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.

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.

The Swedish Malaise Trap Project (SMTP) is one of the most ambitious insect inventories ever attempted. The project was designed to target poorly known insect groups across a diverse range of habitats in Sweden. The field campaign involved the deployment of 73 Malaise traps at 55 localities across the country for three years (2003-2006). Over the past 15 years, the collected material has been hand sorted by trained technicians into over 300 taxonomic fractions suitable for expert attention. The resulting collection is a tremendous asset for entomologists around the world, especially as we now face a desperate need for baseline data to evaluate phenomena like insect decline and climate change. Here, we describe the history, organisation, methodology and logistics of the SMTP, focusing on the rationale for the decisions taken and the lessons learned along the way. The SMTP represents one of the early instances of community science applied to large-scale inventory work, with a heavy reliance on volunteers in both the field and the laboratory. We give estimates of both staff effort and volunteer effort involved. The project has been funded by the Swedish Taxonomy Initiative; in total, the inventory has cost less than 30 million SEK (approximately 3.1 million USD). Based on a subset of the samples, we characterise the size and taxonomic composition of the SMTP material. Several different extrapolation methods suggest that the material comprises around 20 million specimens in total. The material is dominated by Diptera (75% of the specimens) and Hymenoptera (15% of specimens). Amongst the Diptera, the dominant groups are Chironomidae (37% of specimens), Sciaridae (15%), Phoridae (13%), Cecidomyiidae (9.5%) and Mycetophilidae (9.4%). Within Hymenoptera, the major groups are Ichneumonidae (44% of specimens), Diaprioidea (19%), Braconidae (9.6%), Platygastroidea (8.5%) and Chalcidoidea (7.9%). The taxonomic composition varies with latitude and season. Several Diptera and Hymenoptera groups are more common in non-summer samples (collected from September to April) and in the North, while others show the opposite pattern. About 1% of the total material has been processed and identified by experts so far. This material represents over 4,000 species. One third of these had not been recorded from Sweden before and almost 700 of them are new to science. These results reveal the large amounts of taxonomic work still needed on Palaearctic insect faunas. Based on the SMTP experiences, we discuss aspects of planning and conducting future large-scale insect inventory projects using mainly traditional approaches in relation to more recent approaches that rely on molecular techniques.

Halting biodiversity decline is one of the most critical challenges for humanity, but monitoring biodiversity is hampered by taxonomic impediments. One impediment is the large number of undescribed species (here called "dark taxon impediment") whereas another is caused by the large number of superficial species descriptions, that can only be resolved by consulting type specimens ("superficial description impediment"). Recently, Sharkey et al. (2021) proposed to address the dark taxon impediment for Costa Rican braconid wasps by describing 403 species based on COI barcode clusters ("BINs") computed by BOLD Systems. More than 99% of the BINs (387 of 390) were converted into species by assigning binominal names (e.g. BIN "BOLD: ACM9419" becomes Bracon federicomatarritai) and adding a minimal diagnosis (consisting only of a consensus barcode for most species). We here show that many of Sharkey et al.'s species are unstable when the underlying data are analyzed using different species delimitation algorithms. Add the insufficiently informative diagnoses, and many of these species will become the next "superficial description impediment" for braconid taxonomy because they will have to be tested and redescribed after obtaining sufficient evidence for confidently delimiting species. We furthermore show that Sharkey et al.'s approach of using consensus barcodes as diagnoses is not functional because it cannot be applied consistently. Lastly, we reiterate that COI alone is not suitable for delimiting and describing species, and voice concerns over Sharkey et al.'s uncritical use of BINs because they are calculated by a proprietary algorithm (RESL) that uses a mixture of public and private data. We urge authors, reviewers and editors to maintain high standards in taxonomy by only publishing new species that are rigorously delimited with openaccess tools and supported by publicly available evidence.

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...

