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DNA 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.We here introduce a simple workflow that combines rapid DNA extraction with HotSHOT, amplicon production with the aid of NextGenPCR® thermocyclers, and sequencing with low-cost MinION sequencers.We 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® thermocycler, microplate sealer, Qubit, and MinION.We 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.

Current understanding of ecological and evolutionary processes underlying island biodiversity is heavily shaped by empirical data from plants and birds, although arthropods comprise the overwhelming majority of known animal species, and as such can provide key insights into processes governing biodiversity. Novel high throughput sequencing (HTS) approaches are now emerging as powerful tools to overcome limitations in the availability of arthropod biodiversity data, and hence provide insights into these processes. Here, we explored how these tools might be most effectively exploited for comprehensive and comparable inventory and monitoring of insular arthropod biodiversity. We first reviewed the strengths, limitations and potential synergies among existing approaches of high throughput barcode sequencing. We considered how this could be complemented with deep learning approaches applied to image analysis to study arthropod biodiversity. We then explored how these approaches could be implemented within the framework of an island Genomic Observatories Network (iGON) for the advancement of fundamental and applied understanding of island biodiversity. To this end, we identified seven island biology themes at the interface of ecology, evolution and conservation biology, within which collective and harmonized efforts in HTS arthropod inventory could yield significant advances in island biodiversity research., The iGON Working Group was organized by the iBioGen project, which has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 810729.

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.

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.

Synchronous multispecific coral spawning generally occurs annually and forms an integral part of the coral life cycle. Apart from spawning times and species participation, however, much else remains unknown. Here, we applied environmental DNA (eDNA) metabarcoding to study two tropical reef sites of contrasting coral cover before, during and after coral spawning. Using coral-ITS2 and vertebrate-12S markers, we evaluated eDNA as an alternative monitoring tool by assessing its capabilities in detecting spawning species and tracking relative abundances of coral and fish eDNA. Over 3 years, elevated eDNA coral signals during the event (proportional read increase of up to five-fold) were observed, detecting a total of 38 coral and 133 fish species with all but one of the coral species visually observed to be spawning. This is also the first demonstration that eDNA metabarcoding can be used to infer the diurnal partitioning of night- and day-time spawning, spawning in coral species overlooked by visual surveys, and the associated changes in fish trophic structures as an indicator of spawning events. Our study paves the way for applied quantitative eDNA metabarcoding approaches to better study ephemeral and important biological events.

Environmental DNA (eDNA) with metabarcoding or metagenomics will likely become a major biomonitoring tool in the 21st century, perhaps even more so in the face of increased coastal urbanization and its associated effects such as pollution, land reclamation, and seabed dredging. Together, these impacts and the consequent high turbidity pose severe challenges to traditional survey techniques that rely heavily on visual observations. We here demonstrate that eDNA can be used for biomonitoring in turbid waters, using Singapore as a case study. The cytochrome c oxidase subunit I (COI) locus was used to detect 525 metazoan molecular operational taxonomic units (MOTUs) based on eDNA obtained from 52 L of filtered seawater. Of these, 130 MOTUs (24.7%) were identifiable to species, including ecologically significant species that in some cases were invasive or rarely observed. Metazoan signals also enabled discrimination of discrete, but connected, environments from intertidal and subtidal zones. Taxa with known habitat preferences were found to have left trace eDNA at sites that matched putatively suitable habitats. Moreover, no coast‐specific signals were detected in open water samples, which suggest that intermixing of water was limited. The study confirms that eDNA metabarcoding is a viable biomonitoring tool for coastal areas with highly sedimented waters.

New techniques for the species-level sorting of millions of specimens are needed in order to accelerate species discovery, determine how many species live on earth, and develop efficient biomonitoring techniques. These sorting methods should be reliable, scalable, and cost-effective, as well as being largely insensitive to low-quality genomic DNA, given that this is usually all that can be obtained from museum specimens. Mini-barcodes seem to satisfy these criteria, but it is unclear how well they perform for species-level sorting when compared with full-length barcodes. This is here tested based on 20 empirical data sets covering ca. 30,000 specimens (5500 species) and six clade-specific data sets from GenBank covering ca. 98,000 specimens ($>$20,000 species). All specimens in these data sets had full-length barcodes and had been sorted to species-level based on morphology. Mini-barcodes of different lengths and positions were obtained in silico from full-length barcodes using a sliding window approach (three windows: 100 bp, 200 bp, and 300 bp) and by excising nine mini-barcodes with established primers (length: 94–407 bp). We then tested whether barcode length and/or position reduces species-level congruence between morphospecies and molecular operational taxonomic units (mOTUs) that were obtained using three different species delimitation techniques (Poisson Tree Process, Automatic Barcode Gap Discovery, and Objective Clustering). Surprisingly, we find no significant differences in performance for both species- or specimen-level identification between full-length and mini-barcodes as long as they are of moderate length ($>$200 bp). Only very short mini-barcodes ($200 bp and the congruent mOTUs contain ca. 75% of all specimens. Most conflict is caused by ca. 10% of the specimens that can be identified and should be targeted for re-examination in order to efficiently resolve conflict. Our study suggests that large-scale species discovery, identification, and metabarcoding can utilize mini-barcodes without any demonstrable loss of information compared to full-length barcodes. [DNA barcoding; metabarcoding; mini-barcodes; species discovery.]

