act as a minimalist barcode. Here, we used the trnL approach with high-throughput pyrosequencing to study diet from feces in a wild herbivore, the alpine chamois (Rupicapra rupicapra) and showed that the fine resolution in plant determination obtained with this method allows exploring subtle temporal shifts and inter-individual variability in diet composition. First, we built a DNA barcoding database of 475 plants species. Seventy-two percent of plant species can be unambiguously identified to species level, 79% to genus level and 100% to family level using the P6 loop. Second, we analysed 74 feces collected from October to November. Based on 47,896 P6 loop sequences, we identified a total of 110 taxa, 96 in October and 76 in November, with a clear diet shift between October and November. We recognized four and two clusters of feces composition in October and November, respectively, revealing different diet categories among individuals within each month. DNA-based diet analysis is faster and more taxonomically precise than studies based on microhistology, and opens new possibilities for analysing plant-herbivore interactions in the wild.     

基于粪便DNA及宏条形码技术的食肉动物快速调查及食性分析

在陆地生态系统中, 大型食肉动物对于稳定食物网结构和生态系统功能有重要作用。在世界范围内, 由于栖息地丧失和破碎化、猎杀、人类活动干扰以及病原体的传播, 大型食肉动物生存正面临严重威胁, 多种食肉动物地理分布范围及种群数量大幅度缩减。如何有效保护大型食肉动物物种多样性及种群已经成为世界关注的焦点问题和保护生物学的重要研究方向。川西高原地处我国西南山地与青藏高原东缘交界地带, 属于世界生物多样性热点地区, 是世界大型食肉动物物种最丰富的地区之一, 而日益增强的人类活动可能会加剧对当地动植物资源的破坏, 进而威胁野生食肉动物的生存。获得准确的物种多样性信息及食肉动物食性数据有助于深入了解该地区生态系统结构及食物网关系, 对研究物种共存机制及生物多样性保护有重要意义。本研究通过从四川甘孜藏族自治州新龙县和石渠县野外采集的食肉动物粪便样品中提取DNA, 利用DNA条形码进行物种鉴定, 快速获得该地区食肉动物物种构成信息。38份粪便样品经鉴定来自于7种食肉动物, 分别为5种大型食肉动物(狼Canis lupus、棕熊Ursus arctos、豹Panthera pardus、雪豹P. unica、狗Canis lupus familiaris)和2种中小型食肉动物(豹猫Prionailurus bengalensis、赤狐Vulpes vulpes)。进一步利用高通量测序和宏条形码技术对7种食肉动物粪便中的食物DNA进行精准食性分析, 得到包含19种哺乳类、8种鸟类和1种鱼类共计28个不同的食物分子可操作分类单元(molecular operational taxonomic unit, MOTU)。结果显示, 狼、狗、棕熊最主要的食物来源为偶蹄目动物, 其中取食频率最高的物种为家牦牛(Bos grunniens); 而豹猫和赤狐食物中小型哺乳动物如啮齿目和兔形目占重要比例, 其中高原松田鼠(Neodon irene)和高原鼠兔(Ochotona curzoniae)被取食频率最高。豹和雪豹的食物分别为偶蹄目的中华斑羚(Naemorhedus griseus)和岩羊(Pseudois nayaur)。本研究显示了粪便DNA及宏条形码技术在食肉动物多样性快速调查及高通量精确食性分析中的应用前景, 并为此类研究提供了技术路线的有力借鉴。     

Chloroplast DNA evidence for the evolution of Microseris (Asteraceae) in Australia and New Zealand after long-distance dispersal from western North America

Restriction site mutations and trnL(UAA)-trnF(GAA) intergenic spacer length variants in the chloroplast genome were used to investigate the phylogenetic relationships among 53 Australian and New Zealand Microseris populations and to assess their position within their primarily North American genus. The study was performed to enhance understanding of evolutionary processes within this unique example of intercontinental dispersal and subsequent adaptive radiation. A southern blot method using four-base restriction enzymes and fragment separation on polyacryamide gels resulted in 55 mutations of which 30 were potentially phylogenetically informative. Most mutations were small indels of <162 bp, 80% of which were <20 bp. The small indels were useful for phylogenetic reconstruction of Australasian Microseris as judged by the high consistency indexes. The results confirmed the monophyly of the Australian and New Zealand Microseris. The occurrence of “hard” basal polytomies in the most parsimonious trees indicated that rapid radiation has occurred early in the history of the taxon. The monophyly of M. lanceolata, which includes the self-incompatible ecotypes of the Australian mainland, was confirmed. Within this species three clades were found that reflect more geographic distribution than morphological entities, suggesting that migration and possibly introgression between different ecotypes, or parallel evolution of similar adaptations, has occurred. One of the three clades was supported by a 162-bp deletion in the trnL-trnF spacer, while a subgroup of this exhibited also a tandemly repeated trnF exon. The data were inconclusive about the monophyly of the second Australasian species, M. scapigera, which comprises the New Zealand, Tasmanian, and autofertile ecotypes of Australia.