Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods

Terrestrial arthropods comprise the most species‐rich communities on Earth, and grassland flowers provide resources for hundreds of thousands of arthropod species. Diverse grassland ecosystems worldwide are threatened by various types of environmental change, which has led to decline in arthropod diversity. At the same time, monitoring grassland arthropod diversity is time‐consuming and strictly dependent on declining taxonomic expertise. Environmental DNA (eDNA) metabarcoding of complex samples has demonstrated that information on species compositions can be efficiently and non‐invasively obtained. Here, we test the potential of wild flowers as a novel source of arthropod eDNA. We performed eDNA metabarcoding of flowers from several different plant species using two sets of generic primers, targeting the mitochondrial genes 16S rRNA and COI. Our results show that terrestrial arthropod species leave traces of DNA on the flowers that they interact with. We obtained eDNA from at least 135 arthropod species in 67 families and 14 orders, together representing diverse ecological groups including pollinators, parasitoids, gall inducers, predators, and phytophagous species. Arthropod communities clustered together according to plant species. Our data also indicate that this experiment was not exhaustive, and that an even higher arthropod richness could be obtained using this eDNA approach. Overall, our results demonstrate that it is possible to obtain information on diverse communities of insects and other terrestrial arthropods from eDNA metabarcoding of wild flowers. This novel source of eDNA represents a vast potential for addressing fundamental research questions in ecology, obtaining data on cryptic and unknown species of plant‐associated arthropods, as well as applied research on pest management or conservation of endangered species such as wild pollinators.     

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

DNA metabarcoding is a promising approach for rapidly surveying biodiversity and is likely to become an important tool for measuring ecosystem responses to environmental change. Metabarcoding markers need sufficient taxonomic coverage to detect groups of interest, sufficient sequence divergence to resolve species, and will ideally indicate relative abundance of taxa present. We characterized zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) to compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology‐ and DNA‐based identification. COI amplicons sequenced on separate runs showed that operational taxonomic units representing >0.1% of reads per sample were highly reproducible, although slightly more taxa were detected using a lower annealing temperature. Mitochondrial COI and nuclear 18S showed similar taxonomic coverage across zooplankton phyla. However, mitochondrial COI resolved up to threefold more taxa to species compared to 18S. All markers revealed similar patterns of beta‐diversity, although different taxa were identified as the greatest contributors to these patterns for 18S. For calanoid copepod families, all markers displayed a positive relationship between biomass and sequence reads, although the relationship was typically strongest for 18S. The use of COI for metabarcoding has been questioned due to lack of conserved primer‐binding sites. However, our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well‐developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment.     

Promises and pitfalls of using high‐throughput sequencing for diet analysis

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.     

Reconstructing marine plankton food web interactions using DNA metabarcoding

Knowledge of zooplankton in situ diet is critical for accurate assessment of marine ecosystem function and structure, but due to methodological constraints, there is still a limited understanding of ecological networks in marine ecosystems. Here, we used DNA‐metabarcoding to study trophic interactions, with the aim to unveil the natural diet of zooplankton species under temporal variation of food resources. Several target consumers, including copepods and cladocerans, were investigated by sequencing 16S rRNA and 18S rRNA genes to identify prokaryote and eukaryote potential prey present in their guts. During the spring phytoplankton bloom, we found a dominance of diatom and dinoflagellate trophic links to copepods. During the summer period, zooplankton including cladocerans showed a more diverse diet dominated by cyanobacteria and heterotrophic prey. Our study suggests that copepods present trophic plasticity, changing their natural diet over seasons, and adapting their feeding strategies to the available prey spectrum, with some species being more selective. We did not find a large overlap of prey consumed by copepods and cladocerans, based on prey diversity found in their guts, suggesting that they occupy different roles in the trophic web. This study represents the first molecular approach to investigate several zooplankton–prey associations under seasonal variation, and highlights how, unlike other techniques, the diversity coverage is high when using DNA, allowing the possibility to detect a wide range of trophic interactions in plankton communities.     

eDNA metabarcoding of avocado flowers: ‘Hass’ it got potential to survey arthropods in food production systems?

