Heimeier Dorothea, Lavery Shane, Sewell Mary A
Mar Genomics. 2010 Sep-Dec;3(3-4):165-77. doi: 10.1016/j.margen.2010.09.004. Epub 2010 Oct 13.
Ecological studies of the diversity and distribution of marine planktonic larvae are increasingly depending on molecular methods for accurate taxonomic identification. The greater coverage of reference marine species on genetic databases such as GenBank and BoLD (Barcoding of Life Data Systems; www.boldystems.org); together with the decreasing costs for DNA sequencing have made large scale larval identification studies using molecular methods more feasible. Here, we present the development and implementation of a practical molecular approach to identify over 2000 individual marine invertebrate larvae that were collected in the Ross Sea, Antarctica, during the austral summer over five years (2002-2007) as part of the LGP (Latitudinal Gradient Project). Larvae for molecular ID were morphologically identified to belong to the Phyla Mollusca, Echinodermata, Nemertea and Annelida (Class Polychaeta), but also included unidentified early developmental stages which could not be assigned a specific taxon (e.g., eggs, blastulae). The use of a 100μm mesh plankton net makes this one of the first larval identification studies to simultaneously consider both embryos and larvae. Molecular identification methods included amplification of up to three molecular loci for each specimen, a pre-identification step using BLAST with GenBank, phylogenetic reconstructions and cross-validation of assigned Molecular Operational Taxonomic Units (MOTUs). This combined approach of morphological and molecular methods assigned about 700 individuals to 53 MOTUs, which were identified to the lowest possible taxonomic level. During the course of this long-term study we identified several procedural difficulties, including issues with the collection of larvae, locus amplification, contamination, assignment and validation of MOTUs. The practical guidelines that we describe here should greatly assist other researchers to conduct reliable molecular identification studies of larvae in the future.
对海洋浮游幼虫的多样性和分布进行的生态学研究越来越依赖分子方法来进行准确的分类鉴定。诸如GenBank和BoLD（生命条形码数据系统；www.boldystems.org）等遗传数据库中参考海洋物种的覆盖范围不断扩大；再加上DNA测序成本的降低，使得使用分子方法进行大规模幼虫鉴定研究变得更加可行。在此，我们介绍一种实用分子方法的开发与实施，该方法用于鉴定在五年（2002 - 2007年）的南半球夏季期间于南极罗斯海收集的2000多个海洋无脊椎动物幼虫个体，这些幼虫是纬度梯度项目（LGP）的一部分。用于分子鉴定的幼虫在形态学上被鉴定属于软体动物门、棘皮动物门、纽形动物门和环节动物门（多毛纲），但也包括无法归为特定分类单元的未鉴定早期发育阶段（如卵、囊胚）。使用100μm网目的浮游生物网使得本研究成为首批同时考虑胚胎和幼虫的幼虫鉴定研究之一。分子鉴定方法包括为每个样本扩增多达三个分子位点、使用GenBank的BLAST进行预鉴定步骤、系统发育重建以及对指定的分子操作分类单元（MOTU）进行交叉验证。这种形态学和分子方法相结合的方式将约700个个体归为53个MOTU，并将其鉴定到尽可能低的分类水平。在这项长期研究过程中，我们发现了几个程序上的困难，包括幼虫采集、位点扩增、污染、MOTU的指定和验证等问题。我们在此描述的实用指南应能极大地帮助其他研究人员在未来开展可靠的幼虫分子鉴定研究。
