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This is the first comprehensive synopsis of the biodiversity of Diptera, which with more than 150.000 described species contain more than one tenth of all described animal species. The first part is a review of what is already known, with treatments of all the major biogeographical regions and important archipelagoes; the second part contains case studies on open-ended taxa, Diptera as ecological indicators, and how to estimate the still unknown proportion of our fauna; and the third part discusses the digital and molecular tools needed to document the fauna. The book has an emphasis on principles and analytical approaches as well as on practical 'how-to' information and is intended for academicians and other professionals but with a significant outreach to students.

Bees, hoverflies and butterflies are taxa frequently studied as pollinators in agricultural and conservation contexts. Although there are many records of non-syrphid Diptera visiting flowers, they are generally not regarded as important pollinators. We use data from 30 pollen-transport networks and 71 pollinator-visitation networks to compare the importance of various flower-visiting taxa as pollen-vectors. We specifically compare non-syrphid Diptera and Syrphidae to determine whether neglect of the former in the literature is justified. We found no significant difference in pollen-loads between the syrphid and non-syrphid Diptera. Moreover, there was no significant difference in the level of specialization between the two groups in the pollen-transport networks, though the Syrphidae had significantly greater visitation evenness. Flower visitation data from 33 farms showed that non-syrphid Diptera made up the majority of the flower-visiting Diptera in the agricultural studies (on average 82% abundance and 73% species richness), and we estimate that non-syrphid Diptera carry 84% of total pollen carried by farmland Diptera. As important pollinators, such as bees, have suffered serious declines, it would be prudent to improve our understanding of the role of non-syrphid Diptera as pollinators.

Many species groups, including mammals and many insects, determine sex using heteromorphic sex chromosomes. Diptera flies, which include the model Drosophila melanogaster, generally have XY sex chromosomes and a conserved karyotype consisting of six chromosomal arms (five large rods and a small dot), but superficially similar karyotypes may conceal the true extent of sex chromosome variation. Here, we use whole-genome analysis in 37 fly species belonging to 22 different families of Diptera and uncover tremendous hidden diversity in sex chromosome karyotypes among flies. We identify over a dozen different sex chromosome configurations, and the small dot chromosome is repeatedly used as the sex chromosome, which presumably reflects the ancestral karyotype of higher Diptera. However, we identify species with undifferentiated sex chromosomes, others in which a different chromosome replaced the dot as a sex chromosome or in which up to three chromosomal elements became incorporated into the sex chromosomes, and others yet with female heterogamety (ZW sex chromosomes). Transcriptome analysis shows that dosage compensation has evolved multiple times in flies, consistently through up-regulation of the single X in males. However, X chromosomes generally show a deficiency of genes with male-biased expression, possibly reflecting sex-specific selective pressures. These species thus provide a rich resource to study sex chromosome biology in a comparative manner and show that similar selective forces have shaped the unique evolution of sex chromosomes in diverse fly taxa.

Metabarcoding is an emerging genetic tool to rapidly assess biodiversity in ecosystems. It involves high-throughput sequencing of a standard gene from an environmental sample and comparison to a reference database. However, no consensus has emerged regarding laboratory pipelines to screen species diversity and infer species abundances from environmental samples. In particular, the effect of primer bias and the detection limit for specimens with a low biomass has not been systematically examined, when processing samples in bulk. We developed and tested a DNA metabarcoding protocol that utilises the standard cytochrome c oxidase subunit I (COI) barcoding fragment to detect freshwater macroinvertebrate taxa. DNA was extracted in bulk, amplified in a single PCR step, and purified, and the libraries were directly sequenced in two independent MiSeq runs (300-bp paired-end reads). Specifically, we assessed the influence of specimen biomass on sequence read abundance by sequencing 31 specimens of a stonefly species with known haplotypes spanning three orders of magnitude in biomass (experiment I). Then, we tested the recovery of 52 different freshwater invertebrate taxa of similar biomass using the same standard barcoding primers (experiment II). Each experiment was replicated ten times to maximise statistical power. The results of both experiments were consistent across replicates. We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads. Furthermore, we reliably recovered 83% of the 52 taxa used to test primer bias. However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass. Our metabarcoding approach yielded reliable results for high-throughput assessments. However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample. Thus, PCR-based metabarcoding assessments of biodiversity should rely on presence-absence metrics.

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.

The increasing number of Barcode of Life Database (BOLD) records per species and genus leads to contradictory species assignments within Barcode Index Numbers (BINs), serving as identifiers for the BOLD ID engine. To examine these issues, we analyzed a dataset comprising original and curated BOLD records for the genus Tachina (Insecta: Tachinidae), based on a previous publication. This dataset included both published and private records. We were able to assess the performance of the BOLD engine’s species determination algorithm, Refined Single Linkage (RESL), and compare it to Assemble Species by Automatic Partitioning (ASAP). Additionally, we investigated the usage of BINs by the BOLD v4 ID engine. Our analysis confirmed that BOLD queries primarily rely on BINs for species identification, although some cases deviated from this pattern, resulting in species matches inconsistent with the assigned BIN species. ASAP was found to be superior to RESL due to RESL’s adherence to the concept of the DNA barcoding gap. Moreover, we found that taxonomic misassignments, inconsistencies in BIN formation, and missing metadata also contribute significantly to unreliable identifications. These problems appear to stem from both algorithmic limitations and deficiencies in submission and post-submission processes. Moreover, we noted that the default mode of the BOLD v4 ID engine integrates both private and published data, leading to public records based solely on COI-based identifications. However, this issue may now be mitigated, as the BOLD v5 ID engine default mode exclusively employs published data. To enhance BOLD’s reliability, we propose improvements to submission and post-submission processes. Without such amendments, the accumulation of contradictory species assignments within BINs will continue to rise and the reliability of specimen identification by BOLD will decrease.

