Astrid Cornils

DNA Barcoding of Marine Copepods: Assessment of Analytical Approaches to Species Identification

More than 2,500 species of copepods (Class Maxillopoda; Subclass Copepoda) occur in the marine planktonic environment. The exceptional morphological conservation of the group, with numerous sibling species groups, makes the identification of species challenging, even for expert taxonomists. Molecular approaches to species identification have allowed rapid detection, discrimination, and identification of species based on DNA sequencing of single specimens and environmental samples. Despite the recent development of diverse genetic and genomic markers, the barcode region of the mitochondrial cytochrome c oxidase subunit I (COI) gene remains a useful and – in some cases – unequaled diagnostic character for species-level identification of copepods. This study reports 800 new barcode sequences for 63 copepod species not included in any previous study and examines the reliability and resolution of diverse statistical approaches to species identification based upon a dataset of 1,381 barcode sequences for 195 copepod species. We explore the impact of missing data (i.e., species not represented in the barcode database) on the accuracy and reliability of species identifications. Among the tested approaches, the best close match analysis resulted in accurate identification of all individuals to species, with no errors (false positives), and out-performed automated tree-based or BLAST based analyses. This comparative analysis yields new understanding of the strengths and weaknesses of DNA barcoding and confirms the value of DNA barcodes for species identification of copepods, including both individual specimens and bulk samples. Continued integrative morphological-molecular taxonomic analysis is needed to produce a taxonomically-comprehensive database of barcode sequences for all species of marine copepods.

Phylogeny of the Paracalanidae Giesbrecht, 1888 (Crustacea: Copepoda: Calanoida).

The Paracalanidae are ecologically-important marine planktonic copepods that occur in the epipelagic zone in temperate and tropical waters. They are often the dominant taxon - in terms of biomass and abundance - in continental shelf regions. As primary consumers, they form a vital link in the pelagic food web between primary producers and higher trophic levels. Despite the ecological importance of the taxon, evolutionary and systematic relationships within the family remain largely unknown. A multigene phylogeny including 24 species, including representatives for all seven genera, was determined based on two nuclear genes, small-subunit (18S) ribosomal RNA and Histone 3 (H3) and one mitochondrial gene, cytochrome c oxidase subunit I (COI). The molecular phylogeny was well supported by Maximum likelihood and Bayesian inference analysis; all genera were found to be monophyletic, except for Paracalanus, which was separated into two distinct clades: the Paracalanus aculeatus group and Paracalanus parvus group. The molecular phylogeny also confirmed previous findings that Mecynocera and Calocalanus are genera of the family Paracalanidae. For comparison, a morphological phylogeny was created for 35 paracalanid species based on 54 morphological characters derived from published descriptions. The morphological phylogeny did not resolve all genera as monophyletic and bootstrap support was not strong. Molecular and morphological phylogenies were not congruent in the positioning of Bestiolina and the Paracalanus species groups, possibly due to the lack of sufficient phylogenetically-informative morphological characters.

Global phylogeography of Oithona similis s.l. (Crustacea, Copepoda, Oithonidae) – A cosmopolitan plankton species or a complex of cryptic lineages?

Traditionally, many small-sized copepod species are considered to be widespread, bipolar or cosmopolitan. However, these large-scale distribution patterns need to be re-examined in view of increasing evidence of cryptic and pseudo-cryptic speciation in pelagic copepods. Here, we present a phylogeographic study of Oithona similis s.l. populations from the Arctic Ocean, the Southern Ocean and its northern boundaries, the North Atlantic and the Mediterrranean Sea. O. similis s.l. is considered as one of the most abundant species in temperate to polar oceans and acts as an important link in the trophic network between the microbial loop and higher trophic levels such as fish larvae. Two gene fragments were analysed: the mitochondrial cytochrome oxidase c subunit I (COI), and the nuclear ribosomal 28 S genetic marker. Seven distinct, geographically delimitated, mitochondrial lineages could be identified, with divergences among the lineages ranging from 8 to 24%, thus representing most likely cryptic or pseudocryptic species within O. similis s.l. Four lineages were identified within or close to the borders of the Southern Ocean, one lineage in the Arctic Ocean and two lineages in the temperate Northern hemisphere. Surprisingly the Arctic lineage was more closely related to lineages from the Southern hemisphere than to the other lineages from the Northern hemisphere, suggesting that geographic proximity is a rather poor predictor of how closely related the clades are on a genetic level.

