The extraction and characterization of DNA from aquatic environmental samples offers an alternative, noninvasive approach for the detection of rare species. Environmental DNA, coupled with PCR and next-generation sequencing ("metabarcoding"), has proven to be very sensitive for the detection of rare aquatic species. Our study used a custom-designed group-specific primer set and next-generation sequencing for the detection of three species at risk (Eastern Sand Darter, Ammocrypta pellucida; Northern Madtom, Noturus stigmosus; and Silver Shiner, Notropis photogenis), one invasive species (Round Goby, Neogobius melanostomus) and an additional 78 native species from two large Great Lakes tributary rivers in southern Ontario, Canada: the Grand River and the Sydenham River. Of 82 fish species detected in both rivers using capture-based and eDNA methods, our eDNA method detected 86.2% and 72.0% of the fish species in the Grand River and the Sydenham River, respectively, which included our four target species. Our analyses also identified significant positive and negative species co-occurrence patterns between our target species and other identified species. Our results demonstrate that eDNA metabarcoding that targets the fish community as well as individual species of interest provides a better understanding of factors affecting the target species spatial distribution in an ecosystem than possible with only target species data. Additionally, eDNA is easily implemented as an initial survey tool, or alongside capture-based methods, for improved mapping of species distribution patterns.© 2017 John Wiley & Sons Ltd.

The commonly observed high diversity of trees in tropical rain forests and corals on tropical reefs is a nonequilibrium state which, if not disturbed further, will progress toward a low-diversity equilibrium community. This may not happen if gradual changes in climate favor different species. If equilibrium is reached, a lesser degree of diversity may be sustained by niche diversification or by a compensatory mortality that favors inferior competitors. However, tropical forests and reefs are subject to severe disturbances often enough that equilibrium may never be attained.

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High-throughput sequencing ("HTS") platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed "environmental DNA" or "eDNA"). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called "eDNA metabarcoding" and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.© 2017 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Freshwater fauna are particularly sensitive to environmental change and disturbance. Management agencies frequently use fish and amphibian biodiversity as indicators of ecosystem health and a way to prioritize and assess management strategies. Traditional aquatic bioassessment that relies on capture of organisms via nets, traps and electrofishing gear typically has low detection probabilities for rare species and can injure individuals of protected species. Our objective was to determine whether environmental DNA (eDNA) sampling and metabarcoding analysis can be used to accurately measure species diversity in aquatic assemblages with differing structures. We manipulated the density and relative abundance of eight fish and one amphibian species in replicated 206-L mesocosms. Environmental DNA was filtered from water samples, and six mitochondrial gene fragments were Illumina-sequenced to measure species diversity in each mesocosm. Metabarcoding detected all nine species in all treatment replicates. Additionally, we found a modest, but positive relationship between species abundance and sequencing read abundance. Our results illustrate the potential for eDNA sampling and metabarcoding approaches to improve quantification of aquatic species diversity in natural environments and point the way towards using eDNA metabarcoding as an index of macrofaunal species abundance.© 2015 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

The extensive use of salt marsh creeks by nekton is widely recognized, yet few investigations have quantified the extent to which tidal migratory nekton make repeated visits to the same location within creek networks. An improved understanding of nekton movements and fidelity can improve insights into the nursery function and value of creek habitats. We conducted tag-recapture experiments with young fishes to determine movements among intertidal creeks in the North Inlet estuary, South Carolina. In 2008, 3246 fishes were tagged with coded microwire tags. In 2012, 577 fishes were tagged with passive integrated transponder (PIT) tags. Based on percentages of recaptures, creek-specific fidelity was determined for spot (Leiostomus xanthurus) (78%), silver perch (Bairdiella chrysoura) (71%), pinfish (Lagodon rhomboides) (80%), and mullets (Mugil curema and Mugil cephalus) (77%). Recaptures occurred up to 79days after release. Rates of recapture of all tagged fishes combined varied among creeks (74% to 100%). Considering earlier research that demonstrated differences in habitat quality among local intertidal creeks, we suggest that differences in fidelity reflect differences in habitat quality among creeks. Greater rates of repeated use (higher fidelity) could help explain why some creeks consistently support more tidal migratory nekton than other creeks. Changes in the strength of fidelity over time could be a useful measure of changes in habitat quality. We suggest that site fidelity be considered as another metric to use in efforts by resource managers to identify and evaluate essential fish habitat.

