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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.

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.

Morphometric research is being applied to a growing number and variety of organisms. Discoveries achieved via morphometric approaches are often considered highly transferable, in contrast to the tacit and idiosyncratic interpretation of discrete character states. The reliability of morphometric workflows in insect systematics has never been a subject of focused research, but such studies are sorely needed. In this paper, we assess the reproducibility of morphometric studies of ants where the mode of data collection is a shared routine.We compared datasets generated by eleven independent gaugers, that is, collaborators, who measured 21 continuous morphometric traits on the same pool of individuals according to the same protocol. The gaugers possessed a wide range of morphometric skills, had varying expertise among insect groups, and differed in their facility with measuring equipment. We used intraclass correlation coefficients (ICC) to calculate repeatability and reproducibility values (i.e., intra‐ and intergauger agreements), and we performed a multivariate permutational multivariate analysis of variance (PERMANOVA) using the Morosita index of dissimilarity with 9,999 iterations.The calculated average measure of intraclass correlation coefficients of different gaugers ranged from R = 0.784 to R = 0.9897 and a significant correlation was found between the repeatability and the morphometric skills of gaugers (p = 0.016). There was no significant association with the magnification of the equipment in the case of these rather small ants. The intergauger agreement, that is the reproducibility, varied between R = 0.872 and R = 0.471 (mean R = 0.690), but all gaugers arrived at the same two‐species conclusion. A PERMANOVA test revealed no significant gauger effect on species identity (R 2 = 0.69, p = 0.58).Our findings show that morphometric studies are reproducible when observers follow the standard protocol; hence, morphometric findings are widely transferable and will remain a valuable data source for alpha taxonomy., This paper reveals that morphometric findings are reliable and widely transferable in insect research and can represent a valuable data source in the field of ecological and evolutionary studies. The manuscript can also help to educate readers on important issues regarding measurements and measurement error. This is especially important in the field of ecology and systematics today because there is a general lack of formal training in these important concepts in the 21st century biological science curriculum.

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 (

Food mislabelling is a growing world-wide problem that is increasingly addressed through the authentication of ingredients via techniques like mass spectrometry or DNA-sequencing. 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 lower-value 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.

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.ResultsWe 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.).ConclusionsWe 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.

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.

We here argue that data from comparative studies of genome size and karyotypes provide important information for planning comparative research on genome evolution. We document for 39 species of sepsids that there is a four-fold difference in genome size (151-618 Mbp). Mapping genome sizes onto a phylogenetic hypothesis identifies that this range is the result of five genome expansions and four genome contractions that we here define as changes in genome size of more than 50 Mbp. We then generate karyotype data for 10 species and find no changes in chromosome number. The study reveals that the "Oriental" clade of sepsids is a promising system for studying genome evolution because it has experienced three genome expansion events. These events can be compared with an expansion in the "Neotropical" clade in order to reveal the mechanisms that underlie genome expansion in Sepsidae. A review of the literature on genome sizes and karyotypes reveals that they have been poorly documented in Metazoa. This means that researchers interested in the evolution of genome expansions and contractions are currently not being able to identify appropriate target taxa for genome sequencing. We thus argue for more comparative research on genome sizes and karyotypes and point out that historically species were chosen for genome sequencing for reasons not related to genome evolution (e.g. small genome size, model species status, phylogenetic position, interesting phenotypes). We believe that it is now time to use a more genome-centric selection criterion, where species for whole genome sequencing are selected based on their importance for understanding genome evolution.