, Volume 13, Issue 3, pp 261–267 | Cite as

DNA extraction and amplification methods for ephippial cases of Daphnia resting eggs in lake sediments: a novel approach for reconstructing zooplankton population structure from the past

  • Seiji IshidaEmail author
  • Hajime Ohtsuki
  • Tamotsu Awano
  • Narumi K. Tsugeki
  • Wataru Makino
  • Yoshihisa Suyama
  • Jotaro Urabe
Note Rapid communication on important and novel findings


This study describes a method of DNA extraction and amplification for ephippial cases of Daphnia resting eggs from lake sediment. Recent studies have reconstructed succession records of Daphnia species by genetically analyzing Daphnia resting eggs stored in lake sediments and revealed changes in dominant Daphnia species that correspond well with environmental changes. However, this approach is not applicable to lakes where most of the resting eggs in the sediment have already hatched out. We modified conventional methods for DNA extraction and amplification to enable genetic analyses of the ephippial case that envelops and protects the resting eggs, and we compared the performance of the modified method to the conventional one. We confirmed that we could efficiently analyze the sequences of the ephippial cases collected in a sediment core using our modified method. It enables us to reconstruct changes in genetic structure of Daphnia populations regardless of hatching rates of the resting eggs.


Paleolimnology Daphnia Resting eggs Ephippia DNA extraction 



This study was supported, in whole or in part, by the Environmental Research & Technology Development Fund (D-1002) from the Ministry of the Environment of Japan, a Global COE Research Grant (Tohoku University Ecosystem Adaptability), and a Grant-in-Aid for Scientific Research (B) (22370007) and Young Scientists (B) (22770076) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We thank Marcia Kyle for kind review of our manuscript before submitting it.


