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角毛虫中一个非常鲜为人知但规模很大且种类繁多的生物群体。Endomyxa包括商业上重要的原生植物寄生虫和单孢子虫及其近亲，其中包括各种无脊椎动物的寄生虫，最著名的是牡蛎的MSX病。这两个类群的进化关系在很长一段时间内都是未知的(植原体类长期以来被认为是真菌)，但良好的核糖体DNA树表明它们彼此相关(这种关系因形态上的突触而得到加强)，并且一些分子证据表明它们属于真核超类群Rhizaria，特别是作为尾丝虫的一个亚门，但与所谓的核心尾丝虫是分开的。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。其中包括大型遗体海生阿米巴格罗米亚，以及大型网状、裸露的阿米巴：食菌丝虫和捕食性蜘蛛和扁形阿米巴。这些生物体代表着非常不同且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菌寄生虫，例如斑点对虾寄生虫和桡足类寄生虫Paraddium。独立于培养的环境rDNA文库表明，还有大量其他内粘菌支系和谱系仍未确定--我们所拥有的唯一信息是检测到序列的样本的来源。环境图书馆还表明，有非常强的生态结构--到目前为止，许多内粘菌分支仅在相当特定的生境中被发现，例如深海样品、厌氧海洋、厌氧淡水或叶平面群落。这些模式表明了高度的生态专业化，可能涉及到与其他有机体的互动。该项目的目标是尽可能多地鉴定这些新的谱系和支系，无论它们是自由生活的、共生的还是寄生的，使用结合使用各种食物来源和环境图书馆结果的培养条件的密集选择性培养，以及使用荧光探针的荧光原位杂交(FISH)将专门检测选定的未表征的谱系。鱼类还将显示不同生境中内粘菌素谱系的相对丰度，以及它们是否与其他真核细胞相关或在其中被发现，如果是的话，它们与哪些生物共存(因此可能是它们的寄生虫)。将使用一组覆盖所有Endomyxa的重叠的PCR引物，构建多个和不同的SSU rDNA环境文库，通过在每个采样点共同测量的其他生物和非生物变量的背景下分析序列数据，比以前可能的更清楚和真实地显示Endomyxa的真实多样性及其生态性质。文库将同时使用rDNA和从rRNA反转录的crDNA：后者通常被用作细胞活动水平的替代(更活跃的细胞中将存在更多的核糖体)，而不仅仅是rDNA所指示的存在/不存在和生物量。这种方法将比单独的rDNA文库提供更多关于生态特征的信息。环境序列将通过增加树的这一部分的分类单元样本，以及通过提供可用于直接从环境DNA样本中获得代表性基因的LSU rDNA序列的序列数据，来改进Endomyxa的系统发育重建及其与根霉属其他部分的关系。在有培养物可用的地方，其他基因可以被聚合酶链式反应作为靶点，与多基因系统发育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.