Abstract
The Kuroshima Knoll, located in the southern Ryukyu Arc, is known to actively bubble with gas containing methane and hydrogen sulfide from numerous fissures in the large carbonate pavement. Although ecological studies regarding macrobenthos and bacteria from Kuroshima Knoll have been intensively conducted, the community structure and ecological importance of microbial eukaryotes (protists) have not yet been investigated. In the present study, we directly extracted DNA from sediment of the Kuroshima Knoll at a depth of 640 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit ribosomal DNA (SSU rDNA). Although the SSU rDNA sequences of several types of benthic foraminifers were retrieved from the surface of the sediment, all other sequences (just below the sediment surface to approximately 9 cm below sediment surface) were derived from the basidiomycetous yeast Cryptococcus curvatus. Furthermore, sequences of the internal transcribed spacer of rDNA (ITS-rDNA) retrieved from the same sediment were identical to that of C. curvatus originating from terrestrial habitats. The diversity of microbial eukaryotes in the Kuroshima Knoll sediment seems to be extremely low and significantly different from that of other marine environments previously reported.
Avoid common mistakes on your manuscript.
Recently, culture-independent molecular analyses have demonstrated the diversity and ecological importance of microbial eukaryotes (protists) in various marine environments. In particular, there has been increasing interest in the diversity of microbial eukaryotes from extreme marine environments. For example, phylogenetic analyses of eukaryotic small subunit ribosomal DNA (SSU rDNA) retrieved from the sediment and ambient seawater of chemosynthetic ecosystems (i.e. deep-sea hydrothermal vents) have detected unexpectedly diverse phylotypes, including many novel ones at a high-taxonomic level (Edgcomb et al. 2002; López-García et al. 2003). However, some of these high-taxonomic level novel phylotypes have been suggested to be artifacts caused by the failure to detect chimeric sequences, phylogenetic misplacement of fast-evolving sequences, and/or incomplete sampling of described but as of yet unsequenced eukaryotes (Berney et al. 2004). Furthermore, Moreira and López-García (2003), based on these molecular analyses, have argued that parasitic protists inhabiting areas around deep-sea hydrothermal vents, such as Apicomplexa and Kinetoplastida, are possibly hosted by dense animal populations, and these parasites may cause sudden massive mortality of host animals. Our recent molecular survey of anoxic sediment around fumaroles on a submarine caldera floor at a depth of 200 m, with conditions similar to conventional deep-sea hydrothermal vents, also suggested the existence of taxonomically diverse protists, including a few novel eukaryotes at high-taxonomic levels (Takishita et al. 2005). Remarkably, some sequences from this environment were closely related to those of eukaryotic parasites, such as Phytomyxea and Ichthyosporea, although it was not clear whether these organisms occurred in free-living or parasitic forms (Takishita et al. 2005).
However, the genetic diversity of microbial eukaryotes occurring at cold seeps (chemosynthetic ecosystems where energy-rich fluids seep out of the ocean floor due to the geology of the underlying sediments and rock layers) has not yet been investigated. The Kuroshima Knoll, located in the southern Ryukyu Arc near Ishigaki Island, Japan, at a depth of about 650 m, is one of the most well-described cold methane seep sites, characterized by active bubbling from numerous fissures in the large carbonate pavement. The site has large-scale colonies of the vesicomyid clam genus Calyptogena and the mytilid mussel genus Bathymodiolus (Machiyama et al. 2001). Calyptogena spp. and Bathymodiolus spp. harbor sulfur-oxidizing lithotrophic symbionts and methanotrophic symbionts, respectively, and the growth of both symbiont types depends on the bubbles released from the fissures (Inagaki et al. 2004). Furthermore, it has been shown that Kuroshima Knoll sediment contains abundant archaea, including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of Methanosarcinales, as well as epsilon-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occur at this site (Inagaki et al. 2004). In the present study, to obtain information on the diversity of another ecological organism category (in addition to the previously surveyed macrobenthos, bacteria, and archaea), we have analyzed microbial eukaryotes (protists) from the sediment of this chemosynthetic ecosystem at the molecular level.
