DNA Metabarcoding Approach Research Articles

DNA Metabarcoding Across Disciplines: Sequencing Our Way to Greater Understanding Across Scales of Biological Organization.

DNA metabarcoding describes the use of targeted DNA (i.e., amplicon) sequencing to identify community constituents from a complex sample containing genetic material from multiple organisms, such as water, soil, gut contents, microbiomes, or biofilms. This molecular approach for characterizing mixed DNA samples relies on the development of "universal primers" that allow for effective amplification of target sequences across a broad range of taxa. Armed with optimized lab protocols and rigorous bioinformatics tools, DNA metabarcoding can produce a wealth of information about the hidden biodiversity of various sample types by probing for organisms' molecular footprints. DNA metabarcoding has received considerable popular press over the last few years because of gut microbiome studies in humans and beyond. However, there are many other applications that are continually integrating molecular biology with other fields of study to address questions that have previously been unanswerable, for both prokaryotic and eukaryotic targets. For example, we can now sample mostly digested gut contents from virtually any organism to learn about ontogeny and foraging ecology. Water samples collected from different locations can be filtered to extract eDNA (i.e., environmental DNA), revealing the biodiversity of fish and other taxa targeted by carefully selected primer sets. This universal primer metabarcoding approach has even been extended to looking at diverse gene families within single species, which is particularly useful for complex immune system genetics. The purpose of this SICB symposium was to bring together researchers using DNA metabarcoding approaches to (a) showcase the diversity of applications of this technique for addressing questions spanning ecology, evolution, and physiology, and (b) to spark connections among investigators from different fields that are utilizing similar approaches to facilitate optimization and standardization of metabarcoding methods and analyses. The resulting manuscripts from this symposium represent a great diversity of metabarcoding applications and taxonomic groups of interest.

Diet analysis of bats killed at wind turbines suggests large‐scale losses of trophic interactions

AbstractAgricultural practice has led to landscape simplification and biodiversity decline, yet recently, energy‐producing infrastructures, such as wind turbines, have been added to these simplified agroecosystems, turning them into multi‐functional energy‐agroecosystems. Here, we studied the trophic interactions of bats killed at wind turbines using a DNA metabarcoding approach to shed light on how turbine‐related bat fatalities may possibly affect local habitats. Specifically, we identified insect DNA in the stomachs of common noctule bats (Nyctalus noctula) killed by wind turbines in Germany to infer in which habitats these bats hunted. Common noctule bats consumed a wide variety of insects from different habitats, ranging from aquatic to terrestrial ecosystems (e.g., wetlands, farmland, forests, and grasslands). Agricultural and silvicultural pest insects made up about 20% of insect species consumed by the studied bats. Our study suggests that the potential damage of wind energy production goes beyond the loss of bats and the decline of bat populations. Bat fatalities at wind turbines may lead to the loss of trophic interactions and ecosystem services provided by bats, which may add to the functional simplification and impaired crop production, respectively, in multi‐functional ecosystems.

Metabarcoding analysis of different portions of the digestive tract of scorpions (Scorpiones, Arachnida) following a controlled diet regime shows long prey DNA half‐life

AbstractMolecular analysis of gut content is one of the widest used methods to investigate diet in arthropods. Stomach content analysis in some arthropods is particularly difficult, e.g., in arachnids, because they have external digestion and a low foraging frequency. Scorpions have a particularly low feeding frequency, and their diet information is scarce. In this work we explore a DNA metabarcoding approach to detect prey DNA in Vietnamese forest scorpions (Heterometrus laoticus) under a controlled diet regime. A different type of prey (crickets, mealworms, and cockroaches) was offered once every 3 weeks for a total of 9 weeks. To assess the most suitable part of the digestive system and extraction method to use for molecular diet analysis, we separately analyzed three different portions of the digestive tract of scorpions (mid‐gut, hepatopancreas, and hindgut) using two different extraction methods (salt‐out method and a customized beads‐based protocol). We calculated the detectability half‐life of the prey DNA for each digestive tract section. We detected all three targeted prey items, showing that in scorpions multiple predation events can be distinguished in the same specimen within its last 9 weeks of foraging activity. The hepatopancreas was the portion of the digestive tract that provided the best prey detection and the longest DNA detectability half‐life (51 days), followed by the mid‐gut (22 days) and the hindgut (16 days). We found no significant difference between the extraction methods used. However, the salt‐out method was less effective in some of the PCRs and is therefore not recommended for molecular diet analysis.

