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Protist, Vol. 163, 832843, November 2012http://www.elsevier.de/protisPublished online date 23 February 2012
ORIGINAL PAPER
Evolution of the Chloroplast Genome inPhotosynthetic Euglenoids: A Comparison ofEutreptia viridis and Euglena gracilis(Euglenophyta)
Krystle E. Wiegert, Matthew S. Bennett, and Richard E. Triemer1
Michigan State University, Department of Plant Biology, 166 Plant Biology, East Lansing, MI, USA 48824
Submitted August 29, 2011; Accepted January 6, 2012Monitoring Editor: Saul Purton
The chloroplast genome of Eutreptia viridis Perty, a basal taxon in the photosynthetic euglenoid lin-eage, was sequenced and compared with that of Euglena gracilis Ehrenberg, a crown species. Severalcommon gene clusters were identified and gene order, conservation, and sequence similarity wasassessed through comparisons with Euglena gracilis. Significant gene rearrangements were presentbetween Eutreptia viridis and Euglena gracilis chloroplast genomes. In addition, major expansion hasoccurred in the Euglena gracilis chloroplast accounting for its larger size. However, the key chloroplastgenes are present and differ only in the absence of psaM and roaA in Eutreptia viridis, and psaI inEuglena gracilis, suggesting a high level of gene conservation within the euglenoid lineage. Further
comparisons with the plastid genomes of closely related green algal taxa have provided additional sup-port for the hypothesis that a Pyramimonas-like alga was the euglenoid chloroplast donor via secondaryendosymbiosis. 2012 Elsevier GmbH. All rights reserved.
Key words: Eutreptia viridis; chloroplast; Euglenophyta; Euglena gracilis; genome.
Introduction
The euglenoids represent a diverse, ancienteukaryotic lineage. Huber-Pestalozzi (1955)describes over 800 species belonging to about40 genera, of which about two thirds are non-photosynthetic. Molecular phylogenies placeeuglenoids near the base of the eukaryotic treeof life (Adl et al. 2005; Cavalier-Smith 1998;Lane and Archibald 2008; Moreira et al. 2007;Parfrey et al. 2010; Simpson and Roger 2004) withthe phagotrophic forms positioned basal to thephotosynthetic genera. This study will focus on the
Corresponding author; fax +1 517 353 1926-mail [email protected] (R.E. Triemer).
photosynthetic euglenoids and specifically on theacquisition and evolution of the chloroplast.
Euglenoids are believed to have gained theability to photosynthesize through the acquisi-tion of a chloroplast via secondary endosymbiosisof a green alga. This hypothesis originally wasbased on the existence of closely related non-photosynthetic phagotrophic euglenoids and thepresence of three membranes surrounding theresulting chloroplast rather than the two mem-branes typical of green algae and higher plants(Gibbs 1978, 1981). In recent years the sequenc-ing of chloroplast genomes has been used to inferdeep evolutionary relationships among photosyn-thetic lineages (Burger et al. 2007; Nock et al. 2011;Parks et al. 2009; Qiu et al. 2006). This is due in part
2012 Elsevier GmbH. All rights reserved.doi:10.1016/j.protis.2012.01.002
dx.doi.org/10.1016/j.protis.2012.01.002http://www.elsevier.de/protismailto:[email protected]/10.1016/j.protis.2012.01.002
Eutreptia viridis Chloroplast 833
Figure 1. Diagrammatic representation of the cur-rent state of euglenoid phlogeny adapted fromMarin et al. 2003. Peranema is a non-photosyntheticphagotrophic euglenoid found basal to the photo-synthetic euglenoids including Eutreptia viridis andEuglena gracilis.
to the small size and comparative simplicity of thesegenomes relative to their nuclear counterparts andto the ability to analyze multigene datasets withoutdelving into the more complex eukaryotic nucleargenome. Sequencing of the chloroplast genomeis also facilitated by the typically high chloroplastgenome copy number present within cells rela-tive to the nuclear genome and the ability to usemultiplexed, massively parallel sequencing (Parkset al. 2009). At last count a total of 229 chloro-plasts have been sequenced to completion, mostof which are representative or economically impor-tant land plant species (Benson et al. 2011). Feware algal species and most of these are greenalgae with meager representation of other algal lin-eages. Within the photosynthetic euglenoids, theonly chloroplast genome that has been sequencedis from Euglena gracilis (Hallick et al. 1993).