The dive survey was conducted at Kuroshima Knoll in Okinawa Trough using the ROV Hyper-Dolphin on April 24, 2004 (Cruise No. NT04-03, Hyper-Dolphin Dive #299). Sediment samples were collected with an MBARI-type push corer at two sites at a depth of 642 m: Site A (24°7.800′N, 124°11.455′E) and Site B (24°7.810′N, 124°11.385′E). The distance between the two sites was about 120 m. Both of the two core samples smelled strongly of hydrogen sulfide, and Site B’s sample was colored black due to hydrogen sulfide. Both of the two core samples were composed mainly of planktonic foraminiferal ooze. Site A samples at 1, 9, and 16 cm from the sediment surface, and Site B samples at 1, 3, 9, and 17 cm from the sediment surface were fractionated in consideration of sediment facies. Total DNA from each sediment sample was extracted with UltraCleanTM Soil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA). According to the manufacturer’s instructions PCR-amplification using the total DNA as a template was performed with HotStarTaq DNA polymerase (QIAGEN, Tokyo, Japan). Nuclear SSU rDNA were amplified by using different combinations of the eukaryotic SSU rDNA universal primers 18S-42F (5′-CTCAARGAYTAAGCCATGCA-3′), 18S-82F (5′-GAAACTGCGAATGGCTC-3′), 18S-1498R (5′-CACCTACGGAAACCTTGTTA-3′), and 18S-1520R (5′-CYGCAGGTTCACCTAC-3′), under the following thermal cycle conditions: 32 cycles of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C, followed by a final elongation step of 10 min at 72°C. The amplified products were confirmed on 1.0% agarose gel electrophoresis. The PCR-amplified DNA fragments were cloned into the pCR2.1 vector of the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA). Positive transformants (more than 60 clones) for each sediment sample were screened by PCR amplification of inserts with InsertCheck-Ready-Blue (TOYOBO, Osaka, Japan). The clones from the libraries were sequenced with an ABI PRISMTM 3700 DNA Analyzer (PE Biosystems, Foster City, CA, USA) using a BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems). Unexpectedly, all clones of all sediment fractions other than 16 cm of Site A and 17 cm of Site B had sequences identical to that of SSU rDNA from the saprophylic basidiomycetous fungus Cryptococcus curvatus. In addition, electrophoresis patterns of digestions by four base cutter restriction enzymes (HhaI, MspI, and RsaI) of the PCR-amplified products, as well as an insertion cloned into the plasmid vector and subsequently sequenced, showed no variation in results (data not shown), suggesting that the PCR-amplified products were derived from one sequence. No amplified-products were obtained from the samples at 16 cm of Site A and 17 cm of Site B. In the sediment samples from which the sequences of C. curvatus were obtained, yeast-like cells were frequently found during microscopic observation (but their density was considerably low when compared with that of prokaryotic cells). Figure 1 shows the maximum-likelihood (ML) tree based on the alignment including our obtained sequence ‘KUROS1’ (GenBank accession no. AB234889) along with previously reported SSU rDNA sequences from C. curvatus and its related fungal species (19 taxa, 1718 sites) constructed with a general time reversal model (Rodríguez et al. 1990) incorporating invariable sites and a discrete gamma distribution (eight categories) (GTR + I + Γ) using PhyML (Guindon and Gascuel 2003). PhyML bootstrap trees (500 replicates) were constructed using the same parameters as the individual ML trees. Furthermore, the internal transcribed spacer of rDNA (ITS-rDNA) was amplified with a set of primers pITS-F (5′-GTCGTAACAAGGTTAACCTGCGG-3′) and pITS-R (5′-TCCTCCGCTTATTGATATGC-3′) using each sample fraction DNA as templates. PCR conditions, cloning, and sequencing of ITS-rDNA were same to those of SSU rDNA mentioned above. Similarly, all clones (20 clones) of each sediment fraction, except for 16 cm of Site A and 17 cm of Site B, for which no amplified products were obtained, had sequences identical to that of ITS-rDNA from C. curvatus previously reported (accession no. AB035574 and AB035686-AB035696). The ITS-rDNA sequence obtained in this study ‘KUROS2’ was deposited in GenBank (accession no. AB234890).
