Air-quality networks collect environmental DNA with the potential to measure biodiversity at continental scales.

One of the biggest planetary challenges is the accelerating loss of biodiversity threatening ecosystem functioning on a global scale. The WWF Living Planet Report (https://livingplanet.panda.org/) estimates a 69% decline in populations since 1970. The Convention on Biological Diversity and related international treaties ask countries to monitor shifts in community composition and assess rates of species decline to quantify extant biodiversity relative to global targets1. However, quantifying biodiversity is a challenge, and monitoring continual change is impossible at almost any scale due to a lack of standardized data and indicators2,3. A common problem is that the required infrastructure for such global monitoring does not exist. Here, we challenge this notion by analysing environmental DNA (eDNA) captured along with particulate matter by routine ambient air quality monitoring stations in the UK. In our samples, we identified eDNA from >180 vertebrate, arthropod, plant and fungal taxa representative of local biodiversity. We contend that air monitoring networks are in fact gathering eDNA data reflecting local biodiversity on a continental scale, as a result of their routine function. In some regions, air quality samples are stored for decades, presenting the potential for high resolution biodiversity time series. With minimal modification of current protocols, this material provides the best opportunity to date for detailed monitoring of terrestrial biodiversity using an existing, replicated transnational design and it is already in operation.

Out of thin air: surveying tropical bat roosts through air sampling of eDNA.

Understanding roosting behaviour is essential to bat conservation and biomonitoring, often providing the most accurate methods of assessing bat population size and health. However, roosts can be challenging to survey, e.g., physically impossible to access or presenting risks for researchers. Disturbance during monitoring can also disrupt natural bat behaviour and present material risks to the population such as disrupting hibernation cycles. One solution to this is the use of non-invasive monitoring approaches. Environmental (e)DNA has proven especially effective at detecting rare and elusive species particularly in hard-to-reach locations. It has recently been demonstrated that eDNA from vertebrates is carried in air. When collected in semi-confined spaces, this airborne eDNA can provide remarkably accurate profiles of biodiversity, even in complex tropical communities. In this study, we deploy novel airborne eDNA collection for the first time in a natural setting and use this approach to survey difficult to access potential roosts in the neotropics. Using airborne eDNA, we confirmed the presence of bats in nine out of 12 roosts. The identified species matched previous records of roost use obtained from photographic and live capture methods, thus demonstrating the utility of this approach. We also detected the presence of the white-winged vampire bat (Diaemus youngi) which had never been confirmed in the area but was long suspected based on range maps. In addition to the bats, we detected several non-bat vertebrates, including the big-eared climbing rat (Ototylomys phyllotis), which has previously been observed in and around bat roosts in our study area. We also detected eDNA from other local species known to be in the vicinity. Using airborne eDNA to detect new roosts and monitor known populations, particularly when species turnover is rapid, could maximize efficiency for surveyors while minimizing disturbance to the animals. This study presents the first applied use of airborne eDNA collection for ecological analysis moving beyond proof of concept to demonstrate a clear utility for this technology in the wild.

Non-target effects of agri-environmental schemes on solitary bees and fungi in the United Kingdom.

Agri-environmental schemes (AES) are used to enhance pollinator diversity on agricultural farms within the UK. Though the impacts of these schemes on archetypal pollinator species such as the bumblebee (Bombus) and honeybee (Apis) are well-studied, the effects on non-target bee species like solitary bees, in the same environment, are generally lacking. One goal of AES is to alter floral provision and taxonomic composition of plant communities to provide better forage for pollinators, however, this may potentially impact other ecological communities such as fungal diversity associated with plant-bee communities. Fungi are integral in these bee communities as they can impact bee species both beneficially and detrimentally. We test the hypothesis that alteration of the environment through provision of novel plant communities has non-target effects on the fungi associated with solitary bee communities. We analyse fungal diversity and ecological networks formed between fungi and solitary bees present on 15 agricultural farms in the UK using samples from brood cells. The farms were allocated to two categories, low and high management, which differ in the number of agri-environmental measures implemented. Using internal transcribed spacer metabarcoding, we identified 456 fungal taxa that interact with solitary bees. Of these, 202 (approximately 44%) could be assigned to functional groups, the majority being pathotrophic and saprotrophic species. A large proportion was Ascosphaeraceae, a family of bee-specialist fungi. We considered the connectance, nestedness, modularity, nestedness overlap and decreasing fill, linkage density and fungal generality of the farms' bee-fungi ecological networks. We found no difference in the structure of bee-fungi ecological networks between low and high management farms, suggesting floral provision by AES has no significant impact on interactions between these two taxonomic groups. However, bee emergence was lower on the low management farms compared to high management, suggesting some limited non-target effects of AES. This study characterizes the fungal community associated with solitary bees and provides evidence that floral provision through AES does not impact fungal interactions.

