Revisiting the Phylogenetic Position of Synchroma grande

VISHWANATH PATIL,a,b,1 JON BRATE,b,c KAMRAN SHALCHIAN-TABRIZIc and KJETILL S. JAKOBSENb,c

aDepartment of Plant and Environmental Sciences, The Norwegian University of Life Sciences, P.O. Box 5003, N-1432, As, Norway, andbDepartment of Biology, Centre for Ecological and Evolutionary Synthesis (CEES), University of Oslo, P.O. Box 1006 Blindern,

0316 Oslo, Norway, andcMicrobial Evolution Research Group (MERG), Department of Biology, University of Oslo, P.O. Box 1006 Blindern, 0316 Oslo, Norway

ABSTRACT. Two new classes Synchromophyceae and Picophagea, belonging to the heterokonts, have been proposed recently in sep-arate studies of 18S rRNA phylogenies. Here we revise the 18S phylogeny of these classes by including all available sequenced speciesand applying Bayesian and maximum likelihood methods; Synchroma grande groups with the photophagotrophic Chlamydomyxa laby-rinthuloides with high statistical support. This clade is sister to Chrysophyceae, together they share a common ancestry. Our results showthat the creation of class Synchromophyceae by Horn et al. was premature, because they did not include data from the closely relatedC. labyrinthuloides and Picophagus flagellatus species. A revision of these classes should include additional species and most likelymultigene phylogenies.

Key Words. Amoeboid algae, Chlamydomyxa, chromista, heterokonts, Picophagus, 18S ribosomal RNA.

HETEROKONTS (stramenopiles) encompass highly diversegenera and are of ecological and economic importance. Sev-

eral recent studies on heterokont evolution and diversity have re-sulted in phylogenetic rearrangements within the group as well asdescriptions of numerous new genera and species (Andersen, Pot-ter, and Bailey 2002; Bongiorni et al. 2005; Cavalier-Smith andChao 2006; Eikrem et al. 2004; Kawachi, Noel, and Andersen2002a; O’Kelly 2002; Riisberg et al. 2009) and even of severalnew classes of Heterokonta (Kawachi et al. 2002b; Kawaiet al. 2003; Moriya, Nakayama, and Inouye 2002).

Horn et al. (2007) have described Synchromophyceae as a newclass of Heterokonta based on phylogenetic data (18S rRNA andrbcL) and morphological traits. However, they did not includemembers of the class Picophagea (Cavalier-Smith and Chao 2006)in their 18S rRNA analysis. Here we revise the 18S rRNA phy-logeny of the deeply diverging Chrysophyceae and their closestrelatives. We included Chlamydomyxa labyrinthuloides (Wende-roth et al. 1999) and Picophagus flagellatus (Guillou et al. 1999)in the 18S rRNA alignment from Horn et al. (2007) and analyzedthe new alignment with different phylogenetic methods includingmaximum likelihood (ML) and Bayesian analysis. The early di-verging heterokont, Protoopalina japonica (AB175929) was notincluded as the sequence was highly divergent and created noisein preliminary analysis. Two other sequences included in the Hornet al. (2007) alignment, erroneously identified as opalinids due tofungal contamination (Opalina ranarum [AF141970] and Cepe-dea virguloidea [AF141969]), were also removed. The final align-ment consisted of 82 taxa and 1,418 characters.

RESULTS AND DISCUSSION

In all the inferred phylogenetic trees, Synchroma grande wasrecovered along with C. labyrinthuloides supported by highbootstrap and posterior probability values (Bayesian posteriorprobability (PP) 5 1.0, ML bootstrap values 5 100%; Fig. 1). Fur-thermore, they were the closest relatives to the Chrysophyceaewith fairly good support suggesting a common ancestry for theselineages (Fig. 1).

