Biology subjects

Kutty, S. N.

Publications and source records attributed to Kutty, S. N..

5 recordsLinked to original sources

bioRxiv2022

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

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.

ecology↗

bioRxiv2022

Global convergence of dominance and neglect in flying insect diversity

Most of arthropod biodiversity is unknown to science. For this reason, it has been unclear whether insect communities around the world are dominated by the same or different taxa. This question can be answered through standardized sampling of biodiversity followed by estimation of species diversity and community composition with DNA sequences. This approach is here applied to flying insects sampled by 39 Malaise traps placed in five biogeographic regions, eight countries, and numerous habitats (>220,000 specimens belonging to >25,000 species in 463 families). Unexpectedly, we find that 20 insect families account for >50% of local species diversity regardless of continent, climatic region, and habitat type. These consistent differences in family-level dominance explain two-thirds of variation in community composition despite massive levels of species turnover, with most species (>97%) in the top 20 families encountered at a single site only. Alarmingly, the same families that dominate global insect diversity also suffer from extreme taxonomic neglect with little signs of increasing activities in recent years. Tackling the biodiversity of these "dark taxa" thereby emerges as an urgent priority because the arthropod groups comprising most of the global flying insect diversity are particularly poorly known.

ecology↗

bioRxiv2021

Towards environmental DNA-based bioassessment of freshwater reservoirs with small volumes of water: robust molecular protocols

Bioassessment of freshwater quality via eDNA is rapidly developing into a powerful alternative to traditional methods involving collecting, sorting, and identifying macroinvertebrates based on morphology. Particularly attractive would be methods that can use remote-controlled boats for sampling because it would allow for cost-effective, and frequent monitoring at multiple sites. The latter will be particularly important for tropical reservoirs that require year-around surveillance. We here optimize molecular protocols for capturing reservoir-specific differences in metazoan communities based on small water volumes (15 mL). The optimization is based on samples from two freshwater reservoirs with very different water qualities ("reservoir signal"). Each reservoir was sampled at three sites ("biological replicates"). For each water sample, the DNA was extracted twice ("technical replicates"). We then tested how much DNA template (0.1 ng to 15 ng) and how many PCR cycles (25 or 35) minimized variance between technical replicates. We find that 15 mL is sufficient for capturing the reservoir signal regardless of sampling time, template amounts, or PCR cycle numbers. Indeed, extrapolation from our results suggests that <1 mL would be sufficient because only 17 of 59 metazoan mOTUs (mainly planktonic crustaceans and rotifers) detected with a 313bp COI minibarcode were shared. We find that the use of 35 PCR cycles significantly lowered the number of detected species and that template amounts <0.5 ng yielded somewhat higher variance between technical replicates. Despite extensive trials, the variance between technical replicates remained high (Bray-Curtis: 5-20%; Jaccard: 10-40%) and we predict that it will be difficult to reduce this variance further. However, the overall reservoir differences are so strong that all biological and technical replicates can be correctly assigned.

ecology↗

bioRxiv2021

MinION barcodes: biodiversity discovery and identification by everyone, for everyone

BackgroundDNA 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, real-time sequencer MinION. ResultsWe describe cost-effective and rapid procedures for barcoding individual specimens with MinION sequencing. We illustrate how tagged amplicons can be obtained and sequenced with the portable, 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. ConclusionsWe 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.

ecology↗

bioRxiv2019

1D MinION sequencing for large-scale species discovery: 7000 scuttle flies (Diptera: Phoridae) from one site in Kibale National Park (Uganda) revealed to belong to >650 species

BackgroundMore than 80% of all animal species remain unknown to science. Most of these species live in the tropics and belong to animal taxa that combine small body size with high specimen abundance and large species richness. For such clades, using morphology for species discovery is slow because large numbers of specimens must be sorted using detailed microscopic investigations. Fortunately, species discovery could be greatly accelerated if DNA sequences could be used for sorting specimens to species. Morphological verification of such \"molecular Operational Taxonomic Units\" (mOTUs) could then be based on dissection of a small subset of specimens. However, this approach requires cost-effective and low-tech DNA barcoding techniques because well equipped, well-funded molecular laboratories are not readily available in many biodiverse countries.\n\nResultsWe here document how MinION sequencing can be used for large-scale species discovery in a specimen- and species-rich taxon like the hyper-diverse fly family Phoridae (Diptera). We sequenced 7,059 specimens collected in a single Malaise trap in Kibale National Park, Uganda over the short period of eight weeks. We discovered >650 species which exceeded the number of phorid species currently described for the entire Afrotropical region. The barcodes were obtained using an improved low-cost MinION pipeline that increased the barcoding capacity sevenfold from 500 to 3,500 barcodes per flowcell. This was achieved by adopting 1D sequencing, re-sequencing weak amplicons on a used flowcell, and improving demultiplexing. Comparison with Illumina data revealed that the MinION barcodes were very accurate (99.99% accuracy, 0.46% Ns) and thus yielded very similar species units (match ratio: 0.991). Morphological examination of 100 mOTUs also confirmed good congruence with morphology (93% of mOTUs; >99% of specimens) and revealed that 90% of the putative species belong to a neglected, megadiverse genus (Megaselia). We demonstrate for one Megaselia species how the molecular data can guide the description of a new species (Megaselia sepsioides sp. nov.).\n\nConclusionsWe document that one field site in Africa can be home to an estimated 1000 species of phorids and speculate that the Afrotropical diversity could exceed 100,000 species. We furthermore conclude that low-cost MinION sequencers are very suitable for reliable, rapid, and large-scale species discovery in hyperdiverse taxa. MinION sequencing could quickly reveal the extent of the unknown diversity and is especially suitable for biodiverse countries with limited access to capital-intensive sequencing facilities.

evolutionary biology↗