DNA metabarcoding unveils the effects of habitat fragmentation on pollinator diversity, plant-pollinator interactions, and pollination efficiency in Maldive islands.

Habitat fragmentation affects biodiversity, but with unclear effects on pollinators and their interactions with plants in anthropized landscapes. Islands could serve as open air laboratories, suitable to disentangle how land-use alteration impacts pollination ecology. In Maldive islands we investigated how pollinator richness, plant-pollinator interactions and pollination efficiency are influenced by the green area fragmentation (i.e., gardens and semi-natural patches). Moreover, we considered the mediating role of pollinator body size and the plant trait of being invasive in shaping interactions. To do this, we surveyed pollinator insects from 11 islands representing a gradient of green area fragmentation. A DNA metabarcoding approach was adopted to identify the pollen transported by pollinators and characterize the plant-pollinator interactions. We found that intermediate levels of green area fragmentation characterized pollinator communities and increased their species richness, while decreasing interaction network complexity. Invasive plants were more frequently found on pollinator bodies than native or exotic noninvasive ones, indicating a concerningly higher potential for pollen dispersal and reproduction of the former ones. Intriguingly, pollinator body size mediated the effect of landscape alteration on interactions, as only the largest bees expanded the foraging diet in terms of plant richness in the transported pollen at increasing fragmentation. In parallel, the pollination efficiency increased with pollinator species richness in two sentinel plants. This study shows that moderate landscape fragmentation of green areas shapes many aspects of the pollination ecosystem service, where despite interactions being less complex and mediated by pollinator body size, pollinator insect biodiversity and potential plant reproduction are supported.

Current methodologies in construction of plant-pollinator network with emphasize on the application of DNA metabarcoding approach

Current methodologies in construction of plant-pollinator network with emphasize on the application of DNA metabarcoding approach

A Comparison of Fish Diversity in Rocky Reef Habitats by Multi-Mesh Gillnets and Environmental DNA Metabarcoding

This study was undertaken in order to explore the practical effectiveness of the environmental DNA (eDNA) metabarcoding approach in evaluating fish composition and diversity in a high heterogeneous rocky reef habitat. We assessed the fish composition and diversity characteristics of the rocky reef habitat at Dachen Islands, Taizhou and the Zhejiang Province in China in November 2020 by comparing two methods: multi-mesh gillnets and eDNA. A comparative analysis was carried out on the fish composition and diversity characteristics gained under the two methods by using taxonomy, ecotypes and diversity indices. The results showed that there were 28 species of fish collected through gillnets, distributed under 24 genera, 19 families, 6 orders and one class. Among them, 4, 18, and 6 species of near-surface, near groundfish and groundfish were found, respectively, with Thryssa mystax, Johnius belangerii, and Sebastiscus marmoratus being the dominant species in each water layer. A total of 81 species of fish detected by eDNA metabarcoding belonging to 67 genera, 46 families, 15 orders and 2 classes. The near-surface, near groundfish and groundfish species were 17, 42, and 22, with Thryssa vitrirostris, Benthosema pterotum, Harpadon nehereus, and Dasyatis akajei being the dominant species in each water layer. Twenty species (71.4%) and 41 species (50.6%) of reef fish were counted by gillnets and eDNA, respectively. The results showed that multi-mesh gillnets can accurately obtain information on fish composition in rocky reef habitats, but with some selectivity. The eDNA technology can detect species not collected by gillnets, but the number of species detected in areas with fast water velocity is significantly less than other eDNA stations where the water velocity is slow. In summary, the combination of traditional nets and eDNA will provide more information on taxonomic diversity and population biomass, transforming natural resource management and ecological studies of fish communities on a larger spatial and temporal scale.

