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1.
    
Little is known about the diversity patterns of plant pathogens and how they change with land use at a broad scale. We employed DNA metabarcoding to describe the diversity and composition of putative plant pathogen communities in three substrates (soil, roots, and leaves) across five major land uses at a national scale. Almost all plant pathogen communities (fungi, oomycetes, and bacteria) showed strong responses to land use and substrate type. Land use category could explain up to 24% of the variance in composition between communities. Alpha‐diversity (richness) of plant pathogens was consistently lower in natural forests than in agricultural systems. In planted forests, there was also generally low pathogen alpha‐diversity in soil and roots, but alpha‐diversity in leaves was high compared with most other land uses. In contrast to alpha‐diversity, differences in within‐land use beta‐diversity of plant pathogens (the predictability of plant pathogen communities within land use) were subtle. Our results show that large‐scale patterns and distributions of putative plant pathogens can be determined using metabarcoding, allowing some of the first landscape level insights into these critically important communities.  相似文献   

2.
    
Fungi are a key component of tropical biodiversity. However, due to their inconspicuous and largely subterranean nature, they are usually neglected in biodiversity inventories. The goal of this study was to identify the key determinants of fungal richness, community composition, and turnover in tropical rainforests. We tested specifically for the effect of soil properties, habitat, and locality in Amazonia. For these analyses, we used high‐throughput sequencing data of short and long reads of fungal DNA present in soil and organic litter samples, combining existing and novel genomic data. Habitat type (phytophysiognomy) emerges as the strongest factor explaining fungal community composition. Naturally open areas—campinas—are the richest habitat overall. Soil properties have different effects depending on the soil layer (litter or mineral soil) and the choice of genetic marker. We suggest that campinas could be a neglected hotspot of fungal diversity. An underlying cause for their rich diversity may be the overall low soil fertility, which increases the reliance on biotic interactions essential for nutrient absorption in these environments, notably ectomycorrhizal fungi–plant associations. Our results highlight the advantages of using both short and long DNA reads produced through high‐throughput sequencing to characterize fungal diversity. While short reads can suffice for diversity and community comparison, long reads add taxonomic precision and have the potential to reveal population diversity.  相似文献   

3.
    
DNA metabarcoding is an efficient method for measuring biodiversity, but the process of initiating long‐term DNA‐based monitoring programmes, or integrating with conventional programs, is only starting. In marine ecosystems, plankton surveys using the continuous plankton recorder (CPR) have characterized biodiversity along transects covering millions of kilometres with time‐series spanning decades. We investigated the potential for use of metabarcoding in CPR surveys. Samples (n = 53) were collected in two Southern Ocean transects and metazoans identified using standard microscopic methods and by high‐throughput sequencing of a cytochrome c oxidase subunit I marker. DNA increased the number of metazoan species identified and provided high‐resolution taxonomy of groups problematic in conventional surveys (e.g., larval echinoderms and hydrozoans). Metabarcoding also generally produced more detections than microscopy, but this sensitivity may make cross‐contamination during sampling a problem. In some samples, the prevalence of DNA from large plankton such as krill masked the presence of smaller species. We investigated adding a fixed amount of exogenous DNA to samples as an internal control to allow determination of relative plankton biomass. Overall, the metabarcoding data represent a substantial shift in perspective, making direct integration into current long‐term time‐series challenging. We discuss a number of hurdles that exist for progressing DNA metabarcoding from the current snapshot studies to the requirements of a long‐term monitoring programme. Given the power and continually increasing efficiency of metabarcoding, it is almost certain this approach will play an important role in future plankton monitoring.  相似文献   

4.
    
Environmental DNA (eDNA) techniques refer to utilizing the organisms’ DNA extracted from environment samples to genetically identify target species without capturing actual organisms. eDNA metabarcoding via high‐throughput sequencing can simultaneously detect multiple fish species from a single water sample, which is a powerful tool for the qualitative detection and quantitative estimates of multiple fish species. However, sequence counts obtained from eDNA metabarcoding may be influenced by many factors, of which primer bias is one of the foremost causes of methodological error. The performance of 18 primer pairs for COI, cytb, 12S rRNA, and 16S rRNA mitochondrial genes, which are all frequently used in fish eDNA metabarcoding, were evaluated in the current study. The ribosomal gene markers performed better than the protein‐coding gene markers during in silico screening, resulting in higher taxonomic coverage and appropriate barcode lengths. Four primer pairs—AcMDB07, MiFish‐U, Ve16S1, and Ve16S3—designed for various regions of the 12S and 16S rRNA genes were screened for tank metabarcoding in a case study targeting six freshwater fish species. The four primer pairs were able to accurately detect all six species in different tanks, while only MiFish‐U, Ve16S1, and Ve16S3 revealed a significant positive relationship between species biomass and read count for the pooled tank data. The positive relationship could not be found in all species within the tanks. Additionally, primer efficiency differed depending on the species while primer preferential species varied in different fish assemblages. This case study supports the potential for eDNA metabarcoding to assess species diversity in natural ecosystems and provides an alternative strategy to evaluate the performance of candidate primers before application of eDNA metabarcoding in natural ecosystems.  相似文献   

