Toward the Monophyly of Haeckel's Radiolaria: 18S rRNA Environmental Data Support the Sisterhood of Polycystinea and Acantharea

Purificación López-García, Francisco Rodríguez-Valera and David Moreira

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 (1872–1876) of the British oceanographic corvette H.M.S. Challenger (Haeckel 1887Citation ). 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 1983Citation ). This led some contemporary authors to classify, as Haeckel did one century ago, Acantharea, Polycystinea, and Phaeodarea within a common group (Cavalier-Smith 1987Citation ). 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 1997Citation ). 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 gene–based diversity of small protists (0.2–5 µm fraction) inhabiting deep Antarctic waters (250–3,000 m depth), we retrieved a sequence close to the Acantharea (DH147-EKD17, see fig. 1 ) from 2,000 m depth (López-García et al. 2001Citation ). 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 2001Citation ). To complement our previous PCR-based study avoiding possible amplification-induced biases (Wintzingerode, Göbel, and Stackerbrandt 1997Citation ; Polz and Cavanaugh 1998Citation ), 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.



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Fig. 1.—Maximum likelihood (ML) phylogenetic tree of all available Acantharea- and Polycystinea-related 18S rRNA sequences. Two new sequences, HA2 and KW16 (grey branches), were obtained from an environmental genomic DNA library of 500 m depth Antarctic plankton. OLI sequences and DH147-EKD17 are PCR-amplified marine environmental sequences from 75 and 2,000 m depth, respectively (López-García et al. 2001, Moon-van der Staay, De Wachter, and Vaulot 2001Citation ). Eight sequences from heterokonts, alveolates, plants, and fungi were used as the outgroup (grey triangle). The tree was constructed using a heuristic search (options -q -n 2500) with the MOLPHY 2.3 package (Adachi and Hasegawa 1996Citation ) and the branch lengths for the best tree were recalculated using a {Gamma} law (eight rate classes + invariant sites, {alpha} = 1.06) with the program PUZZLE (Strimmer and von Haeseler 1996Citation ). Numbers at the nodes are ML bootstrap values estimated with the RELL method (Kishino, Miyata, and Hasegawa 1990Citation ) upon the 2,500 top-ranking trees using the MOLPHY 2.3 package. Bootstrap values concerning the position of the two new sequences HA2 and KW16 are in bold. The scale bar corresponds to 10 substitutions per 100 positions for a unit branch length

 
Our environmental genomic library was constructed using 3.5 µg of DNA. DNA was mechanically broken by intense shearing and fractionated in a 0.8% low melting point agarose gel. The band corresponding to 35–45 kb DNA fragments was cut off and digested with gelase. After concentration, the DNA was cloned in the 8,179-bp cosmid vector of the pWEB Cloning System (Epicentre Technologies). A total of 6,107 cosmid clones were PCR-screened for the presence of eukaryotic 18S rRNA genes using the eukaryotic specific primers EK-82F (GAAACTGCGAATGGCTC) and EK-1520R (CYGCAGGTTCACCTAC). Three cosmids were positive, and the respective 18S rRNA genes were completely sequenced with previously described internal primers (López-García et al. 2001Citation ). One of them corresponded to an alveolate sequence belonging to the recently described marine Alveolate Group I (López-García et al. 2001Citation ) (not shown). The other two were related to Radiolaria: exhaustive phylogenetic reconstruction using all the available 18S rRNA sequences showed that HA2 branched at the base of the Polycystinea and KW16 at the base of the Acantharea, both with strong statistical support (fig. 1 ).

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 1997Citation ; Cavalier-Smith 1999Citation ). In fact, Polycystinea 18S rRNA sequences display very long branches and a long basal, unbroken, branch (Zettler, Anderson, and Caron 1999Citation ). 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 1978Citation ). 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 1997Citation ) (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 1993Citation ] 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 1993Citation ), 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 1990Citation ), 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 {Gamma} law [Yang 1996Citation ]) 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 1997Citation ). 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).



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Fig. 2.—ML phylogenetic tree of eukaryotic 18S rRNA sequences showing the monophyly of Radiolaria. The tree was obtained by comparing 1,250 unambiguously aligned positions, applying the method described in figure 1 with an {alpha} parameter of 0.39. Bootstrap values are shown only for the radiolarian nodes. For the internal nodes, only ML bootstrap values are indicated. For the node concerning the monophyly of Acantharea and Polycystinea, bootstrap values correspond from top to bottom to: ML, ML applying a {Gamma} law, maximum parsimony (MP), and Neighbor-Joining (NJ). Bootstraps were calculated as described in figure 1 legend for ML values and, for the rest, from 100 replicates using the programs PUZZLEBOOT (Holder and Roger 1999Citation ) and PUZZLE (Strimmer and von Haeseler 1996Citation ) ({Gamma} law-ML), and from 1,000 replicates using PAUP 3.1 (Swofford 1993Citation ) (MP), and the MUST package (Philippe 1993Citation ) (NJ). Triangles correspond to groups of two sequences. The scale bar corresponds to 5 substitutions per 100 positions for a unit branch length

 
As our sequences were obtained directly from environmental samples, without classical identification and characterization of the corresponding microorganisms, it could be argued that they do not belong to the true Radiolaria, so our results are not an evidence for the monophyly of this group. Nevertheless, given the topology of the phylogenetic tree obtained, a simple application of basic cladistic rules strongly suggests that these new organisms should display several radiolarian features, in particular the possession of central capsule and axopodial extracapsular network, and perhaps even the ability to secrete SrSO4. We cannot exclude the possibility of HA2 being a member of the Phaeodarea, given that sequences from known species of this group are not available. However, this does not invalidate our conclusion, as Polycystinea and Acantharea would still be members of a same monophyletic group. Furthermore, if HA2 turns out to be a phaeodarean, this would imply the monophyly of Haeckel's Radiolaria sensu stricto.

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 2001Citation ). 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. 2000Citation ; Moreira, Le Guyader, and Philippe 2000Citation ), and the use of representative slow-evolving sequences (Aguinaldo et al. 1997Citation ). 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 1997Citation ) but, instead, a fast-evolving branch emerging together with Acantharea in the apical region of the eukaryotic tree (the so-called crown [Knoll 1992Citation ]). Polycystinea have also, within Radiolaria, left a vast fossil record (Anderson 1983Citation ). 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 1990Citation ). 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 Back

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 . Back

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Accepted for publication August 27, 2001.