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.

Species Delimitation

Species delimitation is the process of determining whether a group of sampled individuals belong to the same species or to different species. The criteria used to delimit species differ across taxonomic groups, and the methods for delimiting species have changed over time, with a dramatic rise in the popularity of genomic approaches recently. Because inferred species boundaries have ramifications that extend beyond systematics, affecting all fields that rely upon species as a foundational unit, controversy has unsurprisingly surrounded not only the practices used to delimit species boundaries, but also the idea of what species are, which varies across taxa (e.g., the use of subspecies varies across the tree of life). This lack of consensus has no doubt contributed to the appeal of genetic-based delimitation. Specifically, genomic data can be collected from any taxon. Moreover, it can be analyzed in a common statistical framework (as popularized by the multispecies coalescent as a model for species delimitation). With the ease of collecting genetic data, the power of genomics, and the purported standardization for diagnosing species limits, genetic-based species delimitation is displacing traditional time-honored (albeit time-consuming) taxonomic practices of species diagnosis. It has also become an invaluable tool for discovering species in understudied groups, and genetic-based approaches are the foundation of international endeavors to generate a catalogue of DNA barcodes to illuminate biodiversity for all of life on the planet. Yet, genomic applications, and especially the sole reliance upon genetic data for inferring species boundaries, are not without their own set of challenges.

Hydrodroma angelieri (Acari, Hydrachnidia: Hydrodromidae) a new water mite species from Corsica based on morphological and DNA barcode evidence

In the present study we used morphological data and DNA barcodes to describe a new species, Hydrodroma angelieri sp. nov. from Corsica, France. A high genetic distance of 17.3±0.017% K2P from its molecularly most closely related European congener, H. despiciens (Müller, 1776), supports H. angelieri sp. nov. as a distinct species. Morphologically the new species can be identified on the basis of relatively small leg claws, the presence of only one swimming seta on II-L-5 and 4-6 swimming setae on the anterior surface of IV-L-5. An updated key for the European species of Hydrodroma is provided.

Species discrimination of novel chloroplast DNA barcodes and their application for identification of Panax (Aralioideae, Araliaceae)

Certain species within the genus Panax L. (Araliaceae) contain pharmacological precious ginsenosides, also known as ginseng saponins. Species containing these compounds are of high commercial value and are thus of particular urgency for conservation. However, within this genus, identifying the particular species that contain these compounds by morphological means is challenging. DNA barcoding is one method that is considered promising for species level identification. However, in an evolutionarily complex genus such as Panax, commonly used DNA barcodes such as nrITS, matK, psbA-trnH, rbcL do not provide species-level resolution. A recent in silico study proposed a set of novel chloroplast markers, trnQ-rps16, trnS-trnG, petB, and trnE-trnT for species level identification within Panax. In the current study, the discriminatory efficiency of these molecular markers is assessed and validated using 91 reference barcoding sequences and 38 complete chloroplast genomes for seven species, one unidentified species and one sub-species of Panax, and two outgroup species of Aralia L. along with empirical data of Panax taxa present in Vietnam via both distance-based and tree-based methods. The obtained results show that trnQ-rps16 can classify with species level resolution every clade tested here, including the highly valuable Panax vietnamensis Ha et Grushv. We thus propose that this molecular marker to be used for identification of the species within Panax to support both its conservation and commercial trade.

Contribution to a reference library of DNA barcodes of Colombian freshwater fishes

