Phaeogromids of the mesopelagic marine plankton: Temporal variability of concentrations and observations of feeding structures of four species from the mesopelagic in the Mediterranean Sea.

Challengerids, phaeogromids rhizarian protists, are emblematic protists of the deep sea but are also enigmatic as they occur in very low concentrations. In previous studies, we reported on temporal changes in abundance at a near-shore mesopelagic site, but only as part of sampling of the entire microplankton assemblage, not well-suited for examining phaeogromids. Consequently, we turned to using a closing plankton net to provide material from large volumes of seawater, thus allowing for more robust estimates of concentrations and material for observations of living cells, to our knowledge the first made. Here, we report our results on the four most commonly occurring species: Challengeranium diadon, Challengereron willemoesii, Challengeria xiphodon, and Euphysetta lucani. In contrast to our previous report, we found that changes in concentrations were not related to water column stratification, and the four species roughly co-varied with time. Observations of live cells revealed that all four species deploy tentacle-like pseudopods and also very large unstructured webs of fine pseudopods. The similarities in feeding webs suggest similar prey are exploited, and the similar temporal changes in abundances suggest a common factor or factors (unknown at this time) govern their concentrations. Films of live cells are provided in Supplementary Files.

Past President's Address: Protists of the Mesopelagic and a Bit on the Long Path to the Deep Sea.

The deep sea has long been a mysterious and attractive habitat for protistologists. However, logistical difficulties severely limit sampling opportunities. Consequently, our knowledge of the protists in the deep sea, (arguably the largest habitat on earth), is relatively sparse. Here, we present a unique time-series concerning three different protist taxa that share only the characteristics of being relatively large, robust to sampling, and easily identifiable to species level using light microscopy: tintinnid ciliates, phaeogromid cercozoans (e.g. Challengerids) and amphisolenid dinoflagellates. We sampled a near-shore deep water site in the N.W. Mediterranean Sea at 250 m depth over a 2-yr period at approximately weekly intervals from January 2017 to December 2018. To our knowledge, no previous studies have employed sampling on a similar time scale. We found taxa that appear to be restricted to deep waters, distinct seasonal patterns of abundance in some taxa, and in others nonseasonal successional patterns. Based on data from sampling following a flash flood event, the Challengerid population appeared to respond positively to a pulse of terrigenous input. Some of the distinct mesopelagic tintinnid ciliates and amphisolinid dinoflagellates were also found in two samples from the North Atlantic mesopelagic gathered from near the Azores Islands in September 2018. We conclude that there are a variety of protist taxa endemic to the mesopelagic, that the populations are dynamic, and they may be widely distributed in the deep waters of the world ocean.

Community Structure of Tintinnid Ciliates of the Microzooplankton in the South West Pacific Ocean: Comparison of a High Primary Productivity with a Typical Oligotrophic Site.

Transient 'hot spots' of phytoplankton productivity occur in the generally oligotrophic Southern Pacific Ocean and we hypothesized that the population structure of tintinnid ciliates, planktonic grazers, would differ from that of a typical oligotrophic sites. Samples were collected over a 1-wk period at each of two sites between Fiji and Tahiti: one of elevated chlorophyll a concentrations and primary productivity with an abundance of N-fixing cyanobacteria Trichodesmium, and a distant oligotrophic site. Tintinnid abundance differed between the sites by a factor of 2. A single species (Favella sp.), absent from the oligotrophic site, highly dominated the 'hot spot' site. However, total species richness was identical (71 spp.) as well as short-term temporal variability (2-4 d). At both sites, species abundance distributions most closely fit a log-series or log-normal distribution and the abundance distributions of ecological types, forms of distinct lorica oral diameter, were the typical geometric. Morphological diversity was only slightly lower at the high productivity site. We found that communities of these plankton grazers in 'hot spots' of phytoplankton productivity in oligotrophic systems, although harboring different species, differ little from surrounding oligotrophic areas in community structure.

Molecular Phylogeny and Evolutionary Relationships between the Ciliate Genera Peniculistoma and Mytilophilus (Peniculistomatidae, Pleuronematida).

