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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 3
  
I. Secondary Endosymbiotic Origin of Red Algal-Derived Plastids
We have focused on this aim in the last year and have successfully met this objective of the grant. A paper was published in PNAS in 2002 (Yoon et al. 2002a [cover photo for issue]) and a review on plastid endosymbiosiswas published in BioEssays (see below).		  
In addition, it become clear to us that the timing of the chromist secondary endosymbiosis that we estimated at 1260 million year ago (Yoon et al. 2002a) is of great significance with regard to the fossil record and the conflicting hypotheses about the first appearance of eukaryotes (i.e., Paleoproterozoic [e.g., Anbar and Knoll 2002] vs. Neoproterozoic snowball Earth [e.g., Cavalier-Smith 2002] hypotheses). To strengthen our dating work, we developed a 6-gene data set (16S rRNA, psa A, psb A, psa B, rbc L, tuf A) that also includes a cyanobacterium and multiple green algae and land plants to incorporate additional fossil constraints in the analysis. This approach allowed us to generate a broader view of algal evolution. Maximum likelihood clock analyses were used with the best maximum likelihood tree as well as with the credible tree set identified with Bayesian inference, to calculate divergence dates in this analysis. The results of this work unambiguously support the Paleoproterozoic hypothesis for the origin of eukaryotes and has recently been published in Molecular Biology and Evolution (Yoon et al. 2004). Post-doc Hwan Su Yoon has also generated a 7-protein data set for the chromist-red taxa to test the results that were based on DNA analyses. This paper is being prepared for publication. And finally, D. Bhattacharya and Hwan Su prepared a review article for the proceedings of the XVIII International Seaweed Symposium in Bergen Norway (June 20-25, 2004) where DB presented an invited lecture.  
Bhattacharya, D., H. S. Yoon, and J. D. Hackett. 2004. Photosynthetic eukaryotes unite: endosymbiosis
connects the dots. BioEssays 26:50-60.
Yoon, H.S., J. Hackett, C. Ciniglia, G. Pinto, and D. Bhattacharya. 2004. A molecular timeline for the
origin of photosynthetic eukaryotes. Mol. Biol. Evol. 21:809-818.
 

II. Origin of Dinoflagellate Plastids
We are significantly extending our work on dinoflagellate host and plastid evolution and the relationship of dinoflagellate plastids to those in the red algae and chromists. This work tests the chromalveolate hypothesis, helps us understand the eukaryotic tree of life, and the role of endosymbiotic gene transfer in eukaryotic evolution. The research is done in conjunction with our award from the Microbial Genome Sequencing Program (MCB 02-36631). A recent grant from NASA will allow us to broaden our analysis of endosymbiosis and endosymbiotic gene transfer to include glaucophyte algae (EXBO3-0000-0014). We have published several papers as a result of these grants (listed below) and plan to expand this work in 2004-2005. 		 

Hackett, J.D., L. Maranda, H.S. Yoon, and D. Bhattacharya. 2003. Phylogenetic evidence
for the cryptophyte origin of the plastid of Dinophysis (Dinophysiales, Dinophyceae).
J. Phycol. 39:440-448.

Hackett, J.D., H.S. Yoon, M.B. Soares, M.F. Bonaldo, T.L. Casavant, T.E. Scheetz, T. Nosenko,
and D. Bhattacharya. 2004. Migration of the plastid genome to the nucleus in a peridinin
dinoflagellate. Curr. Biol. 14:213-218.

Hackett, J.D., D.M. Anderson, D. Erdner, and D. Bhattacharya. Accepted. Dinoflagellates:
a remarkable evolutionary experiment. Am. J. Bot. Special Issue, "The Plant Tree of Life"
(eds. J.D. Palmer, M. Chase, and D. Soltis).

Zhang, H., D. Bhattacharya, and S. Lin. Submitted. Phylogeny of dinoflagellates based on
mitochondrial cytochrome b and nuclear small subunit rDNA sequence comparisons.
J. Phycol.

Hackett, J.D., H.S. Yoon, M.B. Soares, M.F. Bonaldo, T.L. Casavant, T.E. Scheetz, and
D. Bhattacharya. In preparation. EST analysis of the toxic dinoflagellate Alexandrium
tamarense . Genome.

