Data from: DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples

Although species assignment of scats is important to study carnivoran biology, there is still no standardized assay for the identification of carnivores worldwide, which would allow large-scale routine assessments and reliable cross-comparison of results. Here we evaluate the potential of two short mtDNA fragments (ATP6 [126 bp] and COI [187 bp]) to serve as standard markers for the Carnivora. Samples of 66 species were sequenced for one or both of these segments. Alignments were complemented with archival sequences, and analyzed with three approaches (tree-based, distance-based and character-based). Intraspecific genetic distances were generally lower than between-species distances, resulting in diagnosable clusters for 86% (ATP6) and and 85% (COI) of the species. Notable exceptions were recently diverged species, most of which could still be identified using diagnostic characters, uniqueness of haplotypes, or by reducing the geographic scope of the comparison. In silico comparative analyses were also performed with a 110-bp cytochrome b (cytb) segment, whose identification success was lower (70%), possibly due to the smaller number of informative sites and/or the influence of misidentified sequences obtained from GenBank. Finally, we performed case-studies with faecal samples, which supported the suitability of our two focal markers for poor-quality DNA, and allowed an assessment of prey-DNA co-amplification. No evidence of prey DNA contamination was found for ATP6, while some cases were observed for COI and subsequently eliminated by the design of more specific primers. Overall, our results indicate that these segments hold good potential as standard markers for accurate species-level identification in the Carnivora.

尽管对食肉目（Carnivora）动物粪便样本进行物种鉴定是开展食肉目生物学研究的关键环节，但目前全球范围内仍缺乏标准化的食肉动物鉴定检测方案，难以实现大规模常规检测与结果的可靠跨研究比对。本研究评估了两段短线粒体DNA（mitochondrial DNA, mtDNA）片段——ATP6 [126 bp]与COI [187 bp]——作为食肉目物种鉴定标准化分子标记的潜力。研究团队对66个物种类群的样本进行了上述两段片段中至少一段的扩增测序，序列比对分析辅以公共存档序列数据，并通过三类方法开展数据分析：基于系统发育树的方法、基于遗传距离的方法以及基于特征的方法。种内遗传距离普遍低于种间遗传距离，使得86%（ATP6）与85%（COI）的物种类群均可形成可明确区分的聚类簇。不过存在显著例外：近期分化的物种类群，其中多数仍可通过诊断特征、单倍型唯一性，或是缩小比对的地理范围实现物种鉴定。本研究同时针对一段110 bp的细胞色素b（cytochrome b, cytb）片段开展了计算机模拟（in silico）比对分析，其物种鉴定成功率仅为70%，这可能源于该片段的信息位点数较少，或是受到GenBank（基因银行）数据库中误鉴定序列的干扰。最后，本研究以粪便样本开展了案例研究，结果证实了我们选定的两段标记在降解DNA样本中的适用性，同时可用于评估猎物DNA的共扩增情况。ATP6未检测到猎物DNA污染的情况，而COI则出现了少量此类问题，后续通过设计特异性更强的引物成功消除了该干扰。综上，本研究结果表明，上述两段线粒体DNA片段具备作为食肉目物种精准鉴定标准化分子标记的良好应用潜力。