5.
    
High‐throughput sequencing methods have become a routine analysis tool in environmental sciences as well as in public and private sector. These methods provide vast amount of data, which need to be analysed in several steps. Although the bioinformatics may be applied using several public tools, many analytical pipelines allow too few options for the optimal analysis for more complicated or customized designs. Here, we introduce PipeCraft, a flexible and handy bioinformatics pipeline with a user‐friendly graphical interface that links several public tools for analysing amplicon sequencing data. Users are able to customize the pipeline by selecting the most suitable tools and options to process raw sequences from Illumina, Pacific Biosciences, Ion Torrent and Roche 454 sequencing platforms. We described the design and options of PipeCraft and evaluated its performance by analysing the data sets from three different sequencing platforms. We demonstrated that PipeCraft is able to process large data sets within 24 hr. The graphical user interface and the automated links between various bioinformatics tools enable easy customization of the workflow. All analytical steps and options are recorded in log files and are easily traceable.  相似文献   

6.
刘山林 《生物多样性》2019,27(5):526-367
近年来DNA条形码技术迅速发展, 产生的条形码的数量及其应用范围都呈指数性增长, 现已广泛用于物种鉴定、食性分析、生物多样性评估等方面。本文重点总结并讨论了构建条形码参考数据库和序列聚类相关的信息分析的技术和方法, 包括: 基于高通量测序(high throughput sequencing, HTS)平台以高效并较低的成本获取条形码序列的方法; 同时还介绍了从原始测序序列到分类操作单元(operational taxonomic units, OTUs)过程中的一些计算逻辑以及被广泛采用的软件和技术。这是一个较新并快速发展的领域, 我们希望本文能为读者提供一个梗概, 了解DNA条形码技术在生物多样性研究应用中的方法和手段。  相似文献   

7.
    
Metabarcoding has been used in a range of ecological applications such as taxonomic assignment, dietary analysis and the analysis of environmental DNA. However, after a decade of use in these applications there is little consensus on the extent to which proportions of reads generated corresponds to the original proportions of species in a community. To quantify our current understanding, we conducted a structured review and meta‐analysis. The analysis suggests that a weak quantitative relationship may exist between the biomass and sequences produced (slope = 0.52 ± 0.34, p < 0.01), albeit with a large degree of uncertainty. None of the tested moderators, sequencing platform type, the number of species used in a trial or the source of DNA, were able to explain the variance. Our current understanding of the factors affecting the quantitative performance of metabarcoding is still limited: additional research is required before metabarcoding can be confidently utilized for quantitative applications. Until then, we advocate the inclusion of mock communities when metabarcoding as this facilitates direct assessment of the quantitative ability of any given study.  相似文献   

8.
    
As molecular tools for assessing trophic interactions become common, research is increasingly focused on the construction of interaction networks. Here, we demonstrate three key methods for incorporating DNA data into network ecology and discuss analytical considerations using a model consisting of plants, insects, bats and their parasites from the Costa Rica dry forest. The simplest method involves the use of Sanger sequencing to acquire long sequences to validate or refine field identifications, for example of bats and their parasites, where one specimen yields one sequence and one identification. This method can be fully quantified and resolved and these data resemble traditional ecological networks. For more complex taxonomic identifications, we target multiple DNA loci, for example from a seed or fruit pulp sample in faeces. These networks are also well resolved but gene targets vary in resolution and quantification is difficult. Finally, for mixed templates such as faecal contents of insectivorous bats, we use DNA metabarcoding targeting two sequence lengths (157 and 407 bp) of one gene region and a MOTU, BLAST and BIN association approach to resolve nodes. This network type is complex to generate and analyse, and we discuss the implications of this type of resolution on network analysis. Using these data, we construct the first molecular‐based network of networks containing 3,304 interactions between 762 nodes of eight trophic functions and involving parasitic, mutualistic and predatory interactions. We provide a comparison of the relative strengths and weaknesses of these data types in network ecology.  相似文献   

9.
    
