, Volume 11, Issue 4, pp 563–576 | Cite as

Diversity of microbial eukaryotes in sediment at a deep-sea methane cold seep: surveys of ribosomal DNA libraries from raw sediment samples and two enrichment cultures

  • Kiyotaka Takishita
  • Naoji Yubuki
  • Natsuki Kakizoe
  • Yuji Inagaki
  • Tadashi Maruyama
Original Paper


Recent culture-independent surveys of eukaryotic small-subunit ribosomal DNA (SSU rDNA) from many environments have unveiled unexpectedly high diversity of microbial eukaryotes (microeukaryotes) at various taxonomic levels. However, such surveys were most probably biased by various technical difficulties, resulting in underestimation of microeukaryotic diversity. In the present study on oxygen-depleted sediment from a deep-sea methane cold seep of Sagami Bay, Japan, we surveyed the diversity of eukaryotic rDNA in raw sediment samples and in two enrichment cultures. More than half of all clones recovered from the raw sediment samples were of the basidiomycetous fungus Cryptococcus curvatus. Among other clones, phylotypes of eukaryotic parasites, such as Apicomplexa, Ichthyosporea, and Phytomyxea, were identified. On the other hand, we observed a marked difference in phylotype composition in the enrichment samples. Several phylotypes belonging to heterotrophic stramenopiles were frequently found in one enrichment culture, while a phylotype of Excavata previously detected at a deep-sea hydrothermal vent dominated the other. We successfully established a clonal culture of this excavate flagellate. Since these phylotypes were not identified in the raw sediment samples, the approach incorporating a cultivation step successfully found at least a fraction of the “hidden” microeukaryotic diversity in the environment examined.


Cultivation Diversity Methane seep Microbial eukaryotes SSU rDNA 



We thank Dr. J.D. Reimer (Biological Institute on Kuroshio) for critical reading of the manuscript; Drs. M. Tsuchiya and H. Nomaki (JAMSTEC) for providing unpublished environmental data on the sediments investigated here; Dr. I. Inouye (University of Tsukuba) for valuable advice; and the captain and crew of the R/V Natsushima and the commander, pilots, and operation team of the ROV Hyper-Dolphin for dedicated efforts. This work was supported in part by grants from the Japan Society for the Promotion of Science (No. 17770077 to K. Takishita, No. 18570214 to Y. Inagaki and No. 17370029 to I. Inouye).

Supplementary material


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Copyright information

© Springer 2007

Authors and Affiliations

  • Kiyotaka Takishita
    • 1
  • Naoji Yubuki
    • 2
  • Natsuki Kakizoe
    • 1
    • 3
  • Yuji Inagaki
    • 2
    • 4
  • Tadashi Maruyama
    • 1
  1. 1.Japan Agency for Marine-Earth Science and Technology (JAMSTEC)YokosukaJapan
  2. 2.Graduate School of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan
  3. 3.College of Bioresource SciencesNihon UniversityFujisawaJapan
  4. 4.Center for Computational Sciences and Institute of Biological SciencesUniversity of TsukubaTsukubaJapan

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