Using high-throughput amplicon sequencing to determine diet of generalist lady beetles in agricultural landscapes

Determining feeding relationships is central to understanding biological control potential in the field. However, methods to differentiate actual (or realized) feeding relationships from potential feeding relationships is lacking especially for small, generalist predators such as lady beetles. In this study, we used DNA metabarcoding approaches to characterize actual feeding relationships of lady beetles (Coccinellidae) in the field and validated our methods with a lab study. We first asked whether high-throughput amplicon sequencing (HTS) can characterize diets of lady beetles ranging from monotypic diets to diverse diet mixtures in the lab. We then examined whether diet composition and breadth of lady beetles collected from different habitat types in southern WI varied between monocultures of soybean and corn, diverse tallgrass prairie, and urban habitats. Lastly, we asked whether different body or tissue types (partial-body versus whole-body specimens) would change the likelihood of prey detection for both studies. In our controlled lab study, we found that HTS can accurately assess diet composition and diet breadth for lady beetle populations, but at the individual level, HTS has limitation for individuals feeding on more than three species of prey at any given time. In our field study, we documented lower prey richness and diet breadth in corn compared to soybean and grassland, and greater prey DNA in regurgitants than in whole-body specimens. Finally, we found that reduced diet diversity was associated with an increased prevalence of intraguild predation, but that habitat and prey diversity are not necessarily correlated. The prey detection rates (proportion of specimens with prey DNA) in our study were comparable to other studies (55% in the lab study, 33% in field study) and varied with diet composition and habitat type. The relatively low detection rates suggest that many consumer individuals would need to be assayed to fully assess diet diversity, especially in diverse systems.

A novel approach for reliable qualitative and quantitative prey spectra identification of carnivorous plants combining DNA metabarcoding and macro photography

Prey spectra (the number and composition of captured arthropods) represent a crucial aspect of carnivorous plant ecology, yet remain poorly studied. Traditional morphology-based approaches for prey identification are time-intensive, require specialists with considerable knowledge of arthropod taxonomy, and are hampered by high numbers of unidentifiable (i.e., heavily digested) prey items. We examined prey spectra of three species of closely-related annual Drosera (Droseraceae, sundews) from tropical northern Australia using a novel DNA metabarcoding approach with in-situ macro photography as a plausibility control and to facilitate prey quantity estimations. This new method facilitated accurate analyses of carnivorous plant prey spectra (even of heavily digested prey lacking characteristic morphological features) at a taxonomic resolution and level of completeness far exceeding morphology-based methods and approaching the 100% mark at arthropod order level. Although the three studied species exhibited significant differences in detected prey spectra, little prey specialisation was observed and habitat or plant population density variations were likely the main drivers of prey spectra dissimilarity.

Seasonal progression and differences in major floral resource use by bees and hoverflies in a diverse horticultural and agricultural landscape revealed by DNA metabarcoding

Abstract Gardens are important habitats for pollinators, providing floral resources and nesting sites. There are high levels of public support for growing ‘pollinator‐friendly’ plants but while plant recommendation lists are available, they are usually inconsistent, poorly supported by scientific research and target a narrow group of pollinators. In order to supply the most appropriate resources, there is a clear need to understand foraging preferences, for a range of pollinators, across the season within horticultural landscapes. Using an innovative DNA metabarcoding approach, we investigated foraging preferences of four groups of pollinators in a large and diverse, horticultural and agricultural landscape, across the flowering season and over 2 years, significantly improving on the spatial and temporal scale that can be achieved using observational studies. Bumblebees, honeybees, non‐corbiculate bees and hoverflies visited 191 plant taxa. Overall floral resources were shared between the different types of pollinators, but significant differences were seen between the plants used most abundantly by bees (Hymenoptera) and hoverflies (Diptera). Floral resource use by pollinators is strongly associated with seasonal changes in flowering plants, with pollinators relying on dominant plants found within each season, with preferences consistent across both years. The plants identified were categorised according to their native status to investigate the value of native and non‐native plants. The majority of floral resources used were of native and near‐native origin, but the proportion of horticultural and naturalised plants increased during late summer and autumn. Synthesis and applications. Plant recommendation lists for pollinators should distinguish between bees and hoverflies and provide evidence‐based floral recommendations throughout the year that include native as well as non‐native plants for use in the United Kingdom and Northern Europe. Specific management recommendations include reducing mowing to encourage plants such as dandelion Taraxacum officinale and buttercups Ranunculus spp., and reducing scrub management to encourage bramble Rubus fruticosus.