In general, basidiomycetous yeasts often account for the majority of the total yeast population in oligotrophic oceanic waters, and among the basidiomycetous yeasts, some species of Cryptococccus and its teleomorphs have been found to be widespread across various oceanic regions (Nagahama 2005). In addition, yeast-like cells have been shown to be abundant in deep-sea sediments from quantitative surveys of microorganisms (Burnett 1981; Alongi 1987, 1992). A number of yeasts including the genus Cryptococcus were isolated during surveys of yeast distribution in deep-sea environments around the Pacific Ocean (Nagahama et al. 1999, 2001a, b, 2003a, b). However, based on molecular and other studies there has been no report of C. curvatus exclusively dominating not only yeast communities but also microbial eukaryotic communities in marine environments. Thus, the sediment of Kuroshima Knoll may be a unique marine environment with respect to the community structure of microbial eukaryotes. There has been a longstanding debate surrounding the questions: ‘Are there indigenous marine yeasts?’ and ‘If some yeast species are indigenous, which species are indigenous?’ (Kohlmeyer and Kohlmeyer 1979). According to a review by Nagahama (2005), the candidates for autochthonous marine yeasts are not thought to be ubiquitous, their occurrence being restricted to specific geographical latitudes or to associations with certain macroorganisms. ITS-rDNA sequences (known to be fast-evolving) retrieved from the Kuroshima Knoll sediment were exactly identical to sequences from C. curvatus isolated from terrestrial habitats, suggesting that the C. curvatus cells occurring in the sediment of Kuroshima Knoll are not autochthonous, but allochthonous. Why only allochthonous C. curvatus would be present at Kuroshima Knoll and not other microbial eukaryotes (including fungi) remains unclear. In addition, ecological interactions between C. curvatus and other organisms in the same environment (macrobenthos, bacteria, and archaea) are still enigmatic. C. curvatus is known to be a opportunistic pathogen of animals, including humans (Dromer et al. 1995). Because bivalves were found in the same sediment, C. curvatus may infect these animals. To properly investigate and clarify these questions, culturing, and subsequent physiological experiments of resident C. curvatus are necessary in the future.
In our microscopic observation not only yeast-like cells, but also foraminifer-like cells were found in the surface sediment of our samples. Although it is known that foraminifers are frequently found in various deep-sea sediments (Snider et al. 1984), the foraminifer-like cell density (frequency) at Kuroshima Knoll was extremely low. Because the sequences of the foraminiferal SSU rDNA are exceptionally divergent, eukaryotic SSU rDNA universal primers used in the present study failed to amplify foraminiferal SSU rDNA. Thus, with a set of primers specific to the foraminiferal SSU rDNA; s14f1 (5′-AAGGGCACCACAAGAACGC-3′) and sB (5′-TGATCCTTCTGCAGGTTCACCTAC-3′) (Pawlowski et al. 1996), PCR amplifications were performed using all fraction samples of DNA as templates. PCR conditions were the same as mentioned above except 40 cycle steps were conducted. Products of the expected size (about 1,000 b.p.) were amplified only from samples 1 cm from the sediment surface at both sites. Consequently, three different sequences ‘KUROS3-5’ were obtained by cloning and sequencing (17 clones) (GenBank accession no. AB234891–AB234893). These sequences were aligned with those of various foraminiferal SSU rDNA (24 taxa, 685 sites), and were applied to ML analysis under the GTR + I + Γ model with PhyML. PhyML bootstrap analysis (500 replicates) was also performed. In the phylogenetic tree (Fig. 2) KUROS3 was positioned within the radiation of the calcareous hard-shelled benthic foraminiferal lineage, while KUROS4 and KUROS5 were within the soft-shelled benthic foraminiferal lineage. Thus, it is highly likely that all the three sequences were derived from benthic foraminifers intrinsic in the sediment, and not from sinking planktonic foraminifers. Previously, many foraminiferal species have been found in a methane-seep in Monterey Bay, and hydrocarbon-seeps both at Blake Ridge, Atlantic Ocean and in the Gulf of Mexico (Bernhard et al. 2001; Rathburn et al. 2003; Robinson et al. 2004). However, whether the foraminiferal species having these sequences obtained in this study occur specifically at methane seeps remains unclear, because no other existing molecular information on foraminifers inhabiting other seep sites.
