Search

Large-scale digitization of natural history collections requires automation of image acquisition and processing. Reflecting this fact, various approaches, some highly sophisticated, have been developed to support imaging of museum specimens. However, most of these systems are complex and expensive, restricting their deployment. Here we describe a simple, inexpensive technique for imaging arthropods larger than 5 mm. By mounting a digital SLR camera on a CNC (computer numerical control) motor-drive rig, we created a system that captures high-resolution z-axis stacked images (6960 × 4640 pixels) of 95 specimens in 30 minutes. This system can be assembled inexpensively ($1000 USD without a camera) and it is easy to set-up and maintain. By coupling low cost with high production capacity, it represents a solution for digitizing any natural history collection.

Abstract Novel methods for sampling and characterizing biodiversity hold great promise for re-evaluating patterns of life across the planet. The sampling of airborne spores with a cyclone sampler, and the sequencing of their DNA, have been suggested as an efficient and well-calibrated tool for surveying fungal diversity across various environments. Here we present data originating from the Global Spore Sampling Project, comprising 2,768 samples collected during two years at 47 outdoor locations across the world. Each sample represents fungal DNA extracted from 24 m3 of air. We applied a conservative bioinformatics pipeline that filtered out sequences that did not show strong evidence of representing a fungal species. The pipeline yielded 27,954 species-level operational taxonomic units (OTUs). Each OTU is accompanied by a probabilistic taxonomic classification, validated through comparison with expert evaluations. To examine the potential of the data for ecological analyses, we partitioned the variation in species distributions into spatial and seasonal components, showing a strong effect of the annual mean temperature on community composition.

Abstract Global biodiversity gradients are generally expected to reflect greater species replacement closer to the equator. However, empirical validation of global biodiversity gradients largely relies on vertebrates, plants, and other less diverse taxa. Here we assess the temporal and spatial dynamics of global arthropod biodiversity dynamics using a beta-diversity framework. Sampling includes 129 sampling sites whereby malaise traps are deployed to monitor temporal changes in arthropod communities. Overall, we encountered more than 150,000 unique barcode index numbers (BINs) (i.e. species proxies). We assess between site differences in community diversity using beta-diversity and the partitioned components of species replacement and richness difference. Global total beta-diversity (dissimilarity) increases with decreasing latitude, greater spatial distance and greater temporal distance. Species replacement and richness difference patterns vary across biogeographic regions. Our findings support long-standing, general expectations of global biodiversity patterns. However, we also show that the underlying processes driving patterns may be regionally linked.

Olixon testaceum is a widely distributed species of brachypterous parasitoid wasp (Vespoidea: Rhopalosomatidae) occurring in Meso- and South America, but little is known of its biology. Here, the first known host of O. ? testaceum is identified as the cricket Anaxipha sp. (Grylloidea: Trigonidiidae) through DNA barcoding of six Olixon larvae and their hosts. Barcoding results also indicated substantial genetic diversity within nominal O. testaceum specimens. The number of species and statistical significance of these groups were tested using Maximum Likelihood phylogenies, distance-based cluster analyses, and coalescence models. All analyses revealed at least six distinct lineages, which suggests six or more cryptic species within O. ? testaceum. Combined with what is currently known about Rhopalosoma host use, these results indicate that rhopalosomatids may be generalist rather than specialist parasitoids, and further confirm the benefits of open global collaboration and DNA barcoding in advancing taxonomic knowledge.

Two new genera and one new species of the Braconinae tribe Adeshini are described and illustrated: Crenuladesha Ranjith & Quicke, gen. nov., type species Adesha narendrani Ranjith, 2017, comb. nov. from India, and Protadesha Quicke & Butcher, gen. nov., type species Protadesha intermedia Quicke & Butcher, sp. nov. from South Africa. The former lacks the mid-longitudinal propodeal carina characteristic of the tribe, and the latter displays less derived fore wing venation with two distinct abscissae of vein 2CU. A molecular phylogenetic analysis is included to confirm their correct placement. Since neither of the two new genera displays all of the characters given in the original diagnosis of the Adeshini a revised diagnosis is provided, as well as an illustrated key to the genera.

