Although aquatic macroinvertebrates and freshwater fishes are important indicators for freshwater quality assessments, the morphological identification to species-level is often impossible and thus ...especially in many invertebrate taxa not mandatory during Water Framework Directive monitoring, a pragmatism that potentially leads to information loss. Here, we focus on the freshwater fauna of the River Sieg (Germany) to test congruence and additional value in taxa detection and taxonomic resolution of DNA barcoding vs. morphology-based identification in monitoring routines. Prior generated morphological identifications of juvenile fishes and aquatic macroinvertebrates were directly compared to species assignments using the identification engine of the Barcode of Life Data System. In 18% of the invertebrates morphology allowed only assignments to higher systematic entities, but DNA barcoding lead to species-level assignment. Dissimilarities between the two approaches occurred in 7% of the invertebrates and in 1% of the fishes. The 18 fish species were assigned to 20 molecular barcode index numbers, the 104 aquatic invertebrate taxa to 113 molecular entities. Although the cost-benefit analysis of both methods showed that DNA barcoding is still more expensive (5.30-8.60€ per sample) and time consuming (12.5h), the results emphasize the potential to increase taxonomic resolution and gain a more complete profile of biodiversity, especially in invertebrates. The provided reference DNA barcodes help building the foundation for metabarcoding approaches, which provide faster sample processing and more cost-efficient ecological status determination.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Oxynoemacheilus shehabi, new species, is described from the upper Orontes in southern Syria. It is distinguished from other Oxynoemacheilus species in the Levant by possession of a complete lateral ...line, a deeply emarginate caudal fin, a narrow caudal peduncle, a suborbital groove in male individuals, a well-developed pelvic axillary lobe, and 5-7 dark-brown bars on the flank. Molecular data suggest that the new species is characterised by a minimum K2P distance of 3.8% to O. "seyhanicola" in the COI mtDNA barcode region.
In order to better understand the complex geologic history of the Mediterranean area, we have analysed evolutionary history, phylogeographic structure and molecular diversity of freshwater fishes ...belonging to the genus Telestes. As primary freshwater fishes distributed largely in the Mediterranean basin, this genus represents a suitable model system for investigating the historical biogeography of freshwater drainage systems in southern Europe. In this investigation we have included samples representing all Telestes species and based our analyses on one mitochondrial and one nuclear gene. We have investigated phylogenetic structure inside the genus Telestes, estimated divergence times, reconstructed ancestral distribution ranges and described intraspecific molecular diversity. Diversification of Telestes started in the Early Miocene, when the ancestors of T. souffia, lineage comprising T. croaticus and T. fontinalis, and the one comprising T. pleurobipunctatus and T. beoticus got isolated. The remaining species are genetically more closely related and form a common cluster in the recovered phylogenetic trees. Complex geological history of southern Europe, including formation of continental bridges, fragmentation of landmass, closing of the sea corridor, local tectonic activities, led to complicated biogeographical pattern of this genus, caused by multiple colonization events and passovers between ancient rivers and water basins. Especially pronounced diversity of Telestes found in the Adriatic watershed in Croatia and Bosnia and Herzegovina is a consequence of a triple colonization of this area by different lineages, which led to an existence of genetically distinct species in neighboring areas. Significant intraspecific structuring is present in T. souffia, T. muticellus, T. croaticus and T. pleurobipunctatus. Besides in well-structured species, elevated levels of genetic polymorphism were found inside T. turskyi and T. ukliva, as a consequence of their old origin and unconstrained evolutionary history.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
