Scratching the tip of the iceberg: integrative taxonomy reveals 30 new species records of Microgastrinae (Braconidae) parasitoid wasps for Germany, including new Holarctic distributions.

Substantial parts of the European and German insect fauna still remain largely unexplored, the so-called "dark taxa". In particular, midges (Diptera) and parasitoid wasps (Hymenoptera) are abundant and species-rich throughout Europe, yet are often neglected in biodiversity research. One such dark taxon is Microgastrinae wasps (Hymenoptera: Braconidae), a group of parasitoids of lepidopteran caterpillars with 252 species reported in Germany so far. As part of the German Barcode of Life Project GBOL III: Dark Taxa, reverse DNA barcoding and integrative taxonomic approaches were used to shed some light on the German Fauna of Microgastrinae wasps. In our workflow, DNA barcoding was used for molecular clustering of our specimens in a first step, morphological examination of the voucher specimens in a second step, and host data compared in a third step. Here, 30 species are reported for the first time in Germany, adding more than 10% to the known German fauna. Information for four species is provided in a new Holarctic context, reporting them for the Nearctic or, respectively, Palaearctic region, and 26 additional country records are added from sequenced material available in the collections accessible to us. Molecular clusters that show signs of discrepancies are discussed. Results show that we are just scratching the tip of the iceberg of the unexplored Microgastrinae diversity in Germany.

Discovery of the primary aphid (Hemiptera: Aphidomorpha) and scale insect (Hemiptera: Coccomorpha) type specimens from the collection of Theodor Hartig (18051880).

Theodor Hartigs aphid and scale insect type specimens have been presumed lost or destroyed for the last 140 years. Here we document their discovery at the Bavarian State Collection of Zoology (Zoologische Staatssammlung Mnchen, ZSM), in Munich, Germany. These specimens include primary types for 24 aphid, three adelgid, and two armored scale insect species named by Hartig between 1834 and 1851, as well as other specimens of unknown importance.

Surprisingly high genetic divergence of the mitochondrial DNA barcode fragment (COI) within Central European woodlice species (Crustacea, Isopoda, Oniscidea).

DNA barcoding has become the most popular approach for species identification in recent years. As part of the German Barcode of Life project, the first DNA barcode library for terrestrial and freshwater isopods from Germany is presented. The analyzed barcode library included 38 terrestrial (78% of the documented species of Germany) and five freshwater (63%) species. A total of 513 new barcodes was generated and 518 DNA barcodes were analyzed. This analysis revealed surprisingly high intraspecific genetic distances for numerous species, with a maximum of 29.4% for Platyarthrushoffmannseggii Brandt, 1833. The number of BINs per species ranged from one (32 species, 68%) to a maximum of six for Trachelipusrathkii (Brandt, 1833). In spite of such high intraspecific variability, interspecific distances with values between 12.6% and 29.8% allowed a valid species assignment of all analyzed isopods. The observed high intraspecific distances presumably result from phylogeographic events, Wolbachia infections, atypical mitochondrial DNAs, heteroplasmy, or various combinations of these factors. Our study represents the first step in generating an extensive reference library of DNA barcodes for terrestrial and freshwater isopods for future molecular biodiversity assessment studies.

Peering into the Darkness: DNA Barcoding Reveals Surprisingly High Diversity of Unknown Species of Diptera (Insecta) in Germany.

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.

Out of taxonomic crypsis: A new trans-arctic cryptic species pair corroborated by phylogenetics and molecular evidence.

