A universal tool for marine metazoan species identification: towards best practices in proteomic fingerprinting.

Proteomic fingerprinting using MALDI-TOF mass spectrometry is a well-established tool for identifying microorganisms and has shown promising results for identification of animal species, particularly disease vectors and marine organisms. And thus can be a vital tool for biodiversity assessments in ecological studies. However, few studies have tested species identification across different orders and classes. In this study, we collected data from 1246 specimens and 198 species to test species identification in a diverse dataset. We also evaluated different specimen preparation and data processing approaches for machine learning and developed a workflow to optimize classification using random forest. Our results showed high success rates of over 90%, but we also found that the size of the reference library affects classification error. Additionally, we demonstrated the ability of the method to differentiate marine cryptic-species complexes and to distinguish sexes within species.

Highly structured populations of deep-sea copepods associated with hydrothermal vents across the Southwest Pacific, despite contrasting life history traits.

Hydrothermal vents are extreme environments, where abundant communities of copepods with contrasting life history traits co-exist along hydrothermal gradients. Here, we discuss how these traits may contribute to the observed differences in molecular diversity and population genetic structure. Samples were collected from vent locations across the globe including active ridges and back-arc basins and compared to existing deep-sea hydrothermal vent and shallow water data, covering a total of 22 vents and 3 non-vent sites. A total of 806 sequences of mtDNA from the Cox1 gene were used to reconstruct the phylogeny, haplotypic relationship and demography within vent endemic copepods (Dirivultidae, Stygiopontius spp.) and non-vent-endemic copepods (Ameiridae, Miraciidae and Laophontidae). A species complex within Stygiopontius lauensis was studied across five pacific back-arc basins at eight hydrothermal vent fields, with cryptic species being restricted to the basins they were sampled from. Copepod populations from the Lau, North Fiji and Woodlark basins are undergoing demographic expansion, possibly linked to an increase in hydrothermal activity in the last 10 kya. Highly structured populations of Amphiascus aff. varians 2 were also observed from the Lau to the Woodlark basins with populations also undergoing expansion. Less abundant harpacticoids exhibit little to no population structure and stable populations. This study suggests that similarities in genetic structure and demography may arise in vent-associated copepods despite having different life history traits. As structured meta-populations may be at risk of local extinction should major anthropogenic impacts, such as deep-sea mining, occur, we highlight the importance of incorporating a trait-based approach to investigate patterns of genetic connectivity and demography, particularly regarding area-based management tools and environmental management plans.

Potential of MALDI-TOF MS-based proteomic fingerprinting for species identification of Cnidaria across classes, species, regions and developmental stages.

Morphological identification of cnidarian species can be difficult throughout all life stages due to the lack of distinct morphological characters. Moreover, in some cnidarian taxa genetic markers are not fully informative, and in these cases combinations of different markers or additional morphological verifications may be required. Proteomic fingerprinting based on MALDI-TOF mass spectra was previously shown to provide reliable species identification in different metazoans including some cnidarian taxa. For the first time, we tested the method across four cnidarian classes (Staurozoa, Scyphozoa, Anthozoa, Hydrozoa) and included different scyphozoan life-history stages (polyp, ephyra, medusa) in our dataset. Our results revealed reliable species identification based on MALDI-TOF mass spectra across all taxa with species-specific clusters for all 23 analysed species. In addition, proteomic fingerprinting was successful for distinguishing developmental stages, still by retaining a species specific signal. Furthermore, we identified the impact of different salinities in different regions (North Sea and Baltic Sea) on proteomic fingerprints to be negligible. In conclusion, the effects of environmental factors and developmental stages on proteomic fingerprints seem to be low in cnidarians. This would allow using reference libraries built up entirely of adult or cultured cnidarian specimens for the identification of their juvenile stages or specimens from different geographic regions in future biodiversity assessment studies.

Perspectives of species identification by MALDI-TOF MS in monitoring-Stability of proteomic fingerprints in marine epipelagic copepods.

We analysed the robustness of species identification based on proteomic composition to data processing and intraspecific variability, specificity and sensitivity of species-markers as well as discriminatory power of proteomic fingerprinting and its sensitivity to phylogenetic distance. Our analysis is based on MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry) data from 32 marine copepod species coming from 13 regions (North and Central Atlantic and adjacent seas). A random forest (RF) model correctly classified all specimens to the species level with only small sensitivity to data processing, demonstrating the strong robustness of the method. Compounds with high specificity showed low sensitivity, that is identification was based on complex pattern-differences rather than on presence of single markers. Proteomic distance was not consistently related to phylogenetic distance. A species-gap in proteome composition appeared at 0.7 Euclidean distance when using only specimens from the same sample. When other regions or seasons were included, intraspecific variability increased, resulting in overlaps of intra and inter-specific distance. Highest intraspecific distances (>0.7) were observed between specimens from brackish and marine habitats (i.e., salinity probably affects proteomic patterns). When testing library sensitivity of the RF model to regionality, strong misidentification was only detected between two congener pairs. Still, the choice of reference library may have an impact on identification of closely related species and should be tested before routine application. We envisage high relevance of this time- and cost-efficient method for future zooplankton monitoring as it provides not only in-depth taxonomic resolution for counted specimens but also add-on information, such as on developmental stage or environmental conditions.

Investigating the benthic megafauna in the eastern Clarion Clipperton Fracture Zone (north-east Pacific) based on distribution models predicted with random forest.

The eastern Clarion Clipperton Fracture Zone (CCZ) is a heterogeneous abyssal environment harbouring relatively low abundances of highly diverse megafauna communities. Potential future mining of polymetallic nodules threatens these benthic communities and calls for detailed spatial investigation of megafauna. Based on the predicted probability of occurrence of 68 megafauna morphotypes, a seabed area extending over 62,000 km2 was divided into three assemblages covering an eastern plain area, a deeper western plain area and an area covering both seamount and abyssal hill sites. Richness, estimated as the sum of morphotypes with a predicted probability of occurrence larger than 0.5, amounts to 15.4 of 68 morphotypes. Highest richness was predicted at seamount sites, and lowest richness in the western part of the study area. Combining the predicted probability of megafauna occurrences with bathymetric variables, two seamount habitats and two plain habitats could be defined. One of these megafauna plain habitats corresponds with contiguous nodule fields of high abundance that may be targeted for future mining, showing that prospective nodule fields have a clearly differentiated megafauna assemblage. Monitoring and management schemes, including the delineation of preservation and protection areas within contract areas, need to incorporate this geological and biological heterogeneity.

Recent speciation and hybridization in Icelandic deep-sea isopods: An integrative approach using genomics and proteomics.

The crustacean marine isopod species Haploniscus bicuspis (Sars, 1877) shows circum-Icelandic distribution in a wide range of environmental conditions and along well-known geographic barriers, such as the Greenland-Iceland-Faroe (GIF) Ridge. We wanted to explore population genetics, phylogeography and cryptic speciation as well as investigate whether previously described, but unaccepted subspecies have any merit. Using the same set of specimens, we combined mitochondrial COI sequences, thousands of nuclear loci (ddRAD), and proteomic profiles, plus selected morphological characters using confocal laser scanning microscopy (CLSM). Five divergent genetic lineages were identified by COI and ddRAD, two south and three north of the GIF Ridge. Assignment of populations to the three northern lineages varied and detailed analyses revealed hybridization and gene flow between them, suggesting a single northern species with a complex phylogeographic history. No apparent hybridization was observed among lineages south of the GIF Ridge, inferring the existence of two more species. Differences in proteomic profiles between the three putative species were minimal, implying an ongoing or recent speciation process. Population differentiation was high, even among closely associated populations, and higher in mitochondrial COI than nuclear ddRAD loci. Gene flow is apparently male-biased, leading to hybrid zones and instances of complete exchange of the local nuclear genome through immigrating males. This study did not confirm the existence of subspecies defined by male characters, which probably instead refer to different male developmental stages.

Megafauna of the German exploration licence area for seafloor massive sulphides along the Central and South East Indian Ridge (Indian Ocean).

