Shotgun metagenomics reveal a diverse assemblage of protists in a model Antarctic soil ecosystem.

The soils of the McMurdo Dry Valleys (MDV) of Antarctica are established models for understanding fundamental processes in soil ecosystem functioning (e.g. ecological tipping points, community structuring and nutrient cycling) because the extreme physical environment drastically reduces biodiversity and ecological complexity. Understanding the functioning of MDV soils requires in-depth knowledge of the diversity of MDV soil species. Protists, which contribute significantly to soil ecosystem functioning worldwide, remain poorly characterized in the MDV. To better assess the diversity of MDV protists, we performed shotgun metagenomics on 18 sites representing a variety of landscape features and edaphic variables. Our results show MDV soil protists are diverse at both the genus (155 of 281 eukaryote genera) and family (120) levels, but comprise only 6% of eukaryotic reads. Protists are structured by moisture, total N and distance from the local coast and possess limited richness in arid (< 5% moisture) and at high elevation sites, known drivers of communities in the MDV. High relative diversity and broad distribution of protists in our study promotes these organisms as key members of MDV soil microbiomes and the MDV as a useful system for understanding the contribution of soil protists to the structure of soil microbiomes.

An emergency situation for pen shells in the Mediterranean: The Adriatic Sea, one of the last Pinna nobilis shelters, is now affected by a mass mortality event.

We identified areas with high individual densities of the pen shell, Pinna nobilis, in two areas along the Croatian Adriatic coast. The surveys carried out in 2018 and 2019 showed population densities of approximately 9 to 13 individuals/100 m2. However, in 2019 a mass mortality event (MME) causing 36% to 100% mortality of this bivalve species was observed in the surveyed Croatian bays. The parasite Haplosporidium pinnae was identified by histological and molecular methods in all affected sampled individuals, while Mycobacterium sp. and Gram negative bacilli were detected in some affected and live bivalves. This finding constitutes the first record of these pathogens affecting P. nobilis in the middle Adriatic, confirming the continuous spread of the disease. Previously, the Adriatic water body was considered to be a natural shelter against the MME caused by pathogens in pen shell populations because of its distinct ecological features. The Adriatic Sea is a semi-closed water body with the largest continental shelf in the Mediterranean Sea, and due to its geomorphology and bathymetry, it is a sea with distinct characteristics. Monitoring plans and further studies in the Adriatic bays are now a priority for mitigating the high risk of extinction and working toward the conservation of this protected species.

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.

Distinct communities of Cercozoa at different soil depths in a temperate agricultural field.

Protists are the most important predators of soil microbes like bacteria and fungi and are highly diverse in terrestrial ecosystems. However, the structure of protistan communities throughout the soil profile is still poorly explored. Here, we used Illumina sequencing to track differences in the relative abundance and diversity of Cercozoa, a major group of protists, at two depths; 10-30 cm (topsoil) and 60-75 cm (subsoil) in an agricultural field in Germany. At the two depths, we also distinguished among three soil compartments: rhizosphere, drilosphere (earthworm burrows) and bulk soil. With increasing depth, we found an overall decline in richness, but we were able to detect subsoil specific phylotypes and contrasting relative abundance patterns between topsoil and subsoil for different clades. We also found that the compartment effect disappeared in the subsoil when compared to the topsoil. More studies are now needed to describe and isolate these possibly subsoil specific phylotypes and better understand their ecology and function.

A novel paramyxean parasite, Marteilia tapetis sp. nov. (Cercozoa) infecting the digestive gland of Manila clam Ruditapes philippinarum from the southeast coast of Korea.

Paramyxean parasites in the genus Marteilia deteriorate digestive tissues of the host organisms, resulting in mortality of oysters, cockles, and mussels. Most reports of infection by Marteilia spp. are from Europe, while a new species of Marteilia was identified recently in Japan. Here, we report a previously unidentified species in the genus Marteilia from digestive diverticula of Manila clam Ruditapes philippinarum from the south coast of Korea. Prevalence of the parasite was low, 0.5-3.3% in the study sites. We characterized this species using light and transmission electron microscopy (TEM), and analyzed the 18S rDNA sequence. Light microscopy revealed the sporulation process from uninucleated stage to spore in the epithelial tissues of the digestive gland. TEM revealed that the parasites produced four secondary cells containing four tri-cellular spores. An electron-dense haplosporosome-like structure and striated inclusions were evident in the spore and the primary cells, respectively, while refringent granules were rarely observed in the secondary cells. Phylogenetic analyses of the 18S rDNA sequence placed this isolate in the genus Marteilia, although it is not identical to other known species in the genus. Based on morphological and molecular characters, we describe this species as Marteilia tapetis sp. nov., the second Marteilia species reported parasitizing Manila clams in Asian waters.