Shunning the scoop: Sidestepping the race to publish.

What happens when a researcher finds out that research very similar to their own is already being conducted? What if they find out that the said research is also very close to being published? First, there is probably anxiety and panic. Maybe, there are frantic calls to collaborators. Perhaps Twitter rants about the phenomenon of scooping that plagues all researchers, especially those early-career researchers who often feel they are in a race to get their best work out to the world.

Detection rates of aphid DNA in the guts of larval hoverflies and potential links to the provision of floral resources.

Aphidophagous hoverflies (Diptera, Syrphidae, Syrphinae) are common flower visitors and aphid predators in a range of flowering plants, including fruit crops. Here, we investigate whether aphid prey DNA can be detected in the gut contents of hoverfly larvae from a commercial strawberry field as a proof of concept that a molecular approach can be used to measure agricultural biocontrol. We used high-throughput sequencing (HTS) to target insect DNA and compared the resulting data to reference databases containing aphid and hoverfly DNA sequences. We explored what impact incorporating wildflowers within polythene-clad tunnels may have on aphid DNA detection rates in hoverfly larvae. In a randomized block experiment, coriander (Coriandrum sativum), field forget-me-not (Myosotis arvensis) and corn mint (Mentha arvensis) plants were inserted in rows of strawberries. Their effect on aphid DNA detection rates was assessed. Aphid DNA was found in 55 of 149 specimens (37%) validating the method in principle for measuring agricultural services provided by hoverflies. Interestingly, detection rates were higher near plots with forget-me-not than plots with coriander, though detection rates in control plots did not differ significantly from either wildflower species. These findings confirm that hoverflies predate aphids in UK strawberry fields, and that HTS is a viable method of identifying aphid DNA in predatory hoverflies. We comment on the need for further method development to narrow down identifications of both predator and prey. We furthermore provide some evidence that there is an effect of intercropping strawberry crops with wildflowers which may affect aphid consumption in hoverfly larvae.

The structure of tropical bat-plant interaction networks during an extreme El Niño-Southern Oscillation event.

Interaction network structure reflects the ecological mechanisms acting within biological communities, which are affected by environmental conditions. In tropical forests, higher precipitation usually increases fruit production, which may lead frugivores to increase specialization, resulting in more modular and less nested animal-plant networks. In these ecosystems, El Niño is a major driver of precipitation, but we still lack knowledge of how species interactions change under this influence. To understand bat-plant network structure during an extreme El Niño-Southern Oscillation event, we determined the links between plantivorous bat species and the plants they consume by DNA barcoding seeds and pulp in bat faeces. These interactions were recorded in the dry forest and rainforest of Costa Rica, during the dry and the wet seasons of an extreme El Niño year. From these we constructed seasonal and whole-year bat-plant networks and analysed their structures and dissimilarities. In general, networks had low nestedness, had high modularity, and were dominated by one large compartment which included most species and interactions. Contrary to our expectations, networks were less nested and more modular in drier conditions, both in the comparison between forest types and between seasons. We suggest that increased competition, when resources are scarce during drier seasons and habitats, lead to higher resource partitioning among bats and thus higher modularity. Moreover, we have found similar network structures between dry and rainforests during El Niño and non-El Niño years. Finally, most interaction dissimilarity among networks occurred due to interaction rewiring among species, potentially driven by seasonal changes in resource availability.