Invertebrate biodiversity remains poorly understood although it comprises much of the terrestrial animal biomass, most species and supplies many ecosystem services. The main obstacle is specimen-rich samples obtained with quantitative sampling techniques (e.g., Malaise trapping). Traditional sorting requires manual handling, while molecular techniques based on metabarcoding lose the association between individual specimens and sequences and thus struggle with obtaining precise abundance information. Here we present a sorting robot that prepares specimens from bulk samples for barcoding. It detects, images and measures individual specimens from a sample and then moves them into the wells of a 96-well microplate. We show that the images can be used to train convolutional neural networks (CNNs) that are capable of assigning the specimens to 14 insect taxa (usually families) that are particularly common in Malaise trap samples. The average assignment precision for all taxa is 91.4% (75%-100%). This ability of the robot to identify common taxa then allows for taxon-specific subsampling, because the robot can be instructed to only pick a prespecified number of specimens for abundant taxa. To obtain biomass information, the images are also used to measure specimen length and estimate body volume. We outline how the DiversityScanner can be a key component for tackling and monitoring invertebrate diversity by combining molecular and morphological tools: the images generated by the robot become training images for machine learning once they are labelled with taxonomic information from DNA barcodes. We suggest that a combination of automation, machine learning and DNA barcoding has the potential to tackle invertebrate diversity at an unprecedented scale.

The Sepsidae is, with approximately 300 described species, a relatively small family of cyclorrhaphan flies whose behaviour, morphology, and development have been extensively studied. However, currently the only available tree for Sepsidae is more than 10 years old and was based entirely on morphological characters. Here, we present the results of parsimony and Bayesian analyses based on 75 species, ten genes, and morphology. Parsimony and Bayesian analyses produce largely congruent and well-supported topologies regardless of whether indels are coded as 5th character states, as missing values, or all sites with indels are removed. The tree confirms the monophyly of Sepsidae and identifies the Ropalomeridae as its sister group. With regard to higher-level relationships, we identify widespread conflict between the morphological and the DNA sequence data. The proposed hypothesis based on both partitions largely reflects the signal in the molecular data. Particularly surprising is the rejection of two relationship hypotheses with strong morphological support, namely the sister group relationship between Orygma and the remaining Sepsidae and the monophyly of the Sepsis species group. Our partitioned Bremer support (PBS) analyses imply that indel coding has a stronger effect on the relative performance of individual gene partitions than the exclusion of alignment-ambiguous sequences or the location of a gene on the mitochondrial or nuclear genome. However, these analyses also reveal unexpectedly strong fluctuations in PBS values given that indel treatment has only a minor effect on tree topology and jacknife support. These unexpected fluctuations highlight the need for a comparative study across multiple data sets that investigates the influence of conflict and indel treatment on PBS values. © The Willi Hennig Society 2008.

We present the largest morphological character set ever compiled for Holometabola. This was made possible through an optimized acquisition of data. Based on our analyses and recently published hypotheses based on molecular data, we discuss higherlevel phylogeny and evolutionary changes. We comment on the information content of different character systems and discuss the role of morphology in the age of phylogenomics. Microcomputer tomography in combination with other techniques proved highly efficient for acquiring and documenting morphological data. Detailed anatomical information (356 characters) is now available for 30 representatives of all holometabolan orders. A combination of traditional and novel techniques complemented each other and rapidly provided reliable data. In addition, our approach facilitates documenting the anatomy of model organisms. Our results show little congruence with studies based on rRNA, but confirm most clades retrieved in a recent study based on nuclear genes: Holometabola excluding Hymenoptera, Coleopterida (= Strepsiptera + Coleoptera), Neuropterida excl. Neuroptera, and Mecoptera. Mecopterida (= Antliophora + Amphiesmenoptera) was retrieved only in Bayesian analyses. All orders except Megaloptera are monophyletic. Problems in the analyses are caused by taxa with numerous autapomorphies and ⁄ or inapplicable character states due to the loss of major structures (such as wings). Different factors have contributed to the evolutionary success of various holometabolan lineages. It is likely that good flying performance, the ability to occupy different habitats as larvae and adults, parasitism, liquid feeding, and co-evolution with flowering plants have played important roles. We argue that even in the ‘‘age of phylogenomics’’, comparative morphology will still play a vital role. In addition, morphology is essential for reconstructing major evolutionary transformations at the phenotypic level, for testing evolutionary scenarios, and for placing fossil taxa. � The Willi Hennig Society 2010.

