The application of high-throughput sequencing-based approaches to DNA extracted from environmental samples such as gut contents and faeces has become a popular tool for studying dietary habits of animals. Due to the high resolution and prey detection capacity they provide, both metabarcoding and shotgun sequencing are increasingly used to address ecological questions grounded in dietary relationships. Despite their great promise in this context, recent research has unveiled how a wealth of biological (related to the study system) and technical (related to the methodology) factors can distort the signal of taxonomic composition and diversity. Here, we review these studies in the light of high-throughput sequencing-based assessment of trophic interactions. We address how the study design can account for distortion factors, and how acknowledging limitations and biases inherent to sequencing-based diet analyses are essential for obtaining reliable results, thus drawing appropriate conclusions. Furthermore, we suggest strategies to minimize the effect of distortion factors, measures to increase reproducibility, replicability and comparability of studies, and options to scale up DNA sequencing-based diet analyses. In doing so, we aim to aid end-users in designing reliable diet studies by informing them about the complexity and limitations of DNA sequencing-based diet analyses, and encourage researchers to create and improve tools that will eventually drive this field to its maturity.© 2018 John Wiley & Sons Ltd.

We present the open-source software package DADA2 for modeling and correcting Illumina-sequenced amplicon errors (https://github.com/benjjneb/dada2). DADA2 infers sample sequences exactly and resolves differences of as little as 1 nucleotide. In several mock communities, DADA2 identified more real variants and output fewer spurious sequences than other methods. We applied DADA2 to vaginal samples from a cohort of pregnant women, revealing a diversity of previously undetected Lactobacillus crispatus variants.

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.© 2013 John Wiley & Sons Ltd.

Advances in DNA sequencing technology have revolutionized the field of molecular analysis of trophic interactions, and it is now possible to recover counts of food DNA sequences from a wide range of dietary samples. But what do these counts mean? To obtain an accurate estimate of a consumer's diet should we work strictly with data sets summarizing frequency of occurrence of different food taxa, or is it possible to use relative number of sequences? Both approaches are applied to obtain semi-quantitative diet summaries, but occurrence data are often promoted as a more conservative and reliable option due to taxa-specific biases in recovery of sequences. We explore representative dietary metabarcoding data sets and point out that diet summaries based on occurrence data often overestimate the importance of food consumed in small quantities (potentially including low-level contaminants) and are sensitive to the count threshold used to define an occurrence. Our simulations indicate that using relative read abundance (RRA) information often provides a more accurate view of population-level diet even with moderate recovery biases incorporated; however, RRA summaries are sensitive to recovery biases impacting common diet taxa. Both approaches are more accurate when the mean number of food taxa in samples is small. The ideas presented here highlight the need to consider all sources of bias and to justify the methods used to interpret count data in dietary metabarcoding studies. We encourage researchers to continue addressing methodological challenges and acknowledge unanswered questions to help spur future investigations in this rapidly developing area of research.© 2018 John Wiley & Sons Ltd.

Understanding how biodiversity affects functioning of ecosystems requires integrating diversity within trophic levels (horizontal diversity) and across trophic levels (vertical diversity, including food chain length and omnivory). We review theoretical and experimental progress toward this goal. Generally, experiments show that biomass and resource use increase similarly with horizontal diversity of either producers or consumers. Among prey, higher diversity often increases resistance to predation, due to increased probability of including inedible species and reduced efficiency of specialist predators confronted with diverse prey. Among predators, changing diversity can cascade to affect plant biomass, but the strength and sign of this effect depend on the degree of omnivory and prey behaviour. Horizontal and vertical diversity also interact: adding a trophic level can qualitatively change diversity effects at adjacent levels. Multitrophic interactions produce a richer variety of diversity-functioning relationships than the monotonic changes predicted for single trophic levels. This complexity depends on the degree of consumer dietary generalism, trade-offs between competitive ability and resistance to predation, intraguild predation and openness to migration. Although complementarity and selection effects occur in both animals and plants, few studies have conclusively documented the mechanisms mediating diversity effects. Understanding how biodiversity affects functioning of complex ecosystems will benefit from integrating theory and experiments with simulations and network-based approaches.

