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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 2
  
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 endosymbiosis is in press at BioEssays. These abstracts are shown below.		  
 
PNAS – The Single, Ancient Origin of Chromist Plastids
Hwan Su Yoon1, Jeremiah D. Hackett1, Gabriele Pinto2, and Debashish Bhattacharya1*
1Department of Biological Sciences and Center for Comparative Genomics, University of Iowa, 210 Biology Building, Iowa City, Iowa 52242, United States.
2Dipartimento di Biologia vegetale, Università "Federico II", Via Foria 223, 80139 Napoli, Italy
*Corresponding author
  
 

Abstract: Algae include a diverse array of photosynthetic eukaryotes excluding land plants. Explaining the origin of algal plastids continues to be a major challenge in evolutionary biology. Current knowledge suggests 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 eukaryote then likely gave rise through vertical evolution to the red, green, and glaucophyte algae. 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 to show that a taxonomically 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 (about 1260 million years ago, Ma) secondary endosymbiosis involving a red alga. This finding is consistent with Chromista monophyly and implicates secondary endosymbiosis as an important force in generating eukaryotic biodiversity.

 

  
  BioEssays – Photosynthetic Eukaryotes Unite: Endosymbiosis Connects the Dots
Debashish Bhattacharya*, Hwan Su Yoon, and Jeremiah D. Hackett
Department of Biological Sciences and Center for Comparative Genomics, University of Iowa
210 Biology Building, Iowa City, Iowa 52242-1324.
*Corresponding author
  
 

Abstract: The photosynthetic organelle of algae and plants (the plastid) traces its origin to a primary endosymbiotic event in which a previously non-photosynthetic protist engulfed and enslaved a cyanobacterium. This eukaryote then gave rise to the red, green, and glaucophyte algae. However, many algal lineages, such as the chlorophyll c-containing chromists, have a more complicated evolutionary history involving a secondary endosymbiotic event, in which a protist engulfed an existing eukaryotic alga (in this case, a red alga [Fig. 1]). The dinoflagellates have mastered this process and undergone tertiary (engulfment of a secondary plastid) and even quaternary endosymbioses. In this review, we summarize the existing, primarily phylogenetic data regarding the number of secondary and tertiary endosymbioses that have occurred during eukaryotic evolution and their role in driving genome evolution. This area of research has been advancing rapidly and many long-standing questions such as the validity of the chromalveolate hypothesis and the extent of endosymbiotic gene transfer have recently been clarified.

  
   
 
Fig. 1. Proposed evolutionary tree of the chromalveolates showing the origin and loss of important (mostly plastid) characters. The evidence for chromist plastid monophyly is described in Yoon et al.(2002a). The timing of the tertiary endosymbiosis that gave rise to the haptophyte-type plastid in either the pre-dinoflagellate or only in fucoxanthin-containing taxa remains an open question (large filled circle with a cross). The possible green algal endosymbiosis in the common ancestor of apicomplexans and dinoflagellates (large open circle with a cross) is based on Funes et al. (2002) Kohler et al. (1997), and our preliminary results. Gain or loss of character states is shown with the slashes across the branches. The branches with broken lines at the origin represent uncertainty about the position of these taxa in the host tree, whereas the small filled circles with dashes or the wavy line inside represent plastid loss and plastid degeneration, respectively. pRER is the plastid rough endoplasmic reticulum.
  

In addition, it has 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, psaA, psbA, psaB, rbcL, tufA) 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 support the Paleoproterozoic hypothesis for the origin of eukaryotes. This manuscript is accepted at Molecular Biology and Evolution. The abstract is shown below. 		 
 


