Khedkar Trupti, Sharma Rashmi, Tiknaik Anita, Khedkar Gulab, Naikwade Bhagwat S, Ron Tetsuzan Benny, Haymer David
a Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar Marathwada University , Aurangabad , Maharashtra , India .
b Aurangabad Municipal Corporation Siddharth Zoo , Aurangabad , Maharashtra , India , and.
Mitochondrial DNA A DNA Mapp Seq Anal. 2016;27(1):499-507. doi: 10.3109/19401736.2014.905830. Epub 2015 Sep 4.
Snakes represent a taxonomically underdeveloped group of animals in India with a lack of experts and incomplete taxonomic descriptions being the main deterrents to advances in this area. Molecular taxonomic approaches using DNA barcoding could aid in snake identification as well as studies of biodiversity. Here a non-invasive sampling method using DNA barcoding is tested using skin exuviates. Taxonomically authenticated samples were collected and tested for validation and comparisons to unknown snake exuviate samples. This approach was also used to construct the first comprehensive study targeting the snake species from Maharashtra state in India. A total of 92 skin exuviate samples were collected and tested for this study. Of these, 81 samples were successfully DNA barcoded and compared with unknown samples for assignment of taxonomic identity. Good quality DNA was obtained irrespective of age and quality of the exuviate material, and all unknown samples were successfully identified. A total of 23 species of snakes were identified, six of which were in the list of Endangered species (Red Data Book). Intra- and inter-specific distance values were also calculated, and these were sufficient to allow discrimination among species and between species without ambiguity in most cases. Two samples were suspected to represent cryptic species based on deep K2P divergence values (>3%), and one sample could be identified to the genus level only. Eleven samples failed to amplify COI sequences, suggesting the need for alternative PCR primer pairs. This study clearly documents how snake skin exuviates can be used for DNA barcoding, estimates of diversity and population genetic structuring in a noninvasive manner.
在印度，蛇类是一个分类学研究尚不充分的动物群体，缺乏专家以及分类学描述不完整是该领域取得进展的主要障碍。使用DNA条形码的分子分类学方法有助于蛇类的鉴定以及生物多样性研究。在此，我们使用皮肤蜕落物对一种基于DNA条形码的非侵入性采样方法进行了测试。收集了经过分类学鉴定的样本并进行测试，以进行验证并与未知的蛇类蜕落物样本进行比较。该方法还被用于构建针对印度马哈拉施特拉邦蛇类物种的首个全面研究。本研究共收集了92份皮肤蜕落物样本并进行测试。其中，81个样本成功进行了DNA条形码分析，并与未知样本进行比较以确定分类身份。无论蜕落物材料的年龄和质量如何，都获得了高质量的DNA，并且所有未知样本都被成功鉴定。总共鉴定出23种蛇，其中6种在濒危物种名单（《红色数据手册》）中。还计算了种内和种间距离值，在大多数情况下，这些值足以明确区分物种之间的差异。基于较深的K2P分歧值（>3%），有两个样本被怀疑代表隐存种，还有一个样本仅能鉴定到属级。11个样本未能扩增出COI序列，这表明需要替代的PCR引物对。这项研究清楚地证明了蛇类皮肤蜕落物如何能够以非侵入性方式用于DNA条形码分析、多样性估计和种群遗传结构研究。
