Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
Department of Parasitology and Entomology, Faculty of Public Health, Mahidol University, Bangkok, Thailand.
Acta Trop. 2020 Nov;211:105645. doi: 10.1016/j.actatropica.2020.105645. Epub 2020 Jul 20.
The Angiostrongylus cantonensis lineage (Nematoda: Metastrongyloidea) consists of the closely related species A. cantonensis, Angiostrongylus malaysiensis, and Angiostrongylus mackerrasae. Various genetic markers have been used for species discrimination in molecular phylogenetic studies of this lineage. However, despite showing potential in other organisms, mitochondrial 12S and 16S ribosomal RNA (rRNA) genes have not been used for Angiostrongylus species discrimination. Therefore, this study assessed these genes' suitability for inter- and intraspecies discrimination in the A. cantonensis lineage. The ultimate aim was to provide a novel genetic marker to support existing phylogenies. Sixty adult Angiostrongylus spp. worms from four geographic locations in Thailand were identified morphologically before molecular identification with 12S and 16S rRNA genes. Neighbor-joining and maximum likelihood algorithms were used for phylogenetic analyzes, and sequence variation was calculated to determine whether the genes could be used to discriminate among species. Furthermore, sequence variation was compared among previously used genetic markers to evaluate the robustness of the 12S and 16S rRNA genes as markers. Using both markers, the A. cantonensis lineage formed a monophyletic clade with a clear separation between A. cantonensis, A. malaysiensis, and A. mackerrasae. From our representative A. cantonensis and A. malaysiensis specimens, the genetic distance between the two clades was 6.8% -7.9% and 7.9% -10.0% for 12S and 16S rRNA genes, respectively, which is sufficient interspecific genetic variation for species discrimination. Higher levels of genetic variation were observed for the 16S rRNA gene, with 12 haplotypes and an intraspecific variation ≤2.2%. Thus, as a genetic marker, the 16S rRNA gene is comparable to mitochondrial protein-coding genes, which are commonly used in intra-level Angiostrongylus spp. studies. In conclusion, mitochondrial 12S and 16S rRNA genes can discriminate among closely related species in the A. cantonensis lineage, and they represent novel genetic markers for supporting existing phylogenies and verifying the phylogenetic position of A. mackerrasae.
广州管圆线虫种系（线虫纲：旋尾目）由密切相关的物种广州管圆线虫、马来管圆线虫和麦克氏管圆线虫组成。在该种系的分子系统发育研究中，已经使用了各种遗传标记来进行物种鉴别。然而，尽管在线粒体 12S 和 16S 核糖体 RNA（rRNA）基因在其他生物中具有潜力，但它们尚未用于广州管圆线虫物种鉴别。因此，本研究评估了这些基因在广州管圆线虫种系中用于种间和种内鉴别。最终目的是提供一种新的遗传标记来支持现有的系统发育。从泰国四个地理地点获得的 60 条成年广州管圆线虫成虫在分子鉴定为 12S 和 16S rRNA 基因之前，通过形态学进行了鉴定。使用邻接法和最大似然法进行系统发育分析，并计算序列变异以确定这些基因是否可用于鉴别物种。此外，还比较了以前使用的遗传标记中的序列变异，以评估 12S 和 16S rRNA 基因作为标记的稳健性。使用这两种标记，广州管圆线虫种系形成了一个单系分支，广州管圆线虫、马来管圆线虫和麦克氏管圆线虫之间有明显的分离。从我们的代表性广州管圆线虫和马来管圆线虫标本中，两个分支之间的遗传距离分别为 12S 和 16S rRNA 基因的 6.8%-7.9%和 7.9%-10.0%，这足以进行物种鉴别。16S rRNA 基因观察到更高水平的遗传变异，有 12 个单倍型，种内变异≤2.2%。因此，作为遗传标记，16S rRNA 基因与线粒体蛋白编码基因相当，后者常用于管圆线虫属内水平的研究。总之，线粒体 12S 和 16S rRNA 基因可鉴别广州管圆线虫种系中密切相关的物种，它们代表了支持现有系统发育和验证麦克氏管圆线虫系统发育位置的新遗传标记。
