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Finding copepod footprints: a protocol for molecular identification of diapausing eggs in lake sediments

机译：寻找co足类足印：用于湖泊沉积物中滞育卵分子鉴定的方案

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Even though calanoid copepods produce diapausing eggs that stay alive in lake sediments, these eggs have rarely been used paleolimnologically, as they lack diagnostic morphological features. In this study, we developed a method to identify copepod diapausing eggs in Japan as a clue toward reconstructing past plankton populations. We first determined a 28S ribosomal DNA (rDNA) (i.e., nc28S) regional sequence library (240 bp) of various calanoid copepod species using ethanol-fixed plankton samples collected from across the Japanese archipelago. Then we applied the UltraSHOT method to extract DNA from an individual diapausing egg. Finally, the nc28S region of diapausing eggs collected from various lakes was sequenced and compared with the regional library for species identification. In total, 21 haplotypes of the nc28S region were recovered from planktonic samples of 11 Japanese freshwater calanoid copepod species. Despite the short length of this region, no identical haplotypes were shared among the species analyzed, including the Acanthodiaptomus pacificus complex treated as a species. Even different lineages of A. pacificus could be separated. These results indicate that the nc28S region can be used as a barcode in Japan. A total of 112 diapausing eggs collected from various lakes and ponds was processed, and the nc28S region of each was successfully sequenced. All of these egg sequences matched one or the other of the nc28S haplotypes in the regional library mentioned above. The set of protocols we applied (i.e., preparing a comprehensive regional sequence library and sequencing egg DNA) is thus useful for involving copepod diapausing eggs in paleolimnological studies in lakes. The nc28S region treated in this study has a strong potential to uncover the paleodiversity of copepods, at least in Japan.
机译：尽管cal兰足爪足类动物会产生滞育性的卵，它们仍然活在湖泊沉积物中，但由于缺乏诊断形态学特征，这些卵很少被古生物学应用。在这项研究中，我们开发了一种方法来识别日本的pe足动物滞育卵，以此作为重建过去浮游生物种群的线索。我们首先使用从日本群岛上收集的乙醇固定的浮游生物样品，确定了各种cal类co足类物种的28S核糖体DNA（rDNA）（即nc28S）区域序列库（240 bp）。然后，我们应用UltraSHOT方法从单个滞育卵中提取DNA。最后，对从各个湖泊收集的滞育卵的nc28S区域进行了测序，并与区域库进行了物种鉴定。总共从11种日本淡水Calanoid pe足类物种的浮游样品中回收了nc28S区域的21个单倍型。尽管该区域的长度很短，但在所分析的物种中没有共享相同的单倍型，包括被视为一个物种的棘皮棘阿米目虫。甚至可以分离出太平洋曲霉的不同谱系。这些结果表明，nc28S区域在日本可以用作条形码。总共处理了从各个湖泊和池塘收集的112个致死卵，并对每个卵的nc28S区进行了成功测序。所有这些卵序列均与上述区域文库中的nc28S单倍型匹配。因此，我们应用的协议集（即准备一个全面的区域序列库并对卵DNA进行测序）对于将co足动物滞育卵包括在湖泊的古湖泊学研究中非常有用。在这项研究中处理的nc28S区域，至少在日本，具有发现Japan足类古生物的强大潜力。

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