DNA metabarcoding reveals human impacts on macroinvertebrate communities in polluted headwater streams: Evidence from the Liao River in northeast China

Headwater streams are a hotspot of freshwater biodiversity, carrying indispensable resource pools of aquatic species. However, up to now, there remain many challenges to accurately and efficiently characterize the responses of this vulnerable ecosystem to human-induced changes. Here, we collected macroinvertebrate data from 12 different headwater streams in the Liao River of northeast China by DNA metabarcoding approach, to reveal biodiversity changes and ecological thresholds affected by human beings. Our data showed that the community composition and structure of headwater streams had unique and significant differences under human impacts, and 5-day biological oxygen demand (BOD5) and ammonia nitrogen (NH3–N) were the key variables explaining the variation in community structure. Although α diversity had a unimodal relationship with nutrients and organic loads, β diversity and its turnover component (species replacement) increased significantly. In addition, 22 and 33 indicative taxa were identified to have significant negative responses to BOD5 and NH3–N, respectively, and the change points derived from Threshold Indicator Taxa Analysis (TITAN) for the negative response of their frequency and abundance were BOD5 >3.42 mg/L and NH3–N >0.14 mg/L. Overall, this study reveals the biodiversity changes in headwater streams from the aspects of α and β diversity, and also determines the thresholds of BOD5 and NH3–N pollutants for one reach at one date from 12 headwater streams, suggesting the potential of DNA metabarcoding approach for threshold analyses in headwater streams.

Framing Cutting-Edge Integrative Deep-Sea Biodiversity Monitoring via Environmental DNA and Optoacoustic Augmented Infrastructures

Deep-sea ecosystems are reservoirs of biodiversity that are largely unexplored, but their exploration and biodiscovery are becoming a reality thanks to biotechnological advances (e.g., omics technologies) and their integration in an expanding network of marine infrastructures for the exploration of the seas, such as cabled observatories. While still in its infancy, the application of environmental DNA (eDNA) metabarcoding approaches is revolutionizing marine biodiversity monitoring capability. Indeed, the analysis of eDNA in conjunction with the collection of multidisciplinary optoacoustic and environmental data, can provide a more comprehensive monitoring of deep-sea biodiversity. Here, we describe the potential for acquiring eDNA as a core component for the expanding ecological monitoring capabilities through cabled observatories and their docked Internet Operated Vehicles (IOVs), such as crawlers. Furthermore, we provide a critical overview of four areas of development: (i) Integrating eDNA with optoacoustic imaging; (ii) Development of eDNA repositories and cross-linking with other biodiversity databases; (iii) Artificial Intelligence for eDNA analyses and integration with imaging data; and (iv) Benefits of eDNA augmented observatories for the conservation and sustainable management of deep-sea biodiversity. Finally, we discuss the technical limitations and recommendations for future eDNA monitoring of the deep-sea. It is hoped that this review will frame the future direction of an exciting journey of biodiscovery in remote and yet vulnerable areas of our planet, with the overall aim to understand deep-sea biodiversity and hence manage and protect vital marine resources.

Seasonal variations in the plant diet of the Chinese Monal revealed by fecal DNA metabarcoding analysis

The Chinese Monal (Lophophorus lhuysii) is an alpine-obligate galliform species of global conservation priority. It has been listed as a first class protected wildlife species in China, requiring conservation actions during the 14th Five-Year Plan period. However, the diet composition of Chinese Monal and its seasonal variations have rarely been studied, constraining the effective conservation of the species. Here, we investigated the plant diet composition of the Chinese Monal and its seasonal variations using a DNA metabarcoding approach on fecal samples. We collected 190 fecal samples of the Chinese Monals from the central Qionglai Mountains located in China, and analyzed the plant diet of this species using a DNA metabarcoding approach. Taxonomic profiling of higher plants in the fecal samples was performed using the second internal transcribed spacer (ITS2) amplicon. Downstream analyses, including rarefaction curves, nonmetric multidimensional scaling (NMDS) and permutational multivariate analysis of variance (PERMANOVA), were used to explore the seasonal variations in diet composition. The Chinese Monal foraged a wide range of plant recipes composed of 35 families and 83 genera throughout the year, with Brassicaceae, Apiaceae, and Poaceae as the dominant families, and Cardamine as the dominant genus. The species consumed plants from 62 genera from 28 families during the breeding season (n ​= ​81) and 66 genera from 31 families during the non-breeding season (n ​= ​109). Further, the plant diet composition significantly varied between the breeding and non-breeding seasons, especially for the frequency of occurrence and relative read abundances at genus level. Our study analyzed the plant diet of the Chinese Monal at a high resolution for the first time, and the results revealed that the seasonal variations in its plant diet composition was adapted to plant phenology and foraging strategy. Fritillaria species, a previously confirmed important food resource for the Chinese Monal, were not detected in any fecal samples, potentially due to overharvesting of Fritillaria bulbs for Traditional Chinese Medicine. Therefore, we highly recommend further restriction of herb gathering in Chinese Monal habitats to facilitate the conservation of this endangered species. Altogether, our study enriches essential ecological information for the Chinese Monal and also provides insights into conservation management for this endangered species.

