Accessible Requires Authentication Published by De Gruyter September 4, 2014

Scaly green flagellates from Spanish Atlantic coastal waters: molecular, ultrastructural and pigment analyses

Aitor Alonso-González, Emma Orive, Helena David, Koldo García-Etxebarria, José Luis Garrido, Aitor Laza-Martínez and Sergio Seoane
From the journal Botanica Marina

Abstract

Seven scale-bearing species of prasinophyceans (Chlorophyta) were studied in light and electron microscopy, pigment analysis, and molecular analysis of the small subunit (SSU) rDNA. Unialgal cultures were obtained from samples collected in the Nervión River estuary, Gulf of Biscay, Spanish Atlantic coast. Five of the species belong to the genus Pyramimonas (Prasinophyceae, Pyramimonaceae) and one each to the genera Mamiella (Mamiellophyceae, Mamiellaceae) and Nephroselmis (Nephroselmidophyceae, Nephroselmidaceae). The morphological features of all the strains analyzed agreed closely with the phylogenetic analysis, which in the case of the genus Pyramimonas presented several clusters corresponding to the subgenera Vestigifera (Pyramimonas orientalis, Pyramimonas moestrupii), Punctatae (Pyramimonas robusta), Pyramimonas (Pyramimonas propulsa), and Trichocystis (Pyramimonas grossii). Even though the phylogenetic relationship among these subgenera remains unclear, the results suggested that the Punctatae should remain an independent clade at least until more sequences or genes are analyzed. According to their accessory pigment composition, three groups were distinguished after their pigment ratios were calculated: prasinoxanthin-containing (Mamiella gilva), loroxanthin ester-containing (P. grossii and P. moestrupii), and siphonaxanthin-containing strains (Nephroselmis pyriformis and remaining Pyramimonas species). A high intraspecific variability was found, which highlights the need of an in-depth analysis and multiple technique approach to ensure accurate identification of nanoplanktonic microalgae.


Corresponding author: Emma Orive, Faculty of Science and Technology, Department of Plant Biology and Ecology, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain, e-mail:

Acknowledgments

Alex Diez, Ricardo Andrade, and Jonh Arluzea helped in taking photographs with the electron microscope; Manuel Zapata, who, we are deeply sorry to say, passed away during this work, helped with the pigment analysis, a subject that he honored with his passionate research work throughout his career. Financial support for this research was provided by the Basque Government (project IT-481-10) and the Spanish Ministry of Economy and Competitiveness (projects CGL2010-19016 and GIC07/111-417-07) and the former Ministry of Education and Science (project CMT2006-04570/MAR). Grants from the University of the Basque Country (UPV/EHU) to H. David and from the Ministry of Education and Science to A. Alonso-González also supported this study. TEM work and genetic sequencing were performed in the unit of Analytical and High-Resolution Microscopy in Biomedicine, and the Sequencing and Genotyping Unit from SGIker services of the UPV/EHU, respectively.

Funding: Spanish Ministry of Economy and Competitiveness, (Grant/Award Number: ‘projects CGL2010-19016 and GIC07/111-417-07’), Spanish Ministry of Education and Science, (Grant/Award Number: ‘CMT2006-04570/MAR’), University of the Basque Country (UPV/EHU).

References

Aken, M.E. and R.N. Pienaar. 1981. Observations on the reproduction of Pyramimonas parkeae Norris et Pearson (Prasinophyceae). S. Afr. J. Sci. 77: 330–331. Search in Google Scholar

Bell, T.G. 2008. A taxonomic study of Nephroselmis Stein (Prasinophyceae, Chlorophyta). MSc Thesis, University of Witwatersrand, Johannesburg. pp. 149. Search in Google Scholar

Bérard-Therriault, L., M. Poulin and L. Bossé. 1999. Guide d’identification du phytoplancton marin de l’estuaire et du golfe du Saint-Laurent incluant également certains protozoaires.Publ. Spéc. Can. Sci. Halieut. Aquat. 128: 1–387. Search in Google Scholar

