Environmental DNA最新文献

Environmental DNA (eDNA) metabarcoding typically relies on collecting and characterising a pool of mixed, fragmented DNA from environmental samples for species identification. Here, we introduce environmental metazoan cells (emCells), representing whole individual cells shed by macro-organisms into aquatic ecosystems, and report on a method to successfully isolate and amplifying short amplicons to determine species identity. Using a custom fish probe and a novel multi-factor fluorescence-activated cell sorting (FACS) protocol on mesocosm water samples, we successfully enriched for target emCells, as confirmed by shifts in population density using FACS and imaging flow cytometry. Imaging flow cytometry demonstrated dual nuclear and mitochondrial staining of whole single cells, while multiplexed PCR assays (targeting both mitochondrial and nuclear DNA) confirmed the effective enrichment of fish emCells, with one-quarter of sorted cells identified as fish. Sequences obtained from isolated emCells matched known species in the mesocosm, validating our approach. Despite efforts to exclude non-target cells, diverse single-cell eukaryotes were also recovered, highlighting the need for additional strategies to enrich for target emCells given the abundance and diversity of off-target particles present in aquatic environments, which will be especially important for real-world environments. Isolation and analysis of emCells could provide a versatile complementary approach to current eDNA methodologies by providing genomic information that normally requires direct sampling from live organisms.

The canga of the Serra dos Carajás in the Eastern Amazon (Pará, Brazil) has one of the largest iron ore deposits on the planet and is home to a community of endemic and rare plants. However, conservation and monitoring programs in megadiverse areas, as in the case of the region, are often hampered by the lack of knowledge of the species that inhabit these ecosystems. In this scenario, the comprehensive DNA barcoding effort directed to the complete flora of the canga in the Brazilian Amazon has enabled the implementation of DNA metabarcoding approaches for species monitoring. Here, we assessed the potential of implementing DNA metabarcoding with environmental DNA (eDNA) in future surveys of plant species of the ironstone outcrops of the Serra dos Carajás. After extracting eDNA from soil samples, the nuclear ITS2 region was amplified and sequenced using the Illumina MiSeq platform. With the metabarcoding analyses, we detected 95 species from 72 genera and 35 families, revealing a higher overall diversity than the morphology-based approach, including taxa that were not identified in a traditional floristic survey. The fact that DNA metabarcoding results mostly agreed with the data from the floristic survey indicates the robustness of the molecular approach to be used in monitoring studies of plant diversity in the region. Additionally, we discuss the relevance of our results to guide the development of broader applications of eDNA-based biodiversity monitoring in species-rich environments such as the Serra dos Carajás.

Sympatric speciation is defined as the formation of new species in the absence of geographic barriers, but the genomic and life history strategy mechanisms underpinning sympatric speciation are still far from clear. It has recently been discovered that the cichlid fish Astatotilapia calliptera from crater Lake Masoko in Tanzania have diverged sympatrically into littoral (shallow water) and benthic (deep water) ecotypes, which differ in head and pharyngeal jaw morphology. Carbon stable isotope analysis has also broadly indicated trophic differentiation between ecotypes. Here, we explore trophic niche divergence on a finer scale, using metabarcoding of stomach contents. A combination of the mitochondrial COI region and 18S V4 region from the eukaryotic nuclear small subunit ribosomal DNA was used to target macroinvertebrate and broader eukaryotic taxonomic diversity, respectively, revealing dietary divergence between the ecotypes. Large proportions of Arthropoda (dipterans and copepod) were found in both ecotypes, indicating some food sources common to both microhabitats. However, gut contents of benthic A. calliptera individuals were characterized by an abundance of annelids and diatoms, while Lepidoptera, mayflies, fungi, freshwater mussels, and bivalves were common in littoral ecotypes. The variation observed in the dietary contents of the ecotypes indicates the presence of resource partitioning, facilitating adaptation to unique feeding strategies.

