Biology subjects

Meier, R.

Publications and source records attributed to Meier, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Beyond DNA barcodes: an open-source workflow for recovering and organizing barcoded vouchers for ecological and evolutionary research

1. Most species are neither discovered nor named, let alone included in analyses that require biological information such as trait measurements, images, ecological information and genome-scale data. Specimen-level DNA barcoding can help discover many of these species rapidly, but everything beyond discovery requires vouchers organized into putative species. Yet, existing barcoding workflows lack efficient techniques for voucher recovery, creating a post-barcoding bottleneck that limits the ability of converting barcoded specimens into biological knowledge. 2. Here we present a low-cost, open-source workflow consisting of two stages. The first safeguards barcoded specimens by separating them from DNA extracts and transferring them from microplates into ethanol-filled glass vials. The second converts the resulting voucher collection into a searchable physical resource by linking barcode-derived molecular Operational Taxonomic Unit (mOTU) assignments to vial positions and enabling specimens to be sorted into putative species either manually or automatically using a newly developed open-access robot (SORTER). 3. We evaluated the workflow using 2,024 insect specimens distributed across 21 96-well plates. For the first stage, DNA separation and specimen transfer required approximately 15 minutes per plate. For the second stage, MOTUmapper generated retrieval coordinates in a few seconds, after which the 2,024 vouchers belonging to the 452 putative species could be recovered manually in 5 days or with SORTER in 5 hours. Throughout both stages, specimen identities remained linked to barcode sequences, metadata and storage positions. 4. Vouchers are the Rosetta stones of biology because they connect different kinds of data to the same specimens. By safeguarding these vouchers and making them searchable, the workflow converts barcode projects from one-time molecular surveys into reusable resources for ecological and evolutionary research.

molecular biology↗

bioRxiv2026

Accuracy of occurrence and abundance estimates from insect metabarcoding

1. DNA metabarcoding--high-throughput sequencing of barcode regions from bulk samples--has become a key tool for insect biodiversity assessment. Yet, how methodological choices affect the accuracy of metabarcoding data remains insufficiently explored. In this paper, we ask: (1) How does the lysis method (non-destructive lysis vs. destructive homogenization) affect community recovery? (2) How comprehensively does metabarcoding capture species richness? (3) To what extent can spike-ins improve abundance estimates? (4) How accurately can species abundances be estimated? 2. We evaluated the accuracy of insect metabarcoding using 4,749 bulk samples from a large-scale biodiversity survey subjected to mild lysis. Of these samples, 856 were also homogenized, allowing a systematic comparison of the effect of alternative treatments. To potentially improve abundance estimates, we added six biological spike-ins (i.e., foreign insects) to all samples, and two synthetic spike-ins (artificial DNA fragments) to the homogenization treatment. In addition, we established the contents of 15 samples by individually barcoding all specimens, enabling direct assessment of occurrence and abundance estimates. 3. Our results revealed consistent differences between destructive and non-destructive treatments. While both methods reliably detected the majority of species, small and soft-bodied taxa were more often recovered after mild lysis than after homogenization, while the reverse was true for heavily sclerotized, hairy, and large taxa. Using biological spike-ins for calibration reduced the variance in read numbers per specimen considerably, especially in homogenized samples, while synthetic spike-ins were less effective. In a Bayesian analysis, where species data were matched to the best-fitting spike-in calibration curve, accurate abundance estimates (+/-1 individual) were obtained for 72.9% of species occurrences. 4. Our results show that it is possible to obtain reasonably accurate abundance estimates from metabarcoding data, and that mild lysis and homogenization result in different taxon-specific biases in terms of occurrence data, with neither method outperforming the other. Accuracy is improved by homogenization rather than mild lysis of samples, and by the use of biological rather than synthetic spike-ins. Together, these findings provide a major step towards robust, quantitative biodiversity monitoring using DNA-metabarcoding.

zoology↗

bioRxiv2026

Entomoscope 2.0 and ENIMAS 2.0: An Open-Source, AI-Integrated Platform for Rapid and Affordable Insect Digitization

The rapid decline of global biodiversity necessitates scalable and accessible tools for monitoring insect populations, yet the high cost and slow pace of specimen digitization remain significant bottlenecks. To address this challenge, we present the Entomoscope 2.0, an open-source platform that integrates a low-cost photomicroscope with an AI-integrated software suite, ENIMAS 2.0. The system combines optimized hardware with an end-to-end software module for digital specimen curation. This module comprises automated specimen cropping, background standardization, morphometric analysis using an Oriented Bounding Box (OBB) with a human-in-the-loop (HITL) supervision, and a flexible interface for rapid taxonomic screening using custom AI identification models. With a material cost of only 400 {euro}, the system offers a cost-effective alternative to expensive commercial solutions. We compare results from the Entomoscope 2.0 with a high-end commercial system (Keyence) using 54 insect specimens and demonstrate the efficiency of the proposed AI workflow. Entomoscope 2.0 completed the whole digitization process in an average of 54.6 seconds per specimen, representing a 2.28-fold increase in speed over the Keyence systems multi-step workflow (124.3 seconds). Crucially, all hardware specifications and construction manuals are freely available to support widespread adoption. By lowering financial barriers and accelerating research workflows, the Entomoscope 2.0 platform offers a practical solution to enable high-throughput digitization for researchers, educators, and citizen scientists.

