Towards holistic insect monitoring: species discovery, description, identification and traits for all insects.

Holistic insect monitoring needs scalable techniques to overcome taxon biases, determine species abundances, and gather functional traits for all species. This requires that we address taxonomic impediments and the paucity of data on abundance, biomass and functional traits. We here outline how these data deficiencies could be addressed at scale. The workflow starts with large-scale barcoding (megabarcoding) of all specimens from mass samples obtained at biomonitoring sites. The barcodes are then used to group the specimens into molecular operational taxonomic units that are subsequently tested/validated as species with a second data source (e.g. morphology). New species are described using barcodes, images and short diagnoses, and abundance data are collected for both new and described species. The specimen images used for species discovery then become the raw material for training artificial intelligence identification algorithms and collecting trait data such as body size, biomass and feeding modes. Additional trait data can be obtained from vouchers by using genomic tools developed by molecular ecologists. Applying this pipeline to a few samples per site will lead to greatly improved insect monitoring regardless of whether the species composition of a sample is determined with images, metabarcoding or megabarcoding. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Drivers of arthropod biodiversity in an urban ecosystem.

Our world is becoming increasingly urbanized with a growing human population concentrated around cities. The expansion of urban areas has important consequences for biodiversity, yet the abiotic drivers of biodiversity in urban ecosystems have not been well characterized for the most diverse group of animals on the planet, arthropods. Given their great diversity, comparatively small home ranges, and ability to disperse, arthropods make an excellent model for studying which factors can most accurately predict urban biodiversity. We assessed the effects of (i) topography (distance to natural areas and to ocean) (ii) abiotic factors (mean annual temperature and diurnal range), and (iii) anthropogenic drivers (land value and amount of impervious surface) on the occurrence of six arthropod groups represented in Malaise trap collections run by the BioSCAN project across the Greater Los Angeles Area. We found striking heterogeneity in responses to all factors both within and between taxonomic groups. Diurnal temperature range had a consistently negative effect on occupancy but this effect was only significant in Phoridae. Anthropogenic drivers had mixed though mostly insignificant effects, as some groups and species were most diverse in highly urbanized areas, while other groups showed suppressed diversity. Only Phoridae was significantly affected by land value, where most species were more likely to occur in areas with lower land value. Los Angeles can support high regional arthropod diversity, but spatial community composition is highly dependent on the taxonomic group.

Express barcoding with NextGenPCR and MinION for species-level sorting of ecological samples.

The use of DNA barcoding is well established for specimen identification and large-scale biodiversity discovery, but remains underutilized for time-sensitive applications such as rapid species discovery in field stations, identifying pests, citizen science projects, and authenticating food. The main reason is that existing express barcoding workflows are either too expensive or can only be used in very well-equipped laboratories by highly-trained staff. We here show an alternative workflow combining rapid DNA extraction with HotSHOT, amplicon production with NextGenPCR thermocyclers, and sequencing with low-cost MinION sequencers. We demonstrate the power of the approach by generating 250 barcodes for 285 specimens within 6 h including specimen identification through BLAST. The workflow required only the following major equipment that easily fits onto a lab bench: Thermocycler, NextGenPCR, microplate sealer, Qubit, and MinION. Based on our results, we argue that simplified barcoding workflows for species-level sorting are now faster, more accurate, and sufficiently cost-effective to replace traditional morpho-species sorting in many projects.

FAVIS: Fast and versatile protocol for non-destructive metabarcoding of bulk insect samples.

