DNA obtained from invertebrates (iDNA) can be metabarcoded in order to survey vertebrate communities. However, little attention has been paid to the interaction between the invertebrate and vertebrate species. Here, we tested for specialization by sampling the dung and carrion fly community of a swamp forest remnant along a disturbance gradient (10 sites: 80–310 m from a road). Approximately, 60% of the baited 407 flies yielded 294 vertebrate identifications based on two COI fragments and 16S. A bipartite network analysis found no statistically significant specialization in the interactions between fly and vertebrate species, but uncommon fly species can carry the signal for vertebrate species that are otherwise difficult to detect with iDNA. A spatial analysis revealed that most of the 20 vertebrate species reported in this study could be detected within 150 m of the road (18 spp.) and that the fly community sourced for iDNA was unexpectedly rich (24 species, 3 families). They carried DNA for rare and common species inhabiting different layers of the forest (ground‐dwelling: wild boar, Sunda pangolin, skinks, rats; arboreal: long‐tailed macaque, Raffles' banded langur; flying: pin‐striped tit‐babbler, olive‐winged bulbul). All our results were obtained with a new, greatly simplified iDNA protocol that eliminates DNA extraction by obtaining template directly through dissolving fly faeces and regurgitates with water. Lastly, we show that MinION‐ and Illumina‐based metabarcoding yield similar results. We conclude by urging more studies that use different baits and involve experiments that are capable of revealing the dispersal capabilities of the flies carrying the iDNA.

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Bioassessment of freshwater quality with eDNA is a powerful alternative to traditional methods involving collecting, sorting, and identifying metazoan taxa with morphology (e.g., macroinvertebrates). Particularly attractive for routine monitoring would be an eDNA method that uses a remote-controlled boat for collecting small volumes of water without filtration in order to simplify sampling. Such a method would not likely capture eDNA signals for all metazoan species, but may nevertheless allow for cost-effective, and frequent monitoring of water quality, which is important for tropical waterbodies that require year-round surveillance. We here optimize a molecular protocol for capturing metazoan signatures based on eDNA obtained from 15 ml of water. To test the robustness of the method, we used samples from two tropical reservoirs with known differences in water quality to optimize molecular procedures so that they yield repeatable results. Each reservoir was sampled at three sites ("biological replicates") and each water sample was subsampled twice before extracting the eDNA using ethanol precipitation for both subsamples ("technical replicates"). We then tested how much DNA (0.1 ng to 15 ng) and how many PCR cycles (25 or 35) minimized the variance of the metazoan eDNA signal between biological and technical replicates. For this purpose, we amplified a 313 bp COI minibarcode using a universal metazoan primer pair. We found that regardless of template amounts or PCR cycle numbers, the eDNA signatures for both reservoirs were distinct because only 17 of 59 mOTUs (mainly planktonic crustaceans and rotifers) were shared. We also found that template amounts between 0.5 and 15 ng yielded overall similar results, but the use of 35 PCR cycles significantly depressed the number of detected species (p-value < 0.05). Fortunately, the differences between the detected metazoan community of the reservoirs were so strong that all biological and technical replicates could be assigned unambiguously to their source reservoir although the variance between technical replicates remained high for all PCR experiments (Bray-Curtis dissimilarity: 5%-20%; Jaccard distance: 10%-40%).

Metabarcoding of vertebrate DNA obtained from invertebrates (iDNA) has been used to survey vertebrate communities, but we here show that it can also be used to study species interactions between invertebrates and vertebrates in a spatial context. We sampled the dung and carrion fly community of a swamp forest remnant along a disturbance gradient (10 sites: 80-310m from a road). Approximately, 60% of the baited 407 flies yield 294 vertebrate identifications based on two COI fragments and 16S sequenced with Illumina and/or MinION. A bipartite network analysis finds no specialization in the interaction between flies and vertebrate species, but a spatial analysis revealed that surprisingly 18 of the 20 vertebrate species can be detected within 150m of the road. We show that the fly community sourced for iDNA was unexpectedly rich (24 species, 3 families) and carried DNA for mammals, birds, and reptiles. They included common and rare ground-dwelling (e.g., wild boar, Sunda pangolin), and arboreal species (e.g., long-tailed macaque, Raffles' banded langur) as well as small bodied vertebrates (skinks, rats). All of our results were obtained with a new, greatly simplified iDNA protocol that eliminates DNA extraction by obtaining template directly through dissolving feces and regurgitates from individual flies with water. Lastly, we show that MinION- and Illumina-based metabarcoding yield similar results. Overall, flies from several families (calliphorids, muscids and sarcophagids) should be used in iDNA surveys because we show that uncommon fly species carry the signal for several vertebrate species that are otherwise difficult to detect with iDNA.

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