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.

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.

Environmental DNA (eDNA) reveals potential for interoceanic fish invasions across the Panama Canal.

Interoceanic canals can facilitate biological invasions as they connect the world's oceans and remove dispersal barriers between bioregions. As a consequence, multiple opportunities for biotic exchange arise and the resulting establishment of migrant species often causes adverse ecological and economic impacts. The Panama Canal is a key region for biotic exchange as it connects the Pacific and Atlantic Oceans in Central America. In this study, we used two complementary methods (environmental DNA (eDNA) metabarcoding and gillnetting) to survey fish communities in this unique waterway. Using COI (cytochrome oxidase subunit I) metabarcoding, we detected a total of 142 fish species, including evidence for the presence of sixteen Atlantic and eight Pacific marine fish in different freshwater sections of the Canal. Of these, nine are potentially new records. Molecular data did not capture all species caught with gillnets, but generally provided a more complete image of the known fish fauna as more small-bodied fish species were detected. Diversity indices based on eDNA surveys revealed significant differences across different sections of the Canal reflecting in part the prevailing environmental conditions. The observed increase in the presence of marine fish species in the Canal indicates a growing potential for interoceanic fish invasions. The potential ecological and evolutionary consequences of this increase in marine fishes are not only restricted to the fish fauna in the Canal as they could also impact adjacent ecosystems in the Pacific and Atlantic Oceans.

Proteomic fingerprinting enables quantitative biodiversity assessments of species and ontogenetic stages in Calanus congeners (Copepoda, Crustacea) from the Arctic Ocean.

Species identification is pivotal in biodiversity assessments and proteomic fingerprinting by MALDI-TOF mass spectrometry has already been shown to reliably identify calanoid copepods to species level. However, MALDI-TOF data may contain more information beyond mere species identification. In this study, we investigated different ontogenetic stages (copepodids C1-C6 females) of three co-occurring Calanus species from the Arctic Fram Strait, which cannot be identified to species level based on morphological characters alone. Differentiation of the three species based on mass spectrometry data was without any error. In addition, a clear stage-specific signal was detected in all species, supported by clustering approaches as well as machine learning using Random Forest. More complex mass spectra in later ontogenetic stages as well as relative intensities of certain mass peaks were found as the main drivers of stage distinction in these species. Through a dilution series, we were able to show that this did not result from the higher amount of biomass that was used in tissue processing of the larger stages. Finally, the data were tested in a simulation for application in a real biodiversity assessment by using Random Forest for stage classification of specimens absent from the training data. This resulted in a successful stage-identification rate of almost 90%, making proteomic fingerprinting a promising tool to investigate polewards shifts of Atlantic Calanus species and, in general, to assess stage compositions in biodiversity assessments of Calanoida, which can be notoriously difficult using conventional identification methods.

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.

The crossover from microscopy to genes in marine diversity: from species to assemblages in marine pelagic copepods.

An accurate identification of species and communities is a prerequisite for analysing and recording biodiversity and community shifts. In the context of marine biodiversity conservation and management, this review outlines past, present and forward-looking perspectives on identifying and recording planktonic diversity by illustrating the transition from traditional species identification based on morphological diagnostic characters to full molecular genetic identification of marine assemblages. In this process, the article presents the methodological advancements by discussing progress and critical aspects of the crossover from traditional to novel and future molecular genetic identifications and it outlines the advantages of integrative approaches using the strengths of both morphological and molecular techniques to identify species and assemblages. We demonstrate this process of identifying and recording marine biodiversity on pelagic copepods as model taxon. Copepods are known for their high taxonomic and ecological diversity and comprise a huge variety of behaviours, forms and life histories, making them a highly interesting and well-studied group in terms of biodiversity and ecosystem functioning. Furthermore, their short life cycles and rapid responses to changing environments make them good indicators and core research components for ecosystem health and status in the light of environmental change. This article is part of the theme issue 'Integrative research perspectives on marine conservation'.

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

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Do molecular phylogenies unravel the relationships among the evolutionary young "Brafordian" families (Copepoda; Calanoida)?

