Université Pierre et Marie Curie, Paris
División de Microbiología, Universidad Miguel Hernández, San Juan de Alicante, Spain
In 1887, Ernst Haeckel published a monumental and amazingly illustrated monograph describing several thousand different radiolarian species, which had been collected during the 4-year journey (18721876) of the British oceanographic corvette H.M.S. Challenger (Haeckel 1887
). Radiolaria consist of diverse marine planktonic protists, mostly unicellular, usually endowed with complex, conspicuous mineral skeletons. Haeckel applied the term Radiolaria to three different groups: Acantharea, Polycystinea (Spumellaria and Nassellaria), and Phaeodarea. All of them are united by the possession of a central capsule defining an intracapsular and an extracapsular region in the cytoplasm, and some of them, (all Acantharea and several Spumellaria), also by the ability to secrete strontium sulfate (SrSO4) (Anderson 1983
). This led some contemporary authors to classify, as Haeckel did one century ago, Acantharea, Polycystinea, and Phaeodarea within a common group (Cavalier-Smith 1987
). Nevertheless, a recent study based on the phylogenetic analysis of small subunit ribosomal RNA (18S rRNA) sequences challenged this view and supported the early emergence of Polycystines before Acantharea (Zettler, Sogin, and Caron 1997
). This would render Haeckel's Radiolaria polyphyletic and inappropriate as a taxonomic entity. We present here a phylogenetic analysis, including new Polycystinea- and Acantharea-related 18S rRNA sequences retrieved from an oceanic environmental genomic library (500 m depth, Antarctic Polar Front). Our study strongly supports the monophyly of both groups, which constitutes a first piece of evidence for the legitimacy of Radiolaria sensu Haeckel as a valid taxon.
During a recent study on the 18S rRNA genebased diversity of small protists (0.25 µm fraction) inhabiting deep Antarctic waters (2503,000 m depth), we retrieved a sequence close to the Acantharea (DH147-EKD17, see fig. 1
) from 2,000 m depth (López-García et al. 2001
). Three additional acantharean environmental sequences were determined from surface waters (75 m depth) in the equatorial Pacific by other authors (Moon-van der Staay, De Wachter, and Vaulot 2001
). To complement our previous PCR-based study avoiding possible amplification-induced biases (Wintzingerode, Göbel, and Stackerbrandt 1997
; Polz and Cavanaugh 1998
), we subsequently constructed a cosmid genomic library of the 0.2- to 5-µm biomass fraction from 500-m-deep waters at the same location (59°19'S, 55°45'W). The construction of the environmental genomic libraries has a number of advantages over a direct PCR amplification of a target gene. First, the diversity retrieved is not affected by well-known PCR-related biases. Second, these kinds of libraries offer the possibility of retrieving other gene sequences, in addition to the 18S rRNA, from the same genomic fragment.
|
Besides its basal position, HA2 displays a branch significantly shorter than that of its close polycystinean relatives (fig. 1 ). The possibility that Polycystinea is a fast-evolving lineage has already been advanced (Zettler, Sogin, and Caron 1997
; Cavalier-Smith 1999
). In fact, Polycystinea 18S rRNA sequences display very long branches and a long basal, unbroken, branch (Zettler, Anderson, and Caron 1999
). Therefore, HA2 sequence was potentially very useful to break the long Polycystinea branch that, given its accelerated evolutionary rate, could be affected by phylogenetic reconstruction problems, such as the well-known long-branch attraction (LBA) artifact (Felsenstein 1978
). KW16 could represent a similar situation within the Acantharea, although these display shorter branches than the Polycystinea. A close inspection of the 18S rRNA alignment already showed that both sequences had signatures common to Polycystinea and Acantharea, a trend most remarkable for HA2 (not shown). Indeed, the addition of HA2 and KW16 sequences to the eukaryotic 18S rRNA data set had very important effects on tree reconstruction. Without them Polycystinea still branched before Acantharea, even with a larger taxonomic sampling for both groups than that used in previous studies (Zettler, Sogin, and Caron 1997