BACKGROUND: The growing interest in mineral resources of the deep sea, such as seafloor massive sulphide deposits, has led to an increasing number of exploration licences issued by the International Seabed Authority. In the Indian Ocean, four licence areas exist, resulting in an increasing number of new hydrothermal vent fields and the discovery of new species. Most studies focus on active venting areas including their ecology, but the non-vent megafauna of the Central Indian Ridge and South East Indian Ridge remains poorly known.In the framework of the Indian Ocean Exploration project in the German license area for seafloor massive sulphides, baseline imagery and sampling surveys were conducted yearly during research expeditions from 2013 to 2018, using video sledges and Remotely Operated Vehicles. NEW INFORMATION: This is the first report of an imagery collection of megafauna from the southern Central Indian- and South East Indian Ridge, reporting the taxonomic richness and their distribution. A total of 218 taxa were recorded and identified, based on imagery, with additional morphological and molecular confirmed identifications of 20 taxa from 89 sampled specimens. The compiled fauna catalogue is a synthesis of megafauna occurrences aiming at a consistent morphological identification of taxa and showing their regional distribution. The imagery data were collected during multiple research cruises in different exploration clusters of the German licence area, located 500 km north of the Rodriguez Triple Junction along the Central Indian Ridge and 500 km southeast of it along the Southeast Indian Ridge.

Proteomic fingerprinting facilitates biodiversity assessments in understudied ecosystems: A case study on integrated taxonomy of deep sea copepods.

Accurate and reliable biodiversity estimates of marine zooplankton are a prerequisite to understand how changes in diversity can affect whole ecosystems. Species identification in the deep sea is significantly impeded by high numbers of new species and decreasing numbers of taxonomic experts, hampering any assessment of biodiversity. We used in parallel morphological, genetic, and proteomic characteristics of specimens of calanoid copepods from the abyssal South Atlantic to test if proteomic fingerprinting can accelerate estimating biodiversity. We cross-validated the respective molecular discrimination methods with morphological identifications to establish COI and proteomic reference libraries, as they are a pre-requisite to assign taxonomic information to the identified molecular species clusters. Due to the high number of new species only 37% of the individuals could be assigned to species or genus level morphologically. COI sequencing was successful for 70% of the specimens analysed, while proteomic fingerprinting was successful for all specimens examined. Predicted species richness based on morphological and molecular methods was 42 morphospecies, 56 molecular operational taxonomic units (MOTUs) and 79 proteomic operational taxonomic units (POTUs), respectively. Species diversity was predicted based on proteomic profiles using hierarchical cluster analysis followed by application of the variance ratio criterion for identification of species clusters. It was comparable to species diversity calculated based on COI sequence distances. Less than 7% of specimens were misidentified by proteomic profiles when compared with COI derived MOTUs, indicating that unsupervised machine learning using solely proteomic data could be used for quickly assessing species diversity.

Copepods and ostracods associated with bromeliads in the Yucatán Peninsula, Mexico.

A substantial fraction of the freshwater available in the Neotropical forests is enclosed within the rosettes of bromeliads that form small aquatic islands within a terrestrial landscape. These aquatic oases provide shelter, water, nutrients and resting of aggregation sites for several aquatic organisms, among them crustaceans. However, in comparison with the multitude of studies on open aquatic systems, our knowledge on crustaceans inhabiting semi-terrestrial habitats and phytotelmata is limited and their presence in such environments is poorly understood. The present study was carried out in two natural protected areas of the Yucatán Peninsula aiming to understand the diversity and dispersal strategies of crustaceans living in bromeliads. Sediment and water contained in four species of bromeliads have been collected in order to understand the diversity and dispersal strategies of crustaceans living in such habitats. From a total of 238 bromeliads surveyed, 55% were colonized by crustaceans. Sixteen copepod, three ostracod and one branchiopod species were recorded during this study, however only seven species are considered as true bromeliad inhabitants. Different degrees of association between crustaceans and bromeliad species were assessed with an indicator species analysis, where significant associations were found for all crustaceans. We found significant differences between bromeliad species and reserves and their associated fauna. In order to analyze the genetic diversity of this fauna, we sequenced several individuals of each species with two genetic markers (18S rRNA and COI mtDNA). Bayesian analyses and the Generalized Mixed Yule Coalescent method (GMYC), delimited 7 well supported species. A comparison of the dispersal strategies used by different species, including passive dispersal, phoretic behavior and active dispersal, is included. This study stresses the need of studying meiofauna of phytotelms, which could be used as an indicator of local diversity in Neotropical forests.