SSU-rRNA Gene Sequencing Survey of Benthic Microbial Eukaryotes from Guaymas Basin Hydrothermal Vent.

Microbial eukaryotes have important roles in marine food webs, but their diversity and activities in hydrothermal vent ecosystems are poorly characterized. In this study, we analyzed microbial eukaryotic communities associated with bacterial (Beggiatoa) mats in the 2,000 m deep-sea Guaymas Basin hydrothermal vent system using 18S rRNA gene high-throughput sequencing of the V4 region. We detected 6,954 distinct Operational Taxonomic Units (OTUs) across various mat systems. Of the sequences that aligned with known protistan phylotypes, most were affiliated with alveolates (especially dinoflagellates and ciliates) and cercozoans. OTU richness and community structure differed among sediment habitats (e.g. different mat types and cold sediments away from mats). Additionally, full-length 18S rRNA genes amplified and cloned from single cells revealed the identities of some of the most commonly encountered, active ciliates in this hydrothermal vent ecosystem. Observations and experiments were also conducted to demonstrate that ciliates were trophically active and ingesting fluorescent bacteria or Beggiatoa trichomes. Our work suggests that the active and diverse protistan community at the Guaymas Basin hydrothermal vent ecosystem likely consumes substantial amounts of bacterial biomass, and that the different habitats, often defined by distances of just a few 10s of cm, select for particular assemblages and levels of diversity.

Phylogeny and Classification of Novel Diversity in Sainouroidea (Cercozoa, Rhizaria) Sheds Light on a Highly Diverse and Divergent Clade.

Sainouroidea is a molecularly diverse clade of cercozoan flagellates and amoebae in the eukaryotic supergroup Rhizaria. Previous 18S rDNA environmental sequencing of globally collected fecal and soil samples revealed great diversity and high sequence divergence in the Sainouroidea. However, a very limited amount of this diversity has been observed or described. The two described genera of amoebae in this clade are Guttulinopsis, which displays aggregative multicellularity, and Rosculus, which does not. Although the identity of Guttulinopsis is straightforward due to the multicellular fruiting bodies they form, the same is not true for Rosculus, and the actual identity of the original isolate is unclear. Here we isolated amoebae with morphologies like that of Guttulinopsis and Rosculus from many environments and analyzed them using 18S rDNA sequencing, light microscopy, and transmission electron microscopy. We define a molecular species concept for Sainouroidea that resulted in the description of 4 novel genera and 12 novel species of naked amoebae. Aggregative fruiting is restricted to the genus Guttulinopsis, but other than this there is little morphological variation amongst these taxa. Taken together, simple identification of these amoebae is problematic and potentially unresolvable without the 18S rDNA sequence.

Short-term dynamics and interactions of marine protist communities during the spring-summer transition.

We examined the short-term variability, by daily to weekly sampling, of protist assemblages from March to July in surface water of the San Pedro Ocean Time-series station (eastern North Pacific), by V4 Illumina sequencing of the 18S rRNA gene. The sampling period encompassed a spring bloom followed by progression to summer conditions. Several protistan taxa displayed sharp increases and declines, with whole community Bray-Curtis dissimilarities of adjacent days being 66% in March and 40% in May. High initial abundance of parasitic Cercozoa Cryothecomonas longipes and Protaspis grandis coincided with a precipitous decline of blooming Pseudo-nitzschia diatoms, possibly suggesting their massive infection by these parasites; these cercozoans were hardly detectable afterwards. Canonical correspondence analysis indicated a limited predictability of community variability from environmental factors. This indicates that other factors are relevant in explaining changes in protist community composition at short temporal scales, such as interspecific relationships, stochastic processes, mixing with adjacent water, or advection of patches with different protist communities. Association network analysis revealed that interactions between the many parasitic OTUs and other taxa were overwhelmingly positive and suggest that although sometimes parasites may cause a crash of host populations, they may often follow their hosts and do not regularly cause enough mortality to potentially create negative correlations at the daily to weekly time scales we studied.

High planktonic diversity in mountain lakes contains similar contributions of autotrophic, heterotrophic and parasitic eukaryotic life forms.

