Protist Species Research Articles

Mixotrophic Activity and Diversity of Antarctic Marine Protists in Austral Summer

Identifying putative mixotrophic protist species in the environment is important for understanding their behavior, with the recovery of these species in culture essential for determining the triggers of feeding, grazing rates and overall impact on bacterial standing stocks. In this project, mixotroph abundances determined using tracer ingestion in water and sea ice samples collected in the Ross Sea, Antarctica during the summer of 2011 were compared with data from the spring (Ross Sea) and fall (Arctic) to examine the impacts of bacterivory/mixotrophy. Mixotrophic nanoplankton were usually less abundant than heterotrophs, but consumed more of the bacterial standing stock per day due to relatively higher ingestion rates (1-7 bacteria mixotroph-1 h-1 vs. 0.1-4 bacteria heterotroph-1 h-1). Yet, even with these high rates observed in the Antarctic summer, mixotrophs appeared to have a smaller contribution to bacterivory than in the Antarctic spring. Additionaly, putative mixotroph taxa were identified through incubation experiments accomplished with bromodeoxyuridine labeled bacteria as food, immunoprecipitation (IP) of labeled DNA, and amplification and high throughput sequencing of the eukaryotic ribosomal V9 region. Putative mixotroph OTUs were identified in the IP samples by taxonomic similarity to known phototroph taxa. OTUs that had increased abundance in IP samples compared to the non-IP samples from both surface and chlorophyll maximum (CM) depths were considered to represent active mixotrophy and include ones taxonomically similar to Dictyocha, Gymnodinium, Pentapharsodinium and Symbiodinium. These OTUs represent target taxa for isolation and laboratory experiments on triggers for mixotrophy, to be combined with qPCR to estimate their abundance, seasonal distribution and potential impact.

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species. Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species

Incomplete Co-cladogenesis Between Zootermopsis Termites and Their Associated Protists.

Coevolution is a major driver of speciation in many host-associated symbionts. In the termite-protist digestive symbiosis, the protists are vertically inherited by anal feeding among nest mates. Lower termites (all termite families except Termitidae) and their symbionts have broadly co-diversified over ~170 million yr. However, this inference is based mainly on the restricted distribution of certain protist genera to certain termite families. With the exception of one study, which demonstrated congruent phylogenies for the protist Pseudotrichonympha and its Rhinotermitidae hosts, coevolution in this symbiosis has not been investigated with molecular methods. Here we have characterized the hindgut symbiotic protists (Phylum Parabasalia) across the genus Zootermopsis (Archotermopsidae) using single cell isolation, molecular phylogenetics, and high-throughput amplicon sequencing. We report that the deepest divergence in the Zootermopsis phylogeny (Zootermopsis laticeps [Banks; Isoptera: Termopsidae]) corresponds with a divergence in three of the hindgut protist species. However, the crown Zootermopsis taxa (Zootermopsis angusticollis [Hagen; Isoptera: Termopsidae], Z. nevadensis nevadensis [Hagen; Isoptera: Termopsidae], and Z. nevadensis nuttingi [Haverty & Thorne; Isoptera: Termopsidae]) share the same protist species, with no evidence of co-speciation under our methods. We interpret this pattern as incomplete co-cladogenesis, though the possibility of symbiont exchange cannot be entirely ruled out. This is the first molecular evidence that identical communities of termite-associated protist species can inhabit multiple distinct host species.