Introduced species provide the opportunity to study evolutionary change on short time scales—a key first step to understand and manage the ecosystem-level impact of invasions. This study examined mitochondrial DNA sequence variation in cytochrome c oxidase subunit 1 (COI) for 26 insect species—Coleoptera (9), Hymenoptera (9), and Lepidoptera (8)—introduced to the Nearctic from the Palearctic. A total of 6,302 barcode records were retrieved from BOLD (boldsystems.org) to compare sequence diversity between the native and introduced range. As expected, genetic variation averaged nearly an order of magnitude lower in introduced populations (2.19 × 10−5 substitutions per nucleotide) than in the native range (1.48 ×10−4 substitutions per nucleotide). Nonsynonymous and synonymous changes had a similar incidence in the introduced populations (p-value = 0.83, averaging respectively 1.08 × 10−5 and 1.11 × 10−5 substitutions). By contrast, nonsynonymous changes were ten-fold less frequent than synonymous changes in the native populations (p-value < 0.001, averaging 1.74 × 10−5 and 1.3 × 10−4 substitutions, respectively). Patterns of sequence variation in the introduced range were largely congruent across the three insect orders which suggests that they are produced by general processes. This study explores the molecular evolution of introduced species, a fundamental aspect to improve understanding of their biology and manage their impact on ecosystems.

Abstract The Ponto-Caspian region is an endemicity hotspot that harbours several crustacean radiations, among which amphipods are the most diverse. These poorly known species are severely threatened in their native range, while at the same time they are invading European inland waters with significant ecological consequences. A proper taxonomic knowledge of this fauna is paramount for its conservation within the native region and monitoring outside of it. Here, we assemble a DNA barcode reference library for nearly 60% of all known Ponto-Caspian amphipod species. We use several methods to define molecular operational taxonomic units (MOTUs), based on two mitochondrial markers (COI and 16S), and assess their congruence with current species-level taxonomy based on morphology. Depending on the method, we find that 54–69% of species had congruent morpho-molecular boundaries. The cases of incongruence resulted from lumping distinct morphospecies into a single MOTU (7–27%), splitting a morphospecies into several MOTUs (4–28%), or both (4–11%). MOTUs defined by distance-based methods without a priori divergence thresholds showed the highest congruence with morphological taxonomy. These results indicate that DNA barcoding is valuable for clarifying the diversity of Ponto-Caspian amphipods, but reveals that extensive work is needed to resolve taxonomic uncertainties. Our study advances the DNA barcode reference library for the European aquatic biota, paving the way towards improved taxonomic knowledge needed to enhance monitoring and conservation efforts.

Abstract Background Aquatic ecosystems provide breeding sites for blood-sucking insects such as Culicoides biting midges (Diptera: Ceratopogonidae), but factors affecting their distribution and host choice are poorly understood. A study was undertaken at two nature reserves in northern Spain to examine the abundance, species composition, population dynamics and feeding patterns of biting midges between 2018 and 2019. Methods Culicoides were captured by light suction traps baited with CO2 and by sweep netting vegetation. Blood meals and species identification of blood-fed specimens were determined using cytochrome c oxidase I subunit (COI) DNA barcoding. Multivariate generalized linear models were used to evaluate the associations between the abundance of Culicoides, the species richness and other parameters. Results The 4973 identified specimens comprised 28 species of Culicoides. These included two species reported for the first time in northern Spain, thus raising to 54 the number of Culicoides species described in the region. Specimens of all 28 species and 99.6% of the total specimens collected were caught in suction traps, while sweep netting vegetation revealed just 11 species and 0.4% of the total specimens. Midge abundance peaked in June/early July, with five species comprising > 80% of the captures: Culicoides alazanicus (24.9%), Culicoides griseidorsum (20.3%), Culicoides poperinghensis (16.2%), Culicoides kibunensis (10.7%) and Culicoides clastrieri (9.6%). DNA barcode analysis of blood meals from eight Culicoides species revealed that they fed on 17 vertebrate species (3 mammals and 14 birds). Species in the subgenus Avaritia were primarily ornithophilic, except for C. griseidorsum and C. poperinghensis. Host DNA from blood meals was successfully amplified from 75% of blood-fed females. A pictorial blood meal digestion scale is provided to accurately assess the blood-fed status of female Culicoides. Conclusions The large number of different blood meal sources identified in the midges captured in this study signals the likely importance of wild birds and mammals (e.g. red deer and wild boar) as reservoir/amplifying hosts for pathogens. Available hosts are more exposed to being bitten by biting midge populations in aquatic ecosystems in late spring and early summer.