Cryptic species are a common phenomenon in cosmopolitan marine species. The use of molecular tools has often uncovered cryptic species occupying a fraction of the geographic range of the original morphospecies. Shipworms (Teredinidae) are marine bivalves, living in drift and fixed wood, many of which have a conserved morphology across cosmopolitan distributions. Herein novel and GenBank mitochondrial (cytochrome c oxidase subunit I) and nuclear (18S rRNA) DNA sequences are employed to produce a phylogeny of the Teredinidae and delimit a cryptic species pair in the Psiloteredo megotara complex. The anatomy, biogeography, and ecology of P. megotara, Psiloteredo sp. and Nototeredo edax are compared based on private and historic museum collections and a thorough literature review. Morphological and anatomical characters of P. megotara from the North Atlantic and Psiloteredo sp. from Japan were morphologically indistinguishable, and differ in pallet architecture and soft tissue anatomy from N. edax. The two Psiloteredo species were then delimited as genetically distinct species using four molecular-based methods. Consequently, the Northwest Pacific species, Psiloteredo pentagonalis, first synonymized with N. edax and then with P. megotara, is resurrected. Nototeredo edax, P. megotara and P. pentagonalis are redescribed based upon morphological and molecular characters. Phylogenetic analysis further revealed cryptic species complexes within the cosmopolitan species Bankia carinata and possibly additional cryptic lineages within the cosmopolitan Lyrodus pedicellatus.

Relicts from Glacial Times: The Ground Beetle Pterostichus adstrictus Eschscholtz, 1823 (Coleoptera: Carabidae) in the Austrian Alps.

The last ice age considerably influenced distribution patterns of extant species of plants and animals, with some of them now inhabiting disjunct areas in the subarctic/arctic and alpine regions. This arctic-alpine distribution is characteristic for many cold-adapted species with a limited dispersal ability and can be found in many invertebrate taxa, including ground beetles. The ground beetle Pterostichus adstrictus Eschscholtz, 1823 of the subgenus Bothriopterus was previously known to have a holarctic-circumpolar distribution, in Europe reaching its southern borders in Wales and southern Scandinavia. Here, we report the first findings of this species from the Austrian Ötztal Alps, representing also the southernmost edge of its currently known distribution, confirmed by the comparison of morphological characters to other Bothriopterus species and DNA barcoding data. Molecular data revealed a separation of the Austrian and Finish specimens with limited to no gene flow at all. Furthermore, we present the first data on habitat preference and seasonality of P. adstrictus in the Austrian Alps.

A DNA barcode library for ground beetles of Germany: the genus Pterostichus Bonelli, 1810 and allied taxa (Insecta, Coleoptera, Carabidae).

Species of the ground beetle genus Pterostichus Bonelli, 1810 are some of the most common carabids in Europe. This publication provides a first comprehensive DNA barcode library for this genus and allied taxa including Abax Bonelli, 1810, Molops Bonelli, 1810, Poecilus Bonelli, 1810, and Stomis Clairville, 1806 for Germany and Central Europe in general. DNA barcodes were analyzed from 609 individuals that represent 51 species, including sequences from previous studies as well as more than 198 newly generated sequences. The results showed a 1:1 correspondence between BIN and traditionally recognized species for 44 species (86%), whereas two (4%) species were characterized by two BINs. Three BINs were found for one species (2%), while one BIN for two species was revealed for two species pairs (8%). Low interspecific distances with maximum pairwise K2P values below 2.2% were found for four species pairs. Haplotype sharing was found for two closely related species pairs: Pterostichus adstrictus Eschscholtz, 1823/Pterostichus oblongopunctatus (Fabricius, 1787) and Pterostichus nigrita Paykull, 1790/Pterostichus rhaeticus Heer, 1837. In contrast to this, high intraspecific sequence divergences with values above 2.2% were shown for three species (Molops piceus (Panzer, 1793), Pterostichus panzeri (Panzer, 1805), Pterostichus strenuus (Panzer, 1793)). Summarizing the results, the present DNA barcode library does not only allow the identification of most of the analyzed species, but also provides valuable information for alpha-taxonomy as well as for ecological and evolutionary research. This library represents another step in building a comprehensive DNA barcode library of ground beetles as part of modern biodiversity research.

A DNA barcode library for ground beetles of Germany: the genus Amara Bonelli, 1810 (Insecta, Coleoptera, Carabidae).

