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Secondary Symbiotic Origin of Algal Plastids and
the Phylogeny of the Bangiophycidae (Rhodophyta)
NSF-DEB 01-07754

 

INTRODUCTION: The main objective of this grant is to isolate and sequence nuclear and plastid genes from bangiophyte red algae and algae containing red algal-derived plastids. These data will help us determine the number of red algal secondary endosymbiotic events and lead to the erection of a robust phylogeny of the Bangiophycidae. Our previous results suggest that multiple secondary endosymbioses explain the origin of the red algal–derived plastids of cryptophyte, haptophyte, and stramenopiles (=heterokonts) algae and that there is substantial paraphyly of the Bangiophycidae orders. The primary aims of the grant are:
 		    

1)
Determine the number of secondary symbiotic events that have resulted in the bangiophyte-derived plastids of the cryptophyte, haptophyte, and stramenopiles (together, the Chromista). 		 

This will be done by reconstructing the phylogeny of plastids in the Bangiophycidae and in the Chromista by comparing concatenated plastid SSU rDNA and rbcL genes.

  

2)
Reconstruct Bangiophycidae phylogeny to create a robust systematic scheme for these taxa. 		 
This will be done by analyzing concatenated nuclear and plastid SSU rDNA, rbcL, and mitochondrial coxI coding regions from a broad diversity of Bangiophycidae.  

Project activities and findings - Year 1
  
I. Secondary Endosymbiotic Origin of Red Algal-Derived Plastids
Addressing this aim was the major focus of the PI's lab in the first year. To this end, we studied a number of different plastid genes to assess their usefulness in generating a robust plastid phylogeny (see Section IV below). We sequenced 5 different plastid genes from a total of 36 taxa that span the known taxonomic diversity of red algae and the Chromista. This expanded data set was gathered by project post-doc Hwan Su Yoon who determined a total of 117 new sequences. These organellar data are the most complete ever assembled for the red algae and the Chromista and provide a robust phylogeny of their plastids. The surprising result of this analysis is that all Chromista plastids form a well-supported monophyletic group that is sister to the Cyanidiales and other red algae. This finding supports a single secondary endosymbiotic origin of the Chromista plastid and is consistent with monophyly of chromist host cells (Cavalier-Smith 1986). Our molecular clock calculations using maximum likelihood methods (r8s V1.01, Sanderson 2001) suggest that this endosymbiotic event occurred about 1260 million years ago (Ma), substantially older than previously thought (Cavalier-Smith 2000). A preliminary abstract is provided below as well as a phylogenetic analysis that summarizes the major findings. 		 
 
The Single, Ancient Origin of Chromist Plastids
Hwan Su Yoon, Jeremiah Hackett, Gabriele Pinto, and Debashish Bhattacharya		  
 
Abstract: Phylogenetic comparisons suggest that plastid primary endosymbiosis, in which a single-celled protist engulfs and "enslaves" a cyanobacterium, likely occurred once and resulted in the primordial alga. This photosynthetic cell diversified, through vertical evolution, into the ubiquitous green (Chlorophyta) and red (Rhodophyta) algae, and the more scarce Glaucophyta. However, some modern algal lineages have a more complicated evolutionary history involving a secondary endosymbiotic event, in which a protist engulfed an existing eukaryotic alga (rather than a cyanobacterium), which was then reduced to a secondary plastid. Secondary endosymbiosis explains the majority of algal biodiversity, yet the number and timing of these events is unresolved. Here we analyzed a five-gene plastid data set (16S rDNA, rbcL, psaA, psbA, tufA) to show that a diverse group of chlorophyll c2 -containing protists comprising cryptophyte, haptophyte, and stramenopiles algae (Chromista) share a common plastid that most likely arose from a single, ancient (1261 ± 28 Ma) secondary endosymbiosis involving a red alga (see Fig. 1). This finding is consistent with Chromista monophyly and implicates secondary endosymbiosis as a driving force in early eukaryotic evolution.		  