Using metabarcoding to assess Viridiplantae sequence diversity present in Antarctic glacial ice.

Antarctica contains most of the glacial ice on the planet, a habitat that is largely unexplored by biologists. Recent warming in parts of Antarctica, particularly the Antarctic Peninsula region, is leading to widespread glacial retreat, releasing melt water and, potentially, contained biological material and propagules. In this study, we used a DNA metabarcoding approach to characterize Viridiplantae DNA present in Antarctic glacial ice. Ice samples from six glaciers in the South Shetland Islands and Antarctic Peninsula were analysed, detecting the presence of DNA representing a total of 16 taxa including 11 Chlorophyta (green algae) and five Magnoliophyta (flowering plants). The green algae may indicate the presence of a viable algal community in the ice or simply of preserved DNA, and the sequence diversity assigned included representatives of Chlorophyta not previously recorded in Antarctica. The presence of flowering plant DNA is most likely to be associated with pollen or tissue fragments introduced by humans.

Bacterial assemblages of urban microbiomes mobilized by runoff waters match land use typologies and harbor core species involved in pollutant degradation and opportunistic human infections

Cities are patchworks of urban catchments divided into functional units according to their commercial, residential and industrial activities, and socio-urbanistic patterns. The hypothesis of city surface microbiomes being structured by socio-urbanistic variables leading to an emergence of synurbic taxa was tested. According to the r/K microbial ecology theory, a gradient of well-adapted synurbic K-strategists and of opportunistic –r-strategists should occur over city surfaces. K-strategists would be core components while r-ones would be transiently detected. To resolve these patterns, sub-catchments (n = 21) of an area of high commercial and industrial activities were investigated over three time periods covering one year. The sub-catchments' land use patterns and associated human behaviors were converted into socio-urbanistic variables and groupings. Bacterial cells mobilized by runoffs per sub-catchment were recovered, and analyzed by classical approaches, microbial source tracking DNA assays and DNA meta-barcoding approaches. Relationships between these datasets, the runoff physico-chemical properties, and descriptors of the socio-urbanistic groupings were investigated. 16S rRNA meta-barcoding analyses showed evidence of the occurrence of K- and r-like strategists. Twenty-eight core genera were identified, and correlation networks revealed large bacterial modules organized around actinobacterial taxa involved in hydrocarbon degradation processes. Other bacterial networks were related to the occurrences of hygienic wastes, and involved bacteria originating from fecal contaminations. Several r-strategists like Sulfurospirillum were recorded and found associated to point source pollutions. The tpm-metabarcoding approach deciphered these r / K strategists at the species level among more than ten genera. Nine core K-like Pseudomomas species were identified. The P. aeruginosa human opportunistic pathogen and P. syringae phytopathogens were part of these K-strategists. Other tpm-harboring bacterial pathogens showed r-like opportunistic distribution patterns. Correlation network analyses indicated a strong incidence of hygienic wastes and hydrocarbon-pollutions on tpm-harboring bacteria. These analyses demonstrated the occurrence of core synurbic bacterial K-strategists over city surfaces.