Morphological and genetic description of Syringammina limosa sp. nov., the first xenophyophore (Foraminifera) from the deep Sea of Okhotsk

Foraminifera are one of the most abundant taxa in the deep sea. Our sampling in the Sea of Okhotsk and the adjacent Pacific during the SokhoBio cruise revealed a large number of species confined to this ecosystem. Many Foraminifera below the CCD (carbonate compensation depth) are characterized by the formation of agglutinated tests, made up of material from the surrounding environment. Xenophyophores represent an important group of large agglutinated monothalamous Foraminifera with fragile tests that can measure several centimeters in size. In the deep Kuril Basin of the Sea of Okhotsk, we retrieved an undescribed xenophyophore, dwelling on diatomaceous ooze at a depth of about 3300 m. The species Syringammina limosa sp. nov., described herein, possesses a soft, agglutinated test made up primarily of fine sediment particles, diatom fragments and sponge spicules. It measures about 3 cm in diameter and is characterized by a complex 3D structure consisting of tubular elements proximally fused into continuous, folding plates. In a phylogenetic tree based on small-subunit ribosomal DNA (SSU rDNA) sequences it branches as the sister to Syringammina corbicula. All specimens found were considered alive at the moment of collection, due to the presence of intact cytoplasm-containing strands (granellare) within the test interior. The granellare contained copious amounts of intracellular crystals (granellae) with a wide range of different shapes. Electron microscopy and analysis of the elemental composition identified Si, Al and Fe in the granellae of the holotype, and the xenophyophore-typical barium sulphate (barite) granellae in another specimen. While the presence of minerals, gathered from the environment, is typical for the cytoplasm of xenophyophores, their actual role in the organism remains unclear. Despite many species of xenophyophores having been described recently, the discovery of a new species of these fascinating protists in the Sea of Okhotsk suggests that there is still a large amount of undocumented diversity.

Parasitic amoebae found in water bodies of Ukraine

Two parasitic amoebian species are found in mollusks of the water bodies of Ukraine. Vahlkampfia sp. is found in hepatopankreas of Unio conus Spengler, 1793, and Acanthamoeba sp. is observed in mantle cavity of Viviparus viviparus Linnaeus, 1758. For these protist species, the mollusks are shown to be intermediate hosts where amoebae feed and reproduce. An experimental infection with Vahlkampfia sp. and Acanthamoeba sp. was not successful, no pathological changes in mollusks were observed. These amoebae are successfully cultured in fresh water and agar medium, hence we can safely consider them free-living.

Diatom flagellar genes and their expression during sexual reproduction in Leptocylindrus danicus

BackgroundFlagella have been lost in the vegetative phase of the diatom life cycle, but they are still present in male gametes of centric species, thereby representing a hallmark of sexual reproduction. This process, besides maintaining and creating new genetic diversity, in diatoms is also fundamental to restore the maximum cell size following its reduction during vegetative division. Nevertheless, sexual reproduction has been demonstrated in a limited number of diatom species, while our understanding of its different phases and of their genetic control is scarce.ResultsIn the transcriptome of Leptocylindrus danicus, a centric diatom widespread in the world’s seas, we identified 22 transcripts related to the flagella development and confirmed synchronous overexpression of 6 flagellum-related genes during the male gamete formation process. These transcripts were mostly absent in the closely related species L. aporus, which does not have sexual reproduction. Among the 22 transcripts, L. danicus showed proteins that belong to the Intra Flagellar Transport (IFT) subcomplex B as well as IFT-A proteins, the latter previously thought to be absent in diatoms. The presence of flagellum-related proteins was also traced in the transcriptomes of several other centric species. Finally, phylogenetic reconstruction of the IFT172 and IFT88 proteins showed that their sequences are conserved across protist species and have evolved similarly to other phylogenetic marker genes.ConclusionOur analysis describes for the first time the diatom flagellar gene set, which appears to be more complete and functional than previously reported based on the genome sequence of the model centric diatom, Thalassiosira pseudonana. This first recognition of the whole set of diatom flagellar genes and of their activation pattern paves the way to a wider recognition of the relevance of sexual reproduction in individual species and in the natural environment.

Intestinal Parasitic Infections and Environmental Water Contamination in a Rural Village of Northern Lao PDR.