Large mammalian herbivores (LMH) are abundant in grazing ecosystems and play a pivotal role in shaping vegetation characteristics. However, accurately determining their diets through traditional methods, such as direct observations, remains challenging, particularly in natural communities and mixed-species grazing systems. Recent studies have shown that DNA metabarcoding can effectively identify the plant composition in LMH diets as well as the plant-dwelling arthropods (PDA) incidentally ingested by LMH while grazing. Given the high specificity of herbivorous insects to their host plant, we hypothesize that DNA metabarcoding of arthropods ingested by LMH could offer valuable insights into their feeding preferences. The goal of this study is to evaluate the accuracy of plant and arthropod DNA metabarcoding methods in identifying the diets of sheep and cattle and to compare their performance with direct observations and known dietary patterns from the literature. To test this, we collected fecal samples from sheep and cattle grazing in the northeast Asian grasslands. We amplified arthropod DNA using COI mitochondrial markers and plant DNA using ITS1 markers, followed by Illumina sequencing. Additionally, we conducted field observations to identify plants grazed by sheep and cattle. The DNA metabarcoding methods provided a comprehensive view of the LMH diet. Both DNA metabarcoding methods successfully detected dietary differences between sheep and cattle, with sheep primarily consuming nutrient-rich forbs and cattle predominantly grazing on Poaceae, consistent with known foraging behaviors. While the constant presence of arthropods across multiple samples suggests that DNA of ingested arthropods could provide complementary information regarding LMH foraging behavior, we found such to be rather limited. However, our findings confirm that plant DNA metabarcoding is a reliable and accurate method for identifying LMH diets.

Pollinators play a critical role in ensuring the stability of food systems, yet their populations are in decline. To better understand and promote pollinator biodiversity, this study explored the use of environmental DNA (eDNA) metabarcoding techniques to assess plant–pollinator interactions. We conducted two experiments to optimize eDNA metabarcoding strategies for detecting pollinators on flowering plants. In the first experiment, we compared visual observations and eDNA detection via Illumina sequencing to characterize pollinator visitation on two native plant species in public and private green spaces across the Richmond, Virginia metropolitan area. Our findings revealed notable differences between the two methods, with visual surveys more sensitive to Hymenoptera and eDNA more sensitive to plant pests and other organisms. We devised a second experiment in a controlled environment at the Lewis Ginter Botanical Garden butterfly exhibit. Here, we tested different sampling strategies, primer pairs, and DNA sequencing platform (using Oxford Nanopore Technology). Despite detecting two butterfly species present in the exhibit, the eDNA metabarcoding showed limited sensitivity to the expected Lepidoptera. Our results indicate that while eDNA metabarcoding can detect a broader range of eukaryotic organisms, it may not be as effective for monitoring specific pollinator taxa like Hymenoptera and Lepidoptera. Factors such as sample type, primer bias, sequencing platform, and bioinformatics pipeline may impact detection outcomes. This study underscores the need for combining traditional monitoring techniques with eDNA metabarcoding to gain a comprehensive understanding of plant–pollinator interactions and improve pollinator conservation efforts.