Among the most derived calanoid copepod superfamily Clausocalanoidea about half of the genera belong to the so-called "Bradfordian" families that are defined by the presence of sensory setae at the maxilla and maxilliped. Many of these "Bradfordian" taxa are insufficiently well described, because their taxonomy is complicated and phylogenetic relationships are not completely resolved. We therefore aimed to unravel their phylogenetic relationships using molecular multi-gene analyses. We conducted molecular multi-gene analysis on 26 species from 15 genera representing all seven "Bradfordian" families using five gene fragments, the nuclear ribosomal 18S, 28S and internal transcribed spacer 2 DNA, and mitochondrial cytochrome c oxidase subunit I and cytochrome b. The monophyly of "Bradfordians" as one lineage in the superfamily Clausocalanoidea was supported by Maximum Likelihood and Bayesian Inference multi-gene analyses. Except for the support of species belonging to the same genus and specimens belonging to the same species, no phylogenetic relationships among genera and families were supported. The impossibility of resolving phylogenetic relationships among "Bradfordian" genera and families may be due to the young age or fast radiation of "Bradfordians" within the mostly derived calanoid superfamily Clausocalanoidea. Therefore, mutation rates might be not sufficient to track phylogenetic relationships. Evidence on phylogenetic relationships between genera and families remain unresolved after implementing integrated morphological and molecular taxonomic approaches.

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.

Identification of North Sea molluscs with DNA barcoding.

Sequence-based specimen identification, known as DNA barcoding, is a common method complementing traditional morphology-based taxonomic assignments. The fundamental resource in DNA barcoding is the availability of a taxonomically reliable sequence database to use as a reference for sequence comparisons. Here, we provide a reference library including 579 sequences of the mitochondrial cytochrome c oxidase subunit I for 113 North Sea mollusc species. We tested the efficacy of this library by simulating a sequence-based specimen identification scenario using Best Match, Best Close Match (BCM) and All Species Barcode (ASB) criteria with three different threshold values. Each identification result was compared with our prior morphology-based taxonomic assignments. Our simulation resulted in 87.7% congruent identifications (93.8% when excluding singletons). The highest number of congruent identifications was obtained with BCM and ASB and a 0.05 threshold. We also compared identifications with genetic clustering (Barcode Index Numbers, BINs) computed by the Barcode of Life Datasystem (BOLD). About 68% of our morphological identifications were congruent with BINs created by BOLD. Forty-nine sequences were clustered in 16 discordant BINs, and these were divided in two classes: sequences from different species clustered in a single BIN and conspecific sequences divided in more BINs. Whereas former incongruences were probably caused by BOLD entries in need of a taxonomic update, the latter incongruences regarded taxa requiring further investigations. These include species with amphi-Atlantic distribution, whose genetic structure should be evaluated over their entire range to produce a reliable sequence-based identification system.

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.

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 sequences.

A reliable DNA barcode reference library for the identification of the North 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.

Evolution in the deep sea: biological traits, ecology and phylogenetics of pelagic copepods.

Deep-sea biodiversity has received increasing interest in the last decade, mainly focusing on benthic communities. In contrast, studies of zooplankton in the meso- to bathypelagic zones are relatively scarce. In order to explore evolutionary processes in the pelagic deep sea, the present study focuses on copepods of two clausocalanoid families, Euchaetidae and Aetideidae, which are abundant and species-rich in the deep-sea pelagic realm. Molecular phylogenies based on concatenated-portioned data on 18S, 28S and internal transcribed spacer 2 (ITS2), as well as mitochondrial cytochrome c oxidase subunit I (COI), were examined on 13 species, mainly from Arctic and Antarctic regions, together with species-specific biological traits (i.e. vertical occurrence, feeding behaviour, dietary preferences, energy storage, and reproductive strategy). Relationships were resolved on genus, species and even sub-species levels, the latter two established by COI with maximum average genetic distances ranging from ≤5.3% at the intra-specific, and 20.6% at the inter-specific level. There is no resolution at a family level, emphasising the state of Euchaetidae and Aetideidae as sister families and suggesting a fast radiation of these lineages, a hypothesis which is further supported by biological parameters. Euchaetidae were similar in lipid-specific energy storage, reproductive strategy, as well as feeding behaviour and dietary preference. In contrast, Aetideidae were more diverse, comprising a variety of characteristics ranging from similar adaptations within Paraeuchaeta, to genera consisting of species with completely different reproductive and feeding ecologies. Reproductive strategies were generally similar within each aetideid genus, but differed between genera. Closely related species (congeners), which were similar in the aforementioned biological and ecological traits, generally occurred in different depth layers, suggesting that vertical partitioning of the water column represents an important mechanism in the speciation processes for these deep-sea copepods. High COI divergence between Arctic and Antarctic specimens of the mesopelagic cosmopolitan Gaetanus tenuispinus and the bipolar Aetideopsis minor suggest different geographic forms, potentially cryptic species or sibling species. On the contrary, Arctic and Antarctic individuals of the bathypelagic cosmopolitans Gaetanus brevispinus and Paraeuchaeta barbata were very similar in COI sequence, suggesting more gene flow at depth and/or that driving forces for speciation were less pronounced in bathypelagic than at mesopelagic depths.