New, rapid, accurate, scalable, and cost-effective species discovery and delimitation methods are needed for tackling “dark taxa”, here defined as groups for which &lt;10% of all species are described and the estimated diversity exceeds 1000 species. Species delimitation for these taxa should be based on multiple data sources (“integrative taxonomy”) but collecting multiple types of data risks impeding a discovery process that is already too slow. We here develop Large-scale Integrative Taxonomy (LIT), an explicit method where preliminary species hypotheses are generated based on inexpensive data that can be obtained quickly and cost-effectively. These hypotheses are then evaluated based on a more expensive type of “validation data” that are only obtained for specimens selected based on objective criteria applied to the preliminary species hypotheses. We here use this approach to sort 18 000 scuttle flies (Diptera: Phoridae) into 315 preliminary species hypotheses based on NGS barcode (313 bp) clusters (using Objective Clustering (OC) with a 3% threshold). These clusters are then evaluated with morphology as the validation data. We develop quantitative indicators for predicting which barcode clusters are likely to be incongruent with morphospecies by randomly selecting 100 clusters for in-depth validation with morphology. A linear model demonstrates that the best predictors for incongruence between barcode clusters and morphology are maximum p-distance within the cluster and a newly proposed index that measures cluster stability across different clustering thresholds. A test of these indicators using the 215 remaining clusters reveals that these predictors correctly identify all clusters that are incongruent with morphology. In our study all morphospecies are true or disjoint subsets of the initial barcode clusters so that all incongruence can be eliminated by varying clustering thresholds. This leads to a discussion of when a third data source is needed to resolve incongruent grouping statements. The morphological validation step in our study involved 1039 specimens (5.8% of the total). The formal LIT protocol we propose would only have required the study of 915 (5.1%: 2.5 specimens per species), as we show that clusters without signatures of incongruence can be validated by only studying two specimens representing the most divergent haplotypes. To test the generality of our results across different barcode clustering techniques, we establish that the levels of incongruence are similar across Objective Clustering (OC), Automatic Barcode Gap Discovery (ABGD), Poisson Tree Processes (PTP) and Refined Single Linkage (RESL) (used by Barcode of Life Data System (BOLD) to assign Barcode Index Numbers (BINs)). OC and ABGD achieved a maximum congruence score with morphology of 89% while PTP was slightly less effective (84%). RESL could only be tested for a subset of the specimens because the algorithm is not public. BINs based on 277 of the original 1 714 haplotypes were 86% congruent with morphology while the values were 89% for OC, 74% for PTP, and 72% for ABGD.

Halting biodiversity decline is one of the most critical challenges for humanity, but biodiversity assessment and monitoring are hampered by taxonomic impediments. We here distinguish between a "dark taxon impediment" caused by a large number of undescribed species and a "superficial description impediment" caused by species descriptions so imprecise that type specimens have to be consulted in order to resolve species identities. Recently, Sharkey et al. (2021) proposed to address the dark taxon impediment for Costa Rican braconid wasps by describing 403 species based on barcode clusters ("BINs") computed by BOLD Systems. The default assumption of the revision is that BIN=Species (e.g., BOLD:ACM9419 becomes Bracon federicomatarritai Sharkey, sp. nov.) and therefore the diagnoses of most species consist only of a consensus barcode. We here argue that this type of "minimalist revision" is unnecessary and undesirable. It is unnecessary because barcode clusters (e.g. BINs) already provide grouping statements that overcome many of the obstacles associated with dark taxon impediments. However, minimalist revisions are also undesirable and problematic because the diagnoses are only based on one character system - that in the case of Sharkey et al. was poorly analyzed. Furthermore, the revision relies on units that violate basic rules of reproducibility because the BINs were delimited by a proprietary algorithm (RESL) that is applied to a mixture of public and private data. Here, we illustrate that many of the BINs described as species are unstable when the available public data are reanalyzed, reiterate that COI mostly measures time of divergence, and that BOLD Systems violates key principles of open science. We conclude by urging authors, reviewers, editors, and grantors to only publish and fund projects that adhere to modern standards of reproducibility.

An urban biodiversity study sampling primarily from private backyards in Los Angeles, California (USA), reveals the presence of fifty-six species of Megaselia within the first few months of sampling. Thirty of these are described as new to science: