Chlorophyll c–Containing Plastid Relationships Based on Analyses of a Multigene Data Set with All Four Chromalveolate Lineages

Chlorophyll c–Containing Plastid Relationships Based on Analyses of a Multigene Data Set with All Four Chromalveolate Lineages Tsvetan R. Bachvaroff,1 M. Virginia Sanchez Puerta, and Charles F. Delwiche Department of Cell Biology and Molecular Genetics, University of Maryland Introduction Four groups of photosynthetic eukaryotes, namely, dinoflagellates, heterokonts, haptophytes, and cryptophytes, contain chlorophyll c as a major photosynthetic pigment. In recent years three of these, heterokonts, haptophytes, and cryptophytes, have been grouped together based on their common pigmentation in a phylum called Chromophyta sensu Cavalier-Smith (1986), although Christensen’s original concept of a chromophyte clade also included dinoflagellates (Christensen 1962, 1989). Classification of algae primarily on the basis of plastid pigmentation fell into disfavor when it was understood that these organelles were endosymbiotic and potentially subject to transfer. Based on plastid membrane topology and molecular phylogenies, it has become clear that the chlorophyll c– containing plastids were acquired from another eukaryote in a secondary endosymbiotic event. Moreover, morphological studies and phylogenetic analyses based on nuclear genes showed that nonphotosynthetic organisms were closely related to some of these groups (Van de Peer and De Watcher 1997; Marin, Klinberg, and Melkonian 1998; Gunderson, Goss, and Coats 1999). To reconcile these observations, it has been proposed that the four lineages including the nonphotosynthetic relatives are included in a monophyletic clade called the chromalveolates (Cavalier-Smith 1999, 2002). This hypothesis suggests that a single endosymbiotic event gave rise to the plastids of chromalveolates, after which the four distinct host lineages diverged, and the nonphotosynthetic lineages in the chromalveolates have lost photosynthesis (and in most cases, plastids). Another hypothesis would postulate four separate plastid acquisitions in the four host lineages 1 Present address: Center of Marine Biotechnology, Baltimore, MD. Key words: Chromophyte, dinoflagellate, heterokont, cryptophyte, haptophyte, plastid. E-mail: . Mol. Biol. Evol. 22(9):1772–1782. 2005 doi:10.1093/molbev/msi172 Advance Access publication May 25, 2005 Ó The Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: (Cavalier-Smith, Allsopp, and Chao 1994; Delwiche 1999). Here, we refer to ‘‘chromophyte plastids’’ as heterokont, haptophyte, and cryptophyte plastids and ‘‘chromalveolate plastids’’ or ‘‘chlorophyll c–containing plastids’’ as chromophyte plus dinoflagellate plastids. Even though there is good evidence that all four groups have plastids ultimately derived from the red algal plastid lineage (Delwiche and Palmer 1997; Durnford et al. 1999; Yoon et al. 2002), plastid relationships within the chromalveolates are still uncertain. Conflicting evidence has been reported concerning the monophyly of chlorophyll c–containing plastids (Daugbjerg and Andersen 1997; Fast et al. 2001; Ishida and Green 2002; Yoon, Hackett, and Bhattacharya 2002; Yoon et al. 2002). Unfortunately, most of previous studies have included only a subset of those four lineages and were based on analyses of one or only a few genes. Peridinin-containing dinoflagellates have been problematic because of their highly modified plastid, which, among other peculiarities, apparently only contains a few plastid genes (Barbrook and Howe 2000; Hiller 2001) with a very high rate of sequence evolution (Zhang, Green, and Cavalier-Smith 1999, 2000; Takishita et al. 2003). The haptophytes have not been thoroughly studied in this regard, and only a few genes from their plastid genome have been available for analysis. Recent evidence suggests a relationship between haptophyte and peridinin-containing dinoflagellate plastids (Yoon, Hackett, and Bhattacharya 2002). However, one of the genes used for this analysis may be biased (Inagaki et al. 2004). To address the questions of chlorophyll c–containing plastid monophyly and a specific relationship between haptophyte and dinoflagellate plastids, we undertook phylogenetic analyses designed to include all four chlorophyll c–containing plastid lineages and to make use of a number of concatenated plastid genes. We obtained chloroplast genome data from the haptophytes, a key lineage of chromalveolates, missing from most previous studies (Sanchez-Puerta, Bachvaroff, and Delwiche 2005). Ten plastid genes from representatives of each of the four The chlorophyll c–containing algae comprise four major lineages: dinoflagellates, haptophytes, heterokonts, and cryptophytes. These four lineages have sometimes been grouped together based on their pigmentation, but cytological and rRNA data had suggested that they were not a monophyletic lineage. Some molecular data support monophyly of the plastids, while other plastid and host data suggest different relationships. It is uncontroversial that these groups have all acquired plastids from another eukaryote, probably from the red algal lineage, in a secondary endosymbiotic event, but the number and sequence of such event(s) remain controversial. Understanding chlorophyll c–containing plastid relationships is a first step towards determining the number of endosymbiotic events within the chromalveolates. We report here phylogenetic analyses using 10 plastid genes with representatives of all four chromalveolate lineages. This is the first organellar genomescale analysis to include both haptophytes and dinoflagellates. Concatenated analyses support the monophyly of the chlorophyll c–containing plastids and suggest that cryptophyte plastids are the basal member of the chlorophyll c–containing plastid lineage. The gene psbA, which has at times been used for phylogenetic purposes, was found to differ from the other genes in its placement of the dinoflagellates and the haptophytes, and in its lack of support for monophyly of the green and red plastid lineages. Overall, the concatenated data are consistent with a single origin of chlorophyll c–containing plastids from red algae. However, these data cannot test several key hypothesis concerning chromalveolate host monophyly, and do not preclude the possibility of serial transfer of chlorophyll c–containing plastids among distantly related hosts. Chlorophyll c–Containing Plastid Evolution 1773 groups were analyzed in the context of other plastids, and tests were performed to evaluate support for the monophyly of the chlorophyll c–containing plastids and to understand the phylogenetic relationships among them. These 10 genes represent almost all the protein-coding genes known from peridinin-containing dinoflagellate plastids (Takishita et al. 2003; but see Laatsch et al. 2004); thus this study makes use of almost all of the presumably plastid-encoded data share by these four lineages. To av (...truncated)