In the face of global biodiversity declines, surveys of beneficial and antagonistic arthropod diversity as well as the ecological services that they provide are increasingly important in both natural and agro-ecosystems. Conventional survey methods used to monitor these communities often require extensive taxonomic expertise and are time-intensive, potentially limiting their application in industries such as agriculture, where arthropods often play a critical role in productivity (e.g. pollinators, pests and predators). Environmental DNA (eDNA) metabarcoding of a novel substrate, crop flowers, may offer an accurate and high throughput alternative to aid in the detection of these managed and unmanaged taxa. Here, we compared the arthropod communities detected with eDNA metabarcoding of flowers, from an agricultural species (Persea americana—‘Hass’ avocado), with two conventional survey techniques: digital video recording (DVR) devices and pan traps. In total, 80 eDNA flower samples, 96 h of DVRs and 48 pan trap samples were collected. Across the three methods, 49 arthropod families were identified, of which 12 were unique to the eDNA dataset. Environmental DNA metabarcoding from flowers revealed potential arthropod pollinators, as well as plant pests and parasites. Alpha diversity levels did not differ across the three survey methods although taxonomic composition varied significantly, with only 12% of arthropod families found to be common across all three methods. eDNA metabarcoding of flowers has the potential to revolutionize the way arthropod communities are monitored in natural and agro-ecosystems, potentially detecting the response of pollinators and pests to climate change, diseases, habitat loss and other disturbances.     

Trophic resource partitioning drives fine‐scale coexistence in cryptic bat species

Understanding the processes that enable species coexistence has important implications for assessing how ecological systems will respond to global change. Morphology and functional similarity increase the potential for competition, and therefore, co‐occurring morphologically similar but genetically unique species are a good model system for testing coexistence mechanisms. We used DNA metabarcoding and high‐throughput sequencing to characterize for the first time the trophic ecology of two recently described cryptic bat species with parapatric ranges, Myotis escalerai and Myotis crypticus. We collected fecal samples from allopatric and sympatric regions and from syntopic and allotopic locations within the sympatric region to describe the diets both taxonomically and functionally and compare prey consumption with prey availability. The two bat species had highly similar diets characterized by high arthropod diversity, particularly Lepidoptera, Diptera and Araneae, and a high proportion of prey that is not volant at night, which points to extensive use of gleaning. Diet overlap at the prey item level was lower in syntopic populations, supporting trophic shift under fine‐scale co‐occurrence. Furthermore, the diet of M. escalerai had a marginally lower proportion of not nocturnally volant prey in syntopic populations, suggesting that the shift in diet may be driven by a change in foraging mode. Our findings suggest that fine‐scale coexistence mechanisms can have implications for maintaining broad‐scale diversity patterns. This study highlights the importance of including both allopatric and sympatric populations and choosing meaningful spatial scales for detecting ecological patterns. We conclude that a combination of high taxonomic resolution with a functional approach helps identify patterns of niche shift.     

Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias

Studies of insect assemblages are suited to the simultaneous DNA‐based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR‐amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers [16S and cytochrome oxidase c subunit I (COI)] and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico <75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided >90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR‐amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR‐amplified the blend using five primer sets, targeting either COI or 16S, with high‐throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers.     

DNA宏条形码技术在海洋浮游动物多样性和生态学研究中的应用

浮游动物是海洋生态系统的关键类群,其覆盖门类广泛,多样性高。传统形态鉴定技术需要检测人员具备专业的形态鉴定知识,且费时费力。宏条形码技术无需分离生物个体,而是提取拖网采集到的浮游动物混合样本的总DNA,或者水体中的环境DNA （eDNA）,依托高通量测序平台测序,能够实现对大规模样本快速、准确、经济的分析,在海洋浮游动物生态学研究中得到越来越广泛的应用。分析了DNA宏条形码技术常用的核糖体和线粒体分子标记,在浮游动物多样性和数量研究中的可靠性和不足,并给出在海洋浮游动物群落监测,食物关系分析及生物入侵早期预警等研究中的应用。未来,开发多基因片段组合条形码,发展完备的参考数据库及实现准确的量化研究是DNA宏条形码技术发展的重要方向。     

Metabarcoding for the parallel identification of several hundred predators and their prey: Application to bat species diet analysis