A rich eukaryotic planktonic community exists in high-mountain lakes despite the diluted, oligotrophic and cold, harsh prevailing conditions. Attempts of an overarching appraisal have been traditionally hampered by observational limitations of small, colorless, and soft eukaryotes. We aimed to uncover the regional eukaryotic biodiversity of a mountain lakes district to obtain general conclusions on diversity patterns, dominance, geographic diversification, and food-web players common to oligotrophic worldwide distributed freshwater systems. An unprecedented survey of 227 high-altitude lakes comprising large environmental gradients was carried out using Illumina massive tag sequencing of the 18S rRNA gene. We observed a large Chrysophyceae dominance in richness, abundance and novelty, and unveiled an unexpected richness in heterotrophic phagotrophs and parasites. In particular, Cercozoa and Chytridiomycota showed diversity features similar to the dominant autotrophic groups. The prominent beta-dispersion shown by parasites suggests highly specific interactions and a relevant role in food webs. Interestingly, the freshwater Pyrenean metacommunity contained more diverse specific populations than its closest marine oligotrophic equivalent, with consistently higher beta-diversity. The relevance of unseen groups opens new perspectives for the better understanding of planktonic food webs. Mountain lakes, with remarkable environmental idiosyncrasies, may be suitable environments for the genetic diversification of microscopic eukaryotic life forms.

Diversity of Cercomonad Species in the Phyllosphere and Rhizosphere of Different Plant Species with a Description of Neocercomonas epiphylla (Cercozoa, Rhizaria) a Leaf-Associated Protist.

Cercomonads are among the most abundant and diverse groups of heterotrophic flagellates in terrestrial systems and show an affinity to plants. However, we still lack basic knowledge of plant-associated protists. We isolated 75 Cercomonadida strains from the phyllosphere and rhizosphere of plants from three functional groups: grasses (Poa sp.), legumes (Trifolium sp.) and forbs (Plantago sp.), representing 28 OTUs from the genera Cercomonas, Neocercomonas and Paracercomonas. The community composition differed clearly between phyllosphere and rhizosphere, but was not influenced by plant species identity. From these isolates we describe three novel cercomonad species including Neocercomonas epiphylla that was consistently and exclusively isolated from the phyllosphere. For each new species we provide a detailed morphological description as well as an 18S rDNA gene sequence as a distinct marker of species identity. Our data contribute to a better resolution of the systematics of cercomonads and their association with plants, by describing three novel species and adding gene sequences of 10 new cercomonad genotypes and of nine previously described species. In view of the functional importance of cercozoan communities in the phyllosphere and rhizosphere of plants, a more detailed understanding of their composition, function and predator-prey interactions are clearly required.

Sappinia sp. (Amoebozoa: Thecamoebida) and Rosculus sp. (SAR: Cercozoa) Isolated From King Penguin Guano Collected in the Subantarctic (South Georgia, Salisbury Plain) and their Coexistence in Culture.

Two amoeboid organisms of the genera Sappinia Dangeard, 1896 and Rosculus Hawes, 1963 were identified in a sample containing king penguin guano. This sample, collected in the Subantarctic, enlarges the list of fecal habitats known for the presence of coprophilic amoebae. The two organisms were co-isolated and subcultured for over 6 mo, with continuous efforts being invested to separate each one from the mixed culture. In the mixed culture, Rosculus cells were fast growing, tolerated changes in culturing conditions, formed cysts, and evidently were attracted by Sappinia trophozoites. The separation of the Rosculus strain was accomplished, whereas the Sappinia strain remained intermixed with inseparable Rosculus cells. Sappinia cell populations were sensitive to changes in culturing conditions; they improved with reduction of Rosculus cells in the mixed culture. Thick-walled cysts, reportedly formed by Sappinia species, were not seen. The ultrastructure of both organisms was congruent with the currently accepted generic characteristics; however, some details were remarkable at the species level. Combined with the results of phylogenetic analyses, our findings indicate that the ultrastructure of the glycocalyx and the presence/absence of the Golgi apparatus in differential diagnoses of Sappinia species require a critical re-evaluation.

An Orphan Protist Quadricilia rotundata Finally Finds Its Phylogenetic Home in Cercozoa.

Quadricilia rotundata is a heterotrophic flagellate with four flagella. However, because this species has no clear morphological characteristics or molecular data affiliating it with any known group, Q. rotundata has been treated as a protist incertae sedis, for a long time. Here, we established a clonal culture of Q. rotundata and sequenced its 18S rDNA sequence. Molecular phylogenetic analysis successfully placed Q. rotundata in an environmental clade within Cercozoa, which contributes to expand the morphological and species diversity within Cercozoa. We also discuss morphological evolution within Cercozoa based on this finding.