The genus Amara Bonelli, 1810 is a very speciose and taxonomically difficult genus of the Carabidae. The identification of many of the species is accomplished with considerable difficulty, in particular for females and immature stages. In this study the effectiveness of DNA barcoding, the most popular method for molecular species identification, was examined to discriminate various species of this genus from Central Europe. DNA barcodes from 690 individuals and 47 species were analysed, including sequences from previous studies and more than 350 newly generated DNA barcodes. Our analysis revealed unique BINs for 38 species (81%). Interspecific K2P distances below 2.2% were found for three species pairs and one species trio, including haplotype sharing between Amara alpina/Amara torrida and Amara communis/Amara convexior/Amara makolskii. This study represents another step in generating an extensive reference library of DNA barcodes for carabids, highly valuable bioindicators for characterizing disturbances in various habitats.

From water striders to water bugs: the molecular diversity of aquatic Heteroptera (Gerromorpha, Nepomorpha) of Germany based on DNA barcodes.

With about 5,000 species worldwide, the Heteroptera or true bugs are the most diverse taxon among the hemimetabolous insects in aquatic and semi-aquatic ecosystems. Species may be found in almost every freshwater environment and have very specific habitat requirements, making them excellent bioindicator organisms for water quality. However, a correct determination by morphology is challenging in many species groups due to high morphological variability and polymorphisms within, but low variability between species. Furthermore, it is very difficult or even impossible to identify the immature life stages or females of some species, e.g., of the corixid genus Sigara. In this study we tested the effectiveness of a DNA barcode library to discriminate species of the Gerromorpha and Nepomorpha of Germany. We analyzed about 700 specimens of 67 species, with 63 species sampled in Germany, covering more than 90% of all recorded species. Our library included various morphological similar taxa, e.g., species within the genera Sigara and Notonecta as well as water striders of the genus Gerris. Fifty-five species (82%) were unambiguously assigned to a single Barcode Index Number (BIN) by their barcode sequences, whereas BIN sharing was observed for 10 species. Furthermore, we found monophyletic lineages for 52 analyzed species. Our data revealed interspecific K2P distances with below 2.2% for 18 species. Intraspecific distances above 2.2% were shown for 11 species. We found evidence for hybridization between various corixid species (Sigara, Callicorixa), but our molecular data also revealed exceptionally high intraspecific distances as a consequence of distinct mitochondrial lineages for Cymatia coleoptrata and the pygmy backswimmer Plea minutissima. Our study clearly demonstrates the usefulness of DNA barcodes for the identification of the aquatic Heteroptera of Germany and adjacent regions. In this context, our data set represents an essential baseline for a reference library for bioassessment studies of freshwater habitats using modern high-throughput technologies in the near future. The existing data also opens new questions regarding the causes of observed low inter- and high intraspecific genetic variation and furthermore highlight the necessity of taxonomic revisions for various taxa, combining both molecular and morphological data.

Cryptic species in a well-known habitat: applying taxonomics to the amphipod genus Epimeria (Crustacea, Peracarida).

Taxonomy plays a central role in biological sciences. It provides a communication system for scientists as it aims to enable correct identification of the studied organisms. As a consequence, species descriptions should seek to include as much available information as possible at species level to follow an integrative concept of 'taxonomics'. Here, we describe the cryptic species Epimeria frankei sp. nov. from the North Sea, and also redescribe its sister species, Epimeria cornigera. The morphological information obtained is substantiated by DNA barcodes and complete nuclear 18S rRNA gene sequences. In addition, we provide, for the first time, full mitochondrial genome data as part of a metazoan species description for a holotype, as well as the neotype. This study represents the first successful implementation of the recently proposed concept of taxonomics, using data from high-throughput technologies for integrative taxonomic studies, allowing the highest level of confidence for both biodiversity and ecological research.

DNA barcoding and morphological studies confirm the occurrence of three Atarbolana (Crustacea: Isopoda: Cirolanidae) species along the coastal zone of the Persian Gulf and Gulf of Oman.