Assessing diet variability is of main importance to better understand the biology of bats and design conservation strategies. Although the advent of metabarcoding has facilitated such analyses, this approach does not come without challenges. Biases may occur throughout the whole experiment, from fieldwork to biostatistics, resulting in the detection of false negatives, false positives or low taxonomic resolution. We detail a rigorous metabarcoding approach based on a short COI minibarcode and two‐step PCR protocol enabling the “all at once” taxonomic identification of bats and their arthropod prey for several hundreds of samples. Our study includes faecal pellets collected in France from 357 bats representing 16 species, as well as insect mock communities that mimic bat meals of known composition, negative and positive controls. All samples were analysed using three replicates. We compare the efficiency of DNA extraction methods, and we evaluate the effectiveness of our protocol using identification success, taxonomic resolution, sensitivity and amplification biases. Our parallel identification strategy of predators and prey reduces the risk of mis‐assigning prey to wrong predators and decreases the number of molecular steps. Controls and replicates enable to filter the data and limit the risk of false positives, hence guaranteeing high confidence results for both prey occurrence and bat species identification. We validate 551 COI variants from arthropod including 18 orders, 117 family, 282 genus and 290 species. Our method therefore provides a rapid, resolutive and cost‐effective screening tool for addressing evolutionary ecological issues or developing “chirosurveillance” and conservation strategies.     

High‐throughput metabarcoding of eukaryotic diversity for environmental monitoring of offshore oil‐drilling activities

As global exploitation of available resources increases, operations extend towards sensitive and previously protected ecosystems. It is important to monitor such areas in order to detect, understand and remediate environmental responses to stressors. The natural heterogeneity and complexity of communities means that accurate monitoring requires high resolution, both temporally and spatially, as well as more complete assessments of taxa. Increased resolution and taxonomic coverage is economically challenging using current microscopy‐based monitoring practices. Alternatively, DNA sequencing‐based methods have been suggested for cost‐efficient monitoring, offering additional insights into ecosystem function and disturbance. Here, we applied DNA metabarcoding of eukaryotic communities in marine sediments, in areas of offshore drilling on the Norwegian continental shelf. Forty‐five samples, collected from seven drilling sites in the Troll/Oseberg region, were assessed, using the small subunit ribosomal RNA gene as a taxonomic marker. In agreement with results based on classical morphology‐based monitoring, we were able to identify changes in sediment communities surrounding oil platforms. In addition to overall changes in community structure, we identified several potential indicator taxa, responding to pollutants associated with drilling fluids. These included the metazoan orders Macrodasyida, Macrostomida and Ceriantharia, as well as several ciliates and other protist taxa, typically not targeted by environmental monitoring programmes. Analysis of a co‐occurrence network to study the distribution of taxa across samples provided a framework for better understanding the impact of anthropogenic activities on the benthic food web, generating novel, testable hypotheses of trophic interactions structuring benthic communities.     

In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

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.     

Replicate DNA metabarcoding can discriminate seasonal and spatial abundance shifts in river macroinvertebrate assemblages

The delivery of consistent and accurate fine-resolution data on biodiversity using metabarcoding promises to improve environmental assessment and research. Whilst this approach is a substantial improvement upon traditional techniques, critics note that metabarcoding data are suitable for establishing taxon occurrence, but not abundance. We propose a novel hierarchical approach to recovering abundance information from metabarcoding, and demonstrate this technique using benthic macroinvertebrates. To sample a range of abundance structures without introducing additional changes in composition, we combined seasonal surveys with fish-exclusion experiments at Catamaran Brook in northern New Brunswick, Canada. Five monthly surveys collected 31 benthic samples for DNA metabarcoding divided between caged and control treatments. A further six samples per survey were processed using traditional morphological identification for comparison. By estimating the probability of detecting a single individual, multispecies abundance models infer changes in abundance based on changes in detection frequency. Using replicate detections of 184 genera (and 318 species) from metabarcoding samples, our analysis identified changes in abundance arising from both seasonal dynamics and the exclusion of fish predators. Counts obtained from morphological samples were highly variable, a feature that limited the opportunity for more robust comparison, and emphasizing the difficulty standard methods also face to detect changes in abundance. Our approach is the first to demonstrate how quantitative estimates of abundance can be made using metabarcoding, both among species within sites as well as within species among sites. Many samples are required to capture true abundance patterns, particularly in streams where counts are highly variable, but few studies can afford to process entire samples. Our approach allows study of responses across whole communities, and at fine taxonomic resolution. We discuss how ecological studies can use additional sampling to capture changes in abundance at fine resolution, and how this can complement broad-scale biomonitoring using DNA metabarcoding.     