In the UK, the native European otter (Lutra lutra) and invasive American mink (Neovison vison) have experienced concurrent declines and expansions. Currently, the otter is recovering from persecution and waterway pollution, whereas the mink is in decline due to population control and probable interspecific interaction with the otter. We explored the potential of DNA metabarcoding for investigating diet and niche partitioning between these mustelids. Otter spraints (n = 171) and mink scats (n = 19) collected from three sites (Malham Tarn, River Hull and River Glaven) in northern and eastern England were screened for vertebrates using high-throughput sequencing. Otter diet mainly comprised aquatic fishes (81.0%) and amphibians (12.7%), whereas mink diet predominantly consisted of terrestrial birds (55.9%) and mammals (39.6%). The mink used a lower proportion (20%) of available prey (n = 40 taxa) than the otter and low niche overlap (0.267) was observed between these mustelids. Prey taxon richness of mink scats was lower than otter spraints and beta diversity of prey communities was driven by taxon turnover (i.e. the otter and mink consumed different prey taxa). Considering otter diet only, prey taxon richness was higher in spraints from the River Hull catchment and beta diversity of prey communities was driven by taxon turnover (i.e. the otter consumed different prey taxa at each site). Studies using morphological faecal analysis may misidentify the predator as well as prey items. Faecal DNA metabarcoding can resolve these issues and provide more accurate and detailed dietary information. When scaled up across multiple habitat types, DNA metabarcoding should greatly improve future understanding of resource use and niche overlap between the otter and mink.

In South Korea, the Eurasian otter (Lutra lutra (Linnaeus, 1758)), a semi-aquatic carnivore, is found mainly in lower order streams that tend to have a low abundance of preferred prey fish species. To investigate the relationship between resource use and availability, we used DNA barcoding to identify otter diet items in 24 otter spraints (faeces) from 16 sites along the Nakdong River basin from 4 to 6 June 2014. At these sites fish availability was assessed using scoop nets and casting nets. Fish formed the bulk of otter diet, which included also frogs, mammals, and reptiles. By DNA barcoding (success rate: 72.38%), we identified 79 prey items from 105 bone remains. The diet comprised mostly fish, but frogs, mammals, and reptiles were also identified. The fish fauna and otter diet composition differed significantly. Across the study sites, members of the Cyprinidae dominated in netted samples, but occurred less frequently in otter diet. Because most Cyprinidae are fast swimmers, otters also fed on benthic fishes and frogs, suggesting limited foraging flexibility in otters and specialization on more slowly moving prey.

Knowledge of the effect of habitat type and region on diet and feeding behaviours of a species facilitates a better understanding of factors impacting populations, which contributes to effective conservation management. Using spraint analysis and relative frequency of occurrence data from the literature, we described the dietary patterns of Eurasian otters (Lutra lutra) in 23 study sites within the Pannonian biogeographical region in Hungary. Our results indicated that diet composition varied by habitat type and is therefore context dependant. The differences among habitat types were however lower than expected. We noticed a decline in the fish consumption with a concomitant increase in trophic niche breadth and amphibian consumption in rivers, ponds (fish farms), backwaters, marshes and small watercourses. The main differences in diet were not attributed to the consumption of primary and secondary food types (fish and amphibians), but rather to differences in other, less important food types (mammals, birds). Using hierarchical cluster analysis, rivers and ponds could clearly be separated from other habitat types. We found the main fish diet of otters in most of these areas consisted of small (<100 g), eurytopic, littoral and non-native, mostly invasive species. Dietary studies from 91 sites in six European biogeographical regions showed that fish are consumed most frequently in the Atlantic and Boreal, less in the Continental and Pannonian, and least in the Alpine and Mediterranean regions. Comparative analysis indicated that the Mediterranean region (with frequent crayfish consumption) and Alpine region (frequent amphibian consumption) cluster separate from the other regions.

Three species of otters are known from China; the Eurasian otter Lutra lutra is widespread throughout the country and the smooth-coated Lutrogale perspicillata and Asian small-clawed otters Aonyx cinereus occur in tropical and subtropical regions. We summarize the past status and distribution of otters in China, and provide an update based on a literature review, interviews and field surveys. Otter populations have undergone a dramatic countrywide decline, and are extirpated over much of their former ranges. Relict populations persist, however, in well-protected nature reserves, in sparsely populated headwaters of the Qinghai–Tibetan Plateau, at remote sites along international borders, and in densely populated deltas and floodplains. Recent records were mostly of the Eurasian otter, and we could find no confirmed recent record of the smooth-coated otter. The otters that survive in certain well-protected sites could act as source populations for recolonization if adequate conservation interventions are implemented. Urgent, focused action is needed to protect the remaining populations, and to study the taxonomy and ecology of China's otters.