A field survey studying intestinal parasites in humans and microbial pathogen contamination at environment was performed in a Laotian rural village to identify potential risks for disease outbreaks. A parasitological investigation was conducted in Ban Lak Sip village, Luang Prabang, Lao PDR involving fecal samples from 305 inhabitants as well as water samples taken from 3 sites of the local stream. Water analysis indicated the presence of several enteric pathogens, i.e., Aeromonas spp., Vibrio spp., E. coli H7, E. coli O157: H7, verocytotoxin-producing E. coli (VTEC), Shigella spp., and enteric adenovirus. The level of microbial pathogens contamination was associated with human activity, with greater levels of contamination found at the downstream site compared to the site at the village and upstream, respectively. Regarding intestinal parasites, the prevalence of helminth and protozoan infections were 68.9% and 27.2%, respectively. Eight helminth taxa were identified in fecal samples, i.e., 2 tapeworm species (Taenia sp. and Hymenolepis diminuta), 1 trematode (Opisthorchis sp.), and 5 nematodes (Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis, trichostrongylids, and hookworms). Six species of intestinal protists were identified, i.e., Blastocystis hominis, Cyclospora spp., Endolimax nana, Entamoeba histolytica/E. dispar, Entamoeba coli, and Giardia lamblia. Questionnaires and interviews were also conducted to determine risk factors of infection. These analyses together with a prevailing infection level suggested that most of villagers were exposed to parasites in a similar degree due to limited socio-economic differences and sharing of similar practices. Limited access to effective public health facilities is also a significant contributing factor.

Protist Communities in Moored Long-Term Sediment Traps (Fram Strait, Arctic)–Preservation with Mercury Chloride Allows for PCR-Based Molecular Genetic Analyses

Here we present a pilot study demonstrating, that preservation with mercury chloride allows the application of PCR-based molecular methods for the characterization of marine protist communities collected with moored long-term sediment traps. They can provide information on pelagic protist communities by collecting sinking plankton from the upper water column all year-round, even in remote polar oceans. Assessment of small protist species from the nano- and picoplankton fractions in sedimented material by microscopy is extremely challenging or almost impossible. Hence, comprehensive studies of variability in protist community composition in moored long-term sediment traps are scarce. Considering that marine nano- and picoeukaryotes are ecologically very important, new approaches are urgently needed to investigate protists in the smallest size-fractions of moored long-term sediment trap samples. We applied the quick and cost-effective Terminal Restriction Length Polymorphism (T-RFLP) on a set of selected samples that were collected between 2000 and 2010 in September at a depth of ~ 300 m in the area of the “LTER (Long-Term Ecological Research) site HAUSGARTEN“ in the eastern Fram Strait (Arctic). The results of these analyses suggest a change in the trapped protist community after 2002 in this area. A comparison of 18S sequences obtained via 454-pyrosequencing from samples collected in the water column and mercury chloride preserved sediment traps in 2009 and 2010 suggests, that sediment traps might reflect the pelagic eukaryotic microbial biodiversity qualitatively. Furthermore, we have indication that preservation with mercury chloride does not severely change the nucleotide composition of 18S rRNA genes in long-term sediment traps. Overall, we suggest that preservation with mercury chloride is a key to open the door for molecular genetic analyses of long-term sediment trap samples, and that PCR-based molecular methods have a strong potential to become an important tool for comprehensive taxonomic analyses of protist- and bacterial communities in moored long-term sediment traps.

A tree of life based on ninety-eight expressed genes conserved across diverse eukaryotic species.

