Background: 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 wellequipped, well-funded molecular laboratories are not readily available in many biodiverse countries. Results: We here document how MinION sequencing can be used for large-scale species discovery in a specimenand 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.). Conclusions: 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.

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction ('direct PCR') and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.

In many taxa the morphology of females and immatures is poorly known because species descriptions and identification tools have a male bias. The root causes are problems with matching life-history stages and genders belonging to the same species. Such matching is time-consuming when conventional methods are used (e.g. rearing) and expensive when the stages are matched with DNA barcodes. Unfortunately, the lack of associations is not a trivial problem because it renders a large part of the phenome of insects unexplored, although larvae and females are useful sources of characters for descriptive and phylogenetic purposes. In addition, many collectors intentionally avoid females and immature stages, which skews survey results, interferes with collecting life-history information, and makes it less likely that rare species are discovered. These problems even exist for well-studied taxa like Odonata, where obtaining adult-larva matches relies largely on rearing. Here we demonstrate how the matching problem can be addressed with cost-effective tagged amplicon sequencing of a 313-bp segment of cox1 with next-generation sequencing (NGS) ('NGS barcoding'). We illustrate the value of this approach based on Singapore's odonate fauna which is of a similar size as the European fauna (Singapore, 122 extant species; Europe, 138 recorded species). We match the larvae and adults of 59 species by first creating a barcode database for 338 identified adult specimens representing 83 species. We then sequence 1178 larvae from a wide range of sources. We successfully barcode 1123 specimens, which leads to adult-larva matches for 59 species based on our own barcodes (55) and online barcode databases (4). With these additions, 84 of the 131 species recorded in Singapore have now been associated with a species name. Most common species are now matched (83%), and good progress has been made for vulnerable/near-threatened (55%), endangered (53%), and critically endangered species (38%). We used nondestructive DNA extraction methods in order to be able to use high-resolution imaging of matched larvae to establish a publicly available digital reference collection for odonates which is incorporated into 'Biodiversity of Singapore' (https://singapore.biodiversity.online/). We suggest that the methods described here are suitable for many insect taxa because NGS barcoding allows for fast and low-cost matching of well-studied life-history stages with neglected semaphoronts (eggs, larvae, females). We estimate that the specimen-specific amplicons in this study (c. 1500 specimens) can now be obtained within eight working days and that the laboratory and sequencing cost is c. US$600 (< US$0.40 per specimen).

1Background: 2More than 80% of all animal species remain unknown to science. Most of these species live in 3 the tropics and belong to animal taxa that combine small body size with high specimen 4abundance and large species richness. For such clades, using morphology for species 5 discovery is slow because large numbers of specimens must be sorted using detailed 6 microscopic investigations. Fortunately, species discovery could be greatly accelerated if DNA 7 sequences could be used for sorting specimens to species. Morphological verification of such 8 "molecular Operational Taxonomic Units" (mOTUs) could then be based on dissection of a small 9 subset of specimens. However, this approach requires cost-effective and low-tech DNA 10 barcoding techniques because well equipped, well-funded molecular laboratories are not readily 11 available in many biodiverse countries. 12 Results: 13We here document how MinION sequencing can be used for large-scale species discovery in a 14 specimen-and species-rich taxon like the hyper-diverse fly family Phoridae (Diptera). We 15 sequenced 7,059 specimens collected in a single Malaise trap in Kibale National Park, Uganda 16 over the short period of eight weeks. We discovered >650 species which exceeded the number 17 of phorid species currently described for the entire Afrotropical region. The barcodes were 18 obtained using an improved low-cost MinION pipeline that increased the barcoding capacity 19 sevenfold from 500 to 3,500 barcodes per flowcell. This was achieved by adopting 1D 20 sequencing, re-sequencing weak amplicons on a used flowcell, and improving demultiplexing. 21Comparison with Illumina data revealed that the MinION barcodes were very accurate (99.99% 22 methods. In insects, one of the most widely used methods is Malaise trapping. Such traps 48 routinely collect thousands, or even tens of thousands, of specimens per site and week; i.e., 49 sorting all specimens to species-level virtually never happens and the world's natural history 50 museums store billions of unsorted specimens. Species-level sorting is usually restricted to a 51 few taxa with small to moderate numbers of specimens. It is accomplished in two stages. The 52 first is grouping specimens into easily identifiable major taxa (e.g., major groups of beetles, flies, 53 wasps). This type of pre-sorting is usually accomplished by parataxonomists with basic training 54 in morphology (e.g., students). The main challenge is the second sorting stage; i.e., sorting to 55 species-level. This work is best carried out by taxonomic experts whose techniques are, 56 however, mostly effective for taxa that have fairly small numbers of specimens and species. In 57 contrast, large, hyperdiverse and abundant taxa are ill-suited because they require dissection 58 and microscopic study of many specimens. An alternative to species-level sorting by 59 taxonomists is a hybrid approach that combines rapid pre-sorting to "morpho-species" by 60 parataxonomists with subsequent verification of morpho-species via DNA barcodes that ar...

