Marine Biology

, Volume 155, Issue 4, pp 451–460 | Cite as

Application of temperature gradient gel electrophoresis technique to monitor changes in the structure of the eukaryotic leaf-epiphytic community of Posidonia oceanica

  • F. J. Medina-PonsEmail author
  • J. Terrados
  • R. Rosselló-Móra


Seagrass leaves have been recognized as a suitable substratum in shallow sedimentary environments for the establishment of epiphytic communities. Microscope-based identification of species has been traditionally used to monitor changes in the composition of the eukaryotic leaf-epiphytic community of the seagrass Posidonia oceanica (L.) Delile. Our main goal was to adapt the temperature gradient gel electrophoresis (TGGE) barcoding technique largely used in molecular microbial ecology studies to monitor changes in the composition of P. oceanica epiphytic community. This molecular technique has been successful for handling large amounts of samples in a fast and reproducible manner. To that end, we applied the TGGE technique to study the epiphytic community in two different seasons and compare the results with those provided by the classical microscope approach. The results obtained with both approaches were generally consistent. The complexity of the banding pattern produced by TGGE was mirrored by the taxa richness of the community described using the classical approach. The minimum number of P. oceanica shoots necessary to adequately represent the composition of the eukaryotic leaf-epiphytic community was of the same order of magnitude for both techniques. Partial gene sequences of some selected bands affiliated with sequences of zoo and phytoephytic taxa. Some of them were detected using microscopy. Our results showed that TGGE is an excellent approach for comparative macrobenthic community studies that need parallel treatment of many samples at a time. To the best of our knowledge, this is the first time in which molecular barcoding techniques have been applied to the comparison of eukaryotic epiphytic communities.


Operational Taxonomic Unit Cetyl Trimethyl Ammonium Bromide Rhodophyta Taxon Richness Posidonia Oceanica 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We are very grateful to the Marine Microbiology Group of IMEDEA for facilities and A. Gómez-Garreta and members of ‘Departament de Productes Naturals, Biología Vegetal y Edafología’ of University of Barcelona (UB) for their help with macroalgal epiphyte taxonomy. This study was funded by Ministerio de Educación y Ciencia (Acción Complementaria CTM2005-23775-E), Govern de les Illes Balears (Acció Especial de recerca, desenvolupament tecnològic i innovació, year 2006) and CSIC (Proyectos Intramurales Frontera 200530F0033 and 200530F0182). F.J.M.P would like to thank ‘Conselleria de Economia, Hisenda e Innovació’ of Balearic Government for a PhD grant (FPI05) that supported this work. All experiments done in this study comply with the current laws of Spain.