Two species of Atarbolana (Cirolanidae: Isopoda) from the intertidal zone of the Gulf of Oman and the Persian Gulf were studied and redescribed. The known distribution of this small genus is limited to the northern areas of the Indian Ocean, from the Pakistan coasts to the Persian Gulf. The analyses of DNA barcodes as well as detailed morphological studies clearly support the existence of three distinct Atarbolana species along the coastal zone of the Persian Gulf and northern Arabian Sea. Furthermore, A. dasycolus Yasmeen, 2004 is synonymized with A. setosa Javed and Yasmeen, 1989.

A DNA barcode library for ground beetles (Insecta, Coleoptera, Carabidae) of Germany: The genus Bembidion Latreille, 1802 and allied taxa.

As molecular identification method, DNA barcoding based on partial cytochrome c oxidase subunit 1 (COI) sequences has been proven to be a useful tool for species determination in many insect taxa including ground beetles. In this study we tested the effectiveness of DNA barcodes to discriminate species of the ground beetle genus Bembidion and some closely related taxa of Germany. DNA barcodes were obtained from 819 individuals and 78 species, including sequences from previous studies as well as more than 300 new generated DNA barcodes. We found a 1:1 correspondence between BIN and traditionally recognized species for 69 species (89%). Low interspecific distances with maximum pairwise K2P values below 2.2% were found for three species pairs, including two species pairs with haplotype sharing (Bembidion atrocaeruleum/Bembidion varicolor and Bembidion guttula/Bembidion mannerheimii). In contrast to this, deep intraspecific sequence divergences with distinct lineages were revealed for two species (Bembidion geniculatum/Ocys harpaloides). Our study emphasizes the use of DNA barcodes for the identification of the analyzed ground beetles species and represents an important step in building-up a comprehensive barcode library for the Carabidae in Germany and Central Europe as well.

Identification of North Sea molluscs with DNA barcoding.

Sequence-based specimen identification, known as DNA barcoding, is a common method complementing traditional morphology-based taxonomic assignments. The fundamental resource in DNA barcoding is the availability of a taxonomically reliable sequence database to use as a reference for sequence comparisons. Here, we provide a reference library including 579 sequences of the mitochondrial cytochrome c oxidase subunit I for 113 North Sea mollusc species. We tested the efficacy of this library by simulating a sequence-based specimen identification scenario using Best Match, Best Close Match (BCM) and All Species Barcode (ASB) criteria with three different threshold values. Each identification result was compared with our prior morphology-based taxonomic assignments. Our simulation resulted in 87.7% congruent identifications (93.8% when excluding singletons). The highest number of congruent identifications was obtained with BCM and ASB and a 0.05 threshold. We also compared identifications with genetic clustering (Barcode Index Numbers, BINs) computed by the Barcode of Life Datasystem (BOLD). About 68% of our morphological identifications were congruent with BINs created by BOLD. Forty-nine sequences were clustered in 16 discordant BINs, and these were divided in two classes: sequences from different species clustered in a single BIN and conspecific sequences divided in more BINs. Whereas former incongruences were probably caused by BOLD entries in need of a taxonomic update, the latter incongruences regarded taxa requiring further investigations. These include species with amphi-Atlantic distribution, whose genetic structure should be evaluated over their entire range to produce a reliable sequence-based identification system.

High-Throughput Sequencing-The Key to Rapid Biodiversity Assessment of Marine Metazoa?