Rapid advances in DNA sequencing technologies have resulted in the accumulation of large data sets in the public domain, facilitating comparative studies to provide novel insights into the evolution of life. Phylogenetic studies across the eukaryotic taxa have been reported but on the basis of a limited number of genes. Here we present a genome-wide analysis across different plant, fungal, protist, and animal species, with reference to the 36,002 expressed genes of the rice genome. Our analysis revealed 9831 genes unique to rice and 98 genes conserved across all 49 eukaryotic species analysed. The 98 genes conserved across diverse eukaryotes mostly exhibited binding and catalytic activities and shared common sequence motifs; and hence appeared to have a common origin. The 98 conserved genes belonged to 22 functional gene families including 26S protease, actin, ADP–ribosylation factor, ATP synthase, casein kinase, DEAD-box protein, DnaK, elongation factor 2, glyceraldehyde 3-phosphate, phosphatase 2A, ras-related protein, Ser/Thr protein phosphatase family protein, tubulin, ubiquitin and others. The consensus Bayesian eukaryotic tree of life developed in this study demonstrated widely separated clades of plants, fungi, and animals. Musa acuminata provided an evolutionary link between monocotyledons and dicotyledons, and Salpingoeca rosetta provided an evolutionary link between fungi and animals, which indicating that protozoan species are close relatives of fungi and animals. The divergence times for 1176 species pairs were estimated accurately by integrating fossil information with synonymous substitution rates in the comprehensive set of 98 genes. The present study provides valuable insight into the evolution of eukaryotes.

Environmental warming accelerates extinctions but does not alter extinction debt

Climate change is predicted to contribute to the present wave of extinctions; however, these extinctions may not immediately follow temperature increases and other climate change effects. We examined how predator species composition and increased temperatures affected bacterivorous protist community extinction debt (the number of species that will go extinct) and relaxation time (the amount of time it takes for the extinction debt to be paid off). We found that neither affected relaxation time, but that the presence of a generalist predatory protist species increased extinction debt through its role as a keystone predator. Warming also significantly decreased how long it took communities to lose half of the species that were to eventually go extinct. That this half-relaxation time was shorter in warmed communities than in unwarmed communities in this system indicates that extinctions were front-loaded in the warmed communities compared to the unwarmed communities. Our findings suggest that even if increased temperature is not expected to increase extinction debt, it could still accelerate the rate at which those extinctions will occur. This in turn narrows the time window for taking actions to prevent a large portion of the extinction debt from being paid off.

Microhabitat segregation of heterotrophic protists in the rice (Oryza sativa L.) rhizosphere

The rice rhizosphere is a hotspot for microorganisms, where heterotrophic protists are the major group of microeukaryotes and probably function as microbial predators. In this work, the microscale distribution of soil protists in a rice rhizosphere was studied in a microcosm experiment. Rice seedlings (Oryza sativa L. Nipponbare) were grown in “mini-rhizoboxes” made of thin glass plates and containing a nutrient solution and a diluted suspension of a rice-field soil in soft agar. This enabled nondestructive microscopic observations and the enumeration of heterotrophic protists in the rhizosphere. Different morphotypes of protists (ciliates, flagellates, and amoebae) densely populated the rhizosphere within 3 days after root development with an extended area at the root tips. There was a clear boundary between the rhizosphere and non-rhizosphere in terms of protistan density. Flagellates had the highest frequency and abundance in the colonization. Different protist morphotypes differed in their microscale distribution patterns. Flagellates including amoeboflagellates dominated near the roots, while amoebae inhabited sites adjacent to the flagellates, at a slight distance from the roots and with a clearly segregated habitat. Ciliates were exclusively observed at the tips of the rice roots. Denaturing gradient gel electrophoresis demonstrated that the microeukaryotic community in the rice rhizosphere was distinct from that in the non-rhizosphere. Protistan species inhabiting the rice rhizosphere were affiliated with Cercozoa (Nucleocercomonas sp. and Gymnophrys sp.), Amoebozoa (Echinamoeba sp.), and Ciliophora (Oxytricha sp.). Our results demonstrate that the rhizosphere effect of rice roots on heterotrophic protists differs at a microscale depending on the root segment. They also suggest a close relationship between Cercozoa flagellates and the roots.