This study represents the first comprehensive molecular assessment of freshwater fishes and lampreys from Germany. We analysed COI sequences for almost 80% of the species mentioned in the current ...German Red List. In total, 1056 DNA barcodes belonging to 92 species from all major drainages were used to (i) build a reliable DNA barcode reference library, (ii) test for phylogeographic patterns, (iii) check for the presence of barcode gaps between species and (iv) evaluate the performance of the barcode index number (BIN) system, available on the Barcode of Life Data Systems. For over 78% of all analysed species, DNA barcodes are a reliable means for identification, indicated by the presence of barcode gaps. An overlap between intra‐ and interspecific genetic distances was present in 19 species, six of which belong to the genus Coregonus. The Neighbour‐Joining phenogram showed 60 nonoverlapping species clusters and three singleton species, which were related to 63 separate BIN numbers. Furthermore, Barbatula barbatula, Leucaspius delineatus, Phoxinus phoxinus and Squalius cephalus exhibited remarkable levels of cryptic diversity. In contrast, 11 clusters showed haplotype sharing, or low levels of divergence between species, hindering reliable identification. The analysis of our barcode library together with public data resulted in 89 BINs, of which 56% showed taxonomic conflicts. Most of these conflicts were caused by the use of synonymies, inadequate taxonomy or misidentifications. Moreover, our study increased the number of potential alien species in Germany from 14 to 21 and is therefore a valuable groundwork for further faunistic investigations.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Metabarcoding is a powerful, increasingly popular tool for biodiversity assessment, but it still suffers from some drawbacks (specimen destruction, separation, and size sorting). In the present ...study, we tested a non-destructive protocol that excludes any sample sorting, where the ethanol used for sample preserving is filtered and DNA is extracted from the filter for subsequent DNA metabarcoding. When tested on macroinvertebrate mock communities, the method was widely successful but was unable to reliably detect mollusc taxa. Three different protocols (no treatment, shaking, and freezing) were successfully applied to increase DNA release to the fixative. The protocols resulted in similar success in taxa detection (6.8-7 taxa) but differences in read numbers assigned to taxa of interest (33.8%-93.7%). In comparison to conventional bulk sample metabarcoding of environmental samples, taxa with pronounced exoskeleton and small-bodied taxa were especially underrepresented in ethanol samples. For EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa, which are important for determining stream ecological status, the methods detected 46 OTUs in common, with only 4 unique to the ethanol samples and 10 to the bulk samples. These results indicate that fixative-based metabarcoding is a non-destructive, time-saving alternative for biodiversity assessments focussing on taxa used for ecological status determination. However, for a comprehensive assessment on total invertebrate biodiversity, the method may not be sufficient, and conventional bulk sample metabarcoding should be applied.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Biodiversity patterns are inherently complex and difficult to comprehensively assess. Yet, deciphering shifts in species composition through time and space are crucial for efficient and successful ...management of ecosystem services, as well as for predicting change. To better understand species diversity patterns, Germany participated in the Global Malaise Trap Program, a world-wide collection program for arthropods using this sampling method followed by their DNA barcode analysis. Traps were deployed at two localities: "Nationalpark Bayerischer Wald" in Bavaria, the largest terrestrial Natura 2000 area in Germany, and the nature conservation area Landskrone, an EU habitats directive site in the Rhine Valley. Arthropods were collected from May to September to track shifts in the taxonomic composition and temporal succession at these locations.
In total, 37,274 specimens were sorted and DNA barcoded, resulting in 5,301 different genetic clusters (BINs, Barcode Index Numbers, proxy for species) with just 7.6% of their BINs shared. Accumulation curves for the BIN count versus the number of specimens analyzed suggest that about 63% of the potential diversity at these sites was recovered with this single season of sampling. Diversity at both sites rose from May (496 & 565 BINs) to July (1,236 & 1,522 BINs) before decreasing in September (572 & 504 BINs). Unambiguous species names were assigned to 35% of the BINs (1,868) which represented 12,640 specimens. Another 7% of the BINs (386) with 1,988 specimens were assigned to genus, while 26% (1,390) with 12,092 specimens were only placed to a family. These results illustrate how a comprehensive DNA barcode reference library can identify unknown specimens, but also reveal how this potential is constrained by gaps in the quantity and quality of records in BOLD, especially for Hymenoptera and Diptera. As voucher specimens are available for morphological study, we invite taxonomic experts to assist in the identification of unnamed BINs.
