Search

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

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

Understanding and slowing down biodiversity loss are critical tasks for the survival of our planet. They are difficult because much of the earth’s biodiversity is concentrated in abundant and species-rich groups of invertebrates like insects. Traditionally, bulk samples of insects had to be analyzed manually by experts using morphology. Not only did this require much taxonomic expertise, but it was also error-prone, time-consuming, and almost always involved commercially available microscopes that are too expensive for many biodiverse, tropical countries with limited science funding. An alternative to sorting bulk samples by experts using morphology is the use of DNA barcoding. However, this requires a well-equipped molecular laboratory and a different skill set. We present an alternative solution - the Entomoscope, a low-cost, open-source photomicroscope for taking high-resolution, focus-stacked images that can be used for insect classification. We describe two different versions of the Entomoscope, a standalone version that can be operated without additional hardware and an even simpler version, that is operated via a computer. We show that the optics are of sufficiently high quality to classify specimens with >95% accuracy into 15 different types of insects (mostly ’families’ according to the Linnaean classification). The classifier can be successively extended or individually trained for specific classification tasks. We provide the building instructions, 3D files, and a list of commercially available parts so that everyone can build their own Entomoscope. We predict that open-source DIY hardware like the Entomoscope will facilitate cutting-edge biodiversity research by entomologists around the planet.

Abstract Understanding global patterns of genetic diversity is essential for describing, monitoring, and preserving life on Earth. To date, efforts to map macrogenetic patterns have been restricted to vertebrates, which comprise only a small fraction of Earth’s biodiversity. Here, we construct a global map of predicted insect mitochondrial genetic diversity from cytochrome c oxidase subunit 1 sequences, derived from open data. We calculate the mitochondrial genetic diversity mean and genetic diversity evenness of insect assemblages across the globe, identify their environmental correlates, and make predictions of mitochondrial genetic diversity levels in unsampled areas based on environmental data. Using a large single-locus genetic dataset of over 2 million globally distributed and georeferenced mtDNA sequences, we find that mitochondrial genetic diversity evenness follows a quadratic latitudinal gradient peaking in the subtropics. Both mitochondrial genetic diversity mean and evenness positively correlate with seasonally hot temperatures, as well as climate stability since the last glacial maximum. Our models explain 27.9% and 24.0% of the observed variation in mitochondrial genetic diversity mean and evenness in insects, respectively, making an important step towards understanding global biodiversity patterns in the most diverse animal taxon.

Abstract Bioassessment of freshwater quality with eDNA is a powerful alternative to traditional methods involving collecting, sorting, and identifying metazoan taxa with morphology (e.g., macroinvertebrates). Particularly attractive for routine monitoring would be an eDNA method that uses a remote‐controlled boat for collecting small volumes of water without filtration in order to simplify sampling. Such a method would not likely capture eDNA signals for all metazoan species, but may nevertheless allow for cost‐effective, and frequent monitoring of water quality, which is important for tropical waterbodies that require year‐round surveillance. We here optimize a molecular protocol for capturing metazoan signatures based on eDNA obtained from 15 ml of water. To test the robustness of the method, we used samples from two tropical reservoirs with known differences in water quality to optimize molecular procedures so that they yield repeatable results. Each reservoir was sampled at three sites (“biological replicates”) and each water sample was subsampled twice before extracting the eDNA using ethanol precipitation for both subsamples (“technical replicates”). We then tested how much DNA (0.1 ng to 15 ng) and how many PCR cycles (25 or 35) minimized the variance of the metazoan eDNA signal between biological and technical replicates. For this purpose, we amplified a 313 bp COI minibarcode using a universal metazoan primer pair. We found that regardless of template amounts or PCR cycle numbers, the eDNA signatures for both reservoirs were distinct because only 17 of 59 mOTUs (mainly planktonic crustaceans and rotifers) were shared. We also found that template amounts between 0.5 and 15 ng yielded overall similar results, but the use of 35 PCR cycles significantly depressed the number of detected species (p‐value

Abstract 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.

Abstract 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.