The Barcode of Life initiative was originally motivated by the large number of species, taxonomic difficulties and the limited number of expert taxonomists. Colombia has 1,610 freshwater fish species and comprises the second largest diversity of this group in the world. As genetic information continues to be limited, we constructed a reference collection of DNA sequences of Colombian freshwater fishes deposited in the Ichthyology Collection of the University of Antioquia (CIUA), thus joining the multiple efforts that have been made in the country to contribute to the knowledge of genetic diversity in order to strengthen the inventories of biological collections and facilitate the solution of taxonomic issues in the future. This study contributes to the knowledge on the DNA barcodes and occurrence records of 96 species of Colombian freshwater fishes. Fifty-seven of the species represented in this dataset were already available in the Barcode Of Life Data System (BOLD System), while 39 correspond to new species to the BOLD System. Forty-nine specimens were collected in the Atrato River Basin and 708 in the Magdalena-Cauca asin during the period 2010-2020. Two species (Loricariichthys brunneus (Hancock, 1828) and Poecilia sphenops Valenciennes, 1846) are considered exotic to the Atrato, Cauca and Magdalena Basins and four species (Oncorhynchus mykiss (Walbaum, 1792), Oreochromis niloticus (Linnaeus, 1758), Parachromis friedrichsthalii (Heckel, 1840) and Xiphophorus helleri Heckel, 1848) are exotic to the Colombian hydrogeographic regions. All specimens are deposited in CIUA and have their DNA barcodes made publicly available in the BOLD online database. The geographical distribution dataset can be freely accessed through the Global Biodiversity Information Facility (GBIF).

Three cryptic Anaplecta (Blattodea, Blattoidea, Anaplectidae) species revealed by female genitalia, plus seven new species from China

Morphological characteristics, including male and female genitalia, combined with DNA barcodes were used to identify 470 Anaplecta specimens sampled from China. Ten Anaplecta species are new to science, including three cryptic species: A. paraomei Zhu & Che, sp. nov., A. condensa Zhu & Che, sp. nov., and A. longihamata Zhu & Che, sp. nov., which are distinguished mainly by their female genitalia. The other seven new species are as follows: A. bicruris Zhu & Che, sp. nov., A. spinosa Zhu & Che, sp. nov., A. ungulata Zhu & Che, sp. nov., A. anomala Zhu & Che, sp. nov., A. serrata Zhu & Che, sp. nov., A. bombycina Zhu & Che, sp. nov., and A. truncatula Zhu & Che, sp. nov. This study illustrates that differences in female genitalia can be used to distinguish among species of Anaplecta. The female genitalia of 19 Chinese Anaplecta species are described and illustrated in this paper.

Corrections and Additions to Descriptions of Some Species of the Subgenus Orthocladius s. str. (Diptera, Chironomidae, Orthocladiinae)

The larvae of some species of the subgenus Orthocladius s. str. (Diptera, Chironomidae) are here described for the first time with corrections and additions to the descriptions of adult males and pupal exuviae. The identification of larvae is generally not possible without association with pupal exuviae and/or adult males, so the descriptions here are based only on reared material or on pupae with the associated larval exuviae. Usually, Chironomidae larvae can be separated on the basis of morphometric characters, the most discriminant ones are: (1) the ratio between the width of median tooth of mentum (Dm) and the width of the first lateral tooth (Dl) = mental ratio (DmDl), (2) the ratio between the length of the first antennal segment (A1) and the combined length of segments 2–5 (A2–5) = antennal ratio (AR). The shape of mandible, maxilla, and other body parts are almost identical in all the species considered in this study. The larva of Orthocladius (Symposiocladius) lignicola is very characteristic and can be separated by the shape of mentum and the larvae of all the known species of Symposiocladius are characterized by the presence of large Lauterborn organs on antennae and of tufts of setae on abdominal segments. The larvae of Orthocladius (Orthocladius) oblidens and Orthocladius (Orthocladius) rhyacobius can be distinguished from other species basing on their large Dm and to each other by AR. A principal component analysis was carried out using 5 characters: (1) Dm, (2) Dl, (3) length of A1, (4) width of A1 (A1W), (5) combined length of segments 2–5 (A2–5). The most discriminant characters were Dm and A1, confirming that DmDl and AR can be used to separate species at larval stage, but the large superposition of morphometric characters in different species confirms that association with pupal exuviae is in any case needed to identify larvae. In future perspective, the development of reference DNA barcodes from specimens identified by specialists is recommended since possibly the best tool for larvae identification, but association of barcodes with morphotypes is in any case fundamental.