bioengineering↗

bioRxiv2025

Integrative Taxonomy: From FASTA Pain to Species Gain with the interactive ''IntegraTax''

Here we present IntegraTax, a tool for analysing and managing taxonomic projects that combine DNA data with other evidence such as morphology to arrive at integrative species boundaries. IntegraTax visualizes genetic clusters through single-linkage clustering ("Objective Clustering") and provides an interactive browser interface that allows users to record taxonomic decisions regarding species limits. Projects can be saved at any stage, thus allowing continuous tracking of annotations and taxonomic decisions across many sessions. A typical IntegraTax session starts with a set of sequences that are visualized as a cluster fusion diagram revealing the genetic distances between the sequences and clusters. Users can define an "instability zone" to distinguish clusters that are clearly distinct, likely representing separate species, from those whose status is uncertain based on genetic data alone. Based on the instability zone setting, IntegraTax then suggests which and how many specimens should be studied with a second source of data to validate species hypotheses. This process is facilitated by an interactive html environment that enables detailed specimen-level annotations. For example, a taxonomist can label which specimens have been studied, which clusters have been validated as species, and which species can be identified. By combining clustering, intuitive visualization, and easy annotation in one interactive framework, IntegraTax treats species hypotheses as annotated objects that can be inspected, revised, and exported at any stage, with documentation of examined specimens. It simultaneously allows researchers to manage taxonomy projects with tens of thousands of specimens and hundreds of species. This will become increasingly important as taxonomists start resolving the species boundaries of the millions of undescribed species particularly within hyperdiverse dark taxa.

zoology↗

bioRxiv2025

Balancing Barcoding and Genomics: gDNA Quality in Insect Vouchers after HotSHOT DNA extraction

DNA extraction with an alkaline buffer system called "HotSHOT" is widely used for barcoding because it is rapid, inexpensive, and voucher preserving, but it remains unclear whether sufficient genomic DNA (gDNA) remains in small vouchers for downstream use in genomics. We here evaluate gDNA quality and quantity before and after HotSHOT treatment of 11 insect families representing six orders. Some specimens were flash frozen immediately after collection, while others were kept for one week at tropical temperatures in ethanol to mimic Malaise trap conditions. Encouragingly, we show that gDNA of sufficiently high quality and quantity for genomic sequencing remained in specimens treated with HotSHOT. We also show that DNA integrity was strongly influenced by field storage with specimens exposed to Malaise trap conditions showing such pronounced degradation that the standard HotSHOT treatment no longer significantly altered DNA quality. For control material, HotSHOT treatments involving longer exposure to high temperature led to smaller fragment lengths with the effect apparently being influenced by the degree of specimen sclerotization. Our results thus suggest that optimized HotSHOT treatments, together with carefully controlled pre-extraction storage, preserve voucher gDNA of sufficient quality for downstream genomic analyses with both short-read and possibly even some long-read sequencing technologies. Our protocol selection guidelines improve voucher gDNA preservation in HotSHOT-treated samples. This is particularly important for many species which are only known from one or few specimens discovered during barcoding projects.

molecular biology↗

bioRxiv2025

Automated Specimen Triage for Dark Taxa: Deep Learning Enables Orientation, Sex Identification, and Anatomical Segmentation from Robotic Imaging

Robotic specimen processing is transforming biodiversity discovery by replacing labor-intensive handling with scalable systems that can simultaneously generate high-quality specimen images. We demonstrate that these images can be leveraged by deep learning to efficiently extract key biological information and guide targeted specimen processing. Using a model dark taxon, the Phoridae (Diptera), the workflow performs three core tasks: sex identification, specimen orientation classification, and anatomical segmentation. Sex identification allows selective retention of diagnostically informative specimens, avoiding wasted effort on non-diagnostic individuals. Orientation classification enables specimens in the desired orientation to proceed immediately, while suboptimally oriented specimens can be repositioned for informative processing. Anatomical segmentation allows targeted processing of specimens displaying diagnostic characters or targeted analysis of specific anatomical regions in subsequent workflow steps. Comparative analysis of model architectures shows task-specific selection is crucial: a Convolutional Neural Network achieved an accuracy of 0.94 for orientation, a Vision Transformer achieved 0.88 for sex, and a U-Net precisely segmented nine anatomical regions with a mean IoU of 0.78. These results demonstrate that robotic imaging combined with deep learning provides a validated foundation for high-throughput, targeted specimen processing, maximising efficiency and utility for taxonomic and trait-based analyses, and supporting scalable, sustainable biodiversity workflows.

bioinformatics↗

bioRxiv2025

Weak and inverse latitudinal diversity gradients in the 10 most abundant and diverse flying insect clades

Latitudinal diversity gradients are a key concept in biodiversity science, but the available data are skewed towards large-bodied, charismatic taxa. To address this bias, we analyzed DNA barcode data for 1.35 million specimens of flying insects collected using 101 Malaise traps in 27 countries across six continents. We identify those ten families that consistently contribute more than half of all specimens and species across climates, habitats, and continents. Contrary to canonical expectations, five families have inverse or non-significant latitudinal gradients while the remaining five exhibit only weak to average gradients compared to 470 other terrestrial datasets. Furthermore, precipitation seasonality, rather than temperature, emerged as the strongest climatic correlate. Overall, we thus not only reveal a surprising convergence in the globally dominant insect clades but also illustrate that key textbook concepts in macroecology need testing for poorly studied invertebrate taxa that contribute most of Earths animal biodiversity and may behave unexpectedly.

