DNA metabarcoding in forest ecosystems: current methods and future directions

Bats are the only mammals capable of powered flight and therefore need a high calorie intake to survive. They hunt at night using the echoes made by their own calls to navigate and locate prey. Bats can use different tactics to hunt for food: hawking involves catching prey on the wing and requires fast aerial manoeuvring and more intense echolocation calls, while gleaning involves listening for movements of ground and water dwelling prey as the bat hovers. Some bat species specialise as hawkers or gleaners but maintain the ability to hunt with both methods. With the ever-growing impact of human activities on their habitats, it is important to understand how adaptable bats feeding habits are to changes in their environment. To find out more, Stidsholt et al. studied greater mouse-eared bats, which primarily feed by gleaning. To understand how this species chooses feeding strategies they fitted bats with tiny backpacks that could record the animal’s location and foraging behaviour. They could also monitor prey sizes by recording the sounds of the bats chewing. Stidsholt et al. found that, although these bats tried to catch prey on the ground more often than in the air, they were actually more successful as airborne hunters. Despite this, gleaning was still a more profitable strategy for them, because the body mass of ground prey is higher than for airborne prey. Gleaning gave the bats a higher calorie intake, even though their capture rate was lower. Although feeding habits differed slightly between individual bats on a given night of monitoring, there were much larger changes in behaviour between different feeding nights. This shows that, although this species of bat prefers gleaning, they will switch strategies to hawking as their environment changes, for example if there is more airborne prey, or if rainfall makes it hard to hear movements on the ground. Bats tended to get enough calories for their needs but did not catch more prey than they needed to survive. Stidsholt et al. concluded that greater mouse-eared bats change their feeding strategy based on prey availability and size, as well as the bat’s environment. Their study provides an important insight into how bats fit into the ecosystem and how adaptable bats might be to changes in their habitat.

Bats are the only mammals capable of powered flight and therefore need a high calorie intake to survive. They hunt at night using the echoes made by their own calls to navigate and locate prey. Bats can use different tactics to hunt for food: hawking involves catching prey on the wing and requires fast aerial manoeuvring and more intense echolocation calls, while gleaning involves listening for movements of ground and water dwelling prey as the bat hovers. Some bat species specialise as hawkers or gleaners but maintain the ability to hunt with both methods. With the ever-growing impact of human activities on their habitats, it is important to understand how adaptable bats feeding habits are to changes in their environment. To find out more, Stidsholt et al. studied greater mouse-eared bats, which primarily feed by gleaning. To understand how this species chooses feeding strategies they fitted bats with tiny backpacks that could record the animal’s location and foraging behaviour. They could also monitor prey sizes by recording the sounds of the bats chewing. Stidsholt et al. found that, although these bats tried to catch prey on the ground more often than in the air, they were actually more successful as airborne hunters. Despite this, gleaning was still a more profitable strategy for them, because the body mass of ground prey is higher than for airborne prey. Gleaning gave the bats a higher calorie intake, even though their capture rate was lower. Although feeding habits differed slightly between individual bats on a given night of monitoring, there were much larger changes in behaviour between different feeding nights. This shows that, although this species of bat prefers gleaning, they will switch strategies to hawking as their environment changes, for example if there is more airborne prey, or if rainfall makes it hard to hear movements on the ground. Bats tended to get enough calories for their needs but did not catch more prey than they needed to survive. Stidsholt et al. concluded that greater mouse-eared bats change their feeding strategy based on prey availability and size, as well as the bat’s environment. Their study provides an important insight into how bats fit into the ecosystem and how adaptable bats might be to changes in their habitat.