The applications of traditional morphological and molecular methods for species identification are greatly restricted by processing speed and on a regional or greater scale are generally considered unfeasible. In this context, high-throughput sequencing, or metagenetics, has been proposed as an efficient tool to document biodiversity. Here we evaluated the effectiveness of 454 pyrosequencing in marine metazoan community analysis using the 18S rDNA: V1-V2 region. Multiplex pyrosequencing of the V1-V2 region was used to analyze two pooled samples of DNA, one comprising 118 and the other 37 morphologically identified species, and one natural sample taken directly from a North Sea zooplankton community. A DNA reference library comprising all species represented in the pooled samples was created by Sanger sequencing, and this was then used to determine the optimal similarity threshold for species delineation. The optimal threshold was found at 99% species similarity, with 85% identification success. Pyrosequencing was able to identify between fewer species: 67% and 78% of the species in the two pooled samples. Also, a large number of sequences for three species that were not included in the pooled samples were amplified by pyrosequencing, suggesting preferential amplification of some genotypes and the sensitivity of this approach to even low levels of contamination. Conversely, metagenetic analysis of the natural zooplankton sample identified many more species (particularly gelatinous zooplankton and meroplankton) than morphological analysis of a formalin-fixed sample from the same sampling site, suggesting an increased level of taxonomic resolution with pyrosequencing. The study demonstrated that, based on the V1-V2 region, 454 sequencing does not provide accurate species differentiation and reliable taxonomic classification, as it is required in most biodiversity monitoring. The analysis of artificially prepared samples indicated that species detection in pyrosequencing datasets is complicated by potential PCR-based biases and that the V1-V2 marker is poorly resolved for some taxa.

The Application of DNA Barcodes for the Identification of Marine Crustaceans from the North Sea and Adjacent Regions.

During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for 198 (96.6%) of the analyzed species. Six species were characterized by two BINs (2.9%), and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%). Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%). Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761), underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequences.

Looking back on a decade of barcoding crustaceans.

Species identification represents a pivotal component for large-scale biodiversity studies and conservation planning but represents a challenge for many taxa when using morphological traits only. Consequently, alternative identification methods based on molecular markers have been proposed. In this context, DNA barcoding has become a popular and accepted method for the identification of unknown animals across all life stages by comparison to a reference library. In this review we examine the progress of barcoding studies for the Crustacea using the Web of Science data base from 2003 to 2014. All references were classified in terms of taxonomy covered, subject area (identification/library, genetic variability, species descriptions, phylogenetics, methods, pseudogenes/numts), habitat, geographical area, authors, journals, citations, and the use of the Barcode of Life Data Systems (BOLD). Our analysis revealed a total number of 164 barcoding studies for crustaceans with a preference for malacostracan crustaceans, in particular Decapoda, and for building reference libraries in order to identify organisms. So far, BOLD did not establish itself as a popular informatics platform among carcinologists although it offers many advantages for standardized data storage, analyses and publication.

Building-up of a DNA barcode library for true bugs (insecta: hemiptera: heteroptera) of Germany reveals taxonomic uncertainties and surprises.

During the last few years, DNA barcoding has become an efficient method for the identification of species. In the case of insects, most published DNA barcoding studies focus on species of the Ephemeroptera, Trichoptera, Hymenoptera and especially Lepidoptera. In this study we test the efficiency of DNA barcoding for true bugs (Hemiptera: Heteroptera), an ecological and economical highly important as well as morphologically diverse insect taxon. As part of our study we analyzed DNA barcodes for 1742 specimens of 457 species, comprising 39 families of the Heteroptera. We found low nucleotide distances with a minimum pairwise K2P distance <2.2% within 21 species pairs (39 species). For ten of these species pairs (18 species), minimum pairwise distances were zero. In contrast to this, deep intraspecific sequence divergences with maximum pairwise distances >2.2% were detected for 16 traditionally recognized and valid species. With a successful identification rate of 91.5% (418 species) our study emphasizes the use of DNA barcodes for the identification of true bugs and represents an important step in building-up a comprehensive barcode library for true bugs in Germany and Central Europe as well. Our study also highlights the urgent necessity of taxonomic revisions for various taxa of the Heteroptera, with a special focus on various species of the Miridae. In this context we found evidence for on-going hybridization events within various taxonomically challenging genera (e.g. Nabis Latreille, 1802 (Nabidae), Lygus Hahn, 1833 (Miridae), Phytocoris Fallén, 1814 (Miridae)) as well as the putative existence of cryptic species (e.g. Aneurus avenius (Duffour, 1833) (Aradidae) or Orius niger (Wolff, 1811) (Anthocoridae)).