Ecological specialization and niche overlap of subterranean rodents inferred from DNA metabarcoding diet analysis

Knowledge of how animal species use food resources available in the environment can increase our understanding of many ecological processes. However, obtaining this information using traditional methods is difficult for species feeding on a large variety of food items in highly diverse environments. We amplified the DNA of plants for 306 scat and 40 soil samples, and applied an environmental DNA metabarcoding approach to investigate food preferences, degree of diet specialization and diet overlap of seven herbivore rodent species of the genus Ctenomys distributed in southern and midwestern Brazil. The metabarcoding approach revealed that these species consume more than 60% of the plant families recovered in soil samples, indicating generalist feeding habits of ctenomyids. The family Poaceae was the most common food resource retrieved in scats of all species as well in soil samples. Niche overlap analysis indicated high overlap in the plant families and molecular operational taxonomic units consumed, mainly among the southern species. Interspecific differences in diet composition were influenced, among other factors, by the availability of resources in the environment. In addition, our results provide support for the hypothesis that the allopatric distributions of ctenomyids allow them to exploit the same range of resources when available, possibly because of the absence of interspecific competition.     

Unveiling the Diet of Elusive Rainforest Herbivores in Next Generation Sequencing Era? The Tapir as a Case Study

Characterizing the trophic relationships between large herbivores and the outstanding plant diversity in rainforest is a major challenge because of their elusiveness. This is crucial to understand the role of these herbivores in the functioning of the rainforest ecosystems. We tested a non-invasive approach based on the high-throughput sequencing of environmental samples using small plant plastid sequences (the trnL P6 loop) and ribosomal ITS1 primers, referred to as DNA metabarcoding, to investigate the diet of the largest neotropical herbivore, the lowland tapir. Sequencing was performed on plant DNA extracted from tapir faeces collected at the Nouragues station, a protected area of French Guiana. In spite of a limited sampling, our approach reliably provided information about the lowland tapir''s diet at this site. Indeed, 95.1% and 74.4% of the plant families and genera identified thanks to the trnL P6 loop, respectively, matched with taxa already known to be consumed by tapirs. With this approach we were able to show that two families and eight new genera are also consumed by the lowland tapir. The taxonomic resolution of this method is limited to the plant family and genera. Complementary barcodes, such as a small portion of ITS1, can be used to efficiently narrow identifications down to the species in some problematic families. We will discuss the remaining limitations of this approach and how useful it is at this stage to unravel the diet of elusive rainforest herbivores and better understand their role as engineers of the ecosystem.     

Approaches to integrating genetic data into ecological networks

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.     

DNA metabarcoding quantifies the relative biomass of arthropod taxa in songbird diets: Validation with camera‐recorded diets

Ecological research is often hampered by the inability to quantify animal diets. Diet composition can be tracked through DNA metabarcoding of fecal samples, but whether (complex) diets can be quantitatively determined with metabarcoding is still debated and needs validation using free‐living animals. This study validates that DNA metabarcoding of feces can retrieve actual ingested taxa, and most importantly, that read numbers retrieved from sequencing can also be used to quantify the relative biomass of dietary taxa. Validation was done with the hole‐nesting insectivorous Pied Flycatcher whose diet was quantified using camera footage. Size‐adjusted counts of food items delivered to nestlings were used as a proxy for provided biomass of prey orders and families, and subsequently, nestling feces were assessed through DNA metabarcoding. To explore potential effects of digestion, gizzard and lower intestine samples of freshly collected birds were subjected to DNA metabarcoding. For metabarcoding with Cytochrome Oxidase subunit I (COI), we modified published invertebrate COI primers LCO1490 and HCO1777, which reduced host reads to 0.03%, and amplified Arachnida DNA without significant changing the recovery of other arthropod taxa. DNA metabarcoding retrieved all commonly camera‐recorded taxa. Overall, and in each replicate year (N = 3), the relative scaled biomass of prey taxa and COI read numbers correlated at R = .85 (95CI:0.68–0.94) at order level and at R = .75 (CI:0.67–0.82) at family level. Similarity in arthropod community composition between gizzard and intestines suggested limited digestive bias. This DNA metabarcoding validation demonstrates that quantitative analyses of arthropod diet is possible. We discuss the ecological applications for insectivorous birds.     