Yoon, H.S., J.D. Hackett, K.B. Lidie, F. van Dolah, and D. Bhattacharya. In preparation.
Endosymbiosis driven genome evolution in the dinoflagellates. PNAS. 		 
   
III. Phylogeny and Biodiversity of the Cyanidiales
The thermoacidophilic Cyanidiales is one of the most intriguing bangiophyte red algae. These taxa are thought to be evolutionarily distinct from all other red algae and to be one of the first photosynthetic eukaryotes to have evolved on our planet (Seckbach 1987, Müller et al. 2001, Yoon et al. 2002a). 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, Pinto et al. 2003). We have made significant progress in our work on this basal and fascinating group of red algae (see http://www.biology.uiowa.edu/debweb/html/ThermoacidophilicCyanidiales.php ). With our collaborators in Napoli, Italy we published a paper on the phylogeny and physiology of Galdieria (Pinto et al. 2003). Furthermore, we have established an environmental sampling procedure with these taxa using conserved plastid genes (rbc L, psb A) that allowed us to determine the hidden biodiversity of Cyanidiales from environmental samples. Application of this method, using sediment and water samples from the Phlegrean Fields surrounding Napoli, have revealed a plethora of new species and genera of Cyanidiales. Our prediction that the present understanding of Cyanidiales biodiversity and distribution is limited by the availability of only a handful of cultured strains has been borne out. This work was recently published in Molecular Ecology (see below). Post-doc Claudia Ciniglia from Napoli will again visit our lab this summer to broaden the analysis of Cyanidiales biodiversity at different southern European sites.		  
Ciniglia, C., H.S. Yoon, A. Pollio, G. Pinto, and D. Bhattacharya. 2004. Hidden biodiversity of the
extremophilic Cyanidiales red algae. Mol. Ecol. 13. (see below for details) 		 

Abstract
The Cyanidiales is a group of asexual, unicellular red algae, which thrive in acidic and high temperature conditions around hot springs. These unicellular taxa have a relatively simple morphology and are currently classified in three genera, Cyanidium , Cyanidioschyzon , and Galdieria . Little is known however about the biodiversity of Cyanidiales, their population structure, and their phylogenetic relationships. Here we used a taxonomically broadly sampled 3-gene data set of plastid sequences to infer a robust phylogenetic framework for the Cyanidiales. The phylogenetic analyses support the existence of at least four distinct Cyanidiales lineages: the Galdieria spp. lineage (excluding Galdieria maxima ), the Cyanidium caldarium lineage, a novel monophyletic lineage of mesophilic Cyanidium spp., and the Cyanidioschyzon merolae plus Galdieria maxima lineage (Fig. 1). Our analyses do not support the notion of a mesophilic ancestry of the Cyanidiales and suggest that these algae were ancestrally thermo-acidotolerant. 		 
   
Figure 1. Phylogeny of the Cyanidiales inferred from a minimum evolution (ME) analysis using the LogDet transformation of plastid-encoded rbc L sequences. Results of a ME-LogDet bootstrap analysis are shown above the branches, whereas the bootstrap values from a protein maximum likelihood analysis using the JTT model are shown below the branches. Only bootstrap values > 60% are shown. The thick nodes represent > 95% Bayesian posterior probability for clades using the site-specific GTR model. Sequences from the environmental survey are represented by "Pisciarelli-[followed by] collection site" and are marked with asterisks. The four major Cyanidiales lineages are numbered and the broken lines show tree rearrangements that are rejected by the AU-test.

We also used environmental PCR for the rbc L gene to sample Cyanidiales biodiversity at five ecologically distinct sites at Pisciarelli in the Phlegrean Fields in Italy. This analysis showed a high level of sequence divergence among Cyanidiales species and the partitioning of taxa based on environmental conditions. Our research revealed an unexpected level of genetic diversity among Cyanidiales that revises current thinking about the phylogeny and biodiversity of this group. We predict that future environmental PCR studies will significantly augment known biodiversity that we have discovered and demonstrate the Cyanidiales to be a species-rich branch of red algal evolution. 		 

