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Limnology

, Volume 14, Issue 3, pp 269–282 | Cite as

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

  • Wataru Makino
  • Hajime Ohtsuki
  • Jotaro Urabe
Research paper

Abstract

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.

Keywords

Japanese lakes Biodiversity Calanoid copepods Diapausing eggs 28S ribosomal DNA Molecular identification 

Notes

Acknowledgments

We thank anonymous reviewers for providing useful comments and references. This research was supported by grants from the Ministry of the Environment, Japan (the Environment Research and Technology Development Fund, No. D-1002) to JU and from the Japan Society for the Promotion of Science (KAKENHI, Nos. 16770011, 19770010, and 23570015) to WM. We are grateful to Syuhei Ban, Ryoma Hayashi, Ryuta Himori, Fujio Hyodo, Seiji Ishida, Michinobu Kuwae, Naoko Sasaki, Kohei Omoto, and Narumi K. Tsugeki for their support with some of our samplings. All works in this study comply with the current laws of Japan.

Supplementary material

10201_2013_404_MOESM1_ESM.pdf (384 kb)
Supplementary Fig. S1 (PDF 384 kb)

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Copyright information

© The Japanese Society of Limnology 2013

Authors and Affiliations

  1. 1.Graduate School of Life SciencesTohoku UniversitySendaiJapan

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