Bergesch, M., C. Odebrecht and Ø. Moestrup. 2008. Nanoflagellates from coastal waters of southern Brazil (32°S). Bot. Mar. 51: 35–50. Search in Google Scholar

Butcher, R.W. 1959. An introductory account of the smaller algae of British coastal waters. I. Introduction and Chlorophyceae. Fish. Invest. Lond. Ser.4: 1–74. Search in Google Scholar

Coleman, A.W. 1983. The use of fluorescence microscopy to study cell structure, nuclear ploidy, cell cycles and plastid DNA. In: (C.K. Tseng, ed) Proceedings of the joint US-China phycology symposium. Science Press, Beijing. pp. 291–310. Search in Google Scholar

Daugbjerg, N. 2000. Pyramimonas tychotreta, sp. nov. (Prasinophyceae), a new marine species from Antarctica: light and electron microscopy of the motile stage and notes on growth rates. J. Phycol.36: 160–171. Search in Google Scholar

Daugbjerg, N. and Ø. Moestrup. 1992. Fine structure of Pyramimonas cyclotreta sp. nov. (Prasinophyceae) from Northern Foxe Basin, Arctic Canada, with some observations on growth rates. Eur. J. Protistol. 28: 288–298. Search in Google Scholar

Daugbjerg, N. and Ø. Moestrup. 1993. Four new species of Pyramimonas (Prasinophyceae) from arctic Canada including a light and electron microscopic description of Pyramimonas quadrifolia sp. nov. Eur. J. Phycol.28: 3–16. Search in Google Scholar

Daugbjerg, N., Ø. Moestrup and P. Arctander. 1994. Phylogeny of the genus Pyramimonas (Prasinophyceae, Chlorophyta) inferred from the rbcL gene. J. Phycol. 30: 991–999. Search in Google Scholar

Egeland, E.S., R.R.L. Guillard and S. Liaaen-Jensen. 1997. Additional carotenoid prototype representatives and a general chemosystematic evaluation of carotenoids in Prasinophyceae (Chlorophyta). Phytochemistry 44: 1087–1097. Search in Google Scholar

Egeland, E.S., J.L. Garrido, L. Clementson, K. Andresen, C.S. Thomas, M. Zapata, R. Airs, C.A. Llewellyn, G.L. Newman, F. Rodríguez and S. Roy. 2011. Data sheets aiding identification of phytoplankton carotenoids and chlorophylls. In: (S. Roy, C.A. Llewellyn, E.S. Egeland and G. Johnsen, eds) Phytoplankton pigments: characterization, chemotaxonomy and applications in oceanography. Cambridge University Press, London. pp. 665–822. Search in Google Scholar

Faria, D.G., A. Kato, M.R. de la Peña and S. Suda. 2011. Taxonomy and phylogeny of Nephroselmis clavistella sp. nov. (Nephroselmidophyceae, Chlorophyta). J. Phycol. 47: 1388–1396. Search in Google Scholar

Faria, D.G., A. Kato and S. Suda. 2012. Nephroselmis excentrica sp. nov. (Nephroselmidophyceae, Chlorophyta) from Okinawa-jima, Japan. Phycologia 51: 271–282. Search in Google Scholar

García-Barcina, J.M., J.A. González-Oreja and A. de la Sota. 2006. Assessing the improvement of the Bilbao estuary water quality in response to pollution abatement measures. Water Research40: 951–960. Search in Google Scholar

Garmendia, M., M. Revilla, J. Bald, J. Franco, A. Laza-Martínez, E. Orive, S. Seoane, V. Valencia and A. Borja. 2011. Phytoplankton communities and biomass size structure (fractionated chlorophyll “a”), along trophic gradients of the Basque coast (northern Spain). Biogeochemistry 106: 243–263. Search in Google Scholar