DNA barcoding was proposed in 2003, the Consortium for the Barcode of Life was established in 2004, and the movement has since attracted more than $80 million funding. Here we investigate how many species of multicellular animals have been barcoded. We compare the numbers in a public database (GenBank as of January 2012) with those in the Barcode of Life Database (BOLD) and find that GenBank contains COI (cytochrome c oxidase subunit 1) sequences for ca. 60 000 species while BOLD reports barcodes for ca. 150 000 species. The discrepancy is likely due to a large amount of unpublished data in BOLD. Overall, the species coverage remains sparse, growth rates are low, and the barcode accumulation curve for Metazoa is linear with only 4788 species having been added in 2011. In addition, the vast majority of species in the public database (73%) were barcoded by projects that are unlikely to be related to the DNA barcoding movement. Particularly surprising was the large number of DNA barcodes in GenBank that were not identified to species (Jan 2012: 74%), with insect barcodes often being identified only to order. Of these several hundred thousand have since been suppressed by NCBI because they did not satisfy the iBOL/GenBank early release agreement. Species coverage is considerably better for target taxa of DNA barcoding campaigns (e.g. birds, fishes, Lepidoptera), although it also falls short of published campaign targets. © The Willi Hennig Society 2012

Sciomyzidae is a family of acalyptrate flies with 546 species in 61 genera distributed worldwide and is among the most extensively studied groups of higher Diptera. Most of the known larvae are obligate enemies of Gastropoda. Hundreds of studies published during the last 50 years have resulted in detailed information concerning morphology of adults and immature stages, their biology, development, behaviour, phenology and distribution. However, recent studies of phylogenetic relationships are based almost exclusively on evaluation of morphological characters of adults and no comprehensive molecular-genetic analysis across the family has been published. Here we fill this void by generating and analyzing molecular data for 54 species of Sciomyzidae (22 genera) including Phaeomyiidae (1 genus) and seven representative species of five other families of Sciomyzoidea (Coelopidae, Dryomyzidae, Helcomyzidae, Heteromyzidae and Huttoninidae) as outgroups. The reconstruction is based on morphological characters as well as nucleotide sequences for genes from the mitochondrial (12S, 16S, COI, COII, CytB) and nuclear genome (28S, EF1 alpha). The results are compared with recent morphological analyses. Our analyses support the monophyly of Sciomyzidae + Phaeomyiidae and place Phaeomyiinae as a unique lineage within Sciomyzidae. A modified classification comprising three subfamilies is proposed. The major subfamily, Sciomyzinae, consists of two monophyletic and well separated groups, the tribes Sciomyzini and Tetanocerini.

Many phoretic relationships between insects are understudied because of taxonomic impediments. We here illustrate for avian lice riding on hippoboscid flies how new natural history data on phoretic relationships can be acquired quickly with NGS barcoding. Most avian lice are host-specific, but some can arrive on new hosts by riding hippoboscid flies that feed on bird blood. Our summary of the literature yielded 254 published records which we here show to belong to two large and 13 small interaction networks for birds, flies, and lice. In order to generate new records, we then developed a new protocol based on screening bird carcasses sourced by citizen scientists. The inspection of 131 carcasses from Singapore lead to the first record of a Guimaraesiella louse species riding on Ornithoica momiyamai flies collected from a pitta carcass. Phoresy may explain why this louse species is now known from three phylogenetically disparate hosts (Pitta moluccensis, Ficedula zanthopygia; Pardaliparus elegans). A second new case of phoresy enhances a large interaction network dominated by Ornithophila metallica, a cosmopolitan and polyphagous hippoboscid fly species. Overall, we argue that many other two- and three-way phoretic relationships between arthropods (e.g., mites, pseudoscorpiones, beetles, flies) can be resolved using cost-effective large-scale NGS barcoding, which can be used to pre-sort specimens for taxonomic revision and thus partially overcome some taxonomic impediments.

BackgroundDNA 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 ResultsWe 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.ConclusionsWe 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.

BackgroundDNA 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 ResultsWe describe cost-effective and rapid procedures for barcoding individual specimens with MinION sequencing. We illustrate how tagged amplicons can be obtained and sequenced with the portable, 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.ConclusionsWe 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.