Bats are the only mammals capable of powered flight and therefore need a high calorie intake to survive. They hunt at night using the echoes made by their own calls to navigate and locate prey. Bats can use different tactics to hunt for food: hawking involves catching prey on the wing and requires fast aerial manoeuvring and more intense echolocation calls, while gleaning involves listening for movements of ground and water dwelling prey as the bat hovers. Some bat species specialise as hawkers or gleaners but maintain the ability to hunt with both methods. With the ever-growing impact of human activities on their habitats, it is important to understand how adaptable bats feeding habits are to changes in their environment. To find out more, Stidsholt et al. studied greater mouse-eared bats, which primarily feed by gleaning. To understand how this species chooses feeding strategies they fitted bats with tiny backpacks that could record the animal’s location and foraging behaviour. They could also monitor prey sizes by recording the sounds of the bats chewing. Stidsholt et al. found that, although these bats tried to catch prey on the ground more often than in the air, they were actually more successful as airborne hunters. Despite this, gleaning was still a more profitable strategy for them, because the body mass of ground prey is higher than for airborne prey. Gleaning gave the bats a higher calorie intake, even though their capture rate was lower. Although feeding habits differed slightly between individual bats on a given night of monitoring, there were much larger changes in behaviour between different feeding nights. This shows that, although this species of bat prefers gleaning, they will switch strategies to hawking as their environment changes, for example if there is more airborne prey, or if rainfall makes it hard to hear movements on the ground. Bats tended to get enough calories for their needs but did not catch more prey than they needed to survive. Stidsholt et al. concluded that greater mouse-eared bats change their feeding strategy based on prey availability and size, as well as the bat’s environment. Their study provides an important insight into how bats fit into the ecosystem and how adaptable bats might be to changes in their habitat.

With forests covering roughly half of the Austrian land area, forest ecosystems have been monitored and characterized for centuries. While some aspects remain the same long-term, we are about to experience rapid changes due to climate change and loss of species. At the same time, emerging technologies like high throughput sequencing allow us to have deeper insights into the occurrence and diversity of species. For the hidden subsoil, metabarcoding of environmental DNA can uncover invisible soil life. This includes under-explored mycorrhizal fungi, especially species which do not regularly form fruiting bodies.Supported by the Austrian biodiversity fund, the project „Zurück in die Zukunft“ (Back to the future) analyses archived forest soil samples from the past 32 years in order to capture diversity of ectomycorrhizal fungi. The dataset comprises samples from all federal states reaching from meadow forests of Pannonian regions to montane forests of the inner alps. Initial analyses detected >7000 fungal species, including >800 species of ectomycorrhizal fungi. The results provide an addition to already existing fungi databases of fruiting-body records. Fungal community composition is highly dependent on site-specific factors, which is thought to be explored using records of bio-geochemical data, vegetation and climate. By comparing the fungal communities of the 1990s, early 2000s and 2024, long-term changes and trends can be identified. The status of endangered species and potential neobiota in different habitats will be evaluated. We also aim to get a glance of future developments regarding forest ecosystems and their ecosystem functions.

An understanding of the food web in forest ecosystems is essential to ensuring that society lives in harmony with nature; however, this can be challenging in areas mainly composed of forest environments, such as in the Japanese Archipelago. Examining fecal samples collected from the forest edge can aid in determining the ecological roles of host species. In this study, a DNA barcoding method using original primers was applied to identify the carnivoran host species from fecal samples. DNA metabarcoding using ITS2 and COI markers was then conducted to elucidate the plant and invertebrate diets of the Japanese marten, Martes melampus (Carnivora, Mustelidae). The dietary analyses revealed that M. melampus consumed a diverse array of plants and animals. Most of the consumed plant species were fresh fruits, reflecting the fruiting season of the detected plants. This implies a role for M. melampus in seed dispersal and thus in forest maintenance. Considering the activity seasons, we also found that various adult-stage insects (beetles, cicadas, sphinx moths, and grasshoppers) contributed to the marten's diet, together with invertebrates (earthworms, etc.), which are easily digested and therefore difficult to detect through traditional methods. Although the COI marker used was designed for invertebrate species, one bird species, the brown-eared bulbul, Microscelis amaurotis, was found to make up a small part of the winter to early spring diet. These results show that, while M. melampus mainly consumes seasonal fruits, it can adapt its diet in response to environmental changes, such as by including invertebrates and small vertebrates.