Euglena gracilis is the model organism for pho-tosynthetic euglenoids due to its ease in culturingand the ability to achieve high cell densities. How-ever, Euglena gracilis is not a typical representativephotosynthetic euglenoid due to the diversity withinthe lineage, and it is not closely related to thephagotrophic forms believed to have been thehost for the endosymbiont. The diversity of theeuglenoid lineage warrants further exploration intotheir chloroplast evolution, which could elucidateunderstanding of this key basal eukaryotic lineage.
To date, all inferences regarding the chloro-plast donor taxon have relied on the chloroplastsequence of Euglena gracilis. Based on establishedphylogenetic assessment of the photosyntheticeuglenoids, Euglena gracilis is consistently a crownspecies of the lineage (Fig. 1; Linton et al. 2010;Marin et al. 2003; Triemer et al. 2006).
Although euglenoids are thought to haveobtained their chloroplast from a green alga,the chloroplast of Euglena gracilis shows somesignificant changes. Most notably, the Euglenagracilis chloroplast genome is not divided intolarge and small single copy regions separatedby inverted repeats containing the rRNA genes(as well as a few other genes). Not only doesEuglena gracilis lack the inverted repeats, but theribosomal operon is organized in at least threeand a half tandemly arranged copies (5S/23S/16S:5S/23S/16S:5S/23S/16S:16S, Hallick et al. 1993).The Euglena gracilis gene content appears consis-tent with other sequenced green algal chloroplastgenomes, but the arrangement is not (Turmelet al. 2009). This raises several questions withregard to chloroplast evolution within the photo-synthetic euglenoids. Is the chloroplast genome ofEuglena gracilis representative of all photosyntheticeuglenoids? Will the chloroplast genome of morebasal taxa look more like that of green algae? Whatchanges have occurred during the evolution of thechloroplast in the euglenoid lineage?
To address these questions the chloroplastgenome of Eutreptia viridis was sequenced.Eutreptia viridis is the type species of the genusEutreptia which consistently falls basal to photo-synthetic euglenoids in phylogenetic assessmentsof both nuclear and chloroplast ribosomal genesas well as nuclear encoded protein coding genes(Fig. 1; Linton et al. 2010; Marin et al. 2003;Triemer et al. 2006). A diagrammatic representa-tion of euglenoid phylogeny based on analysis byMarin et al. in 2003 demonstrates the position ofEutreptia viridis relative to Euglena gracilis andthe non-photosynthetic euglenoids (Fig. 1). Fur-thermore, Eutreptia viridis differs morphologicallyfrom Euglena gracilis by the presence of two emer-gent flagella versus one. This is notable in thatthe closest extant phagotrophic euglenoids suchas Peranema also have two emergent flagella.This study will assess and compare gene con-tent, arrangement, size, and sequence similaritybetween these two photosynthetic euglenoids.
Results and Discussion
The Eutreptia viridis chloroplast genome wasassembled from reads obtained by sequencing totalgenomic DNA using Roche 454 sequencing andde novo assembly. Because the ribosomal genesform a tandem array rather than inverted repeatsas in the green algae, it was difficult to determinethe exact number of ribosomal operons and the 3
834 K. E. Wiegert et al.
Figure 2. Circular map of the Eutreptia viridis chloroplast genome. Filled boxes of different colors rep-resent genes of various functional groups (Green: photosystems/photosynthesis; Yellow: large ribosomalproteins, rpl genes; Red: small ribosomal proteins, rps genes; Blue: rpo genes, tufA; Gray: ribosomal rRNAs;Orange: atp genes; Black: miscellaneous, ycf, orfs, tRNAs). Boxes are proportional to their sequence lengthincluding any introns present and those positioned on the outside of the circle are considered on the positivestrand while those inside the circle are on the negative strand. Genes were annotated based on the best availablenomenclature and tRNAs specified by their single letter code with associated anticodon in parentheses.
connection to the rest of the genome. Based onread coverage of the ribosomal operon compo-nents (5S, 23S, 16S) compared to single copyprotein coding genes, it appears that at least twocopies of the operon are present. Although a lackof overlap in sequence following the ribosomalgenes to that before psaC does not allow the com-pletion of the circularized genome, it is believedthat all genes are present based on comparisonswith Euglena gracilis. The chloroplast genome wasassembled into a single contig of 65,513 bp (Fig. 2)and is represented in circular form to facilitate
comparison
