Network analysis with either Illumina or MinION reveals that detecting vertebrate species requires metabarcoding of iDNA from a diverse fly community.

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.

ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone.

BACKGROUND: DNA barcodes are a useful tool for discovering, understanding, and monitoring biodiversity which are critical tasks at a time of rapid biodiversity loss. However, widespread adoption of barcodes requires cost-effective and simple barcoding methods. We here present a workflow that satisfies these conditions. It was developed via "innovation through subtraction" and thus requires minimal lab equipment, can be learned within days, reduces the barcode sequencing cost to < 10 cents, and allows fast turnaround from specimen to sequence by using the portable MinION sequencer. RESULTS: We describe how tagged amplicons can be obtained and sequenced with the real-time MinION sequencer in many settings (field stations, biodiversity labs, citizen science labs, schools). We also provide amplicon coverage recommendations that are based on several runs of the latest generation of MinION flow cells ("R10.3") which suggest that each run can generate barcodes for > 10,000 specimens. Next, we present a novel software, ONTbarcoder, which overcomes the bioinformatics challenges posed by MinION reads. The software is compatible with Windows 10, Macintosh, and Linux, has a graphical user interface (GUI), and can generate thousands of barcodes on a standard laptop within hours based on only two input files (FASTQ, demultiplexing file). We document that MinION barcodes are virtually identical to Sanger and Illumina barcodes for the same specimens (> 99.99%) and provide evidence that MinION flow cells and reads have improved rapidly since 2018. CONCLUSIONS: We propose that barcoding with MinION is the way forward for government agencies, universities, museums, and schools because it combines low consumable and capital cost with scalability. Small projects can use the flow cell dongle ("Flongle") while large projects can rely on MinION flow cells that can be stopped and re-used after collecting sufficient data for a given project.

First Report of Co-invasion by the Reptile Nematode Ozolaimus megatyphlon (Nematoda: Pharyngodonidae) with Invasive Green Iguanas (Iguana iguana) in the Asia-Pacific.

PURPOSE: Co-invasion of naïve ecosystems by non-native parasites is a serious threat to global biodiversity, though such events are difficult to detect early in the invasion process. Green iguanas (Iguana iguana) are an emerging invasive species and have colonised several countries in the Asia-Pacific. A survey was undertaken to determine whether parasites of the green iguana had co-invaded naïve ecosystems with their introduced host. METHODS: Over a 10-month period, wild green iguanas were trapped and euthanised in Singapore. All animals were necropsied and sampled for parasites. Parasites were then identified morphologically and subsequently characterised molecularly at the cytochrome c oxidase I (COI) locus. RESULTS: The reptile nematode Ozolaimus megatyphlon was found in 38% of the sampled green iguanas, with burdens of 100 + worms in all infected animals. This represents the first recorded co-invasion of this species with wild green iguanas in the Asia-Pacific. Based on the molecular characterisation of the cytochrome c oxidase I (COI) locus, the first DNA barcode is provided for O. megatyphlon. CONCLUSION: For the first time, the reptile nematode Ozolaimus megatyphlon is shown to be invasive and to have colonised the Asia-Pacific region with its introduced host, the green iguana. The DNA barcode provided here will facilitate future monitoring programmes as O. megatyphlon invades new habitats and countries.