Rediscovery of the Testate Amoeba Genus Penardeugenia (Thaumatomonadida, Imbricatea).

The genus Penardeugenia DEFLANDRE 1958 accommodates a single species which was described as Pamphagus bathybioticus PENARD 1904. Although this species seems to be well characterized in its description, it has never been recorded again. Despite its possession of silica scales, it was assigned to the scale-lacking Chlamydophryidae (Thecofilosea, Cercozoa). We here present morphological data of three isolates. One of them was successfully cultured and enabled the amplification of its SSU rDNA sequence. Molecular analyses revealed the evolutionary origin of Penardeugenia in the scale-bearing flagellate class Thaumatomonadida, which is backed up by morphological similarities of their scales. We consider Penardeugenia to represent a closely related genus to Thaumatomastix, which apparently lost its ability to form flagella. We describe two new species from Germany, P. huelsmannii and P. hausmannii.

Phytocercomonas venanatans, a New Species of Cercozoa Associated with Chlorotic Streak of Sugarcane.

Chlorotic streak is a global disease of commercial sugarcane (Saccharum spp. hybrids). The disease is transmitted by wet soil, water, as well as in diseased planting material. Although first recognized almost 90 years ago and despite significant research effort, the identity of the causal agent has been elusive. Metagenomic high throughput sequencing (HTS) facilitated the discovery of novel protistan ribosomal and nuclear genes in chlorotic streak-infected sugarcane. These sequences suggest a possible causal agent belonging to the order Cercomonadida (Rhizaria, phylum Cercozoa). An organism with morphological features similar to cercomonads (=Cercomonadida) was isolated into pure axenic culture from internal stalk tissues of infected sugarcane. The isolated organism contained DNA sequences identical to those identified in infected plants by HTS. The DNA sequences and the morphology of the organism did not match any known species. Here we present a new genus and species, Phytocercomonas venanatans, which is associated with chlorotic streak of sugarcane. Amplicon sequencing also supports that P. venanatans is associated with this disease. This is the first reported member from Cercomonadida showing a probable pathogenic association with higher plants.

Protists are an integral part of the Arabidopsis thaliana microbiome.

Although protists occupy a vast range of habitats and are known to interact with plants among other things via disease suppression, competition or growth stimulation, their contributions to the 'phytobiome' are not well described. To contribute to a more comprehensive picture of the plant holobiont, we examined cercozoan and oomycete taxa living in association with the model plant Arabidopsis thaliana grown in two different soils. Soil, roots, leaves and wooden toothpicks were analysed before and after surface sterilization. Cercozoa were identified using 18S rRNA gene metabarcoding, whereas the Internal Transcribed Spacer 1 was used to determine oomycetes. Subsequent analyses revealed strong spatial structuring of protist communities between compartments, although oomycetes appeared more specialized than Cercozoa. With regards to oomycetes, only members of the Peronosporales and taxa belonging to the genus Globisporangium were identified as shared members of the A. thaliana microbiome. This also applied to cercozoan taxa belonging to the Glissomonadida and Cercomonadida. We identified a strong influence by edaphic factors on the rhizosphere, but not for the phyllosphere. Distinct differences of Cercozoa found preferably in wood or fresh plant material imply specific niche adaptations. Our results highlight the importance of micro-eukaryotes for the plant holobiont.

Confirmation that the Novel Cercozoa Phytocercomonas venanatans Is the Cause of the Disease Chlorotic Streak in Sugarcane.

A cercomonad, named Phytocercomonas venanatans, is confirmed as the cause of the sugarcane disease chlorotic streak. This was achieved by establishing aseptic liquid cultures of the pathogen isolated from internal pieces of sugarcane stalk tissue. Actively motile cultures of the pathogen were inoculated into sugarcane roots, stalks, and leaf whorls. Infected plants subsequently developed the characteristic symptoms of chlorotic streak. Infection was confirmed by PCR screening of plant tissues and by reisolation of the pathogen into aseptic culture followed by PCR and microscopic confirmation. P. venanatans is the first reported pathogenic cercomonad able to systemically infect higher plants and the first plant pathogenic cercozoan able to be successfully grown in axenic culture on common microbiological media.

Application of a competitive real time PCR for detection of Marteilia refringens genotype "O" and "M" in two geographical locations: The Ebro Delta, Spain and the Rhine-Meuse Delta, the Netherlands.