1. Despite implications for top-down and bottom-up control and the stability of food webs, understanding the links between consumers and their diets remains difficult, particularly in remote tropical locations where food resources are usually abundant and variable and seasonal hydrology produces alternating patterns of connectivity and isolation. 2. We used a large scale survey of freshwater biota from 67 sites in three catchments (Daly River, Northern Territory; Fitzroy River, Western Australia; and the Mitchell River, Queensland) in Australia's wet-dry tropics and analysed stable isotopes of carbon (δ(13) C) to search for broad patterns in resource use by consumers in conjunction with known and measured indices of connectivity, the duration of floodplain inundation, and dietary choices (i.e. stomach contents of fish). 3. Regression analysis of biofilm δ(13) C against consumer δ(13) C, as an indicator of reliance on local food sources (periphyton and detritus), varied depending on taxa and catchment. 4. The carbon isotope ratios of benthic invertebrates were tightly coupled to those of biofilm in all three catchments, suggesting assimilation of local resources by these largely nonmobile taxa. 5. Stable C isotope ratios of fish, however, were less well-linked to those of biofilm and varied by catchment according to hydrological connectivity; the perennially flowing Daly River with a long duration of floodplain inundation showed the least degree of coupling, the seasonally flowing Fitzroy River with an extremely short flood period showed the strongest coupling, and the Mitchell River was intermediate in connectivity, flood duration and consumer-resource coupling. 6. These findings highlight the high mobility of the fish community in these rivers, and how hydrological connectivity between habitats drives patterns of consumer-resource coupling.© 2011 The Authors. Journal of Animal Ecology © 2011 British Ecological Society.

Complex microbial communities shape the dynamics of various environments, ranging from the mammalian gastrointestinal tract to the soil. Advances in DNA sequencing technologies and data analysis have provided drastic improvements in microbiome analyses, for example, in taxonomic resolution, false discovery rate control and other properties, over earlier methods. In this Review, we discuss the best practices for performing a microbiome study, including experimental design, choice of molecular analysis technology, methods for data analysis and the integration of multiple omics data sets. We focus on recent findings that suggest that operational taxonomic unit-based analyses should be replaced with new methods that are based on exact sequence variants, methods for integrating metagenomic and metabolomic data, and issues surrounding compositional data analysis, where advances have been particularly rapid. We note that although some of these approaches are new, it is important to keep sight of the classic issues that arise during experimental design and relate to research reproducibility. We describe how keeping these issues in mind allows researchers to obtain more insight from their microbiome data sets.

Environmental DNA (eDNA) has been used in a variety of ecological studies and management applications. The rate at which eDNA decays has been widely studied but at present it is difficult to disentangle study-specific effects from factors that universally affect eDNA degradation. To address this, a systematic review and meta-analysis was conducted on aquatic eDNA studies. Analysis revealed eDNA decayed faster at higher temperatures and in marine environments (as opposed to freshwater). DNA type (mitochondrial or nuclear) and fragment length did not affect eDNA decay rate, although a preference for < 200 bp sequences in the available literature means this relationship was not assessed with longer sequences (e.g. > 800 bp). At present, factors such as ultraviolet light, pH, and microbial load lacked sufficient studies to feature in the meta-analysis. Moving forward, we advocate researching these factors to further refine our understanding of eDNA decay in aquatic environments.This article is protected by copyright. All rights reserved.

为探究黄河三角洲盐沼土壤碳氮含量在潮沟水系中的时空分布特征,选取黄河口一条典型的潮沟系统,采集一、二、三级潮沟表层土壤,探寻土壤有机碳、总氮与土壤容重、盐度、pH等理化因子的相关关系。结果表明：土壤有机碳和总氮在时空尺度上表现出极大的异质性特征。时间尺度上,土壤有机碳和总氮出现先上升后下降的趋势。空间尺度上,一级潮沟土壤有机碳和总氮平均值（2.9 g&#x000b7;kg^-1、0.36 g&#x000b7;kg^-1）大于二级（1.4 g&#x000b7;kg^-1、0.18 g&#x000b7;kg^-1）、三级（1.6 g&#x000b7;kg^-1、0.21 g&#x000b7;kg^-1）潮沟。相关分析表明,土壤有机碳和总氮与盐度呈显著正相关（P&#x0003C;0.01）,与容重呈显著负相关（P&#x0003C;0.01）。盐沼湿地土壤碳氮含量受土壤水盐条件的影响,而潮沟水系的树状结构对水盐条件的影响是导致土壤碳氮含量时空差异分布的重要因素。

We reviewed the current methodology and practices of the DNA metabarcoding approach using a universal PCR primer pair MiFish, which co-amplifies a short fragment of fish DNA (approx. 170 bp from the mitochondrial 12S rRNA gene) across a wide variety of taxa. This method has mostly been applied to biodiversity monitoring using environmental DNA (eDNA) shed from fish and, coupled with next-generation sequencing technologies, has enabled massively parallel sequencing of several hundred eDNA samples simultaneously. Since the publication of its technical outline in 2015, this method has been widely used in various aquatic environments in and around the six continents, and MiFish primers have demonstrably outperformed other competing primers. Here, we outline the technical progress in this method over the last 5 years and highlight some case studies on marine, freshwater, and estuarine fish communities. Additionally, we discuss various applications of MiFish metabarcoding to non-fish organisms, single-species detection systems, quantitative biodiversity monitoring, and bulk DNA samples other than eDNA. By recognizing the MiFish eDNA metabarcoding strengths and limitations, we argue that this method is useful for ecosystem conservation strategies and the sustainable use of fishery resources in “ecosystem-based fishery management” through continuous biodiversity monitoring at multiple sites.

