New insights on the evolutionary relationships between the major lineages of Amoebozoa.

The supergroup Amoebozoa unites a wide diversity of amoeboid organisms and encompasses enigmatic lineages that have been recalcitrant to modern phylogenetics. Deep divergences, taxonomic placement of some key taxa and character evolution in the group largely remain poorly elucidated or controversial. We surveyed available Amoebozoa genomes and transcriptomes to mine conserved putative single copy genes, which were used to enrich gene sampling and generate the largest supermatrix in the group to date; encompassing 824 genes, including gene sequences not previously analyzed. We recovered a well-resolved and supported tree of Amoebozoa, revealing novel deep level relationships and resolving placement of enigmatic lineages congruent with morphological data. In our analysis the deepest branching group is Tubulinea. A recent proposed major clade Tevosa, uniting Evosea and Tubulinea, is not supported. Based on the new phylogenetic tree, paleoecological and paleontological data as well as data on the biology of presently living amoebozoans, we hypothesize that the evolution of Amoebozoa probably was driven by adaptive responses to a changing environment, where successful survival and predation resulted from a capacity to disrupt and graze on microbial mats-a dominant ecosystem of the mid-Proterozoic period of the Earth history.

Ecosystem state change in the Arabian Sea fuelled by the recent loss of snow over the Himalayan-Tibetan Plateau region.

The recent trend of global warming has exerted a disproportionately strong influence on the Eurasian land surface, causing a steady decline in snow cover extent over the Himalayan-Tibetan Plateau region. Here we show that this loss of snow is undermining winter convective mixing and causing stratification of the upper layer of the Arabian Sea at a much faster rate than predicted by global climate models. Over the past four decades, the Arabian Sea has also experienced a profound loss of inorganic nitrate. In all probability, this is due to increased denitrification caused by the expansion of the permanent oxygen minimum zone and consequent changes in nutrient stoichiometries. These exceptional changes appear to be creating a niche particularly favorable to the mixotroph, Noctiluca scintillans which has recently replaced diatoms as the dominant winter, bloom forming organism. Although Noctiluca blooms are non-toxic, they can cause fish mortality by exacerbating oxygen deficiency and ammonification of seawater. As a consequence, their continued range expansion represents a significant and growing threat for regional fisheries and the welfare of coastal populations dependent on the Arabian Sea for sustenance.

Atmospheric respiratory CO2 efflux by aquatic suspended particle-bound microbial communities: A laboratory experimental study.

Natural sources of atmospheric CO2 are of increasing interest as possible contributors to global climate warming. This study documents the amount of respiratory CO2 contributed by microbial communities associated with suspended particulates in aquatic water columns. Microcosms containing three different sources of water (pond freshwater, NY East River estuary and Hudson River estuary) were used to experimentally determine the atmospheric respiratory CO2 released from particle-associated microbes. Two different approaches were used. In the first, finely powdered dried cereal leaves (alfalfa) were added to each of the three microcosms as a consistent source of particulate organic matter (POM). In the second, only Hudson River estuary water samples were used with natural densities of POM. Respiration rates associated with two sizes of particles were assessed: 1) ≥ 200 µm and 2) ≥ 50 µm but less than 200 µm. The total respiration rate for the three microcosms with cereal leaf POM ranged from 5.09 to 14.87 µmol CO2 min-1 L-1. Of this, the amount contributed by larger particulates was in the range of 55-63%; and for smaller particulates ranged from 18 to 32 %. Data for microcosms containing water from the Hudson River estuary, with natural particulates, was as follows: total respiration ranged from ∼3 µmol CO2 min-1 L-1 to ∼3.73 µmol CO2 min-1 L-1. Larger particulates contributed approximately 40% of total respiration, and that of smaller particulates was substantially less (4-5% of total). Overall, these results indicate that microbial communities associated with particulates in the water column (especially larger particulates) may contribute substantial amounts of CO2 to the atmosphere.

Stenamoeba polymorpha, a New Species Isolated from Domesticated Horse Equus ferus caballus.