Chlamydomyxa labyrinthuloides and P. flagellatus were placedin the class Picophagea by Cavalier-Smith and Chao (2006).However, our analyses of these species, as well as S. grande,show with high support that this class is paraphyletic (Fig. 1).Thus, a higher order taxonomic revision for the heterokont classesPicophagea and Synchromophyceae is needed. Some of the mor-phological characters used by Horn et al. (2007) for establishingthe new class Synchromophyceae cannot be fully justified. Oneunique feature used in the formal description of their new class isthe arrangement of chloroplasts into groups sharing an outer pairof membranes. A similar description of chloroplast grouping hasbeen previously reported for C. labyrinthuloides (Wenderoth et al.1999) and Chlamydomyxa montana (Pearlmutter and Timpano1984). Further, the lack of stomatocysts is another criterion for theSynchromophyceae (Horn et al. 2007), but lack of stomatocystshas already been used as a criterion for class Picophagea (Cava-lier-Smith and Chao 2006). Altogether, the phylogenetic analysesand similarities in morphological descriptions from the literaturedemonstrate that the amoeboid marine alga S. grande is closelyrelated to the photophagotrophic C. labyrinthuloides.

Our analysis confirms that the amoeboid algae S. grande andC. labyrinthuloides, previously assigned to Picophagea/Sync-hromophyceae, are related to the chrysophytes with the heterotro-phic P. flagellatus as their closest relative. The paraphyly of thesetaxa makes it difficult to group them into a single class. Our data

Fig. 1. Maximum likelihood (ML) tree based on 18S rRNA geneanalysis of heterokonts rooted with rhodophytes and cryptophytes. TheRAxML v. 7.0.4 program (Stamatakis 2006) with rapid hill climbing modewas run using the General Time Reversible (GTR) evolutionary modelwith a gamma distribution (G) (four discrete rate categories), and a pro-portion of invariable sites (I). The selected tree topology had the highestlikelihood score out of 100 heuristic tree searches, and the tree robustnesswas estimated by 100 bootstrap replicates, all analyses run from a randomstarting tree. Bayesian phylogenetic analyses were done with the programPhylobayes v. 2.3 (Lartillot and Philippe 2004). The program was run withthe GTR1G model and two independent Markov chains, which lasted for7,000 cycles. The log-likelihood values were used to set the burn-in and todetermine whether the two chains had converged. The largest discrepancy(maxdiff) between the two chains was o0.1 and we therefore concludedthat the two chains had converged. Shown at the branches are ML boot-strap values and Bayesian posterior probability (PP) values separated byslashes (ML/PP). PP values are only shown for branches that received MLvalues higher than 50%. Thick branches indicate maximum support inboth analyses and branching pattern not retrieved in both analyses aremarked with a hyphen (-). An asterisk (�) indicates branch length halfed.Synchromophyceae and Picophagea species are in bold.

1Present Address: Borregaard, Biorefinery R & D, P.O. Box 162,1701 Sarpsborg, Norway.

Corresponding Author: Kjetill S. Jakobsen, Department of Biology,Centre for Ecological and Evolutionary Synthesis (CEES), University ofOslo, P.O. Box 1006 Blindern, 0316 Oslo, Norway—Telephone num-ber: 147 22854601; FAX number: 147 22854001; e-mail: k.s.jakobsen@bio.uio.no

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J. Eukaryot. Microbiol., 56(4), 2009 pp. 394–396r 2009 The Author(s)Journal compilation r 2009 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2009.00389.x

395PATIL ET AL.—18S RRNA PHYLOGENY OF SYNCHROMA GRANDE

are supported by the heterokont phylogeny by Kai et al. (2008) intheir study on Aurearenophyceae classis. Although our work sug-gests revisions of the classes Synchromophyceae and Picophagea,additional data from other related species and multigene phylo-genies are likely needed to resolve this part of the heterokontphylogeny.

ACKNOWLEDGMENTS

We thank Susanne Horn for providing the alignment, the Re-search Council of Norway for funding (Project nr. 172572/S40),and the Bioportal platform (http://www.bioportal.uio.no) for pro-viding computer resources.

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Received: 04/22/08, 10/21/08, 11/25/08; accepted: 12/06/08

396 J. EUKARYOT. MICROBIOL., 56, NO. 4, JULY–AUGUST 2009