II. Origin of Dinoflagellate Plastids
As it became clear to us that we had solved a major aim of our grant and addressed a central issue in plastid origin, the piece of the puzzle that still remained was the dinoflagellates. The primary lineage of dinoflagellates containing the unique carotenoid peridinin also contain a plastid of red algal origin (Zhang et al. 1999) but did it originate through secondary or tertiary endosymbiosis? Hwan Su familiarized himself with the literature regarding dinoflagellate plastids and we sequenced a minimum number of genes to gain insights into the position of two fundamentally different dinoflagellate plastids in our tree. This work was designed to test Tom Cavalier-Smith’s idea (Cavalier-Smith 2000) that not only do chromist plastids (and therefore the host cells) have a single origin but that the alveolates (dinoflagellates, ciliates, and apicomplexans) are also ancestrally photosynthetic and share the same plastid as the Chromista (i.e., together the super assemblage, Chromalveolates). To address this issue, we sequenced eight psbA, psaA, and rbcL genes from select dinoflagellates to test their evolutionary position in our now large and broadly sampled red algal and chromist plastid tree. Again, to our surprise, we found strong support for Chromalveolate monophyly but with an interesting twist. Our data suggest that dinoflagellates ancestrally contained a plastid of haptophyte, not red algal, origin (i.e., through an ancient tertiary replacement of a secondary red plastid), and peridinin, plus a number of other distinctive characters (e.g., mini-circle plastid genes, nuclear-encoded "Form II" rbcL), evolved later in evolution. These findings have been described in a manuscript that has recently appeared in PNAS. The abstract and a relevant figure is shown below.
In related work, graduate student Jeremiah Hackett in the PI’s lab has completed a side-project with collaborator Lucie Maranda (University of Rhode Island) aimed at clarifying the origin of the plastid in the toxic dinoflagellate Dinophysis. Jeremiah has shown that this plastid resulted from a tertiary replacement involving a cryptophyte. 		 
 
A Single Origin of the Peridinin-, and Fucoxanthin-Containing Plastids in Dinoflagellates Through Tertiary Endosymbiosis
Hwan Su Yoon, Jeremiah D. Hackett, and Debashish Bhattacharya		  
 
Abstract: The most widely distributed dinoflagellate plastid contains chlorophyll c2 and peridinin as the major carotenoid. A second plastid type, found in taxa such as Karlodinium micrum and Karenia spp., contains chlorophylls c1+c2 and 19’-hexanoyloxy-fucoxanthin and/or 19’-butanoyloxy-fucoxanthin but lacks peridinin. Because the presence of chlorophylls c1 + c2 and fucoxanthin is typical of haptophyte algae, the second plastid type is believed to have originated from a haptophyte tertiary endosymbiosis in an ancestral peridinin-containing dinoflagellate. This hypothesis has, however, never been thoroughly tested in plastid trees that contain genes from both peridinin- and fucoxanthin- containing dinoflagellates. To address this issue, we sequenced the plastid-encoded psaA, psbA, and "Form I" rbcL genes from various red and dinoflagellate algae. The combined psaA + psbA tree shows significant support for the monophyly of peridinin- and fucoxanthin-containing dinoflagellates as sister to the haptophytes. The monophyly with haptophytes is robustly recovered in the psbA phylogeny (Fig. 2) in which we increased the sampling of dinoflagellates to 14 species. As expected from previous analyses, the fucoxanthin-containing dinoflagellates formed a well-supported sister group with haptophytes in the rbcL tree (results not shown). Based on these analyses, we postulate that the plastid of peridinin- and fucoxanthin-containing dinoflagellates originated from a haptophyte tertiary endosymbiosis that occurred before the split of these lineages. Our findings imply that the presence of chlorophylls c1+c2 and fucoxanthin, and the plastid-encoded "Form I" rbcL gene are in fact the primitive (not derived, as widely believed) condition in dinoflagellates.		  

Minimum evolution (ME) tree (LogDet distances) of psbA sequences from red algal and red algal-derived plastids. A total of 957 nt were considered. The LogDet bootstrap values (2000 replications) are shown above and ME-GTR+I+Γ bootstrap values are shown below the branches. The thick branches denote >95% posterior probability for groups to the right, resulting from a Bayesian inference using Markov chain Monte Carlo sampling of phylogenies. The GTR+Γ model was used in the Bayesian inference with separate alpha parameters for each of the codon positions. Peridinin-containing dinoflagellates are underlined. The blue circle marks the monophyly of all dinoflagellate plastids, whereas the red circle marks the monophyly of plastids containing peridinin.