Applicability of massively parallel sequencing on monitoring harmful algae at Varna Bay in the Black Sea

In this study the plankton diversity in 13 environmental samples from Varna Bay (in the western Black Sea) was analyzed using massively parallel sequencing (MPS). This preliminary study was undertaken to assess the potential of this technology for future implementation in monitoring programs in the Black Sea. Amplicon sequences of the 18S rRNA gene (V4-5 regions) were obtained using the Illumina MiSeq 250PE platform. A total of 1137 operational taxonomic units (OTUs) were obtained among which 242 OTUs with >0.990 BLAST top hit similarity (21.3% of all detected OTUs) closely related to sequences belonging to −protists. A large portion (175 OTUs=72.3%) was identified at the species levels, including species typical for the Bulgarian Black Sea plankton community, as well as many that haven’t been reported earlier in the Bulgarian Black Sea coast (124 OTUs=51.2%). Dinoflagellates were represented by the highest species number (77 OTUs comprising 31.8% of protist species), with dominant genera Gyrodinium and Heterocapsa. The present survey revealed the presence of 12 species listed as harmful, some of which have been previously overlooked, such as Cochlodinium polykrikoides, Karenia bicuneiformis, and Karlodinium veneficum. Species identification was possible for 10.3–36.0% of the detected OTUs in the six major supergroups. The frequency in Rhizaria was significantly lower than that in other major groups (p<0.05–0.01), implying difficulties in the classification from morphology-based observations. The metagenetic data had an insufficient resolution of the 18S rRNA gene for species identification in many genera. These issues may hamper the implementation of MPS-based surveys for plankton monitoring, especially for detecting harmful algal blooms (HAB). The sequencing technology is steadily improving and it is expected that sequence length and quality issues will be resolved in the near future. The ongoing efforts to register taxonomic information and quality controls in the international nucleotide sequence databases (INSDs) will be essential for improving taxonomic identification power.

Transition boundaries for protistan species turnover in hypersaline waters of different biogeographic regions.

The identification of environmental barriers which govern species distribution is a fundamental concern in ecology. Even though salt was previously identified as a major transition boundary for micro- and macroorganisms alike, the salinities causing species turnover in protistan communities are unknown. We investigated 4.5 million high-quality protistan metabarcodes (V4 region of the SSU rDNA) obtained from 24 shallow salt ponds (salinities 4%-44%) from South America and Europe. Statistical analyses of protistan community profiles identified four salinity classes, which strongly selected for different protistan communities: 4-9%, 14-24%, 27-36% and 38-44%. The proportion of organisms unknown to science is highest in the 14-24% salinity class, showing that environments within this salinity range are an unappreciated reservoir of as yet undiscovered organisms. Distinct higher-rank taxon groups dominated in the four salinity classes in terms of diversity. As increasing salinities require different cellular responses to cope with salt, our results suggest that different evolutionary lineages of protists have evolved distinct haloadaptation strategies. Salinity appears to be a stronger selection factor for the structuring of protistan communities than geography. Yet, we find a higher degree of endemism in shallow salt ponds compared with less isolated ecosystems such as the open ocean. Thus, rules for biogeographic structuring of protistan communities are not universal, but depend on the ecosystem under consideration.

Inside Front Cover, Volume 23 Issue 6

The cover image, by Katrina M. Pagenkopp Lohan et al., is based on the Biodiversity Research Molecular characterisation of protistan species and communities in ships’ ballast water across three U.S. coasts, DOI: 10.1111/ddi.12550. Photo Credit: Kimberly K. Holzer.