) (not shown). When incorporated into the data set, Polycystinea and Acantharea emerged as sister groups (that is, the monophyly of two of the three groups of Radiolaria was retrieved) for all distance (neighbor-joining [NJ] with the Tamura-Nei [Tamura and Nei 1993
] model of sequence evolution), maximum parsimony (MP, with heuristic search), and maximum likelihood (ML) phylogenetic reconstruction methods (see fig. 2
for a ML phylogenetic tree). The statistical support for this node, measured as bootstrap proportions (BP), was medium to very high depending on the reconstruction method employed. In fact, for the methods more prone to LBA, NJ, and MP, the BP were 58% and 79%, respectively, while for ML, generally the most robust method (Hasegawa and Fujiwara 1993
), the BP rose up to 92% (fig. 2
). This strongly suggested that LBA was responsible for an artifactual early emergence of Polycystinea in previous studies. Nevertheless, ML bootstrap values, calculated using the RELL method (Kishino, Miyata, and Hasegawa 1990
), might be overestimated. However, true bootstrap values calculated using ML with a more sophisticated and realistic model of sequence evolution (accounting for among-site rate variation with a
law [Yang 1996
]) still increased the support for the monophyly of Radiolaria, up to 96% (fig. 2
). The monophyly of Polycystinea and Acantharea appears stable as it was retrieved using different combinations of species. For instance, the inclusion of the Mastigamoeba balamuthi sequence in our analyses did not affect the tree topology or the statistical support (fig. 2
) in contrast to previous analyses (Zettler, Sogin, and Caron 1997
). In addition, when HA2 was included as the only representative for the Polycystinea group, all reconstruction methods retrieved its sisterhood to the Acantharea with a BP of 100% (not shown).
|
In any case, HA2 and KW16 have a strong phylogenetic impact, bringing together Polycystinea and Acantharea with strong statistical support. Actually, the use of environmental sequences has already proved useful to stabilize other problematic branches of the eukaryotic tree (Moreira, López-García, and Rodríguez-Valera 2001
). At present, there are three major strategies to solve these unstable regions of the eukaryotic tree, namely, the retention of good supported relationships retrieved from the analyses of different markers, the use of large multigene fusions (Baldauf et al. 2000
; Moreira, Le Guyader, and Philippe 2000
), and the use of representative slow-evolving sequences (Aguinaldo et al. 1997
). Whereas massive genome sequencing will provide the necessary data for comparative phylogeny and gene-fusion analyses, molecular ecology can be an invaluable source of slow-evolving genes.
The recovered monophyly of Polycystinea and Acantharea has important consequences. From an evolutionary point of view, Polycystinea do not appear to be an ancient lineage as previously inferred from certain phylogenetic analyses (Zettler, Sogin, and Caron 1997
) but, instead, a fast-evolving branch emerging together with Acantharea in the apical region of the eukaryotic tree (the so-called crown [Knoll 1992
]). Polycystinea have also, within Radiolaria, left a vast fossil record (Anderson 1983
). Polycystinean siliceous skeletons are easily fossilizable, in contrast to the very soluble SrSO4 skeletons of acanthareans, and have recorded the history of some environments on Earth (in particular the deep sea, as calcareous fossils are rare at very high depth) and paleoclimate (as climatic oscillations induce alterations in the morphology of the skeletons) (Steineck and Casey 1990
). Accordingly, the Radiolaria fossil record should be reinterpreted in the light of this newly validated phylogenetic framework.
Acknowledgements
This work was supported by the European MIDAS project. The Hésperides campaign He052 was financed by the Spanish Research Council (CSIC). We thank Tom Cavalier-Smith for critical reading of the manuscript. Sequences have been deposited in GenBank under accession numbers AF382824 and AF382825.
Footnotes
Geoffrey McFadden, Reviewing Editor
Keywords: Radiolaria
Haeckel
molecular ecology
18S rRNA
phylogeny
Address for correspondence and reprints: David Moreira, Université Pierre et Marie Curie, UMR 7622, 9 quai St Bernard, 75005 Paris, France. david.moreira{at}snv.jussieu.fr
.
