Ecological and morphological characterisation, and phylogeny of Endomyxa: poorly known but widespread and diverse protozoan parasites and omnivores

内粘虫是原生动物门尾虫门中一个非常鲜为人知但庞大且多样的生物群。内粘虫包括商业上重要的疟原虫植物寄生虫，以及单孢子虫和亲戚，其中包括广泛的无脊椎动物的寄生虫，最著名的是牡蛎的MSX疾病。这两个群体的进化关系在很长一段时间内都是未知的（疟原虫被认为是真菌很长一段时间），但良好的核糖体DNA树显示，它们是相互关联的（形态学的共形性加强了这种关系）和几条分子证据表明，它们属于真核生物超类群Rhizaria，特别是作为Cercozoa内的一个亚门，但与所谓的“核心”尾藻分开。最近，通过培养/细胞分离和rDNA测序发现了这些寄生虫的自由生活亲属。这些包括大型有遗嘱的海洋丝状变形虫Gromia，和大型网状裸变形虫：食菌的Filoreta和捕食性的Arachnula和Platyreta。这些生物体代表了非常独特和研究很少的原生动物形态类型，其生态作用几乎完全未知。其他研究揭示了更多的内粘液寄生虫，例如斑虾寄生虫和桡足类寄生虫。不依赖于培养的环境rDNA文库表明，还有大量的其他内粘多糖分支和谱系尚未被鉴定-我们对它们的唯一信息是检测到序列的样品的来源。环境图书馆还显示，有非常强大的生态结构-许多endomyxan分支到目前为止只发现在相当特定的栖息地，例如深海样品，厌氧海洋，厌氧淡水，或phylloplane社区。这些模式表明高度的生态专业化，可能涉及与其他生物的相互作用。该项目的目的是确定尽可能多的这些新的谱系和分支，无论他们是自由生活，共生，或寄生，使用密集的选择性培养的组合使用的食物来源和文化条件的多样性通知的结果的环境库，和荧光原位杂交（FISH），使用荧光探针，将专门检测选定的非特征谱系。FISH还将显示不同栖息地中内粘液谱系的相对丰度，以及它们是否与其他真核生物细胞相关或在其他真核生物细胞内发现，如果是，它们与哪些生物共存（因此可能是寄生虫）。多重和不同的SSU rDNA环境文库将构建，使用一套重叠的PCR引物覆盖所有Endomyxa，显示更清楚和现实的比以前可能的真正多样性Endomyxa，和他们的生态性质，通过分析在其他生物和非生物变量的背景下，在每个采样点共同测量的序列数据。文库将使用rDNA和从rRNA逆转录的crDNA两者来制备：后者通常被视为细胞活性水平的替代物（更多的核糖体将存在于更活跃的细胞中），而不仅仅是存在/不存在和生物量，如rDNA所示。这种方法将提供更多的信息比单独的rDNA文库的生态特性。环境序列将被用来提高系统发育重建Endomyxa和它们的关系，其余的根瘤菌都增加了这部分的树的分类单元采样，并通过提供序列数据，可用于获得LSU rDNA序列的代表性基因型直接从环境DNA样品。在培养物可用的情况下，可以通过PCR靶向其他基因，以与多基因遗传学中的现有数据相结合。

Endomyxa are a very poorly known but large and diverse group of organisms in the protozoan phylum Cercozoa. Endomyxa includes the commercially important plasmodiophorid plant parasites, and haplosporidia and relatives, which include parasites of a wide range of invertebrates, most famously MSX disease of oysters. The evolutionary relationships of both of these groups were for a long time unknown (plasmodiophorids were considered fungi for a long time), but good ribosomal DNA trees show that they are related to each other (a relationship reinforced by morphological synapomorphies) and several lines of molecular evidence show that they belong within the eukaryotic supergroup Rhizaria, specifically as a subphylum within Cercozoa, but separately from the so-called 'core' Cercozoa. More recently, free-living relatives of these parasites have been found through culturing/cell isolation and rDNA sequencing. These include the large testate marine filose amoeba Gromia, and large reticulose, naked amoebae: the bacterivorous Filoreta and the predatory Arachnula and Platyreta. Such organisms represent very distinct and poorly studied protozoan morphotypes whose ecological roles are almost completely unknown. Other research has revealed more endomyxan parasites, for example the spot prawn parasite, and Paradinium, a parasite of copepods. Culture-independent environmental rDNA libraries show that there are a large diversity of other endomyxan clades and lineages which remain uncharacterised - the only information we have for them is the provenance of the samples in which the sequences were detected. The environmental libraries also show that there is very strong ecological structuring - many endomyxan clades have so far only been found in quite specific habitats, for example deep-sea samples, anaerobic marine, anaerobic freshwater, or phylloplane communities. These patterns suggest high levels of ecological specialization, perhaps involving interactions with other organisms. This project aims to identify as many of these novel lineages and clades as possible, whether they are free-living, symbiotic, or parasitic, using a combination of intensive selective culturing using a diversity of food sources and culture conditions informed by the results of the environmental libraries, and fluorescent in situ hybridization (FISH) using fluorescent probes that will specifically detect chosen uncharacterized lineages. FISH will also show the relative abundance of endomyxan lineages in different habitats, and whether they are associated with, or are found inside, other eukaryote cells, and if so, which organisms they co-occur with (and are therefore perhaps parasites of). Multiple and diverse SSU rDNA environmental libraries will be constructed, using a set of overlapping PCR primers covering all Endomyxa, to show much more clearly and realistically than previously possible the true diversity of Endomyxa, and their ecological nature by analyzing the sequence data in the context of other biotic and abiotic variables co-measured at each sampling site. The libraries will be made using both rDNA and crDNA reverse transcribed from rRNA: the latter is generally taken as a surrogate for the level of activity of the cells (more ribosomes will be present in more active cells), rather than just presence/absence and biomass as indicated by rDNA. This approach will provide more informative about the ecological characteristics than rDNA libraries alone. The environmental sequences will be used to improve phylogenetic reconstructions of Endomyxa and their relationships to the rest of Rhizaria both by increasing taxon sampling of this part of the tree, and by providing sequence data that can be used to obtain LSU rDNA sequences for representative genotypes directly from environmental DNA samples. Where cultures are available, other genes can be targeted by PCR, to be combined with existing data in multigene phylogenies.