On the origin of Acochlidia and other enigmatic euthyneuran gastropods, with implications for the systematics of Heterobranchia

<p>Abstract</p> <p>Background</p> <p>A robust phylogenetic hypothesis of euthyneuran gastropods, as a basis to reconstructing their evolutionary history, is still hindered by several groups of aberrant, more or less worm-like slugs with unclear phylogenetic relationships. As a traditional "order" in the Opisthobranchia, the Acochlidia have a long history of controversial placements, among others influenced by convergent adaptation to the mainly meiofaunal habitats. The present study includes six out of seven acochlidian families in a comprehensive euthyneuran taxon sampling with special focus on minute, aberrant slugs. Since there is no fossil record of tiny, shell-less gastropods, a molecular clock was used to estimate divergence times within Euthyneura.</p> <p>Results</p> <p>Our multi-locus molecular study confirms Acochlidia in a pulmonate relationship, as sister to Eupulmonata. Previous hypotheses of opisthobranch relations, or of a common origin with other meiofaunal Euthyneura, are clearly rejected. The enigmatic amphibious and insectivorous Aitengidae <it>incerta sedis </it>clusters within Acochlidia, as sister to meiofaunal and brackish Pseudunelidae and limnic Acochlidiidae. Euthyneura, Opisthobranchia and Pulmonata as traditionally defined are non-monophyletic. A relaxed molecular clock approach indicates a late Palaeozoic diversification of Euthyneura and a Mesozoic origin of the major euthyneuran diversity, including Acochlidia.</p> <p>Conclusions</p> <p>The present study shows that the inclusion of small, enigmatic groups is necessary to solve deep-level phylogenetic relationships, and underlines that "pulmonate" and "opisthobranch" phylogeny, respectively, cannot be solved independently from each other. Our phylogenetic hypothesis requires reinvestigation of the traditional classification of Euthyneura: morphological synapomorphies of the traditionally defined Pulmonata and Opisthobranchia are evaluated in light of the presented phylogeny, and a redefinition of major groups is proposed. It is demonstrated that the invasion of the meiofaunal habitat has occurred several times independently in various euthyneuran taxa, leading to convergent adaptations previously misinterpreted as synapomorphies. The inclusion of Acochlidia extends the structural and biological diversity in pulmonates, presenting a remarkable flexibility concerning habitat choice.</p

Crabs, scallops, fish, and more: barcoding the marine fauna of the North Sea

Background: During the last years, the effectiveness of DNA barcoding for animal species identification has been proven in many studies, analyzing both vertebrate and invertebrate taxa. In terms of marine organisms, however, most barcoding studies typically focus on economically relevant species, for example, fish, as well asonthedocumentationof hotspots of species diversity, for example, tropical coral reefs or regions of the almost unexplored deep sea regions. In contrast to this, species diversity of “well-known” habitats is nearly neglected. As part of our running project we started to build up a comprehensive DNA barcode library for the metazoan taxa of the North Sea, one of the most extensively studied ecosystems of the world. The North Sea is characterized by a highamountof anthropogenic pressure such as intensive fishing and ship traffic as well as offshore installations. Environmental parameters (e.g., depth, sediment characteristics, temperature, and salinity) of this semi-enclosed shelf sea follow a distinct pattern: high seasonal fluctuations can be observed in southern areas, but low fluctuations are given in the northern regions. This heterogeneity is also displayed in macrobenthic community structures, with a lower number of species in the shallow southern parts (i.e., the German Bight) and more species in the central and northern North Sea. In addition to this, species with a typical Mediterranean-Lusitanean distribution are also known to occur in parts of the North Sea where oceanic influences prevail. Results: Our barcode library includes a broad variety of taxa, including typical taxa of marine barcoding studies, for example, fish or decapod crustaceans. Our on-growing library also includes groups that are often ignored as cnidarians, parasitic crustaceans, echinoderms, mollusks, pantopods, polychaets, and others. In total, our library includes more than 4200 DNA barcodes of more than 600 species at the moment. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for more than 90% of the analyzed species. Significance: Our data represent a first step towards the establishment of a comprehensive DNA barcode library of the Metazoa of the North Sea. Despite the fact that various taxa are still missing or are currently underrepresented, our results clearly underline the usefulness of DNA barcodes to discriminate the vast majority of the analyzed species. It should be also kept in mind that the benefits of DNA barcoding are not restricted to taxonomic or systematic research only. The rise of modern high-throughput sequencing technologies will change biomonitoring applications and surveys significantly in the coming years. Following this, reference datasets such as ours will become essential for a correct identification of specimens sequenced as part of a metabarcoding study. This is especially true for the North Sea, a marine region that has been massively affected by cargo ship traffic, the exploitation of oil and gas resources, offshore wind parks, and in particular extensive long-term fisheries

