Biodiversity and environmental distribution of apusozoan Protozoa and the deep phylogeny of bikont eukaryotes

原生动物是一种单细胞生物，在所有环境中都非常丰富。与细菌不同，它们拥有一个细胞核，使它们成为真核生物的一部分，真核生物是多细胞植物、真菌和动物所属的一组活有机体。形态和分子研究(基于对不同生物的基因序列的比较)允许将原生动物划分为11个主要类群(门)。这些门中的大多数可以归入真核生物的6个主要组合中的一个，其中3个只包含原生动物。不同原生动物门的物种多样性、丰富度和生态意义差异很大，但很难直接评估。在一些门中，许多物种是已知的，由于它们数量丰富和/或在形态上容易区分，它们的存在或不存在很容易在不同的栖息地被确定。在其他门中，只有几个物种被描述，可能是因为它们几乎没有明显的形态特征，而且几乎对它们的生态一无所知。后者真的是物种贫乏的门吗？它们的成员真的很稀有吗？或者这是因为人们对这个群体缺乏兴趣，或者缺乏可用来区分物种的特征？我们现在可以用分子方法来回答这些问题，以检查不同生境中选定的有机体群体的存在或不存在以及多样性，使用基于已知基因序列的群体特异性遗传探针。无孢子虫是最近定义的一个门，由于它缺乏明显的医学或经济重要性，并且包含非常少的原生动物谱系，因此尚未受到太多关注。然而，它们的多样性可能比目前所知的要多得多，而Amastigomonas是一种已知的非常古老的细胞类型，比整个动物界都要古老，像其他一些无脊椎动物一样，在海洋沉积物中非常广泛。为数不多的无孢子虫物种是已知的，关于这个门的进化统一性也是不确定的：可能有5到6个同样古老的原生动物谱系属于这个门。无孢子虫引起人们特别的兴趣有两个原因。首先，他们是一个完美的模型组，用来测试独立于栽培的分子方法进行生态推断的效用。关于无孢子虫的物种多样性和生态重要性，人们知之甚少。我们将使用特定的遗传探针来扩增编码核糖体RNA的基因片段(广泛用于遗传比较)，以确定(1)每个谱系中可以定义多少不同的遗传类型，(2)在哪些栖息地可以找到不同的无孢子虫谱系，以及(3)某些谱系或物种是否可能特定于给定的栖息地。与新的基因类型匹配的特定遗传探针也将被用来尝试从采样的栖息地中分离并培养基因序列所属的生物体。我们将从蛋白质编码基因中获得大量序列，以测试这些基因的突变是否像高等生物一样通过有性繁殖进行重组，或者这些无孢子是否像细菌一样是非有性繁殖的。我们还将使用这些蛋白质序列来确定每个细胞是只有一个基因组拷贝还是两个拷贝(像动物一样)，以及它们是否从食物的DNA中获得了新的基因。无孢子虫的第二个也是关键的兴趣是它们对于理解真核生物整体进化的关键重要性。核糖体RNA比较表明，它们不属于到目前为止定义的真核生物的六个主要组合中的任何一个。相反，它们可能形成一个独立的群体，非常接近所有真核生物的最后共同祖先。利用100多个蛋白质基因序列，我们还将构建一个独立于核糖体RNA的更好的进化树，以更准确地确定已被认为属于无孢子虫的不同谱系是否真的形成了一个连贯的进化单位，以及它们与其他真核生物的确切关系。

Protozoa are single-celled organisms very abundant in all environments. Unlike bacteria, they possess a nucleus, making them part of eukaryotes, the group of living organisms to which multicellular plants, fungi, and animals belong. Morphological and molecular studies (based on comparing gene sequences from different organisms) allow the division of Protozoa into 11 main groups (phyla). Most of these phyla can be placed in one of 6 major assemblages of eukaryotes, three of which contain only protozoans. The species diversity, abundance and ecological significance of the different protozoan phyla are highly variable but can be difficult to assess directly. In some phyla very many species are known, and as they are abundant and/or easy to distinguish morphologically, their presence or absence can readily be ascertained in different habitats. In other phyla only a few species have been described perhaps because they possess very few distinguishing morphological characters, and virtually nothing is known of their ecology. Are the latter phyla truly species-poor and their members rare? Or is this an artefact due to a lack of interest in the group or the paucity of available characters for distinguishing species? We can now answer these questions using molecular methods to check the presence or absence and diversity of selected groups of organisms in different habitats using group-specific genetic probes based on known gene sequences. Apusozoa is a recently defined phylum that not yet been given much attention because it lacks obvious medical or economic importance and comprises very poorly studied lineages of protozoans. However, they could be considerably more diverse than is currently realised and Amastigomonas is known to be a very ancient cell type, older than the whole animal kingdom, and very widespread in marine sediments as are some other Apusozoa. Few apusozoan species are known, and there is uncertainty about the evolutionary unity of the phylum: 5 or 6 probably equally ancient lineages of protozoans may belong to this phylum. Apusozoa are of particular interest for two reasons. First, they are a perfect model group to test the utility of cultivation-independent, molecular approaches for ecological inferences. The species diversity and ecological importance of Apusozoa are extremely poorly known. We will use specific genetic probes to amplify pieces of the gene encoding ribosomal RNA (widely used for genetic comparisons) to determine (1) how many distinct genetic types can be defined in each lineage, (2) in which habitats the different apusozoan lineages can be found, and (3) whether some lineages or species may be specific to a given habitat. Specific genetic probes matching new genotypes will also be used to try and isolate from the sampled habitat and culture the organisms to which the gene sequences belong. We will obtain large numbers of sequences from protein-coding genes to test whether mutations in these genes are being recombined by sexual reproduction as in higher organisms, or whether instead these Apusozoa are non-sexual like bacteria. We will also use these protein sequences to decide whether each cell has one copy only of the genome or two (like animals) and whether they have acquired novel genes from the DNA of their food. The second and key interest of Apusozoa is their pivotal importance for understanding the evolution of eukaryotes as a whole. Ribosomal RNA comparisons imply that they do not belong to any of the six major assemblages of eukaryotes defined up to now. Rather, they might form an independent group very close to the last common ancestor of all eukaryotes. Using over 100 protein gene sequences, we will also construct a better evolutionary tree independent of that from ribosomal RNA to determine more precisely whether the different lineages that have been suggested to belong to Apusozoa truly form a coherent evolutionary unit, and what their exact relationships are to other eukaryotes.