Studies of insect mating behaviour usually focus on what happens before and during copulation. Few pay close attention to the actions needed to end copulation. However, genital separation after copulation is likely to be an important cause of mechanical stress and injuries because it often involves the withdrawal of heavily armoured male intromittent organs from membranous female reproductive tracts. Difficult and/or slow separations can also reduce male and female fitness by increasing their exposure to predation. We here report the results of a comparative study of separation behaviour in 48 species of Sepsidae (Diptera) and one outgroup. We find a surprising amount of qualitative and quantitative behavioural variability within and between species. We characterize and reconstruct three types of behaviours: 1) The sepsid ancestor likely used ‘back-off; a gentle separation technique that does not involve any pulling or twisting (https://youtu.be/EbkJvOaubZ0). 2) This potentially gave rise to the most common ‘pull’ technique where the male turns 180 degrees and pulls in an opposite direction from the female (https://youtu.be/oLf4xGpkk1s). This separation can be quick and straightforward, but in some species the ‘pull’ is slow and protracted and we routinely find dead males and/or females attached to their living partners in the latter (difficult: https://youtu.be/MbYPbXN6jr0; failure: https://youtu.be/leTiXefFzCc). 3) Finally, several species use ‘twist’, a new technique where the male rotates >360 degrees from the initial mounting position (https://youtu.be/WMUXbIPyLbk). We document that species capable of using ‘twist’ have shorter and less variable separation times than those using “pull”. However, many species capable of ‘twist’ also retain the ability to use ‘pull’ (‘back-off’/’pull’= 8%; ‘pull’ only= 41%; ‘twist’/ ‘pull’= 24%; ‘twist’ only = 27%). Overall, our study suggests that separation behaviour can vary among closely related species and highlights the significance of studying variable behavioural traits in a phylogenetic context.

Invertebrate biodiversity remains poorly explored although it comprises much of the terrestrial animal biomass, more than 90% of the species-level diversity, supplies many ecosystem services. Increasing anthropogenic threads also require regular monitoring of invertebrate communities. The main obstacle is specimen- and species-rich samples consisting of thousands of small specimens. Traditional sorting techniques require manual handling based on morphology and are too slow and labor-intensive. Molecular techniques based on metabarcoding struggle with obtaining reliable abundance information. We here present a fully automated sorting robot for small specimens that are detected in the mixed sample using a convolutional neural network. Each specimen is then moved from the mixed sample to a well of a 96-well microplate in preparation for DNA barcoding. Prior to movement, the specimen is being photographed and assigned to 14 particularly common “classes” of insects in Malaise trap samples. The average assignment precision for the classes is 91.4 % (75-100 %) based on a preliminary neural network that is expected to improve further as more images are used for training. In order to obtain biomass information, the specimen images are also used to measure the specimen length and estimate the body volume. We outline how the “DiversityScanner” robot can be a key component for tackling and monitoring invertebrate diversity by generating large numbers of images that become training sets for species-, genus-, or family-level convolutional neural networks, once the imaged specimens are classified with DNA barcodes. The robot also allows for taxon-specific subsampling of large invertebrate samples. We conclude that the combination of automation, machine learning, and DNA barcoding has the potential to tackle invertebrate diversity at an unprecedented scale.

Background: The black soldier fly (Hermetia illucens) is the most promising insect candidate for nutrient-recycling through bioconversion of organic waste into biomass, thereby improving sustainability of protein supplies for animal feed and facilitating transition to a circular economy. Contrary to conventional livestock, genetic resources of farmed insects remain poorly characterised. We present the first comprehensive population genetic characterisation of H. illucens. Based on 15 novel microsatellite markers, we genotyped and analysed 2862 individuals from 150 wild and captive populations originating from 57 countries on seven subcontinents. Results: We identified 16 well-distinguished genetic clusters indicating substantial global population structure. The data revealed genetic hotspots in central South America and successive northwards range expansions within the indigenous ranges of the Americas. Colonisations and naturalisations of largely unique genetic profiles occurred on all non-native continents, either preceded by demographically independent founder events from various single sources or involving admixture scenarios. A decisive primarily admixed Polynesian bridgehead population serially colonised the entire Australasian region and its secondarily admixed descendants successively mediated invasions into Africa and Europe. Conversely, captive populations from several continents traced back to a single North American origin and exhibit considerably reduced genetic diversity, although some farmed strains carry distinct genetic signatures. We highlight genetic footprints characteristic of progressing domestication due to increasing socio-economic importance of H. illucens, and ongoing introgression between domesticated strains globally traded for large-scale farming and wild populations in some regions. Conclusions: We document the dynamic population genetic history of a cosmopolitan dipteran of South American origin shaped by striking geographic patterns. These reflect both ancient dispersal routes, and stochastic and heterogeneous anthropogenic introductions during the last century leading to pronounced diversification of worldwide structure of H. illucens. Upon the recent advent of its agronomic commercialisation, however, current human-mediated translocations of the black soldier fly largely involve genetically highly uniform domesticated strains, which meanwhile threaten the genetic integrity of differentiated unique local resources through introgression. Our in-depth reconstruction of the contemporary and historical demographic trajectories of H. illucens emphasises benchmarking potential for applied future research on this emerging model of the prospering insect-livestock sector. This work was primarily supported by the Swiss Federal Office for Agriculture (grant no. 627000824 to CS), as well as by the R4D programme of the Swiss Agency for Development and Cooperation and the Swiss National Science Foundation (grant no. IZ01Z0_147278 to CS, and grant no. 400540_152154 to MKen), the Austrian Science Foundation (grant no. FWF P32275 to MKap), the Canadian International Development Research Centre (IDRC) and the Australian Centre for International Agricultural Research (ACIAR) (grant no. 108866-001 to CMT), and the National Research Foundation, South Africa (reference no. CSRP170506229933 to CR).

