MBE Advance Access originally published online on September 6, 2007
Molecular Biology and Evolution 2007 24(11):2358-2361; doi:10.1093/molbev/msm186
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Letters |
Phylogeny of Nuclear-Encoded Plastid-Targeted Proteins Supports an Early Divergence of Glaucophytes within Plantae
Department of Biological Sciences and Roy J. Carver Center for Comparative Genomics, University of Iowa
E-mail: debashi-bhattacharya{at}uiowa.edu.
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Abstract |
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The phylogenetic position of the glaucophyte algae within the eukaryotic supergroup Plantae remains to be unambiguously established. Here, we assembled a multigene data set of conserved nuclear-encoded plastid-targeted proteins of cyanobacterial origin (i.e., through primary endosymbiotic gene transfer) from glaucophyte, red, and green (including land plants) algae to infer the branching order within this supergroup. We find strong support for the early divergence of glaucophytes within the Plantae, corroborating 2 important putatively ancestral characters shared by glaucophyte plastids and the cyanobacterial endosymbiont that gave rise to this organelle: the presence of a peptidoglycan deposition between the 2 organelle membranes and carboxysomes. Both these traits were apparently lost in the common ancestor of red and green algae after the divergence of glaucophytes.
Key Words: Cyanophora paradoxa • glaucophytes • endosymbiosis • Plantae • plastid-targeted proteins
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Introduction |
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TOPAbstractIntroductionSupplementary MaterialAcknowledgementsReferences |
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Eukaryotic photosynthesis traces its origin to an ancient (e.g., Yoon et al. 2004
), putative single primary endosymbiotic event in which a phagotrophic protist (the host), engulfed and retained permanently an oxygenic photosynthetic cyanobacterium (the endosymbiont; Cavalier-Smith and Lee 1985; Bhattacharya et al. 2004). The captured cyanobacterium evolved into a 2-membrane-bound photosynthetic organelle. The result of the endosymbiosis was the emergence of the first algae that ultimately gave rise to the green (including land plants), red, and glaucophyte algae. These lineages form the putative supergroup Plantae (or Archaeplastida; Cavalier-Smith 1998; Adl et al. 2005) and are considered to be monophyletic based on nuclear and plastid phylogenies (e.g., Rodriguez-Ezpeleta et al. 2005; Hackett et al. 2007 [but see Nozaki et al. 2007; Stiller 2007 and discussion in Rodriguez-Ezpeleta et al. 2007]), shared components of the plastid protein import system (McFadden and van Dooren 2004), common gene replacements for plastid pathways (e.g., Fast et al. 2001; Reyes-Prieto and Bhattacharya 2007), and the presence of a family of genes involved in plastid solute transport that putatively originated in the Plantae ancestor (Weber et al. 2006; Tyra HM, Linka M, Weber APM, Bhattacharya D, unpublished data).
The biflagellate Cyanophora paradoxa is the best-studied glaucophyte, particularly with respect to the plastid import machinery (Steiner and Loffelhardt 2005), the cyanelle peptidoglycan layer (CPL) (Pfanzagl et al. 1996), and other aspects of plastid function (e.g., Gross et al. 1994; Nickol et al. 2000). To establish branching order within Plantae, we generated a 19-protein tree of nuclear-encoded plastid-targeted proteins using complete genome and expressed sequence tag data (see table 1 and Methods in Supplementary Material online) that included C. paradoxa and another glaucophyte Glaucocystis nostochinearum. Based on the abundant evidence cited above, we assumed that the Plantae form a monophyletic group that shares not only the plastid of cyanobacterial derivation but also the set of plastid-targeted proteins that reside in their nucleus and have originated through endosymbiotic gene transfer from the prokaryote (see Martin et al. 2002; Reyes-Prieto et al. 2006). Under this scenario, the phylogeny of the ancestrally shared nuclear-encoded plastid-targeted proteins recapitulates the host tree.
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The maximum likelihood (RAxML) 19-protein phylogeny (fig. 1A) supports the expected separation of the Plantae (RAxML bootstrap support, RBS = 100%; PHYML bootstrap support, PBS = 100%; Bayesian posterior probability, BPP = 1.0) from the cyanobacteria in our data set as well as the monophyly of each of its constituent groups. The red and green algae are united with high bootstrap and Bayesian support (RBS = 97%, PBS = 100%, BPP = 1.0) identifying the glaucophytes as the earliest diverging Plantae. This branching order is consistent with some analyses of plastid (e.g., Martin et al. 1998
; Yoon et al. 2004) and nuclear (Hackett et al. 2007) genes in Plantae but conflicts with the basal position of red algae found with a recent analysis of nuclear genes (Rodriguez-Ezpeleta et al. 2005; albeit with low maximum likelihood bootstrap support = 64%, see also Rodriguez-Ezpeleta et al. 2007). To test our result, we used the approximately unbiased (AU) test to assess the likelihoods of all alternative positions of glaucophytes in the tree shown in figure 1A. This analysis shows that the early divergence of glaucophytes has the highest probability among these set of alternative rearrangements (P = 0.99). The other 2 key alternative positions as sister to red or green algae were rejected by the AU test (P = 0.012 and P = 0.0002, respectively; see table 2). All other alternative topologies (see Methods, Supplementary Material online) were rejected at P < 0.05. To further test these results, we removed the class of fastest evolving amino acid sites from the multiprotein alignment and reran the phylogenetic analyses and the AU test. This operation resulted in a RAxML tree that was identical to figure 1A (see fig. 1B) and now more strongly supported the early divergence of glaucophytes within Plantae (RBS, PBS = 100%; BPP = 1.0). Consistent with this result, the AU test now provided significant support for the "glaucophytes first" hypothesis (P = 0.997) and rejected the alternative positions of this clade as sister to red and green algae at a higher significance level (P = 0.005 and P = 0.006, respectively) as well as all other alternative positions in the tree (see table 2).
