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.

Three New Freshwater Cochliopodium Species (Himatismenida, Amoebozoa) from the Southeastern United States.

Cochliopodium is a lens-shaped genus of Amoebozoa characterized by a flexible layer of microscopic dorsal scales. Recent taxonomic and molecular studies reported cryptic diversity in this group and suggested that the often-used scale morphology is not a reliable character for species delineation in the genus. Here, we described three freshwater Cochliopodium spp. from the southeastern United States based on morphological, immunocytochemistry (ICC), and molecular data. A maximum-likelihood phylogenetic analysis and pairwise comparison of COI sequences of Cochliopodium species showed that each of these monoclonal cultures were genetically distinct from each other and any described species with molecular data. Two of the new isolates, "crystal UK-YT2" (Cochliopodium crystalli n. sp.) and "crystal-like UK-YT3" (C. jaguari n. sp.), formed a clade with C. larifeili, which all share a prominent microtubule organizing center (MTOC) and have cubical-shaped crystals. The "Marrs Spring UK-YT4" isolate, C. marrii n. sp., was 100% identical to "Cochliopodium sp. SG-2014 KJ569724." These sequences formed a clade with C. actinophorum and C. arabianum. While the new isolates can be separated morphologically, most of the taxonomic features used in the group show plasticity; therefore, Cochliopodium species can only be reliably identified with the help of molecular data.

Phylogenomics of Thecamoebida (Discosea, Amoebozoa) with the Description of Stratorugosa tubuloviscum gen. nov. sp. nov., a Freshwater Amoeba with a Perinuclear MTOC.

Thecamoebida Smirnov and Cavalier-Smith, 2011 (Discosea, Amoebozoa) has been molecularly understudied. The group until recently consisted of three genera containing species that live in terrestrial or aquatic environments. Here, we describe a fourth genus, Stratorugosa tubuloviscum gen. nov. sp. nov., which was isolated from a freshwater Amoeba proteus Ward's Science culture. Although this species most closely morphologically resembles a large, rugose Thecamoeba, S. tubuloviscum gen. nov. sp. nov. can be differentiated from Thecamoeba spp. by the following: 1) the presence of definitive finger-like (lobate-like) subpseudopodia extending at both the anterior and lateral parts of the cell during locomotion; 2) a peculiar locomotive mechanism with two sections, frontal and back, of the cells moving in a pulling and piggyback movement, respectively; 3) the presence of fibrillar cytoplasmic microtubules (MTs) organized by a prominent, perinuclear microtubule-organizing center (MTOC). A phylogenomic analysis of 511 genes assembled from transcriptomic data showed that this new genus was highly supported as sister to Stenamoeba. Despite the variance in gross morphology, Stenamoeba and S. tubuloviscum gen nov. sp. nov. both have MTOCs unlike two Thecamoeba spp., which display dot-like cytoplasmic MTs and lack an MTOC.

A practical implementation of large transcriptomic data analysis to resolve cryptic species diversity problems in microbial eukaryotes.

BACKGROUND: Transcriptome sequencing has become a method of choice for evolutionary studies in microbial eukaryotes due to low cost and minimal sample requirements. Transcriptome data has been extensively used in phylogenomic studies to infer ancient evolutionary histories. However, its utility in studying cryptic species diversity is not well explored. An empirical investigation was conducted to test the applicability of transcriptome data in resolving two major types of discordances at lower taxonomic levels. These include cases where species have the same morphology but different genetics (cryptic species) and species of different morphologies but have the same genetics. We built a species comparison bioinformatic pipeline that takes into account the nature of transcriptome data in amoeboid microbes exemplifying such discordances. RESULT: Our analyses of known or suspected cryptic species yielded consistent results regardless of the methods of culturing, RNA collection or sequencing. Over 95% of the single copy genes analyzed in samples of the same species sequenced using different methods and cryptic species had intra- and interspecific divergences below 2%. Only a minority of groups (2.91-4.87%) had high distances exceeding 2% in these taxa, which was likely caused by low data quality. This pattern was also observed in suspected genetically similar species with different morphologies. Transcriptome data consistently delineated all taxa above species level, including cryptically diverse species. Using our approach we were able to resolve cryptic species problems, uncover misidentification and discover new species. We also identified several potential barcode markers with varying evolutionary rates that can be used in lineages with different evolutionary histories. CONCLUSION: Our findings demonstrate that transcriptome data is appropriate for understanding cryptic species diversity in microbial eukaryotes.

Genetic Evidence for Sexuality in Cochliopodium (Amoebozoa).

Microbial eukaryotes, including amoeboids, display diverse and complex life cycles that may or may not involve sexual reproduction. A recent comprehensive gene inventory study concluded that the Amoebozoa are ancestrally sexual. However, the detection of sex genes in some lineages known for their potentially sexual life cycle was very low. Particularly, the genus Cochliopodium, known to undergo a process of cell fusion, karyogamy, and subsequent fission previously described as parasexual, had no meiosis genes detected. This is likely due to low data representation, given the extensive nuclear fusion observed in the genus. In this study, we generate large amounts of transcriptome data for 2 species of Cochliopodium, known for their high frequency of cellular and nuclear fusion, in order to study the genetic basis of the complex life cycle observed in the genus. We inventory 60 sex-related genes, including 11 meiosis-specific genes, and 31 genes involved in fusion and karyogamy. We find a much higher detection of sex-related genes, including 5 meiosis-specific genes not previously detected in Cochliopodium, in this large transcriptome data. The expressed genes form a near-complete recombination machinery, indicating that Cochliopodium is an actively recombining sexual lineage. We also find 9 fusion-related genes in Cochliopodium, although no conserved fusion-specific genes were detected in the transcriptomes. Cochliopodium thus likely uses lineage specific genes for the fusion and depolyploidization processes. Our results demonstrate that Cochliopodium possess the genetic toolkit for recombination, while the mechanism involving fusion and genome reduction remains to be elucidated.

