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.

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.

Description of three new species of Bradyidius (Copepoda: Calanoida), the new aetideids from the deep Pacific Ocean, with notes on the genera Bradyidius and Aetideopsis.

Three new aetideid species, Bradyidius abyssalis sp. nov., Bradyidius parabyssalis sp. nov., and B. kurilokamchaticus sp. nov. are described from female specimens collected near the seafloor in the abyss of the Pacific and Atlantic Oceans. Specimens of Bradyidius parabyssalis sp. nov. were obtained in both the Atlantic and Pacific Oceans (Argentine Basin, area of the Meteor Seamount and the Kurile-Kamchatka Trench). Bradyidius abyssalis sp. nov. was found only in the Atlantic Ocean, (Brazil and Guinea Basins and area of the Meteor Seamount) and Bradyidius kurilokamchaticus sp. nov. was recorded from the Kurile-Kamchatka Trench of the Pacific Ocean. Three new herein described Bradyidius species constitute the first documented records of the genus from the abyss of the World Ocean. In addition, three Bradyidius species from the Weddell Sea, the Atlantic Ocean and the Kurile-Kamchatka Trench, are briefly described without biological names due to their bad condition. Bradyidius parabyssalis sp. nov. and B. abyssalis sp. nov. are distinguished from all known congeners by the presence of 3 setae at the basis of the mandible and morphological details of the prosome posterior corners and P1. They show close resemblance to each other but differ in body size, rostrum structure, P4 coxa armament and length of the setae of the antennule ancestral segment I and the mandible basis. Bradyidius kurilokamchaticus sp. nov. shares with B. curtus Markhaseva, 1993, B. pacificus (Brodsky, 1950) and B. arnoldi Fleminger, 1957 a rostrum with non-divergent or parallel points, but differs from these species in the size, the well developed lateral spine on exopod segment 1, in the number of setae at the antenna exopod segment 1 and some morphological details of the prosome posterior corners. Characters that define the genus Bradyidius Giesbrecht, 1897 from Aetideopsis Sars, 1903, i.e. the shape of lateral spines of P1exopod segments 1 and 2; the endopod of P2 segmentation and the setation of the antennule ancestral segments XII, XV and XVII are discussed.

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.

Description of Bradyetes paramatthei sp. nov. (Copepoda: Calanoida), a new aetideid species from the deep Pacific Ocean with notes on the genus Bradyetes.

A new and rare aetideid species of the benthopelagic genus Bradyetes Farran, 1905 is described from female specimens collected near the seafloor from the abyss of the Pacific, Atlantic and Southern Oceans between 2000 and 2014. The new species, Bradyetes paramatthei sp. nov., is described from the Kurile-Kamchatka Trench (Pacific Ocean) and is additionally reported from the Angola and Guinea basins and from the Meteor Seamount of the Atlantic Ocean. Bradyetes paramatthei sp. nov. is related to the species B. matthei Johannessen, 1976, but differs in the shape of the prosome posterior corners, which are oval-rounded, and in the proximal segment of the antennal exopod, which is supplied with one seta. These two species are shown to constitute a separate species group within the genus Bradyetes. The other species group contains the remaining congeners including Bradyetes inermis Farran, 1905, for which morphological variability is discussed. Specimens of this species show diverse morphology and comprise 3 morphotypes distinguished by the P1, with a developed or absent lateral lobe , the P1 basal medial seta, which is either nude or supplied with setules, and by the different number of setae of the maxillule praecoxal arthrite.

Repositories for Taxonomic Data: Where We Are and What is Missing.

Natural history collections are leading successful large-scale projects of specimen digitization (images, metadata, DNA barcodes), thereby transforming taxonomy into a big data science. Yet, little effort has been directed towards safeguarding and subsequently mobilizing the considerable amount of original data generated during the process of naming 15,000-20,000 species every year. From the perspective of alpha-taxonomists, we provide a review of the properties and diversity of taxonomic data, assess their volume and use, and establish criteria for optimizing data repositories. We surveyed 4113 alpha-taxonomic studies in representative journals for 2002, 2010, and 2018, and found an increasing yet comparatively limited use of molecular data in species diagnosis and description. In 2018, of the 2661 papers published in specialized taxonomic journals, molecular data were widely used in mycology (94%), regularly in vertebrates (53%), but rarely in botany (15%) and entomology (10%). Images play an important role in taxonomic research on all taxa, with photographs used in >80% and drawings in 58% of the surveyed papers. The use of omics (high-throughput) approaches or 3D documentation is still rare. Improved archiving strategies for metabarcoding consensus reads, genome and transcriptome assemblies, and chemical and metabolomic data could help to mobilize the wealth of high-throughput data for alpha-taxonomy. Because long-term-ideally perpetual-data storage is of particular importance for taxonomy, energy footprint reduction via less storage-demanding formats is a priority if their information content suffices for the purpose of taxonomic studies. Whereas taxonomic assignments are quasifacts for most biological disciplines, they remain hypotheses pertaining to evolutionary relatedness of individuals for alpha-taxonomy. For this reason, an improved reuse of taxonomic data, including machine-learning-based species identification and delimitation pipelines, requires a cyberspecimen approach-linking data via unique specimen identifiers, and thereby making them findable, accessible, interoperable, and reusable for taxonomic research. This poses both qualitative challenges to adapt the existing infrastructure of data centers to a specimen-centered concept and quantitative challenges to host and connect an estimated $ \le $2 million images produced per year by alpha-taxonomic studies, plus many millions of images from digitization campaigns. Of the 30,000-40,000 taxonomists globally, many are thought to be nonprofessionals, and capturing the data for online storage and reuse therefore requires low-complexity submission workflows and cost-free repository use. Expert taxonomists are the main stakeholders able to identify and formalize the needs of the discipline; their expertise is needed to implement the envisioned virtual collections of cyberspecimens. [Big data; cyberspecimen; new species; omics; repositories; specimen identifier; taxonomy; taxonomic data.].

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.

Blue pigmentation of neustonic copepods benefits exploitation of a prey-rich niche at the air-sea boundary.

The sea-surface microlayer (SML) at the air-sea interface is a distinct, under-studied habitat compared to the subsurface and copepods, important components of ocean food webs, have developed key adaptations to exploit this niche. By using automated SML sampling, high-throughput sequencing and unmanned aerial vehicles, we report on the distribution and abundance of pontellid copepods in relation to the unique biophysicochemical signature of the SML. We found copepods in the SML even during high exposure to sun-derived ultraviolet radiation and their abundance was significantly correlated to increased algal biomass. We additionally investigated the significance of the pontellids' blue pigmentation and found that the reflectance peak of the blue pigment matched the water-leaving spectral radiance of the ocean surface. This feature could reduce high visibility at the air-sea boundary and potentially provide camouflage of copepods from their predators.