Measuring biodiversity from DNA in the air.

The crisis of declining biodiversity1 exceeds our current ability to monitor changes in ecosystems. Rapid terrestrial biomonitoring approaches are essential to quantify the causes and consequences of global change. Environmental DNA2 has revolutionized aquatic ecology,3 permitting population monitoring4 and remote diversity assessments matching or outperforming conventional methods of community sampling.3-5 Despite this model, similar methods have not been widely adopted in terrestrial ecosystems. Here, we demonstrate that DNA from terrestrial animals can be filtered, amplified, and then sequenced from air samples collected in natural settings representing a powerful tool for terrestrial ecology. We collected air samples at a zoological park, where spatially confined non-native species allowed us to track DNA sources. We show that DNA can be collected from air and used to identify species and their ecological interactions. Air samples contained DNA from 25 species of mammals and birds, including 17 known terrestrial resident zoo species. We also identified food items from air sampled in enclosures and detected taxa native to the local area, including the Eurasian hedgehog, endangered in the United Kingdom. Our data demonstrate that airborne eDNA concentrates around recently inhabited areas but disperses away from sources, suggesting an ecology to airborne eDNA and the potential for sampling at a distance. Our findings demonstrate the profound potential of air as a source of DNA for global terrestrial biomonitoring.

Cascading effects of habitat loss on ectoparasite-associated bacterial microbiomes.

Suitable habitat fragment size, isolation, and distance from a source are important variables influencing community composition of plants and animals, but the role of these environmental factors in determining composition and variation of host-associated microbial communities is poorly known. In parasite-associated microbial communities, it is hypothesized that evolution and ecology of an arthropod parasite will influence its microbiome more than broader environmental factors, but this hypothesis has not been extensively tested. To examine the influence of the broader environment on the parasite microbiome, we applied high-throughput sequencing of the V4 region of 16S rRNA to characterize the microbiome of 222 obligate ectoparasitic bat flies (Streblidae and Nycteribiidae) collected from 155 bats (representing six species) from ten habitat fragments in the Atlantic Forest of Brazil. Parasite species identity is the strongest driver of microbiome composition. To a lesser extent, reduction in habitat fragment area, but not isolation, is associated with an increase in connectance and betweenness centrality of bacterial association networks driven by changes in the diversity of the parasite community. Controlling for the parasite community, bacterial network topology covaries with habitat patch area and exhibits parasite-species specific responses to environmental change. Taken together, habitat loss may have cascading consequences for communities of interacting macro- and microorgansims.

Dung beetles as samplers of mammals in Malaysian Borneo-a test of high throughput metabarcoding of iDNA.

Invertebrate-derived DNA (iDNA) sampling in biodiversity surveys is becoming increasingly widespread, with most terrestrial studies relying on DNA derived from the gut contents of blood-feeding invertebrates, such as leeches and mosquitoes. Dung beetles (superfamily Scarabaeoidea) primarily feed on the faecal matter of terrestrial vertebrates and offer several potential benefits over blood-feeding invertebrates as samplers of vertebrate DNA. Importantly, these beetles can be easily captured in large numbers using simple, inexpensive baited traps, are globally distributed, and occur in a wide range of habitats. To build on the few existing studies demonstrating the potential of dung beetles as sources of mammalian DNA, we subjected the large-bodied, Bornean dung beetle (Catharsius renaudpauliani) to a controlled feeding experiment. We analysed DNA from gut contents at different times after feeding using qPCR techniques. Here, we first describe the window of DNA persistence within a dung beetle digestive tract. We found that the ability to successfully amplify cattle DNA decayed over relatively short time periods, with DNA copy number decreasing by two orders of magnitude in just 6 h. In addition, we sampled communities of dung beetles from a lowland tropical rainforest in Sabah, Malaysia, in order to test whether it is possible to identify vertebrate sequences from dung beetle iDNA. We sequenced both the gut contents from large dung beetle species, as well as whole communities of smaller beetles. We successfully identified six mammalian species from our samples, including the bearded pig (Sus barbatus) and the sambar deer (Rusa unicolor)-both vulnerable species on the IUCN red list. Our results represent the first use of dung beetle iDNA to sample Southeast Asian vertebrate fauna, and highlight the potential for dung beetle iDNA to be used in future biodiversity monitoring surveys.