The Neotropical members formerly included in Earinus Wesmael, 1837 are transferred to a new genus, Chilearinus Sharkey gen. nov. Presently three Nearctic species of Earinus are recognized, i.e., Earinus erythropoda Cameron, 1887, Earinus limitaris Say,1835, and Earinus zeirapherae Walley, 1935, and these are retained in Earinus. Earinus chubuquensis Berta, 2000 and Earinus scitus Enderlein, 1920 are transferred to Chilearinus, i.e., C. chubuquensis, and C. scitus, comb. nov. One other species is transferred to Chilearinus, i.e., Microgaster rubricollis Spinola, 1851, Chilearinus rubricollis, comb. nov. Two other Neotropical species, Earinus hubrechtae Braet, 2002 and Earinus bourguignoni Braet, 2002 were described under the genus Earinus but are here transferred to Lytopylus, L. hubrechtae, and L. bourguignoni comb. nov. Two new species of Chilearinus are described, C. covidchronos and C. janbert spp. nov. The status of Agathis laevithorax Spinola,1851, Agathis rubricata Spinola,1851, and Agathis areolata Spinola, 1851 is discussed. A neotype is designated for Earinus limitaris (Say, 1835) and diagnosed with a COI barcode. Earinus austinbakeri and Earinus walleyi spp. nov. are described. The status of both Earinus and Chilearinus in the Americas is discussed. A revised key to the genera of Agathidinae of the Americas is presented.

We report the results of DNA barcoding week-long Malaise trap catches from 11 sites in three Thai conservation areas, concentrating on the parasitoid Hymenoptera, particularly the superfamily Ichneumonoidea. From a total of 15,338 parasitoid wasp sequences, 13,473 were barcode compliant and could be assigned to a family based on morphology and sequence data. These collectively represented 4917 unique BINs (putative species) in 46 families, with the Scelionidae, Ichneumonidae, Eulophidae, Braconidae and Platygastridae being, by far, the most abundant. Spatial proximity had a strong positive effect on the numbers of BINs shared between traps.

The nuclear genomes of most animal species include NUMTs, segments of the mitogenome incorporated into their chromosomes. Although NUMT counts are known to vary greatly among species, there has been no comprehensive study of their frequency/attributes in the most diverse group of terrestrial organisms, insects. This study examines NUMTs derived from a 658 bp 5’ segment of the cytochrome c oxidase I (COI) gene, the barcode region for the animal kingdom. This assessment is important because unrecognized NUMTs can elevate estimates of species richness obtained through DNA barcoding and derived approaches (eDNA, metabarcoding). This investigation detected nearly 10,000 COI NUMTs ≥ 100 bp in the genomes of 1,002 insect species (range = 0–443). Variation in nuclear genome size explained 56% of the mitogenome-wide variation in NUMT counts. Although insect orders with the largest genome sizes possessed the highest NUMT counts, there was considerable variation among their component lineages. Two thirds of COI NUMTs possessed an IPSC (indel and/or premature stop codon) allowing their recognition and exclusion from downstream analyses. The remainder can elevate species richness as they showed 10.1% mean divergence from their mitochondrial homologue. The extent of exposure to “ghost species” is strongly impacted by the target amplicon’s length. NUMTs can raise apparent species richness by up to 22% when a 658 bp COI amplicon is examined versus a doubling of apparent richness when 150 bp amplicons are targeted. Given these impacts, metabarcoding and eDNA studies should target the longest possible amplicons while also avoiding use of 12S/16S rDNA as they triple NUMT exposure because IPSC screens cannot be employed.