ecology↗

bioRxiv2025

UITOTO: a new software for molecular diagnoses in species descriptions

Millions of species remain undescribed, and each eventually will require a species description that includes a diagnosis. However, no software currently exists that fully integrates the derivation and validation of state-specific and contrastive molecular diagnoses. Here we introduce UITOTO which addresses this gap and facilitates the identification, testing, and visualization of Diagnostic Molecular Combinations (DMCs). The software uses a weighted random sampling algorithm based on the Jaccard Index for building candidate DMCs. It then selects DMCs with the highest specificity stability, meeting user-defined thresholds for exclusive character states. If multiple optimal DMCs are identified, UITOTO derives a majority-consensus DMC. To verify whether the newly generated DMCs are contrastive, UITOTO includes a validation module that tests DMCs against database sequences, efficiently handling up to hundreds of thousands of aligned or unaligned sequences. In this manuscript we propose and assess UITOTOs performance with DMCs obtained from other software (e.g., MOLD) for three large empirical datasets: i) Megaselia (Diptera: Phoridae: 69 species, 2,229 training and 30,289 testing barcodes); ii) Mycetophilidae, (Diptera: 118 species, 1,456 training and 60,349 testing barcodes); and iii) European Lepidoptera (49 species, 591 training and 21,483 testing barcodes). Based on several classification metrics (e.g., Accuracy, F1 Score), UITOTOs DMCs outcompete DMCs obtained with other software. We furthermore provide guidelines for generating molecular diagnoses and facilitate obtaining DMCs by providing a user-friendly Shiny App GUI and a module for obtaining publication-quality DMC visualizations in RStudio. Overall, our study confirms that the biggest challenge for generating molecular and morphological diagnoses is similar, i.e., balancing specificity and length. Short diagnoses often lack specificity, while excessively long DMCs are often so specific that they do not accommodate intraspecific variation.

bioinformatics↗

bioRxiv2024

RESOLVING BIOLOGY'S DARK MATTER: SPECIES RICHNESS, SPATIOTEMPORAL DISTRIBUTION, AND COMMUNITY COMPOSITION OF A DARK TAXON

BackgroundZoologys dark matter comprises hyperdiverse, poorly known taxa that are numerically dominant but largely unstudied, even in temperate regions where charismatic taxa are well understood. It is everywhere, but high diversity, abundance, and small size have historically stymied its study. We demonstrate how entomological dark matter can be elucidated using high-throughput DNA barcoding ("megabarcoding"). We reveal the high abundance and diversity of scuttle flies (Diptera: Phoridae) in Sweden using 31,800 specimens from 37 sites across four seasonal periods. We investigate the number of scuttle fly species in Sweden and the environmental factors driving community changes across time and space. ResultsSwedish scuttle fly diversity is much higher than previously known, with 549 mOTUs (putative species) detected, compared to 374 previously recorded species. Hierarchical Modelling of Species Communities reveals that scuttle fly communities are highly structured by latitude and strongly driven by climatic factors. Large dissimilarities between sites and seasons are driven by turnover rather than nestedness. Climate changes are predicted to significantly affect the 47% of species that show significant responses to mean annual temperature. Results were robust whether using haplotype diversity or species-proxies (mOTUs) as response variables. Additionally, species-level models of common taxa adequately predict overall species richness. ConclusionsUnderstanding the bulk of the diversity around us is imperative during an era of biodiversity loss. We show that dark insect taxa can be efficiently characterized and surveyed with megabarcoding. Undersampling of rare taxa and choice of operational taxonomic units do not alter the main ecological inferences, making it an opportune time to tackle zoologys dark matter.

ecology↗

bioRxiv2024

Megabarcoding reveals a tale of two very different dark taxa along the same elevational gradient

AbstractOur planet is entering the sixth mass extinction without detailed spatio-temporal distribution and abundance information for most insect species. Particularly lacking is this information for dark taxa although they contain most animal species. This problem can be addressed with high-throughput single specimen barcoding (megabarcoding), but it remain unknown to what extent one dark taxon can be used as a proxy for another because of similar biodiversity patterns. This is here tested and we show that two Diptera taxa (Phoridae, Mycetophilidae) have very different spatio-temporal distributions across the same elevational gradient. We applied COI megabarcoding to samples collected every 14 days over three seasons at six elevations (800 to 1,800 m) in a temperate forest ecosystem in Northern China (Baihua Mountain Reserve). Elevation, precipitation, and temperature were used as environmental variables to understand the data obtained for 17,179 specimens representing 492 mOTUs of Phoridae and 2,281 specimens of 148 mOTUs of Mycetophilidae. Congruent is that the alpha diversity for both taxa increased in the summer and there is evidence for a mid-domain effect and phylogenetic clustering in high elevations. However, the extent and pattern of seasonal variation differed considerably. Phorid communities exhibited shifts between spring, summer and autumn seasons, but surprisingly little difference between elevations. In contrast, mycetophilid communities were structured along the elevational gradient and dependent on precipitation. Our study highlights the urgent need for obtaining detailed spatio-temporal information for the most diverse dark taxa, but also illustrates that these data can be readily obtained with megabarcoding.