  1. Alasaad S, Rossi L, Maione S, Sartore S, Soriguer RC, Pérez JM, Rasero R, Zhu XQ, Soglia D (2008) HotSHOT Plus ThermalSHOCK, a new and efficient technique for preparation of PCR-quality mite genomic DNA. Parasitol Res 103:1455–1457PubMedCrossRefGoogle Scholar
  2. Allen MR (2010) Genetic and environmental factors influence survival and hatching of diapausing eggs. Limnol Oceanogr 55:549–559CrossRefGoogle Scholar
  3. Appleby PG, Oldfield F (1978) The calculation of lead-210 dates assuming a constant rate of supply of unsupported 210Pb to the sediment. Catena 5:1–8CrossRefGoogle Scholar
  4. Benzie JAH (2005) Cladocera: the genus Daphnia (including Daphniopsis) (Anomopoda: Daphniidae). Guides to the identification of the microinvertebrates of the conntinental waters of the world, vol 21. Kenobi Productions, GhentGoogle Scholar
  5. Brede N, Sandrock C, Straile D, Spaak P, Jankowski T, Streit B, Schwenk K (2009) The impact of human-made ecological changes on the genetic architecture of Daphnia species. Proc Natl Acad Sci USA 106:4758–4763PubMedCrossRefGoogle Scholar
  6. Caceres C (1998) Interspecific variation in the abundance, production, and emergence of Daphnia diapausing eggs. Ecology 79:1699–1710Google Scholar
  7. Caceres CE, Tessier AJ (2003) How long to rest: the ecology of optimal dormancy and environmental constraint. Ecology 84:1189–1198CrossRefGoogle Scholar
  8. Cousyn C, De Meester L, Colbourne JK, Brendonck L, Verschuren D, Volckaert F (2001) Rapid, local adaptation of zooplankton behavior to changes in predation pressure in the absence of neutral genetic changes. Proc Natl Acad Sci USA 98:6256–6260PubMedCrossRefGoogle Scholar
  9. Decaestecker E, Gaba S, Raeymaekers JAM, Stoks R, Van Kerckhoven L, Ebert D, De Meester L (2007) Host-parasite ‘Red Queen’ dynamics archived in pond sediment. Nature 450:870–873. doi: 10.1038/nature06291 PubMedCrossRefGoogle Scholar
  10. Faustová M, Petrusek A, Černý M (2004) Status of Daphnia resting egg banks in Bohemian forest lakes affected by acidification. Hydrobiologia 526:23–31CrossRefGoogle Scholar
  11. Hairston NG, Lampert W, Caceres CE, Holtmeier CL, Weider LJ, Gaedke U, Fischer JM, Fox JA, Post DM (1999) Lake ecosystems—rapid evolution revealed by dormant eggs. Nature 401:446–446CrossRefGoogle Scholar
  12. Hairston NG, Holtmeier CL, Lampert W, Weider LJ, Post DM, Fischer JM, Cáceres CE, Fox JA, Gaedke U (2001) Natural selection for grazer resistance to toxic cyanobacteria: evolution of phenotypic plasticity? Evolution 55:2203–2214PubMedCrossRefGoogle Scholar
  13. Hofreiter M, Mead JI, Martin P, Poinar HN (2003) Molecular caving. Curr Biol 13:R693–R695PubMedCrossRefGoogle Scholar
  14. Ishida S, Kotov A, Taylor D (2006) A new divergent lineage of Daphnia (Cladocera : Anomopoda) and its morphological and genetical differentiation from Daphnia curvirostris Eylmann, 1887. Zool J Linn Soc 146:385–405CrossRefGoogle Scholar
  15. Jankowski T, Straile D (2003) A comparison of egg-bank and long-term plankton dynamics of two Daphnia species, D. hyalina and D. galeata: potentials and limits of reconstruction. Limnol Oceanogr 48:1948–1955CrossRefGoogle Scholar
  16. Jeppesen E, Leavitt P, De Meester L, Jensen JP (2001) Functional ecology and palaeolimnology: using cladoceran remains to reconstruct anthropogenic impact. Trends Ecol Evol 16:191–198PubMedCrossRefGoogle Scholar
  17. Marková S, Černý M, Rees DJ, Stuchlík E (2006) Are they still viable? Physical conditions and abundance of Daphnia pulicaria resting eggs in sediment cores from lakes in the Tatra Mountains. Biologia 61:S135–S146CrossRefGoogle Scholar
  18. Meeker ND, Hutchinson SA, Ho L, Trede NS (2007) Method for isolation of PCR-ready genomic DNA from zebrafish tissues. Biotechniques 43:610–614PubMedCrossRefGoogle Scholar
  19. Montero-Pau J, Gomez A, Munoz J (2008) Application of an inexpensive and high-throughput genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing eggs. Limnol Oceanogr Methods 6:218–222CrossRefGoogle Scholar