Abstract 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 10,000 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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

The schizophoran superfamily Ephydroidea (Diptera: Cyclorrhapha) includes eight families, ranging from the well-known vinegar flies (Drosophilidae) and shore flies (Ephydridae), to several small, relatively unusual groups, the phylogenetic placement of which has been particularly challenging for systematists. An extraordinary diversity in life histories, feeding habits and morphology are a hallmark of fly biology, and the Ephydroidea are no exception. Extreme specialization can lead to “orphaned” taxa with no clear evidence for their phylogenetic position. To resolve relationships among a diverse sample of Ephydroidea, including the highly modified flies in the families Braulidae and Mormotomyiidae, we conducted phylogenomic sampling. Using exon capture from Anchored Hybrid Enrichment and transcriptomics to obtain 320 orthologous nuclear genes sampled for 32 species of Ephydroidea and 11 outgroups, we evaluate a new phylogenetic hypothesis for representatives of the superfamily. These data strongly support monophyly of Ephydroidea with Ephydridae as an early branching radiation and the placement of Mormotomyiidae as a family-level lineage sister to all remaining families. We confirm placement of Cryptochetidae as sister taxon to a large clade containing both Drosophilidae and Braulidae–the latter a family of honeybee ectoparasites. Our results reaffirm that sampling of both taxa and characters is critical in hyperdiverse clades and that these factors have a major influence on phylogenomic reconstruction of the history of the schizophoran fly radiation.

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.

Abstract 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.

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. : Genetics; Molecular Biology; Evolutionary Biology; Developmental Biology Subject Areas: Genetics, Molecular Biology, Evolutionary Biology, Developmental Biology

Despite more than 250 years of taxonomic research, we still have only a vague idea about the true size and composition of the faunas and floras of the planet. Many biodiversity inventories provide limited insight because they focus on a small taxonomic subsample or a tiny geographic area. Here, we report on the size and composition of the Swedish insect fauna, thought to represent roughly half of the diversity of multicellular life in one of the largest European countries. Our results are based on more than a decade of data from the Swedish Taxonomy Initiative and its massive inventory of the country's insect fauna, the Swedish Malaise Trap Project The fauna is considered one of the best known in the world, but the initiative has nevertheless revealed a surprising amount of hidden diversity: more than 3,000 new species (301 new to science) have been documented so far. Here, we use three independent methods to analyze the true size and composition of the fauna at the family or subfamily level: (1) assessments by experts who have been working on the most poorly known groups in the fauna; (2) estimates based on the proportion of new species discovered in the Malaise trap inventory; and (3) extrapolations based on species abundance and incidence data from the inventory. For the last method, we develop a new estimator, the combined non-parametric estimator, which we show is less sensitive to poor coverage of the species pool than other popular estimators. The three methods converge on similar estimates of the size and composition of the fauna, suggesting that it comprises around 33,000 species. Of those, 8,600 (26%) were unknown at the start of the inventory and 5,000 (15%) still await discovery. We analyze the taxonomic and ecological composition of the estimated fauna, and show that most of the new species belong to Hymenoptera and Diptera groups that are decomposers or parasitoids. Thus, current knowledge of the Swedish insect fauna is strongly biased taxonomically and ecologically, and we show that similar but even stronger biases have distorted our understanding of the fauna in the past. We analyze latitudinal gradients in the size and composition of known European insect faunas and show that several of the patterns contradict the Swedish data, presumably due to similar knowledge biases. Addressing these biases is critical in understanding insect biomes and the ecosystem services they provide. Our results emphasize the need to broaden the taxonomic scope of current insect monitoring efforts, a task that is all the more urgent as recent studies indicate a possible worldwide decline in insect faunas.

Abstract 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.

Abstract Background Macroinvertebrates such as non-biting midges (Chironomidae: Diptera) are important components of freshwater ecosystems. However, they are often neglected in biodiversity and conservation research because invertebrate species richness is difficult and expensive to quantify with traditional methods. We here demonstrate that Next Generation Sequencing barcodes (“NGS barcodes”) can provide relief because they allow for fast and large-scale species-level sorting of large samples at low cost. Results We used NGS barcoding to investigate the midge fauna of Singapore’s swamp forest remnant (Nee Soon Swamp Forest). Based on > 14.000 barcoded specimens, we find that the swamp forest maintains an exceptionally rich fauna composed of an observed number of 289 species (estimated 336 species) in a very small area (90 ha). We furthermore barcoded the chironomids from three surrounding reservoirs that are located in close proximity. Although the swamp forest remnant is much smaller than the combined size of the freshwater reservoirs in the study (90 ha vs. > 450 ha), the latter only contains 33 (estimated 61) species. We show that the resistance of the swamp forest species assemblage is high because only 8 of the 314 species are shared despite the close proximity. Moreover, shared species are not very abundant (3% of all specimens). A redundancy analysis revealed that ~ 21% of the compositional variance of midge communities within the swamp forest was explained by a range of variables with conductivity, stream order, stream width, temperature, latitude (flow direction), and year being significant factors influencing community structure. An LME analysis demonstrates that the total species richness decreased with increasing conductivity. Conclusion Our study demonstrates that midge diversity of a swamp forest can be so high that it questions global species diversity estimates for Chironomidae, which are an important component of many freshwater ecosystems. We furthermore demonstrate that small and natural habitat remnants can have high species turnover and can be very resistant to the invasion of species from neighboring reservoirs. Lastly, the study shows how NGS barcodes can be used to integrate specimen- and species-rich invertebrate taxa in biodiversity and conservation research.