Rapid species level identification of fish eggs by proteome fingerprinting using MALDI-TOF MS.

Quantifying spawning biomass of commercially relevant fish species is important to generate fishing quotas. This will mostly rely on the annual or daily production of fish eggs. However, these have to be identified precisely to species level to obtain a reliable estimate of offspring production of the different species. Because morphological identification can be very difficult, recent developments are heading towards application of molecular tools. Methods such as COI barcoding have long handling times and cause high costs for single specimen identifications. In order to test MALDI-TOF MS, a rapid and cost-effective alternative for species identification, we identified fish eggs using COI barcoding and used the same specimens to set up a MALDI-TOF MS reference library. This library, constructed from two different MALDI-TOF MS instruments, was then used to identify unknown eggs from a different sampling occasion. By using a line of evidence from hierarchical clustering and different supervised identification approaches we obtained concordant species identifications for 97.5% of the unknown fish eggs, proving MALDI-TOF MS a good tool for rapid species level identification of fish eggs. At the same time we point out the necessity of adjusting identification scores of supervised methods for identification to optimize identification success. SIGNIFICANCE: Fish products are commercially highly important and many societies rely on them as a major food resource. Over many decades stocks of various relevant fish species have been reduced due to unregulated overfishing. Nowadays, to avoid overfishing and threatening of important fish species, fish stocks are regularly monitored. One component of this monitoring is the monitoring of spawning stock sizes. Whereas this is highly dependent on correct species identification of fish eggs, morphological identification is difficult because of lack of morphological features.

The BenBioDen database, a global database for meio-, macro- and megabenthic biomass and densities.

Benthic fauna refers to all fauna that live in or on the seafloor, which researchers typically divide into size classes meiobenthos (32/64 µm-0.5/1 mm), macrobenthos (250 µm-1 cm), and megabenthos (>1 cm). Benthic fauna play important roles in bioturbation activity, mineralization of organic matter, and in marine food webs. Evaluating their role in these ecosystem functions requires knowledge of their global distribution and biomass. We therefore established the BenBioDen database, the largest open-access database for marine benthic biomass and density data compiled so far. In total, it includes 11,792 georeferenced benthic biomass and 51,559 benthic density records from 384 and 600 studies, respectively. We selected all references following the procedure for systematic reviews and meta-analyses, and report biomass records as grams of wet mass, dry mass, or ash-free dry mass, or carbon per m2 and as abundance records as individuals per m2. This database provides a point of reference for future studies on the distribution and biomass of benthic fauna.

Genus level molecular phylogeny of Aegisthidae Gisbrecht, 1893 (Copepoda: Harpacticoida) reveals morphological adaptations to deep-sea and plagic habitats.

BACKGROUND: The family Aegisthidae is known as typical component of deep-sea hyperbenthic waters that gradually colonized other marine environments. The phylogenetic relationships within this family have been examined here including hyperbenthic, planktonic, benthic forms and two associated Aegisthidae species. RESULTS: Ninety four specimens belong to 14 genera were studied using 18S and 28S rRNA and COI mtDNA. Bayesian analysis supports the monophyly of 10 genera whereas Andromastax, Jamstecia, Nudivorax and Aegisthus revealed to be paraphyletic. The first offshoot of the phylogenetic tree is a clade consists of the undescribed genus Aegisthidae gen.1 sister to the two monophyletic genera Cerviniella and Hase, whereas the other Cerviniinae members (represented by Cervinia and Expansicervinia) assemble a monophylum, sister to the hyperbenthic and planktonic aegisthid genera, resulting in the paraphyly of the subfamily Cerviniinae. Hence, we defined the new subfamily Cerviniellinae subfam. nov. encompassing the three benthic genera Cerviniella, Hase and Eucanuella. The subfamily Cerviniinae has been re-defined to include Cervinia, Expansicervinia and Paracerviniella. Members of the subfamily Pontostratiotinae were clustered into two clades, one consists of the genus Stratiopontotes sister to an undescribed genus + Cerviniopsis and Siphonis. The second contains Pontostratiotes sister to the members of the planktonic subfamily Aegisthinae, resulting in the paraphyly of the Pontostratiotinae. Therefore, the Pontostratiotinae has been re-defined to include only members of the genus Pontostratiotes; whereas the subfamily Cerviniopseinae has been re-erected and re-defined containing Stratiopontotes, Cerviniopsis, Siphonis, Aegisthidae gen. 2, Herdmaniopsis, Hemicervinia and Tonpostratiotes. Within this subfamily, the associated Siphonis clusters as sister to the Cerviniopsis represents an example of convergent evolution in which the possession of a stylet-like mandible and an oral cone reminiscent of the Siphonostomatoida. The planktonic Aegisthus, Andromastax, Jamstecia, Nudivorax and Scabrantenna confirm the monophylom Aegisthinae, sister to the Pontostratiotinae. CONCLUSIONS: Our DNA based phylogeny reveals the deep-sea origin of Aegisthidae by placing benthic Aegisthidae gen.1 and Cerviniellinae subfam. nov. as the most basal lineages. Secondary adaptations to hyperbenthic and planktonic realms, as well as associated lifestyle were discovered here by the derived positions of Pontostratiotinae, Aegisthinae and Siphonis respectively.

