Future of DNA-based insect monitoring.

Insects are crucial for ecosystem health but climate change and pesticide use are driving massive insect decline. To mitigate this loss, we need new and effective monitoring techniques. Over the past decade there has been a shift to DNA-based techniques. We describe key emerging techniques for sample collection. We suggest that the selection of tools should be broadened, and that DNA-based insect monitoring data need to be integrated more rapidly into policymaking. We argue that there are four key areas for advancement, including the generation of more complete DNA barcode databases to interpret molecular data, standardisation of molecular methods, scaling up of monitoring efforts, and integrating molecular tools with other technologies that allow continuous, passive monitoring based on images and/or laser imaging, detection, and ranging (LIDAR).

Two centuries of biodiversity discovery and loss in Singapore.

There is an urgent need for reliable data on the impacts of deforestation on tropical biodiversity. The city-state of Singapore has one of the most detailed biodiversity records in the tropics, dating back to the turn of the 19th century. In 1819, Singapore was almost entirely covered in primary forest, but this has since been largely cleared. We compiled more than 200 y of records for 10 major taxonomic groups in Singapore (>50,000 individual records; >3,000 species), and we estimated extinction rates using recently developed and novel statistical models that account for "dark extinctions," i.e., extinctions of undiscovered species. The estimated overall extinction rate was 37% (95% CI [31 to 42%]). Extrapolating our Singapore observations to a future business-as-usual deforestation scenario for Southeast Asia suggests that 18% (95% CI [16 to 22%]) of species will be lost regionally by 2100. Our extinction estimates for Singapore and Southeast Asia are a factor of two lower than previous estimates that also attempted to account for dark extinctions. However, we caution that particular groups such as large mammals, forest-dependent birds, orchids, and butterflies are disproportionately vulnerable.

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

Most arthropod species are undescribed and hidden in specimen-rich samples that are difficult to sort to species using morphological characters. For such samples, sorting 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 using the portable and inexpensive MinION sequencer produced by Oxford Nanopore Technologies (ONT) could be useful for presorting specimen-rich samples with DNA barcodes because it requires little space and is inexpensive. However, similarly important is user-friendly and reliable software for analysis of the ONT data. It is here provided in the form of ONTbarcoder 2.0 that is suitable for all commonly used operating systems and includes a Graphical User Interface (GUI). Compared with an earlier version, ONTbarcoder 2.0 has three key improvements related to the higher read quality obtained with ONT's latest flow cells (R10.4), chemistry (V14 kits) and basecalling model (super-accuracy model). First, the improved read quality of ONT's 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 the primer cost and can potentially improve PCR success rates. Second, ONTbarcoder now delivers real-time barcoding to complement ONT's real-time sequencing. This means that the first barcodes are obtained within minutes of starting a sequencing run; i.e. flow cell use can be optimized by terminating sequencing runs when most barcodes have already been obtained. The only input needed by ONTbarcoder 2.0 is a demultiplexing sheet and sequencing data (raw or basecalled) generated by either a Mk1B or a Mk1C. Thirdly, we demonstrate that the availability of R10.4 chemistry for the low-cost Flongle flow cell is an attractive option for users who require only 200-250 barcodes at a time.

Size rather than complexity of sexual ornaments prolongs male metamorphosis and explains sexual size dimorphism in sepsid flies.

Male sexual ornaments often evolve rapidly and are thought to be costly, thus contributing to sexual size dimorphism. However, little is known about their developmental costs, and even less about costs associated with structural complexity. Here, we quantified the size and complexity of three morphologically elaborate sexually dimorphic male ornaments that starkly differ across sepsid fly species (Diptera: Sepsidae): (i) male forelegs range from being unmodified, like in most females, to being adorned with spines and large cuticular protrusions; (ii) the fourth abdominal sternites are either unmodified or are converted into complex de novo appendages; and (iii) male genital claspers range from small and simple to large and complex (e.g. bifurcated). We tracked the development of 18 sepsid species from egg to adult to determine larval feeding and pupal metamorphosis times of both sexes. We then statistically explored whether pupal and adult body size, ornament size and/or ornament complexity are correlated with sex-specific development times. Larval growth and foraging periods of male and female larvae did not differ, but the time spent in the pupal stage was ca 5% longer for sepsid males despite emerging 9% smaller than females on average. Surprisingly, we found no evidence that sexual trait complexity prolongs pupal development beyond some effects of trait size. Evolving more complex traits thus does not incur developmental costs at least in this system.

Multispecies environmental DNA metabarcoding sheds light on annual coral spawning events.