Acletoxenus is a small genus of Drosophilidae with only four described species that are closely associated with whiteflies (adults and larvae). Here, the first video recordings of larvae feeding on whiteflies (Aleurotrachelus trachoides) are presented. Typical morphological adaptations for predation by schizophoran larvae are also described: the larval pseudocephalon lacks a facial mask and the cephaloskeleton is devoid of cibarial ridges that could be used for saprophagy via filtration. Despite being a predator, Acletoxenus is unlikely to be a good candidate for biological control of whiteflies because the life cycle is fairly long (24 days), lab cultures could not be established, and the puparia have high parasitization rates by a pteromalid wasp (Pachyneuron leucopiscida). Unfortunately, a confident identification of the Singapore Acletoxenus population to species was not possible because species identification and description in the genus overemphasize coloration characters of the mesonotum which are shown to be unsuitable because the Singapore population has flies with coloration patterns matching three of the four described species. Based on morphology and DNA sequences, the population from Singapore is tentatively assigned to Acletoxenus indicus or a closely related species.

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.

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.

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’ population connectivity nor favour asexual reproduction.

Specimens of the enigmatic, monotypic European genus Zuskamira Pont, 1987 (Sepsidae) were initially collected only from the lower central Swedish provinces of Darlana, Uppland and Västmanland. However, the same species was subsequently found much more south in Lower-Saxony and Schleswig-Holstein although Germany is overall well sampled for sepsid flies. Here we report a further (longitudinal) range expansion based on new localities in Southern Finland. New localities for Finland and Sweden are here added and we discuss briefly the habitat requirements of the species.

Fauna Europaea provides a public web-service with an index of scientific names (including important synonyms) of all extant multicellular European terrestrial and freshwater animals and their geographical distribution at the level of countries and major islands (east of the Urals and excluding the Caucasus region). The Fauna Europaea project comprises about 230,000 taxonomic names, including 130,000 accepted species and 14,000 accepted subspecies, which is much more than the originally projected number of 100,000 species. Fauna Europaea represents a huge effort by more than 400 contributing taxonomic specialists throughout Europe and is a unique (standard) reference suitable for many user communities in science, government, industry, nature conservation and education. The Diptera–Brachycera is one of the 58 Fauna Europaea major taxonomic groups, and data have been compiled by a network of 55 specialists. Within the two-winged insects (Diptera), the Brachycera constitute a monophyletic group, which is generally given rank of suborder. The Brachycera may be classified into the probably paraphyletic 'lower brachyceran grade' and the monophyletic Eremoneura. The latter contains the Empidoidea, the Apystomyioidea with a single Nearctic species, and the Cyclorrhapha, which in turn is divided into the paraphyletic 'aschizan grade' and the monophyletic Schizophora. The latter is traditionally divided into the paraphyletic 'acalyptrate grade' and the monophyletic Calyptratae. Our knowledge of the European fauna of Diptera–Brachycera varies tremendously among families, from the reasonably well known hoverflies (Syrphidae) to the extremely poorly known scuttle flies (Phoridae). There has been a steady growth in our knowledge of European Diptera for the last two centuries, with no apparent slow down, but there is a shift towards a larger fraction of the new species being found among the families of the nematoceran grade (lower Diptera), which due to a larger number of small-sized species may be considered as taxonomically more challenging. Most of Europe is highly industrialised and has a high human population density, and the more fertile habitats are extensively cultivated. This has undoubtedly increased the extinction risk for numerous species of brachyceran flies, yet with the recent re-discovery of Thyreophora cynophila (Panzer), there are no known cases of extinction at a European level. However, few national Red Lists have extensive information on Diptera. For the Diptera–Brachycera, data from 96 families containing 11,751 species are included in this paper.