As the apex predator of plateau ecosystems in Central Asia and the Qinghai-Tibet Plateau, the snow leopard (Panthera uncia) plays an essential role in maintaining food-web structure and ecosystem stability. Learning the diet composition and dynamics of the snow leopard is important for understanding its role in ecosystem functioning and interspecific interactions. Previous diet analyses of the snow leopard have been based mainly on morphological identification of food debris in the feces, though the accuracy of this practice has been broadly debated. The Qionglai Mountains are located at the southeast edge of the snow leopard range, harboring a small and relatively isolated population of snow leopards that are barely studied. Using non-invasive sampling, we collected 38 putative snow leopard fecal samples in the Wolong National Nature Reserve in the Qionglai Mountains. To identify the fecal origin, we extracted the fecal DNA and amplified the mitochondrial DNA 16S rRNA gene fragment. Twenty-two fecal samples were identified as originating from snow leopards. Subsequently, vertebrate universal primers and a snow leopard-specific blocking oligo were used to amplify the food components in the fecal DNA, and then high-throughput sequencing was performed to analyze the diet composition of snow leopards. The blue sheep (Pseudois nayaur) was detected in 67% of the samples and was found to be the main staple food of snow leopards’ diet. The domestic yak (Bos grunniens) appeared in 33% of the fecal samples, also accounting for a high proportion of the snow leopard diet. In addition, pikas (Ochotona spp.) and birds were found in a small number of fecal samples. Therefore, wild prey was found to be the main food source for snow leopards in Wolong. However, livestock (yak) also accounted for a relatively large proportion of their diet.

作为中亚和青藏高原山地生态系统中的顶级捕食者, 雪豹(Panthera uncia)对于维持食物网结构和生态系统稳定性有重要作用。了解雪豹的食性组成和变化对于理解其生态系统功能和物种间相互作用有重要意义。以往的雪豹食性分析多基于对其粪便中食物残渣的形态学鉴定, 但准确度受人员经验和主观因素影响较大。邛崃山脉位于雪豹分布区东南缘, 该区域的雪豹种群规模小且相对孤立, 研究匮乏。本研究基于非损伤性取样, 在邛崃山脉的卧龙国家级保护区采集疑似雪豹粪便样品38份, 首先提取粪便DNA, 并扩增线粒体DNA 16S rRNA基因片段进行分子物种鉴定, 确定其中22份为雪豹粪便样品。随后, 利用脊椎动物通用引物和雪豹特异性阻抑引物扩增粪便DNA中的食物成分, 并进行高通量测序, 分析雪豹食性构成。食性分析结果显示岩羊(Pseudois nayaur)是卧龙地区雪豹最主要的食物, 在67%的样品中均有检出。家牦牛(Bos grunniens)在33%的样品中出现, 也在雪豹食性中占较高比例。此外, 鼠兔(Ochotona spp.)和鸟类也在少量样品中发现。可见, 野生猎物是卧龙地区雪豹的主要食物资源; 与大世界大多数其他地区的雪豹食性相同, 野生大型有蹄类是雪豹最重要的食物。然而家畜(牦牛)在卧龙雪豹食谱中有相当高的占比, 显示该区域内可能存在较为严重的由雪豹捕食散养家畜引起的人兽冲突问题。