Insects are diverse and sustain essential ecosystem functions, yet remain understudied. Recent reports about declines in insect abundance and diversity have highlighted a pressing need for comprehensive large-scale monitoring. Metabarcoding (high-throughput bulk sequencing of marker gene amplicons) offers a cost-effective and relatively fast method for characterizing insect community samples. However, the methodology applied varies greatly among studies, thus complicating the design of large-scale and repeatable monitoring schemes. Here we describe a non-destructive metabarcoding protocol that is optimized for high-throughput processing of Malaise trap samples and other bulk insect samples. The protocol details the process from obtaining bulk samples up to submitting libraries for sequencing. It is divided into four sections: 1) Laboratory workspace preparation; 2) Sample processing-decanting ethanol, measuring the wet-weight biomass and the concentration of the preservative ethanol, performing non-destructive lysis and preserving the insect material for future work; 3) DNA extraction and purification; and 4) Library preparation and sequencing. The protocol relies on readily available reagents and materials. For steps that require expensive infrastructure, such as the DNA purification robots, we suggest alternative low-cost solutions. The use of this protocol yields a comprehensive assessment of the number of species present in a given sample, their relative read abundances and the overall insect biomass. To date, we have successfully applied the protocol to more than 7000 Malaise trap samples obtained from Sweden and Madagascar. We demonstrate the data yield from the protocol using a small subset of these samples.

Towards Large-Scale Integrative Taxonomy (LIT): Resolving the Data Conundrum for Dark Taxa.

New, rapid, accurate, scalable, and cost-effective species discovery and delimitation methods are needed for tackling "dark taxa," here defined as groups for which $<$10$\%$ of all species are described and the estimated diversity exceeds 1,000 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 is 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 next-generation sequencing 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 1,039 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 OC, Automatic Barcode Gap Discovery (ABGD), Poisson Tree Processes (PTP), and Refined Single Linkage (RESL) (used by Barcode of Life Data System to assign Barcode Index Numbers [BINs]). OC and ABGD achieved a maximum congruence score with the 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. [Biodiversity discovery; dark taxa; DNA barcodes; integrative taxonomy.].

A re-analysis of the data in Sharkey et al.'s (2021) minimalist revision reveals that BINs do not deserve names, but BOLD Systems needs a stronger commitment to open science.

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 open-access tools and supported by publicly available evidence.

ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone.

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: A 15 Year Retrospective on a Countrywide Insect Inventory.

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.

Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing.

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 well-equipped, 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 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.). 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.

Temperature accounts for the biodiversity of a hyperdiverse group of insects in urban Los Angeles.

The urban heat island effect is a worldwide phenomenon that has been linked to species distributions and abundances in cities. However, effects of urban heat on biotic communities are nearly impossible to disentangle from effects of land cover in most cases because hotter urban sites also have less vegetation and more impervious surfaces than cooler sites within cities. We sampled phorid flies, one of the largest, most biologically diverse families of true flies (Insecta: Diptera: Phoridae), at 30 sites distributed within the central Los Angeles Basin, where we found that temperature and the density of urban land cover are decoupled. Abundance, richness, and community composition of phorids inside urban Los Angeles were most parsimoniously accounted for by mean air temperature in the week preceding sampling. Sites with intermediate mean temperatures had more phorid fly individuals and higher richness. Communities were more even at urban sites with lower minimum temperatures and sites located further away from natural areas, suggesting that communities separated from natural source populations may be more homogenized. Species composition was best explained by minimum temperature. Inasmuch as warmer areas within cities can predict future effects of climate change, phorid fly communities are likely to shift nonlinearly under future climates in more natural areas. Exhaustive surveys of biotic communities within cities, such as the one we describe here, can provide baselines for determining the effects of urban and global climate warming as they intensify.

A new white-spotted Megaselia Rondani (Diptera: Phoridae) from western North America.

BACKGROUND: The phorid fly genus Megaselia Rondani is a large, poorly-known taxon whose species are found worldwide. NEW INFORMATION: A new species of Megaselia Rondani, M. simunorum, is described from both urban and rural sites in southern California. With a large area of white colour on the posterior part of the abdominal dorsum, it closely resembles the much more common species M. sulphurizona, but M. simunorum has distinctly thicker ventral setae on the abdomen and a differently-shaped white spot.

Mystery mushroom malingerers: Megaselia marquezi Hartop et al. 2015 (Diptera: Phoridae).