ecology↗

bioRxiv2024

Implementing high-throughput insect barcoding in microbiome studies: impact of non-destructive DNA extraction on microbiome reconstruction

BackgroundSymbiotic relationships with diverse microorganisms are crucial for many aspects of insect biology. However, while our understanding of insect taxonomic diversity and the distribution of insect species in natural communities is limited, we know much less about their microbiota. In the era of rapid biodiversity declines, as researchers increasingly turn towards DNA-based monitoring, developing and broadly implementing approaches for high-throughput and cost-effective characterization of both insect and insect-associated microbial diversity is essential. We need to verify whether approaches such as high-throughput barcoding, a powerful tool for identifying wild insects, would permit subsequent microbiota reconstruction in these specimens. MethodsHigh-throughput barcoding ("megabarcoding") methods often rely on non-destructive approaches for obtaining template DNA for PCR amplification by leaching DNA out of insect specimens using alkaline buffers such as HotSHOT. This study investigated the impact of HotSHOT on microbial abundance estimates and the reconstructed bacterial community profiles. We addressed this question by comparing quantitative 16S rRNA amplicon sequencing data for HotSHOT-treated or untreated specimens of 16 insect species representing six orders and selected based on the expectation of limited variation among individuals. ResultsWe find that in 13 species, the treatment significantly reduced microbial abundance estimates, corresponding to an estimated 15-fold decrease in amplifiable 16S rRNA template on average. On the other hand, HotSHOT pre-treatment had a limited effect on microbial community composition. The reconstructed presence of abundant bacteria with known significant effects was not affected. On the other hand, we observed changes in the presence of low-abundance microbes, those close to the reliable detection threshold. Alpha and beta diversity analyses showed compositional differences in only a few species. ConclusionOur results indicate that HotSHOT pre-treated specimens remain suitable for microbial community composition reconstruction, even if abundance may be hard to estimate. These results indicate that we can cost-effectively combine barcoding with the study of microbiota across wild insect communities. Thus, the voucher specimens obtained using megabarcoding studies targeted at characterizing insect communities can be used for microbiome characterizations. This can substantially aid in speeding up the accumulation of knowledge on the microbiomes of abundant and hyperdiverse insect species.

microbiology↗

bioRxiv2023

An integrative taxonomic treatment of the Mycetophilidae (Diptera: Bibionomorpha) from Singapore reveals 115 new species on 730km2

