More 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 based on 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.
We here document how MinION sequencing can be used for large-scale species discovery in a specimen- and species-rich taxon like the hyperdiverse fly family Phoridae (Diptera). We sequenced 7059 specimens collected in a single Malaise trap in Kibale National Park, Uganda, over the short period of 8 weeks. We discovered > 650 species which exceeds 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 3500 barcodes per flowcell. This was achieved by adopting 1D sequencing, resequencing 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 the 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.).
We 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 200,000 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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Sarcophagidae (flesh flies) comprise a large and widely distributed radiation within the Calyptratae (Diptera). Larval feeding habits are ecologically diverse and include sarcosaprophagy, ...coprophagy, herbivory, invertebrate and vertebrate predation, and kleptoparasitism. To elucidate the geographic origin and evolution of flesh fly life‐history, we inferred a backbone phylogeny based on transcriptomic data from 26 sarcophagid species covering all three subfamilies plus 15 outgroups. The phylogeny was inferred using maximum parsimony and maximum likelihood methods based on a series of supermatrices, one set with overall information content improved by MARE (2290 loci), one set with 100% gene coverage for all included species (587 loci), and the last set including mitochondrial and nuclear genes (589 loci) and additional taxa. In order to obtain a more detailed hypothesis, we utilized the supertree approach to combine results from the present study with previously published hypotheses. This resulted supertree covers 84 of the one hundred currently recognized sarcophagid genera and formed the basis for the ancestral state reconstructions. The monophyletic Sarcophagidae is well‐supported as sister to {Mystacinobiidae + Oestridae}, and relationships at the subfamily level are inferred as {Sarcophaginae, (Paramacronychiinae + Miltogramminae)}. The Sarcophagidae and each subfamily originated in the Americas, with Sarcophaginae diversifying mainly in the Neotropics, whereas the major radiation of both Miltogramminae and Paramacronychiinae occurred in the Palaearctic. Sarcosaprophagy is reconstructed as the ancestral larval feeding habit of the family Sarcophagidae and each subfamily. The ancestral sarcophagid larva probably utilized dead invertebrates as food, and the food spectrum expanded together with the diversification of breeding strategies. Particularly, kleptoparasitism in Miltogramminae is derived from sarcosaprophagy and may be seen as having derived from the breeding biology of ‘lower’ miltogrammines, the larvae of which feed on buried vertebrate carrion.
Diffuse optical measurement is an evolving optical modality providing a fast and portable solution for microcirculation assessment. Diffuse optics in static and dynamic modalities are combined here ...in a system to assess hemodynamics in skin tissues of control and diabetic subjects. The in‐house developed system consists of a laser source, fiber optic probe, a low‐cost avalanche photodiode, a finite element model (FEM) derived static optical property estimator, and a software correlator for continuous flow monitoring through microvasculature. The studies demonstrated that the system quantifies the changes in blood flow rate in the immediate skin subsurface. The system is calibrated with in vitro flow models and a proof‐of‐concept was demonstrated on a limited number of subjects in a clinical environment. The flow changes in response to vasoconstrictive and vasodilative stimuli were analyzed and used to classify different stages of diabetes, including diabetic neuropathy.
The system is proposed and demonstrated for estimating skin perfusion at lower extremities subjects with varying stages of diabetes mellitus. The novel concept of employing diffuse correlation spectroscopy at short source to detector separation and utilization of a static parameter estimator from a pre‐defined look‐up table modelled from human skin tissue parameters using FEM provides a viable alternative to detect the onset of diabetic neuropathy via assessing microcirculation.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Culicomorpha is a particularly species‐rich clade within Diptera (true flies) that comprises c. 10% of the described diversity, including many medically important flies. Morphological studies – even ...when all life stages are included – yield relationships different from those derived from molecular data, notably with regard to the position of Chironomidae. Congruence amongst molecular studies has been weak due to limitations in gene‐ and family‐level taxon coverage. Here we use a whole‐transcriptome shotgun phylogenomic approach to clarify the relationships among all families of Culicomorpha. The dataset comprised 30 species (27 ingroup) and 364 888 amino acid residues for 1233 single‐copy protein‐encoding genes. Likelihood and parsimony analyses produce robust and highly congruent phylogenetic trees, with only one node in conflict. The superfamily Culicoidea is well supported and comprises Dixidae + Corethrellidae + (Chaoboridae + Culicidae). As suggested previously, Chironomoidea is not monophyletic. The well supported Thaumaleidae + Simuliidae is sister group to Culicoidea, with the weakly supported Chironomidae + Ceratopogonidae probably being the sister group of all remaining Culicomorpha. We used random addition concatenation analysis (RADICAL) and four‐cluster likelihood mapping (FcLM) to assess the strengths of nodal support. The sister‐group relationship between Chironomidae + Ceratopogonidae is consistent with the FcLM results but support for this relationship emerges only when 1150 of the 1233 loci are analysed. We discuss briefly nodes that remain poorly supported even with thousands of genes and mention problems with vouchering in transcriptomic studies.
We reconstruct the relationships among the eight constituent families of the species‐rich Culicomorpha (Diptera) clade using a phylogenomic approach.