In summary, Kuroshima Knoll sediment is very unique with respect to the community composition of microbial eukaryotes, the fungus C. curvatus being dominant to a depth of 9 cm below the sediment surface, and with a minority of several benthic foraminiferal species just below the sediment surface.
References
Alongi DM (1987) The distribution and composition of deep-sea microbenthos in a bathyal region of the western Coral Sea. Deep Sea Res 34:1245–1254
Alongi DM (1992) Bathymetric patterns of deep-sea benthic communities from bathyal to abyssal depths in the western South Pacific (Solomon and Coral Seas). Deep Sea Res 39:549–565
Berney C, Fahrni J, Pawlowski J (2004) How many novel eukaryotic ‘kingdoms’? Pitfalls and limitations of environmental DNA surveys. BMC Biol 2:13
Bernhard JM, Buck KR, Barry JP (2001) Monterey Bay cold-seep biota: assemblages, abundance, and ultrastructure of living foraminifera. Deep Sea Res I 48:2233–2249
Burnett BR (1981) Quantitative sampling of nanobiota (microbiota) of the deep-sea benthos-III. The bathyal San Diego trough. Deep Sea Res 28:649–663
Dromer F, Moulignier A, Dupont B, Gueho E, Baudrimont M, Improvisi L, Provost F, Gonzalez-Canali G (1995) Myeloradiculitis due to Cryptococcus curvatus in AIDS. AIDS 9:395–396
Edgcomb VP, Kysela DT, Teske A, de Vera Gomez A, Sogin ML (2002) Benthic eukaryotic diversity in the Guaymas Basin hydrothermal vent environment. Proc Natl Acad Sci USA 99:7658–7662
Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704
Inagaki F, Tsunogai U, Suzuki M, Kosaka A, Machiyama H, Takai K, Nunoura T, Nealson KH, Horikoshi K (2004) Characterization of C1-metabolizing prokaryotic communities in methane seep habitats at the Kuroshima Knoll, southern Ryukyu Arc, by analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA genes. Appl Environ Microbiol 70:7445–7455
Kohlmeyer J, Kohlmeyer E (1979) Yeast. In: Kohlmeyer J, Kohlmeyer E (eds) Marine mycology: the higher fungi. Academic, New York pp 556–606
López-García P, Philippe H, Gail F, Moreira D (2003) Autochthonous eukaryotic diversity in hydrothermal sediment and experimental microcolonizers at the Mid-Atlantic ridge. Proc Natl Acad Sci USA 100:697–702
Machiyama H, Matsumoto T, Matsumoto R, Hattori M, Okano M, Iwase R, Tomaru H (2001) Outline of Shinkai 2000 dive surveys on the Kuroshima Knoll, off Ishigaki Island. JAMSTEC J Deep Sea Res 19:45–60
Moreira D, López-García P (2003) Are hydrothermal vents oases for parasitic protists? Trends Parasitol 19:556–558
Nagahama T, Hamamoto M, Nakase T, Horikoshi K (1999) Kluyveromyces nonfermentans sp. nov., a new yeast species isolated from the deep sea. Int J Syst Bacteriol 49:1899–1905
Nagahama T, Hamamoto M, Nakase T, Horikoshi K (2001a) Rhodotorula lamellibrachii sp. nov., a new yeast species from a tubeworm collected at the deep-sea floor in Sagami bay and its phylogenetic analysis. Antonie Van Leeuwenhoek 80:317–323