A new species of lobosean amoeba, Stenamoeba polymorpha n. sp., was isolated from the diarrheic stool of a domesticated horse in Great Falls Virginia, U.S. It shares characteristics with the five other described Stenamoeba species. However, electron microscopy revealed S. polymorpha has a substantially thickened cell surface lamina. Under light microscopy, the amoebae had a dynamic polymorphic appearance because hyaloplasm readily formed and resorbed subpseudopodia from any peripheral region of the cell. While in locomotion, the amoebae produced subpseudopodia that led and alternated the direction of movement with an apparent zigzag path. Sometimes, stationary amoebae had a vertical cell posture that was substantially taller than wide or long. The new species description is also supported by small subunit ribosomal ribonucleic acid gene analyses using phylogenies with both broad and narrow taxon sampling, pairwise nucleotide comparisons, and in-silico secondary structure predictions. The latter suggested Stenamoeba spp. discriminatory motifs in variable region eight. Stenamoeba polymorpha n. sp. is the first of its genus to be recovered from mammal feces, the first to yield a potential group one intron and the second described from North America. Here, the phylogenies suggest Stenamoeba limacina is this new species' nearest known relative.

Phylogenomics of 'Discosea': A new molecular phylogenetic perspective on Amoebozoa with flat body forms.

The majority of amoeboid lineages with flattened body forms are placed under a taxonomic hypothetical class 'Discosea' sensu Smirnov et al. (2011), which encompasses some of the most diverse morphs within Amoebozoa. However, its taxonomy and phylogeny is poorly understood. This is partly due to lack of support in studies that are based on limited gene sampling. In this study we use a phylogenomic approach including newly-generated RNA-Seq data and comprehensive taxon sampling to resolve the phylogeny of 'Discosea'. Our analysis included representatives from all orders of 'Discosea' and up to 550 genes, the largest gene sampling in Amoebozoa to date. We conducted extensive analyses to assess the robustness of our resulting phylogenies to effects of missing data and outgroup choice using probabilistic methods. All of our analyses, which explore the impact of varying amounts of missing data, consistently recover well-resolved and supported groups of Amoebozoa. Our results neither support the monophyly nor dichotomy of 'Discosea' as defined by Smirnov et al. (2011). Rather, we recover a robust well-resolved clade referred to as Eudiscosea encompassing the majority of discosean orders (seven of the nine studied here), while the Dactylopodida, Thecamoebida and Himatismenida, previously included in 'Discosea,' are non-monophyletic. We also recover novel relationships within the Eudiscosea that are largely congruent with morphology. Our analyses enabled us to place some incertae sedis lineages and previously unstable lineages such as Vermistella, Mayorella, Gocevia, and Stereomyxa. We recommend some phylogeny-based taxonomic amendments highlighting the new findings of this study and discuss the evolution of the group based on our current understanding.

The Role of Heterotrophic Microbial Communities in Estuarine C Budgets and the Biogeochemical C Cycle with Implications for Global Warming: Research Opportunities and Challenges.

Estuaries are among the most productive and economically important marine ecosystems at the land-ocean interface and contribute significantly to exchange of CO2 with the atmosphere. Estuarine microbial communities are major links in the biogeochemical C cycle and flow of C in food webs from primary producers to higher consumers. Considerable attention has been given to bacteria and autotrophic eukaryotes in estuarine ecosystems, but less research has been devoted to the role of heterotrophic eukaryotic microbes. Current research is reviewed here on the role of heterotrophic eukaryotic microbes in C biogeochemistry and ecology of estuaries, with particular attention to C budgets, trophodynamics, and the metabolic fate of C in microbial communities. Some attention is given to the importance of these processes in climate change and global warming, especially in relation to sources and sinks of atmospheric CO2 , while also documenting the current paucity of research on the role of eukaryotic microbes that contribute to this larger question of C biogeochemistry and the environment. Some recommendations are made for future directions of research and opportunities of applying newer technologies and analytical approaches to a more refined analysis of the role of C in estuarine microbial community processes and the biogeochemical C cycle.

Experimental Evidence that Fungi are Dominant Microbes in Carbon Content and Growth Response to Added Soluble Organic Carbon in Moss-rich Tundra Soil.