IV. Identifying Plastid Genes for Multi-Gene Analyses
A critical tool in the evolving field of genomics is the use of multiple genes in phylogenetic reconstruction. Despite the widely held belief that more genes are better than less, there is little comparative work available with real data sets to guide gene choice or to decide when enough sequence has been gathered to robustly estimate a phylogeny (e.g., Goldman 1998). We have in review at Molecular Phylogenetics and Evolution a research paper that provides some clear insights into the utility of plastid genes in phylogenetic analysis. 		 

Yoon, H.S., S.B. Heard, J.D. Hackett, and D. Bhattacharya. In review. The utility of different plastid proteins
in phylogeny reconstruction. Mol. Phylogenet. Evol.


V. Bangiophyte and Red Algal Phylogeny and Systematics
The basic framework of the Bangiophycidae phylogeny has been resolved using a dataset of 5 plastid genes (results not shown). We identified five major red algal clades, the Cyanidiales that is sister to the Bangiales + Florideophycidae, Compsopogonales + Erythropeltidales + Rhodochaetales, and three unclassified groups of Porphyridiales. We have now focused on a more broadly based sampling program of Bangiophycidae to meet our grant objective. Co-PIs Müller and Sheath have provided us with field-collected bangiophytes and Dr. Franklin Ott has made available to us cultures from his personal collection that are not available commercially. These taxa include Compsopogon hookeri , C. oishii , Chroothece mobilis , Kylinella latvica , Porphyridium sordidum , and Boldia maxima . As a result, we are now gaining a better understanding of the breadth of the bangophyte phylogeny. The sampling program is on-going and we expect to have a comprehensive bangiophyte molecular tree by the end of the grant period in August 2005.
   

Figure 2. Phylogeny of Bangiophycidae (Rhodophyta) inferred from a Bayesian analysis of plastid-encoded psa A. The results of a minimum evolution (ME) analysis using the HKY85 model, Bayesian posterior probabilities, and maximum parsimony (MP) analyses are shown. Results of the bootstrap ME analysis (1000 replicates) are shown above the branches, whereas the MP analysis of 1000 bootstrap data sets are shown below the branches. Only bootstrap values > 50% are shown. The thick branches represent posterior probabilities greater than 95%.


VI. Other Projects that Impinge on Red Algal Phylogeny or Endosymbiosis
 
1. A paper describing the complete plastid genome sequence of the florideophyte red alga Gracilaria tenuistipitata has been recently accepted in the Journal of Molecular Evolution . This work was done in collaboration with Mariana Oliveira at the University of Sao Paulo, Brazil and Fulbright scholar, Jonathan Hagopian, who worked in Mariana's lab.  

Hagopian, J.C., M. Reis, J.P. Kitajima, D. Bhattacharya, and M.C. de Oliveira. Accepted.
Comparative analysis of the complete plastid genome sequence of the red alga Gracilaria
tenuistipitata var. liui : insights on the evolution of rhodoplasts and their relationship to other
plastids. J. Mol. Evol.

 
2. Our EST project with the toxic dinoflagellate Alexandrium tamarense and the haptophyte Emiliania huxleyi if of direct relevance to this grant. These two projects are in fact part of the same set of aims; i.e., creating robust phylogenetic hypotheses of red algal hosts and plastids (including those derived from red algae) to resolve the systematics of this group and to understand the ramifications of endosymbiosis to nuclear genome evolution. We have generated nearly 8,000 unique ESTs from Alexandrium and 3,000 from Emiliania and these data provide key insights into endosymbiosis that are presently being analyzed in detail.  
3. We have recently received a 3-year grant (June 2004 - May 2007) from the Exobiology and Evolutionary Biology Program at NASA to generate 10,000 ESTs from the glaucophyte Cyanophora paradoxa . We will use phylogenetic analyses to assess the extent of ancient endosymbiotic gene transfer from the cyanobacterial plastid endosymbiont to the nucleus of this early diverging alga.  

VII. Invited Talks on Algal Evolution by the PI in2003/ 2004

Bhattacharya, D. A molecular timeline for the origin of photosynthetic eukaryotes. The Third European Phycological Congress, July 21-26, Queen's University, Belfast, Northern Ireland.