Using DNA metabarcoding to decipher the diet plant component of mammals from the Eastern Mediterranean region

Longevity of species populations depends largely on interactions among animals and plants in an ecosystem. Predation and seed dispersal are among the most important interactions necessary for species conservation and persistence, and diet analysis is a prerequisite tool to evaluate these interactions. Understanding these processes is crucial for identifying conservation targets and for executing efficient reforestation and ecological restoration. In this study, we applied a scat DNA metabarcoding technique using the P6-loop of thetrnL(UAA) chloroplastic marker to describe the seasonal plant diet composition of 15 mammal species from a highly biodiverse Lebanese forest in the Eastern Mediterranean. We also recovered plant seeds, when present, from the scats for identification. The mammal species belong to 10 families from 5 different orders. More than 133 plant species from 54 plant families were detected and identified. Species from the Rosaceae, Poaceae, Apiaceae, Fabaceae, Fagaceae and Berberidaceae families were consumed by the majority of the mammals and should be taken into consideration in future reforestation and conservation projects. Our results showed that the DNA metabarcoding approach provides a promising method for tracking the dietary plant components of a wide diversity of mammals, yielding key insights into plant-animal interactions inside Lebanon’s forests.

Comparing whole-genome shotgun sequencing and DNA metabarcoding approaches for species identification and quantification of pollen species mixtures.

Molecular identification of mixed‐species pollen samples has a range of applications in various fields of research. To date, such molecular identification has primarily been carried out via amplicon sequencing, but whole‐genome shotgun (WGS) sequencing of pollen DNA has potential advantages, including (1) more genetic information per sample and (2) the potential for better quantitative matching. In this study, we tested the performance of WGS sequencing methodology and publicly available reference sequences in identifying species and quantifying their relative abundance in pollen mock communities. Using mock communities previously analyzed with DNA metabarcoding, we sequenced approximately 200Mbp for each sample using Illumina HiSeq and MiSeq. Taxonomic identifications were based on the Kraken k‐mer identification method with reference libraries constructed from full‐genome and short read archive data from the NCBI database. We found WGS to be a reliable method for taxonomic identification of pollen with near 100% identification of species in mixtures but generating higher rates of false positives (reads not identified to the correct taxon at the required taxonomic level) relative to rbcL and ITS2 amplicon sequencing. For quantification of relative species abundance, WGS data provided a stronger correlation between pollen grain proportion and sequence read proportion, but diverged more from a 1:1 relationship, likely due to the higher rate of false positives. Currently, a limitation of WGS‐based pollen identification is the lack of representation of plant diversity in publicly available genome databases. As databases improve and costs drop, we expect that eventually genomics methods will become the methods of choice for species identification and quantification of mixed‐species pollen samples.

Adaptation to the hottest spot on earth: Dietary ecology of an enigmatic desert gecko based on DNA metabarcoding

The Lut Desert, southeastern Iran, regularly has the largest contiguous area of surface temperatures above 65°C compared to anywhere else on Earth. Despite these harsh conditions, some specialized species thrive in this extreme habitat. Misonne's spider gecko, Rhinogecko misonnei de Witte, 1973 (Reptilia, Squamata, Gekkonidae) is an endemic species that is native to the central part of the Lut Desert. This research aimed to explore the dietary gut content and ecology of Misonne's spider gecko populations inhabiting the Lut Desert. A DNA metabarcoding approach was applied to obtain data from the stomach content of six collected specimens. A total of 147,451 amplicon reads representing a total of 94 OTU (=Operational Taxonomic Units) for the CO1-5P barcode region were sequenced. Of 94 OTUs, 50 (53.2%) identically matched ≥97% with BOLD and/or NCBI database and were identified at the genus level. Blasting in both databases revealed the presence of three phyla including Chordata (55.23%), Arthropoda (44.72%), and Annelida (0.05). 99.54% of Chordata DNA belonged to Rhinogecko misonnei mainly coming from stomach tissue of the examined specimens. Within the arthropods, 87.64% of the OTUs matched Insecta and 12.08% Arachnida. Our findings indicate that Rhinogecko misonnei is a generalist gecko that feeds on a variety of food items. About 81% of Rhinogecko food items in the central Lut are migratory insects that come from adjacent areas of the Lut District, although the arachnid food items originate from the central Lut. This study emphasizes the importance of the insect fauna of adjacent areas of the Lut Desert for the stability of the Desert food web.

