Global arthropod beta-diversity is spatially and temporally structured by latitude.

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.

Repeated subsamples during DNA extraction reveal increased diversity estimates in DNA metabarcoding of Malaise traps.

With increased application of DNA metabarcoding in biodiversity assessment, various laboratory protocols have been optimized, and their further evaluation is subject of current research. Homogenization of bulk samples and subsequent DNA extraction from a subsample of destructed tissue is a common first stage of the metabarcoding process. This can either be conducted using sample material soaked in a storage fixative, e.g., ethanol (here referred to as "wet" treatment) or from dried individuals ("dry"). However, it remains uncertain if perfect mixing and equal distribution of DNA within the tube is ensured during homogenization and to what extent incomplete mixing and resulting variations in tissue composition affect diversity assessments if only a fraction of the destructed sample is processed in the downstream metabarcoding workflow. Here we investigated the efficiency of homogenization under wet and dry conditions and tested how variations in destructed tissue composition might affect diversity assessments of complex arthropod samples. We considered five time intervals of Malaise trap bulk samples and process nine different subsamples of homogenized tissue (20 mg each) in both treatments. Results indicate a more consistent diversity assessment from dried material, but at the cost of a higher processing time. Both approaches detected comparable OTU diversity and revealed similar taxa compositions in a single tissue extraction. With an increased number of tissue subsamples during DNA extraction, OTU diversity increased for both approaches, especially for highly diverse samples obtained during the summer. Here, particularly the detection of small and low-biomass taxa increased. The processing of multiple subsamples in the metabarcoding protocol can therefore be a helpful procedure to enhance diversity estimates and counteract taxonomic bias in biodiversity assessments. However, the process induces higher costs and time effort and the application in large-scale biodiversity assessment, e.g., in monitoring schemes needs to be considered on project-specific prospects.

A practical approach to revise the Oxynoemacheilus bergianus species group (Teleostei: Nemacheilidae).

The Oxynoemacheilus bergianus species group is revised based on tree topology (ML, NJ, MP), distance (K2P and ASAP) and Poisson tree process analyses of DNA barcode data tested against morphometric and morphological characters including colour patterns. The O. bergianus species group is distinguished from other Oxynoemacheilus groups based on morphological characters: its constituent species have a slender caudal peduncle, a suborbital flap in the male, a mottled or blotched colour pattern, and lack bold, black spots on the caudal-fin base. It is also supported as a monophyletic unit in our molecular analysis. The O. bergianus group includes 10 molecular clades following congruently well-supported NJ, MP and ML based entities. Species described as O. bergianus, O. banarescui, O. erdali, O. fatsaensis, O. samanticus, and O. simavicus from Turkey, O. lenkoranensis from Azerbaijan, and O. longipinnis and O. parvinae from Iran belong to this species group. The group includes also four unnamed molecular clades. We were unable to detect external differences between any of the molecular clades in colour pattern or any morphometric or morphological characters examined. In the 10 molecular clades in the O. bergianus species group, the intraclade K2P distance ranges from 0.01.8% while the distances between molecular clades ranges from 0.65.9%. To resolve the species diversity of this group, we also analysed the intraspecific and interspecific variability in the K2P distance of DNA barcode data from 53 other Oxynoemacheilus species. Here, the intraspecific variability ranges from 0.02.4% while the interspecific K2P distance ranges from 1.220.8%. In the O. bergianus species group, only four groups are detected by the mPTP species delimitation approach distinguished by a K2P distance of 2.9% or more. We treat these four groups as valid species, corresponding to O. banarescui, O. bergianus, O. fatsaensis, and O. simavicus. Oxynoemacheilus samanticus from the Kzlrmak and Seyhan drainages, O. lenkoranensis from the Caspian basin, O. erdali from the Euphrates, and O. longipinnis and O. parvinae from the Tigris drainage are treated as synonyms of O. bergianus. Fishes from an unnamed molecular clade from the upper Tigris, and from a second unnamed clade from the upper Euphrates, are both identified as O. bergianus. Oxynoemacheilus bergianus might be a junior synonym of O. bergi from the Kura. The distribution range of O. simavicus, described from the Simav drainage in the Marmara basin, is expanded to the east and two molecularly differentiated population groups occur in the Sakarya drainage, the Byk Melen River and potentially in other adjacent coastal streams. Oxynoemacheilus fatsaensis, described from the coastal stream Eleki in northern Anatolia, is also widespread in the Yeilrmak drainage. Morphological characters proposed to distinguish O. fatsaensis from the other species of the O. bergianus group could not be confirmed by our data on fishes from the Yeilrmak. This study also discusses the theoretical background, our reasons for conducting this revision in the way we did, and what the alternatives would be.