Many species descriptions, especially older ones, consist mostly of text and have few illustrations. Only the most conspicuous morphological features needed for species diagnosis and delimitation at the time of description are illustrated. Such descriptions can quickly become inadequate when new species or characters are discovered. We propose that descriptions should become more data-rich by presenting a large amount of images and illustrations to cover as much morphology as possible; these descriptions are more likely to remain adequate over time because their large amounts of visual data could capture character systems that may become important in the future. Such an approach can now be quickly and easily achieved given that high-quality digital photography is readily available. Here, we re-describe the sepsid fly Perochaeta orientalis (de Meijere 1913) (Diptera, Sepsidae) which has suffered from inadequate descriptions in the past, and use photomicrography, scanning electron microscopy and videography to document its external morphology and mating behaviour. All images and videos are embedded within the electronic publication. We discuss briefly benefits and problems with our approach.

Freshwater habitats are of high conservation value and provide a wide range of ecosystem services. Effective management requires regular monitoring. However, conventional methods based on direct observation or specimen collection are so invasive, expensive and labour-intensive that frequent monitoring is uncommon. Here, we test whether the evaluation of environmental DNA (eDNA) from water based on a simple protocol can be used for assessing biodiversity. We use universal metazoan primers for characterizing water eDNA across horizontal and vertical spatial dimensions in two reservoirs with known species diversity for two key taxa. eDNA obtained directly from 42 samples × 15 ml water (total = 630 ml) per reservoir yielded DNA signatures for more than 500 metazoan species, of which 105 could be identified to species/genus based on DNA barcodes. We show that eDNA can be used to assign each water sample to its reservoir of origin, and that eDNA outperforms conventional survey methods in single-sample richness comparisons, while revealing evidence for hundreds of unknown species that are undetected by conventional bioassessment methods. eDNA also confirms the presence of a recently discovered invasive snail species and provides evidence for the continued survival of a rare native species of goby not sighted in that habitat since 2007. eDNA thus promises to be a useful addition to the bioassessment toolbox for freshwater systems.

The ornamental pet trade is often considered a key culprit for conservation problems such as the introduction of invasive species (including infectious diseases) and overharvesting of rare species. Here, we present the first assessment of the biodiversity of freshwater molluscs in the ornamental pet trade in Singapore, one of the most important global hubs of the ornamental aquarium trade, and discuss associated conservation concerns. We recorded freshwater molluscs from ornamental pet shops and major exporters including non-ornamental species (e.g., hitchhikers, molluscs sold as fish feed). We recorded an unexpectedly high diversity-59 species-of freshwater bivalves and gastropods, with the majority (38 species or 64%) being from the Oriental region. In addition to morphological examination, we sequenced the DNA barcode region of mitochondrial CO1 and 16S genes to provide molecular data for the confirmation of the identification and for future re-identification. DNA barcodes were obtained for 50 species, and all but four were separated by > 3% uncorrected pairwise distances. The trade has been considered a main introduction pathway for non-native species to Singapore, and we found that out of 15 species in the trade as well as in the wild in Singapore, 12 are either introduced or of unknown origin, representing almost half of the known non-native freshwater molluscs in Singapore. Particularly prevalent are non-ornamental species: six hitchhikers on aquarium plants and six species sold as fish feed. We found that a quarter of the trade species have a history of introduction, which includes 11 known or potentially invasive species. We conclude that potential overharvesting is difficult to assess because only half of the trade species have been treated by IUCN. Of these, 21 species are of Least Concern and three are Data Deficient. Our checklist, with accompanying DNA barcodes, images, and museum vouchers, provides an important reference library for future monitoring, and constitutes a step toward creating a more sustainable ornamental pet trade.

A recent collecting trip to Vietnam yielded three new species and two new records of Sepsidae (Diptera) for the country. Here we describe two new species in the species-poor genus Perochaeta (Perochaeta cuirassa sp. n. and P. lobo sp. n.) and one to the largest sepsid genus Sepsis (Sepsis spura sp. n.) which is also found in Sumatra and Sulawesi. Two additional Sepsis species are new records for Vietnam (Sepsis sepsi Ozerov, 2003; Sepsis monostigma Thompson, 1869). We conclude with a discussion of the distribution of Perochaeta and the three Sepsis species.