Bhattacharya, D. Insights into endosymbiosis and algal evolution using comparative genomic and phylogenetic methods. October 27, MIFAB: International Symposium on Genomic Studies of Toxic Microalgae , October 27, Santiago, Chile.

Bhattacharya, D., Endosymbiosis and algal evolution: a phylogenetic and genomic perspective. February 27, University of Maine, School of Marine Sciences, Orono.

Bhattacharya, D., A genomic and phylogenetic perspective on algal evolution. March 1-2, "Updating the Tree of Life" Symposium, Geneva, Switzerland.

Bhattacharya, D., A genomic and phylogenetic perspective on the endosymbiotic origin of algal plastids. April 8, University of Texas, Austin.

Bhattacharya, D., Genomic analysis and evolution of the toxic dinoflagellate Alexandrium tamarense . April 23, NOAA-National Ocean Service, Charleston, South Carolina.

Bhattacharya, D. Genome evolution in photosynthetic eukaryotes. June 3, Special NSF-funded symposium, "Genome Evolution in Microbial Eukaryotes", at the Annual Meeting of the Society of Protozoologists, Bryant College, Rhode Island.

Bhattacharya, D. Plastid based phylogeny of photosynthetic eukaryotes. June 6-10, Gordon Research Conference Series: Marine Microbes - Picophytoplankton, from Ecology to Genomics, Roscoff, France.

Bhattacharya, D. A genomic and phylogenetic perspective on algal origin. June 20-25, XVII International Seaweed Symposium, Bergen, Norway.

 

VIII. Planned Conference Presentations in 2004 by Iowa Grant Participants 		 
Genomes & Evolution 2004, June 17-20, University Park PA
Jeremiah D. Hackett* and Debashish Bhattacharya. Migration of a Plastid Genome to the Nucleus
in a Peridinin Dinoflagellate.
Hwan Su Yoon* and Debashish Bhattacharya. The Origin of Minicircle Genes in the Dinoflagellate Algae *speaker 		 

References. 		 

Albertano, P., Ciniglia, C., Pinto, G. & Pollio, A. The taxonomic position of Cyanidium , Cyanidioschyzon and Galdieria : an update. Hydrobiologia 433, 137-143 (2000).

Anbar, A.D. & Knoll, A.H. Proterozoic ocean chemistry and evolution: a bioinorganic bridge. Science 297, 1137-1142 (2002).

Cavalier-Smith, T. The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa. Int. J. Syst. Evol. Microbiol. 52, 297-354 (2002).

Goldman, N. Phylogenetic information and experimental design in molecular systematics. Proc. R. Soc. Lond. B 265, 1779-1786 (1998).

Huelsenbeck, J.P., & Ronquist, F. MrBayes: Bayesian inference of phylogeny. Bioinformatics 17, 754-755 (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).

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, 2001).

Hackett, J.D., Maranda, L., Yoon, H.S., Bhattacharya, D. Phylogenetic evidence for the cryptophyte origin of the plastid of Dinophysis (Dinophysiales, Dinophyceae). J. Phycol. 39 , in press (2003).

Morden, C.W. & Sherwood, A.R. Continued evolutionary surprises among dinoflagellates. Proc. Natl. Acad. Sci. USA 99, 11558-11560 (2002).

Pinto, G., Albertano, P., Ciniglia, C., Cozzolino, S., Pollio, A., Yoon, H.S., Bhattacharya, D. Comparative approaches to the taxonomy of the genus Galdieria Merola (Cyanidiales, Rhodophyta). Crypt. Algol. 24(1), 13-32 (2003).

Yoon, H.S., Hackett, J., Pinto, G., Bhattacharya, D. The single, ancient origin of chromist plastids. Proc. Natl. Acad. Sci. USA 99, 15507-15512 (2002a).

Yoon, H.S., Hackett, J., Bhattacharya, D. A single origin of the peridinin-, and fucoxanthin-containing plastids in dinoflagellates through tertiary endosymbiosis. Proc. Natl. Acad. Sci. USA 99, 11724-11729 (2002b).

 

Algal Molecular Evolution: Year one activities Year two activities Year three activities