New insights into global biogeography, population structure and natural selection from the genome of the epipelagic copepod Oithona

In the epipelagic ocean, the genus Oithona is considered as one of the most abundant and widespread copepods and plays an important role in the trophic food web. Despite its ecological importance, little is known about Oithona and cyclopoid copepods genomics. Therefore, we sequenced, assembled and annotated the genome of Oithona nana. The comparative genomic analysis integrating available copepod genomes highlighted the expansions of genes related to stress response, cell differentiation and development, including genes coding Lin12‐Notch‐repeat (LNR) domain proteins. The Oithona biogeography based on 28S sequences and metagenomic reads from the Tara Oceans expedition showed the presence of O. nana mostly in the Mediterranean Sea (MS) and confirmed the amphitropical distribution of Oithona similis. The population genomics analyses of O. nana in the Northern MS, integrating the Tara Oceans metagenomic data and the O. nana genome, led to the identification of genetic structure between populations from the MS basins. Furthermore, 20 loci were found to be under positive selection including four missense and eight synonymous variants, harbouring soft or hard selective sweep patterns. One of the missense variants was localized in the LNR domain of the coding region of a male‐specific gene. The variation in the B‐allele frequency with respect to the MS circulation pattern showed the presence of genomic clines between O. nana and another undefined Oithona species possibly imported through Atlantic waters. This study provides new approaches and results in zooplankton population genomics through the integration of metagenomic and oceanographic data.

Unravelling diversity of deep-sea copepods using integrated morphological and molecular techniques

Accurate species identification is crucial for ecological studies. For copepods, this is usually based on a few diagnostic morphological characters, which can be highly conserved, resulting in an underestimation of species diversity in many copepod families. We elucidate species richness in the morphologically challenging and ecologically important deep-sea copepod family Spinocalanidae in the tropical Atlantic by applying an integrated taxonomic approach combining morphology, DNA-sequence analyses and proteomic fingerprinting. In total, 28 morphospecies could be discriminated, while 39 putative species were detected using DNA-sequence analyses and 42 using proteomic fingerprinting. This outcome verifies proteomic fingerprinting to simplify and accelerate future biodiversity studies of copepods with high taxonomic resolution. Our findings demonstrate the power of this integrated morphological and molecular taxonomic approach by revealing high numbers of cryptic or pseudocryptic species and thus uncovering the incompleteness of taxonomic guides for this group in the poorly explored mesopelagic realm. Furthermore, our analyses reveal a close relationship of Mospicalanus and Spinocalanus group A and indicate that the genus Spinocalanus may be polyphyletic. The underestimated species diversity suggests complex ecological interactions in terms of predator–prey relationships, interspecific competition and species-specific specializations in the vast, but under-studied mesopelagic realm.

Mare Incognitum: A Glimpse into Future Plankton Diversity and Ecology Research

With global climate change altering marine ecosystems, research on plankton ecology is likely to navigate uncharted seas. Yet, a staggering wealth of new plankton observations, integrated with recent advances in marine ecosystem modelling, may shed light on marine ecosystem structure and functioning. A EuroMarine foresight workshop on the “Impact of climate change on the distribution of plankton functional and phylogenetic diversity” (PlankDiv) identified five grand challenges for future plankton diversity and macroecology research: 1) What can we learn about plankton communities from the new wealth of high-throughput ‘omics’ data? 2) What is the link between plankton diversity and ecosystem function? 3) How can species distribution models be adapted to represent plankton biogeography? 4) How will plankton biogeography be altered due to anthropogenic climate change? and 5) Can a new unifying theory of macroecology be developed based on plankton ecology studies? In this review, we discuss potential future avenues to address these questions, and challenges that need to be tackled along the way.