Species belonging to the genus Marteilia are protozoan parasites of bivalves. The species Marteilia refringens, jeopardizing the health of European bivalves, is included on the list of OIE notifiable pathogens. Two genotypes of Marteilia refringens are distinguished: type "O" affecting mainly oysters, and type "M" affecting mainly mussels. Historically, detection of Marteilia species is primarily carried out by histology. In recent years molecular assays are more frequently used for the detection of mollusc pathogens, also in routine monitoring. In the present work, a competitive real-time PCR assay was developed for rapid and sensitive detection of M. refringens and discrimination between "M" and "O" genotypes of M. refringens. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability and efficiency. Subsequent application of the assay on collected bivalves from two geographical locations, the Ebro Delta in Mediterranean Spain and the Rhine-Meuse Delta in the Netherlands resulted in detection of M. refringens type M in Mytilus galloprovincialis and M. refringens type O in Ostrea edulis from Spain. In two O. edulis specimen both M. refringens type O and type M were detected. In the Netherlands M. refringens was not observed in any of the tested Mytilus edulis and O. edulis. The results obtained by real time PCR were in correspondence with the results obtained by histopathology and a substantial agreement with the results obtained by conventional PCR. In conclusion, the developed real time PCR assay facilitates rapid detection and subtyping of M. refringens and could be applied for further studies on epidemiology of the parasite, geographical distribution and host specificity.

Rhogostomidae (Cercozoa) from soils, roots and plant leaves (Arabidopsis thaliana): Description of Rhogostoma epiphylla sp. nov. and R. cylindrica sp. nov.

Cercozoa are a highly diverse protist phylum in soils and in the phyllosphere of plants. Many families are still poorly described and the vast majority of species are still unknown. Although testate amoebae are among the better-studied protists, only little quantitative information exists on the morphology, phylogeny and ecology of cercozoan Rhogostomidae. We cultured four different strains of Rhogostoma spp. isolated from Arabidopsis leaves, agricultural soil and rhizosphere soil of Ocimum basilicum and Nicotiana sp. We describe Rhogostoma epiphylla sp. nov. and R. cylindrica sp. nov. and present their morphology, studied their food spectra in food range experiments and obtained two SSU rDNA gene sequences resulting in an updated thecofilosean phylogeny. Short generation times, desiccation resistance and the ability to prey on a wide range of algae and yeasts from the phyllosphere were seen as crucial traits for the phyllosphere colonization by Rhogostoma. In contrast, the soil-dwelling R. cylindrica did not feed on eukaryotes in our experiment.

A Novel Lineage of 'Naked Filose Amoebae'; Kraken carinae gen. nov. sp. nov. (Cercozoa) with a Remarkable Locomotion by Disassembly of its Cell Body.

The term 'filose amoebae' describes a highly polyphyletic assemblage of protists whose phylogenetic placement can be unpredictable based on gross morphology alone. We isolated six filose amoebae from soils of two European countries and describe a new genus and species of naked filose amoebae, Kraken carinae gen. nov. sp. nov. We provide a morphological description based on light microscopy and small subunit rRNA gene sequences (SSU rDNA). In culture, Kraken carinae strains were very slow-moving and preyed on bacteria using a network of filopodia. Phylogenetic analyses of SSU sequences reveal that Kraken are core (filosan) Cercozoa, branching weakly at the base of the cercomonad radiation, most closely related to Paracercomonas, Metabolomonas, and Brevimastigomonas. Some Kraken sequences are >99% similar to an environmental sequence obtained from a freshwater lake in Antarctica, indicating that Kraken is not exclusively soil dwelling, but also inhabits freshwater habitats.

High Diversity Revealed in Leaf-Associated Protists (Rhizaria: Cercozoa) of Brassicaceae.

The largest biological surface on earth is formed by plant leaves. These leaf surfaces are colonized by a specialized suite of leaf-inhabiting microorganisms, recently termed "phyllosphere microbiome". Microbial prey, however, attract microbial predators. Protists in particular have been shown to structure bacterial communities on plant surfaces, but virtually nothing is known about the community composition of protists on leaves. Using newly designed specific primers targeting the 18S rDNA gene of Cercozoa, we investigated the species richness of this common protist group on leaves of four Brassicaceae species from two different locations in a cloning-based approach. The generated sequences revealed a broad diversity of leaf-associated Cercozoa, mostly bacterial feeders, but also including known plant pathogens and a taxon of potential endophytes that were recently described as algal predators in freshwater systems. This initial study shows that protists must be regarded as an integral part of the microbial diversity in the phyllosphere of plants.