Global warming significantly affects Arctic tundra, including permafrost thaw and soluble C release that may differentially affect tundra microbial growth. Using laboratory experiments, we report some of the first evidence for the effects of soluble glucose-C enrichment on tundra soil prokaryotes (bacteria and archaea) and fungi, with comparisons to microbial eukaryotes. Fungal increase in C-biomass was equivalent to 10% (w/w) of the added glucose-C, and for prokaryote biomass 2% (w/w), the latter comparable to prior published results. The C-gain after 14 d was 1.3 mg/g soil for fungi, and ~200 µg/g for prokaryotes.

Cochliopodium arabianum n. sp. (Amorphea, Amoebozoa).

A new species of Cochliopodium isolated from freshwater at Arabia Lake in Lithonia, GA, USA is described based on light microscopic morphology, fine structure, and molecular genetic evidence. Cochliopodium arabianum n. sp., previously labeled as "isolate Con1" in prior publications, has been shown to group within the genus Cochliopodium in our molecular phylogenetic analysis. Light microscopy and fine structure evidence indicates the new isolate not only shares characters of the genus but also unique distinctive features. Cochliopodium arabianum n. sp. is typically round when stationary; or oval to sometimes broadly flabellate or triangular in shape during locomotion, with average length of 35 µm and breadth of 51 µm. Fine structure evidence indicates C. arabianum n. sp. has tower-like scales, lacking a terminal spine, sharing high similarity with its closest relative C. actinophorum. However, the scales of C. arabianum n. sp. are unique in height and the breadth of the base plate. Both morphological and molecular data, including SSU-rDNA and COI, indicate that this new species falls in a clade sufficiently different from other species to suggest that it is a valid new species.

Shifts in Cyanobacterial Strain Dominance during the Onset of Harmful Algal Blooms in Florida Bay, USA.

Cyanobacteria are fundamental components of aquatic phytoplankton communities and some taxa can cause harmful blooms in coastal ecosystems. Harmful cyanobacterial blooms are typically comprised of multiple strains of a single genus or species that cannot be resolved microscopically. Florida Bay, USA, has experienced harmful cyanobacterial blooms that have been associated with the loss of eelgrass, spiny lobsters, and general food web disruption for more than two decades. To identify the strain or strains of cyanobacteria forming blooms in Florida Bay, samples were collected across the system over an annual cycle and analyzed via DNA sequencing using cyanobacterial-specific 16S rRNA gene primers, flow cytometry, and scanning electron microscopy. Analyses demonstrated that the onset of blooms in Florida Bay was coincident with a transformation of the cyanobacterial populations. When blooms were absent, the cyanobacterial population in Florida Bay was dominated by phycoerythrin-containing Synechococcus cells that were most similar to strains within Clade III. As blooms developed, the cyanobacterial community transitioned to dominance by phycocyanin-containing Synechococcus cells that were coated with mucilage, chain-forming, and genetically most similar to the coastal strains within Clade VIII. Clade VIII strains of Synechococcus are known to grow rapidly, utilize organic nutrients, and resist top-down control by protozoan grazers and viruses, all characteristics consistent with observations of cyanobacterial blooms in Florida Bay. Further, the strains of Synechococcus blooming in this system are genetically distinct from the species previously thought to cause blooms in Florida Bay, Synechococcus elongatus. Collectively, this study identified the causative organism of harmful cyanobacterial blooms in Florida Bay, demonstrates the dynamic nature of cyanobacterial stains within genera in an estuary, and affirms factors promoting Synechococcus blooms.

Diverse protist grazers select for virulence-related traits in Legionella.