Accurate analyses of the diets of predators are key to understand trophic interactions and defining conservation strategies. Diets are commonly assessed through analysis of non-invasively collected scats, and the use of faecal DNA (fDNA) analysis can reduce the species misidentifications that could lead to biased ecological inference. We review the scientific literature since publication of the first paper on amplifying fDNA, in order to assess trends in the use of genetic non-invasive sampling (gNIS) for predator species identification in scat-based diet studies of North American and European terrestrial mammalian carnivores (Carnivora). We quantify error rates in morphology-based predator species identification. We then provide an overview of how applying gNIS would improve research on trophic interactions and other areas of carnivore ecology. We found that carnivore species identity was verified by using gNIS in only 8% of 400 studies of carnivore diets based on scats. The median percentage of false positives (i.e. samples wrongly identified as belonging to the target species) in morphology-based studies was 18%, and was consistent regardless of species' body size. We did not find an increasing trend in the use of gNIS over time, despite the existing technical capability to identify almost all carnivore species. New directions for fDNA studies include employing high-throughput sequencing (HTS) and DNA metabarcoding to identify the predator species, the individual predator, the entire assemblage of consumed items, and the microbiome of the predator and pathogens. We conclude that HTS protocols and metagenomic approaches hold great promise for elevating gNIS as a fundamental cornerstone for future research in ecology and conservation biology of mammals.

Genetic analysis using noninvasively collected samples such as faeces continues to pose a formidable challenge because of unpredictable variation in the extent to which usable DNA is obtained. We investigated the influence of multiple variables on the quantity of DNA extracted from faecal samples from wild mountain gorillas and chimpanzees. There was a small negative correlation between temperature at time of collection and the amount of DNA obtained. Storage of samples either in RNAlater solution or dried using silica gel beads produced similar results, but significantly higher amounts of DNA were obtained using a novel protocol that combines a short period of storage in ethanol with subsequent desiccation using silica.

The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.© 2011 Blackwell Publishing Ltd.

VSEARCH is an open source and free of charge multithreaded 64-bit tool for processing and preparing metagenomics, genomics and population genomics nucleotide sequence data. It is designed as an alternative to the widely used USEARCH tool (Edgar, 2010) for which the source code is not publicly available, algorithm details are only rudimentarily described, and only a memory-confined 32-bit version is freely available for academic use.

Quality and quantity of food items consumed has a crucial effect on phenotypes. In addition to direct effects mediated by nutrient resources, an individual's diet can also affect the phenotype indirectly by altering its gut microbiota, a potent modulator of physiological, immunity and cognitive functions. However, most of our knowledge of diet-microbiota interactions is based on mammalian species, whereas little is still known about these effects in other vertebrates. We developed a metabarcoding procedure based on cytochrome c oxidase I high-throughput amplicon sequencing and applied it to describe diet composition in breeding colonies of an insectivorous bird, the barn swallow (Hirundo rustica). To identify putative diet-microbiota associations, we integrated the resulting diet profiles with an existing dataset for faecal microbiota in the same individual. Consistent with previous studies based on macroscopic analysis of diet composition, we found that Diptera, Hemiptera, Coleoptera and Hymenoptera were the dominant dietary components in our population. We revealed pronounced variation in diet consumed during the breeding season, along with significant differences between nearby breeding colonies. In addition, we found no difference in diet composition between adults and juveniles. Finally, our data revealed a correlation between diet and faecal microbiota composition, even after statistical control for environmental factors affecting both diet and microbiota variation. Our study suggests that variation in diet induce slight but significant microbiota changes in a non-mammalian host relying on a narrow spectrum of items consumed.© 2022. The Author(s).

Large carnivores play an important role in the regulation of food-web structure and ecosystem functioning. However, large carnivores face serious threats that have caused declines in their populations and geographic ranges due to habitat loss and degradation, hunting, human disturbance and pathogen transmission. Conservation of large carnivore species richness and population size has become a pressing issue and an important research focus of conservation biology. The western Sichuan Plateau, located at the intersection of the mountains of southwest China and the eastern margin of the Tibetan Plateau, is a global biodiversity hotspot and has high carnivore species richness. However, increasing human activities may exacerbate the destruction of local flora and fauna, thereby threatening the survival of wild carnivores. Information on species composition and dietary habits can improve our understanding of the structure and function of the ecosystem and food-web relationships in the study area. In addition, species composition and dietary habits are of great significance for understanding multi-species coexistence mechanisms and preserving biodiversity. This study collected carnivore fecal samples from Xinlong and Shiqu counties in the Ganzi Tibetan Autonomous Prefecture, Sichuan Province. DNA was then extracted from the samples and the species was identified based on DNA sequences and DNA barcoding techniques. Seven carnivores were identified, including five large carnivores (Canis lupus, Ursus arctos, Panthera pardus, P. uncia and Canis lupus familiaris) and two medium and small-sized carnivores (Prionailurus bengalensis and Vulpes vulpes). Using fecal DNA, high-throughput sequencing and metabarcoding, we conducted diet analysis for the seven carnivores and found 28 different food molecular operational taxonomic units (MOTUs), including 19 mammals, eight birds and one fish species. The predominant prey categories of wolves, dogs and brown bears were ungulates. The domestic yak (Bos grunniens) was the most frequently identified prey species. Small mammals such as rodents and lagomorphs accounted for a significant proportion in the diets of leopard cats and red foxes, The most frequent prey of this category of carnivore were the Chinese scrub vole (Neodon irene) and plateau pika (Ochotona curzoniae). In addition, leopards and snow leopards mainly fed on the Chinese goral (Naemorhedus griseus) and blue sheep (Pseudois nayaur), respectively. Our study highlights the utility of fecal DNA and metabarcoding technique in fast carnivore surveys and high-throughput diet analysis, and provides a technical reference and guidance for future biodiversity surveys and food-web studies.