Altered structure of bat-prey interaction networks in logged tropical forests revealed by metabarcoding.

Habitat degradation is pervasive across the tropics and is particularly acute in Southeast Asia, with major implications for biodiversity. Much research has addressed the impact of degradation on species diversity; however, little is known about how ecological interactions are altered, including those that constitute important ecosystem functions such as consumption of herbivores. To examine how rainforest degradation alters trophic interaction networks, we applied DNA metabarcoding to construct interaction networks linking forest-dwelling insectivorous bat species and their prey, comparing old-growth forest and forest degraded by logging in Sabah, Borneo. Individual bats in logged rainforest consumed a lower richness of prey than those in old-growth forest. As a result, interaction networks in logged forests had a less nested structure. These network structures were associated with reduced network redundancy and thus increased vulnerability to perturbations in logged forests. Our results show how ecological interactions change between old-growth and logged forests, with potentially negative implications for ecosystem function and network stability.

eDNAir: proof of concept that animal DNA can be collected from air sampling.

Environmental DNA (eDNA) is one of the fastest developing tools for species biomonitoring and ecological research. However, despite substantial interest from research, commercial and regulatory sectors, it has remained primarily a tool for aquatic systems with a small amount of work in substances such as soil, snow and rain. Here we demonstrate that eDNA can be collected from air and used to identify mammals. Our proof of concept successfully demonstrated that eDNA sampled from air contained mixed templates which reflect the species known to be present within a confined space and that this material can be accessed using existing sampling methods. We anticipate this demonstration will initiate a much larger research programme in terrestrial airDNA sampling and that this may rapidly advance biomonitoring approaches. Lastly, we outline these and potential related applications we expect to benefit from this development.

Exploratory analysis reveals arthropod consumption in 10 lemur species using DNA metabarcoding.

Arthropods (insects, spiders, etc.) can fulfill major nutritional requirements for primates, particularly in terms of proteins, fats, vitamins, and minerals. Yet, for many primate species we know very little about the frequency and importance of arthropod consumption. Traditional methods for arthropod prey identification, such as behavioral observations and fecal dissections, offer limited taxonomic resolution and, as a result, underestimate true diversity. Metabarcoding arthropod DNA from primate fecal samples provides a promising but underused alternative. Here, we inventoried arthropod prey diversity in wild lemurs by sequencing two regions of the CO1 gene. Samples were collected opportunistically from 10 species of lemurs inhabiting three national parks in southern Madagascar using a combination of focal animal follows and live trapping. In total, we detected arthropod DNA in 98 of the 170 fecal samples analyzed. Although all lemur species included in these analyses showed evidence of arthropod consumption, those within the family Cheirogaleidae appeared to consume the highest frequency and diversity of arthropods. To our knowledge, this study presents the first evidence of arthropod consumption in Phaner pallescens, Avahi peyrierasi, and Propithecus verreauxi, and identifies 32 families of arthropods as probable food items that have not been published as lemur dietary items to date. Our study emphasizes the importance of arthropods as a nutritional source and the role DNA metabarcoding can play in elucidating an animal's diet.

Assessing the impact of taxon resolution on network structure.