Abstract"Open ended" or "dark taxa" are species-rich clades that are so abundant and diverse that conventional taxonomic methods tend to struggle with the onslaught of specimens and species. New approaches based on presorting specimens to putative species with low-cost DNA barcodes may make tackling these taxa manageable. However, this will still require limiting the geographic scope of taxonomic revisions, given that most countries and biogeographic regions will have too many specimens and species for comprehensive coverage. We demonstrate the power of this approach by carrying out a revision of the Mycetophilidae fungus gnats (Diptera) of Singapore. The material revised here was obtained from 496 samples collected with 71 Malaise traps placed at 107 sites in different habitats in Singapore: mangroves, swamp forests, freshwater swamps, primary rainforests, and different types of secondary forests (old, maturing, young, urban). Based on molecular and morphological data for 1,454 specimens, we delimit 120 species of 23 genera. Of these, only five were species previously described. The remaining 115 species are new to science and described here. We name, however, only 98 of these species since 14 species are currently only known from females and we cannot prepare a fully satisfying morphological diagnosis (Manota spp. A-G and Neoempheria spp. A-G), and three species lack molecular data (Epicypta sp. A, Epicypta sp. B, and Neoempheria sp. H). To assess congruence between species delimited with DNA barcodes (3% clusters) and morphology, we determined a match ratio and found it to be overall high (95%) with even higher match ratios (99%) observed for MOTUs clustered at 5% with Objective Clustering and MOTUs obtained with ABGD set to (P=0.060). Overall, the ratio of undescribed to described is an astonishing 20:1. Only revising the Singapore fauna increases the number of described species of Oriental Mycetophilidae by about 25%, highlighting the size of the taxonomic impediment for fungus gnats. Most of the Singapore Mycetophilidae diversity belongs to three genera--Neoempheria Osten-Sacken (31 species), Epicypta Winnertz (29 species) and Manota Williston (14 species), but we also describe a new genus-- Integricypta, gen. n., which is the putative sistergroup of Aspidionia Colless--belonging to the Mycetophilinae Mycetophilini based on three species. The species sequenced, illustrated, and named are: Leptomorphus rafflesi, sp. n.; Monoclona simhapura, sp. n.; Azana demeijeri, sp. n.; Azana leekongchiani, sp. n. (Sciophilinae); Tetragoneura crawfurdi, sp. n.; Tetragoneura chola, sp. n.; Tetragoneura dayuan, sp. n.; Tetragoneura farquhari, sp. n.; Ectrepesthoneura johor, sp. n. (Tetragoneurinae); Mohelia zubirsaidi, sp. n.; Allactoneura tumasik, sp. n.; Allactoneura limbosengi, sp. n.; Manota banzu, sp. n.; Manota tantocksengi, sp. n.; Manota bukittimah, sp. n.; Manota chiamassie, sp. n.; Manota danmaxi, sp. n.; Manota mahuan, sp. n.; Manota temenggong, sp. n.; Clastobasis sritribuana, sp. n.; Clastobasis bugis, sp. n.; Clastobasis oranglaut, sp. n. (Leiinae); Parempheriella mait, sp. n.; Parempheriella longyamen, sp. n.; Parempheriella peranakan, sp. n.; Mycomya sachmatich, sp. n.; Neoempheria merlio, sp. n.; Neoempheria sabana, sp. n.; Neoempheria sangabo, sp. n.; Neoempheria shicheng, sp. n.; Neoempheria ujong, sp. n.; Neoempheria subaraji, sp. n.; Neoempheria kokoiyeeae, sp. n.; Neoempheria mandai, sp. n.; Neoempheria malacca, sp. n.; Neoempheria sinkapho, sp. n.; Neoempheria singapura, sp. n.; Neoempheria xinjiapo, sp. n.; Neoempheria puluochung, sp. n.; Neoempheria merdeka, sp. n.; Neoempheria neesoon, sp. n.; Neoempheria pulau, sp. n.; Neoempheria cinkappur, sp. n.; Neoempheria temasek, sp. n.; Neoempheria polunini, sp. n.; Neoempheria fajar, sp. n.; Neoempheria riatanae, sp. n. (Mycomyinae); Brachycampta glorialimae, sp. n.; Brachycampta murphyi, sp. n.; Brachycampta limtzepengi, sp. n.; Rymosia teopohlengi, sp. n.; Exechia tanswiehiani, sp. n.; Exechia alinewongae, sp. n.; Mycetophila chngseoktinae, sp. n.; Mycetophila georgettechenae, sp. n.; Mycetophila aishae, sp. n.; Platyprosthiogyne phanwaithongae, sp. n.; Platyprosthiogyne gohsookhimae, sp. n.; Platyprosthiogyne rahimahae, sp. n.; Platyprosthiogyne lynetteseahae, sp. n.; Platyprosthiogyne neilaae, sp. n.; Platyprosthiogyne snehalethaae, sp. n.; Platurocypta adeleneweeae, sp. n.; Platurocypta tanhoweliangi, sp. n.; Epicypta constancesingamae, sp. n.; Epicypta jennylauae, sp. n.; Epicypta limchiumeiae, sp. n.; Epicypta janetyeeae, sp. n.; Epicypta kohkhenglianae, sp. n.; Epicypta daintoni, sp. n.; Epicypta holltumi, sp.n.; Epicypta ridleyi, sp. n.; Epicypta chezaharaae, sp. n.; Epicypta tanjiakkimi, sp. n.; Epicypta gehminae, sp. n.; Epicypta jackieyingae, sp. n.; Epicypta khatijunae, sp. n.; Epicypta purchoni, sp. n.; Epicypta foomaoshengi, sp. n.; Epicypta ganengsengi, sp. n.; Epicypta nanyangu, sp. n.; Epicypta nus, sp. n.; Epicypta peterngi, sp. n.; Epicypta maggielimae, sp. n.; Epicypta yupeigaoae, sp. n.; Epicypta annwee, sp. n.; Epicypta wallacei, sp. n.; Epicypta lamtoongjini, sp. n.; Epicypta catherinelimae, sp. n.; Epicypta grootaerti, sp. n.; Epicypta joaquimae, sp. n.; Aspidionia cheesweeleeae, sp. n.; Aspidionia janetjesudasonae, sp. n.; Aspidionia fatimahae, sp. n.; Integricypta fergusondavie, sp. n.; Integricypta teosoonkimae, sp. n.; Integricypta shirinae, sp. n.; Integricypta hoyuenhoeae, sp. n. (Mycetophilinae). The previously described species are Metanepsia malaysiana Kallweit, Eumanota racola Soli, Parempheriella defectiva (Edwards), Neoempheria dizonalis (Edwards) (all known from Sumatra and/or the Malaysian peninsula), and Chalastonepsia hokkaidensis Kallweit a species spread in east Asia. The mycomyine genus Vecella Wu & Yang is here proposed as a new synonym to Parempheriella, with P. guadunana (Wu & Yang), n. comb. corresponding to an additional Palearctic species of Parempheriella. Barcodes for a second set of 1,493 Singapore mycetophilid specimens suggest the presence of an additional 18 MOTUs. We thus estimate that approximately 85% of all the Singapore species that routinely enter Malaise traps are identified or described here. The revision concludes with a discussion of the biogeography and generic composition of the mycetophilid fauna at the southern end of the Malay Peninsula. Zusammenfassung"Open-ended" oder "Dark Taxa" sind artenreiche Klade, die so abundant und arteinreich sind, dass die herkommlichen taxonomischen Methoden angesichts