Abstract Although mites (Acari) are abundant in many terrestrial and freshwater ecosystems, their diversity is poorly understood. Since most mite species can be distinguished by variation in the DNA barcode region of cytochrome c oxidase I, the Barcode Index Number (BIN) system provides a reliable species proxy that facilitates large-scale surveys. Such analysis reveals many new BINs that can only be identified as Acari until they are examined by a taxonomic specialist. This study demonstrates that the Barcode of Life Datasystem’s identification engine (BOLD ID) generally delivers correct ordinal and family assignments from both full-length DNA barcodes and their truncated versions gathered in metabarcoding studies. This result was demonstrated by examining BOLD ID’s capacity to assign 7021 mite BINs to their correct order (4) and family (189). Identification success improved with sequence length and taxon coverage but varied among orders indicating the need for lineage-specific thresholds. A strict sequence similarity threshold (86.6%) prevented all ordinal misassignments and allowed the identification of 78.6% of the 7021 BINs. However, higher thresholds were required to eliminate family misassignments for Sarcoptiformes (89.9%), and Trombidiformes (91.4%), consequently reducing the proportion of BINs identified to 68.6%. Lineages with low barcode coverage in the reference library should be prioritized for barcode library expansion to improve assignment success.

Vlad Dincă, Leonardo Dapporto, and colleagues provide a continental-scale DNA barcode library for 97% of the 473 European butterfly species and examine patterns of haplotype diversity. Results of mitochondrial diversity show elevated haplotype richness in southern European refugia, and this dataset provides a valuable resource for specimen identification, conservation, and research.

Although Pakistan has rich biodiversity, many groups are poorly known, particularly insects. To address this gap, we employed DNA barcoding to survey its insect diversity. Specimens obtained through diverse collecting methods at 1,858 sites across Pakistan from 2010–2019 were examined for sequence variation in the 658 bp barcode region of the cytochrome c oxidase 1 (COI) gene. Sequences from nearly 49,000 specimens were assigned to 6,590 Barcode Index Numbers (BINs), a proxy for species, and most (88%) also possessed a representative image on the Barcode of Life Data System (BOLD). By coupling morphological inspections with barcode matches on BOLD, every BIN was assigned to an order (19) and most (99.8%) were placed to a family (362). However, just 40% of the BINs were assigned to a genus (1,375) and 21% to a species (1,364). Five orders (Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera) accounted for 92% of the specimens and BINs. More than half of the BINs (59%) are so far only known from Pakistan, but others have also been reported from Bangladesh (13%), India (12%), and China (8%). Representing the first DNA barcode survey of the insect fauna in any South Asian country, this study provides the foundation for a complete inventory of the insect fauna in Pakistan while also contributing to the global DNA barcode reference library.

DNA metabarcoding has the potential to greatly advance understanding of soil biodiversity, but this approach has seen limited application for the most abundant and species-rich group of soil fauna–the arthropods. This study begins to address this gap by comparing information on species composition recovered from metabarcoding two types of bulk samples (specimens, soil) from a temperate zone site and from bulk soil samples collected at eight sites in the Arctic. Analysis of 22 samples (3 specimen, 19 soil) revealed 410 arthropod OTUs belonging to 112 families, 25 orders, and nine classes. Studies at the temperate zone site revealed little overlap in species composition between soil and specimen samples, but more overlap at higher taxonomic levels (families, orders) and congruent patterns of α- and β-diversity. Expansion of soil analyses to the Arctic revealed locally rich, highly dissimilar, and spatially structured assemblages compatible with dispersal limited and environmentally driven assembly. The current study demonstrates that DNA metabarcoding of bulk soil enables rapid, large-scale assessments of soil arthropod diversity. However, deep sequence coverage is required to adequately capture the species present in these samples, and expansion of the DNA barcode reference library is necessary to improve taxonomic resolution of the sequences recovered through this approach.