This study summarizes results of a DNA barcoding campaign on German Diptera, involving analysis of 45,040 specimens. The resultant DNA barcode library includes records for 2,453 named species ...comprising a total of 5,200 barcode index numbers (BINs), including 2,700 COI haplotype clusters without species‐level assignment, so called “dark taxa.” Overall, 88 out of 117 families (75%) recorded from Germany were covered, representing more than 50% of the 9,544 known species of German Diptera. Until now, most of these families, especially the most diverse, have been taxonomically inaccessible. By contrast, within a few years this study provided an intermediate taxonomic system for half of the German Dipteran fauna, which will provide a useful foundation for subsequent detailed, integrative taxonomic studies. Using DNA extracts derived from bulk collections made by Malaise traps, we further demonstrate that species delineation using BINs and operational taxonomic units (OTUs) constitutes an effective method for biodiversity studies using DNA metabarcoding. As the reference libraries continue to grow, and gaps in the species catalogue are filled, BIN lists assembled by metabarcoding will provide greater taxonomic resolution. The present study has three main goals: (a) to provide a DNA barcode library for 5,200 BINs of Diptera; (b) to demonstrate, based on the example of bulk extractions from a Malaise trap experiment, that DNA barcode clusters, labelled with globally unique identifiers (such as OTUs and/or BINs), provide a pragmatic, accurate solution to the “taxonomic impediment”; and (c) to demonstrate that interim names based on BINs and OTUs obtained through metabarcoding provide an effective method for studies on species‐rich groups that are usually neglected in biodiversity research projects because of their unresolved taxonomy.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
DNA barcodes (COI) of Troglocobitis starostini, endemic to a single site in Turkmenistan, were analysed and put into the taxonomic context of the large group of nemacheilid loaches known from Western ...and Central Asia. All applied phylogenetic tree-based analyses place the species into the genus Paracobitis. This finding supports previous morphological studies. While the exact position of Troglocobitis starostini within Paracobitis was not resolved unambiguously, it was constantly recovered within Paracobitis, irrespective of the tree reconstruction method applied. With a minimum interspecific K2P distance of 7.19% P. persa was the closest hit in our dataset, which comprised a total of ten species of Paracobitis, which showed an average interspecific K2P distance of 5.43% (range 2.78-9.44%).
Mitochondrial DNA (mtDNA) sequences are often found as byproducts in next‐generation sequencing (NGS) datasets that were originally created to capture genomic or transcriptomic information of an ...organism. These mtDNA sequences are often discarded, wasting this valuable sequencing information.
We developed MitoGeneExtractor, an innovative tool which allows to extract mitochondrial protein coding genes (PCGs) of interest from NGS libraries through multiple sequence alignments of sequencing reads to amino acid references. General references, for example on order level are sufficient for mining mitochondrial PCGs. In a case study, we applied MitoGeneExtractor to recently published genomic datasets of 1993 birds and were able to extract complete or nearly complete sequences for all 13 mitochondrial PCGs for a large proportion of libraries. Compared to an existing assembly guided sequence reconstruction algorithm, MitoGeneExtractor was faster and substantially more sensitive.
We compared COI sequences mined with MitoGeneExtractor to COI databases. Mined sequences show a high sequence similarity and correct taxonomic assignment between the recovered sequence and the assigned morphospecies in most samples. In some cases of incongruent taxonomic assignments, we found evidence for contamination in NGS libraries.
MitoGeneExtractor allows a fast extraction of mitochondrial PCGs from a wide range of NGS datasets. We recommend to routinely harvest and curate mitochondrial sequence information from genomic resources. MitoGeneExtractor output can be used to identify contaminated NGS libraries and to validate the species identity of the sequenced animal based on the extracted COI sequences.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
For successful material deployment in tissue engineering, the material itself, its mechanical properties, and the microscopic geometry of the product are of particular interest. While silk is a ...widely applied protein‐based tissue engineering material with strong mechanical properties, the size and shape of artificially spun silk fibers are limited by existing processes. This study adjusts a microfluidic spinneret to manufacture micron‐sized wet‐spun fibers with three different materials enabling diverse geometries for tissue engineering applications. The spinneret is direct laser written (DLW) inside a microfluidic polydimethylsiloxane (PDMS) chip using two‐photon lithography, applying a novel surface treatment that enables a tight print‐channel sealing. Alginate, polyacrylonitrile, and silk fibers with diameters down to 1 µm are spun, while the spinneret geometry controls the shape of the silk fiber, and the spinning process tailors the mechanical property. Cell‐cultivation experiments affirm bio‐compatibility and showcase an interplay between the cell‐sized fibers and cells. The presented spinning process pushes the boundaries of fiber fabrication toward smaller diameters and more complex shapes with increased surface‐to‐volume ratio and will substantially contribute to future tailored tissue engineering materials for healthcare applications.
A 3D‐printed microfluidic wet‐spinning platform is developed to fabricate silk fibers with diameters down to 1 µm as tissue for cell culture. The fibers shape is tailored by the nozzle‐geometry and the mechanical fiber properties are controlled by the spinning process parameters.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
PDF