References
Adachi J., M. Hasegawa, 1996 MOLPHY Version 2.3: programs for molecular phylogenetics based on maximum likelihood Comput. Sci. Monogr 28:1-150
Aguinaldo A. M., J. M. Turbeville, L. S. Linford, M. C. Rivera, J. R. Garey, R. A. Raff, J. A. Lake, 1997 Evidence for a clade of nematodes, arthropods and other moulting animals Nature 387:489-493[ISI][Medline]
Anderson R. O., 1983 Radiolaria Springer, New York
Baldauf S. L., A. J. Roger, I. Wenk-Siefert, W. F. Doolittle, 2000 A kingdom-level phylogeny of eukaryotes based on combined protein data Science 290:972-977
Cavalier-Smith T., 1987 The origin of eukaryotic and archaebacterial cells Ann. N. Y. Acad. Sci 503:17-54[ISI][Medline]
. 1999 Principles of protein and lipid targeting in secondary symbiogenesis: euglenoid, dinoflagellate, and sporozoan plastids origin and the eukaryote family tree J. Eukaryot. Microbiol 46:347-366[ISI]
Felsenstein J., 1978 Cases in which parsimony or compatibility methods will be positively misleading Syst. Zool 27:401-410[ISI]
Haeckel E., 1887 Report on radiolaria collected by H. M. S. Challenger during the years 18731876 Rep. Sci. Res. Voyage H.M.S. Challenger 18731876 18:1-1803
Hasegawa M., M. Fujiwara, 1993 Relative efficiencies of the maximum likelihood, maximum parsimony, and neighbor-joining methods for estimating protein phylogeny Mol. Phylogenet. Evol 2:1-5[Medline]
Holder A., M. Roger, 1999 PUZZLEBOOT Marine Biological Laboratory, Woods Hole, Mass
Kishino H., T. Miyata, M. Hasegawa, 1990 Maximum likelihood inference of protein phylogeny, and the origin of chloroplasts J. Mol. Evol 31:151-160[ISI]
Knoll A. H., 1992 The early evolution of eukaryotes: a geological perspective Science 256:622-627[ISI][Medline]
López-García P., F. Rodríguez-Valera, C. Pedr, D. Moreira, 2001 Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton Nature 409:603-607[ISI][Medline]
Moon-van der Staay S. Y., R. De Wachter, D. Vaulot, 2001 Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity Nature 409:607-610[ISI][Medline]
Moreira D., H. Le Guyader, H. Philippe, 2000 The origin of red algae and the evolution of chloroplasts Nature 405:69-72[ISI][Medline]
Moreira D., P. López-Garca, F. Rodrguez-Valera, 2001 New insights on the phylogenetic position of diplonemids: environmental sequences, GC content bias and differences of evolutionary rate Int. J. Syst. Evol. Microbiol 51:2211-2219
Philippe H., 1993 MUST, a computer package of management utilities for sequences and trees Nucleic Acids Res 21:5264-5272[Abstract]
Polz M. F., C. M. Cavanaugh, 1998 Bias in template-to-product ratios in multitemplate PCR Appl. Environ. Microbiol 64:3724-3730
Steineck P. L., R. E. Casey, 1990 Ecology and paleobiology of Foraminifera and Radiolaria Pp. 89138 in G. M. Capriulo, ed. Ecology of marine protozoa. Oxford University Press, Oxford
Strimmer K., A. von Haeseler, 1996 Quartet puzzling: a quartet maximum likelihood method for reconstructing tree topologies Mol. Biol. Evol 13:964-969
Swofford D. L., 1993 PAUP: phylogenetic analysis using parsimony Version 3.1.1. Illinois Natural History Survey, Champaign, Illinois
Tamura K., M. Nei, 1993 Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees Mol. Biol. Evol 10:512-526[Abstract]
Wintzingerode F., U. B. Göbel, E. Stackebrandt, 1997 Determination if microbial diversity in environmental samples: pitfalls of PCR-based analysis FEMS Microbiol. Rev 21:213-229[ISI][Medline]
Yang Z., 1996 Among-site rate variation and its impact on phylogenetic analyses Trends Ecol. Evol 11:367-370[ISI]
Zettler L. A., O. R. Anderson, D. A. Caron, 1999 Towards a molecular phylogeny of colonial spumellarian radiolaria Mar. Micropaleontol 36:67-79[ISI]
Zettler L. A., M. L. Sogin, D. A. Caron, 1997 Phylogenetic relationships between the Acantharea and the Polycystinea: a molecular perspective on Haeckel's Radiolaria Proc. Natl. Acad. Sci. USA 94:11411-11416