  20. Parducci L, Suyama Y, Lascoux M, Bennett KD (2005) Ancient DNA from pollen: a genetic record of population history in Scots pine. Mol Ecol 14:2873–2882PubMedCrossRefGoogle Scholar
  21. Petrusek A, Hobaek A, Nilssen JP, Skage M, Černý M, Brede N, Schwenk K (2008) A taxonomic reappraisal of the European Daphnia longispina complex (Crustacea, Cladocera, Anomopoda). Zool Scr 37:507–519CrossRefGoogle Scholar
  22. Rychlik W (2007) OLIGO 7 Primer Analysis Software. Methods Mol Biol 402:35–60PubMedCrossRefGoogle Scholar
  23. Schwartz SS, Hebert PDN (1987) Methods for the activation of the resting eggs of Daphnia. Freshwat Biol 17:373–379CrossRefGoogle Scholar
  24. Schwenk K, Posada D, Hebert PD (2000) Molecular systematics of European Hyalodaphnia: the role of contemporary hybridization in ancient species. Proc R Sci Lond B 267:1833–1842CrossRefGoogle Scholar
  25. Smol JP, Douglas MS (2007) From controversy to consensus: making the case for recent climate change in the Arctic using lake sediments. Front Ecol Environ 5:466–474CrossRefGoogle Scholar
  26. Suyama Y, Kawamuro K, Kinoshita I, Yoshimura K, Tsumura Y, Takahara H (1996) DNA sequence from a fossil pollen of Abies spp. from Pleistocene peat. Genes Genet Syst 71:145–149PubMedCrossRefGoogle Scholar
  27. Taylor DJ, Hebert PD (1993) Habitat-dependent hybrid parentage and differential introgression between neighboringly sympatric Daphnia species. Proc Natl Acad Sci USA 90:7079–7083PubMedCrossRefGoogle Scholar
  28. Taylor DJ, Hebert PD, Colbourne JK (1996) Phylogenetics and evolution of the Daphnia longispina group (Crustacea) based on 12S rDNA sequence and allozyme variation. Mol Phyl Evol 5:495–510CrossRefGoogle Scholar
  29. Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML (2000) Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques 29:52–54PubMedGoogle Scholar
  30. Tsugeki NK, Urabe J (2009) Paleolimnological approach as a tool for reconstructing lake ecosystems in the past (in Japanese). Seibutsu no Kagaku Iden 63:66–72Google Scholar
  31. Tsugeki N, Oda H, Urabe J (2003) Fluctuation of the zooplankton community in Lake Biwa during the 20th century: a paleolimnological analysis. Limnology 4:101–107CrossRefGoogle Scholar
  32. Tsugeki NK, Ishida S, Urabe J (2008) Sedimentary records of reduction in resting egg production of Daphnia galeata in Lake Biwa during the 20th century: a possible effect of winter warming. J Paleolimnol 42:155–165CrossRefGoogle Scholar
  33. Weider L, Lampert W, Wessels M, Colbourne J, Limburg P (1997) Long-term genetic shifts in a microcrustacean egg bank associated with anthropogenic changes in the Lake Constance ecosystem. Proc R Soc Lond Ser B Biol sci 264:1613–1618CrossRefGoogle Scholar
  34. Willerslev E, Hansen AJ, Binladen J, Brand TB, Gilbert MTP, Shapiro B, Bunce M, Wiuf C, Gilichinsky DA, Cooper A (2003) Diverse plant and animal genetic records from Holocene and Pleistocene sediments. Science 300:791–795PubMedCrossRefGoogle Scholar
  35. Willerslev E, Cappellini E, Boomsma W, Nielsen R, Hebsgaard MB, Brand TB, Hofreiter M, Bunce M, Poinar HN, Dahl-Jensen D, Johnsen S, Steffensen JP, Bennike O, Schwenninger J-L, Nathan R, Armitage S, de Hoog C-J, Alfimov V, Christl M, Beer J, Muscheler R, Barker J, Sharp M, Penkman KEH, Haile J, Taberlet P, Gilbert MTP, Casoli A, Campani E, Collins MJ (2007) Ancient biomolecules from deep ice cores reveal a forested southern Greenland. Science 317:111–114PubMedCrossRefGoogle Scholar

Copyright information

© The Japanese Society of Limnology 2012

Authors and Affiliations

  • Seiji Ishida
    • 1
    • 2
    Email author
  • Hajime Ohtsuki
    • 2
  • Tamotsu Awano
    • 2
  • Narumi K. Tsugeki
    • 3
  • Wataru Makino
    • 2
  • Yoshihisa Suyama
    • 4
  • Jotaro Urabe
    • 2
  1. 1.International Advanced Research and Education OrganizationTohoku UniversitySendaiJapan
  2. 2.Graduate School of Life SciencesTohoku UniversityMiyagiJapan
  3. 3.Senior Research Fellow CenterEhime UniversityMatsuyamaJapan
  4. 4.Field Science Center, Graduate School of Agricultural ScienceTohoku UniversityOsakiJapan

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