Nagahama T, Hamamoto M, Nakase T, Takami H, Horikoshi K (2001b) Distribution and identification of red yeasts in deep-sea environments around the northwest Pacific Ocean. Antonie Van Leeuwenhoek 80:101–110
Nagahama T, Hamamoto M, Nakase T, Horikoshi K (2003a) Rhodotorula benthica sp. nov and Rhodotorula calyptogenae sp. nov, novel yeast species from animals collected from the deep-sea floor, and Rhodotorula lysiniphila sp. nov., which is related phylogenetically. Int J Syst Evol Microbiol 53:897–903
Nagahama T, Hamamoto M, Nakase T, Takaki Y, Horikoshi K (2003b) Cryptococcus surugaensis sp. nov., a novel yeast species from sediment collected on the deep-sea floor of Suruga Bay. Int J Syst Evol Microbiol 53:2095–2098
Nagahama T (2005) Yeast biodiversity in freshwater, marine and deep-sea environments. In: Rosa C, Peter G (eds) The yeast handbook—volume ‘Biodiversity and ecophysiology of Yeasts’. Springer, Berlin Heidelberg New York, pp 241–262
Pawlowski J, Bolivar I, Fahrni JF, Cavalier-Smith T, Gouy M (1996) Early origin of foraminifera suggested by SSU rRNA gene sequences. Mol Biol Evol 13:445–450
Rathburn AE, Perez ME, Martin JB, Day SA, Mahn C, Gieskes J, Ziebis W, Williams D, Bahls A (2003) Relationships between the distribution and stable isotopic composition of living benthic foraminifera and cold methane seep biogeochemistry in Monterey Bay, California. Geochem Geophys Geosyst 4:1–28
Robinson CA, Bernhard JM, Levin LA, Mendoza GF, Blanks JK (2004) Surficial hydrocarbon seep infauna from the lake ridge (Atlantic Ocean, 2150 m) and the Gulf of Mexico (690–2240 m). Mar Ecol 25:313–336
Rodríguez F, Oliver JL, Marín A, Medina JR (1990) The general stochastic model of nucleotide substitutions. J Theor Biol 142:485–501
Snider LJ, Burnett BR, Hessler RR (1984) The composition and distribution of meiofauna and nanobiota in a central North Pacific deep-sea area. Deep Sea Res 31:1225–1249
Takishita K, Miyake H, Kawato M, Maruyama T (2005) Genetic diversity of microbial eukaryotes in anoxic sediment around fumaroles on a submarine caldera floor based on the small-subunit rDNA phylogeny. Extremophiles 9:185–196
Acknowledgements
We thank Dr. M. Nishijima (NCIMB Japan) for useful information on yeast literature; Mr. M. Kawato (JAMSTEC) for technical assistance; 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 performed as part of the project ‘Study for Understanding of Function and Structure of the Marine Ecosystems in the Earth Systems’ conducted at JAMSTEC, and was supported in part by grants from the Japan Society for the Promotion of Science (No. 17770077 to K. Takishita).
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by K. Horikoshi
Rights and permissions
About this article
Cite this article
Takishita, K., Tsuchiya, M., Reimer, J.D. et al. Molecular evidence demonstrating the basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sediment at the Kuroshima Knoll methane seep. Extremophiles 10, 165–169 (2006). https://doi.org/10.1007/s00792-005-0495-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00792-005-0495-7