For the first time, a nearly complete barcode library for European Gelechiidae is provided. DNA barcode sequences (COI gene – cytochrome c oxidase 1) from 751 out of 865 nominal species, belonging to 105 genera, were successfully recovered. A total of 741 species represented by specimens with sequences ≥ 500bp and an additional ten species represented by specimens with shorter sequences were used to produce 53 NJ trees. Intraspecific barcode divergence averaged only 0.54% whereas distance to the Nearest-Neighbour species averaged 5.58%. Of these, 710 species possessed unique DNA barcodes, but 31 species could not be reliably discriminated because of barcode sharing or partial barcode overlap. Species discrimination based on the Barcode Index System (BIN) was successful for 668 out of 723 species which clustered from minimum one to maximum 22 unique BINs. Fifty-five species shared a BIN with up to four species and identification from DNA barcode data is uncertain. Finally, 65 clusters with a unique BIN remained unidentified to species level. These putative taxa, as well as 114 nominal species with more than one BIN, suggest the presence of considerable cryptic diversity, cases which should be examined in future revisionary studies.

The present study provides a DNA barcode library for the world Zygaenidae (Lepidoptera). This study reports 1031 sequence data of the COI gene DNA barcodes for more than 240 species in four of the five subfamilies of the family Zygaenidae. This is about 20% of the world Zygaenidae species. Our results demonstrate the specificity of the COI gene sequences at the species level in most of the studied Zygaenidae and agree with already established taxonomic opinions. The study confirms the effectiveness of DNA barcoding as a tool for determination of most Zygaenidae species. However, some of the results are contradictory. Some cases of shared barcodes have been found, as well as cases of deep intraspecific sequence divergence in species that are well separated by morphological and biological characters. These cases are discussed in detail. Overall, when combined with morphological and biochemical data, as well as biological and ecological observations, DNA barcoding results can be a useful support for taxonomic decisions.

Here we elucidate and justify a DNA barcode approach to insect species description that can be applied to name tens of thousands of species of Ichneumonoidea and many other species-rich taxa. Each description consists of a lateral habitus image of the specimen, a COI barcode diagnosis, and the holotype specimen information required by the International Code of Zoological Nomenclature. We believe this approach, or a slight modification of it, will be useful for many other underdescribed hyperdiverse taxa, especially in the tropics. Due to the extreme species-richness of the Ichneumonoidea, the very low percentage of described species, and the lack of detailed biological information for most described species, the standard taxonomic approach is inefficient and overwhelmingly time consuming. A DNA barcode-based approach to initial description will provide a solid foundation of species hypotheses from which more comprehensive descriptions can be developed as other data, time, and budgets permit. Here we elucidate this view and detailed methodology that can generally be applied to species-rich underdescribed taxa. A real example is given by describing species in two genera, Hemichoma and Zelomorpha, reared from the Área de Conservación Guanacaste in northwestern Costa Rica. The generic type species Zelomorpha arizonensis is given a DNA barcode diagnosis and the following new species are described: Zelomorpha angelsolisi, Zelomorpha bobandersoni, Zelomorpha danjohnsoni, Zelomorpha donwindsori, Zelomorpha effugia, Zelomorpha johnchemsaki, Zelomorpha kellyanneae, Zelomorpha larrykirkendalli, Zelomorpha mariyavladmirovnae, Zelomorpha mikeiviei, Zelomorpha myricagaleae, Zelomorpha noahjaneae, Zelomorpha paulgoldsteini, Zelomorpha terryerwini, Zelomorpha willsflowersi, Hemichoma donwhiteheadi, Hemichoma frankhovorei, and Hemichoma johnkingsolveri.