der gro{beta}en Anzahl von Exemplaren und Arten nicht gut funktionieren. Neue Ansatze, die auf das Vorsortierung von Exemplaren mit DNA Barcodes bis zum Artniveau basieren, erlauben es jetzt aber solche Taxa zu revidieren. Allerdings kann auch mit DNA Barcodes, nur die Fauna eines vergleichsweise kleinen Gebietes bearbeiten werden, weil fur die meisten Lander und biogeografischen Regionen zu viele Exemplare und Arten abgedeckt werden mussten. Wir demonstrieren hier wie eine solche Revision durchgefuhrt werden kann. Wir revidieren hier die Pilzmucken (Diptera: Mycetophilidae) von Singapur, die mit Malaisefallen gefangen werden konnen. Das hier uberarbeitete Material stammt aus 496 Malaise-Fallenproben, die mit 71 Malaise-Fallen an 107 Sammelstellen in verschiedenen Habitaten gefangen wurden: Mangroven, Sumpfwalder, Su{beta}wassersumpfe, Primarregenwalder und Sekundarwalder. Basierend auf molekularen und morphologischen Daten fur mehr als 1454 Tiere grenzen wir 120 Arten mit molekularen und morphologischen Daten ab, wobei nur 5 dieser Arten bereits beschrieben sind. Die verbleibenden 115 werden hier beschrieben. Allerdings benennen wir nur 98 Arten, da fur zwei Arten molekulare Daten fehlen und fur 14 weitere Arten derzeit nur Weibchen bekannt sind. Daher konnen wir derzeit keine zufriedenstellende morphologische Diagnose erstellen. Daruber hinaus fehlen molekulare Daten fur drei Arten (Epicypta sp. A, Epicypta sp. B und Neoempheria sp. H). Was die Artgrenzen betrifft, so stimmen die molekularen und morphologischen Daten in den meisten Fallen uberein (match ratio: 95% fur 3% MOTUs). Eine noch hohere "match ratio" von 99% wird fur 5% und ABGD MOTUs (P=0,060) beobachtet. Insgesamt ist das Verhaltnis zwischen unbeschrieben und beschrieben erstaunlich hoch (20:1), und die Uberarbeitung der singapurer Fauna erhoht die Anzahl der beschriebenen Arten in der Orientalischen Region um uber 25%. Dies unterstreicht den Ausma{beta} des "taxonomic impediments" fur Pilzmucken. Die meisten der Mycetophilidenarten in Singapur gehoren zu drei von 22 Gattungen - Neoempheria Osten-Sacken (31 Arten), Epicypta Winnertz (29 Arten) und Manota Williston (14 Arten), aber wir beschreiben hier auch eine neue Gattung, Integricypta, gen. n. basierend auf drei Arten. Die Gattung gehort zu den Mycetophilinae Mycetophilini und ist die mutma{beta}liche Schwestergruppe von Aspidionia Colless. Die sequenzierten, illustrierten und benannten Arten sind: Leptomorphus rafflesi, sp. n.; Monoclona simhapura, sp. n.; Azana demeijeri, sp. n.; Azana leekongchiani, sp. n. (Sciophilinae); Tetragoneura crawfurdi, sp. n.; Tetragoneura chola, sp. n.; Tetragoneura dayuan, sp. n.; Tetragoneura farquhari, sp. n.; Ectrepesthoneura johor, sp. n. (Tetragoneurinae); Mohelia zubirsaidi, sp. n.; Allactoneura tumasik, sp. n.; Allactoneura limbosengi, sp. n.; Manota banzu, sp. n.; Manota tantocksengi, sp. n.; Manota bukittimah, sp. n.; Manota chiamassie, sp. n.; Manota danmaxi, sp. n.; Manota mahuan, sp. n.; Manota temenggong, sp. n.; Clastobasis sritribuana, sp. n.; Clastobasis bugis, sp. n.; Clastobasis oranglaut, sp. n. (Leiinae); Parempheriella mait, sp. n.; Parempheriella longyamen, sp. n.; Parempheriella peranakan, sp. n.; Mycomya sachmatich, sp. n.; Neoempheria merlio, sp. n.; Neoempheria sabana, sp. n.; Neoempheria sangabo, sp. n.; Neoempheria shicheng, sp. n.; Neoempheria ujong, sp. n.; Neoempheria subaraji, sp. n.; Neoempheria kokoiyeeae, sp. n.; Neoempheria mandai, sp. n.; Neoempheria malacca, sp. n.; Neoempheria sinkapho, sp. n.; Neoempheria singapura, sp. n.; Neoempheria xinjiapo, sp. n.; Neoempheria puluochung, sp. n.; Neoempheria merdeka, sp. n.; Neoempheria neesoon, sp. n.; Neoempheria pulau, sp. n.; Neoempheria cinkappur, sp. n.; Neoempheria temasek, sp. n.; Neoempheria polunini, sp. n.; Neoempheria fajar, sp. n.; Neoempheria riatanae, sp. n. (Mycomyinae); Brachycampta glorialimae, sp. n.; Brachycampta murphyi, sp. n.; Brachycampta limtzepengi, sp. n.; Rymosia teopohlengi, sp. n.; Exechia tanswiehiani, sp. n.; Exechia alinewongae, sp. n.; Mycetophila chngseoktinae, sp. n.; Mycetophila georgettechenae, sp. n.; Mycetophila aishae, sp. n.; Platyprosthiogyne phanwaithongae, sp. n.; Platyprosthiogyne gohsookhimae, sp. n.; Platyprosthiogyne rahimahae, sp. n.; Platyprosthiogyne lynetteseahae, sp. n.; Platyprosthiogyne neilaae, sp. n.; Platyprosthiogyne snehalethaae, sp. n.; Platurocypta adeleneweeae, sp. n.; Platurocypta tanhoweliangi, sp. n.; Epicypta constancesingamae, sp. n.; Epicypta jennylauae, sp. n.; Epicypta limchiumeiae, sp. n.; Epicypta janetyeeae, sp. n.; Epicypta kohkhenglianae, sp. n.; Epicypta daintoni, sp. n.; Epicypta holltumi, sp.n.; Epicypta ridleyi, sp. n.; Epicypta chezaharaae, sp. n.; Epicypta tanjiakkimi, sp. n.; Epicypta gehminae, sp. n.; Epicypta jackieyingae, sp. n.; Epicypta khatijunae, sp. n.; Epicypta purchoni, sp. n.; Epicypta foomaoshengi, sp. n.; Epicypta ganengsengi, sp. n.; Epicypta nanyangu, sp. n.; Epicypta nus, sp. n.; Epicypta peterngi, sp. n.; Epicypta maggielimae, sp. n.; Epicypta yupeigaoae, sp. n.; Epicypta annwee, sp. n.; Epicypta wallacei, sp. n.; Epicypta lamtoongjini, sp. n.; Epicypta catherinelimae, sp. n.; Epicypta grootaerti, sp. n.; Epicypta joaquimae, sp. n.; Aspidionia cheesweeleeae, sp. n.; Aspidionia janetjesudasonae, sp. n.; Aspidionia fatimahae, sp. n.; Integricypta fergusondavie, sp. n.; Integricypta teosoonkimae, sp. n.; Integricypta shirinae, sp. n.; Integricypta hoyuenhoeae, sp. n. (Mycetophilinae). Die Tiere, die zu den bereits beschriebenen Arten gehoren, gehoren zu den folgenden Arten: Metanepsia malaysiana Kallweit, Eumanota racola Soli, Parempheriella defectiva (Edwards), Neoempheria dizonalis (Edwards) (alle Arten sind derzeit aus Sumatra und/oder der malaysischen Halbinsel bekannt), und Chalastonepsia hokkaidensis Kallweit, eine in Ostasien verbreitete Art. Die Gattung Vecella Wu & Yang wird hier als neues Synonym fur Parempheriella vorgeschlagen, wobei P. guadunana (Wu & Yang), n. comb., wobei die Gattung damit eine zusatzlichen palaarktischen Art erhalt. Die DNA Barcodes fur eine zweite Stichprobe mit 1.493 Pilzmucken deuten darauf hin, dass in Singapur 18 zusatzliche Arten mit Malaisefallen gefangen werden konnen. Es sieht aber dennoch so aus, als wurden wir bereits hier circa 85% aller Arten beschreiben, die regelma{beta}ig in Malaise-Fallen geraten. Die Revision endet mit einer Diskussion der Biogeographie und taxonomische Zusammensetzung der Mycetophilidenfauna im sudlichen Teil der Malaiischen Halbinsel.

