Phylogeny of Parasitic Parabasalia and Free-Living Relatives Inferred from Conventional Markers vs. Rpb1, a Single-Copy Gene

BackgroundParabasalia are single-celled eukaryotes (protists) that are mainly comprised of endosymbionts of termites and wood roaches, intestinal commensals, human or veterinary parasites, and free-living species. Phylogenetic comparisons of parabasalids are typically based upon morphological characters and 18S ribosomal RNA gene sequence data (rDNA), while biochemical or molecular studies of parabasalids are limited to a few axenically cultivable parasites. These previous analyses and other studies based on PCR amplification of duplicated protein-coding genes are unable to fully resolve the evolutionary relationships of parabasalids. As a result, genetic studies of Parabasalia lag behind other organisms. Principal FindingsComparing parabasalid EF1α, α-tubulin, enolase and MDH protein-coding genes with information from the Trichomonas vaginalis genome reveals difficulty in resolving the history of species or isolates apart from duplicated genes. A conserved single-copy gene encodes the largest subunit of RNA polymerase II (Rpb1) in T. vaginalis and other eukaryotes. Here we directly sequenced Rpb1 degenerate PCR products from 10 parabasalid genera, including several T. vaginalis isolates and avian isolates, and compared these data by phylogenetic analyses. Rpb1 genes from parabasalids, diplomonads, Parabodo, Diplonema and Percolomonas were all intronless, unlike intron-rich homologs in Naegleria, Jakoba and Malawimonas. Conclusions/SignificanceThe phylogeny of Rpb1 from parasitic and free-living parabasalids, and conserved Rpb1 insertions, support Trichomonadea, Tritrichomonadea, and Hypotrichomonadea as monophyletic groups. These results are consistent with prior analyses of rDNA and GAPDH sequences and ultrastructural data. The Rpb1 phylogenetic tree also resolves species- and isolate-level relationships. These findings, together with the relative ease of Rpb1 isolation, make it an attractive tool for evaluating more extensive relationships within Parabasalia.

背景 副滴虫门（Parabasalia）是一类单细胞真核生物（原生生物（protists）），其类群主要涵盖白蚁与木蜚蠊的内共生体、肠道共生物、人类或兽医寄生虫以及自由生活物种。以往针对副滴虫的系统发育分析通常以形态学特征与18S核糖体RNA基因序列数据（rDNA）为依据，而相关生化或分子研究仅局限于少数可体外纯培养的寄生虫。此前的此类分析以及基于重复蛋白编码基因PCR扩增的其他研究，均无法完全解析副滴虫的进化关系。因此，副滴虫门的遗传学研究进度落后于其他生物类群。
主要研究结果 将副滴虫的延伸因子1α（EF1α）、α-微管蛋白（α-tubulin）、烯醇化酶（enolase）及苹果酸脱氢酶（MDH）蛋白编码基因与阴道毛滴虫（Trichomonas vaginalis）基因组信息进行比对后发现，若不借助重复基因，难以区分物种或分离株的演化历史。在阴道毛滴虫及其他真核生物中，保守的单拷贝基因编码RNA聚合酶II的最大亚基（Rpb1）。本研究直接对10个副滴虫类群的Rpb1简并PCR产物进行了测序，其中涵盖多株阴道毛滴虫分离株与鸟类分离株，并通过系统发育分析对这些数据进行了比较。副滴虫、双滴虫纲（diplomonads）、副波多虫属（Parabodo）、双滴虫属（Diplonema）及Percolomonas的Rpb1基因均不含内含子，而耐格里属（Naegleria）、雅可巴虫属（Jakoba）及马拉威单胞虫属（Malawimonas）的同源基因则富含内含子。
结论与意义 对寄生性与自由生活副滴虫的Rpb1基因进行系统发育分析，并结合保守的Rpb1插入序列，结果支持毛滴虫亚纲（Trichomonadea）、三毛滴虫亚纲（Tritrichomonadea）及假毛滴虫亚纲（Hypotrichomonadea）为单系群。该结果与此前基于rDNA、甘油醛-3-磷酸脱氢酶（GAPDH）序列及超微结构数据的分析结果一致。Rpb1系统发育树还可解析物种及分离株水平的演化关系。上述发现，加之Rpb1分离操作相对简便，使其成为解析副滴虫门内更广泛演化关系的理想工具。