Chitin distribution in the Oithona digestive and reproductive systems revealed by fluorescence microscopy

Among copepods, which are the most abundant animals on Earth, the genus Oithona is described as one of the most numerous and plays a major role in the marine food chain and biogeochemical cycles, particularly through the excretion of chitin-coated fecal pellets. Despite the morphology of several Oithona species is well known, knowledge of its internal anatomy and chitin distribution is still limited. To answer this problem, Oithona nana and O. similis individuals were stained by Wheat Germ Agglutinin-Fluorescein IsoThioCyanate (WGA-FITC) and DiAmidino-2-PhenylIndole (DAPI) for fluorescence microscopy observations. The image analyses allowed a new description of the organization and chitin content of the digestive and reproductive systems of Oithona male and female. Chitin microfibrils were found all along the digestive system from the stomach to the hindgut with a higher concentration at the peritrophic membrane of the anterior midgut. Several midgut shrinkages were observed and proposed to be involved in faecal pellet shaping and motion. Amorphous chitin structures were also found to be a major component of the ducts and seminal vesicles and receptacles. The rapid staining protocol we proposed allowed a new insight into the Oithona internal anatomy and highlighted the role of chitin in the digestion and reproduction. This method could be applied to a wide range of copepods in order to perform comparative anatomy analyses.

Pontellid copepods, Labidocera spp., affected by ocean acidification: A field study at natural CO2 seeps

CO2 seeps in coral reefs were used as natural laboratories to study the impacts of ocean acidification on the pontellid copepod, Labidocera spp. Pontellid abundances were reduced by ∼70% under high-CO2 conditions. Biological parameters and substratum preferences of the copepods were explored to determine the underlying causes of such reduced abundances. Stage- and sex-specific copepod lengths, feeding ability, and egg development were unaffected by ocean acidification, thus changes in these physiological parameters were not the driving factor for reduced abundances under high-CO2 exposure. Labidocera spp. are demersal copepods, hence they live amongst reef substrata during the day and emerge into the water column at night. Deployments of emergence traps showed that their preferred reef substrata at control sites were coral rubble, macro algae, and turf algae. However, under high-CO2 conditions they no longer had an association with any specific substrata. Results from this study indicate that even though the biology of a copepod might be unaffected by high-CO2, Labidocera spp. are highly vulnerable to ocean acidification.

Population genetic structure of Calanoides natalis (Copepoda, Calanoida) in the eastern Atlantic Ocean and Benguela upwelling system

The population genetic structure of Calanoides natalis (ex Calanoides carinatus; Copepoda, Calanoida), an ecologically important component of African upwelling systems, was studied in order to (i) search for potential cryptic species, (ii) describe spatial patterns in the distribution of genetic variance and (iii) identify potential barriers to gene flow. Samples were obtained in the eastern Atlantic Ocean from the Iberian Peninsula to Namibia. Analysis of mitochondrial (cytochrome c oxidase subunit I; COI) and nuclear (citrate synthase; CS) marker genes revealed a genetically cohesive population of C. natalis with a prevalent shift in allele frequencies. The discovery of a deep split solely present in the mitochondrial dataset does not point to cryptic speciation, but rather suggests the occurrence of nuclear mitochondrial pseudogenes or incomplete reproductive isolation upon secondary contact. Genetic differentiation between the northern and southern hemisphere was significant, which may point to a potential, but permeable barrier close to the equator. No vertical genetic structuring was detected in the northern Benguela implying that horizontal differentiation was more pronounced than vertical structuring. Retention mechanisms and the oxygen minimum zone did not have a strong impact on genetic differentiation of C. natalis in the Benguela region.

Corrigendum: Mare Incognitum: A Glimpse into Future Plankton Diversity and Ecology Research

Francois Guilhaumon was not included as an author in the published article. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.