Molecular marker sequences of cattle <i>Cooperia</i> species identify <i>Cooperia spatulata</i> as a morphotype of <i>Cooperia punctata</i>

The genus Cooperia includes important parasites of ruminants and currently contains 34 accepted species. However, even for those species infecting livestock, there is a considerable lack of molecular information and many species are only identifiable using subtle morphological traits. The present study aimed to provide molecular data to allow diagnosis of Cooperia species infecting cattle. Partial sequences of two mitochondrial (cytochrome oxidase 2, 12S rRNA gene) and two nuclear genes (isotype 1 β tubulin gene including two introns, internal transcribed spacers (ITS) were obtained from morphologically identified specimens of Cooperia pectinata, Cooperia punctata and Cooperia spatulata as well as from larvae of pure Cooperia oncophora and C. punctata laboratory isolates. Pairwise identity of ITS-2 sequences was very high and it was the only region able to identify a specimen as Cooperia sp. However, the ITS-2 was unreliable for diagnosis at the species level. All other marker sequences could not unequivocally be allocated to the genus Cooperia but allowed clear species identification with the exception of the pair C. punctata/C. spatulata for which no significant differences were found for any marker sequence. Maximum-likelihood phylogenetic analyses of individual genes as well as a multi-locus analysis covering all four sequences confirmed that specimen identified as C. spatulata were randomly distributed throughout the C. punctata cluster and formed no group of their own. In contrast, the other Cooperia species formed clearly separated and statistically supported clusters. These data indicate that C. spatulata is most likely only a morphotype of C. punctata and the name should be considered a synonym. Combinations of nuclear and mitochondrial markers should be used to identify morphotypes or cryptic species to benefit from excellent barcoding properties of the latter but allowing proper phylogenetic analyses and controlling for lineage sorting that might occur for mitochondrial genotypes within a species.古柏线虫属（Cooperia）是反刍动物的重要寄生线虫类群，目前已收录34个公认有效物种。然而，即便对于感染家畜的该属物种而言，当前仍存在分子信息严重匮乏的问题，多数物种仅能通过细微的形态特征进行鉴别。本研究旨在获取分子数据，以实现对感染牛只的古柏线虫属物种的精准鉴定。研究人员从经形态学鉴定的梳状古柏线虫（Cooperia pectinata）、点状古柏线虫（Cooperia punctata）与匙形古柏线虫（Cooperia spatulata）标本，以及纯培养的牛古柏线虫（Cooperia oncophora）和点状古柏线虫实验室分离株的幼虫中，扩增得到两个线粒体基因（细胞色素氧化酶亚基2（cytochrome oxidase 2）、12S核糖体RNA基因（12S rRNA gene））和两个核基因（含两个内含子的同工型1 β微管蛋白基因、内部转录间隔区（internal transcribed spacers, ITS））的部分序列。ITS-2序列的两两同源性极高，且它是唯一可将标本鉴定为古柏线虫未定种的分子标记区域，但ITS-2无法可靠地用于物种水平的鉴定。其余所有分子标记序列均无法明确将标本归属至古柏线虫属，但可实现除点状古柏线虫/匙形古柏线虫之外的物种的清晰鉴定——针对该物种对，所有标记序列均未发现显著差异。对单基因序列及包含全部四个标记序列的多位点分析开展的最大似然系统发育分析结果证实：被鉴定为匙形古柏线虫的标本随机分布于点状古柏线虫的聚类簇中，并未形成独立演化支。与之相反，其余古柏线虫属物种均形成了界限清晰且具有统计学支持度的独立聚类簇。上述研究结果表明，匙形古柏线虫极有可能仅为点状古柏线虫的一个形态型，其分类学名称应视为同物异名。应结合使用核基因与线粒体分子标记来鉴定形态型或隐存物种：既可以利用线粒体标记优异的条形码识别特性，又可开展可靠的系统发育分析，同时规避物种内线粒体基因型可能出现的谱系分选效应带来的干扰。