Garrido, J.L., F. Rodríguez and M. Zapata. 2009. Occurrence of loroxanthin, loroxanthin decenoate, and loroxanthin dodecenoate in Tetraselmis species (Prasinophyceae, Chlorophyta). J. Phycol. 45: 366–374. Search in Google Scholar

Granéli, E. and M.O. Moreira. 1990. Effects of river water of different origin on the growth of marine dinoflagellates and diatoms in laboratory cultures. J. Exp. Mar. Biol. Ecol. 136: 89–106. Search in Google Scholar

Guillard, R.R.L. and J.H. Ryther. 1962. Studies of marine planktonic diatoms: I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can. J. Microbiol.8: 229–239. Search in Google Scholar

Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41: 95–98. Search in Google Scholar

Hallegraeff, G.M., C.J.S. Bolch, D.R.A. Hill, I. Jameson, J.-M. LeRoi, A. McMinn, S. Murray, M.F. de Salas and K. Saunders. 2010. Algae of Australia: phytoplankton of temperate coastal waters. Australian Biological Resources Study, Canberra and CSIRO Publishing, Melbourne. pp. 432. Search in Google Scholar

Hargraves, P.E. and W.E. Gardiner. 1980. The life history of Pyramimonas amylifera Conrad (Prasinophyceae). J. Plankton Res. 2: 99–108. Search in Google Scholar

Ianson, D., S. Pond and T. Parsons. 2001. The spring phytoplankton bloom in the coastal temperate ocean: growth criteria and seeding from shallow embayments. J. Oceanogr. 57: 723–734. Search in Google Scholar

Inouye, I. and T. Horiguchi. 1982. Notes on microalgae in Japan (6). Pyramimonas aff. amylifera Conrad (Prasinophyceae). Jap. J. Phycol. 30: 154. Search in Google Scholar

Inouye, I. and R.N. Pienaar. 1984. Light and electron microscope observations on Nephroselmis astigmatica sp. nov. (Prasinophyceae). Nord. J. Bot. 4: 409–423. Search in Google Scholar

Inouye, I., T. Hori and M. Chihara. 1985. Ultrastructural characters of Pyramimonas (Prasinophyceae) and their possible relevance in taxonomy. In: (H. Hara, ed) Origin and evolution of diversity in plants and plant communities. Academia Scientific Book Inc., Tokyo. pp. 314–327. Search in Google Scholar

Katoh, K. and H. Toh. 2008. Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform.9: 286–298. Search in Google Scholar

Latasa, M., K. van Lenning, J.L. Garrido, R. Scharek, M. Estrada, F. Rodríguez and M. Zapata. 2001. Losses of chlorophylls and carotenoids in aqueous acetone and methanol extracts prepared for RPHPLC analysis of pigments. Chromatographia 53: 385–391. Search in Google Scholar

Latasa, M., R. Scharek, F. Le Gall and L. Guillou. 2004. Pigment suites and taxonomic groups in Prasinophyceae. J. Phycol.40: 1149–1155. Search in Google Scholar

Laza-Martínez, A. 2012. Urgorri complanatus gen. et sp. nov. (Cryptophyceae), a red-tide-forming species in brackish waters. J. Phycol. 48: 423–435. Search in Google Scholar

Laza-Martínez, A., S. Seoane, M. Zapata and E. Orive. 2007. Phytoplankton pigment patterns in a temperate estuary: from unialgal cultures to natural assemblages. J. Plankton Res.29: 913–929. Search in Google Scholar

Laza-Martínez, A., J. Arluzea, I. Miguel and E. Orive. 2012. Morphological and molecular characterization of Teleaulax gracilis sp. nov. and T. minuta sp. nov. (Cryptophyceae). Phycologia 51: 649–661. Search in Google Scholar