Constructing ecological networks has become an indispensable approach in understanding how different taxa interact. However, the methods used to generate data in network research vary widely among studies, potentially limiting our ability to compare results meaningfully. In particular, methods of classifying nodes vary in their precision, likely altering the architecture of the network studied. For example, rather than being classified as Linnaean species, taxa are regularly assigned to morphospecies in observational studies, or to molecular operational taxonomic units (MOTUs) in molecular studies, with the latter defined based on an arbitrary threshold of sequence similarity. Although the use of MOTUs in ecological networks holds great potential, especially for allowing rapid construction of large data sets of interactions, it is unclear how the choice of clustering threshold can influence the conclusions obtained. To test the impact of taxonomic precision on network architecture, we obtained and analyzed 16 data sets of ecological interactions, inferred from metabarcoding and observations. Our comparisons of networks constructed under a range of sequence thresholds for assigning taxa demonstrate that even small changes in node resolution can cause wide variation in almost all key metric values. Moreover, relative values of commonly used metrics such as robustness were seen to fluctuate continuously with node resolution, thereby potentially causing error in conclusions drawn when comparing multiple networks. In observational networks, we found that changing node resolution could, in some cases, lead to substantial changes to measurements of network topology. Overall, our findings highlight the importance of classifying nodes to the greatest precision possible, and demonstrate the need for caution when comparing networks that differ with respect to node resolution, even where taxonomic groups and interaction types are similar. In such cases, we recommend that comparisons of networks should focus on relative differences rather than absolute values between the networks studied.

Differential effects of fertilisers on pollination and parasitoid interaction networks.

Grassland fertilisation drives non-random plant loss resulting in areas dominated by perennial grass species. How these changes cascade through linked trophic levels, however, is not well understood. We studied how grassland fertilisation propagates change through the plant assemblage into the plant-flower-visitor, plant-leaf miner and leaf miner-parasitoid networks using a year's data collection from a long-term grassland fertiliser application experiment. Our experiment had three fertiliser treatments each applied to replicate plots 15 m2 in size: mineral fertiliser, farmyard manure, and mineral fertiliser and farmyard manure combined, along with a control of no fertiliser. The combined treatment had the most significant impact, and both plant species richness and floral abundance decreased with the addition of fertiliser. While insect species richness was unaffected by fertiliser treatment, fertilised plots had a significantly higher abundance of leaf miners and parasitoids and a significantly lower abundance of bumblebees. The plant-flower-visitor and plant-herbivore networks showed higher values of vulnerability and lower modularity with fertiliser addition, while leaf miner-parasitoid networks showed a rise in generality. The different groups of insects were impacted by fertilisers to varying degrees: while the effect on abundance was the highest for leaf miners, the vulnerability and modularity of flower-visitor networks was the most affected. The impact on the abundance of leaf miners was positive and three times higher than the impact on parasitoids, and the impact on bumblebee abundance was negative and double the magnitude of impact on flower abundance. Overall, our results show that while insect species richness was unaffected by fertilisers, network structure changed significantly as the replacement of forbs by grasses resulted in changes in relative abundance across trophic levels, with the direction of change depending on the type of network. Synthesis. By studying multiple networks simultaneously, we were able to rank the relative impact of habitat change on the different groups of species within the community. This provided a more holistic picture of the impact of agricultural intensification and provides useful information when deciding on priorities for mitigation.

Leech blood-meal invertebrate-derived DNA reveals differences in Bornean mammal diversity across habitats.

The application of metabarcoding to environmental and invertebrate-derived DNA (eDNA and iDNA) is a new and increasingly applied method for monitoring biodiversity across a diverse range of habitats. This approach is particularly promising for sampling in the biodiverse humid tropics, where rapid land-use change for agriculture means there is a growing need to understand the conservation value of the remaining mosaic and degraded landscapes. Here we use iDNA from blood-feeding leeches (Haemadipsa picta) to assess differences in mammalian diversity across a gradient of forest degradation in Sabah, Malaysian Borneo. We screened 557 individual leeches for mammal DNA by targeting fragments of the 16S rRNA gene and detected 14 mammalian genera. We recorded lower mammal diversity in the most heavily degraded forest compared to higher quality twice logged forest. Although the accumulation curves of diversity estimates were comparable across these habitat types, diversity was higher in twice logged forest, with more taxa of conservation concern. In addition, our analysis revealed differences between the community recorded in the heavily logged forest and that of the twice logged forest. By revealing differences in mammal diversity across a human-modified tropical landscape, our study demonstrates the value of iDNA as a noninvasive biomonitoring approach in conservation assessments.

Wing morphology predicts individual niche specialization in Pteronotus mesoamericanus (Mammalia: Chiroptera).

