Yusseff-Vanegas Sohath Z, Agnarsson Ingi
Department of Biology, University of Vermont, Burlington, VT, United States of America.
PeerJ. 2017 Jul 25;5:e3516. doi: 10.7717/peerj.3516. eCollection 2017.
Correct identification of forensically important insects, such as flies in the family Calliphoridae, is a crucial step for them to be used as evidence in legal investigations. Traditional identification based on morphology has been effective, but has some limitations when it comes to identifying immature stages of certain species. DNA-barcoding, using COI, has demonstrated potential for rapid and accurate identification of Calliphoridae, however, this gene does not reliably distinguish among some recently diverged species, raising questions about its use for delimitation of species of forensic importance. To facilitate DNA based identification of Calliphoridae in the Caribbean we developed a vouchered reference collection from across the region, and a DNA sequence database, and further added the nuclear ITS2 as a second marker to increase accuracy of identification through barcoding. We morphologically identified freshly collected specimens, did phylogenetic analyses and employed several species delimitation methods for a total of 468 individuals representing 19 described species. Our results show that combination of COI + ITS2 genes yields more accurate identification and diagnoses, and better agreement with morphological data, than the mitochondrial barcodes alone. All of our results from independent and concatenated trees and most of the species delimitation methods yield considerably higher diversity estimates than the distance based approach and morphology. Molecular data support at least 24 distinct clades within Calliphoridae in this study, recovering substantial geographic variation for and , probably indicating several cryptic species. In sum, our study demonstrates the importance of employing a second nuclear marker for barcoding analyses and species delimitation of calliphorids, and the power of molecular data in combination with a complete reference database to enable identification of taxonomically and geographically diverse insects of forensic importance.
正确识别具有法医学重要性的昆虫，如丽蝇科的苍蝇，是它们在法律调查中用作证据的关键一步。基于形态学的传统鉴定方法一直很有效，但在识别某些物种的未成熟阶段时存在一些局限性。使用细胞色素氧化酶亚基I（COI）的DNA条形码技术已显示出快速准确鉴定丽蝇科的潜力，然而，该基因无法可靠地区分一些最近分化的物种，这引发了其在界定具有法医学重要性的物种方面的应用问题。为了便于在加勒比地区基于DNA鉴定丽蝇科，我们从该地区建立了一个有凭证的参考标本集和一个DNA序列数据库，并进一步添加了核内转录间隔区2（ITS2）作为第二个标记，以提高条形码鉴定的准确性。我们对新采集的标本进行形态学鉴定，进行系统发育分析，并对代表19个已描述物种的468个个体采用了几种物种界定方法。我们的结果表明，与单独的线粒体条形码相比，COI + ITS2基因的组合能产生更准确的鉴定和诊断结果，并且与形态学数据的一致性更好。我们从独立树和串联树得到的所有结果以及大多数物种界定方法得出的多样性估计值都比基于距离的方法和形态学得出的结果高得多。分子数据支持本研究中丽蝇科内至少24个不同的进化枝，发现 和 存在显著的地理变异，这可能表明存在几个隐存物种。总之，我们的研究证明了采用第二个核标记进行条形码分析和丽蝇科物种界定的重要性，以及分子数据与完整参考数据库相结合在鉴定具有法医学重要性的分类学和地理分布多样的昆虫方面的作用。