  1. The objective of this study was to assess barcoding of environmental DNA as a method for monitoring invertebrate ecosystem service providers in soil samples.
  2. We selected 26 invertebrate ecosystem service providers that occur in New Zealand kiwifruit or apple orchards and produced mitochondrial cytochrome c oxidase gene subunit I (cytochrome oxidase I) and/or 28S ribosomal DNA sequences for each. Specific barcode primers were designed for each invertebrate ecosystem service provider and tested, along with generic barcoding cytochrome oxidase I primers, for their ability to detect DNA from invertebrate ecosystem service providers that had been added to sterilized and unsterilized soil samples.
  3. Although the specific primers accurately detected the invertebrate ecosystem service providers in more than 96% of the samples, the generic cytochrome oxidase I primers detected only 37% of the invertebrate ecosystem service providers added to the sterilized samples and 2.5% in the unsterilized samples.
  4. In a field test, we compared metabarcoding with traditional invertebrate trapping methods to detect the invertebrate ecosystem service providers in 10 kiwifruit and 10 apple orchards. All invertebrate ecosystem service providers were collected in traps in at least one orchard, but very few were identified by metabarcoding of soil environmental DNA.
  5. Although the specific primers can be used as a tool for monitoring invertebrate ecosystem service providers in soil samples, methodological improvements are needed before metabarcoding of soil environmental DNA can be used to monitor these taxa.
  相似文献   

10.
    
The purpose of this review is to present the most common and emerging DNA‐based methods used to generate data for biodiversity and biomonitoring studies. As environmental assessment and monitoring programmes may require biodiversity information at multiple levels, we pay particular attention to the DNA metabarcoding method and discuss a number of bioinformatic tools and considerations for producing DNA‐based indicators using operational taxonomic units (OTUs), taxa at a variety of ranks and community composition. By developing the capacity to harness the advantages provided by the newest technologies, investigators can “scale up” by increasing the number of samples and replicates processed, the frequency of sampling over time and space, and even the depth of sampling such as by sequencing more reads per sample or more markers per sample. The ability to scale up is made possible by the reduced hands‐on time and cost per sample provided by the newest kits, platforms and software tools. Results gleaned from broad‐scale monitoring will provide opportunities to address key scientific questions linked to biodiversity and its dynamics across time and space as well as being more relevant for policymakers, enabling science‐based decision‐making, and provide a greater socio‐economic impact. As genomic approaches are continually evolving, we provide this guide to methods used in biodiversity genomics.  相似文献   

11.
    
The diversity of centrohelids in inland saline waters was studied with metabarcoding for the first time. The fragment of V6–V7 regions of 18S rDNA was sequenced with newly designed primers. Obtained OTUs were identified with molecular phylogenetic analysis and comparison of the signatures in 39es9 hairpin of V7. The obtained data included some OTUs, which could be attributed to four described species, but the majority belonged to previously established or novel environmental clades. Along with some presumably marine/brackish clades and freshwater/low salinity (0–2 ppt) clades, seven presumable species demonstrating broad (from 1–2 up to 78 ppt) salinity tolerance were detected. A number of OTUs belonged to Raphidocystis contractilis, which is known from three independent findings in brackish habitats only. Thus, it was assumed that this species is stenohaline and specifically adapted to salinity 5–15 ppt. The high level of salinity tolerance was suggested for centrohelids before based on morphology, which was used to justify their cosmopolitan distribution. Later these views were criticized based on environmental sequencing, but the results of the current survey indicate, that at least some species are present at salinities from almost freshwater (1–2 ppt) to twice oceanic (78 ppt) and are presumably capable of overcoming oceanic salinity barriers for their distribution.  相似文献   

12.
    