We explored the phylogenetic utility and limits of the individual and concatenated mitochondrial genes for reconstructing the higher-level relationships of teleosts, using the complete (or nearly complete) mitochondrial DNA sequences of eight teleosts (including three newly determined sequences), whose relative phylogenetic positions were noncontroversial. Maximum-parsimony analyses of the nucleotide and amino acid sequences of 13 protein-coding genes from the above eight teleosts, plus two outgroups (bichir and shark), indicated that all of the individual protein-coding genes, with the exception of ND5, failed to recover the expected phylogeny, although unambiguously aligned sequences from 22 concatenated transfer RNA (tRNA) genes (stem regions only) recovered the expected phylogeny successfully with moderate statistical support. The phylogenetic performance of the 13 protein-coding genes in recovering the expected phylogeny was roughly classified into five groups, viz. very good (ND5, ND4, COIII, COI), good (COII, cyt b), medium (ND3, ND2), poor (ND1, ATPase 6), and very poor (ND4L, ND6, ATPase 8). Although the universality of this observation was unclear, analysis of successive concatenation of the 13 protein-coding genes in the same ranking order revealed that the combined data sets comprising nucleotide sequences from the several top-ranked protein-coding genes (no 3rd codon positions) plus the 22 concatenated tRNA genes (stem regions only) best recovered the expected phylogeny, with all internal branches being supported by bootstrap values >90%. We conclude that judicious choice of mitochondrial genes and appropriate data weighting, in conjunction with purposeful taxonomic sampling, are prerequisites for resolving higher-level relationships in teleosts under the maximum-parsimony optimality criterion.Copyright 2000 Academic Press.

Environmental DNA (eDNA) consists of DNA fragments shed from organisms into the environment, and can be used to identify species presence and abundance. This study aimed to reveal the dispersion and degradation processes of eDNA in the sea. Caged fish were set off the end of a pier in Maizuru Bay, the Sea of Japan, and their eDNA was traced at sampling stations located at the cage and 10, 30, 100, 300, 600 and 1000m distances from the cage along two transect lines. Sea surface water was collected at each station at 0, 2, 4, 8, 24 and 48h after setting the cage, and again after removing the cage. Quantitative PCR analyses using a species-specific primer and probe set revealed that the target DNA was detectable while the cage was present and for up to 1h after removing the cage, but not at 2h or later. Among the 57 amplified samples, 45 (79%) were collected within 30m from the cage. These results suggest that eDNA can provide a snapshot of organisms present in a coastal marine environment.

Freshwater ecosystems are linked at various spatial and temporal scales by movements of biota adapted to life in water. We review the literature on movements of aquatic organisms that connect different types of freshwater habitats, focusing on linkages from streams and wetlands to downstream waters. Here, streams, wetlands, rivers, lakes, ponds, and other freshwater habitats are viewed as dynamic freshwater ecosystem mosaics (FEMs) that collectively provide the resources needed to sustain aquatic life. Based on existing evidence, it is clear that biotic linkages throughout FEMs have important consequences for biological integrity and biodiversity. All aquatic organisms move within and among FEM components, but differ in the mode, frequency, distance, and timing of their movements. These movements allow biota to recolonize habitats, avoid inbreeding, escape stressors, locate mates, and acquire resources. Cumulatively, these individual movements connect populations within and among FEMs and contribute to local and regional diversity, resilience to disturbance, and persistence of aquatic species in the face of environmental change. Thus, the biological connections established by movement of biota among streams, wetlands, and downstream waters are critical to the ecological integrity of these systems. Future research will help advance our understanding of the movements that link FEMs and their cumulative effects on downstream waters.