DNA barcode data confirm the placement of subterranean Noemacheilus (Troglocobitis) starostini Parin 1983 in the genus Paracobitis (Teleostei, Nemacheilidae).

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%).

The Glyptothorax catfishes of the Euphrates and Tigris with the description of a new species (Teleostei: Sisoridae).

The Glyptothorax species inhabiting the Euphrates and Tigris drainages are reviewed and six species are recognised, one of which is described herein as new species. Glyptothorax armeniacus is endemic to headwater streams in the Euphrates drainage. Glyptothorax kurdistanicus is endemic to the upper Tigris downstream to the Lesser Zab drainage. Glyptothorax cous and G. steindachneri are riverine species widespread in both the Euphrates and Tigris drainages. Glyptothorax silviae is endemic to Iran. Glyptothorax daemon, new species, from the Greater Zab and Yanarsu in the upper Tigris drainage, is distinguished by having the thoracic adhesive apparatus strongly elevated, 1.11.2 times longer than wide, without tubercles on the head, well developed anteromedial striae, the medial pit without striae, and a short adipose fin. Glyptothorax daemon is separated into two mitochondrial lineages, externally indistinguishable and separated by a minimum K2P distance of 2.0% in the DNA barcode region. These lineages are paraphyletic in our analysis indicating past introgressive hybridisation with G. cous. All six species are diagnosed and all, except unstudied G. steindachneri, form distinct mitochondrial clades with between 1.2% and 3.4% minimum K2P distance between them. Species from the Euphrates and Tigris form a monophyletic mitochondrial group separated from 53 other Glyptothorax species studied from India and areas further east.

emOxynoemacheilus/em emphasicus/em, a new nemacheilid loach from the eastern Black Sea basin with some remarks on other Caucasian emOxynoemacheilus/em (Teleostei: Nemacheilidae).

Oxynoemacheilus phasicus, new species, is described from the Rioni and Enguri River drainages in Georgia. It is distinguished from other Oxynoemacheilus species in the O. brandtii group by a mottled or marbled flank pattern in adults, a slightly emarginate caudal fin, and a deep caudal peduncle. Molecular data suggest that the new species is characterized by a minimum K2P distance of 7.5% from O. brandtii from the Kura drainage in the mtDNA COI barcode region. Oxynoemacheilus brandtii and O. elsae are re-diagnosed. A very slender Oxynoemacheilus from the Aras drainage clusters as sister to O. elsae in our molecular analysis and not with O. brandtii from the Kura River. However, it is identified as O. brandtii as it is indistinguishable from this species in morphological characters.

Oxynoemacheilus shehabi, a new nemacheilid loach from the upper Orontes in southern Syria (Teleostei: Nemacheilidae).

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.

Adding DNA barcoding to stream monitoring protocols - What's the additional value and congruence between morphological and molecular identification approaches?

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.

Revision of the genus Seminemacheilus, with the description of three new species (Teleostei: Nemacheilidae).

The nemacheilid genus Seminemacheilus is revised. Six species are recognised, three of them described herein as new. All species are endemic to Central Anatolia. Seminemacheilus lendlii from the Sakarya River drainage and the endorheic Lake Aksehir and Eber basins, S. ispartensis from Lake Egirdir basin and S. ahmeti from Sultan Sazligi are valid species. Although Seminemacheilus lendlii and S. ispartensis have almost identical COI sequences, they are distinguished by the shape of the caudal peduncle and the presence of scales on the caudal peduncle in S. ispartensis (vs. absent in S. lendlii). Seminemacheilus attalicus, new species, from Kirkgöz drainage, is distinguished by having a slightly emarginated caudal fin, a central pore in the supratemporal canal and a marbled flank pattern. Seminemacheilus ekmekciae, new species, from Lake Tuz basin is distinguished by having a roundish caudal fin, a short post-dorsal length and large brown blotches fused into stripes on the flank. Seminemacheilus tubae, new species, from Lake Beysehir basin, is distinguished by having a truncate caudal fin and 2-5 (6) supraorbital head pores. Seminemacheilus dursunavsari from the Göksu River drainage is not a valid name and this population is identified as S. tubae.

Pseudophoxinus cilicicus, a new spring minnow from southern Anatolia (Teleostei: Leuciscidae).

