Abstract Identification of taxa that are morphologically very similar is a standing problem in ecology and is particularly important for invasive species that may differ in their dispersal rates and environmental and economic costs. Three similar pheretimoid earthworms (genera of Amynthas, Metaphire) are important invasive species in North American forests. They often lack the diagnostic morphological characters and their hatchlings, juveniles, and cocoons are impossible to identify to species. We present a multiplex PCR protocol that accurately scores the three species and is inexpensive compared to other molecular methods. Multiplex PCR identification was as accurate as mitochondrial COI barcoding and better than morphological scoring. The method uses unique PCR fragments of different lengths for each species from the COI gene. The multiplex PCR correctly identified embryos within cocoons, juveniles, and adults of Amynthas agrestis, Amynthas tokioensis and Metaphire hilgendorfi, with 100% accuracy. Comparisons of COI sequences (GenBank) with other populations of the same species (including specimens from their native range in Japan), and many other species of earthworms showed the primers always amplify only the three target species. The multiplex PCR method is rapid and costs a fraction of standard COI barcoding. Also, tiny scratches of cells from living specimens can be entered directly into the PCR for easy identification, reducing costs even further by avoiding DNA extraction, and allows the earthworms to be conserved for ecological experiments. To show the utility of the method we present the hatching phenology of the three species which could not be done by morphology. The three species begin hatching at the same time with A. tokioensis producing the most abundant juveniles early in the season. The method facilitates studies on biogeography, phenology, life histories, and resource partitioning among the three co-occurring earthworm species.

中文翻译：

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识别无法识别的：通过形态学和条形码识别验证的用于诊断侵入性类拟蚯蚓物种的 PCR 多重协议

摘要 识别形态上非常相似的分类群是生态学中的一个长期问题，对于可能在其扩散率以及环境和经济成本方面不同的入侵物种尤其重要。三种类似的类拟蚯蚓（Amynthas、Metaphire 属）是北美森林中重要的入侵物种。它们通常缺乏诊断形态特征，并且它们的幼体、幼体和茧无法识别到物种。我们提出了一种多重 PCR 协​​议，可以准确地对三个物种进行评分，并且与其他分子方法相比价格低廉。多重 PCR 鉴定与线粒体 COI 条形码一样准确，并且优于形态学评分。该方法对来自 COI 基因的每个物种使用不同长度的独特 PCR 片段。多重 PCR 正确识别了 Amynthas agrestis、Amynthas tokioensis 和 Metaphire hilgendorfi 的茧、幼虫和成虫内的胚胎，准确度为 100%。COI 序列 (GenBank) 与同一物种的其他种群（包括来自日本本土范围的标本）和许多其他蚯蚓物种的比较表明，引物总是仅扩增三个目标物种。多重 PCR 方法快速且成本仅为标准 COI 条形码的一小部分。此外，活体样本细胞的微小划痕可以直接进入 PCR 以便于识别，通过避免 DNA 提取进一步降低成本，并允许蚯蚓保存用于生态实验。为了显示该方法的实用性，我们展示了无法通过形态学完成的三个物种的孵化物候学。这三个物种同时开始孵化，A. tokioensis 在本季早期产生最丰富的幼鱼。该方法促进了对三种共生蚯蚓物种之间的生物地理学、物候学、生活史和资源分配的研究。