CeO2 nanoparticles alter the outcome of species interactions

Despite considerable research on the environmental impacts of nanomaterials, we know little about how they influence interactions between species. Here, we investigated the acute (12 d) and chronic (64 d) toxicities of cerium oxide nanoparticles (CeO2 NPs) and bulk particles (0–200 mg/L) to three ciliated protist species (Loxocephalus sp., Paramecium aurelia, and Tetrahymena pyriformis) in single-, bi-, and multispecies microcosms. The results show that CeO2 NPs strongly affected the interactions between ciliated protozoan species. When exposed to the highest CeO2 NPs (200 mg/L), the intrinsic growth rates of Loxocephalus and Paramecium were significantly decreased by 18.87% and 88.27%, respectively, while their carrying capacities declined by more than 90%. However, CeO2 NP exposure made it difficult to predict outcomes of interspecific competition between species. At higher NP exposure (100 and 200 mg/L), competition led to the extinction of both species in the Loxocephalus and Paramecium microcosms that survived in the absence of competitors or CeO2 NPs. Further, the presence of potential competitors improved the survival of Loxocephalus to hundreds of individuals per milliliter in microcosms with Tetrahymena where Loxocephalus would otherwise not be able to tolerate high levels of NP exposure. This result could be attributed to weakened NP adsorption on the cell surface due to competitor-caused reduction of NP surface charge (from −18.52 to −25.17 mV) and intensified NP aggregation via phagocytosis of NPs by ciliate cells. Our results emphasize the need to explicitly consider species interactions for a more comprehensive understanding of the ecological consequences of NP exposure.

Response of aquatic protists to electric field exposure

Purpose: To test the effects of short-term exposure of aquatic organisms to electric field (EF) with negligible magnetic component.Materials and methods: We built a plate capacitor that served as a source of EF of strengths that can be found in nature near transmission lines. We exposed two cultured protist species Euglena viridis and Paramecium caudatum to EFs for 24 hours and monitored their abundance, morphology, intracellular superoxide anion (by dihydroethidium [DHE]), hydrogen peroxide by (H2DCF) and lipid peroxidation (MDA) contents, catalase (CAT) and superoxide dismutase (SOD) activity.Results: We found that even short-term exposure to low strength EF causes changes in population abundance, morphology and oxidative stress response in both species. As the EF strength increased, abundance of both species decreased. However, at weaker EFs, fission rates were seemingly promoted. We noted a decrease in size in both organisms in directions perpendicular to their fission planes correlated with EF strength. DHE and H2DCF fluorescence intensity and SOD activity were higher in organisms exposed to the stronger EFs.Conclusions: We suggest that the electric component of the field, rather than the magnetic, is the main cause of all the noted effects. As a result, aquatic organisms should be given greater importance in studies assessing the effects of EMFs in spite of the attenuating effects of water to EF strengths.

An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads

Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of protist diversity while minimizing non-protist DNA representation. We optimized protocols for detecting zoonotic protists in raw sewage samples, with a focus on trichomonad taxa. First, we investigated the utility of two commonly used variable regions of the 18S rRNA marker gene, V4 and V9, by amplifying and Sanger sequencing 23 different eukaryotic species, including 16 protist species such as Cryptosporidium parvum, Giardia intestinalis, Toxoplasma gondii, and species of trichomonad. Next, we optimized wet-lab methods for sample processing and Illumina sequencing of both regions from raw sewage collected from a private apartment building in New York City. Our results show that both regions are effective at identifying several zoonotic protists that may be present in sewage. A combination of small extractions (1 mL volumes) performed on the same day as sample collection, and the incorporation of a vertebrate blocking primer, is ideal to detect protist taxa of interest and combat the effects of metazoan DNA. We expect that the robust, standardized methods presented in our workflow will be applicable to investigations of protists in other environmental samples, and will help facilitate large-scale investigations of protistan diversity.

Molecular characterisation of protistan species and communities in ships’ ballast water across three U.S. coasts