It is generally accepted that selection for resistance to grazing by protists has contributed to the evolution of Legionella pneumophila as a pathogen. Grazing resistance is becoming more generally recognized as having an important role in the ecology and evolution of bacterial pathogenesis. However, selection for grazing resistance presupposes the existence of protist grazers that provide the selective pressure. To determine whether there are protists that graze on pathogenic Legionella species, we investigated the existence of such organisms in a variety of environmental samples. We isolated and characterized diverse protists that graze on L. pneumophila and determined the effects of adding L. pneumophila on the protist community structures in microcosms made from these environmental samples. Several unrelated organisms were able to graze efficiently on L. pneumophila. The community structures of all samples were markedly altered by the addition of L. pneumophila. Surprisingly, some of the Legionella grazers were closely related to species that are known hosts for L. pneumophila, indicating the presence of unknown specificity determinants for this interaction. These results provide the first direct support for the hypothesis that protist grazers exert selective pressure on Legionella to acquire and retain adaptations that contribute to survival, and that these properties are relevant to the ability of the bacteria to cause disease in people. We also report a novel mechanism of killing of amoebae by one Legionella species that requires an intact Type IV secretion system but does not involve intracellular replication. We refer to this phenomenon as 'food poisoning'.

Sapocribrum chincoteaguense n. gen. n. sp.: A Small, Scale-bearing Amoebozoan with Flabellinid Affinities.

The isolate American Type Culture Collection (ATCC)® 50979™ is a small amoebozoan whose actin gene was previously characterized, but did not allow a stable phylogenetic placement. This isolate was originally mis-identified upon deposition, and subsequently mis-illustrated in a recent publication. Here, we provide both a detailed morphological description as well as additional molecular analyses in order to clarify the isolate's phylogenetic relationships. The amoeba is minute (less than 5 µm), and presents the behavior of staying in a fixed location, while emitting one or two thin pseudopods. Transmission electron microscopy reveals that the cell is covered in a layer with embedded scales, giving the cell an armored appearance. Molecular phylogenetic analyses of data (actin, alpha- and beta-tubulin, elongation factor 2, and 14-3-3) from transcriptomes of this and four other isolates reveals that ATCC® 50979(™) is closely related to the recently described Squamamoeba japonica and in a novel, stable clade. Due to the unique nature of the scale covering, as well as other gross morphological characters and the molecular phylogenetic analyses, we formally describe the isolate as Sapocribrum chincoteaguense n. gen. n. sp.

Evidence of parasexual activity in "asexual amoebae" Cochliopodium spp. (Amoebozoa): extensive cellular and nuclear fusion.

The majority of microbial eukaryotes have long been considered asexual, though new evidence indicates sex, or sexual-like (parasexual) behaviors that deviate from the usual union of two gametes, among other variant aspects. Over a dozen amoebozoans are implicated to have sexual stages. However, the exact mechanism by which sex occurs in these lineages remains elusive. This is mainly due to the diverse quality and cryptic nature of their life cycle. In this study we present evidence of some previously unreported aspects of the life cycle of an amoeba, Cochliopodium, that undergoes unusual intraspecific interactions using light microscopy and immunocytochemistry. Similar to other amoebozoans, Cochliopodium, is considered asexual with no published reports of sex or parasexuality. We also investigated environmental conditions that govern the observed intraspecific interactions. Both light microscopic and immunocytochemistry evidence demonstrates Cochliopodium undergoes cellular fusion (plasmogamy) and nuclear fusion (karyogamy). Large plasmodia eventually undergo karyogamy and contain large fused, polyploid, nuclei. These are observed to fragment, subsequently, by karyotomy (nuclear fission) and cytoplasmic fission to yield uninucleated amoebae. This process could lead to a non-meiotic, parasexual exchange of chromosomes in Cochliopodium. These findings strongly suggest that Cochliopodium is involved in parasexual activity and should no longer be considered strictly asexual.

Assessment of a novel module for training dental students in child abuse recognition and reporting.

This study assessed the merits of introducing a novel, online interactive training module designed to positively engage dental students and teach them to recognize and report signs of child abuse and neglect. The study aimed to determine if the online training module educated the students equivalently or better than a lecture presentation of the same content. Seventy-two students from Columbia University College of Dental Medicine's class of 2015 (90 percent of the class) agreed to participate and were randomly assigned to either a traditional lecture-based presentation or the online training module. Study participants were given a twenty-question multiple-choice pretest on their knowledge of child abuse recognition and reporting prior to the start of the study. The same instrument was administered as a posttest. At the end of the training, questionnaires were also given to both groups to assess students' perceptions of the two educational methodologies. The results showed that the interactive online training module was more effective than the lecture-based method. Results of the posttest comparison of the two groups were statistically significant (p<0.05) in favor of the online training group. Additionally, the students reported that the interactive online training module was engaging and a helpful resource, but on average they did not prefer it as a total replacement for the lecture-based approach.