Aims: We used eDNA metabarcoding to detect fish diversity in three river systems of Beijing during summer and autumn. The aim of this study is to explore new methods applicable to monitor and protect fish diversity and study the spatio-temporal patterns of fish community in Beijing. Methods: A total of 34 sites were surveyed simultaneously using the eDNA metabarcoding and ground cage methods. The main processes of the eDNA method are water sample collection and enrichment, DNA extraction with DNeasy Tissue and Blood DNA, PCR amplification with prism of MiFish-U, high-throughput sequencing by Illumina Miseq and bioinformatics analysis. The data of eDNA metabarcoding, environmental factors and human activity were used for statistical analysis between fish communities and influencing factors. The main processes of the ground cage with three replications were placed at each site waiting 24 h to be collected for species identification, and the species with number were recorded for subsequent analysis. Results: The result showed that 55 fish species were detected by eDNA metabarcoding was higher than that captured by the ground cage (35 species). The fish community composition was dominated by Cypriniformes and Perciformes. The diversity of clearwater fish in mountain rivers is significantly higher than urban rivers. Urban rivers (the Beiyun River System) were dominated by pollution-tolerant species such as Carassius auratus, Pseudorasbora parva and Misgurnus anguillicaudatus, with a relatively homogeneous community structure. Mountain rivers (Chaobai River System and Daqing River System) are dominated by Zacco platypus, Rhynchocypris lagowskii and Opsariichthys uncirostris. The community structure of clearwater fish was affected by electrical conductivity and total dissolved solids in summer but by altitude and temperature in autumn. The correlation between the abundance of clearwater fish with environmental factors and human activities showed that the abundance of clearwater fish decreased significantly with the increase by turbidity of water and human activities, and was significant correlation with altitude and temperature. Conclusion: The study used eDNA metabarcoding to show the diversity and spatio-temporal patterns of major rivers in Beijing, with a focus on the distribution of clearwater fish. The results demonstrate the feasibility of eDNA metabarcoding for monitoring fish diversity and spatial-temporal distribution in Beijing.

使用eDNA宏条形码(eDNA metabarcoding)和地笼法检测了北京市3条水系在夏季和秋季两个季节的鱼类多样性, 旨在研究北京市鱼类群落的空间格局特征, 探索适用于北京鱼类生物多样性监测及保护的新方法。结果表明: 在北京市的34个采样点中, 利用eDNA metabarcoding共检测出鱼类55种, 显著高于传统方法所捕获的鱼类种数(35种), 鱼类组成以鲤形目和鲈形目为主。山区河流清水鱼的多样性要显著高于城区河流, 城区河流(北运河水系)群落结构较为单一, 以鲫(Carassius auratus)、麦穗鱼(Pseudorasbora parva)、泥鳅(Misgurnus anguillicaudatus)等耐污种为优势种; 山区河流(潮白河水系及大清河水系)以宽鳍鱲(Zacco platypus)、拉氏鱥(Rhynchocypris lagowskii)、马口鱼(Opsariichthys uncirostris)等为优势种。不同季节影响清水鱼群落结构的环境因子不同, 夏季主要是总溶解固体和电导率, 秋季主要是海拔和温度。清水鱼丰富度与环境因子及人类活动的相关性表明, 清水鱼的丰富度随着总溶解固体及灯光指数增加而显著降低, 且均与海拔、温度等存在显著相关性。本研究证明了eDNA metabarcoding方法用于监测北京市鱼类多样性及其时空分布的可行性。

Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present in the environment. In this study, we tested whether an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI = 0.90-0.99) vs. 0.58 (CI = 0.50-0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA-based approach has the potential to become the next-generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems.© 2015 John Wiley & Sons Ltd.

滨海湿地处于海陆的交错地带,在人类活动和气候变化的双重作用下导致湿地水文连通受阻。选取黄河口滨海湿地三条水文连通强度差异明显的潮沟,调查不同潮沟的大型底栖动物群落,分析水文连通强度对大型底栖动物分布和生物多样性的影响。结果表明：记录到的大型底栖动物共有52种,优势类群为多毛类和软体动物;总体来看随着水文连通强度的增强,大型底栖动物的总密度呈上升趋势;中等水文连通强度的区域,生物多样性较高;大型底栖动物不同类群的数量和分布对水文连通引起的环境因子变化的响应也有很大的差异,多毛类和寡毛类在水文连通弱的区域占优势,软体动物在水文连通较强营养物质含量丰富的区域占优势,甲壳类在靠近潮沟源头水文连通最强的区域占优势。

Environmental DNA (eDNA) metabarcoding has emerged as a potentially powerful tool to assess aquatic community structures. However, the method has hitherto lacked field tests that evaluate its effectiveness and practical properties as a biodiversity monitoring tool. Here, we evaluated the ability of eDNA metabarcoding to reveal fish community structures in species-rich coastal waters. High-performance fish-universal primers and systematic spatial water sampling at 47 stations covering similar to 11 km(2) revealed the fish community structure at a species resolution. The eDNA metabarcoding based on a 6-h collection of water samples detected 128 fish species, of which 62.5% (40 species) were also observed by underwater visual censuses conducted over a 14-year period. This method also detected other local fishes (>= 23 species) that were not observed by the visual censuses. These eDNA metabarcoding features will enhance marine ecosystem-related research, and the method will potentially become a standard tool for surveying fish communities.