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Why then do other analyses with larger nuclear protein data sets have difficulties in resolving the branching order within Plantae (Rodriguez-Ezpeleta et al. 2005
, 2007)? A limited taxon sampling is likely a major issue here. A recent in-depth analysis suggests that the position of Cyanidioschyzon merolae (Cyanidiales), which is often used to represent the red algae in eukaryotic host trees based on genome data is strongly affected by long-branch attraction (LBA) artifacts (Rodriguez-Ezpeleta et al. 2007). The attraction of the red algae to other long branches in the eukaryotic tree results in their position either at the base of the Plantae or outside of this supergroup altogether (see Nozaki et al. 2007; Rodriguez-Ezpeleta et al. 2007). This is not surprising because Cyanidiales are highly specialized extremophilic algae that live in hot acidic springs and fumaroles and have undergone extensive genome reduction (Barbier et al. 2005). Although not formally tested, our data set of 19 nuclear-encoded plastid-targeted proteins appears to not suffer from LBA (i.e., compare the relative branch lengths among Plantae in fig. 1B) and provides robust bootstrap and AU test support for the glaucophytes first hypothesis (table 2). Phylogenetic analyses in which we excluded C. merolae (supplementary figs. S1A and B, Supplementary Material online) or both Cyanidales (supplementary figs. S1C and D, Supplementary Material online) from the data set also produced trees with high support for the early branching of glaucophytes (RBS, PBS > 95%; BPP = 1.0). Other alternative positions for glaucophytes using these data sets were rejected by the AU test at P < 0.05 (table 2). Furthermore, phylogenetic analyses that included only those taxa with >50% sequence data available (see table 1) supported the basal position of glaucophytes (i.e., Cyanophora) within the Plantae (RBS, PBS > 95%; BPP = 1.0; table 2 and supplementary figs. S1E and F [Supplementary Material online]). These results suggest that our analyses are not significantly misled by missing data or by the impact of the long-branched Cyanidiales red algae. And finally, because our work made the key assumption of Plantae monophyly, the multigene data analyzed here provided only a few reasonable alternative positions (i.e., table 2) for the glaucophytes with the distantly related cyanobacteria as the outgroup. In contrast, multigene nuclear trees often include many more closely related eukaryotic outgroup taxa and therefore many more nodes where the glaucophyte algae could potentially diverge. This simplifying feature (given our hypothesis of Plantae monophyly holds) may make our data set less prone to stochastic error that is typical of anciently diverged sequences.
The topology shown in figure 1 is consistent with the presence of 2 glaucophyte characters that have long been postulated as ancestral for Plantae plastids (e.g., see Helmchen et al. 1995). The most important of these so-called "primitive" traits is the CPL (Pfanzagl et al. 1996) that is located between the 2 plastid membranes. Biochemical analyses of the CPL in C. paradoxa support a comparable role in this alga to its involvement in cyanobacterial fission (Berenguer et al. 1987). It has been suggested that the CPL may be involved in osmolarity and volume regulation of the cyanelle, similar to the cell wall in cyanobacteria (Raven 2003). The second primitive feature of cyanelles is the presence of carboxysomes (ß-carboxysomes). Carboxysomes are present in cyanobacteria (and other bacteria) and are accumulations of RuBisCO and carbonic anhydrase that play a role in carbon concentration. Given our results, presumably both of these key traits were lost from the ancestor of red and green algae after the glaucophyte divergence. Plastid fructose-1,6-bisphosphate aldolase (FBA) in Plantae provides another important piece of evidence in this puzzle. Glaucophytes possess the presumed ancestral (cyanobacterial) FBA type II (Nickol et al. 2000), whereas the red and green algae contain the of type I isozyme (Gross et al. 1999). Plastid FBA type I may be of host (Gross et al. 1999; Rogers and Keeling 2004) or cyanobacterial (endosymbiotic) origin (Reyes-Prieto and Bhattacharya 2007). Whichever the case for the latter gene, the presence of FBA type I in green and red algae suggests the replacement of the plastid FBA type II in the common ancestor of green and red algae after the divergence of glaucophytes (Rogers and Keeling 2004; Reyes-Prieto and Bhattacharya 2007). Other ancestral glaucophyte characters that fail to provide decisive insights into the branching order within Plantae include the presence of phycobilins (phycobilisomes) and nonstacked thylakoidal membranes (also present in some cyanobacteria) that is shared with red algae (Kies and Kremer 1990). The unique presence of chlorophyll b in green algal and land plant plastids suggests that the cyanobacterial plastid ancestor likely contained both phycobilins and chlorophyll b for light harvesting (Tomitani et al. 1999) and the latter were lost independently from glaucophytes and red algae. In this case, the more parsimonious scenario of a single loss of chlorophyll b in a putative glaucophyte–rhodophyte ancestor is inconsistent with our results.
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Supplementary Material |
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TOPAbstractIntroductionSupplementary MaterialAcknowledgementsReferences |
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The Methods section and supplementary figures are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org).
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Acknowledgements |
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TOPAbstractIntroductionSupplementary MaterialAcknowledgementsReferences |
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This work was supported by grants from the National Science Foundation and the National Aeronautics and Space Administration awarded to D.B. (EF 04-31117, NNG04GM17G). We thank 2 anonymous reviewers for their helpful comments.
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Footnotes |
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Martin Embley, Associate Editor
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