DNA Metabarcoding Methods for the Study of Marine Benthic Meiofauna: A Review

Meiofaunal animals, roughly between 0.045 and 1 mm in size, are ubiquitous and ecologically important inhabitants of benthic marine ecosystems. Their high species richness and rapid response to environmental change make them promising targets for ecological and biomonitoring studies. However, diversity patterns of benthic marine meiofauna remain poorly known due to challenges in species identification using classical morphological methods. DNA metabarcoding is a powerful tool to overcome this limitation. Here, we review DNA metabarcoding approaches used in studies on marine meiobenthos with the aim of facilitating researchers to make informed decisions for the implementation of DNA metabarcoding in meiofaunal biodiversity monitoring. We found that the applied methods vary greatly between researchers and studies, and concluded that further explicit comparisons of protocols are needed to apply DNA metabarcoding as a standard tool for assessing benthic meiofaunal community composition. Key aspects that require additional consideration include: (1) comparability of sample pre-treatment methods; (2) integration of different primers and molecular markers for both the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear 18S rRNA genes to maximize taxon recovery; (3) precise and standardized description of sampling methods to allow for comparison and replication; and (4) evaluation and testing of bioinformatic pipelines to enhance comparability between studies. By enhancing comparability between the various approaches currently used for the different aspects of the analyses, DNA metabarcoding will improve the long-term integrative potential for surveying and biomonitoring marine benthic meiofauna.

DNA metabarcoding reveals fine scale geographical differences of consumed algae in the Galápagos marine iguanas (Amblyrhynchus cristatus)

Abstract Galápagos marine iguanas are primarily associated with the marine environment and show special nutritional adaptations. They are the only lizards worldwide that forage on marine macroalgae. Until now, consumed algae have been identified by direct observations during their feeding activities and microscopic identification in faeces samples. In this study, we use a novel DNA metabarcoding approach to identify consumed algal species from the faeces of marine iguanas. We developed primers for the ribulose-bisphosphate carboxylase (rbcL) gene and applied a metabarcoding approach to 25 individual faeces samples collected in four representative sites of two subspecies (Amblyrhynchus cristatus mertensi and A. c. godzilla), found in the San Cristóbal Island. We detected 18 consistently occurring macroalgal operational taxonomic units (OTUs). Most of the OTUs were assigned to Rhodophyta (red algae) and only one OTU to Chlorophyta (green algae). Despite the number of consumed algal species did not differ between two subspecies (OTU richness; P = 0.383), diet overlap level between A. c. mertensi and A. c. godzilla was low (Schoener index = 0.345), suggesting that both subspecies consumed different algal species in their natural environment. Further studies are needed to understand whether the difference of consumed algae reflects disparities in the abundance of algal species between sites, or whether iguanas of the two genetically differentiated subspecies prefer distinct algal species.

Impact of land use intensification and local features on plants and pollinators in Sub-Saharan smallholder farms

Sub-Saharan African crop production largely relies on smallholder farms, located both in urban and agricultural landscapes. In this context, the investigation of plant and pollinator diversity and their interactions is of primary importance since both these factors are threatened by land use intensification and the consequent loss of natural habitats. In this study, we evaluated for the first time how plant and pollinator insect assemblages and interactions in Sub-Saharan farming conditions are shaped by land use intensification. To do that, we complemented biodiversity field surveys in Northern Tanzania with a modern DNA metabarcoding approach to characterize the foraged plants and thus built networks describing plant-pollinator interactions at the individual insect level. Moreover, we coupled this information with quantitative traits of landscape composition and floral availability surrounding each farm. We found that pollinator richness decreased with increasing impervious and agricultural cover in the landscape, whereas the flower density at each farm correlated with pollinator richness. The intensification of agricultural land use and urbanization correlated with a higher foraging niche overlap among pollinators due to convergence of individuals’ flower visiting strategies. Furthermore, within farms, the higher availability of floral resources drove lower niche overlap among individuals, while a greater flower visitors abundance shaped higher generalization at the networks level (H2′), possibly due to increased competition. These mechanistic understandings leading to individuals’ foraging niche overlap and generalism at the network level, could imply stability of interactions and of the pollination ecosystem service. Our integrative survey proved that plant-pollinator systems are largely affected by land use intensification and by local factors in smallholder farms of Sub-Saharan Africa. Thus, policies promoting nature-based solutions, among which the introduction of more pollinator-friendly practices by smallholder farmers, could be effective in mitigating the intensification of both urban and rural landscapes in this region, as well as in similar Sub-Saharan contexts.