There are ~240 species of Culicidae in Mexico, of which some are vectors of arthropod-borne viruses such as Zika virus, dengue virus, chikungunya virus, and West Nile virus. Thus, the identification of mosquito feeding preferences is paramount to understanding of vector–host–pathogen interactions that, in turn, can aid the control of disease outbreaks. Typically, DNA and RNA are extracted separately for animal (insects and blood meal hosts) and viral identification, but this study demonstrates that multiple organisms can be analyzed from a single RNA extract. For the first time, residual DNA present in standard RNA extracts was analyzed by DNA barcoding in concert with Sanger and next-generation sequencing (NGS) to identify both the mosquito species and the source of their meals in blood-fed females caught in seven sylvan communities in Chiapas State, Mexico. While mosquito molecular identification involved standard barcoding methods, the sensitivity of blood meal identification was maximized by employing short primers with NGS. In total, we collected 1,634 specimens belonging to 14 genera, 25 subgenera, and 61 morphospecies of mosquitoes. Of these, four species were new records for Mexico (Aedes guatemala, Ae. insolitus, Limatus asulleptus, Trichoprosopon pallidiventer), and nine were new records for Chiapas State. DNA barcode sequences for >300 bp of the COI gene were obtained from 291 specimens, whereas 130 bp sequences were recovered from another 179 specimens. High intraspecific divergence values (>2%) suggesting cryptic species complexes were observed in nine taxa: Anopheles eiseni (5.39%), An. pseudopunctipennis (2.79%), Ae. podographicus (4.05%), Culex eastor (4.88%), Cx. erraticus (2.28%), Toxorhynchites haemorrhoidalis (4.30%), Tr. pallidiventer (4.95%), Wyeomyia adelpha/Wy. guatemala (7.30%), and Wy. pseudopecten (4.04%). The study increased the number of mosquito species known from 128 species to 138 species for Chiapas State, and 239 for Mexico as a whole. Blood meal analysis showed that Aedes angustivittatus fed on ducks and chicken, whereas Psorophora albipes fed on humans. Culex quinquefasciatus fed on diverse hosts including chicken, human, turkey, and Mexican grackle. No arbovirus RNA was detected by reverse transcriptase–polymerase chain reaction in the surveyed specimens. This study demonstrated, for the first time, that residual DNA present in RNA blood meal extracts can be used to identify host vectors, highlighting the important role of molecular approaches in both vector identification and revealing host–vector–pathogen interactions.

Mosquitoes (Diptera: Culicidae) are regarded as annoying biting pests and vectors of disease-causing agents to humans and other vertebrates worldwide. Factors that affect their distribution and host choice are not well understood. Here, we assessed the species abundance, community composition, and feeding patterns of mosquitoes in an urban-to-wild habitat gradient in northern Spain. Adult mosquitoes from four habitats (urban, periurban, rural, and wild) were collected by aspiration from mid-July to mid-September, 2019. Thirteen species were represented among the 268 specimens (132 females and 136 males) trapped, including six new records reported for the first time in the region. Culex pipiens was the most abundant species in all habitats except in the wild, where Culex territans was dominant. The highest mosquito diversity was recorded in the wild habitat [species richness (S) = 10 and Shannon/Margalef-Diversity Indices (H’/MI = 1.51/1.36)] and the lowest in the urban habitat (S = 3; H’/MI = 0.24/0.41). Blood-engorged specimens (n = 65) represented 49.2% of the total female collections. Eighty percent of the blood-meals (n = 52) were successfully identified based on cytochrome c oxidase I subunit (COI) DNA barcoding. Nine species of birds were identified in blood meals from the three ecological forms of Cx. pipiens (n = 48), Culiseta fumipennis (n = 3), and Culiseta morsitans (n = 1) collected along the four sampling habitats. Four dominant bird species were recorded in Cx. pipiens, i.e., Parus major (35.4%), Turdus merula (18.7%), Pica pica (18.7%), and Passer domesticus (10.4%). Despite the availability of dog and human hosts in the sampling sites located in the urban habitat, Cx. pipiens seemed to have a preference to feed on birds. Culiseta fumipennis blood-meal host records are reported for first time in Europe. These findings on mosquito blood-feeding preferences and habitat community changes will help to better understand vector-host associations and pathogen transmission paths.