Pollination is essential for maintaining biodiversity and ensuring food security, and in Europe it is primarily mediated by four insect orders (Coleoptera, Diptera, Hymenoptera, Lepidoptera). However, traditional monitoring methods are costly and time consuming. Although recent automation efforts have focused on butterflies and bees, flies, a diverse and ecologically important group of pollinators, have received comparatively little attention, likely due to the challenges posed by their subtle morphological differences. In this study, we investigate the application of Convolutional Neural Networks (CNNs) for classifying 15 European pollinating fly families and quantifying the associated classification uncertainty. In curating our dataset, we ensured that the images of Diptera captured diverse visual characteristics relevant for classification, including wing morphology and general body habitus. We evaluated the performance of three CNNs, ResNet18, MobileNetV3, and EfficientNetB4 and estimated the prediction confidence using Monte Carlo methods, combining test-time augmentation and dropout to approximate both aleatoric and epistemic uncertainty. We demonstrate the effectiveness of these models in accurately distinguishing fly families. We achieved an overall accuracy of up to 95.61%, with a mean relative increase in accuracy of 5.58% when comparing uncropped to cropped images. Furthermore, cropping images to the Diptera bounding boxes not only improved classification performance across all models but also increased mean prediction confidence by 8.56%, effectively reducing misclassifications among families. This approach represents a significant advance in automated pollinator monitoring and has promising implications for both scientific research and practical applications.

The Eumerus triangularis group (Diptera: Syrphidae: Merodontini), which is endemic to the Afrotropical Region, has been thoroughly reviewed. This study identifies nine species within the group, with four of them redescribed: E . rubidus Hull, 1964, E . tessellatus Hull, 1964, E . triangularis Hervé-Bazin, 1913, and E . villeneuvei Hervé-Bazin, 1913 and with the description of four new species – E . argentipedicellus Radenković, Vujić & Grković sp. nov., E . brunnipennis Radenković, Vujić & Grković sp. nov., E . clavicercus Radenković, Vujić & Grković sp. nov., and E . setifemoratus Radenković, Vujić & Grković sp. nov. Furthermore, the previously unknown male of E . rubidus and the female of E . tessellatus are described. This study includes illustrated identification keys for males and females, along with detailed drawings of male terminalia for all nine species. DNA sequences of two regions of the mitochondrial DNA, cytochrome c oxidase subunit 1 (COI) gene and cytochrome b, and a region of the nuclear ribosomal 28rRNA gene are used to confirm morphological identification. Both molecular and morphological analyses indicate the presence of two distinct clades within the E . triangularis species group.

The potential for reduced pollination ecosystem service due to global declines of bees and other pollinators is cause for considerable concern. Habitat degradation, destruction and fragmentation due to agricultural intensification have historically been the main causes of this pollinator decline. However, despite increasing and accelerating levels of global urbanization, very little research has investigated the effects of urbanization on pollinator assemblages. We assessed changes in the diversity, abundance and species composition of bee and hoverfly pollinator assemblages in urban, suburban, and rural sites across a UK city. Bees and hoverflies were trapped and netted at 24 sites of similar habitat character (churchyards and cemeteries) that varied in position along a gradient of urbanization. Local habitat quality (altitude, shelter from wind, diversity and abundance of flowers), and the broader-scale degree of urbanization (e.g. percentage of built landscape and gardens within 100 m, 250 m, 500 m, 1 km, and 2.5 km of the site) were assessed for each study site. The diversity and abundance of pollinators were both significantly negatively associated with higher levels of urbanization. Assemblage composition changed along the urbanization gradient with some species positively associated with urban and suburban land-use, but more species negatively so. Pollinator assemblages were positively affected by good site habitat quality, in particular the availability of flowering plants. Our results show that urban areas can support diverse pollinator assemblages, but that this capacity is strongly affected by local habitat quality. Nonetheless, in both urban and suburban areas of the city the assemblages had fewer individuals and lower diversity than similar rural habitats. The unique development histories of different urban areas, and the difficulty of assessing mobile pollinator assemblages in just part of their range, mean that complementary studies in different cities and urban habitats are required to discover if these findings are more widely applicable.

The composition and quantity of insect cuticular hydrocarbons (CHCs) can be species-specific as well as sexually dimorphic within species. CHC analysis has been previously used for identification and ageing purposes for several insect orders including true flies (Diptera). Here, we analysed the CHC chemical profiles of adult males and females of eleven species of flesh flies belonging to the genus Sarcophaga Meigen (Sarcophagidae), namely Sarcophaga africa (Wiedemann), S. agnata Rondani, S. argyrostoma Robineau-Desvoidy, S. carnaria (Linnaeus), S. crassipalpis Macquart, S. melanura Meigen, S. pumila Meigen, S. teretirostris Pandellé, S. subvicina Rohdendorf, S. vagans Meigen and S. variegata (Scopoli). Cuticular hydrocarbons extracted from pinned specimens from the collections of the Natural History Museum, London using a customised extraction technique were analysed using Gas Chromatography–Mass Spectrometry. Time of preservation prior to extraction ranged between a few weeks to over one hundred years. CHC profiles (1) allowed reliable identification of a large majority of specimens, (2) differed between males and females of the same species, (3) reliably associated males and females of the same species, provided sufficient replicates (up to 10) of each sex were analysed, and (4) identified specimens preserved for up to over one hundred years prior to extraction.