Dental student perceptions of the educational value of a comprehensive, multidisciplinary OSCE.

The aim of this study was to examine student perceptions of the educational value of a comprehensive, multidisciplinary objective structured clinical examination (OSCE). The examination is administered after the second year of the curriculum, prior to the beginning of clinical training at the Columbia University College of Dental Medicine. The quantitative component of the study consisted of a survey administered to students who had taken the OSCE (N=78). Due to the ordinal level of data produced by the Likert-scale survey, statistical analysis was performed through calculation of the median and interquartile range (IQR). Overall, student perceptions of the educational value of the OSCE, as measured using a five-point scale (1=strongly disagree to 5=strongly agree) were positive, demonstrating students' agreement that the exam required the ability to think critically and problem-solve (median=4, IQR=1), assessed clinically relevant skills (median=5, IQR=1), and was a learning experience (median=5, IQR=1). A statistically significant chi-square value (p<0.001) was found for all questions. The qualitative component consisted of phenomenological examination of student focus group interviews (N=15). Four main themes were identified including that the OSCE was an authentic assessment that required integration and application of knowledge.

Microbial communities associated with tree bark foliose lichens: a perspective on their microecology.

Tree-bark, foliose lichens occur widely on a global scale. In some locales, such as forests, they contribute a substantial amount of biomass. However, there are few research reports on microbial communities including eukaryotic microbes associated with foliose lichens. Lichens collected from tree bark at 11 locations (Florida, New York State, Germany, Australia, and the Arctic) were examined to determine the density and C-biomass of bacteria and some eukaryotic microbes, i.e. heterotrophic nanoflagellates (HNF) and amoeboid protists. A rich microbial diversity was found, including large plasmodial slime molds, in some cases exceeding 100 µm in size. The densities of HNF and amoeboid protists were each positively correlated with densities of bacteria, r = 0.84 and 0.80, respectively (p < 0.01, N = 11 for each analysis) indicating a likely bacterial-based food web. Microbial densities (number/g lichen dry weight) varied markedly across the geographic sampling sites: bacteria (0.7-13.1 × 10(8) ), HNF (0.2-6.8 × 10(6) ) and amoeboid protists (0.4-4.6 × 10(3) ). The ranges in C-biomass (µg/g lichen dry weight) across the 11 sites were: bacteria (8.8-158.5), HNF (0.03-0.85), and amoeboid protists (0.08-540), the latter broad range was due particularly to absence or presence of large slime mold plasmodia.

A revision of the order Pellitida Smirnov et al., 2011 (Amoebozoa, Discosea) based on ultrastructural and molecular evidence, with description of Endostelium crystalliferum n. sp.

We present the results of an ultrastructural re-investigation of two amoebae strains that can be identified morphologically as previously described species of the genus Pellita, as well as the first molecular phylogenetic analysis of these amoebae based on SSU rRNA and actin gene sequences. The results obtained show close relationships between the genera Pellita, Gocevia, and Endostelium. These relationships are further supported by the description of Endostelium crystalliferum n. sp., which shares morphological characters simultaneously with Pellita spp. and Endostelium zonatum. The three genera form a robust clade that branches deeply within Amoebozoa, among either Flabellinia, or Longamoebia, depending on taxon sampling. The results suggest that Gocevia and Endostelium should not be included in the Himatismenida; therefore, we transfer the family Goceviidae into Pellitida. The type of cell organisation that was considered to be typical of Himatismenida (a lens-shaped cell covered dorsally with a flexible layer of organic material) has most probably evolved in some of these amoebae independently of Cochliopodiidae and Parvamoebidae. The robustness of the Pellitida clade in the molecular trees is consistent with the fine structure cytoplasmic evidence for these taxa, in particular, the presence of a centrosphere (a dictyosome-associated lamellar MTOC) in all these genera.