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Most of arthropod biodiversity is unknown to science. Consequently, 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 barcodes. Here this approach is applied to flying insects sampled by 39 Malaise traps placed in five biogeographic regions, eight countries and numerous habitats (>225,000 specimens belonging to >25,000 species in 458 families). We find that 20 insect families (10 belonging to Diptera) account for >50% of local species diversity regardless of clade age, continent, climatic region and habitat type. 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 insect diversity are ‘dark taxa’ in that they suffer from extreme taxonomic neglect, with little signs of increasing activities in recent years. Taxonomic neglect tends to increase with diversity and decrease with body size. Identifying and tackling the diversity of ‘dark taxa’ with scalable techniques emerge as urgent priorities in biodiversity science.

Background The world’s fast disappearing mangrove forests have low plant diversity and are often assumed to also have a species-poor insect fauna. We here compare the tropical arthropod fauna across a freshwater swamp and six different forest types (rain-, swamp, dry-coastal, urban, freshwater swamp, mangroves) based on 140,000 barcoded specimens belonging to ca. 8500 species. Results We find that the globally imperiled habitat “mangroves” is an overlooked hotspot for insect diversity. Our study reveals a species-rich mangrove insect fauna (>3000 species in Singapore alone) that is distinct (>50% of species are mangrove-specific) and has high species turnover across Southeast and East Asia. For most habitats, plant diversity is a good predictor of insect diversity, but mangroves are an exception and compensate for a comparatively low number of phytophagous and fungivorous insect species by supporting an unusually rich community of predators whose larvae feed in the productive mudflats. For the remaining tropical habitats, the insect communities have diversity patterns that are largely congruent across guilds. Conclusions The discovery of such a sizeable and distinct insect fauna in a globally threatened habitat underlines how little is known about global insect biodiversity. We here show how such knowledge gaps can be closed quickly with new cost-effective NGS barcoding techniques.

Food mislabelling is a growing world-wide problem that is increasingly addressed through the authentication of ingredients via techniques like mass spectrometry or DNAsequencing. However, traditional DNA sequencing methods are slow, expensive, and require well-equipped laboratories. We here test whether these problems can be overcome through the use of Nanopore sequencing. We sequenced 92 single and 13 mixed-species samples bought in supermarkets and restaurants in Singapore which has a large and diverse seafood trade. We successfully obtained DNA barcodes for 94% and 100% of the single-and mixed-species products after correcting the numerous sequencing errors of MinION reads with a correction pipeline optimized for DNA barcodes. We find comparatively low levels of clear-cut mislabelling for single-species samples (7.6 %) while the rates are higher for mixed-species samples (38.5 %). These low rates are somewhat deceptive, however, because of the widespread use of vague common species names that do not allow for a precise assessment of the expected ingredients. With regard to the clearly mislabelled single-species products, higher-value products (e.g., prawn roe, wild-caught Atlantic salmon, halibut) are replaced with lowervalue ingredients (e.g., fish roe, Pacific salmon, arrowtooth flounder) while more serious problems are observed for mixed-species samples. Cuttlefish and prawn balls repeatedly contained pig DNA and 100% of all mixed samples labelled as containing crustaceans ('crab', 'prawn', 'lobster') only yielded fish barcodes. We conclude that there is a need for more regular testing of seafood samples and suggest that due to speed and low-cost, MinION would be a good instrument for this purpose. We also emphasize the need for developing clearer labelling guidelines..