Heterotachy Processes in Rhodophyte-Derived Secondhand Plastid Genes: Implications for Addressing the Origin and Evolution of Dinoflagellate Plastids

Kamran Shalchian-Tabrizi 3 Marianne Skanseng 3 Fredrik Ronquist 2 Dag Klaveness Tsvetan R. Bachvaroff 1 4 Charles F. Delwiche 4 Andreas Botnen k Torstein Tengs 0 3 Kjetill S. Jakobsen 3 0 National Veterinary Institute, Department of Microbiology , Ulleva lsveien 68, Oslo , Norway 1 Center of Marine Biotechnology , Baltimore, Maryland 2 Florida State University, Department of Biological Science 3 Centre for Ecological and Evolutionary Synthesis, Department of Biology, University of Oslo , Oslo , Norway; Evolutionary Biology Centre, Department of Systematic Zoology, Uppsala University , Uppsala , Sweden; Program for Plankton Biology, Department of Biology, University of Oslo , Oslo , Norway 4 Cell Biology and Molecular Genetics, University of Maryland , College Park Serial transfer of plastids from one eukaryotic host to another is the key process involved in evolution of secondhand plastids. Such transfers drastically change the environment of the plastids and hence the selection regimes, presumably leading to changes over time in the characteristics of plastid gene evolution and to misleading phylogenetic inferences. About half of the dinoflagellate protists species are photosynthetic and unique in harboring a diversity of plastids acquired from a wide range of eukaryotic algae. They are therefore ideal for studying evolutionary processes of plastids gained through secondary and tertiary endosymbioses. In the light of these processes, we have evaluated the origin of 2 types of dinoflagellate plastids, containing the peridinin or 19#-hexanoyloxyfucoxanthin (19#-HNOF) pigments, by inferring the phylogeny using ''covarion'' evolutionary models allowing the pattern of among-site rate variation to change over time. Our investigations of genes from secondary and tertiary plastids derived from the rhodophyte plastid lineage clearly reveal ''heterotachy'' processes characterized as stationary covarion substitution patterns and changes in proportion of variable sites across sequences. Failure to accommodate covarion-like substitution patterns can have strong effects on the plastid tree topology. Importantly, multigene analyses performed with probabilistic methods using among-site rate and covarion models of evolution conflict with proposed single origin of the peridinin- and 19#-HNOF-containing plastids, suggesting that analysis of secondhand plastids can be hampered by convergence in the evolutionary signature of the plastid DNA sequences. Another type of sequence convergence was detected at protein level involving the psaA gene. Excluding the psaA sequence from a concatenated protein alignment grouped the peridinin plastid with haptophytes, congruent with all DNA trees. Altogether, taking account of complex processes involved in the evolution of dinoflagellate plastid sequences (both at the DNA and amino acid level), we demonstrate the difficulty of excluding independent, tertiary origin for both the peridinin and 19#-HNOF plastids involving engulfment of haptophyte-like algae. In addition, the refined topologies suggest the red algal order, Porphyridales, as the endosymbiont ancestor of the secondary plastids in cryptophytes, haptophytes, and heterokonts. Introduction It is widely accepted that rhodophytes, glaucophytes, and virideplantae obtained their plastids from an association with endosymbiontic cyanobacteria (Goksyr 1967). The plastids of these algae are therefore termed primary. All other algae have probably obtained their plastids secondarily by engulfing eukaryotic algae (Delwiche 1999; Palmer 2003). In this manner, rhodophyte plastids have become incorporated into cryptophytes, heterokonts, haptophytes (defined as the chromists; Cavalier-Smith 1999) as well as into dinoflagellates and apicomplexa (CavalierSmith 1999; Fast et al. 2001; Patron et al. 2004). About half of the dinoflagellates are photosynthetic, harboring endosymbionts and plastids from several eukaryotic phyla (Watanabe et al. 1990; Chesnick et al. 1997; Tengs et al. 2000; Takishita et al. 2002). The vast majority of the dinoflagellate plastids use chlorophyll c and the pigment peridinin, usually regarded as the ancestral plastid form (Hoek et al. 1995; Saunders et al. 1997; Saldarriaga et al. 2001). The other small groups of dinoflagellates with aberrant pigmentation have until recently been regarded as representatives of newer plastid lineages that replaced the peridinincontaining plastid during the radiation of dinoflagellates (Saldarriaga et al. 2001; Cavalier-Smith 2003; Patron et al. 2006). One of these replacement events involved a tertiary endosymbiosis of a haptophyte, resulting in dinoflagellates with 19#-hexanoyloxyfucoxanthin, the characteristic carotenoid of haptophytes (hereafter 19#-HNOF; Tengs et al. 2000). This evolutionary scenario for the plastids among dinoflagellates would imply several independent plastid acquisitions, but phylogenetic inference by Yoon et al. (2002) indicated a single origin for the peridininand 19#-HNOFcontaining plastids involving engulfment of a haptophyte alga. In contrast to this study, subsequent analysis of protein characters applying 5 plastidencoding genes divided the 2 dinoflagellate plastid groups (Yoon et al. 2005) and placed the peridinin plastids weakly within the heterokonts. Using 9 (and 10) plastid genes, Bachvaroff et al. (2005) showed the peridinin plastid and the haptophytes as sister groups with moderate to weak to support. Thus, the inferences of the dinoflagellate plastids have so far generated incongruent results. When plastids are transferred from one algal host to another, one might expect significant effects on the nature of the evolutionary process. If such changes are not accommodated in the model of sequence evolution, and if taxa independently have come to evolve under similar conditions (i.e., being endosymbionts in similar host cells), this could lead to erroneous phylogenetic inferences. Three important aspects of molecular evolution are reflected in the standard nucleotide substitution models: differences in substitution rates, differences in nucleotide frequencies, and rate variation across sites. Of these, the rate variation component usually has a strong impact on the model fit (Yang 1996). Therefore, it is logical to explore evolutionary heterogeneity by focusing on changes in the pattern of among-site rate variation. To some extent, heterogeneous spatial substitution processes can be accommodated under a stationary reversible model, such as by a rates across sites (RASs) model (Yang 1996) or a covarion models (Tuffley and Steel 1998; Galtier 2001; Huelsenbeck 2002). These latter models are somewhat different from the original covarion model of Fitch and Markowitz (1970), but they do allow sites that are variable in some taxa to be invariable in other taxa. Like the model of Fitch and Markowitz (1970), these models all have the restrictive assumption that the proportion of variable sites (Pvar) must be the same in (...truncated)