Molecular techniques for identifying North Sea fauna

Accelerated biodiversity assessment is the key to understanding the relationship between biodiversity and ecosystem functioning, especially in times of rapid climate change and habitat destruction. For the marine fauna of the North Sea, morphological species identification is impaired by the small size of many taxa, morphological convergence, intraspecific variation and larval stages which often elude morphological identification. Accordingly, the use of molecular methods presents highly promising tools for fast and accurate species identification. The aim of the new established research group “molecular taxonomy of marine organisms” at the German Centre of Marine Biodiversity Research is to test and develop molecular methods for the identification of the marine fauna of the North Sea, aiding efforts to monitor biodiversity patterns and changes. The research will focus on the analysis and identification of specimens using DNA barcodes, and environmental samples, in particular zooplankton, using next-generation DNA sequencing techniques. In addition it is planned to develop molecular methods for a fast and routine identification of larvae of selected invertebrate and vertebrate taxa of economic value

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 sequence

Effective monitoring of freshwater fish

Freshwater ecosystems constitute only a small fraction of the planet’s water resources, yet support much of its diversity, with freshwater fish accounting for more species than birds, mammals, amphibians, or reptiles. Fresh waters are, however, particularly vulnerable to anthropogenic impacts, including habitat loss, climate and land use change, nutrient enrichment, and biological invasions. This environmental degradation, combined with unprecedented rates of biodiversity change, highlights the importance of robust and replicable programmes to monitor freshwater fish assemblages. Such monitoring programmes can have diverse aims, including confirming the presence of a single species (e.g. early detection of alien species), tracking changes in the abundance of threatened species, or documenting long-term temporal changes in entire communities. Irrespective of their motivation, monitoring programmes are only fit for purpose if they have clearly articulated aims and collect data that can meet those aims. This review, therefore, highlights the importance of identifying the key aims in monitoring programmes, and outlines the different methods of sampling freshwater fish that can be used to meet these aims. We emphasise that investigators must address issues around sampling design, statistical power, species’ detectability, taxonomy, and ethics in their monitoring programmes. Additionally, programmes must ensure that high-quality monitoring data are properly curated and deposited in repositories that will endure. Through fostering improved practice in freshwater fish monitoring, this review aims to help programmes improve understanding of the processes that shape the Earth's freshwater ecosystems, and help protect these systems in face of rapid environmental change

Data from: A reliable DNA barcode reference library for the identification of the 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