Molecular evidence for the retention of the Thaumatopsyllidae in the order Cyclopoida (Copepoda) and establishment of four suborders and two families within the Cyclopoida.

The classification of the Thaumatopsyllidae within the Copepoda has been an issue of ongoing discussion since the discovery of Thaumatopsyllus paradoxus G.O. Sars, 1913 from the Norwegian coast. The family has been formally placed in the Monstrilloida, the Cyclopoida and even in its own order, the Thaumatopsylloida, based on different morphological criteria. We examined for the first time, the phylogenetic position of the Thaumatopsyllidae using gene sequences of 28S and 18S rRNA, as well as COI mtDNA, obtained from two thaumatopsyllid species occurring off the coast of southern California. We also fortuitously explored the phylogenetic relationships of the Cyclopoida in more detail than Khodami et al. (2017) by including a wider sample of key families such as the Erebonasteridae and Gisellinidae. Both Maximum Likelihood and Bayesian analyses revealed the Thaumatopsyllidae is nested in the Cyclopoida and is related to the marine Speleoithonidae. In addition, 16 families of the Cyclopoida are supported to be monophyletic, but surprisingly, the Cyclopidae is paraphyletic. The Cyclopicinidae is the first monophyletic offshoot of the cyclopoid tree, followed by two derived clades. The first clade contains a monophylum consisting of the Schminkepinellidae + Giselinidae which is sister to a clade including the monophyletic Erebonasteridae and all other poecilostome families. The second clade is divided into two main, well-supported family clusters. One includes the Cyclopidae encompassing two subfamilies (Eucylopinae and Cyclopinae), but unexpectedly the parasitic Lernaeidae cluster as a sister-group to the brackish water Halicyclops (subfamily Halicyclopinae) and the Euryteinae is the sister to all the rest of Cyclopidae s. l., making the Cyclopidae paraphyletic. To resolve this conundrum, we erected two families, Euryteidae and Halicyclopidae. The Cyclopidae s. str. retains the subfamilies Eucyclopinae and Cyclopinae, although our phylogeny does not support the reciprocal monophyly of these subfamilies. Our results support the gradual invasion of fresh water by the four families in this cluster. The highly supported monophyletic marine Euryteidae is the first offshoot followed by the brackish-water, free-living Halicyclopidae and the freshwater, parasitic Lernaeidae. The Cyclopidae fulfilled the colonization of freshwater bodies. The other clade of families comprises 12 monophyletic families recovered by our analysis, including the Pterinopsyllidae (at first offshoot), the Smirnovipinidae sister to the Hemicyclopinidae + Psammocyclopinidae, the Thaumatopsyllidae + Speleoithonidae, an undescribed family sister to the Archinotodelphyidae + Notodelphyidae and the Cyclopinidae sister to the Oithonidae + Cyclopettidae. We propose suborder ranks for each of the four main phylogenetic subdivisions of the Cyclopoida. These are named Cyclopicinida, Ergasilida, Cyclopida and Oithonida after the type genus of the oldest described family in the respective group.

Metabarcoding of marine environmental DNA based on mitochondrial and nuclear genes.