Morphological variation between individuals can increase niche segregation and decrease intraspecific competition when heterogeneous individuals explore their environment in different ways. Among bat species, wing shape correlates with flight maneuverability and habitat use, with species that possess broader wings typically foraging in more cluttered habitats. However, few studies have investigated the role of morphological variation in bats for niche partitioning at the individual level. To determine the relationship between wing shape and diet, we studied a population of the insectivorous bat species Pteronotus mesoamericanus in the dry forest of Costa Rica. Individual diet was resolved using DNA metabarcoding, and bat wing shape was assessed using geometric morphometric analysis. Inter-individual variation in wing shape showed a significant relationship with both dietary dissimilarity based on Bray-Curtis estimates, and nestedness derived from an ecological network. Individual bats with broader and more rounded wings were found to feed on a greater diversity of arthropods (less nested) in comparison to individuals with triangular and pointed wings (more nested). We conclude that individual variation in bat wing morphology can impact foraging efficiency leading to the observed overall patterns of diet specialization and differentiation within the population.

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.

Chemical characterisation of potential pheromones from the shoulder gland of the Northern yellow-shouldered-bat, Sturnira parvidens (Phyllostomidae: Stenodermatinae).

Bats of the genus Sturnira (Family Phyllostomidae) are characterised by shoulder glands that are more developed in reproductively mature adult males. The glands produce a waxy secretion that accumulates on the fur around the gland, dyeing the fur a dark colour and giving off a pungent odour. These shoulder glands are thought to play a role in their reproductive behaviour. Using gas chromatography-mass spectrometry, we analysed solvent extracts of fur surrounding the shoulder gland in the northern-shouldered bat, Sturnira parvidens to (i) characterise the chemical composition of shoulder gland secretions for the first time, and (ii) look for differences in chemical composition among and between adult males, sub-adult/juvenile males and adult females. Fur solvent extracts were analysed as liquids and also further extracted using headspace solid-phase microextraction to identify volatile components in the odour itself. Odour fingerprint analysis using non-metric multidimensional scaling plots and multivariate analysis revealed clear and significant differences (P < 0.001) between adult males vs both juvenile males and adult females. The chemical components of the shoulder gland secretion included terpenes and phenolics, together with alcohols and esters, most likely derived from the frugivorous diet of the bat. Many of the compounds identified were found exclusively or in elevated quantities among adult (reproductive) males compared with adult females and non-reproductive (juvenile) males. This strongly suggests a specific role in male-female attraction although a function in male-male competition and/or species recognition is also possible.

Molecular diet analysis finds an insectivorous desert bat community dominated by resource sharing despite diverse echolocation and foraging strategies.

Interspecific differences in traits can alter the relative niche use of species within the same environment. Bats provide an excellent model to study niche use because they use a wide variety of behavioral, acoustic, and morphological traits that may lead to multi-species, functional groups. Predatory bats have been classified by their foraging location (edge, clutter, open space), ability to use aerial hawking or substrate gleaning and echolocation call design and flexibility, all of which may dictate their prey use. For example, high frequency, broadband calls do not travel far but offer high object resolution while high intensity, low frequency calls travel further but provide lower resolution. Because these behaviors can be flexible, four behavioral categories have been proposed: (a) gleaning, (b) behaviorally flexible (gleaning and hawking), (c) clutter-tolerant hawking, and (d) open space hawking. Many recent studies of diet in bats use molecular tools to identify prey but mainly focus on one or two species in isolation; few studies provide evidence for substantial differences in prey use despite the many behavioral, acoustic, and morphological differences. Here, we analyze the diet of 17 sympatric species in the Chihuahuan desert and test the hypothesis that peak echolocation frequency and behavioral categories are linked to differences in diet. We find no significant correlation between dietary richness and echolocation peak frequency though it spanned close to 100 kHz across species. Our data, however, suggest that bats which use both gleaning and hawking strategies have the broadest diets and are most differentiated from clutter-tolerant aerial hawking species.

Counting with DNA in metabarcoding studies: How should we convert sequence reads to dietary data?

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.