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”) while another is caused by the large number of superficial species descriptions which 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 barcode clusters (“BINs”) computed by BOLD Systems. More than 99% of the BINs (387 of 390) are converted into species by assigning binominal names (e.g., BIN “BOLD:ACM9419” becomes Bracon federicomatarritai) and adding a minimal diagnosis (usually consisting only of a consensus barcode). 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 consistently applied. 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.

New, rapid, accurate, scalable, and cost-effective species discovery and delimitation methods are needed for tackling “dark taxa”, that we here define as clades for which

Background The world’s fast disappearing mangrove forests have low plant diversity and are often assumed to also have a species-poor insect fauna. We here compare the tropical arthropod fauna across a freshwater swamp and six different forest types (rain-, swamp, dry-coastal, urban, freshwater swamp, mangroves) based on 140,000 barcoded specimens belonging to ca. 8500 species. Results We find that the globally imperiled habitat “mangroves” is an overlooked hotspot for insect diversity. Our study reveals a species-rich mangrove insect fauna (>3000 species in Singapore alone) that is distinct (>50% of species are mangrove-specific) and has high species turnover across Southeast and East Asia. For most habitats, plant diversity is a good predictor of insect diversity, but mangroves are an exception and compensate for a comparatively low number of phytophagous and fungivorous insect species by supporting an unusually rich community of predators whose larvae feed in the productive mudflats. For the remaining tropical habitats, the insect communities have diversity patterns that are largely congruent across guilds. Conclusions The discovery of such a sizeable and distinct insect fauna in a globally threatened habitat underlines how little is known about global insect biodiversity. We here show how such knowledge gaps can be closed quickly with new cost-effective NGS barcoding techniques.

Background Blowflies are ubiquitous insects, often shiny and metallic, and the larvae of many species provide important ecosystem services (e.g., recycling carrion) and are used in forensics and debridement therapy. Yet, the taxon has repeatedly been recovered to be para- or polyphyletic, and the lack of a well-corroborated phylogeny has prevented a robust classification. Results We here resolve the relationships between the different blowfly subclades by including all recognized subfamilies in a phylogenomic analysis using 2221 single-copy nuclear protein-coding genes of Diptera. Maximum likelihood (ML), maximum parsimony (MP), and coalescent-based phylogeny reconstructions all support the same relationships for the full data set. Based on this backbone phylogeny, blowflies are redefined as the most inclusive monophylum within the superfamily Oestroidea not containing Mesembrinellidae, Mystacinobiidae, Oestridae, Polleniidae, Sarcophagidae, Tachinidae, and Ulurumyiidae. The constituent subfamilies are re-classified as Ameniinae (including the Helicoboscinae, syn. nov.), Bengaliinae, Calliphorinae (including Aphyssurinae, syn. nov., Melanomyinae, syn. nov., and Toxotarsinae, syn. nov.), Chrysomyinae, Luciliinae, Phumosiinae, Rhiniinae stat. rev., and Rhinophorinae stat. rev. Metallic coloration in the adult is shown to be widespread but does not emerge as the most likely ground plan feature. Conclusions Our study provides the first phylogeny of oestroid calyptrates including all blowfly subfamilies. This allows settling a long-lasting controversy in Diptera by redefining blowflies as a well-supported monophylum, and blowfly classification is adjusted accordingly. The archetypical blowfly trait of carrion-feeding maggots most likely evolved twice, and the metallic color may not belong to the blowfly ground plan.

Sea anemones are sedentary marine animals that tend to disperse via planktonic larvae and are predicted to have high population connectivity in undisturbed habitats. We test whether two sea anemone species living in two different tidal zones of a highly disturbed marine environment can maintain high genetic connectivity. More than 1000 loci with single-nucleotide polymorphisms (SNPs) were obtained with double-digest RADseq for 81 Stichodactyla haddoni and 99 Entacmaea quadricolor individuals to test for population genetic structure. We find evidence that both species predominantly propagate via sexual reproduction, and asexual reproduction is limited. We observe panmixia that indicates the absence of effective dispersal barriers for these species living in a highly anthropogenically disturbed environment. This is positive news for both species that are also found in the aquarium trade. More fundamentally, our results suggest that inhabiting different parts of a shallow reef may not affect a species&rsquo, population connectivity nor favour asexual reproduction.