zoology↗

bioRxiv2023

"Dark taxonomy": a new protocol for overcoming the taxonomic impediments for dark taxa and broadening the taxon base for biodiversity assessment

We are entering the 6th mass extinction with little data for "dark taxa" although they comprise most species. Much of the neglect is due to the fact that conventional taxonomic methods struggle with handling thousands of specimens belonging to hundreds of species. We thus here propose a new strategy that we call "dark taxonomy." It addresses (1) taxonomic impediments, (2) lack of biodiversity baselines, (3) and low impact of revisionary research. Taxonomic impediments are reduced by carrying out revisions at small geographic scales to keep the number of specimens low. The risk of taxonomic error is kept low by delimiting species based on two types of data. We then show that dark taxonomy can yield important biodiversity baseline data because it uses samples obtained with biomonitoring traps. Lastly, we argue that the impact of research can be improved by publishing two manuscripts addressing different readerships. The principles of dark taxonomy are illustrated by our taxonomic treatment of Singapores fungus gnats (Mycetophilidae) based only on Malaise trap samples. We show that a first batch of specimens (N=1,454) contains 120 species, of which 115 are new to science thus reducing taxonomic impediments by increasing the number of described Oriental species by 25%. Species delimitation started with using DNA barcodes to estimate the number of MOTUs before "LIT" (Large-scale Integrative Taxonomy) was used to obtain the species boundaries for the 120 species by integrating morphological and molecular data. To test the taxonomic completeness of the revision, we then analyzed a second batch of 1,493 specimens and found that >97% belonged to the 120 species described based on the first batch. Indeed, the second batch only contained 18 new and rare MOTUs; i.e. our study suggests that a single revision can simultaneously yield the names for all important species and relevant biodiversity baseline data. Overall, we believe that "dark taxonomy" can quickly ready a large unknown taxon for biomonitoring.

zoology↗

bioRxiv2023

Rapid species discovery and identification with real-time barcoding facilitated by ONTbarcoder 2.0 and Oxford Nanopore R10.4