Naked amoebae in biofilms collected from a temperate freshwater pond.

Biofilms collected on Plexiglass substrates, from a freshwater pond in northern New York State, were examined microscopically for naked amoebae densities, sizes, diversity, and estimated C-biomass. Five samples were obtained during summer 2006 and 2007. The densities ranged from 109 to 136/cm(2) biofilm surface and 285 to 550/mg biofilm dry weight. Sizes ranged from 13 to 200 µm. Diversities ranged from 4.23 to 4.55. C-biomass ranged from 64 to 543 ng C/cm(2) and 125 to 1,700 µg C/g dry weight. Thirty morphospecies were identified among the five samples, including very large amoebae in the range of 100-200 µm. Large amoebae (≥ 50 µm) accounted for the largest proportion of the C-biomass.

Reliability and predictive validity of a comprehensive preclinical OSCE in dental education.

This study examined the reliability and predictive validity of a comprehensive, multidisciplinary objective structured clinical examination (OSCE) used to assess the transition from preclinical to clinical education in a dental school. The examination is administered prior to clinical training at the Columbia University College of Dental Medicine to assess student preparedness for patient care activity. The study population consisted of seventy-five students in the Class of 2010 and seventy students in the Class of 2011. The total reliability of the exam was assessed by Cronbach's coefficient alpha. Using polynomial regression, the predictive validity of the examination was evaluated by correlating student scores on the OSCE with clinical performance as measured by successfully completed clinical procedures during the first year of clinical training. Findings indicate a highly reliable exam (α=0.86 and α=0.80) with a moderately high correlation predicting future clinical performance (r=0.614, p<0.0001; r=0.540, p<0.0001) for the Classes of 2010 and 2011, respectively. The findings suggest that an OSCE can serve as a reliable and predictive assessment during the transition from preclinical to clinical education in the predoctoral dental curriculum.

Leaf- and cell-level carbon cycling responses to a nitrogen and phosphorus gradient in two Arctic tundra species.

PREMISE OF THE STUDY: Consequences of global climate change are detectable in the historically nitrogen- and phosphorus-limited Arctic tundra landscape and have implications for the terrestrial carbon cycle. Warmer temperatures and elevated soil nutrient availability associated with increased microbial activity may influence rates of photosynthesis and respiration. • METHODS: This study examined leaf-level gas exchange, cellular ultrastructure, and related leaf traits in two dominant tundra species, Betula nana, a woody shrub, and Eriophorum vaginatum, a tussock sedge, under a 3-yr-old treatment gradient of nitrogen (N) and phosphorus (P) fertilization in the North Slope of Alaska. • KEY RESULTS: Respiration increased with N and P addition-the highest rates corresponding to the highest concentrations of leaf N in both species. The inhibition of respiration by light ("Kok effect") significantly reduced respiration rates in both species (P < 0.001), ranged from 12-63% (mean 34%), and generally decreased with fertilization for both species. However, in both species, observed rates of photosynthesis did not increase, and photosynthetic nitrogen use efficiency generally decreased under increasing fertilization. Chloroplast and mitochondrial size and density were highly sensitive to N and P fertilization (P < 0.001), though species interactions indicated divergent cellular organizational strategies. • CONCLUSIONS: Results from this study demonstrate a species-specific decoupling of respiration and photosynthesis under N and P fertilization, implying an alteration of the carbon balance of the tundra ecosystem under future conditions.

Advancing the educational training of dental educators: review of a model program.

Very few dental educators have formal pedagogical training, and the availability of degree-granting programs for dental educators is very limited. A joint D.D.S.-M.A. program for predoctoral students and a Ph.D. program for faculty members have been established at Columbia University's College of Dental Medicine in conjunction with the Graduate School of Education, Teachers College. Six students have completed the joint degree program, five are currently enrolled, and an additional three have taken courses at Teachers College without pursuing a degree. One faculty member has completed the Ph.D. program, and one is enrolled. This article describes the details of both programs and discusses preliminary outcomes. The model described here may serve as an example for other dental schools that may choose to implement degree programs in education for dental educators.