LeRoi, J.-M. and G.M. Hallegraeff. 2006. Scale-bearing nanoflagellates from southern Tasmanian coastal waters, Australia. II. Species of Chrysophyceae (Chrysophyta), Prymnesiophyceae (Haptophyta, excluding Chrysochromulina) and Prasinophyceae (Chlorophyta). Bot. Mar.49: 216–235. Search in Google Scholar

Manton, I. 1969. Tubular trichocysts in a species of Pyramimonas (P. grossii Parke). Österr. Bot. Z. 116: 378–392. Search in Google Scholar

Manton, I., K. Oates and M. Parke. 1963. Observations on the fine structure of the Pyramimonas stage of Halosphaera and preliminary observations on three species of Pyramimonas. J. Mar. Biol. Ass. UK43: 225–238. Search in Google Scholar

Marin, B. and M. Melkonian. 1994. Flagellar hairs in prasinophytes (Chlorophyta): ultrastructure and distribution on the flagellar surface. J. Phycol.30: 659–678. Search in Google Scholar

Marin, B. and M. Melkonian. 2010. Molecular phylogeny and classification of the Mamiellophyceae class. nov. (Chlorophyta) based on sequence comparisons of the nuclear- and plastid-encoded rRNA operons. Protist 161: 304–336. Search in Google Scholar

McFadden, G.I., Ø. Moestrup and R. Wetherbee. 1982. Pyramimonas gelidicola sp. nov. (Prasinophyceae), a new species isolated from Antarctic sea ice. Phycologia21: 103–111. Search in Google Scholar

McFadden, G.I., D.R.A. Hill and R. Wetherbee. 1986. A study of the genus Pyramimonas (Prasinophyceae) from southeastern Australia. Nord. J. Bot.6: 209–234. Search in Google Scholar

McFadden, G.I., D.R.A. Hill and R. Wetherbee. 1987. Electron microscopic observations on Pyramimonas olivacea N. Carter (Prasinophyceae, Chlorophyta). Phycologia26: 322–327. Search in Google Scholar

Moestrup, Ø. 1983. Further studies on Nephroselmis and its allies (Prasinophyceae). I. The question of the genus Bipedinomonas. Nord. J. Bot.3: 609–627. Search in Google Scholar

Moestrup, Ø. 1984. Further studies on Nephroselmis and its allies (Prasinophyceae). II. Mamiella gen. nov., Mamiellaceae fam. nov., Mamiellales ord. nov. Nord. J. Bot.4: 109–121. Search in Google Scholar

Moestrup, Ø. and D.R.A. Hill. 1991. Studies on the genus Pyramimonas (Prasinophyceae) from Australian and European waters: P. propulsa sp. nov. and P. mitra sp. nov. Phycologia30: 534–546. Search in Google Scholar

Moestrup, Ø. and H.A. Thomsen. 1974. An ultrastructural study of the flagellate Pyramimonas orientalis with particular emphasis on Golgi apparatus activity and the flagellar apparatus. Protoplasma81: 247–269. Search in Google Scholar

Moestrup, Ø., T. Hori and A. Kristiansen. 1987. Fine structure of Pyramimonas octopus sp. nov., an octoflagellated benthic species of Pyramimonas (Prasinophyceae), with some observations on its ecology. Nord. J. Bot. 7: 339–352. Search in Google Scholar

Moro, I., N. La Rocca, L.D. Valle, E. Moschin, E. Negrisolo and C. Andreoli. 2002. Pyramimonas australis sp. nov. (Prasinophyceae, Chlorophyta) from Antarctica: fine structure and molecular phylogeny. Eur. J. Phycol.37: 103–114. Search in Google Scholar

Nakayama, T., S. Watanabe, K. Mitsui, H. Uchida and I. Inouye. 1996. The phylogenetic relationship between the Chlamydomonadales and Chlorococcales inferred from 18S rDNA sequence data. Phycol. Res. 44: 47–55. Search in Google Scholar