A significant number of Southeast Asian mammal species described in the 19th and 20th century were subsequently synonymized and are now considered subspecies. Many are affected by rapid habitat loss which creates an urgent need to re-assess the conservation status based on species boundaries established with molecular data. However, such data are lacking and difficult to obtain for many populations and subspecies. We document via a literature survey and empirical study how shotgun sequencing of faecal DNA is a still underutilized but powerful tool for accelerating such evaluations. We obtain 11 mitochondrial genomes for three subspecies in the langur genus Presbytis through shotgun sequencing of faecal DNA (P. femoralis femoralis, P. f. percura, P. siamensis cf. cana). The genomes support the resurrection of all three subspecies to species based on multiple species delimitation algorithms (PTP, ABGD, Objective Clustering) applied to a dataset covering 40 species and 43 subspecies of Asian colobines. For two of the newly recognized species (P. femoralis, P. percura), the results lead to an immediate change in IUCN status to Critically Endangered due to small population sizes and fragmented habitats. We conclude that faecal DNA should be more widely used for clarifying species boundaries in endangered mammals.

Polymorphic Batesian mimics exhibit multiple protective morphs that each mimic a different noxious model. Here, we study the genomic transitions leading to the evolution of different mimetic wing patterns in the polymorphic Mocker Swallowtail Papilio dardanus. We generated a draft genome (231 Mb over 30 chromosomes) and re-sequenced individuals of three morphs. Genome-wide single nucleotide polymorphism (SNP) analysis revealed elevated linkage disequilibrium and divergence between morphs in the regulatory region of engrailed, a developmental gene previously implicated in the mimicry switch. The diverged region exhibits a discrete chromosomal inversion (of 40 kb) relative to the ancestral orientation that is associated with the cenea morph, but not with the bottom-recessive hippocoonides morph or with non-mimetic allopatric populations. The functional role of this inversion in the expression of the novel phenotype is currently unknown, but by preventing recombination, it allows the stable inheritance of divergent alleles enabling geographic spread and local coexistence of multiple adaptive morphs.

Background: The black soldier fly (Diptera: Stratiomyidae, Hermetia illucens) is renowned for its bioconversion ability of organic matter, and is the worldwide most widely used source of insect protein. Despite varying extensively in morphology, it is widely assumed that all black soldier flies belong to the same species, Hermetia illucens. We here screened about 600 field-collected and cultured flies from 39 countries and six biogeographic regions to test this assumption based on data for three genes (mitochondrial COI, nuclear ITS2 & 28S rDNA) and in order to gain insights into the phylogeography of the species. Results: Our study reveals a surprisingly high level of intraspecific genetic diversity for the mitochondrial barcoding gene COI (divergences up to 4.9%). This level of variability is often associated with the presence of multiple species, but tested nuclear markers (ITS2 and 28S rDNA) were invariant and fly strain hybridization experiments under laboratory conditions revealed reproductive compatibility. COI haplotype diversity is not only very high in all biogeographic regions (56 distinct haplotypes in total), but also in breeding facilities and research centers from six continents (10 haplotypes: divergences up to 4.3%). The high genetic diversity in fly-breeding facilities is mostly likely due to many independent acquisitions of cultures via sharing and/or establishing new colonies from field-collected flies. However, explaining some of the observed diversity in several biogeographic regions is difficult given that the origin of the species is considered to be New World (32 distinct haplotypes) and one would expect severely reduced genetic diversity in the putatively non-native populations in the remaining biogeographic regions. However, distinct, private haplotypes are known from the Australasian (N = 1), Oriental (N = 4), and the Eastern Palearctic (N = 4) populations. We reviewed museum specimen records and conclude that the evidence for introductions is strong for the Western Palearctic and Afrotropical regions which lack distinct, private haplotypes. Conclusions: Based on the results of this paper, we urge the black soldier fly community to apply molecular characterization (genotyping) of the fly strains used in artificial fly-breeding and share these data in research publications as well as when sharing cultures. In addition, fast-evolving nuclear markers should be used to reconstruct the recent invasion history of the species. This work was funded by the H2020 Research and Innovation Staff Exchange Programme of the European Commission (RISE), project 645636: ‘Insect-plant relationships: insights into biodiversity and new applications’ (FlyHigh), and the grants R-154-000-B46–114 and R-154-000-A94–592 to RM, as well as a grant of the Swiss Federal Office for Agriculture to CS (‘Efficiency and sustainability of the production and feeding of insect based feedstuff to fish and poultry’).

Summary Distal-less has been repeatedly co-opted for the development of many novel traits. Here, we document its curious role in the development of a novel abdominal appendage (“sternite brushes”) in sepsid flies. CRISPR/Cas9 deletions in the homeodomain result in losses of sternite brushes, demonstrating that Distal-less is necessary for their development. However, deletions in the upstream coding exon (Exon 2) produce losses or gains of brushes. A dissection of Exon 2 reveals that the likely mechanism for gains involves a deletion in an exon-splicing enhancer site that leads to exon skipping. Such contradictory phenotypes are also observed in butterflies, suggesting that mutations in the conserved upstream regions have the potential to generate phenotypic variability in insects that diverged 300 million years ago. Our results demonstrate the importance of Distal-less for the development of a novel abdominal appendage in insects and highlight how site-specific mutations in the same exon can produce contradictory phenotypes., Graphical Abstract, Highlights • Distal-less is necessary for the development of a novel abdominal appendage • CRISPR/Cas9 editing produced both losses and gains of novel abdominal appendages • Gains of appendages result from mutations in exonic splicing enhancer (ESEs) sites • ESE mutations likely led to exon skipping and an altered Distal-less protein, Genetics; Molecular Biology; Evolutionary Biology; Developmental Biology