During the most recent decade, environmental DNA metabarcoding approaches have been both developed and improved to minimize the biological and technical biases in these protocols. However, challenges remain, notably those relating to primer design. In the current study, we comprehensively assessed the performance of ten COI and two 16S primer pairs for eDNA metabarcoding, including novel and previously published primers. We used a combined approach of in silico, in vivo‐mock community (33 arthropod taxa from 16 orders), and guano‐based analyses to identify primer sets that would maximize arthropod detection and taxonomic identification, successfully identify the predator (bat) species, and minimize the time and financial costs of the experiment. We focused on two insectivorous bat species that live together in mixed colonies: the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy's bat (Myotis emarginatus). We found that primer degeneracy is the main factor that influences arthropod detection in silico and mock community analyses, while amplicon length is critical for the detection of arthropods from degraded DNA samples. Our guano‐based results highlight the importance of detecting and identifying both predator and prey, as guano samples can be contaminated by other insectivorous species. Moreover, we demonstrate that amplifying bat DNA does not reduce the primers' capacity to detect arthropods. We therefore recommend the simultaneous identification of predator and prey. Finally, our results suggest that up to one‐third of prey occurrences may be unreliable and are probably not of primary interest in diet studies, which may decrease the relevance of combining several primer sets instead of using a single efficient one. In conclusion, this study provides a pragmatic framework for eDNA primer selection with respect to scientific and methodological constraints.  相似文献   

13.
    
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14.
    
Environmental DNA (eDNA) extracted from water samples has recently shown potential as a valuable source of population genetic information for aquatic macroorganisms. This approach offers several potential advantages compared with conventional tissue‐based methods, including the fact that eDNA sampling is noninvasive and generally more cost‐efficient. Currently, eDNA approaches have been limited to single‐marker studies of mitochondrial DNA (mtDNA), and the relationship between eDNA haplotype composition and true haplotype composition still needs to be thoroughly verified. This will require testing of bioinformatic and statistical software to correct for erroneous sequences, as well as biases and random variation in relative sequence abundances. However, eDNA‐based population genetic methods have far‐reaching potential for both basic and applied research. In this paper, we present a brief overview of the achievements of eDNA‐based population genetics to date, and outline the prospects for future developments in the field, including the estimation of nuclear DNA (nuDNA) variation and epigenetic information. We discuss the challenges associated with eDNA samples as opposed to those of individual tissue samples and assess whether eDNA might offer additional types of information unobtainable with tissue samples. Lastly, we provide recommendations for determining whether an eDNA approach would be a useful and suitable choice in different research settings. We limit our discussion largely to contemporary aquatic systems, but the advantages, challenges, and perspectives can to a large degree be generalized to eDNA studies with a different spatial and temporal focus.  相似文献   

15.
    
Next‐generation sequencing (NGS) is increasingly used for diet analyses; however, it may not always describe diet samples well. A reason for this is that diet samples contain mixtures of food DNA in different amounts as well as consumer DNA which can reduce the food DNA characterized. Because of this, detections will depend on the relative amount and identity of each type of DNA. For such samples, diagnostic PCR will most likely give more reliable results, as detection probability is only marginally dependent on other copresent DNA. We investigated the reliability of each method to test (a) whether predatory beetle regurgitates, supposed to be low in consumer DNA, allow to retrieve prey sequences using general barcoding primers that co‐amplify the consumer DNA, and (b) to assess the sequencing depth or replication needed for NGS and diagnostic PCR to give stable results. When consumer DNA is co‐amplified, NGS is better suited to discover the range of possible prey, than for comparing co‐occurrences of diet species between samples, as retested samples were repeatedly different in prey detections with this approach. This shows that samples were incompletely described, as prey detected by diagnostic PCR frequently were missed by NGS. As the sequencing depth needed to reliably describe the diet in such samples becomes very high, the cost‐efficiency and reliability of diagnostic PCR make diagnostic PCR better suited for testing large sample‐sets. Especially if the targeted prey taxa are thought to be of ecological importance, as diagnostic PCR gave more nested and consistent results in repeated testing of the same sample.  相似文献   

16.
    
The biodiversity and biogeography of protists inhabiting many ecosystems have been intensely studied using different sequencing approaches, but tropical ecosystems are relatively under‐studied. Here, we sampled planktonic waters from 32 lakes associated with four different river–floodplains systems in Brazil, and sequenced the DNA using a metabarcoding approach with general eukaryotic primers. The lakes were dominated by the largely free‐living Discoba (mostly the Euglenida), Ciliophora, and Ochrophyta. There was low community similarity between lakes even within the same river–floodplain. The protists inhabiting these floodplain systems comprise part of the large and relatively undiscovered diversity in the tropics.  相似文献   

17.
    