Peniculistoma mytili and Mytilophilus pacificae are placed in the pleuronematid scuticociliate family Peniculistomatidae based on morphology and ecological preference for the mantle cavity of mytiloid bivalves. We tested this placement with sequences of the small subunit rRNA (SSUrRNA) and cytochrome c oxidase subunit 1 (cox1) genes. These species are very closely related sister taxa with no distinct genetic difference in the SSUrRNA sequence but about 21% genetic difference for cox1, supporting their placement together but separation as distinct taxa. Using infection frequencies, M. pacificae, like its sister species P. mytili, does not interact with Ancistrum spp., co-inhabitants of the mantle cavity. On the basis of these ecological similarities, the fossil record of host mussels, and features of morphology and stomatogenesis of these two ciliates, we argue that M. pacificae derived from a Peniculistoma-like ancestor after divergence of the two host mussels. Our phylogenetic analyses of pleuronematid ciliates includes the SSUrRNA gene sequence of Sulcigera comosa, a Histiobalantium-like ciliate from Lake Baikal. We conclude: (i) that the pleuronematids are a monophyletic group; (ii) that the genus Pleuronema is paraphyletic; and (iii) that S. comosa is a Histiobalantium species. We transfer S. comosa to Histiobalantium and propose a new combination Histiobalantium comosa n. comb.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy.

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR(2), http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

Morphological and ribosomal DNA-based characterization of six Antarctic ciliate morphospecies from the Amundsen Sea with phylogenetic analyses.

We characterized six tintinnid ciliates from Antarctic waters using molecular markers and morphological traits: Amphorellopsis quinquealata, Codonellopsis gaussi, Cymatocylis convallaria, Cy. calyciformis, Cy. drygalskii, and Laackmanniella prolongata. The 100% similarity in SSU-ITS1-5.8S rDNA-ITS2-partial LSU rDNA sequences among Cy. convallaria, Cy. calyciformis, and Cy. drygalskii is supportive of synonymy. Codonellopsis gaussi and L. prolongata also showed high levels of similarity in SSU rDNA (99.83%) and the D2 domain of LSU rDNA (95.77%), suggesting that they are closely related. Phylogenetic analysis placed Cymatocylis in the Rhabdonellidae, Amphorellopsis in the Tintinnidae and L. prolongata/Co. gaussi within the Dictyocystidae.

The saga of the false fossil foram Eozoon.

Eozoon canadense, 'the dawn animal of Canada', a large foraminifera, was announced in 1864 as the oldest fossil organism known. Camps soon formed into disbelievers of its fossil nature, agnostics, and "Eozoonists". Eozoon would number among its proponents major figures of the time. The saga of Eozoon, or more precisely the dispute as to its actual nature, spawned hundreds of publications. Here the story is told with a new focus, one on the stature and roles of the major personalities involved, and the evidence they presented. Eozoon is considered to have been 'de-bunked' in the late 19th century. However, it will be shown that it was never indisputably proven to be inorganic. Rather Eozoon simply faded away after its most ardent defenders died. As late as 1947, it was shown as the primordial organism in a biology textbook. The saga of Eozoon remains as a valuable cautionary tale. It is an example of an artifact accepted as fact because it filled a troubling void in knowledge, i.e., at that time, the first traces of life before Cambrian, and it endured because it was promoted by only a few, but powerful, figures in the scientific establishment of the era.

The odd couple of protistology: Edward Heron-Allen (1861-1943) and Arthur Earland (1866-1958).

Edward Heron-Allen and Arthur Earland were among the last great amateur foraminifera researchers. Their partnership began in 1907 and ended in 1932. While close in age to one another, they shared little more than a fascination for forams and a lack of any university training. In most other aspects, the two men were completely different. Heron-Allen was a famous upper class polymath, expert not only on forams, but also on the Persian language, violins, palm reading, history, asparagus, and barnacles. He was also an accomplished novelist and poet, who frequented literary circles. In contrast to the flamboyant Heron-Allen, Earland was a discrete civil servant who admitted to working on forams as a relief from the monotony of his job. Hence, the two were improbable partners. However, together they produced 39 substantial works on forams. Their studies concerned assemblages from Southern Ocean to the North Sea and they are today credited with the original description of 186 species. Here the distinct lives of the two men are presented, and their contributions to protistology, as partners as well as individuals, are reviewed. The case is made for considering Earland's work as neglected relative to that of Heron-Allen, except perhaps by foram taxonomists.

Southern ocean biogeography of tintinnid ciliates of the marine plankton.