Pseudophoxinus cilicicus, new species, is described from the Arsuz, Ceyhan and Seyhan river drainages in the Gulf of Iskenderun. It is distinguished from other members of the Pseudophoxinus zeregi species group by having a complete lateral line with 38-45 + 2-3 scales, the lower lip usually slightly projecting beyond the tip of the upper lip, a prominent black stripe along the flank, and no black pigments below the lateral line. Pseudophoxinus cilicicus is distinguished from P. zekayi by a minimum K2P distance of 3.8% based on the mitochondrial DNA barcode region. Pseudophoxinus atropatenus and P. sojuchbulagi are returned to the genus Rutilus.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.

A DNA barcode library for 5,200 German flies and midges (Insecta: Diptera) and its implications for metabarcoding-based biomonitoring.

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.

Paracobitis abrishamchiani, a new crested loach from the southern Caspian Sea basin (Teleostei: Nemacheilidae).

Paracobitis abrishamchiani, new species, is described from the southern Caspian Sea basin, where it is found in the Babol, Haraz, Siah, Tajan, Talar and Kashpal River drainages. It is distinguished from P. hircanica and P. atrakensis, the two other Paracobitis species known from the Caspian Sea basin, by having scales on the flank posterior to the dorsal-fin origin and from its congeners in the Middle East by the position of its dorsal-fin origin, having a roundish posterior narial opening, and a colour pattern consisting of many widely spaced brown spots and blotches. It is further characterized by six diagnostic nucleotide substitutions and a minimum K2P distance of 4.3% to P. persa and 4.6% to P. malapterura in the mtDNA COI barcode region.

DNA metabarcoding from sample fixative as a quick and voucher-preserving biodiversity assessment method 1.

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.

Alburnus goekhani, a new species of bleak from the Anatolian Black Sea basin (Teleostei: Leuciscidae).

Alburnus goekhani, new species, is described from the Yesilirmak and Kizilirmak River drainages. It belongs to the A. alburnus species group and is distinguished from other species in this group in Anatolia by having 48-56 + 2-3 scales in the lateral-line, 12-15 gill rakers, a distinct dark lateral stripe along the flank on live and preserved specimens and the anal-fin origin situated below the branched dorsal-fin ray 6-8. Alburnus goekhani is also well distinguished from other Alburnus species by its DNA barcode sequence.

Ancient connections among the European rivers and watersheds revealed from the evolutionary history of the genus Telestes (Actinopterygii; Cypriniformes).

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.

Testing the Global Malaise Trap Program - How well does the current barcode reference library identify flying insects in Germany?

BACKGROUND: 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. NEW INFORMATION: 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.

Eidinemacheilus proudlovei, a new subterranean loach from Iraqi Kurdistan (Teleostei; Nemacheilidae).

Eidinemacheilus proudlovei, new species, is described from subterranean waters in the Little Zab River drainage in Iraqi Kurdistan. After the discovery of E. smithi in 1976, E. proudlovei is the second troglomorphic nemacheilid loach found in the Middle East and the second species placed in Eidinemacheilus. Eidinemacheilus proudlovei is distinguished from E. smithi by having 8+8 or 8+7 branched caudal-fin rays, no adipose keel on the caudal peduncle, enlarged jaws and a fully developed head canal system. It furthers differs substantially in its DNA barcode (>8% K2P distance) from all other nemacheilid loaches in the Middle East, Europe and Western India.

Squalius namak, a new chub from Lake Namak basin in Iran (Teleostei: Cyprinidae).

Squalius namak, new species, from the endorheic Lake Namak and Kavir basins in Iran, is distinguished from the species of the genus Squalius in the Persian Gulf and the southern Caspian Sea basins by having a wide and thick symphysial knob on the lower jaw, a convex posterior anal-fin margin, a bold, dark-grey or brown, roundish or crescent-shaped blotch at the posterior tip of each flank scale and orange caudal-, anal- and pelvic-fin rays in life. Squalius namak is also characterized by four fixed, diagnostic nucleotide substitutions in the mtDNA COI barcode region.

Garra sindhi, a new species from the Jebel Samhan Nature Reserve in Oman (Teleostei: Cyprinidae).

Garra sindhi, new species, is described from Wadi Andhur in the Dhofar province of the Sultanate of Oman. It is closely related to G. dunsirei and G. smarti, two other species from the same Omani province. It differs from these species by the following combination of characters: breast covered by scales, a shallow hump on back, eyes normally developed and 8-12 gill rakers on the lower limb of the first gill arch.