Droughts in forest ecosystems are a central concern for current and future biodiversity loss, carbon sequestration, and ecosystem functioning. Trees rely on symbiotic relationships with fungi to enhance nutrient uptake and improve stress tolerance, but the impacts of drought on plant-fungal relationships remain unclear and vary across different tree species compositions. This study investigated how inter- and intraspecific interactions among three prominent tree species in European forests—Fagus sylvatica (European beech), Quercus petraea (Sessile oak), and Tilia cordata (small-leaved lime)—shift under simulated drought conditions in relation to their rhizosphere fungal communities. We hypothesized that drought would shift the diversity and functional capacity of fungal communities, with these effects being dependent on the tree species and competitive context. To test this, we set up raised-bed experiments with seedlings of the three species as mono- or polycultures, exposing them to ambient rainfall conditions or two years of reduced precipitation using plastic roofing. We assessed tree seedling growth and development, and at the end of the experiment, we sampled rhizosphere soils from individual trees to characterize fungal diversity using full-length ITS DNA metabarcoding on an Oxford Nanopore Technology PromethION platform. Intraspecific versus interspecific competition provided more favourable conditions for tree growth under drought conditions. Our results show that fungal communities were responsive to variations in plant species, competitive context, and drought, and that fungal biodiversity explained unique patterns in plant growth responses to drought and competition, particularly for plant-symbiotic ectomycorrhizal fungi. This study highlights the variable effects of drought on fungal communities and underscores the importance of species-specific interactions in forest ecosystem responses to climate stress. These findings contribute to our understanding of the ecological role of fungi in forest species' resilience to climate change and may inform future forest management strategies aimed at mitigating the effects of drought in temperate regions.

Insects and other terrestrial invertebrates are declining in species richness and abundance. This includes the invertebrates associated with herbivore dung, which have been negatively affected by grazing abandonment and the progressive loss of large herbivores since the Late Pleistocene. Importantly, traditional monitoring of these invertebrates is time‐consuming and requires considerable taxonomic expertise, which is becoming increasingly scarce. In this study, we investigated the potential of environmental DNA (eDNA) metabarcoding of cow dung samples for biomonitoring of dung‐associated invertebrates. From eight cowpats we recovered eDNA from 12 orders, 29 families, and at least 54 species of invertebrates (mostly insects), representing several functional groups. Furthermore, species compositions differed between the three sampled habitats of dry grassland, meadow, and forest. These differences were in accordance with the species’ ecology; for instance, several species known to be associated with humid conditions or lower temperatures were found only in the forest habitat. We discuss potential caveats of the method, as well as directions for future study and perspectives for implementation in research and monitoring.

Islands make up a large proportion of Earth's biodiversity, yet are also some of the most sensitive systems to environmental perturbation. Biogeographic theory predicts that geologic age, area, and isolation typically drive islands' diversity patterns, and thus potentially impact non-native spread and community homogenization across island systems. One limitation in testing such predictions has been the difficulty of performing comprehensive inventories of island biotas and distinguishing native from introduced taxa. Here, we use DNA metabarcoding and statistical modelling as a high throughput method to survey community-wide arthropod richness, the proportion of native and non-native species, and the incursion of non-natives into primary habitats on three archipelagos in the Pacific - the Ryukyus, the Marianas and Hawaii - which vary in age, isolation and area. Diversity patterns largely match expectations based on island biogeography theory, with the oldest and most geographically connected archipelago, the Ryukyus, showing the highest taxonomic richness and lowest proportion of introduced species. Moreover, we find evidence that forest habitats are more resilient to incursions of non-natives in the Ryukyus than in the less taxonomically rich archipelagos. Surprisingly, we do not find evidence for biotic homogenization across these three archipelagos: the assemblage of non-native species on each island is highly distinct. Our study demonstrates the potential of DNA metabarcoding to facilitate rapid estimation of biogeographic patterns, the spread of non-native species, and the resilience of ecosystems.

The Brazilian Atlantic Forest and its associated ecosystems are highly biodiverse but still understudied, especially with respect to eukaryotic microbes. Protists represent the largest proportion of eukaryotic diversity and play important roles in nutrient cycling and maintenance of the ecosystems in which they occur. However, much of protist diversity remains unknown, particularly in the Neotropics. Understanding the taxonomic and functional diversity of these organisms is urgently needed, not only to fill this gap in our knowledge, but also to enable the development of public policies for biological conservation. This is the first study to investigate the taxonomic and trophic diversity of the major protist groups in freshwater systems and brackish coastal lagoons located in fragments of the Brazilian Atlantic Forest by DNA metabarcoding, using high-throughput sequencing of the gene coding for the V4 region of the 18S rRNA gene. We compared α and β diversity for all protist communities and assessed the relative abundance of phototrophic, consumer, and parasitic taxa. We found that the protist communities of coastal lagoons are as diverse as the freshwater systems studied in terms of α diversity, although differed significantly in terms of taxonomic composition. Our results still showed a notable functional homogeneity between the trophic groups in freshwater environments. Beta diversity was higher among freshwater samples, suggesting a greater level of heterogeneity within this group of samples concerning the composition and abundance of OTUs.Ciliophora was the most represented group in freshwater, while Diatomea dominated diversity in coastal lagoons.