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.

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

DNA obtained from invertebrates (iDNA) can be metabarcoded in order to survey vertebrate communities. However, little attention has been paid to the interaction between the invertebrate and vertebrate species. Here, we tested for specialization by sampling the dung and carrion fly community of a swamp forest remnant along a disturbance gradient (10 sites: 80-310 m from a road). Approximately, 60% of the baited 407 flies yielded 294 vertebrate identifications based on two COI fragments and 16S. A bipartite network analysis found no statistically significant specialization in the interactions between fly and vertebrate species, but uncommon fly species can carry the signal for vertebrate species that are otherwise difficult to detect with iDNA. A spatial analysis revealed that most of the 20 vertebrate species reported in this study could be detected within 150 m of the road (18 spp.) and that the fly community sourced for iDNA was unexpectedly rich (24 species, 3 families). They carried DNA for rare and common species inhabiting different layers of the forest (ground-dwelling: wild boar, Sunda pangolin, skinks, rats; arboreal: long-tailed macaque, Raffles' banded langur; flying: pin-striped tit-babbler, olive-winged bulbul). All our results were obtained with a new, greatly simplified iDNA protocol that eliminates DNA extraction by obtaining template directly through dissolving fly faeces and regurgitates with water. Lastly, we show that MinION- and Illumina-based metabarcoding yield similar results. We conclude by urging more studies that use different baits and involve experiments that are capable of revealing the dispersal capabilities of the flies carrying the iDNA.

Collective and harmonized high throughput barcoding of insular arthropod biodiversity: Toward a Genomic Observatories Network for islands.

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.

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

New, rapid, accurate, scalable, and cost-effective species discovery and delimitation methods are needed for tackling "dark taxa," here defined as groups for which $<$10$\%$ of all species are described and the estimated diversity exceeds 1,000 species. Species delimitation for these taxa should be based on multiple data sources ("integrative taxonomy") but collecting multiple types of data risks impeding a discovery process that is already too slow. We here develop large-scale integrative taxonomy (LIT), an explicit method where preliminary species hypotheses are generated based on inexpensive data that can be obtained quickly and cost-effectively. These hypotheses are then evaluated based on a more expensive type of "validation data" that is only obtained for specimens selected based on objective criteria applied to the preliminary species hypotheses. We here use this approach to sort 18,000 scuttle flies (Diptera: Phoridae) into 315 preliminary species hypotheses based on next-generation sequencing barcode (313 bp) clusters (using objective clustering [OC] with a 3$\%$ threshold). These clusters are then evaluated with morphology as the validation data. We develop quantitative indicators for predicting which barcode clusters are likely to be incongruent with morphospecies by randomly selecting 100 clusters for in-depth validation with morphology. A linear model demonstrates that the best predictors for incongruence between barcode clusters and morphology are maximum p-distance within the cluster and a newly proposed index that measures cluster stability across different clustering thresholds. A test of these indicators using the 215 remaining clusters reveals that these predictors correctly identify all clusters that are incongruent with morphology. In our study, all morphospecies are true or disjoint subsets of the initial barcode clusters so that all incongruence can be eliminated by varying clustering thresholds. This leads to a discussion of when a third data source is needed to resolve incongruent grouping statements. The morphological validation step in our study involved 1,039 specimens (5.8$\%$ of the total). The formal LIT protocol we propose would only have required the study of 915 (5.1$\%$: 2.5 specimens per species), as we show that clusters without signatures of incongruence can be validated by only studying two specimens representing the most divergent haplotypes. To test the generality of our results across different barcode clustering techniques, we establish that the levels of incongruence are similar across OC, Automatic Barcode Gap Discovery (ABGD), Poisson Tree Processes (PTP), and Refined Single Linkage (RESL) (used by Barcode of Life Data System to assign Barcode Index Numbers [BINs]). OC and ABGD achieved a maximum congruence score with the morphology of 89$\%$ while PTP was slightly less effective (84$\%$). RESL could only be tested for a subset of the specimens because the algorithm is not public. BINs based on 277 of the original 1,714 haplotypes were 86$\%$ congruent with morphology while the values were 89$\%$ for OC, 74$\%$ for PTP, and 72$\%$ for ABGD. [Biodiversity discovery; dark taxa; DNA barcodes; integrative taxonomy.].

Boosting natural history research via metagenomic clean-up of crowdsourced feces.

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.