  1. Acinas SG, Klepac-Ceraj V, Hunt DE, Pharino C, Ceraj I, Distel DL et al (2004) Fine-scale phylogenetic architecture of a complex bacterial community. Nature 430(6999):551–554. doi: 10.1038/nature02649 CrossRefGoogle Scholar
  2. Antolic B (1986) Epiphytic flora on leaves of Posidonia oceanica (L.) Delile from the area of Dubrovnik (South Adriatic). Acta Adriat 27(1-2):37–49Google Scholar
  3. Ballesteros E (1987) Estructura i dinàmica del poblament algal de les fulles de Posidonia oceanica (L.) Delile als herbeis de Tossa de Mar (Girona). Bullt Inst Cat Hist Nat 54:13–30Google Scholar
  4. Battiato A, Cinelli F, Cormaci M, Furnari G, Mazzella L (1982) Studio preliminare della macroflora epifita della Posidonia oceanica (L.) Delile di una prateria di Ischia (Golfo di Napoli) (Potamogetonaceae, Helobiae). Nat Sicil 1:15–27Google Scholar
  5. Borowitzka MA, Lavery PS, Van Keulen M (2006) Epiphytes of seagrasses. In: Larkum AWD, Orth RJ, Duarte CM (eds) Seagrasses: biology, ecology and conservation. Springer, Dordrecht, pp 441–461Google Scholar
  6. Buia MC, Cormaci M, Furnari G, Mazzella L (1989) Posidonia oceanica off Capo Passero (Sicily, Italy): leaf phenology and leaf algal epiphytic community. GIS. Posidonie Publ Fr, 2: 127–143Google Scholar
  7. Clarke KR, Gorley RN (2001) Primer (Plymouth Routines In Multivariate Ecological Research) V5: User Manual/Tutorial. Primer-E Ltd, PlymouthGoogle Scholar
  8. Dahllöf I (2002) Molecular community analysis of microbial diversity. Curr Opin Biotechnol 13:213–217. doi: 10.1016/S0958-1669(02)00314-2 CrossRefGoogle Scholar
  9. Díez B, Pedrós-Alió C, Marsh TL, Massana R (2001a) Application of denaturing gradient gel electrophoresis (DGGE) to study the diversity of marine picoeukaryotic assemblages and comparison of DGGE with other molecular techniques. Appl Environ Microbiol 67(7):2942–2951. doi: 10.1128/AEM.67.7.2942-2951.2001 CrossRefGoogle Scholar
  10. Díez B, Pedrós-Alió C, Massana R (2001b) Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing. Appl Environ Microbiol 67(7):2932–2941. doi: 10.1128/AEM.67.7.2932-2941.2001 CrossRefGoogle Scholar
  11. Duarte CM (1991) Seagrass depth limits. Aquat Bot 40(4):363–377. doi: 10.1016/0304-3770(91)90081-F CrossRefGoogle Scholar
  12. Eckenrode VK, Arnold J, Meagher RB (1985) Comparison of the nucleotide sequence of soybean 18S rRNA with the sequences of other small-subunit rRNAs. J Mol Evol 21(3):259–269. doi: 10.1007/BF02102358 CrossRefGoogle Scholar
  13. Eickbush TH, Eickbush DG (2007) Finely orchestrated movements: evolution of the ribosomal RNA genes. Genetics 175:477–485. doi: 10.1534/genetics.107.071399 CrossRefGoogle Scholar
  14. Foucher A, Wilson M (2002) Development of a polymerase chain reaction-based denaturing gradient gel electrophoresis technique to study nematode species biodiversity using the 18s rDNA gene. Mol Ecol Notes 2(1):45–48. doi: 10.1046/j.1471-8286.2002.00141.x CrossRefGoogle Scholar
  15. Furnari G, Giaccone G, Cormaci M, Alongi G, Serio D (2003) Biodiversità marina delle coste italiane. Catalogo del Macrofitobenthos. Biol Mar Medit 10(1):1–483Google Scholar
  16. Gadanho M, Sampaio JP (2004) Application of temperature gradient gel electrophoresis to the study of yeast diversity in the estuary of the Tagus river, Portugal. FEMS Yeast Res 5(3):253–261. doi: 10.1016/j.femsyr.2004.09.001 CrossRefGoogle Scholar
  17. Ganley ARD, Kobayashi T (2007) Highly efficient concerted evolution in the ribosomal DNA repeats: total rDNA repeat variation revealed by whole-genome shotgun sequence data. Genome Res 17:184–191. doi: 10.1101/gr.5457707 CrossRefGoogle Scholar
  18. Giribet G, Okusu A, Lindgren AR, Huff SW, Schrödl M, Nishiguchi MK (2006) Evidence for a clade composed of molluscs with serially repeated structures: monoplacophorans are related to chitons. Proc Natl Acad Sci USA 103(20):7723–7728CrossRefGoogle Scholar
  19. Head IM, Saunders JR, Pickup RW (1998) Microbial evolution, diversity, and ecology: a decade of ribosomal RNA analysis of uncultivated microorganisms. Microb Ecol 35(1):1–21. doi: 10.1007/s002489900056 CrossRefGoogle Scholar
  20. Heuer H, Krsek M, Baker P, Smalla K, Wellington EMH (1997) Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl Environ Microbiol 63(8):3233–3241PubMedPubMedCentralGoogle Scholar