We used DNA metabarcoding to assess the seasonal diets of the large Japanese wood mouse, Apodemus speciosus (Muridae, Rodentia), in forest edges adjacent to citrus orchards on Innoshima Island, Japan. We used one chloroplast and three mitochondrial DNA barcoding markers to determine mouse diets. Among the various plant and invertebrate diets, A. speciosus typically consumed Chinese cork oak (Quercus variabilis) in early spring (likely acorns preserved during winter) and gypsy moths (Lymantria dispar, a forest pest) in late spring and summer. In addition, we found that A. speciosus also preyed on orchard pests, including the gutta stink bug and other potentially harmful invertebrates. The season during which A. speciosus preyed on stink bugs corresponded with the harvest of orchard products. This study revealed several of the ecological roles of A. speciosus within the boundary zone between forest and human ecosystems. Furthermore, based on the performance of various mitochondrial markers in dietary profiling of invertebrate food items, we recommend the multi-locus DNA metabarcoding method to comprehensively assess the diet of A. speciosus.

The data have been presented on the microbial biomass content and on the abundances and taxonomic structure of bacterial and archaeal communities in the upper horizons of gray forest (Eutric Retisol (Loamic, Cutanic, Humic)) and alluvial-meadow (Fluvic Gleyic Phaeozem (Loamic, Pachic)) soils of the autonomous, transitional, and accumulative positions of the slope landscape, corresponding to the fallow, small-leaved forest, and meadow ecosystems. Soil microbial biomass was measured by DNA quantification and chloroform fumigation-extraction; the archaeal and bacterial abundances were estimated using the FISH method; and the structure of microbial communities was analyzed via high-throughput 16S rRNA gene amplicon sequencing (DNA metabarcoding). The microbial biomass and the abundances of metabolically active bacterial and archaeal cells correlated with the organic carbon (Corg) and total nitrogen (Ntot) contents, increasing along the slope from the autonomous to accumulative landscapes, and decreasing with depth throughout soil profiles. The alluvial-meadow soil of the accumulative landscape was characterized by a sharp decrease in the relative abundance of Verrucomicrobia, as well as by the increase in the relative abundance of Proteobacteria and Firmicutes compared to the gray forest soil of the autonomous and transitional positions. The number of operational taxonomic units and α-diversity indices correlated significantly with C/N, Corg, and Ntot and were almost independent of the soil pH. Thus, location in landscape (catena) determined not only the physicochemical but also the microbiological properties of soil, forming the spatial heterogeneity of the microbial community structure and abundance.

BackgroundThis publication presents a dataset of saproxylic (dead wood) fungi which addresses the limited understanding of saproxylic fungal diversity, community structure and colonisation dynamics in fine woody debris (FWD). This knowledge gap is largely due to the microscopic or cryptic nature of most fungal species, which often exist primarily as mycelium. To overcome this challenge, the study employs metabarcoding of DNA extracted from pins representing FWD which were experimentally placed for decomposition in northern Finland. The dataset can be used to investigate the composition and progression of fungal communities across different stages of wood decay. It includes communities from distinct biotopes: one forest site protected from reindeer grazing, one exposed to reindeer and a forest area frequented by tourists. The use of standardised decomposition experiments combined with high-throughput eDNA analysis represents a notable methodological approach in characterising saproxylic fungal communities.New informationThe dataset was generated using the novel MycoPins method, published by the authors in 2023, in which sterilised wooden pins were embedded beneath the forest litter and left to decompose for one year, while being periodically inspected. This innovative decomposition experiment was conducted at the Oulanka Research Station in Kuusamo, Northern Finland. As the first study in this region to investigate saproxylic fungi in fine woody debris (FWD) using MycoPins and DNA metabarcoding, it adds new knowledge to the fungal biodiversity data of the region. The resulting dataset of DNA-derived occurrences has been published through the Global Biodiversity Information Facility (GBIF), offering valuable insights into fungal diversity across different stages of decomposition. Agaricomycetes, a class of fungi strongly associated with their hosts via ectomycorrhiza, was selected from the dataset for comparison with fungal datasets from neighbouring regions. Remarkably, the MycoPins method revealed a high proportion of unique Agaricomycetes taxa not captured in existing species checklists from GBIF.org. These findings highlight the study's contribution to advancing biodiversity assessment. The results demonstrate the potential of this approach to enhance our understanding of fungal community dynamics in boreal forest ecosystems.