III. Phylogeny of the Cyanidiales
The thermoacidophilic Cyanidiales is one of the most interesting and important bangiophyte red algae. These taxa are thought to evolutionarily distinct from all other red algae and to be one of the first photosynthetic eukaryotes to have evolved on our planet (Seckbach 1987). The phylogeny and systematics of the Cyanidiales is, however, in a state of confusion because of their simple morphology combined with difficulties in collecting and culturing these extremophiles (Albertano et al. 2000). A critical advance for this grant was the establishment of a collaboration with the group of Gabriele Pinto in Napoli, Italy. Gabriele has an extensive, worldwide collection of Cyanidiales in culture, is an expert on their physiology and ultrastructure, and has joined us in studying their phylogeny and evolution. Our labs have recently prepared a paper on Galdieria evolution (see abstract below).		  
 
Comparative Approaches to the Taxonomy of the Genus Galdieria Merola (Cyanidiales, Rhodophyta)
Gabriele Pinto, Patrizia Albertano, Claudia Ciniglia, Salvatore Cozzolino, Antonino Pollio, Hwan Su Yoon, Debashish Bhattacharya		  
 


Abstract: Ecophysiological, morphological, ultrastructural, and phylogenetic methods were used to study 9 strains of Galdieria isolated from different geographical regions in order to clarify the taxonomy of the genus and to understand the phylogeny of the Cyanidiales. The studied characters were often conserved in all the isolates. Confocal laser scanning microscopy (CLSM) observations confirmed the presence of a single plastid and allowed its 3-D reconstruction. Analysis of rbcL sequences showed high sequence identity among three Russian strains, G. daedala, G. maxima, and G. partita and among different strains of G. sulphuraria. On the basis of these morphological and phylogenetic data (results not shown), we suggest that Galdieria sulphuraria is the unique species within the genus Galdieria and we emend its description. We also suggest, on the basis of phylogenetic evidence, the emendment of both the order Cyanidiales and the family Cyanidiaceae.

  

TEM micrographs of cells of G. daedala strain P508. (1) sporangium. (2) young cell; arrowhead showing the protrusions of the innermost layer of the cell wall (transfer-cell like structure); arrow indicates dictyosomes. (3) mature cell; arrow indicates osmiophilic globules in the plastid stroma. (4) a detail of a mature cell showing the protrusions of the inner layer of the cell wall (arrowheads). Abbreviations: m = mitochondrion; n = nucleus; v = vacuole; vs = vesicles.

IV. Bangiophyte and Red Algal Phylogeny and Systematics
The extensive sequencing that was done in the first year of the grant has not only provided many important insights into plastid endosymbiosis but has also revealed unexpected structure in the red algal phylogeny. A plastid five-gene tree of the red algae shows that there are 5 recognizable clades, the Cyanidiales, Bangiales+Florideophycidae, Compsopogonales+Erythropeltidales+ Rhodochaetales, Porphyridiales 3, and Porphyridiales 1+2. These groups are often suggested in single gene analyses using, for example, nuclear 18S rDNA or rbcL (Müller et al. 2001) but without convincing bootstrap support. We expect to consolidate further these groups with the addition of more red algal sequences. The major goal of the grant in the second year will be to expand the taxon sampling in this tree and limit the analysis to 3 excellent phylogenetic markers, 16S rDNA, psaA, and nuclear 18S rDNA. 		 