Nakayama, T., S. Suda, M. Kawachi and I. Inouye. 2007. Phylogeny and ultrastructure of Nephroselmis and Pseudoscourfieldia (Chlorophyta), including the description of Nephroselmis anterostigmatica sp. nov. and a proposal for the Nephroselmidales ord. nov. Phycologia46: 680–697. Search in Google Scholar

Norris, R.E. 1964. Studies on phytoplankton in Wellington Harbour. N. Z. J. Bot. 2: 258–278. Search in Google Scholar

Orive, E., J. Franco, I. Madariaga and M. Revilla. 2004. Bacterioplankton and phytoplankton communities. In: (A. Borja and M. Collins, eds) Oceanography and marine environment of the Basque Country. Elsevier, Amsterdam. pp. 367–393. Search in Google Scholar

Orive, E., A. Laza-Martínez, S. Seoane, A. Alonso, R. Andrade and I. Miguel. 2010. Diversity of Pseudo-nitzschia in the southeastern Bay of Biscay. Diatom Res.25: 125–145. Search in Google Scholar

Parke, M. 1949. Studies on marine flagellates. J. Mar. Biol. Ass. UK28: 255–288. Search in Google Scholar

Parke, M. and D.G. Rayns. 1964. Studies on marine flagellates VII. Nephroselmis gilva sp. nov. and some allied forms. J. Mar. Biol. Ass. UK44: 209–217. Search in Google Scholar

Pennick, N.C. 1978. Studies on external morphology of Pyramimonas 5. P. amylifera Conrad. Arch. Protistenk.120: 142–147. Search in Google Scholar

Pennick, N.C. 1984. Comparative ultrastructure and occurrence of scales in Pyramimonas (Chlorophyta, Prasinophyceae). Arch. Protistenk.128: 3–11. Search in Google Scholar

Pennick, N.C. and K.J. Clarke. 1976. Studies on the external morphology of Pyramimonas 3. Pyramimonas grossii Parke. Arch. Protistenk. 118: 285–290. Search in Google Scholar

Pienaar, R.N. 1978. The ultrastructure of the scale bearing flagellate Pyramimonas aff. amylifera Conrad (Prasinophyceae). Proc. S. Afr. Electron Microsc. Soc.8: 75–76. Search in Google Scholar

Pienaar, R.N. and S.D. Sym. 1997. Pyramimonas robusta sp. nov., a new punctate species, and a reappraisal of the subgenus Punctatae. Can. J. Bot.75: 1965–1977. Search in Google Scholar

Pienaar, R.N. and S.D. Sym. 2002. The genus Pyramimonas (Prasinophyceae) from southern African inshore waters. S. Afr. J. Bot.68: 283–298. Search in Google Scholar

Reize, I.B. and M. Melkonian. 1989. A new way to investigate living flagellated/ciliated cells in the light microscope: immobilization of cells in agarose. Bot. Acta102: 145–151. Search in Google Scholar

Ronquist, F. and J.P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics19: 1572–1574. Search in Google Scholar

Seoane, S., A. Laza, I. Urrutxurtu and E. Orive. 2005. Phytoplankton assemblages and their dominant pigments in the Nervion River estuary. Hydrobiologia 549: 1–13. Search in Google Scholar

Seoane, S., A. Laza and E. Orive. 2006. Monitoring phytoplankton assemblages in estuarine waters: The application of pigment analysis and microscopy to size-fractionated samples. Estuar. Coast. Shelf Sci.67: 343–354. Search in Google Scholar

Seoane, S., M. Zapata and E. Orive. 2009a. Growth rates and pigment patterns of haptophytes isolated from estuarine waters. J. Sea Res. 62: 286–294. Search in Google Scholar

Seoane, S., W. Eikrem, J. Arluzea and E. Orive. 2009b. Haptophytes of the Nervión River estuary, northern Spain. Bot. Mar. 52: 47–59. Search in Google Scholar

Seoane, S., W. Eikrem, R. Pienaar and B. Edvardsen. 2009c. Chrysochromulina palpebralis sp. nov. (Prymnesiophyceae): a haptophyte, possessing two alternative morphologies. Phycologia48: 165–176. Search in Google Scholar