DNA metabarcoding provides insights into seasonal diet variations in Chinese mole shrew (Anourosorex squamipes) with potential implications for evaluating crop impacts

Diet analysis of potential small mammals pest species is important for understanding feeding ecology and evaluating their impact on crops and stored foods. Chinese mole shrew (Anourosorex squamipes), distributed in Southwest China, has previously been reported as a farmland pest. Effective population management of this species requires a better understanding of its diet, which can be difficult to determine with high taxonomic resolution using conventional microhistological methods. In this study, we used two DNA metabarcoding assays to identify 38 animal species and 65 plant genera from shrew stomach contents, which suggest that A. squamipes is an omnivorous generalist. Earthworms are the most prevalent (>90%) and abundant (>80%) food items in the diverse diet of A. squamipes. Species of the Fabaceae (frequency of occurrence [FO]: 88%; such as peanuts) and Poaceae (FO: 71%; such as rice) families were the most common plant foods identified in the diet of A. squamipes. Additionally, we found a seasonal decrease in the diversity and abundance of invertebrate foods from spring and summer to winter. Chinese mole shrew has a diverse and flexible diet throughout the year to adapt to seasonal variations in food availability, contributing to its survival even when food resources are limited. This study provides a higher resolution identification of the diet of A. squamipes than has been previously described and is valuable for understanding shrew feeding ecology as well as evaluating possible species impacts on crops.     

DNA metabarcoding unveils multiscale trophic variation in a widespread coastal opportunist

A thorough understanding of ecological networks relies on comprehensive information on trophic relationships among species. Since unpicking the diet of many organisms is unattainable using traditional morphology‐based approaches, the application of high‐throughput sequencing methods represents a rapid and powerful way forward. Here, we assessed the application of DNA metabarcoding with nearly universal primers for the mitochondrial marker cytochrome c oxidase I in defining the trophic ecology of adult brown shrimp, Crangon crangon, in six European estuaries. The exact trophic role of this abundant and widespread coastal benthic species is somewhat controversial, while information on geographical variation remains scant. Results revealed a highly opportunistic behaviour. Shrimp stomach contents contained hundreds of taxa (>1,000 molecular operational taxonomic units), of which 291 were identified as distinct species, belonging to 35 phyla. Only twenty ascertained species had a mean relative abundance of more than 0.5%. Predominant species included other abundant coastal and estuarine taxa, including the shore crab Carcinus maenas and the amphipod Corophium volutator. Jacobs’ selectivity index estimates based on DNA extracted from both shrimp stomachs and sediment samples were used to assess the shrimp's trophic niche indicating a generalist diet, dominated by crustaceans, polychaetes and fish. Spatial variation in diet composition, at regional and local scales, confirmed the highly flexible nature of this trophic opportunist. Furthermore, the detection of a prevalent, possibly endoparasitic fungus (Purpureocillium lilacinum) in the shrimp's stomach demonstrates the wide range of questions that can be addressed using metabarcoding, towards a more robust reconstruction of ecological networks.     

Metabarcoding reveals diet diversity in an ungulate community in Thailand

The diverse large mammal communities found in Asian dry forests and savannas should segregate based on their diet selection. We examined the diet composition of sympatric ungulate species using metabarcoding to determine whether their diet was segregated and whether obvious attributes (i.e., body size, phylogeny, ecology) explained the structure. We collected fecal samples from eight ungulate species in Huai Kha Khaeng Wildlife Sanctuary in the western forest complex of Thailand. The fecal collections occurred around a plot where all woody species were codified within a genetic barcode library, and this library was supplemented with samples from plant species known to be consumed by these species. Of 273 plant species tested, at least 93 were found within the fecal samples. Over half of the identified species were not previously known by experts as forage species. All ungulate species showed a strong consumption of grasses and forbs. For the three species with sufficient sample size (sambar, banteng, and guar), there were seasonal differences in their diet, with each showing increased occurrence of woody plants during the dry season. The pattern of forage consumption did not follow obvious paradigms of body size or taxonomy, with significant diet differences found in two similar‐sized bovids (gaur, banteng), while the diet of sambar was more similar to bovids than to the other deer species. Asian ungulates differ in their forage consumption and metabarcoding should allow for testing of diet shifts in response to seasonal rains and fires which dominate the phenology of Asian dry forests and savannas.