We establish the new approach of environmental DNA (eDNA) analyses for the North Sea. Our study uses a multigene approach, including the mitochondrial cytochrome-c-oxidase subunit I (COI) gene for analyzing species composition and the nuclear hypervariable region V8 of 18S rDNA for analyzing supraspecific biodiversity. A new minibarcode primer (124 bp) was created on the basis of a metazoan COI barcode library with 506 species and tested in silico, in vitro, and in situ. We applied high throughput sequencing to filtrates of 23 near-bottom water samples taken at three seasons from 14 stations. The set of COI primers allowed amplification of mitochondrial minibarcodes for diverse metazoan phyla and the differentiation at the species level for more than 99% of the specimens in the dataset. Our results revealed that the number of sequences is not consistent with proportions in the given DNA mixture. Altogether, environmental sequences could be assigned to 114 species and to 12 metazoan phyla. A spatial distribution of taxa recovered by eDNA was congruent with known distributions. Finally, the successful detection of species and biodiversity depends on a comprehensive sequence reference database. Our study offers a powerful tool for future biodiversity research, including the detection of nonnative species.

Molecular Phylogeny and Revision of Copepod Orders (Crustacea: Copepoda).

For the first time, the phylogenetic relationships between representatives of all 10 copepod orders have been investigated using 28S and 18S rRNA, Histone H3 protein and COI mtDNA. The monophyly of Copepoda (including Platycopioida Fosshagen, 1985) is demonstrated for the first time using molecular data. Maxillopoda is rejected, as it is a polyphyletic group. The monophyly of the major subgroups of Copepoda, including Progymnoplea Lang, 1948 (=Platycopioida); Neocopepoda Huys and Boxshall, 1991; Gymnoplea Giesbrecht, 1892 (=Calanoida Sars, 1903); and Podoplea Giesbrecht, 1892, are supported in this study. Seven copepod orders are monophyletic, including Platycopioida, Calanoida, Misophrioida Gurney, 1933; Monstrilloida Sars, 1901; Siphonostomatoida Burmeister, 1834; Gelyelloida Huys, 1988; and Mormonilloida Boxshall, 1979. Misophrioida (=Propodoplea Lang, 1948) is the most basal Podoplean order. The order Cyclopoida Burmeister, 1835, is paraphyletic and now encompasses Poecilostomatoida Thorell, 1859, as a sister to the family Schminkepinellidae Martinez Arbizu, 2006. Within Harpacticoida Sars, 1903, both sections, Polyarthra Lang, 1948, and Oligoarthra Lang, 1948, are monophyletic, but not sister groups. The order Canuelloida is proposed while maintaining the order Harpacticoida s. str. (Oligoarthra). Cyclopoida, Harpacticoida and Cyclopinidae are redefined, while Canuelloida ordo. nov., Smirnovipinidae fam. nov. and Cyclopicinidae fam. nov are proposed as new taxa.

Resilience of benthic deep-sea fauna to mining activities.

With increasing demand for mineral resources, extraction of polymetallic sulphides at hydrothermal vents, cobalt-rich ferromanganese crusts at seamounts, and polymetallic nodules on abyssal plains may be imminent. Here, we shortly introduce ecosystem characteristics of mining areas, report on recent mining developments, and identify potential stress and disturbances created by mining. We analyze species' potential resistance to future mining and perform meta-analyses on population density and diversity recovery after disturbances most similar to mining: volcanic eruptions at vents, fisheries on seamounts, and experiments that mimic nodule mining on abyssal plains. We report wide variation in recovery rates among taxa, size, and mobility of fauna. While densities and diversities of some taxa can recover to or even exceed pre-disturbance levels, community composition remains affected after decades. The loss of hard substrata or alteration of substrata composition may cause substantial community shifts that persist over geological timescales at mined sites.

Mitochondrial DNA Analyses Indicate High Diversity, Expansive Population Growth and High Genetic Connectivity of Vent Copepods (Dirivultidae) across Different Oceans.