Background The males of some sepsid species (Sepsidae: Diptera) have abdominal appendages that are remarkable in several ways. They are sexually dimorphic, have a complex evolutionary history of gain and loss, and can be jointed and thus highly mobile. The sternite brushes are used extensively in complex courtship behaviors that differ considerably between species and during mating. The abdominal appendages have a novel developmental pathway developing from histoblast nests rather than imaginal discs. Results We focus on the evolution of cell number, nest area, and segment length in both sexes to understand how this tissue relates to the formation of novel abdominal appendages. We map histoblast nest size of wandering-phase larvae of 17 species across 10 genera to a phylogenetic tree of Sepsidae and demonstrate that abdominal appendages require significant increases of histoblast nest size and cell number in most species while one species produces small appendages even without such modifications. In species with particularly large appendages, not only the nests on the fourth, but nests in neighboring segments are enlarged (Themira biloba, Themira putris). The loss of abdominal appendages corresponds to the loss of an enlarged fourth histoblast nest, although one species showed an exception to this pattern. One species that constitutes an independent origin of abdominal appendages (Perochaeta dikowi) uses an unusual developmental mechanism in that the histoblast nest sizes are not sexually dimorphic. Conclusions The surprisingly high diversity in histoblast size and degree of sexual dimorphism suggests that the developmental mechanism used for abdominal appendage formation in sepsids is highly adaptable. The presence of appendages usually correlate with increased histoblast cell number and in most cases appendage loss results in a return to ancestral histoblast morphology. However, we also identify several exceptions that indicate the abdominal appendages have a malleable developmental origin that is responsive to selection. Electronic supplementary material The online version of this article (10.1186/s12862-018-1265-3) contains supplementary material, which is available to authorized users.

DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well-equipped molecular laboratory, is time-consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION™ and demonstrate that one flowcell can generate barcodes for ∼500 specimens despite high base-call error rates of MinION™. The pipeline overcomes the errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. These barcodes are overall mismatch-free but retain indel errors that are concentrated in homopolymeric regions. We thus complement the barcode caller with an optional error correction pipeline that uses conserved amino-acid motifs from publicly available barcodes to correct the indel errors. The effectiveness of this pipeline is documented by analysing reads from three MinION™ runs that represent three different stages of MinION™ development. They generated data for (1) 511 specimens of a mixed Diptera sample, (2) 575 specimens of ants, and (3) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION barcodes for 490 specimens which were assessed against reference Sanger barcodes (N=471). Overall, the MinION barcodes have an accuracy of 99.3%-100% and the number of ambiguities ranges from 90%). We estimate that up to 1000 barcodes can be generated in one flowcell and that the cost of a MinION barcode can be

In many taxa the morphology of females and immatures is poorly known because species descriptions and identification tools have a male bias. The root causes are problems with matching life‐history stages and genders belonging to the same species. Such matching is time‐consuming when conventional methods are used (e.g. rearing) and expensive when the stages are matched with DNA barcodes. Unfortunately, the lack of associations is not a trivial problem because it renders a large part of the phenome of insects unexplored, although larvae and females are useful sources of characters for descriptive and phylogenetic purposes. In addition, many collectors intentionally avoid females and immature stages, which skews survey results, interferes with collecting life‐history information, and makes it less likely that rare species are discovered. These problems even exist for well‐studied taxa like Odonata, where obtaining adult–larva matches relies largely on rearing. Here we demonstrate how the matching problem can be addressed with cost‐effective tagged amplicon sequencing of a 313‐bp segment of cox1 with next‐generation sequencing (NGS) (‘NGS barcoding’). We illustrate the value of this approach based on Singapore's odonate fauna which is of a similar size as the European fauna (Singapore, 122 extant species; Europe, 138 recorded species). We match the larvae and adults of 59 species by first creating a barcode database for 338 identified adult specimens representing 83 species. We then sequence 1178 larvae from a wide range of sources. We successfully barcode 1123 specimens, which leads to adult–larva matches for 59 species based on our own barcodes (55) and online barcode databases (4). With these additions, 84 of the 131 species recorded in Singapore have now been associated with a species name. Most common species are now matched (83%), and good progress has been made for vulnerable/near‐threatened (55%), endangered (53%), and critically endangered species (38%). We used nondestructive DNA extraction methods in order to be able to use high‐resolution imaging of matched larvae to establish a publicly available digital reference collection for odonates which is incorporated into ‘Biodiversity of Singapore’ (https://singapore.biodiversity.online/). We suggest that the methods described here are suitable for many insect taxa because NGS barcoding allows for fast and low‐cost matching of well‐studied life‐history stages with neglected semaphoronts (eggs, larvae, females). We estimate that the specimen‐specific amplicons in this study (c. 1500 specimens) can now be obtained within eight working days and that the laboratory and sequencing cost is c. US$600 (