  21. Hong YK, Sohn CH, Lee KW, Kim HG (1997) Nucleic acid extraction from seaweed tissues for polymerase chain reaction. J Mar Biotechnol 5(2–3):95–99Google Scholar
  22. Huys R, Llewelyn-Hughes J, Olson PD, Nagasawa K (2006) Small subunit rDNA and Bayesian inference reveal Pectenophilus ornatus (Copepoda incertae sedis) as highly transformed Mytilicolidae, and support assignment of Chondracanthidae and Xarifiidae to Lichomolgoidea (Cyclopoida). Biol J Linn Soc Lond 87:403–425. doi: 10.1111/j.1095-8312.2005.00579.x CrossRefGoogle Scholar
  23. Kendrick GA, Lavery PS (2001) Assessing biomass, assemblage structure and productivity of algal epiphytes on seagrasses. In: Short FT, Coles RG, Short CA (eds) Global Seagrass Research Methods. Elsevier, Amsterdam, The Netherlands, pp 199–222CrossRefGoogle Scholar
  24. Lefèvre E, Bardot C, Noel C, Carrias JF, Viscogliosi E, Amblard C et al (2007) Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake. Environ Microbiol 9(1):61–71. doi: 10.1111/j.1462-2920.2006.01111.x CrossRefGoogle Scholar
  25. Lepoint G, Havelange S, Gobert S, Bouquegneau JM (1999) Fauna vs flora contribution to the leaf epiphytes biomass in a Posidonia oceanica seagrass bed (Revellata Bay, Corsica). Hydrobiologia 394:63–67. doi: 10.1023/A:1003557303904 CrossRefGoogle Scholar
  26. Lepoint G, Jacquemart J, Bouquegneau JM, Demoulin V, Gobert S (2007) Field measurements of inorganic nitrogen uptake by epiflora components of the seagrass Posidonia oceanica (Monocotyledons, Posidoniaceae). J Phycol 43:208–218. doi: 10.1111/j.1529-8817.2007.00322.x CrossRefGoogle Scholar
  27. Ludwig W, Strunk O, Westram R, Richter L, Meier H Yadhukumar, Buchner A, Lai T, Steppi S, Jobb G, Forster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, Konig A, Liss T, Lussmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer KH (2004) ARB: a software environment for sequence data. Nucl Acids Res 32: 1363–1371. CrossRefGoogle Scholar
  28. Massana R, Guillou L, Diez B, Pedros-Alio C (2002) Unveiling the organisms behind novel eukaryotic ribosomal DNA sequences from the ocean. Appl Environ Microbiol 68(9):4554–4558. doi: 10.1128/AEM.68.9.4554-4558.2002 CrossRefGoogle Scholar
  29. Mazzella L, Scipione MB, Buia MC (1989) Spatio-temporal distribution of algal and animal communities in a Posidonia oceanica meadow. Mar Ecol Evol Perspect 10:107–129CrossRefGoogle Scholar
  30. Muyzer G (1999) DGGE/TGGE a method for identifying genes from natural ecosystems. Curr Opin Microbiol 2(3):317–322. doi: 10.1016/S1369-5274(99)80055-1 CrossRefGoogle Scholar
  31. Muyzer G, Smalla K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek 73(1):127–141. doi: 10.1023/A:1000669317571 CrossRefGoogle Scholar
  32. Nübel U, Garcia-Pichel F, Kuhl M, Muyzer G (1999) Quantifying microbial diversity: Morphotypes, 16S rRNA genes, and carotenoids of oxygenic phototrophs in microbial mats. Appl Environ Microbiol 65(2):422–430PubMedPubMedCentralGoogle Scholar
  33. Ovreas L, Forney L, Daae FL, Torsvik V (1997) Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Appl Environ Microbiol 63(9):3367–3373PubMedPubMedCentralGoogle Scholar
  34. Panayotidis P, Boudouresque CF (1981) Vegetation marine de l’Ile de Port-Cros (Parc National) XXI. Aire Minimale et patchiness de la flore epiphyte des feuilles de Posidonia oceanica. Trav Sci Parc Nation Port-Cros 7:71–84Google Scholar
  35. Passamaneck Y, Halanych KM (2006) Lophotrochozoan phylogeny assessed with LSU and SSU data: evidence of lophophorate polyphyly. Mol Phylogenet Evol 40:20–28. doi: 10.1016/j.ympev.2006.02.001 CrossRefGoogle Scholar
  36. Porebski S, Bailey LG, Baum BR (1997) Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep 15(1):8–15. doi: 10.1007/BF02772108 CrossRefGoogle Scholar
  37. Procaccini G, Buia MC, Gambi MC, Pérez M, Pergent G, Pergent-Martini C et al (2003) The seagrasses of the Western Mediterranean. In: Green EP, Short FT (eds) World Atlas of Seagrasses. UNEP World Conservation Monitoring Centre. University of California Press, Berkeley, pp 48–58Google Scholar
  38. Pruesse E, Quast C, Knittel K, Fuchs B, Ludwig W, Peplies J et al (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35(21):7188–7196. doi: 10.1093/nar/gkm864 CrossRefGoogle Scholar