Seabirds are recognized as one of the most vulnerable groups of birds, with around a third of species identified as globally threatened. The conservation of seabirds is often linked with their feeding and diet, due to undesirable interactions with human-related fishing activities and fisheries depletion/climate change impacting food resources. Therefore, understanding the diet of seabirds is often a critical first step towards identifying effective conservation measures. DNA metabarcoding and hard parts analyses provide a foundation for assessing the diet of predatory seabird species, giving insight into predator–prey relationships and ecosystem-wide food webs. Congruency between these two methods would increase confidence, providing validation that either method provides a reliable representation of the diet. This study on the diet of the endangered New Zealand king shag (Leucocarbo carunculatus) compared the frequency of occurrence of fishes detected from the same regurgitated pellets (n = 191) using both hard parts and DNA metabarcoding methodologies. The number of pellets with overlapping fish families showed a significant positive correlation between methods (r = 0.96; p < 0.001), with 50 out of 191 pellets showing complete alignment and only two pellets without any alignment. Both methods confirmed the predominance of Bothidae (DNA: 71% of pellets, hard parts: 77%, total: 80%) and Rhombosoleidae (DNA: 45%, hard parts: 51%, total: 59%) taxa in the diet of king shags, while also revealing the consumption of a diversity of other fish species. Overall, this study demonstrates that the two methods provide a complementary approach for revealing the dominant fish prey species in the diet, as well as providing an overview of the diversity of prey species (DNA: 14 unique species, hard parts: 8, total: 28). However, the inexact alignment between the two methods for detecting every taxon in every pellet suggests differences in detection, especially for less common taxa and for DNA metabarcoding, where species-level resolution is dependent on adequate DNA database sequence entries of taxa in the local area. In addition, filtering thresholds for DNA metabarcoding further influenced alignment. Overall, the results indicate that both methods provide consistent detection of major prey items; however, reliably capturing the full diversity of prey species with either method is reliant on a sufficient sample size.

This paper provides a pioneering study case on monitoring fish biodiversity in ports through the eDNA and citizen science approach. eDNA samples were collected in the spring and in fall 2022 in the ports of Calvi, L'Île-Rousse, STARESO, Saint-Florent. Samples collected led to the identification of 73 taxa. These ports appeared to harbor at least 20% of the known teleost biodiversity in Corsica and 11% of the Mediterranean teleost biodiversity. The ports of Calvi and L'Île-Rousse displayed the highest taxonomic, phylogenetic, and functional diversities and appeared the most similar. However, taxonomic turnover highlighted that none of the 4 ports was a subset of any of the others. In August 2022, an extreme climate event (ECE) struck Corsica, offering a unique opportunity to collect data under abnormal conditions. Although it is not possible to distinguish the seasonal effect from the ECE effect in the fall, we detected in all ports but Saint-Florent an increase in taxonomic richness, phylogenetic, and functional diversity: we did not only detect new species but also showed that these species led to an increase in the local representativeness of phylogenetic diversity, most likely correlated with new functional traits. The port of Saint-Florent displayed the highest relative phylogenetic diversity, that is, a smaller but evolutionarily more distinct group of species. Our study demonstrated the robustness and relevance of eDNA citizen science coupled with relevant indicators for port biodiversity monitoring and emphasized the need for more research and targeted conservation efforts to better understand and mitigate the ecological impacts of ports while exploring their potential as habitats.

Freshwater ecosystems are globally threatened by habitat loss, pollution, and invasive species, all of which are particularly acute in urban areas. To assess the impacts of urbanization on freshwater biodiversity—specifically the effects of alien species on native primary aquatic vertebrates—we investigated the World Heritage Site, Lake Xochimilco in Mexico City. Focusing on fishes and amphibians, we applied environmental DNA metabarcoding using primer pairs targeting mitochondrial 12S and 16S across the remnant lake and collected 14 aquatic environmental variables for sampled sites. Our survey recovered ca. 60% of Lake Xochimilco's historically recorded fish and amphibian species, including rare species and novel taxa not detected by past traditional surveys. However, our findings imply a severely degraded wetland, with alpha diversity indices indicating a low-diversity ecosystem dominated by alien fishes. Beta diversity analysis revealed a heterogeneous ecosystem that may be driven partially by the presence of alien fish, particularly cyprinids. Environmental variables linked to pollution predicted the presence of non-native fish families. We also found evidence that some species prefer to occupy different water bodies within the lake remnant. Despite the ongoing degradation of this ecosystem, native and endemic fauna are persisting, although detections were typically rare. We found no evidence of the Critically Endangered axolotl salamanders (Ambystoma sp.) from wild sites; however, we detected their presence in one wildlife refuge, highlighting the potential of refuges to prevent complete extinction in the wild. We also found evidence of cryptic taxonomic diversity in Lithobates frogs and evidence of endemic genera, including the threatened mexclapique fish (Girardinicthys viviparus). These fishes are considered extirpated, suggesting remnant populations persist undetected by traditional surveys. Despite clear evidence of an ecosystem under extreme decline compared to historical biological records, our study demonstrates the potential for restoration, given the presence of native freshwater species and the success of wildlife refuges.