The kingdom Fungi is a megadiverse group represented in all ecosystem types. The global diversity and distribution of fungal taxa are poorly known, in part due to the limitations related to traditional fruit-body survey methods. These previous hurdles are now being overcome by rapidly developing DNA-based surveys. Past fungal DNA surveys have predominantly examined soil samples, which capture high species diversity but represent only the local soil community. Recent work has shown that DNA samples collected from the air with cyclone samplers provide information on fungal diversity at the scale of some tens of kilometers around the sampling location. To test the feasibility of air sampling for investigating global patterns of fungal diversity, we established a new initiative called the Global Spore Sampling Project (GSSP). The GSSP currently involves 50 sampling locations distributed on all continents, with each location collecting two 24-h samples per week. Here we describe the GSSP methodology, including the sampling, DNA extraction and sequencing protocols, and the bioinformatics pipeline. We further report results based on 75 pilot samples from five locations, of which three in Europe, one in Australia, and one in Greenland. The results show highly consistent patterns, suggesting that GSSP holds much promise for systematic global fungal monitoring. The GSSP provides highly standardized sampling across space and time, enabling much-improved estimation of total fungal diversity, the global distribution of different fungal groups, fungal fruiting phenology, and the extent of long-distance dispersal in fungi.

Determining the size of the German insect fauna requires better knowledge of several megadiverse families of Diptera and Hymenoptera that are taxonomically challenging. This study takes the first step in assessing these “dark taxa” families and provides species estimates for four challenging groups of Diptera (Cecidomyiidae, Chironomidae, Phoridae, and Sciaridae). These estimates are based on more than 48,000 DNA barcodes (COI) from Diptera collected by Malaise traps that were deployed in southern Germany. We assessed the fraction of German species belonging to 11 fly families with well-studied taxonomy in these samples. The resultant ratios were then used to estimate the species richness of the four “dark taxa” families (DT families hereafter). Our results suggest a surprisingly high proportion of undetected biodiversity in a supposedly well-investigated country: at least 1800–2200 species await discovery in Germany in these four families. As this estimate is based on collections from one region of Germany, the species count will likely increase with expanded geographic sampling.

Abstract Background Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system. Results By examining templates from more than 5000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL can reduce greatly reduce sequencing costs in comparison to first (Sanger) and second generation platforms (Illumina, Ion). Conclusions SMRT analysis generates high-fidelity sequences from amplicons with varying GC content and is resilient to homopolymer tracts. Analytical costs are low, substantially less than those for first or second generation sequencers. When implemented on the SEQUEL platform, SMRT analysis enables massive amplicon characterization because each instrument can recover sequences from more than 5 million DNA extracts a year.

Lipolexis is a small genus in the subfamily Aphidiinae represented by one species in Europe (Lipolexis gracilis Förster) and by four in Asia (Lipolexis wuyiensis Chen, L. oregmae Gahan, L. myzakkaiae Pramanik and Raychaudhuri and L. pseudoscutellaris Pramanik and Raychaudhuri). Although L. oregmae is employed in biological control programs against pest aphids, the last morphological study on the genus was completed over 50 years ago. This study employs an integrative approach (morphology and molecular analysis (COI barcode region)), to examine Lipolexis specimens that were sampled worldwide, including specimens from BOLD database. These results establish that two currently recognized species of Lipolexis (L. gracilis, L. oregmae) are actually a species complex and also reveal phylogenetic relationships within the genus. Six new species are described and a global key for the identification of Lipolexis species is provided.

Members of the Monoctonina subtribe have long been neglected in applied studies of the subfamily Aphidiinae, due to their low economic importance, as they do not parasitize pests of cultivated plants. Consequently, data about this group are scarce, including its taxonomy and phylogeny. In the present study, we explore inter- and intraspecific genetic variation of Monoctonina species, including genera Monoctonus Haliday 1833, Monoctonia Starý 1962, Falciconus Mackauer 1959 and Harkeria Cameron 1900. We employ two molecular markers, the barcode region of the mitochondrial cytochrome c oxidase subunit I (COI) and the D2 region of the 28S nuclear gene (28S rDNA), to analyze genetic structuring and phylogeny of all available Monoctonina species, and combine them with morphological data for an integrative approach. We report one new species, and three potentially new species which can be formally described when further specimens are available. Analysis of phylogenetic relationships within the subtribe shows a basal position for the genera Falciconus and Monoctonia, and the close relatedness of Harkeria and Monoctonus.