Communities in spatially fragmented deep-sea hydrothermal vents rich in polymetallic sulfides could soon face major disturbance events due to deep-sea mineral mining, such that unraveling patterns of gene flow between hydrothermal vent populations will be an important step in the development of conservation policies. Indeed, the time required by deep-sea populations to recover following habitat perturbations depends both on the direction of gene flow and the number of migrants available for re-colonization after disturbance. In this study we compare nine dirivultid copepod species across various geological settings. We analyze partial nucleotide sequences of the mtCOI gene and use divergence estimates (FST) and haplotype networks to infer intraspecific population connectivity between vent sites. Furthermore, we evaluate contrasting scenarios of demographic population expansion/decline versus constant population size (using, for example, Tajima's D). Our results indicate high diversity, population expansion and high connectivity of all copepod populations in all oceans. For example, haplotype diversity values range from 0.89 to 1 and FST values range from 0.001 to 0.11 for Stygiopontius species from the Central Indian Ridge, Mid Atlantic Ridge, East Pacific Rise, and Eastern Lau Spreading Center. We suggest that great abundance and high site occupancy by these species favor high genetic diversity. Two scenarios both showed similarly high connectivity: fast spreading centers with little distance between vent fields and slow spreading centers with greater distance between fields. This unexpected result may be due to some distinct frequency of natural disturbance events, or to aspects of individual life histories that affect realized rates of dispersal. However, our statistical performance analyses showed that at least 100 genomic regions should be sequenced to ensure accurate estimates of migration rate. Our demography parameters demonstrate that dirivultid populations are generally large and continuously undergoing population growth. Benthic and pelagic species abundance data support these findings.

High-Throughput Sequencing-The Key to Rapid Biodiversity Assessment of Marine Metazoa?

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.

A reverse taxonomic approach to assess macrofaunal distribution patterns in abyssal Pacific polymetallic nodule fields.

Heightened interest in the exploitation of deep seafloor minerals is raising questions on the consequences for the resident fauna. Assessing species ranges and determination of processes underlying current species distributions are prerequisites to conservation planning and predicting faunal responses to changing environmental conditions. The abyssal central Pacific nodule belt, located between the Clarion and Clipperton Fracture Zones (CCZ), is an area prospected for mining of polymetallic nodules. We examined variations in genetic diversity and broad-scale connectivity of isopods and polychaetes across the CCZ. Faunal assemblages were studied from two mining claims (the eastern German and French license areas) located 1300 km apart and influenced by different productivity regimes. Using a reverse taxonomy approach based on DNA barcoding, we tested to what extent distance and large-scale changes in environmental parameters lead to differentiation in two macrofaunal taxa exhibiting different functions and life-history patterns. A fragment of the mitochondrial gene Cytochrome Oxidase Subunit 1 (COI) was analyzed. At a 97% threshold the molecular operational taxonomic units (MOTUs) corresponded well to morphological species. Molecular analyses indicated high local and regional diversity mostly because of large numbers of singletons in the samples. Consequently, variation in composition of genotypic clusters between sites was exceedingly large partly due to paucity of deep-sea sampling and faunal patchiness. A higher proportion of wide-ranging species in polychaetes was contrasted with mostly restricted distributions in isopods. Remarkably, several cryptic lineages appeared to be sympatric and occurred in taxa with putatively good dispersal abilities, whereas some brooding lineages revealed broad distributions across the CCZ. Geographic distance could explain variation in faunal connectivity between regions and sites to some extent, while assumed dispersal capabilities were not as important.

Spatially structured populations with a low level of cryptic diversity in European marine Gastrotricha.

Species of the marine meiofauna such as Gastrotricha are known to lack dispersal stages and are thus assumed to have low dispersal ability and low levels of gene flow between populations. Yet, most species are widely distributed, and this creates a paradox. To shed light on this apparent paradox, we test (i) whether such wide distribution may be due to misidentification and lumping of cryptic species with restricted distributions and (ii) whether spatial structures exist for the phylogeography of gastrotrichs. As a model, we used the genus Turbanella in NW Europe. DNA taxonomy using a mitochondrial and a nuclear marker supports distinctness of four traditional species (Turbanella ambronensis, T. bocqueti, T. mustela and T. cornuta) and provides evidence for two cryptic species within T. hyalina. An effect of geography on the within-species genetic structure is indeed present, with the potential for understanding colonization processes and for performing phylogeographic inference from microscopic animals. On the other hand, the occurrence of widely distributed haplotypes indicates long-distance dispersal as well, despite the assumed low dispersal ability of gastrotrichs.