Longamoebia is not monophyletic: Phylogenomic and cytoskeleton analyses provide novel and well-resolved relationships of amoebozoan subclades.

Longamoebia is one of the most morphologically diverse member of Amoebozoa. It includes the human pathogen Acanthamoeba, which causes minor skin and serious eye infections as well as fatal central nervous system complications. The taxonomy and phylogeny of Longamoebia is poorly understood partly due to the growing number of molecular studies that report unsuspected affiliations of lineages with extremely different morphotypes in the group. A recent molecular study questioned the monophyly of Longamoebia. In this study, we conducted a more comprehensive phylogenomic analysis including all of putative members of Longamoebia to assess its monophyly. We conducted extensive analyses to see effects of outgroup choice, missing data, and gene and taxon sampling on resulting phylogenies. We also collected morphological characters derived from the cytoskeleton using immunocytochemistry to assess homologies of pseudopodia at a finer scale. Our phylogenomic analysis yielded a well-resolved tree of Amoebozoa and highly supported novel relationships. Discosea is recovered as a monophyletic group with all of its known taxonomic orders. However, its within-group relationships dramatically differed from those originally proposed. Our study strongly demonstrates that Longamoebia sensu Smirnov et al. (2011) is not monophyletic and an invalid taxon. Thecamoebida forms a strongly supported sister group relationship with clade Flabellinea (Dactylopodida and Vannellida), while Dermamoebida (Mayorella+Dermamoeba) form an independent branch basal to other members of Discosea. The remaining groups including members of Centramoebida form a consistently well-supported clade that was shown to form a sister group relationship with Himatismenida. This robust clade shares the unique cytoskeletal features of coiled cytoplasmic microtubule network and F-actin characters. Our analyses demonstrated that placement of unstable taxa in large-scale analysis with varying levels of missing data might be compromised by some confounding factors such as outgroup choice and gene and taxon sampling.

Amoebozoans Are Secretly but Ancestrally Sexual: Evidence for Sex Genes and Potential Novel Crossover Pathways in Diverse Groups of Amoebae.

Sex is beneficial in eukaryotes as it can increase genetic diversity, reshuffle their genomes, and purge deleterious mutations. Yet, its evolution remains a mystery. The eukaryotic clade supergroup Amoebozoa encompasses diverse lineages of polymorphic amoeboid forms, including both free-living and parasitic lineages. The group is generally believed to be asexual, though recent studies show that some of its members are implicated in cryptic forms of sexual cycles. In this study, we conduct a comprehensive inventory and analysis of genes involved in meiosis and related processes, in order to investigate the evolutionary history of sex in the clade. We analyzed genomic and transcriptomic data of 39 amoebozoans representing all major subclades of Amoebozoa. Our results show that Amoebozoa possess most of the genes exclusive to meiosis but lack genes encoding synaptonemal complex (SC). The absence of SC genes is discussed in the context of earlier studies that reported ultrastructural evidence of SC in some amoebae. We also find interclade and intrageneric variation in sex gene distribution, indicating diversity in sexual pathways in the group. Particularly, members of Mycetozoa engage in a novel sexual pathway independent of the universally conserved meiosis initiator gene, SPO11. Our findings strongly suggest that not only do amoebozoans possess sex genes in their genomes, but also, based on the transcriptome evidence, the present sex genes are functional. We conclude that Amoebozoa is ancestrally sexual, contrary to the long held belief that most of its members are asexual. Thus, asexuality in Amoebozoa, if confirmed to be present, is a derived-trait that appeared later in their evolution.

National community pharmacy NHS influenza vaccination service in Wales: a primary care mixed methods study.

BACKGROUND: Influenza is a significant cause of morbidity and excess mortality, yet vaccine coverage in the UK remains below target. Community pharmacies are increasingly being promoted as an alternative to vaccination by GPs. AIM: To explore and verify the factors that influence the relative performance of pharmacies providing NHS influenza vaccinations. DESIGN AND SETTING: A mixed methods study utilising qualitative, semi-structured interviews and quantitative analysis of predictors of vaccination numbers in community pharmacies in Wales. METHOD: Interviews were conducted with 16 pharmacists who participated in the Welsh national pharmacy influenza service in 2013-2014. A purposive sampling strategy was used. Qualitative findings were analysed using framework analysis. Potential predictors of vaccination numbers were identified from interviews and a literature review, and included in a multivariable regression model. RESULTS: The contribution of community pharmacies towards vaccination in Wales is small. Findings suggest that community pharmacies reach younger at-risk individuals, in whom vaccine uptake is low, in greater proportion than influenza vaccination programmes as a whole. Extended opening hours and urban locations were positively associated with the number of vaccinations given, although pharmacists reported that workload, vaccine costs, unforeseen delays, lack of public awareness, and GPs' views of the service limited their contribution. Pharmacists, aware of the potential for conflict with GPs, moderated their behaviour to mitigate such risk. CONCLUSION: Before community pharmacies take greater responsibility for delivering healthcare services, obstacles including increasing pharmacist capacity, vaccine procurement, health service delays, managing GP-pharmacy relationships, and improving public awareness must be overcome.