Recent years have seen increased survey and sampling expeditions to the Clarion-Clipperton Zone (CCZ), central Pacific Ocean abyss, driven by commercial interests from contractors in the potential extraction of polymetallic nodules in the region. Part of the International Seabed Authority (ISA) regulatory requirements are that these contractors undertake environmental research expeditions to their CCZ exploration claims following guidelines approved by the ISA Legal and Technical Commission (ISA, 2010). Section 9 (e) of these guidelines instructs contractors to " . . . collect data on the sea floor communities specifically relating to megafauna, macrofauna, meiofauna, microfauna, nodule fauna and demersal scavengers". There are a number of methodological challenges to this, including the water depth (4000-5000 m), extremely warm surface waters (~28˝C) compared to bottom water (~1.5˝C) and great distances to ports requiring a large and long seagoing expedition with only a limited number of scientists. Both scientists and regulators have recently realized that a major gap in our knowledge of the region is the fundamental taxonomy of the animals that live there; this is essential to inform our knowledge of the biogeography, natural history and ultimately our stewardship of the region. Recognising this, the ISA is currently sponsoring a series of taxonomic workshops on the CCZ fauna and to assist in this process we present here a series of methodological pipelines for DNA taxonomy (incorporating both molecular and morphological data) of the macrofauna and megafauna from the CCZ benthic habitat in the recent ABYSSLINE cruise program to the UK-1 exploration claim. A major problem on recent CCZ cruises has been the collection of high-quality samples suitable for both morphology and DNA taxonomy, coupled with a workflow that ensures these data are made available. The DNA sequencing techniques themselves are relatively standard, once good samples have been obtained. The key to quality taxonomic work on macrofaunal animals from the tropical abyss is careful extraction of the animals (in cold, filtered seawater), microscopic observation and preservation of live specimens, from a variety of sampling devices by experienced zoologists at sea. Essential to the long-term iterative building of taxonomic knowledge from the CCZ is an "end-to-end" methodology to the taxonomic science that takes into account careful sampling design, at-sea taxonomic identification and fixation, post-cruise laboratory work with both DNA and morphology and finally a careful sample and data management pipeline that results in specimens and data in accessible open museum collections and online repositories.A key goal of our sampling protocol for the CCZ is the study of both the DNA sequences and morphology of the animals. Both the genetic sequences of animals, and their external shape and morphology hold huge amounts of useful data for understanding their biology. Over the last 250 years since Linnaeus, humans have been busy building a remarkable and vas...

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.

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 Clarion-Clipperton Zone (CCZ), located in the abyssal equatorial Pacific, has been subject to intensive international exploration for polymetallic nodule mining over the last four decades. Many studies have investigated the potential effects of mining on deep-sea ecosystems and highlighted the importance of defining environmental baseline conditions occurring at potential mining sites. However, current information on biodiversity and species distributions in the CCZ is still scarce and hampers the ability to effectively manage and reduce the potential impacts of mining activities. As part of the regulatory regimes adopted by the International Seabed Authority, concession holders are required to conduct an environmental impact assessment and gather baseline data on biodiversity and community structure in relation to their license areas. In the present study, we used an integrative molecular and morphological approach to assess species richness and genetic variation of deep-sea scavenging amphipods collected in two nodule-mining exploration areas (UK-1 and OMS-1 areas) and one Area of Particular Environmental Interest (APEI-6) in the eastern part of the CCZ. We analyzed the DNA sequences of the cytochrome c oxidase subunit I gene of 645 specimens belonging to ten distinct morphospecies. Molecular data uncover potential cryptic diversity in two investigated species, morphologically identified as Paralicella caperesca Shulenberger & Barnard, 1976 and Valettietta cf. anacantha (Birstein & Vinogradov, 1963). Our study highlights the importance of using molecular tools in conjunction with traditional morphological methods for modern biodiversity assessment studies, particularly to evaluate morphologically similar individuals and incomplete specimens. The results of this study can help determine species identity and ranges, information which can feed into environmental management.

During the expedition ANT XIX/3 meiofauna samples were collected from the German research vessel Polarstern near the Shackleton Fracture Zone. During sorting of the samples 86 tantulus larvae were found. Extensive examination of the larvae revealed a high diversity of tantulocaridans in the Southern Ocean deep sea (33 species). A remarkable proportion of these were new species of Tantulacus Huys, Andersen & Kristensen, 1992. The present paper reports the discovery of three new Antarctic tantulocarids which are referred to Tantulacus. The affiliation of T. longispinosus n. sp., T. karolae n. sp. and T. dieteri n. sp. to Tantulacus is straightforward: all representatives of the Tantulocarida are characterised by the presence of 1-2 slender setae on the endopod of the second to fifth thoracopods, but in none of the hitherto described genera, other than Tantulacus, are these elements modified. Tantulacus hoegi Huys, Andersen & Kristensen, 1992 and the three new species share the possession of a distal rigid spine on the endopod of the second to fifth thoracopods as a synapomorphy and thus can be readily distinguished from other tantulocaridans. This is the first record of free-living sediment-inhabiting tantulus larvae from this area, although this probably reflects the degree of undersampling.