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。

Amphibians form a major component of the diet of the otter Lutra lutra in several areas of its wide geographic range. Yet, amphibian remains are rarely identified to species level and therefore information on the diversity of this food resource is generally scarce. The aims of this study were: 1) to assess the overall pattern and trends in the use of amphibians as a resource by otters at the range scale, and 2) to highlight current knowledge on the diversity of amphibians taken as prey by otters. Additionally, we carried out osteological identification of amphibian remains in otter spraints (faeces) from southern Italy, with the aim of demonstrating how this method may improve our knowledge on predator-prey relationships. The frequency of occurrence of amphibians in 64 dietary studies averaged 12%. Predation of amphibians by otters increased with longitude and was the highest in the Alpine biogeographical region. Predation by otters was reported on 28 amphibian species (35% of European species). Peaks in their frequency of use were reported for all seasons, mostly in winter and spring. In southern Italy, we identified 355 individuals belonging to at least seven amphibian taxa (64% of available species; Rana italica, Rana dalmatina/italica, Pelophylax kl. bergeri/hispanicus, Hyla intermedia, Bufo bufo, Bufotes balearicus, and Lissotriton italicus), and pointed out male-biased predation within the Order Anura (frogs). We conclude that the contribution of amphibians to the richness of the otter's prey community is far higher than commonly perceived, and that osteological analyses allow the detailed investigation of the feeding behaviour of this top predator of freshwater habitats.

Virtually all empirical ecological studies require species identification during data collection. DNA metabarcoding refers to the automated identification of multiple species from a single bulk sample containing entire organisms or from a single environmental sample containing degraded DNA (soil, water, faeces, etc.). It can be implemented for both modern and ancient environmental samples. The availability of next-generation sequencing platforms and the ecologists' need for high-throughput taxon identification have facilitated the emergence of DNA metabarcoding. The potential power of DNA metabarcoding as it is implemented today is limited mainly by its dependency on PCR and by the considerable investment needed to build comprehensive taxonomic reference libraries. Further developments associated with the impressive progress in DNA sequencing will eliminate the currently required DNA amplification step, and comprehensive taxonomic reference libraries composed of whole organellar genomes and repetitive ribosomal nuclear DNA can be built based on the well-curated DNA extract collections maintained by standardized barcoding initiatives. The near-term future of DNA metabarcoding has an enormous potential to boost data acquisition in biodiversity research.© 2012 Blackwell Publishing Ltd.

Freshwater ecosystems are among the most endangered habitats on Earth, with thousands of animal species known to be threatened or already extinct. Reliable monitoring of threatened organisms is crucial for data-driven conservation actions but remains a challenge owing to nonstandardized methods that depend on practical and taxonomic expertise, which is rapidly declining. Here, we show that a diversity of rare and threatened freshwater animals--representing amphibians, fish, mammals, insects and crustaceans--can be detected and quantified based on DNA obtained directly from small water samples of lakes, ponds and streams. We successfully validate our findings in a controlled mesocosm experiment and show that DNA becomes undetectable within 2 weeks after removal of animals, indicating that DNA traces are near contemporary with presence of the species. We further demonstrate that entire faunas of amphibians and fish can be detected by high-throughput sequencing of DNA extracted from pond water. Our findings underpin the ubiquitous nature of DNA traces in the environment and establish environmental DNA as a tool for monitoring rare and threatened species across a wide range of taxonomic groups.© 2011 Blackwell Publishing Ltd.