With the continual improvement in high‐throughput sequencing technology and constant updates to fungal reference databases, the use of amplicon‐based DNA markers as a tool to reveal fungal diversity and composition in various ecosystems has become feasible. However, both primer selection and the experimental procedure require meticulous verification. Here, we computationally and experimentally evaluated the accuracy and specificity of three widely used or newly designed internal transcribed spacer (ITS) primer sets (ITS1F/ITS2, gITS7/ITS4 and 5.8S‐Fun/ITS4‐Fun). In silico evaluation revealed that primer coverage varied at different taxonomic levels due to differences in degeneracy and the location of primer sets. Using even and staggered mock community standards, we identified different proportions of chimeric and mismatch reads generated by different primer sets, as well as great variation in species abundances, suggesting that primer selection would affect the results of amplicon‐based metabarcoding studies. Choosing proofreading and high‐fidelity polymerase (KAPA HiFi) could significantly reduce the percentage of chimeric and mismatch sequences, further reducing inflation of operational taxonomic units. Moreover, for two types of environmental fungal communities, plant endophytic and soil fungi, it was demonstrated that the three primer sets could not reach a consensus on fungal community composition or diversity, and that primer selection, not experimental treatment, determines observed soil fungal community diversity and composition. Future DNA marker surveys should pay greater attention to potential primer effects and improve the experimental scheme to increase credibility and accuracy.  相似文献   

18.
    
  1. We present a performance evaluation of environmental DNA (eDNA) metabarcoding with MiFish‐U/E primers to investigate local and regional diversities of stream fish species to examine potential effectiveness, limits and future remedies of this technique in large‐scale monitoring. We hypothesised that eDNA inferences are more consistent with fish assemblages observed upstream than downstream due to a directional flow of river water.
  2. River water was sampled at 102 sites in 51 rivers around Lake Biwa in the central part of Honshu Island, Japan, within 10 person‐days, and fish species compositions inferred from eDNA and existing observational data were compared. Observation sites were chosen from the observational data that were within a certain distance (buffer range) of a water‐sampling site along a river trajectory. The hypothesis of the detection bias of eDNA towards upstream assemblage was tested by comparing results with all of the observational data, data from a higher elevation and data from a lower elevation. The Jaccard dissimilarity index was plotted between the observational data and the eDNA estimates against the buffer range; the buffer range with minimum dissimilarity was chosen.
  3. When using existing observational data from within 6 km upstream of the eDNA sampling sites, the eDNA results were the most consistent with the observational data and inferred 86.4% of the species reported (38/44), as well as two additional species. eDNA results also showed patterns consistent with known upstream–downstream turnover of related species and biogeographical assemblage patterns of certain species.
  4. Our 10‐person‐days survey using the metabarcoding technique enabled us to obtain as much regional fish diversity data including the hypothesised pattern of eDNA detection with an upstream bias as the accumulated observational data obtained through greater amounts of time, money and labour. The problems regarding false‐positive/negative detection were suggested in our survey; however, these should be decreased or removed by modifying the sampling methods and experimental procedures in future works. Therefore, we concluded this new tool to enable monitoring that has never been implemented, such as cross‐nation, and even whole‐Earth monitoring with the data at yearly, seasonal or finer temporal scales.
  相似文献   

19.
    
Genetic taxonomic assignment can be more sensitive than morphological taxonomic assignment, particularly for small, cryptic or rare species. Sequence processing is essential to taxonomic assignment, but can also produce errors because optimal parameters are not known a priori. Here, we explored how sequence processing parameters influence taxonomic assignment of 18S sequences from bulk zooplankton samples produced by 454 pyrosequencing. We optimized a sequence processing pipeline for two common research goals, estimation of species richness and early detection of aquatic invasive species (AIS), and then tested most optimal models’ performances through simulations. We tested 1,050 parameter sets on 18S sequences from 20 AIS to determine optimal parameters for each research goal. We tested optimized pipelines’ performances (detectability and sensitivity) by computationally inoculating sequences of 20 AIS into ten bulk zooplankton samples from ports across Canada. We found that optimal parameter selection generally depends on the research goal. However, regardless of research goal, we found that metazoan 18S sequences produced by 454 pyrosequencing should be trimmed to 375–400 bp and sequence quality filtering should be relaxed (1.5 ≤ maximum expected error ≤ 3.0, Phred score = 10). Clustering and denoising were only viable for estimating species richness, because these processing steps made some species undetectable at low sequence abundances which would not be useful for early detection of AIS. With parameter sets optimized for early detection of AIS, 90% of AIS were detected with fewer than 11 target sequences, regardless of whether clustering or denoising was used. Despite developments in next‐generation sequencing, sequence processing remains an important issue owing to difficulties in balancing false‐positive and false‐negative errors in metabarcoding data.  相似文献   

20.
Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys.  相似文献   

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