Continued evolutionary surprises among dinoflagellates.

I t is well established that chloroplasts in green and red algae are derived from a primary endosymbiotic event between a cyanobacterium and a eukaryotic organism 1 billion years ago (Fig. 1; refs. 1 and 2). Although these two groups account for many of the world’s photosynthetic species, most other major taxonomic groups of photosynthetic organisms (stramenopiles—including diatoms, phaeophytes, chrysophytes—and haptophytes) have plastids derived from a photosynthetic eukaryote implying a secondary endosymbiosis (1, 2). Still other groups, such as the dinoflagellates, have more complicated associations believed to be derived from tertiary endosymbioses involving the engulfment of a secondary endosymbiont. Each endosymbiotic event has characteristic structural changes associated with it, the most notable of which is the addition of two membranes surrounding the plastid (the inner representing the cell membrane of the engulfed organism and the outer representing the phagocytosis vacuole membrane) (2). Dinoflagellates, although believed to be tertiary endosymbionts, have only 3 membranes surrounding their plastids (1, 2), suggesting that the acquisition of too many membranes may be functionally unstable and can cause some to be lost. Dinoflagellates are fascinating organisms that have intrigued researchers for many years. They are most well known for toxic blooms associated with red tides and symbiotic relationships with corals (zooxanthellae) (2). They contain an astounding array of unique features that has been the impetus for continued evolutionary studies. One is their close phylogenetic link with apicomplexans, organisms that are best known for causing some of our most deadly infectious diseases (3, 4). Another is the diverse array of light harvesting pigments within the group. Peridinin is a xanthophyll found exclusively in dinoflagellates and, together with chl a (ubiquitous among photosynthetic organisms), makes up the light harvesting complex found in most species. Dinoflagellates with other combinations of plastid pigments are also known, including chl b (also in green algae), fucoxanthin, chl c1 and c2 (also in stramenopiles and haptophytes) and chl c1 and phycobilins (also in cryptophytes), and are believed to be the products of further endosymbioses with species from those groups (5–8). In this issue of PNAS, Yoon et al. (9) provide startling new evidence that implicates dinof lagellate plastids containing fucoxanthin and chl c1 and c2 (derived from a haptophyte ancestor) as being ancestral to those with peridinin. This new paradigm in the relationship of these species forces us to rethink many aspects of dinoflagellate evolution. In many respects, the findings by Yoon et al. (9) allow a more parsimonious view of dinof lagellate evolution. Previously long-held theories suggested that primitive dinoflagellates were heterotrophic and the addition of a peridinin-containing plastid occurred relatively recently (10), which was borne out by the fact that approximately 50% of dinoflagellate species are heterotrophic (1) and that many photosynthetic dinoflagellates have retained heterotrophic behavior (11). However, recent studies have demonstrated that photosynthetic dinoflagellates are clearly ancestral, and that heterotrophy has been independently derived numerous times within the group (8). Fucoxanthin and chl c1 and c2 are the predominant form of light harvesting pigments among stramenopiles and haptophytes. With the finding that haptophytes are the sister group to dinoflagellates in phylogenetic analyses and that dinoflagellates with fucoxanthin and chl c1 and c2 are apparently ancestral to the peridinin-containing species, there is no need to hypothesize an independent origin of these pigments nor a later endosymbiosis of a haptophyte in dinoflagellates. Selection of the proper gene for examination of deep phylogenetic relationships was critical for carrying out this study. The gene encoding the carbon assimilation protein Rubisco, rbcL, has frequently been used to examine deep phylogenetic relationships (12–14). However, recent studies have demonstrated that there are several problems associated with using rbcL for this purpose. First, there appears to have been rampant horizontal gene transfer among various algal groups (14, 15). Reliance on such a gene to determine potential endosymbiotic relations would be fraught with potential misinterpretations. Second, peridinin-containing dinoflagellates have a nuclear encoded form II Rubisco (L2 structure rather than the L8S8 structure typical of most eukarotic plastids) that is similar in sequence homology to anaerobic bacteria (16–18). Third, multiple copies of Rubisco have been found in some organisms (14). Although identical or nearly identical copies of photosynthesis related genes are often found in cyanobacteria (19–21), both form I and form II Rubisco have been found in some species. The genes chosen by Yoon et al. (9) were psaA (encoding the photosystem I P700 apoprotein A1) and psbA (encoding the photosystem II thylacoid protein D1). These genes are intimately tied to the photosystem reaction centers and have a low rate of base substitution (nonsynonomous replacement rate KA 0.022 and 0.009 for psaA and psbA, respectively; ref. 22) compared with other genes such as rbcL (KA 0.041, considered slowly evolving; ref. 22), so that significant phylogenetic signal should be expected even at these deep evolutionary positions. The resulting phylogenies were rigorously examined using various positions of the protein codons, amino acid sequences, and