BackgroundPoorly regulated international trade in ornamental fishes poses risks to both biodiversity and economic activity via invasive alien species and exotic pathogens. Border security officials need robust tools to confirm identifications, often requiring hard-to-obtain taxonomic literature and expertise. DNA barcoding offers a potentially attractive tool for quarantine inspection, but has yet to be scrutinised for aquarium fishes. Here, we present a barcoding approach for ornamental cyprinid fishes by: (1) expanding current barcode reference libraries; (2) assessing barcode congruence with morphological identifications under numerous scenarios (e.g. inclusion of GenBank data, presence of singleton species, choice of analytical method); and (3) providing supplementary information to identify difficult species.Methodology/principal findingsWe sampled 172 ornamental cyprinid fish species from the international trade, and provide data for 91 species currently unrepresented in reference libraries (GenBank/Bold). DNA barcodes were found to be highly congruent with our morphological assignments, achieving success rates of 90-99%, depending on the method used (neighbour-joining monophyly, bootstrap, nearest neighbour, GMYC, percent threshold). Inclusion of data from GenBank (additional 157 spp.) resulted in a more comprehensive library, but at a cost to success rate due to the increased number of singleton species. In addition to DNA barcodes, our study also provides supporting data in the form of specimen images, morphological characters, taxonomic bibliography, preserved vouchers, and nuclear rhodopsin sequences. Using this nuclear rhodopsin data we also uncovered evidence of interspecific hybridisation, and highlighted unrecognised diversity within popular aquarium species, including the endangered Indian barb Puntius denisonii.Conclusions/significanceWe demonstrate that DNA barcoding provides a highly effective biosecurity tool for rapidly identifying ornamental fishes. In cases where DNA barcodes are unable to offer an identification, we improve on previous studies by consolidating supplementary information from multiple data sources, and empower biosecurity agencies to confidently identify high-risk fishes in the aquarium trade.

The circadian clock is a core molecular mechanism that allows organisms to anticipate daily environmental changes and adapt the timing of behaviors to maximize efficiency. In social insects, the ability to maintain the appropriate temporal order is thought to improve colony efficiency and fitness. We used the newly sequenced fire ant (Solenopsis invicta) genome to characterize the first ant circadian clock. Our results reveal that the fire ant clock is similar to the clock of the honeybee, a social insect with an independent evolutionary origin of sociality. Gene trees for the eight core clock genes, period, cycle, clock, cryptochrome-m, timeout, vrille, par domain protein 1 & clockwork orange, show ant species grouping closely with honeybees and Nasonia wasps as an outgroup to the social Hymenoptera. Expression patterns for these genes suggest that the ant clock functions similar to the honeybee clock, with period and cry-m mRNA levels increasing during the night and cycle and clockwork orange mRNAs cycling approximately anti-phase to period. Gene models for five of these genes also parallel honeybee models. In particular, the single ant cryptochrome is an ortholog of the mammalian-type (cry-m), rather than Drosophila-like protein (cry-d). Additionally, we find a conserved VPIFAL C-tail region in clockwork orange shared by insects but absent in vertebrates. Overall, our characterization of the ant clock demonstrates that two social insect lineages, ants and bees, share a similar, mammalian-like circadian clock. This study represents the first characterization of clock genes in an ant and is a key step towards understanding socially-regulated plasticity in circadian rhythms by facilitating comparative studies on the organization of circadian clockwork.

The gene expression pattern specified by an animal regulatory sequence is generally viewed as arising from the particular arrangement of transcription factor binding sites it contains. However, we demonstrate here that regulatory sequences whose binding sites have been almost completely rearranged can still produce identical outputs. We sequenced the even-skipped locus from six species of scavenger flies (Sepsidae) that are highly diverged from the model species Drosophila melanogaster, but share its basic patterns of developmental gene expression. Although there is little sequence similarity between the sepsid eve enhancers and their well-characterized D. melanogaster counterparts, the sepsid and Drosophila enhancers drive nearly identical expression patterns in transgenic D. melanogaster embryos. We conclude that the molecular machinery that connects regulatory sequences to the transcription apparatus is more flexible than previously appreciated. In exploring this diverse collection of sequences to identify the shared features that account for their similar functions, we found a small number of short (20-30 bp) sequences nearly perfectly conserved among the species. These highly conserved sequences are strongly enriched for pairs of overlapping or adjacent binding sites. Together, these observations suggest that the local arrangement of binding sites relative to each other is more important than their overall arrangement into larger units of cis-regulatory function.