A Timeline for the Origin of Photosynthetic Eukaryotes
Hwan Su Yoon1, Jeremiah D. Hackett1, Claudia Ciniglia2, Debashish Bhattacharya1*
1Department of Biological Sciences and Center for Comparative Genomics, University of Iowa, 210 Biology Building, Iowa City, Iowa 52242, United States.
2Dipartimento di Biologia vegetale, Università "Federico II", Via Foria 223, 80139 Napoli, Italy
*Corresponding author

  
  Abstract: The appearance of photosynthetic eukaryotes (algae and plants) dramatically altered the Earth’s ecosystem, making possible all vertebrate life on land, including humans. Dating algal origin is, however, frustrated by a meager fossil record. We generated a plastid multi-gene phylogeny with Bayesian inference and then used maximum likelihood molecular clock methods to estimate algal divergence times. The plastid tree was used as a surrogate for algal host evolution because of recent phylogenetic evidence supporting the vertical ancestry of the plastid in the red, green, and glaucophyte algae. Nodes in the plastid tree were constrained with 6 reliable fossil dates and a maximum age of 3500 million years ago (Ma) based on the earliest known eubacterial fossil. Our analyses support an ancient (late Paleoproterozoic) origin of photosynthetic eukaryotes with the primary endosymbiosis that gave rise to the first alga having occurred after the split of the Plantae (i.e., red, green, and glaucophyte algae plus land plants) from the opisthokonts sometime before 1558 Ma. The split of the red and green algae is calculated to have occurred about 1500 Ma and the putative single red algal secondary endosymbiosis that gave rise to the plastid in the cryptophyte, haptophyte, and stramenopile algae (chromists) occurred about 1300 Ma. These dates, which are consistent with fossil evidence for putative marine algae (i.e., acritarchs) from the early Mesoproterozoic (1500 Ma) and with a major eukaryotic diversification in the very late Mesoproterozoic and Neoproterozoic, provide a molecular timeline for understanding algal evolution.  

II. Origin of Dinoflagellate Plastids
We published two papers on plastid evolution in dinoflagellates last year (Yoon et al. 2002b, Hackett et al. 2003). These data supported a successful grant application to the NSF-USDA Microbial Genome Sequencing Project (MCB 02-36631). The first paper appeared in PNAS (Yoon et al. 2002b), whereas the second appeared in the Journal of Phycology (abstract shown below). A companion review paper highlighting our findings was published in PNAS (Morden and Sherwood 2002) with the Yoon et al. (2002b) manuscript. The EST data of the dinoflagellate Alexandrium tamarense and our understanding of algal endosymbiosis have together led us to some remarkable insights into dinoflagellate nuclear and plastid genome evolution. A manuscript describing these data are provisionally acccepted at Current Biology.		  
 
Phylogenetic evidence for the cryptophyte origin of the plastid of Dinophysis (Dinophysiales, Dinophyceae)
 Jeremiah D. Hackett1, Lucie Maranda2, Hwan Su Yoon1, and Debashish Bhattacharya1*
1University of Iowa, Department of Biological Sciences and Center for Comparative Genomics, 210 Biology Building, Iowa City, Iowa, 52242, USA
2Graduate School of Oceanography, University of Rhode Island, Narragansett, Rhode Island, 02882-1197, USA
*Corresponding author
  
  Abstract: Photosynthetic members of the genus Dinophysis Ehrenberg contain a plastid of uncertain origin. Ultrastructure and pigment analyses suggest that the two-membrane-bound plastid of Dinophysis spp. has been acquired through endosymbiosis from a cryptophyte. However, these organisms do not survive in culture, raising the possibility that Dinophysis spp. have a transient kleptoplast. To test the origin and permanence of the plastid of Dinophysis, we sequenced plastid-encoded psbA and SSU rDNA from single cell isolates of D. acuminata Claparède et Lachman, D. acuta Ehrenberg, and D. norvegica Claparède et Lachman. Phylogenetic analyses confirm the cryptophyte origin of the plastid. Plastid sequences from different populations isolated at different times are monophyletic with robust support and show limited polymorphism. DNA sequencing also revealed plastid sequences of florideophyte origin, indicating that Dinophysis may be feeding on red algae.  

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 recently 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 (rbcL, psbA) that allows 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. These strains have been isolated and analyzed by our colleagues in Napoli using TEM and physiological measurements. Our prediction is that the present understanding of Cyanidiales biodiversity and distribution has been limited by the availability of only a handful of cultured strains. The environmental samples promise to radically revise our understanding of Cyanidiales evolution and lead to a major revision of their taxonomy. This work also forms the basis for using the thermoacidophilic Cyanidiales as a biotechnological resource and for understanding the nature and ramifications of thermoacido-adaptation in eukaryotes. In the short term, we have radically changed our understanding of Cyanidiales biodiversity and systematics. For example, at least 5 new species or genera have been identified from the initial analysis of one thermophilic site (Phlegrean Fields). Much of this work was done by post-doc Claudia Ciniglia (Napoli) during a 2-month stay in our lab in Iowa City where she was trained in molecular biology and phylogenetic methods (by project-funded post-doc Hwan Su Yoon). The research was partially funded by this grant. We currently have a manuscript (with our collaborators in Napoli) under review at Molecular Ecology.
 

