Finding copepod footprints: a protocol for molecular identification of diapausing eggs in lake sediments
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.
KeywordsJapanese lakes Biodiversity Calanoid copepods Diapausing eggs 28S ribosomal DNA Molecular identification
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.
- Ban S (1992) Seasonal distribution, abundance and viability of dapause eggs of Eurytemora affinis (Copepoda: Calanoida) in the sediment of Lake Ohnuma, Hokkaido. Bull Plankton Soc Jpn 39:41–48Google Scholar
- Ban S, Minoda T (1991) The effect of temperature on the development and hatching of diapausing and subitaneous eggs in Eurytemora affinis (Copepoda: Calanoida) in Lake Ohnuma, Hokkaido, Japan. Bull Plankton Soc Jpn Spec Vol:299–308Google Scholar
- Dussart BH, Defaye D (1983) Répertoire mondial des crustacés copépodes eaux intérieures. I. Calanoïdes. CNRS, ParisGoogle Scholar
- Dussart BH, Defaye D (1995) Copepoda. Introduction to the Copepoda. SPB Academic Publishing, AmsterdamGoogle Scholar
- Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotech 3:294–299Google Scholar
- Frey DG (1964) Remains of animals in quaternary lake and bog sediments and their interpretation. Ergebn Limnol 2:1–116Google Scholar
- Hutchinson GE (1967) A treatise on limnology, vol. 2. Introduction to lake biology and the limnoplankton. John Wiley and Sons, New YorkGoogle Scholar
- Kiesling TL, Wilkinson E, Rabalais J, Ortner PB, McCabe MM, Fell JW (2002) Rapid identification of adult and naupliar stages of copepods using DNA hybridization methodology. Mar Biotech 4:30–39Google Scholar
- Lampert W, Sommer U (1997) Limnoecology. The ecology of lakes and streams. Oxford University Press, OxfordGoogle Scholar
- Mizuno T, Takahashi E (eds) (2000) An illustrated guide to freshwater zooplankton in Japan (in Japanese). Tokai University Press, TokyoGoogle Scholar