Logging activities degrade forest habitats across large areas of the tropics, but the impacts on trophic interactions that underpin forest ecosystems are poorly understood. DNA metabarcoding provides an invaluable tool to investigate such interactions, allowing analysis at a far greater scale and resolution than has previously been possible. We analysed the diet of the insectivorous fawn leaf-nosed bat Hipposideros cervinus across a forest disturbance gradient in Borneo, using a dataset of ecological interactions from an unprecedented number of bat-derived faecal samples. Bats predominantly consumed insects from the orders Lepidoptera, Diptera, Blattodea, and Coleoptera, and the taxonomic composition of their diet remained relatively consistent across sites regardless of logging disturbance. There was little difference in the richness of prey consumed per-bat in each logging treatment, indicating potential resilience of this species to habitat degradation. In fact, bats consumed a high richness of prey items, and intensive sampling is needed to reliably compare feeding ecology over multiple sites. Multiple bioinformatic parameters were used, to assess how they altered our perception of sampling completeness. While parameter choice altered estimates of completeness, a very high sampling effort was always required to detect the entire prey community.

Invasive species within North America, particularly wild pigs ( Sus scrofa ), pose a serious threat to native ecosystems through both direct and indirect impacts. Wild pigs are a large‐bodied omnivorous species native to Eurasia and introduced to North America. Using DNA metabarcoding of fecal samples, we investigated the diet of wild pigs at a bottomland hardwood forest ecosystem within Felsenthal National Wildlife Refuge in southern Arkansas. We found that the wild pig diet was diverse and included at least 74 plant families and 106 genera, dominated by plant matter with Fagaceae (oaks and hickories), Poaceae (grasses), and Asteraceae (asters, sunflowers, and daisies) comprising over 50% of total dietary detections. Hard mast was a primary food source in fall and winter, while herbaceous vegetation peaked in spring. We documented DNA from 23 species of vertebrates in wild pig fecal samples, including mammals, birds, fish, frogs, and turtles. Vertebrate matter was most frequently detected in autumn. Vertebrate consumption constituted ~17% of wild pig diet by relative read abundance. Our results highlighted the potential long‐term ecological consequences of wild pig foraging behaviors, particularly competition for critical food resources with native wildlife and impact on forest regeneration through the consumption of mast. Understanding dietary dynamics is crucial for managing wild pig populations and mitigating impacts on vulnerable ecosystems and wildlife.

ABSTRACTThe Asian elephant (Elephas maximus) is a flagship species of the tropical forest ecosystem in Asia, playing a crucial role in maintaining ecological stability. Investigating the dietary composition of Asian elephants is essential for developing effective conservation and management strategies. In this study, a total of 107 fecal samples from different Asian elephant populations in China were analyzed using chloroplast rbcL DNA metabarcoding to systematically examine the dietary composition and diversity of the species. The results show that the foraged resources of the Asian elephant encompass eight classes, 43 orders, 77 families, and 154 genera. At the order level, Poales, Fabales, Rosales, and Zingiberales have the highest proportions, whereas at the family level, Poaceae, Fabaceae, Cyperaceae, Moraceae, and Musaceae dominate. Diversity and ecological niche width analyses indicate that there are differences among populations, with geographical variations in diet that are likely related to the availability of habitat resources. This study reveals the dietary composition and differences among different populations of Asian elephants, providing important scientific evidence and practical guidance for optimizing the food structure of captive populations and the development of food resource bases.