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 submitted a paper to Molecular Phylogenetics and Evolution that provides some clear insights into the utility of plastid genes in phylogenetic analysis. The abstract from this manuscript is shown below.

  
  The Utility of Different Plastid Proteins in Phylogeny Reconstruction
Hwan Su Yoon1, Stephen B. Heard2, Jeremiah D. Hackett1, and Debashish Bhattacharya1*
1Department of Biological Sciences and Center for Comparative Genomics, University of Iowa, 210 Biology Building, Iowa City, IA 52242-1324, USA
2Department of Biology and Canadian Rivers Institute, University of New Brunswick, Bag Service 45111, Fredericton, New Brunswick, Canada E3B 6E1
*Corresponding author
 
  Abstract: Plastid genomes offer a wide array of markers for resolving both recent and ancient splits in the plant-algal phylogeny. To assess the utility of plastid coding regions in phylogeny reconstruction, we used Bayesian inference to generate a posterior distribution of trees from 22 shared proteins, individually and as concatenated data, found in 22 publicly available plastid genome sequences. We used the contraction-decontraction metric to assess the accuracy of reconstruction by comparing the pool of trees inferred with each protein data set to a “model” 22-plastid phylogeny. Phylogenetic utility varied broadly across plastid proteins. Using our approach, rpoC2, psaA, rps7, rpoC1, and atpA were found to be relatively accurate phylogenetic markers. Sequence length explained much of the variation in tree error (r2 = 0.36) for the different sequences but phylogenetic utility was not dependent solely on protein size. Some small proteins such as rps7 (154 aa) were remarkably accurate, whereas relatively large proteins such as psaB (733 aa) performed poorly. Our results illustrate a possible method for choosing markers in phylogenetics and identify a set of genes that can be applied to a broad diversity of plastids to infer their evolutionary history. They also shed some light on two issues in plant/algal systematics: the monophyly of Chromista plastids and the divergence order among early-branching streptophytes.  

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. We identify five major red algal clades, the Cyanidiales that is sister to the Bangiales + Florideophycidae, Compsopogonales + Erythropeltidales + Rhodochaetales, and three unclassified groups of Porphyridiales. We will submit a paper in Spring 2004 that describes the backbone of the Bangiophycidae phylogeny. Usage of our three target genes (16S rRNA, psaA, 18S rRNA) results in a nearly identical phylogeny (results not shown) with somewhat lower bootstrap support values for the nodes. We plan to expand the 3-gene data set to many other taxa to enlarge our coverage of Bangiophycidae. Most intriguing to us is the analysis of the extremophilic Cyanidiales, about which little had previously been known. The combination of Cyanidiales population/strain analysis from the Phlegrean Fields and our multi-gene analysis of representative members of this group has significantly expanded our understanding of their biodiversity and phylogeny.
 
Of the remaining species on our list, two groups require the most work, the Porphyridiales and the Erythropeltidales. Co-PIs Müller and Sheath received supplemental funding last Fall that they will used this Spring – Summer, 2004 to collect members of these orders. They will send the tissues to Iowa City for processing and will isolate the cells and generate cultures at their labs. We hope to have the analysis of these new taxa finished by the end of 2004. The data set for the chromist algae is essentially complete.  

VI. Other Projects that Impinge on Red Algal Phylogeny or Endosymbiosis  
1. The first draft of a paper describing the complete plastid genome sequence of the florideophyte red alga Gracilaria tenuistipitata has been submitted to Genome Research. This is work done in collaboration with Mariana Oliveira at the University of Sao Paulo, Brazil and Fulbright scholar, Jonathan Hagopian, who is working in Mariana’s lab.  
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 5,000 unique ESTs from Alexandrium and these data provide key insights into endosymbiosis that are presently being analyzed in detail.  

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.  
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).  
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).  

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