Smith, P.J. and L.A. Hobson. 1994. Temporal variation in taxonomic composition of flagellated nanoplankton in a temperate fjord. J. Phycol. 30: 369–375. Search in Google Scholar

Stamatakis, A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics22: 2688–2690. Search in Google Scholar

Suda, S. 2003. Light microscopy and electron microscopy of Nephroselmis spinosa sp. nov. (Prasinophyceae, Chlorophyta). J. Phycol.39: 590–599. Search in Google Scholar

Suda, S. 2004. Taxonomic characterization of Pyramimonas aurea sp. nov. (Prasinophyceae, Chlorophyta). Phycologia43: 682–692. Search in Google Scholar

Sym, S.D. and R.N. Pienaar. 1991. Ultrastructure of Pyramimonas norrisii sp. nov. Br. Phycol. J.26: 51–66. Search in Google Scholar

Sym, S.D. and R.N. Pienaar. 1993. Further observations on Trichocystis, a subgenus of Pyramimonas (Prasinophyceae, Chlorophyta). Phycologia 32: 338–350. Search in Google Scholar

Sym, S.D. and R.N. Pienaar. 1995. Taxonomy of Pyramimonas obovata and other observations on the subgenus Vestigifera of Pyramimonas (Prasinophyceae, Chlorophyta). Phycol. Res. 43: 17–32. Search in Google Scholar

Sym, S.D. and R.N. Pienaar. 1997. Further observations on the type subgenus of Pyramimonas (Prasinophyceae), with particular reference to a new species, P. chlorina, and the flagellar apparatus of P. propulsa. Can. J. Bot. 75: 2196–2215. Search in Google Scholar

Sym, S.D. and R.N. Pienaar. 1999. An additional punctate species of Pyramimonas, P. formosa, sp. nov., and its impact on the subgenera Punctatae and Pyramimonas (Prasinophyceae, Chlorophyta). J. Phycol.35: 1313–1321. Search in Google Scholar

Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei and S. Kumar. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28: 2731–2739. Search in Google Scholar

Thomsen, H.A. and K.R. Buck. 1998. Nanoflagellates of the central California waters: taxonomy, biogeography and abundances of primitive, green flagellates (Pedinophyceae, Prasinophyceae). Deep-Sea Res. II 45: 1687–1707. Search in Google Scholar

Throndsen, J. 1978. The dilution culture method. In: (A. Sournia, ed) Phytoplankton manual. UNESCO Monographs on Oceanographic Methodology 6. Unesco, Paris. pp. 218–224. Search in Google Scholar

Throndsen, J., G.R. Hasle and K. Tangen. 2007. Phytoplankton of Norwegian coastal waters. Almater Forlag AS, Oslo. pp. 343. Search in Google Scholar

van den Hoff, J. and J.M. Ferris. 2009. Pleomorphism in the Antarctic flagellate Pyramimonas gelidicola (Prasinophyceae, Chlorophyta). Polar Res. 28: 426–432. Search in Google Scholar

Yamaguchi, H., S. Suda, T. Nakayama, R.N. Pienaar, M. Chihara and I. Inouye. 2011. Taxonomy of Nephroselmis viridis sp. nov. (Nephroselmidophyceae, Chlorophyta), a sister marine species to freshwater N. olivacea. J. Plant Res.24: 49–62. Search in Google Scholar

Zapata, M. and J.L. Garrido. 1991. Influence of injection conditions in reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids. Chromatographia 31: 589–594. Search in Google Scholar

Zapata, M., F. Rodríguez and J.L. Garrido. 2000. Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases. Mar. Ecol. Prog. Ser. 195: 29–45. Search in Google Scholar

Received: 2013-12-7
Accepted: 2014-8-5
Published Online: 2014-9-4
Published in Print: 2014-10-1

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