, Volume 594, Issue 1, pp 19–32 | Cite as

Intra-specific rDNA-ITS restriction site variation and an improved protocol to distinguish species and hybrids in the Daphnia longispina complex

  • Morten SkageEmail author
  • Anders Hobæk
  • Štĕpánka Ruthová
  • Barbara Keller
  • Adam Petrusek
  • Jaromír Sed’a
  • Piet Spaak


A collaborative research effort was undertaken to evaluate the robustness of a recently developed genetic tool for species identification of members in the morphologically variable Daphnia longispina species complex. This genetic method, based on restriction fragment length polymorphism (RFLP) of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA (rDNA) with restriction enzymes Mwo I and Sau96 I [Billiones et al., 2004. Hydrobiologia 526: 43–53], was applied to many different European populations. Results were compared with two or more independently obtained characters (morphology, allozymes, mitochondrial DNA (mtDNA), or cloned rDNA-ITS sequences). Individuals of most taxa were readily identified, but unexpected ITS-RFLP patterns were found in many individuals indicated by other markers to be D. galeata or one of its hybrids. Among 43 investigated D. galeata populations (902 specimen analysed by ITS-RFLP), deviant RFLP fragment patterns occurred in 26 (i.e., more than half) of the populations. The deviant patterns could be attributed to the loss of one single restriction site in the ITS2 region. This loss made the distinction of D. galeata from other species unreliable, and F1 hybrids could not be identified. Future users should be aware of this shortcoming of the Billions et al. [2004. Hydrobiologia 526: 43–53] protocol. As a solution to this problem, we present an improved genetic identification protocol based on a simple double digestion of the rDNA-ITS region with the restriction enzymes BsrB I and EagI. Sequence analyses of rDNA-ITS clones and preliminary testing indicate that the new protocol is unaffected by the rDNA variation which troubled the Mwo I/Sau96 I protocol. Further, the new protocol identifies all European species of the D. longispina complex, as well as their F1 hybrids. However, a wider screening is required to verify its general utility for all species, since yet unknown variation may occur.


Daphnia longispina species complex ITS-RFLP Ribosomal DNA (rDNA) ITS Intragenomic Interspecific hybrids 



We thank Colleen Durkin, Piotr Madej, Marina Manca, Esther Keller, Christoph Tellenbach and Justyna Wolinska for help to obtain zooplankton samples of Swiss and Italian lakes and help in the laboratory. This study was supported by the University of Bergen, Norwegian Institute of Water Research, the Norwegian Research Council (Grant 121181/720), the Czech Science Foundation (project 206/04/0190), the Grant Agency of the Charles University (GA 114807) and the Czech Ministry of Education (MSM0021620828).


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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Morten Skage
    • 1
    Email author
  • Anders Hobæk
    • 2
  • Štĕpánka Ruthová
    • 3
  • Barbara Keller
    • 4
    • 5
  • Adam Petrusek
    • 3
  • Jaromír Sed’a
    • 6
  • Piet Spaak
    • 4
    • 5
  1. 1.Department of BiologyUniversity of BergenBergenNorway
  2. 2.Norwegian Institute of Water ResearchRegional office BergenBergenNorway
  3. 3.Department of EcologyCharles University in Prague, Faculty of SciencePrague 2Czechia
  4. 4.Eawag, Swiss Federal Institute of Aquatic Science and TechnologyDubendorfSwitzerland
  5. 5.Institute of Integrative BiologyZurichSwitzerland
  6. 6.Biological Centre ASCRInstitute of HydrobiologyCeske BudejoviceCzechia

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