Effective sample collection is a pivotal step in environmental DNA (eDNA) workflows. For aquatic eDNA applications, this typically requires water filtration and cold storage, which present logistical challenges in remote or resource-limited settings. Metal–organic frameworks (MOFs) are porous materials composed of metal ions coordinated with organic linkers that can form around biological molecules in solution. By directly encapsulating and preserving eDNA in situ within a collected water sample, MOFs may simplify field sampling without the need for specialized equipment. In this study, eDNA capture and preservation from seawater samples using the MOF Zeolitic Imidazolate Framework-8 (ZIF-8) was compared with the performance of conventional filtration through mixed cellulose ester (MCE) filters. ZIF-8 samples were stored at ambient temperature for 2 weeks, while MCE filters were either frozen or preserved in a lysis buffer for 5 days. The performance of each method was assessed by high-throughput DNA sequencing and a metabarcoding assay targeting the 16S rRNA gene of fish. The MCE filter method detected, at present, a greater number of fish amplicon sequence variants (ASVs) and taxa than our trial application of the MOF method. However, community composition analyses (PERMANOVA and NMDS ordination) revealed no significant differences between the methods, demonstrating that despite yielding lower DNA quantities, ZIF-8 collection effectively replicates the marine fish community structure. Analysis of taxon abundance showed that MOFs captured dominant taxa effectively but were less sensitive to rarer taxa. With further optimisation to enhance eDNA capture efficiency by MOFs beyond this trial application, MOFs could serve as a practical, field-friendly alternative for eDNA sampling, especially where filtration is difficult.

Biodiversity monitoring in difficult-to-access areas, such as rugged mountain ranges, is currently challenging and thus often absent. Environmental DNA (eDNA) offers new opportunities to monitor remote or strictly protected areas, as rivers integrate the biodiversity information of entire catchments. Environmental samples can be analyzed either with metabarcoding or using species-specific assays. Species-specific assays like quantitative polymerase chain reaction assays do not require a fully-equipped laboratory and thus can be used in settings with limited resources and are especially suited to monitoring elusive or threatened species of management concern. Recently developed molecular tools, such as CRISPR-based diagnostic systems (CRISPR-Dx), provide new avenues to facilitate eDNA analysis through species-specific assays. Here, we combine multispecies primers with CRISPR-Dx to detect terrestrial mammal species in parallel with one amplification to detect multiple species with CRISPR-Dx. Given the short length of metabarcoding amplicons, designing species-specific assays within them can be challenging. We designed species-specific CRISPR-Dx for eight terrestrial mammals within a commonly used metabarcoding amplicon ~59 base pairs in length and tested the assays on eDNA samples collected in high-alpine catchments. Additionally, we compared the detections from CRISPR-Dx with metabarcoding results of the same samples and with catchment-based species inventories obtained through traditional monitoring. First, we show that designing species-specific CRISPR-Dx within a short amplicon allows terrestrial mammal detection in eDNA. Second, we demonstrate that CRISPR-Dx assays combined with multispecies primers are comparable in sensitivity to metabarcoding and thus can bridge a gap between species-specific assays and community analysis without requiring fully equipped laboratories. Third, we highlight that catchment-based eDNA sampling can be used to monitor terrestrial mammals in remote or protected areas. Overall, we demonstrate that eDNA and particularly CRISPR-Dx are a promising tool to monitor inaccessible and/or protected areas and to detect rare species across large spatiotemporal scales, thereby promoting biodiversity conservation.