A new species of Cymodoce Leach, 1814 (Crustacea: Isopoda: Sphaeromatidae) based on morphological and molecular data, with a key to the Northern Indian Ocean species.

Cymodoce waegelei sp. nov. is described from the subtidal zone of the Iranian coasts of the Persian Gulf using both morphological and molecular data. C. waegelei sp. nov. is most similar to C. tribullis Harrison & Holdich, 1984 from Australia, Vietnam and Singapore. Analysis of DNA barcodes and nuclear 28S rDNA: D8 expansion segments clearly support the existence of two distinct species. Cymodoce waegelei sp. nov morphologically differs from C. tribullis by lacking two continuous rows of tubercles on the pereonites 3 and 4. Moreover, the pleotelson has numerous scattered tubercles between two large prominent bosses, and small lateral tubercles rather than two prominent tubercles in C. tribullis. Based on our results we redescribe Cymodoce tribullis using specimens sampled from the type locality, Magnetic Island, Queensland. Cymodoce lirella Schotte & Kensley, 2005 from the Seychelles is placed in synonymy with C. tribullis Harrison & Holdich 1984. Furthermore we provide a key to the northern Indian Ocean species of this genus.

A reliable DNA barcode reference library for the identification of the North European shelf fish fauna.

Valid fish species identification is an essential step both for fundamental science and fisheries management. The traditional identification is mainly based on external morphological diagnostic characters, leading to inconsistent results in many cases. Here, we provide a sequence reference library based on mitochondrial cytochrome c oxidase subunit I (COI) for a valid identification of 93 North Atlantic fish species originating from the North Sea and adjacent waters, including many commercially exploited species. Neighbour-joining analysis based on K2P genetic distances formed nonoverlapping clusters for all species with a ≥99% bootstrap support each. Identification was successful for 100% of the species as the minimum genetic distance to the nearest neighbour always exceeded the maximum intraspecific distance. A barcoding gap was apparent for the whole data set. Within-species distances ranged from 0 to 2.35%, while interspecific distances varied between 3.15 and 28.09%. Distances between congeners were on average 51-fold higher than those within species. The validation of the sequence library by applying BOLDs barcode index number (BIN) analysis tool and a ranking system demonstrated high taxonomic reliability of the DNA barcodes for 85% of the investigated fish species. Thus, the sequence library presented here can be confidently used as a benchmark for identification of at least two-thirds of the typical fish species recorded for the North Sea.

Isolation and characterization of nine polymorphic microsatellite markers for the deep-sea shrimp Nematocarcinus lanceopes (Crustacea: Decapoda: Caridea).

BACKGROUND: The shrimp Nematocarcinus lanceopes Bate, 1888 is found in the deep sea around Antarctica and sub-Antarctic islands. Previous studies on mitochondrial data and species distribution models provided evidence for a homogenous circum-Antarctic population of N. lanceopes. However, to analyze the fine-scale population genetic structure and to examine influences of abiotic environmental conditions on population composition and genetic diversity, a set of fast evolving nuclear microsatellite markers is required. FINDINGS: We report the isolation and characterization of nine polymorphic microsatellite markers from the Antarctic deep-sea shrimp species Nematocarcinus lanceopes (Crustacea: Decapoda: Caridea). Microsatellite markers were screened in 55 individuals from different locations around the Antarctic continent. All markers were polymorphic with 9 to 25 alleles per locus. The observed heterozygosity ranged from 0.545 to 0.927 and the expected heterozygosity from 0.549 to 0.934. CONCLUSIONS: The reported markers provide a novel tool to study genetic structure and diversity in Nematocarcinus lanceopes populations in the Southern Ocean and monitor effects of ongoing climate change in the region on the populations inhabiting these.