The diet of top predators is vital information needed to determine their ecological function and for their conservation management. However, the elusive habit and low population density of many predators constrains determination of their diets. While the morphological identification of scat contents is the traditional method, DNA metabarcoding has lately proven a more efficient and accurate method of identifying prey taxa. We applied DNA metabarcoding to analyzing the diet of the Eurasian otter (Lutra lutra), a top predator in freshwater ecosystems, using 12S and 16S rRNA mitochondrial primers target vertebrate prey. Diet did not vary among different data removal thresholds of 0.1, 1, 3, and 5%, comprising fishes (>90%), amphibians and birds (>2%), and occasionally mammals (<2%). Both 12S and 16S primers revealed similar otter diets, indicating that a single set of primers with a higher threshold is cost-effective for detecting the main prey taxa. Using 12S primers and a 5% threshold, we found no seasonal variation of otter diet in the Tangjiahe National Nature Reserve. A different prey community was found outside the reserve, which resulted in different prey composition for otters. However, prey taxon richness was not different between otters in- and outside the reserve. Otters preferred Schizothorax spp., the largest-sized fish species in the reserve, whereas they mainly preyed on Triplophysa bleekeri, a small-sized fish species, outside the reserve. Otters’ flexible feeding strategy reflect their high adaptability. However, greater human disturbance outside the reserve may present significant challenges to otters by altering prey communities and reducing prey profitability. Combining fecal DNA metabarcoding and local fish survey will provide opportunities for more detailed studies on the impact of different levels of human disturbances on prey communities and otters.

We describe methods for the preservation, extraction and amplification of DNA from faeces that facilitate field applications of faecal DNA technology. Mitochondrial, protein encoding and microsatellite nuclear DNA extracted and amplified from faeces of Malayan sun bears and North American black bears is shown to be identical to that extracted and amplified from the same individual's tissue or blood. A simple drying agent, silica beads, is shown to be a particularly effective preservative, allowing easy and safe transport of samples from the field. Methods are also developed to eliminate the risk of faecal DNA contamination from hair present in faeces.

Dietary information is lacking in most of small to mid-sized carnivores due to their elusive predatory behaviour and versatile feeding habits. The leopard cat (LPC; Prionailurus bengalensis) and the Asiatic golden cat (AGC; Catopuma temminckii) are two important yet increasingly endangered carnivore species in the temperate mountain forest ecosystem in Southwest China, a global biodiversity hotspot and a significant reservoir of China's endemic species. We investigated the vertebrate prey of the two sympatric felids using faecal DNA and a next-generation sequencing (NGS)/metabarcoding approach. Forty vertebrate prey taxa were identified from 93 LPC and 10 AGC faecal samples; 37 taxa were found in the LPC diet, and 20 were detected in the AGC diet. Prey included 27 mammalian taxa, 11 birds, one lizard and one fish, with 73% (29/40) of the taxa assigned to the species level. Rodents and pikas were the most dominant LPC prey categories, whereas rodents, pheasant, fowl and ungulates were the main AGC prey. We also analysed the seasonal and altitudinal variations in the LPC diet. Our results provide the most comprehensive dietary data for these felids and valuable information for their conservation planning.