Ciliate microzooplankton are important grazers in most pelagic ecosystems and among them, tintinnids, with their largely species-specific loricas, allow relatively easy assessment of questions of diversity and distributions. Herein, we present the results of a survey of species records of tintinnids from the Southern Ocean (locations below 40°S) reported in 56 publications yielding 2,047 species records (synonyms included) from 402 locations. The 192 species reported can be parsed into two main groups: 32 endemic Southern Ocean species, known only from 40°S and further south, and a second group of 181 widespread species, forms with extensive geographic ranges extending into the Southern Ocean. Widespread species reported from the Southern Ocean can be further divided into a group of 81 species, each recorded multiple times in the Southern Ocean waters and 70 apparent "stray" species which have only been found but once. The endemic and widespread species of the Southern Ocean show both distinct distributional patterns and morphological differences. The assemblage of Southern Ocean endemics is found mostly within the Antarctic zone delimited by the average location of the Polar Front and contains a relatively large portion of wide-mouthed forms. We give suggestions for future study.

Re-visiting the ridiculed rival of Leeuwenhoek: Louis Joblot (1645-1723).

Louis Joblot published one of the first manuals of microscopy in 1718, just a few years before both he and Leeuwenhoek died. It contained Joblot's microscope designs and his extensive observations on microorganisms including experiments on spontaneous generation. Joblot's work and his observations have often been overlooked, misdated, and denigrated. This is due to attention given to a few apparently fanciful drawings of microorganisms, and the identification of his work as appearing in a posthumous 1754 edition. The second edition not only placed Joblot's work as decades after Leeuwenhoek's death, but was also expanded by the publisher to include unattributed material from famous sources. Here an attempt is made to shine a light on Joblot's work and bring it out of Leeuwenhoek's shadow.

Jacob Whitman Bailey (1811-1857): Ehrenberg's ambassador to America.

Science in general, and microscopy in particular, lagged far behind Europe in early 19th century America. Jacob Bailey was one of the very few American microscopists. In eulogies he was called 'the Ehrenberg of America' and 'the founder of microscopical research'. He was a major figure in the scientific community of America in his time and instrumental in promoting microscopy and the study of microscopic organisms. His name maybe familiar to specialists who focus on diatoms, foraminifera, or radiolaria. However, Bailey's important contributions to protistology have received very little attention and he is rarely mentioned in histories of protistology. Here Jacob Bailey's life, his protistological works, as well as his roles in the development of microscopy in America and the 19th century debate over life in the deep sea, are reviewed.

On Charles Atwood Kofoid (1865-1947): The good, the bad, and the ugly.

Charles Atwood Kofoid was a scientist of considerable stature and a key figure in the development of protistology in the United States of America during first half of the 20th century. Today he is known mainly for his detailed taxonomic monographs on protists of the marine plankton, specifically dinoflagellates and tintinnid ciliates. Lesser known today is the wide range of Kofoid's work in protistology. Little known is his responsibility for one of the great mistakes of protistology: the existence of a protistan nervous system, the "neuromortorium". Largely unknown is Kofoid's enthusiastic involvement in the eugenics movement. Here, following a brief biography, I will provide a review of Kofoid's enduring contribution to protistology, "the good", then the story of the neuromotorium, "the bad", and finally an account of Kofoid's implication in the eugenics movement, "the ugly".

Marine ecology: different measures of biodiversity.

In their analysis of global trends of diversity in marine plankton, Irigoien et al. find that taxonomic diversity of zooplankton (consumer) is a unimodal function of community biomass that is unrelated to phytoplankton (producer) diversity. Their results are unexpected because in terrestrial organisms primary-producer diversity is a good predictor of consumer diversity. I contend that this apparent uncoupling of producer and consumer diversity in marine plankton is likely to be an artefact due to the authors' use of different measures of diversity for producers and for consumers.

On Edouard Claparède and Johannes Lachmann (Clap & Lach) and their "Etudes sur les Infusoires et les Rhizopodes".

Accounts are given of the lives and careers of Edouard Claparède (1832-1871) and Johannes Lachmann (1832-1860), the authors of the landmark work of 19th century protistology "Etudes sur les Infusoires et les Rhizopodes", published in 3 parts in 1859, 1860 and 1861. Accounts are also given on the origin of the monograph, the relationship of Claparède and Lachmann with Ernst Haeckel, and Claparède's role as a promoter of Darwin's theories. Suggestions as to how to properly cite the monograph of Claparède and Lachmann are provided, as well as a supplementary file listing the protist species currently accepted as having been first described in their monograph.