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

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

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

BACKGROUND: DNA barcodes are a useful tool for discovering, understanding, and monitoring biodiversity which are critical tasks at a time of rapid biodiversity loss. However, widespread adoption of barcodes requires cost-effective and simple barcoding methods. We here present a workflow that satisfies these conditions. It was developed via "innovation through subtraction" and thus requires minimal lab equipment, can be learned within days, reduces the barcode sequencing cost to < 10 cents, and allows fast turnaround from specimen to sequence by using the portable MinION sequencer. RESULTS: We describe how tagged amplicons can be obtained and sequenced with the real-time MinION sequencer in many settings (field stations, biodiversity labs, citizen science labs, schools). We also provide amplicon coverage recommendations that are based on several runs of the latest generation of MinION flow cells ("R10.3") which suggest that each run can generate barcodes for > 10,000 specimens. Next, we present a novel software, ONTbarcoder, which overcomes the bioinformatics challenges posed by MinION reads. The software is compatible with Windows 10, Macintosh, and Linux, has a graphical user interface (GUI), and can generate thousands of barcodes on a standard laptop within hours based on only two input files (FASTQ, demultiplexing file). We document that MinION barcodes are virtually identical to Sanger and Illumina barcodes for the same specimens (> 99.99%) and provide evidence that MinION flow cells and reads have improved rapidly since 2018. CONCLUSIONS: We propose that barcoding with MinION is the way forward for government agencies, universities, museums, and schools because it combines low consumable and capital cost with scalability. Small projects can use the flow cell dongle ("Flongle") while large projects can rely on MinION flow cells that can be stopped and re-used after collecting sufficient data for a given project.

Monophyletic blowflies revealed by phylogenomics.

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.

Mangroves are an overlooked hotspot of insect diversity despite low plant diversity.

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.

Beyond Drosophila: resolving the rapid radiation of schizophoran flies with phylotranscriptomics.

BACKGROUND: The most species-rich radiation of animal life in the 66 million years following the Cretaceous extinction event is that of schizophoran flies: a third of fly diversity including Drosophila fruit fly model organisms, house flies, forensic blow flies, agricultural pest flies, and many other well and poorly known true flies. Rapid diversification has hindered previous attempts to elucidate the phylogenetic relationships among major schizophoran clades. A robust phylogenetic hypothesis for the major lineages containing these 55,000 described species would be critical to understand the processes that contributed to the diversity of these flies. We use protein encoding sequence data from transcriptomes, including 3145 genes from 70 species, representing all superfamilies, to improve the resolution of this previously intractable phylogenetic challenge. RESULTS: Our results support a paraphyletic acalyptrate grade including a monophyletic Calyptratae and the monophyly of half of the acalyptrate superfamilies. The primary branching framework of Schizophora is well supported for the first time, revealing the primarily parasitic Pipunculidae and Sciomyzoidea stat. rev. as successive sister groups to the remaining Schizophora. Ephydroidea, Drosophila's superfamily, is the sister group of Calyptratae. Sphaeroceroidea has modest support as the sister to all non-sciomyzoid Schizophora. We define two novel lineages corroborated by morphological traits, the 'Modified Oviscapt Clade' containing Tephritoidea, Nerioidea, and other families, and the 'Cleft Pedicel Clade' containing Calyptratae, Ephydroidea, and other families. Support values remain low among a challenging subset of lineages, including Diopsidae. The placement of these families remained uncertain in both concatenated maximum likelihood and multispecies coalescent approaches. Rogue taxon removal was effective in increasing support values compared with strategies that maximise gene coverage or minimise missing data. CONCLUSIONS: Dividing most acalyptrate fly groups into four major lineages is supported consistently across analyses. Understanding the fundamental branching patterns of schizophoran flies provides a foundation for future comparative research on the genetics, ecology, and biocontrol.

Global population genetic structure and demographic trajectories of the black soldier fly, Hermetia illucens.

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.

Longer is Not Always Better: Optimizing Barcode Length for Large-Scale Species Discovery and Identification.

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 ($>$20,000 species). All specimens in these data sets had full-length barcodes and had been sorted to species-level based on morphology. Mini-barcodes of different lengths and positions were obtained in silico from full-length barcodes using a sliding window approach (three windows: 100 bp, 200 bp, and 300 bp) and by excising nine mini-barcodes with established primers (length: 94-407 bp). We then tested whether barcode length and/or position reduces species-level congruence between morphospecies and molecular operational taxonomic units (mOTUs) that were obtained using three different species delimitation techniques (Poisson Tree Process, Automatic Barcode Gap Discovery, and Objective Clustering). Surprisingly, we find no significant differences in performance for both species- or specimen-level identification between full-length and mini-barcodes as long as they are of moderate length ($>$200 bp). Only very short mini-barcodes (<200 bp) perform poorly, especially when they are located near the 5$^\prime$ end of the Folmer region. The mean congruence between morphospecies and mOTUs was ca. 75% for 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.].