Different analysis strategies produce robust and highly congruent phylogenetic trees, with only the positions of the Chironomidae and Ceratopogonidae in conflict.
Culicoidea is monophyletic and comprises Dixidae, Corethrellidae, Chaoboridae and Culicidae; Thaumaleidae + Simuliidae is the sister group of the Culicoidea.
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.
The Calyptratae, one of the most species‐rich fly clades, only originated and diversified after the Cretaceous–Palaeogene extinction event and yet exhibit high species diversity and a diverse array ...of life history strategies including predation, phytophagy, saprophagy, haematophagy and parasitism. We present the first phylogenomic analysis of calyptrate relationships. The analysis is based on 40 species representing all calyptrate families and on nucleotide and amino acid data for 1456 single‐copy protein‐coding genes obtained from shotgun sequencing of transcriptomes. Topologies are overall well resolved, robust and largely congruent across trees obtained with different approaches (maximum parsimony, maximum likelihood, coalescent‐based species tree, four‐cluster likelihood mapping). Many nodes have 100% bootstrap and jackknife support, but the true support varies by more than one order of magnitude Bremer support from 3 to 3427; random addition concatenation analysis (RADICAL) gene concatenation size from 10 to 1456. Analyses of a Dayhoff‐6 recoded amino acid dataset also support the robustness of many clades. The backbone topology Hippoboscoidea+(Fanniidae+(Muscidae+((Anthomyiidae–Scathophagidae)+Oestroidea))) is strongly supported and most families are monophyletic (exceptions: Anthomyiidae and Calliphoridae). The monotypic Ulurumyiidae is either alone or together with Mesembrinellidae as the sister group to the rest of Oestroidea. The Sarcophagidae are sister to Mystacinobiidae+Oestridae. Polleniinae emerge as sister group to Tachinidae and the monophyly of the clade Calliphorinae+Luciliinae is well supported, but the phylogenomic data cannot confidently place the remaining blowfly subfamilies (Helicoboscinae, Ameniinae, Chrysomyinae). Compared to hypotheses from the Sanger sequencing era, many clades within the muscoid grade are congruent but now have much higher support. Within much of Oestroidea, Sanger era and phylogenomic data struggle equally with regard to finding well‐supported hypotheses.
Spatially resolved diffuse reflectance spectroscopy (SRDRS) is a non‐invasive optical technique that helps in clinical diagnosis of various tissue microcirculation and skin pigmentation disorders ...based on collected backscattered light from multi‐layered tissue. The extraction of the optical properties from the reflectance spectrum using analytical solutions is laborious. Model‐based light tissue interaction studies help in quantifying the optical properties. This work presents the use of finite element models of light tissue interaction for this purpose. A bilayer model mimicking human skin was considered and the diffused reflectance spectra at multiple detector points were generated using finite element modelling for varying melanin concentration, epidermal thickness, blood volume fraction, oxygen saturation and scattering components. The reflectance value based on varying optical parameters from multiple detection points lead to the generation of a look‐up table (LUT), which is further used for finding the tissue parameters that contribute to the spatially resolved reflectance values. The tissue parameters estimated after inverse modelling showed a high degree of agreement with the expected tissue parameters for a test dataset different from the training dataset.
Propagation of visible light is realised through a skin tissue finite element model, and the resulting reflectance spectra were simulated for varying tissue optical properties. A reflectance look‐up table was constructed based on various combinations of inhomogeneous tissue optical properties and source‐detector distances. An algorithm was developed to retrieve the optical properties for unknown models different from the training set. We achieved a reduction in the time of computation and errors when compared to existing methods for similar applications.
•Short-span time-varying fluorescence analysis of FAD and NADH are studied in different concentration levels.•The unique interaction behavior of FAD and NADH leading to simultaneous emission changes ...in a range of concentrations and redox ratios is demonstrated here for the first time.•This study elucidates that the short-time fluorescence intensity variations occur because of molecules’ interaction, not photobleaching.•A 100% accuracy in discriminating the cancer from normal samples is achieved using PCA and LDA analysis of time-varying integrated fluorescence intensity.•Similar trend were observed reported metabolite concentration sin Urine.•Our approach has broad applicability on blood, plasma, and urine samples with various FAD-NADH concentration ranges.
Body fluids carry several biomarkers and genetic material that enable the detection of carcinogenesis and tumor metastasis. Owing to this, liquid biopsy is gaining importance and entering clinical practice as an adjunct modality to the current standard of practice in cancer detection. Liquid biopsy involving specimens such as urine is non-invasive and has biomarkers reflecting the tumor microenvironment. We investigate the normal and altered levels of cellular metabolic by-products in liquid phantoms and demonstrate their photon-induced excited-state interaction behaviour for potential diagnostic applications. Short-span temporal analysis of metabolic by-products FAD and NADH in liquid phantoms with fiber optic fluorescence spectroscopy is carried out. The mixed liquid phantom of FAD and NADH, showed excited state interactions that manifested as simultaneous inverse variation in the fluorescence emission from FAD and NADH in varying pH environments. Linear discriminant analysis of the time-varying integrated fluorescence intensity resulted in a 100% accuracy in classifying liquid samples between high and low metabolite concentrations. An extension of application of the experiment model for analysis of body fluids such as urine is also discussed.
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