AbstractAimWe characterised protistan taxa in ships’ ballast water (BW) arriving to port systems across three U.S. coasts. Our goals were to compare (1) diversity and (2) community composition of protists in BW among and between port systems.LocationChesapeake Bay in Virginia (Port of Hampton Roads), Galveston Bay in Texas (Ports of Texas City, Houston and Bayport) and Prince William Sound in Alaska (Port of Valdez).MethodsWe collected 61 BW samples from 39 vessels from May to August 2013. We conducted amplicon‐based high‐throughput sequencing (HTS) using the hypervariable V4 domain of the small subunit (SSU) gene of the ribosomal RNA (rRNA) complex to identify protistan taxa.ResultsWe detected 8,561 OTUs from protistan taxa, including 35 taxonomic groups. We found high protistan diversity in the BW entering all three port systems, with the dominant taxa belonging to the Alveolata, Rhizaria and Stramenopiles. Thirty‐eight taxa were found in high relative abundance (&gt;10,000 sequences), and some were recovered from multiple samples within and across ports, indicating both a high relative abundance and frequency of introduction events. The composition of the protistan communities entering each of the port systems appeared to vary depending on BW source, with those entering Valdez being consistently more similar to each other than those entering other port systems.Main conclusionsThis study is the most comprehensive assessment of protistan diversity in BW and has important implications for microbial biogeography and invasions. Ships’ BW is importing distinct and diverse protistan communities to U.S. coasts with high propagule pressure for some taxa, creating opportunity for invasion by novel taxa. Further, our results demonstrate the broad‐scale movement of marine protists that has occurred through BW, highlighting a discrepancy between the large magnitude of introductions and limited number of known microbial invasions.

Changes in bacterial community composition and soil respiration indicate rapid successions of protist grazers during mineralization of maize crop residues

Decomposition of organic matter is crucial for ecosystem functioning. Microorganisms, which are responsible for the mineralization of organic matter, are usually treated as a homogeneous functional guild, despite mineralization capacity can differ profoundly between taxa. In addition, a significant part of the microbial community is top-down controlled by microbial grazers, such as protist. Since protist grazing is selective, and selectivity differs among species, we hypothesized that protist taxa complement each other in grazing intensity and thereby affect bacterial community structure and mineralization rate. In a laboratory experiment the species richness of protist communities was manipulated in an arable field soil and the mineralization rate of maize litter residues followed during the decomposition of the labile (4 days) and recalcitrant (3 weeks) carbon (C) fractions. Mineralization rate overall increased in the presence of protists. Changes in microbial function could be related to changes in microbial community composition (measured by phospholipid fatty acids pattern). During microbial decomposition, different protist grazers gained influence on mineralization rates over consecutive time intervals, indicating that a succession of protists caused an enhanced bacterial C-mineralization of plant detritus. Protist identity and species richness affected the microbial community composition, but not the magnitude of its mineralization function. In general, protist identity appeared to be more relevant for the composition of the microbial communities at the beginning of decomposition while the protist species richness appeared to be more critical in the later, slow phase of decomposition. This study provides an example that the overall outcome of ecosystem processes, such as mineralization rate is regulated by the sum of positive and negative effects of complex species interactions operating at a very fine spatial and temporal scales.

Single Cell Transcriptomics, Mega-Phylogeny, and the Genetic Basis of Morphological Innovations in Rhizaria.

The innovation of the eukaryote cytoskeleton enabled phagocytosis, intracellular transport, and cytokinesis, and is largely responsible for the diversity of morphologies among eukaryotes. Still, the relationship between phenotypic innovations in the cytoskeleton and their underlying genotype is poorly understood. To explore the genetic mechanism of morphological evolution of the eukaryotic cytoskeleton, we provide the first single cell transcriptomes from uncultured, free-living unicellular eukaryotes: the polycystine radiolarian Lithomelissa setosa (Nassellaria) and Sticholonche zanclea (Taxopodida). A phylogenomic approach using 255 genes finds Radiolaria and Foraminifera as separate monophyletic groups (together as Retaria), while Cercozoa is shown to be paraphyletic where Endomyxa is sister to Retaria. Analysis of the genetic components of the cytoskeleton and mapping of the evolution of these on the revised phylogeny of Rhizaria reveal lineage-specific gene duplications and neofunctionalization of α and β tubulin in Retaria, actin in Retaria and Endomyxa, and Arp2/3 complex genes in Chlorarachniophyta. We show how genetic innovations have shaped cytoskeletal structures in Rhizaria, and how single cell transcriptomics can be applied for resolving deep phylogenies and studying gene evolution in uncultured protist species.