Protist diversity and function in the dark ocean - Challenging the paradigms of deep-sea ecology with special emphasis on foraminiferans and naked protists.

The dark ocean and the underlying deep seafloor together represent the largest environment on this planet, comprising about 80% of the oceanic volume and covering more than two-thirds of the Earth's surface, as well as hosting a major part of the total biosphere. Emerging evidence suggests that these vast pelagic and benthic habitats play a major role in ocean biogeochemistry and represent an "untapped reservoir" of high genetic and metabolic microbial diversity. Due to its huge volume, the water column of the dark ocean is the largest reservoir of organic carbon in the biosphere and likely plays a major role in the global carbon budget. The dark ocean and the seafloor beneath it are also home to a largely enigmatic food web comprising little-known and sometimes spectacular organisms, mainly prokaryotes and protists. This review considers the globally important role of pelagic and benthic protists across all protistan size classes in the deep-sea realm, with a focus on their taxonomy, diversity, and physiological properties, including their role in deep microbial food webs. We argue that, given the important contribution that protists must make to deep-sea biodiversity and ecosystem processes, they should not be overlooked in biological studies of the deep ocean.

Protistological science dissemination.

It is rare to meet protistologists who are not passionate about their study subject. The vast majority of people, however, never get the chance to hear about the work of these researchers. Although every researcher working on protists is likely to be aware of this situation, efforts made and tools employed for dissemination of knowledge are rarely documented. Following a proposal by the Italian Society of Protistology, a workshop at the 2019 VIII European Congress of Protistology in Rome, Italy, was dedicated to protistological knowledge dissemination. Through the many interventions, we discovered the diversity of efforts to reveal the protistan world to the general public, including museum exhibitions and activities, public understanding of science events, citizen science projects, specific book publications, the use of protists in teaching at all levels from primary school children to university undergraduate students, and to a global audience via social media. The participation of the workshop delegates in the discussions indicated that presentations on the wonderful world of protists to the public not only increase the visibility and accessibility of protistology research but are also very important for the scientific community. Here we report on some of the key aspects of the presentations given in the dissemination workshop.

Unmasking "The Eldest Son of The Father of Protozoology": Charles King.

In 1703 two articles appeared in the Transactions of the Royal Society, authored by an unnamed gentleman. The articles, with deference to Leeuwenhoeck, described recent observations made with a microscope. Clifford Dobell, in his biography of Leeuwenhoeck, remarked at length on the extraordinary quality of the illustrations and descriptions of "animalcules". He declared the anonymous author to be the scion and master draughtsman of Leeuwenhoeck's followers. Still today, one of the illustrations is credited with being the first unambiguous depiction of a diatom. Here I present evidence that the anonymous author was Charles King of Staffordshire and evidence of his talent. John Hill is often credited for the first naming and illustrating Paramecium and other ciliates in his 1752 book, but it has been claimed repeatedly that he copied the anonymous 1703 illustrations without attribution. Here, the illustrations from 1703 and 1752 are given, and casual examination suffices to show not only that the illustrations were copied, but also that the 1703 illustrations (and text descriptions) of Charles King are of a far higher quality than those of John Hill. Although very little is known about Charles King, he deserves recognition as a pioneer of protistology.

From the Popularization of Microscopy in the Victorian Age: A Lesson for Today's "Outreach".

In the latter half of the Victorian Age (1837-1901) microscopy was introduced as popular past-time. Many books were published aimed at general audiences, both adult and juvenile, on microscopy. Here I consider 5 of these popular books of particular interest to protistologists as they included presentations of 'infusoria' or 'animalcules'. I focus on the scientific backgrounds of the authors, from what we know of them, and the approaches taken to engage the reader based on their texts and illustrations. The possible lesson to be drawn from this exercise concerns our oft-mandated efforts in "Outreach". The methods used by 19th century popularizes of the 'wonders of the microscopic world' can likely be used today. They appealed to the imagination, to empowerment, and gave very practical instructions on how to see the invisible. I conclude that we should likely target the very young and describe our organisms with the enthusiasm that brought us to Protistology to begin with, but which we often conceal.