Most arthropod species are undescribed and hidden in biodiversity samples that are difficult to sort to species using morphological characters. Sorting specimens to putative species with DNA barcodes is an attractive alternative, but needs cost-effective techniques that are suitable for use in many laboratories around the world. Barcoding with Oxford Nanopores portable and inexpensive MinION sequencers could be a good presorting approach because it reduces the space and capital cost of a fully functional barcoding laboratory. Similarly important would be user-friendly and reliable software for analysis of the ONT data. It is here provided in the form of an updated ONTbarcoder that is available for all commonly used operating systems and includes a GUI. The new version of ONTbarcoder has three key improvements related to the higher read quality obtained with ONTs latest basecallers and chemistry (R10.4 flow cells, Q20+ kits). Firstly, ONTbarcoder can now deliver real-time barcoding to complement ONTs real-time sequencing. This means that the first barcodes are obtained within minutes of starting a sequencing run and a significant proportion of barcodes are available before sequencing completes. This real-time feature of ONTbarcoder can be configured to handle both Mk1B and Mk1C sequencers. The input is a demultiplexing sheet and sequencing data (raw or basecalled). Secondly, the improved read quality of ONTs latest flow cells (R10.4) allows for the use of primers with shorter indices than those previously needed (9 bp vs. 12-13 bp). This decreases primer cost and improves PCR success rates. Thirdly, we demonstrate that the availability of R10.4 chemistry in the low-cost Flongle flow cell is an attractive option for users who require only 200-250 barcodes at a time.

ecology↗

bioRxiv2023

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

O_LIDNA barcodes are useful for species-level sorting of specimen samples, but rarely used in time-sensitive projects that require species richness estimates or identification of pest or invasive species within hours. The main reason is that existing express barcoding workflows are either too expensive or can only be carried out in very well equipped laboratories by highly trained staff. C_LIO_LIWe here introduce a simple workflow that combines rapid DNA extraction with HotSHOT, amplicon production with the aid of NextGenPCR(R) thermocyclers, and sequencing with low-cost MinION sequencers. C_LIO_LIWe demonstrate the power of the approach by generating and identifying 250 barcodes for 285 specimens within 6 hours. The workflow only required the following major equipment that easily fits onto a lab bench: Conventional thermocycler, NextGenPCR(R) thermocycler, microplate sealer, Qubit, and MinION. C_LIO_LIWe argue that species-level sorting with simplified barcoding workflows is now faster, more accurate, and sufficiently cost-effective that it can and should replace morphospecies sorting in many projects. C_LI

ecology↗

bioRxiv2022

Network analysis with either Illumina or MinION reveals that detecting vertebrate species requires metabarcoding of iDNA from a diverse fly community

Metabarcoding of vertebrate DNA obtained from invertebrates (iDNA) has been used to survey vertebrate communities, but we here show that it can also be used to study species interactions between invertebrates and vertebrates in a spatial context. We sampled the dung and carrion fly community of a swamp forest remnant along a disturbance gradient (10 sites: 80-310m from a road). Approximately, 60% of the baited 407 flies yield 294 vertebrate identifications based on two COI fragments and 16S sequenced with Illumina and/or MinION. A bipartite network analysis finds no specialization in the interaction between flies and vertebrate species, but a spatial analysis revealed that surprisingly 18 of the 20 vertebrate species can be detected within 150m of the road. We show that the fly community sourced for iDNA was unexpectedly rich (24 species, 3 families) and carried DNA for mammals, birds, and reptiles. They included common and rare ground-dwelling (e.g., wild boar, Sunda pangolin), and arboreal species (e.g., long-tailed macaque, Raffles banded langur) as well as small bodied vertebrates (skinks, rats). All of our results were obtained with a new, greatly simplified iDNA protocol that eliminates DNA extraction by obtaining template directly through dissolving feces and regurgitates from individual flies with water. Lastly, we show that MinION- and Illumina-based metabarcoding yield similar results. Overall, flies from several families (calliphorids, muscids and sarcophagids) should be used in iDNA surveys because we show that uncommon fly species carry the signal for several vertebrate species that are otherwise difficult to detect with iDNA.

ecology↗

bioRxiv2022

Global convergence of dominance and neglect in flying insect diversity

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.

ecology↗

bioRxiv2022

Global determinants of the distribution of insect genetic diversity

Understanding global patterns of genetic diversity (GD) is essential for describing, monitoring, and preserving life on Earth. To date, efforts to map macrogenetic patterns have been restricted to vertebrates, which comprise only a small fraction of Earths biodiversity. Here, we construct the first global map of predicted insect mitochondrial (COI) GD, derived from publicly available data. We calculate the mitochondrial GD mean (GDM) and evenness (GDE) of insect assemblages across the globe, identify their environmental correlates, and make predictions of mitochondrial GD levels in unsampled areas based on environmental data. Using the largest single-locus genetic dataset yet assembled, we find that mitochondrial GDE follows a quadratic latitudinal gradient peaking in the subtropics. Both mitochondrial GDM and GDE positively correlate with seasonally hot temperatures, as well as climate stability since the last glacial maximum. Our models explain 27.9% and 24.0% of the observed variation in mitochondrial GDM and GDE in insects, respectively, making an important step towards understanding global biodiversity patterns in the most diverse animal taxon.

evolutionary biology↗