V. Conference Presentations in 2002 by Grant Participants  
International Society of Evolutionary Protistology, June 19-24, 2002, Vancouver, Canada  
Hackett, Jeremiah D.1*, Lucie Maranda2, and Debashish Bhattacharya1. 1Department of Biological Sciences,  
  University of Iowa, Iowa City, IA 52242; 2Graduate School of Oceanography, University of Rhode Island,  
  Narragansett, RI 02882-1197. – The plastid of Dinophysis (Dinophyceae): Phylogenetic evidence for  
  a permanent replacement.  
Yoon, Hwan Su, Jeremiah Hackett, and Debashish Bhattacharya.* University of Iowa, Department of Biological  
  Sciences, 239 Biology Building, Iowa City, IA 52242, United States. – The phylogeny of red algae and  
  red algal-derived plastids.  
Botany 2002, Aug. 4-7, Madison, WI  
Hackett, Jeremiah D.1*, Lucie Maranda2, and Debashish Bhattacharya1. 1Department of Biological Sciences,  
  University of Iowa, Iowa City, IA 52242; 2Graduate School of Oceanography, University of Rhode Island,  
  Narragansett, RI 02882-1197. – The plastid of Dinophysis (Dinophyceae): Phylogenetic evidence for  
  a permanent replacement.  
Yoon, Hwan Su*, Jeremiah D. Hackett, and Debashish Bhattacharya. Department of Biological Sciences, University  
  of Iowa, Iowa City, IA 52242 USA. - The monophyletic origin of the peridinin-, and fucoxanthin-containing  
  dinoflagellate plastid through tertiary replacement.  
Yoon, Hwan Su1, Jeremiah Hackett1, Gabriele Pinto2, and Debashish Bhattacharya1.* 1University of Iowa,  
  Department of Biological Sciences, 239 Biology Building, Iowa City, IA 52242, United States;  
  2Universita Federico II, Dipartimento di Biologia vegetale, Via Foria 223, 80139 Napoli, Italy. - The single,  
  ancient origin of chromist plastids.  
Culture Collections of Algae: Increasing Accessibility and Exploring Algal Biodiversity, Sept. 2-6, 2002, Göttingen, Germany  
Bhattacharya, Debashish*, Hwan Su Yoon, and Jeremiah D. Hackett. Department of Biological Sciences and Center  
  for Comparative Genomics, University of Iowa, Iowa City, IA 52242 USA. - Secondary endosymbiosis and the  
  ancient birth of algae. (Plenary talk)  
*presenter.  
     

References.  
Albertano, P., Ciniglia, C., Pinto, G. & Pollio, A. The taxonomic position of Cyanidium, Cyanidioschyzon and Galdieria: an update.  
  Hydrobiologia 433, 137-143 (2000).  
Baldauf, S. L., Roger, A. J., Wenk-Siefert, I. & Doolittle, W. F. A kingdom-level phylogeny of eukaryotes based on combined protein data.  
  Science 290, 972-977 (2000).  
Cavalier-Smith, T. In Progress in Phycological Research (Vol. 4) (eds Round F. E. & Chapman, D. J.) 309-347 (Biopress, Bristol, 1986).  
Cavalier-Smith, T. Membrane heredity and early chloroplast evolution. Trends Plant Sci. 5, 174-182 (2000).  
Goldman, N. Phylogenetic information and experimental design in molecular systematics. Proc. R. Soc. Lond. B 265, 1779-1786 (1998).  
Huelsenbeck, J. P., Ronquist, F., Nielsen, R. & Bollback, J. P. Bayesian inference of phylogeny and its impact on evolutionary biology.  
  Science 294, 2310-2314 (2001).  
Müller, K. M., Oliveira, M. C., Sheath, R. & Bhattacharya, D. Ribosomal DNA phylogeny of the Bangiophycidae (Rhodophyta) and the  
  origin of secondary plastids. Am. J. Bot. 88, 1390-1400 (2001).  
Sanderson, M. J. r8s V1.01 (beta) (University of California, Davis, 2001).  
Seckbach, J. in Endocytobiology III (eds Lee, J. J. & Frederick, J. F.) 424-437 (Ann. N.Y. Acad. Sci., 1987).  
Swofford, D. L. PAUP*. Phylogenetic analysis using parsimony (*and other methods) (Sinauer, Sunderland, Massachusetts, 2002).  
Valentin, K., & Zetsche, K. Rubisco genes indicate a close phylogenetic relation between the plastids of Chromophyta and Rhodophyta.  
  Plant Mol. Biol. 15, 575-584 (1990).  
Zhang, Z., Green, B. R. & Cavalier-Smith, T. Single gene circles in dinoflagellate chloroplast genomes. Nature 400,155-159 (1999).  

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