  39. Regensbogenova M, Pristas P, Javorsky P, Moon-van der Staay SY, van der Staay GWM, Hackstein JHP et al (2004) Assessment of ciliates in the sheep rumen by DGGE. Lett Appl Microbiol 39(2):144–147. doi: 10.1111/j.1472-765X.2004.01542.x CrossRefGoogle Scholar
  40. Reyes J, Sansón M (1997) Temporal distribution and reproductive phenology of the epiphytes on Cymodocea nodosa leaves in the Canary Islands. Bot Mar 40(3):193–201Google Scholar
  41. Reyes J, Sansón M, Afonso-Carrillo J (1998) Distribution of the epiphytes along the leaves of Cymodocea nodosa in the Canary Islands. Bot Mar 41(6):543–551Google Scholar
  42. Romero J (1988) Epífitos de las hojas de Posidonia oceanica: variaciones estacionales y batimétricas de biomasa en la pradera de las islas Medes (Girona). Oecol Aquat 9:19–25Google Scholar
  43. Rosselló-Mora R, Amann R (2001) The species concept for prokaryotes. FEMS Microbiol Rev 25(1):39–67CrossRefGoogle Scholar
  44. Rosselló-Mora R, López-López A (2008) The least common denominator—species or OTU’s. In: Zengler K (ed) Accessing uncultivated microorganisms. From the environment to organisms and genomes and back. ASM press, WashingtonGoogle Scholar
  45. Rota E, Martin P, Erséus C (2001) Soil-dwelling polychaetes: enigmatic as ever? Some hints on their phylogenetic relationships as suggested by a maximum parsimony analysis of 18S rRNA gene sequences. Contrib Zool 70(3):127–138Google Scholar
  46. Rowan R, Powers DA (1991a) A molecular genetic identification of zooxanthellae and the evolution of animal-algal symbioses. Science 251:1348–1351. doi: 10.1126/science.251.4999.1348 CrossRefGoogle Scholar
  47. Rowan R, Powers DA (1991b) Molecular genetic identification of symbiotic dinoflagellates (zooxanthellae). Mar Ecol Prog Ser 71:65–73. doi: 10.3354/meps071065 CrossRefGoogle Scholar
  48. Sambrook J, Russell DW (2001) In vitro amplification of DNA by PCR. Molecular cloning: a laboratory manual, 3rd edn. vol II, Chapter 3, Section 8.1 Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  49. Sequencher v 4.7 (2006) Gene Codes Corporation, Ann Arbor, Michigan.
  50. Science SPSS (2002) SigmaPlot 8.0 User’s Guide, USA.
  51. StatSoft Inc (2005) STATISTICA (data analysis software system), version 7.1.
  52. Struck TH, Purschke G, Halanych KM (2005) A scaleless scale worm: Molecular evidence for the phylogenetic placement of Pisione remota (Pisionidae, Annelida). Mar Biol Res 1(4):243–253. doi: 10.1080/17451000500261951 CrossRefGoogle Scholar
  53. Trautman DA, Borowitzka MA (1999) Distribution of the epiphytic organisms on Posidonia australis and P.sinuosa, two seagrasses with differing leaf morphology. Mar Ecol Prog Ser 179:215–229. doi: 10.3354/meps179215 CrossRefGoogle Scholar
  54. Tsirika A, Skoufas G, Haritonidis S (2007) Seasonal and bathymetric variations in epiphytic macroflora on Posidonia oceanica (L.) Delile leaves in the National Marine Park of Zakynthos (Greece). Mar Ecol (Berl) 28(Suppl 1):146–153CrossRefGoogle Scholar
  55. Uku J, Bjork M, Bergman B, Diez B (2007) Characterization and comparison of prokaryotic epiphytes associated with three east african seagrasses. J Phycol 43(4):768–779. doi: 10.1111/j.1529-8817.2007.00371.x CrossRefGoogle Scholar
  56. Van der Ben D (1971) Les épiphytes des feuilles de Posidonia oceanica Delile sur les côtes françaises de la Méditerranée. Mem Inst R Sci Nat Belg 168:1–101Google Scholar
  57. Van Dillewijn P, Villadas PJ, Toro N (2002) Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa. Appl Environ Microbiol 68(9):4201–4208. doi: 10.1128/AEM.68.9.4201-4208.2002 CrossRefGoogle Scholar
  58. Vanderklift MA, Lavery PS (2000) Patchiness in assemblages of epiphytic macroalgae on Posidonia coriacea at a hierarchy of spatial scales. Mar Ecol Prog Ser 192:127–135. doi: 10.3354/meps192127 CrossRefGoogle Scholar
  59. Vidal R, Meneses I, Smith M (2002) Enhanced DNA extraction and PCR amplification of SSU ribosomal genes from crustose coralline algae. J Appl Phycol 14(3):223–227. doi: 10.1023/A:1019975409640 CrossRefGoogle Scholar
  60. Zhou JZ, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62(2):316–322PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • F. J. Medina-Pons
    • 1
    Email author
  • J. Terrados
    • 1
  • R. Rosselló-Móra
    • 1
  1. 1.Institut Mediterrani d’Estudis Avançats (CSIC-UIB) Miquel MarquésEsporles, MajorcaSpain

Personalised recommendations