The Nee Soon stream drainage in the Central Catchment Nature Reserve is virtually the last remaining fragment of primary freshwater swamp forest in Singapore. The forest type has been poorly studied i

Biologists frequently sort specimen-rich samples to species. This process is daunting when based on morphology, and disadvantageous if performed using molecular methods that destroy vouchers (e.g., metabarcoding). An alternative is barcoding every specimen in a bulk sample and then presorting the specimens using DNA barcodes, thus mitigating downstream morphological work on presorted units. Such a "reverse workflow" is too expensive using Sanger sequencing, but we here demonstrate that is feasible with an next-generation sequencing (NGS) barcoding pipeline that allows for cost-effective high-throughput generation of short specimen-specific barcodes (313 bp of COI; laboratory cost

New species from well-studied taxa such as Sepsidae (Diptera) are rarely described from localities that have been extensively explored and one may think that New York City belongs to this category. Yet, a new species of Themira (Diptera: Sepsidae) was recently discovered which is currently only known to reside in two of New York City’s largest urban parks. Finding a new species of Themira in these parks was all the more surprising because the genus was revised in 1998 and is not particularly species-rich (13 species). Its status is confirmed as a new species based on morphology, DNA sequences, and reproductive isolation tests with a closely related species, and is described as Themira lohmanus Ang, sp. n. The species breeds on waterfowl dung and it is hypothesized that this makes the species rare in natural environments. However, it thrives in urban parks where the public feeds ducks and geese. The mating behavior of Themira lohmanus was recorded and is similar to the behavior of its closest relative T. biloba.

Spolien – intentional wiederverwendete Bauglieder – referenzieren auf etwas nicht mehr Vorhandenes und machen dieses zugleich materiell präsent. Als einstiger Teil des Ab- wesenden verweisen sie im neuen Kontext zurück auf ihre Herkunftsobjekte. Sie verkörpern abstrakte Konzepte, Autorisierung und Authentisierung. In den präsentierten Bei- spielen werden verschiedene Ähnlichkeitsbezüge zum Herkunftsmonument diskutiert, die von formalen Referenzen über die exzessive Verkörperung zur formlosen Verarbeitung des Materials in einer neuer Oberfläche reichen., Spoils – intentionally reused architectural fragments – refer to something that no longer exists and re-present it materially. As a former part of the absent, they refer back to their original objects in their new context. They embody abstract concepts, authorization and authentication. In the examples presented, different ways of referring to the original monuments are discussed, ranging from formal references to excessive embodiment and complete loss of shape.

Few tropical marine sites have been thoroughly characterised for their animal species, even though they constitute the largest proportion of multicellular diversity. A number of focused biodiversity sampling programmes have amassed immense collections to address this shortfall, but obstacles remain due to the lack of identification tools and large proportion of undescribed species globally. These problems can be partially addressed with DNA barcodes (“biocodes”), which have the potential to facilitate the estimation of species diversity and identify animals to named species via barcode databases. Here, we present the first results of what is intended to be a sustained, systematic study of the marine fauna of Singapore’s first marine park, reporting more than 365 animal species, determined based on DNA barcodes and/or morphology represented by 931 specimens (367 zooplankton, 564 macrofauna including 36 fish). Due to the lack of morphological and molecular identification tools, only a small proportion could be identified to species solely based on either morphology (24.5%) or barcodes (24.6%). Estimation of species numbers for some taxa was difficult because of the lack of sufficiently clear barcoding gaps. The specimens were imaged and added to “Biodiversity of Singapore” (http://singapore.biodiversity.online), which now contains images for > 13,000 species occurring in the country.

Biodiversity is in crisis due to habitat destruction and climate change. The conservation of many noncharismatic species is hampered by the lack of data. Yet, natural history research—a major source of information on noncharismatic species—is in decline. We here suggest a remedy for many mammal species, i.e., metagenomic clean-up of fecal samples that are “crowdsourced” during routine field surveys. Based on literature data, we estimate that this approach could yield natural history information for circa 1,000 species within a decade. Metagenomic analysis would simultaneously yield natural history data on diet and gut parasites while enhancing our understanding of host genetics, gut microbiome, and the functional interactions between traditional and new natural history data. We document the power of this approach by carrying out a “metagenomic clean-up” on fecal samples collected during a single night of small mammal trapping in one of Alfred Wallace’s favorite collecting sites., Natural history research is in crisis and non-charismatic species are increasingly ignored; this Community Page article argues and demonstrates that shotgun sequencing of serendipitously obtained faecal samples could reverse this trend for 1000 mammal species within 10 years.