A mysterious female phorid fly, known for many years to be associated with fungal sporophores ("mushrooms") is identified as Megaselia marquezi Hartop et al. 2015. Male and female flies were collected emerging from the fungus Psathyrella candolleana (Fr.) Maire, and females were observed swarming over the sporophores.

Baby Killers: Documentation and Evolution of Scuttle Fly (Diptera: Phoridae) Parasitism of Ant (Hymenoptera: Formicidae) Brood.

BACKGROUND: Numerous well-documented associations occur among species of scuttle flies (Diptera: Phoridae) and ants (Hymenoptera: Formicidae), but examples of brood parasitism are rare and the mechanisms of parasitism often remain unsubstantiated. NEW INFORMATION: We present two video-documented examples of ant brood (larvae and pupae) parasitism by scuttle flies. In footage from Estação Biológica de Boracéia in Brazil, adult females of Ceratoconus setipennis Borgmeier can be seen attacking workers of Linepithema humile (Mayr) species group while they are carrying brood, and ovipositing directly onto brood in the nest. In another remarkable example, footage from the Soltis Center, near Peñas Blancas in Costa Rica, shows adult females of an unidentified species of the Apocephalus grandipalpus Borgmeier group mounting Pheidole Westwood brood upside-down and ovipositing while the brood are being transported by workers. Analysis of evolutionary relationships (in preparation) among Apocephalus Coquillett species shows that this is a newly derived behavior within the genus, as the A. grandipalpus group arises within a group of adult ant parasitoids. In contrast, relationships of Ceratoconus Borgmeier have not been studied, and the lifestyles of the other species in the genus are largely unknown.

Flies from L.A., The Sequel: A further twelve new species of Megaselia (Diptera: Phoridae) from the BioSCAN Project in Los Angeles (California, USA).

BACKGROUND: Presented are continued results from the BioSCAN Project, an urban biodiversity study sampling primarily from private backyards in Los Angeles, California (USA). Presented are continued results from the BioSCAN Project, an urban biodiversity study sampling primarily from private backyards in Los Angeles, California (USA). NEW INFORMATION: Twelve new species of Megaselia (Diptera: Phoridae) are described: M. baileyae, M. friedrichae, M. gonzalezorum, M. joanneae, M. losangelensis, M. phyllissunae, M. pongsaiae, M. shatesae, M. stoakesi, M. studentorum, M. voluntariorum, M. wongae.

Opportunity in our Ignorance: Urban Biodiversity Study Reveals 30 New Species and One New Nearctic Record for Megaselia (Diptera: Phoridae) in Los Angeles (California, USA).

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: M. armstrongorum, M. bradyi, M. brejchaorum, M. carthayensis, M. ciancii, M. creasoni, M. defibaughorum, M. donahuei, M. francoae, M. fujiokai, M. hardingorum, M. heini, M. hentschkeae, M. hoffmanorum, M. hoggorum, M. hoguei, M. isaacmajorum, M. kelleri, M. lombardorum, M. marquezi, M. mikejohnsoni, M. oxboroughae, M. pisanoi, M. renwickorum, M. rodriguezorum, M. sacatelensis, M. seaverorum, M. sidneyae, M. steptoeae, and M. wiegmanae. M. largifrontalis is newly reported from the Nearctic Region. The implications these findings have for future taxonomic work in Megaselia, particularly in urban areas, are discussed.

The tip of the iceberg: a distinctive new spotted-wing Megaselia species (Diptera: Phoridae) from a tropical cloud forest survey and a new, streamlined method for Megaselia descriptions.

A new Megaselia species, M.shadeae, with a large, central, pigmented and bubble-like wing spot and a greatly enlarged radial wing vein fork, is described from Zurquí de Moravia, Costa Rica. As part of the Zurquí All Diptera Biodiversity Inventory (ZADBI) project, it represents the first of an incredible number of new phorid species to be described from this one Costa Rican cloud forest site. A new, streamlined method of description for species of this enormous genus of phorid flies is presented.