Contribution to understanding the evolution of holometaboly: transformation of internal head structures during the metamorphosis in the green lacewing Chrysopa pallens (Neuroptera: Chrysopidae).

BACKGROUND: Metamorphosis remains one of the most complicated and poorly understood processes in insects. This is particularly so for the very dynamic transformations that take place within the pupal sheath of holometabolous insects. Only few studies address these transformations especially with regard to cranial structures of those holometabolous species where the larval and adult forms have a similar diet. It thus remains unclear to what extent the internal structures undergo histolysis and rebuilding. Here, the development of the brain and skeleto-muscular system of the head of Chrysopa pallens (Rambur, 1838) is studied. This species is a predator of aphids in the larval and adult stage. RESULTS: We used micro-computed-tomography (µ-CT) to study the transformations of the larval, prepupal and pupal head within the cocoon. We first assessed the morphological differences and similarities between the stages. We then determined the point in time when the compound eyes appear and describe the re-orientation of the head capsule which transforms the prognathous larva into a hypognathous adult. The internal head muscles are distinctly more slender in larvae than adults. In addition, the adults have a significantly larger brain which is likely needed for the processing of the signals obtained by the adults vastly expanded sensory organs that are presumably needed for dispersal and mating. Our study shows that the histolysis and modification of the inner muscles and skeletal elements take place within the prepupa. The central nervous system persists throughout metamorphosis but its morphology changes significantly. CONCLUSION: Our study reveals that not only the inner structures, but also the outer morphology continues to change after the final larval moult. The adult cuticle and internal structures form gradually within the cocoon. The histolysis and rebuilding begin with the skeletal elements and is followed by changes in the central nervous system before it concludes with modifications of the musculature. This order of events is likely ancestral for Holometabola because it is also known from Hymenoptera, Diptera, Mecoptera, and Coleoptera.

The puzzling mitochondrial phylogeography of the black soldier fly (Hermetia illucens), the commercially most important insect protein species.

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.

Mimicry diversification in Papilio dardanus via a genomic inversion in the regulatory region of engrailed-invected.

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.

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

BACKGROUND: More than 80% of all animal species remain unknown to science. Most of these species live in the tropics and belong to animal taxa that combine small body size with high specimen abundance and large species richness. For such clades, using morphology for species discovery is slow because large numbers of specimens must be sorted based on detailed microscopic investigations. Fortunately, species discovery could be greatly accelerated if DNA sequences could be used for sorting specimens to species. Morphological verification of such "molecular operational taxonomic units" (mOTUs) could then be based on dissection of a small subset of specimens. However, this approach requires cost-effective and low-tech DNA barcoding techniques because well-equipped, well-funded molecular laboratories are not readily available in many biodiverse countries. RESULTS: We here document how MinION sequencing can be used for large-scale species discovery in a specimen- and species-rich taxon like the hyperdiverse fly family Phoridae (Diptera). We sequenced 7059 specimens collected in a single Malaise trap in Kibale National Park, Uganda, over the short period of 8 weeks. We discovered > 650 species which exceeds the number of phorid species currently described for the entire Afrotropical region. The barcodes were obtained using an improved low-cost MinION pipeline that increased the barcoding capacity sevenfold from 500 to 3500 barcodes per flowcell. This was achieved by adopting 1D sequencing, resequencing weak amplicons on a used flowcell, and improving demultiplexing. Comparison with Illumina data revealed that the MinION barcodes were very accurate (99.99% accuracy, 0.46% Ns) and thus yielded very similar species units (match ratio 0.991). Morphological examination of 100 mOTUs also confirmed good congruence with morphology (93% of mOTUs; > 99% of specimens) and revealed that 90% of the putative species belong to the neglected, megadiverse genus Megaselia. We demonstrate for one Megaselia species how the molecular data can guide the description of a new species (Megaselia sepsioides sp. nov.). CONCLUSIONS: We document that one field site in Africa can be home to an estimated 1000 species of phorids and speculate that the Afrotropical diversity could exceed 200,000 species. We furthermore conclude that low-cost MinION sequencers are very suitable for reliable, rapid, and large-scale species discovery in hyperdiverse taxa. MinION sequencing could quickly reveal the extent of the unknown diversity and is especially suitable for biodiverse countries with limited access to capital-intensive sequencing facilities.