<p id="p00005"><strong>Aims:</strong> The otter is an indicator and flagship species of aquatic ecosystems. Its populations have undergone a drastic decline in China, and have become locally extinct in some regions due to anthropogenic disturbance. However, the current literature on otters in China is inadequate, which has subsequently affected conservation of the species. We aim to assess the potential distribution and conservation priority areas of the Eurasian otter (<i>Lutra lutra</i>) in Northeast China.</p><p id="p00010"><strong>Methods:</strong> In this study, we integrated species distribution models with otter survey data from 2016 to 2020 to assess Eurasian otter distribution. Then, we used the geographic information system and core-area zonation algorithm in Zonation 4.00 to identify conservation priority areas for otter protection. Using the anthropogenic pressures layer, we also evaluated anthropogenic stressors in each province. Then, we analyzed the conservation status of the otter based on the spatial distribution of national nature reserves. Furthermore, we used Inner Mongolia Forest Industry Group, Daxing&#x02019;anling Forestry Group and Yichun Forest Industry Group as case studies to analyze the role of state-owned forest areas for otter conservation. </p><p id="p00015"><strong>Results:</strong> The results demonstrate that potential distribution and conservation priority areas cover 104,515.04 km²and 45,448.99 km², respectively. Large swathes of conservation priority areas remain for the otter in Daxing&#x02019;anling which are connected with conservation priority areas in Xiaoxing&#x02019;anling and there is no obvious geographical boundary between them. Therefore, these regions are mandatory to conserve to maintain the stability of otter populations in Northeast China. Otters in the Liaoning Province faced the greatest anthropogenic pressures, followed by the Jilin Province and Heilongjiang Province. Otters in the Inner Mongolia Autonomous Region faced the lowest amount of pressure. There is potential distribution for the otter in 63 nature reserves and conservation priority areas in 32 nature reserves out of the 110 reserves in the region. The study area includes 11.64% of the otter&#x02019;s potential distribution and 10.88% of conservation priority areas. Three state-owned forest industry groups cover 71.18% of the potential distribution of the otter (74,390.89 km²) and 79.26% of the conservation priority areas (36,022.22 km²). </p><p id="p00020"><strong>Conclusions:</strong> This study indicates that state-owned forest areas may play a greater role in the protection of biodiversity following a comprehensive ban on logging in natural forests. Therefore, we propose that areas with important conservation value in state-owned forest land should be gradually included under the protections of national parks in order to achieve systematic and complete protection of biodiversity. Finally, we suggest: (1) strengthening the management of river pollutions; (2) reducing fishing intensity; (3) a long-term field monitoring network be established to study population changes in otters; (4) increasing the investment in scientific research on otters; and (5) raising awareness of otter conservation through public education.</p>

水獭是水生生态系统重要的指示种和旗舰种, 由于强烈的人为干扰, 中国的水獭种群数量大幅下降, 部分区域已局部灭绝。然而目前国内对水獭的调查和研究非常有限, 本底不清的状况已经严重影响到水獭的野外保育工作。本文以东北地区的欧亚水獭指名亚种(Lutra lutra lutra)为研究对象, 基于2016-2020年的调查数据, 使用组合建模的方法评估了水獭的潜在分布区; 利用地理信息系统和系统保护规划软件分析了水獭的保护优先区并计算了各省级行政区内水獭潜在分布区和保护优先区面临的人类压力; 结合国家级自然保护区的空间布局分析了水獭的保护现状, 并以内蒙古森工集团、大兴安岭林业集团、伊春森工集团三大国有林区为例分析了重点国有林区在水獭保护中的作用。结果表明: (1)水獭潜在分布区和保护优先区面积分别为104,515.04 km²和45,448.99 km², 其中大兴安岭的水獭保护优先区集中连片, 并与小兴安岭的保护优先区相连, 栖息地之间没有明显地理隔离, 是维持东北地区水獭种群稳定的重中之重; (2)水獭面临的人类压力大小依次为: 辽宁 &gt; 吉林 &gt; 黑龙江 &gt; 内蒙古; (3)研究区内110个国家级自然保护区中有63个包含水獭潜在分布区, 覆盖面积为12,168.93 km², 仅占水獭潜在分布区面积的11.64%, 其中32个国家级自然保护区包含水獭保护优先区, 占水獭保护优先区面积的10.88%; (4)三大国有林区涵盖了71.18%的水獭潜在分布区和79.26%的保护优先区(面积分别为74,390.89 km²和36,022.22 km²)。由此可见, 尽管水獭潜在分布区中国家级自然保护区占比较低, 但是在天然林全面禁伐的背景下, 重点国有林区可能在未来东北地区的生物多样性保护中发挥更大作用, 因此我们建议将重点国有林区中具有重要保护价值的区域逐步纳入以国家公园为主体的自然保护地体系中, 以实现生物多样性的系统性和完整性保护。最后, 本文结合研究结果和实地调研提出以下保护建议: (1)加强对河流污染物的管理; (2)控制渔民捕鱼强度; (3)开展全面的水獭专项调查并建立长期的监测体系; (4)加大对水獭的科研投入; (5)加强宣传力度, 提升公众保护意识。