AlignmentBNEAF

Sequence alignment of 860 fish DNA barcode

Phyllodromica iberica Published 2007

Phyllodromica iberica morph #2 (Figs 6 A–I, 7 A–H, 8 A–G, 17) Phyllodromica subaptera morph #2. —Knebelsberger & Bohn, 2003. Material. Numerous specimens from the following localities: Spain. Sp 265, a, 266, a, 267, a, b, c, d, 268, a, 269a, 364, 365, a, 366, 512, a. Description. Size. Length of pronotum: ♂ 1.46–1.59 (mean 1.53) mm; ♀ 1.76–1.93 (mean 1.83) mm. Legs. Distal end of mid tibia with 5 spines. Male Tergites. —Tergite 6. Emargination of the posterior border narrower than in all other representatives of the group and deeper than in morph #1 (Figs 6 C, 8 A–D). Immediately behind the transversal ridge cuticle elevated to a narrow torus running in parallel to the transversal ridge (Fig. 7 A–C). The torus is interrupted in the middle and fades away laterally, shortly before the ridge disappears. Along the torus the bristles are arranged in an extraordinary density. Posteriorly the torus declines medio-laterally to two shallow longitudinal depressions (white arrows in Fig. 7 A). Tergite 7. Median lobe somewhat longer than in morph #1, but shorter than distance between bristle fields and the posterior border of the tergite, almost without glandular pores (Fig. 6 D). Trough (Figs 6 D, 7 D) less deep than in morph #1, anterior wall scarcely hollowed out anteriorly, medially relatively steep. Mound low and small, less broad than the median lobe (Fig. 6 D). Bristle fields small, longitudinally oval, immediately adjacent, rarely separated by a very narrow bristleless ridge (Fig. 6 D). The bristle fields are on both sides of the ridge deepened to shallow grooves with relative strongly declining posterior wall, appearing as a crescent-shaped black shadow posteriorly of each of the two bristle fields (white arrow heads in Fig. 6 D), but less deep than in morph #1. Tergite 8. Similar as in morph #1, but central mound lower, sinusoidal edge anteriorly less steeply declining, mound not broader than the lateral pits, with a smaller conelike process; median gap of bristles smaller (Figs 7 F–H). Tergite 10, supraanal plate triangular, more rounded than in morph #1 (Fig. 6 G). Sternites. —Paraproct. Medio-anterior process of right paraproct without bulge (Fig. 6 G). Colouration. —Tergites 2–5. Posterior extensions of the dark area usually weakly developed (Fig. 6 B) or missing. Tergite 6. Dark area not reaching the posterior border of the tergite leaving a light margin (Fig. 6 C), often with small lightenings forming a central Y - or v -shaped figure (Fig. 8 A, C) occasionally with prolonged transversal arms along the tori (Fig. 8 B, D). Tergite 7 (Fig. 6 D). Very similar to morph #1, but very dark (brown) colouration posteriorly and laterally of the mound, often extended latero-posteriorly. Tergite 8 (Fig. 6 E). Very similar to morph #1, but dark area posteriorly less extended with median and mediolateral extensions. Conical process lighter coloured than in morph #1. Female (Fig. 8 F, G) For description of legs, genitalia and colouration see description of P. subaptera. Genetic Data. 1597 basepairs of the mitochondrial genome including the complete cytochrome c oxidase subunit I (COI) gene were analysed in two specimens (Sp 267d/M5, Sp 267d/M6). DNA and associated parts of body are stored in the DNATAX collection of the ZSM under storage numbers DNATAX02863 (Sp 267d/ M5) and DNATAX02864 (Sp 267d/M6). The sequences were submitted to GenBank under the accession numbers: AM 600687 (DNATAX02863) and AM 600688 (DNATAX02864). The sequences of P. iberica morph #2_ Sp 267d/M5_1597bp and P. iberica morph #2 Sp 267d/ M6_1597bp differ in one position (Appendix 5). The complete sequences are shown in Appendix 4. Geographical distribution (Fig. 17). Small distribution area in eastern Spain, running ± parallel to the coast between Rio Ebro and Rio Túria. Other species of the subaptera -group found. P subaptera: Sp 365. Other morphs of P.iberica found. Morph #1: Sp 366, 512; morph #3: Sp 512.Published as part of KNEBELSBERGER, THOMAS & MILLER, MICHAEL A., 2007, Revision and phylogeny of the subaptera-group of Phyllodromica (Blattoptera: Blattellidae: Ectobiinae), including a parthenogenetic species and the evaluation of COI sequences for species identification (DNA barcoding), pp. 1-68 in Zootaxa 1522 (1) on pages 15-16, DOI: 10.11646/zootaxa.1522.1.1, http://zenodo.org/record/508776

How reliably can northeast Atlantic sand lances of the genera Ammodytes and Hyperoplus be distinguished? A comparative application of morphological and molecular methods

Accurate stock assessments for each of the dominant species of sand lances in the northeast Atlantic Ocean and adjacent areas are not available due to the lack of a reliable identification procedure; therefore, appropriate measures of fisheries management or conservation of sand lances cannot be implemented. In this study, detailed morphological and molecular features are assessed to discriminate between four species of sand lances belonging to the genera Ammodytes and Hyperoplus. Morphological characters described by earlier authors as useful for identification of the genera are confirmed, and two additional distinguishing characters are added. A combination of the following morphological characters is recommended to distinguish between the genera Hyperoplus and Ammodytes: the protrusibility of the premaxillae, the presence of hooked ends of the prevomer, the number of dermal plicae, and the pectoral-fin length as a percentage of the standard length. The discriminant function analysis revealed that morphometric data are not very useful to distinguish the species of each of the two genera. The following meristic characters improve the separation of H. lanceolatus from H. immaculatus: the number of lower arch gill rakers, total number of gill rakers, numbers of caudal vertebrae and total vertebrae, and numbers of dorsal-fin and anal-fin rays. It is confirmed that A. tobianus differs from A. marinus by its belly scales that are organised in tight chevrons, scales which are present over the musculature at the base of the caudal fin, as well as by the lower numbers of dermal plicae, dorsal-fin rays, and total vertebrae. In contrast to the morphological data, mitochondrial COI sequences (DNA barcodes) failed to separate unambiguously the four investigated species. Ammodytes tobianus and H. lanceolatus showed an overlap between intraspecific and interspecific K2P genetic distances and cannot be reliably distinguished using the common DNA barcoding approach. Ammodytes marinus and H. immaculatus exhibited gaps between intraspecific and interspecific K2P distances of 2.73 and 3.34% respectively, indicating that their DNA barcodes can be used for species identification. As an alternative, short nuclear Rhodopsin sequences were analysed and one diagnostic character was found for each of the species A. marinus, H. lanceolatus, and H. immaculatus. Ammodytes tobianus can be characterised by the lack of species-specific mutations when compared to the other three species. In contrast to COI, the short nuclear sequences represent a useful alternative for rapid species identification whenever an examination of morphological characters is not available