J Mol Evol (1995) 41:203-210 JOUrnAL o, MOLECULAR [EVOLUTION © Springer-Verlag New YorkInc. 1995 Analyses of Ribosomal RNA Sequences from Glaucocystophyte Cyanelles Provide New Insights into the Evolutionary Relationships of Plastids Thomas A. Helmchen, Debashish Bhattacharya, Michael Melkonian Botanisches Institut, Lehrstuhl I, Universit~it zu K/Jln, Gyrhofstrage 15, D-50931, K61n, Germany Received: 30 June 1994 / Accepted: 14 October 1994 Abstract. Glaucocystophyte algae (sensu Kies, Bed. Deutsch. Bot. Ges. 92, 1979) contain plastids (cyanelles) that retain the peptidoglycan wall of the putative cyano- bacterial endosymbiont; this and other ultrastructural characters (e.g., unstacked thylakoids, phycobilisomes) have suggested that cyanelles are "primitive" plastids that may represent undeveloped associations between heterotrophic "host" cells (i.e., glaucocystophytes) and cyanobacteria. To test the monophyly of glaucocysto- phyte cyanelles and to determine their evolutionary re- lationship to other plastids, complete 16S ribosomal RNA sequences were determined for Cyanophora para- doxa, Glaucocystis nostochinearum, Glaucosphaera vacuolata, and Gloeochaete wittrockiana. Plastid rRNAs were analyzed with the maximum-likelihood, maximum- parsimony, and neighbor-joining methods. The phyloge- netic analyses show that the cyanelles of C. paradoxa, G. nostochinearum, and G. wittrockiana form a distinct evolutionary lineage; these cyanelles presumably share a monophyletic origin. The rDNA sequence of G. vacuo- lata was positioned within the nongreen plastid lineage. This result is consistent with analyses of nuclear- encoded rRNAs that identify G. vacuolata as a rhodo- phyte and support its removal from the Glaucocysto- phyta. Results of a global search with the maximum- likelihood method suggest that cyanelles are the first divergence among all plastids; this result is consistent with a single loss of the peptidoglycan wall in plastids after the divergence of the cyanelles. User-defined tree analyses with the maximum-likelihood method indicate, Correspondence to: D. Bhattacharya however, that the position of the cyanelles is not stable within the rRNA phylogenies. Both maximum- parsimony and neighbor-joining analyses showed a close evolutionary relationship between cyanelles and non- green plastids; these phylogenetic methods were sensi- tive to inclusion/exclusion of the G. wittrockiana cy- anelle sequence. Base compositional bias within the G. wittrockiana 16S rRNA may explain this result. Taken together the phylogenetic analyses are interpreted as sup- porting a near-simultaneous radiation of cyanelles and green and nongreen plastids; these organelles are all rooted within the cyanobacteria. Key words: Cyanelles -- Cyanophora paradoxa -- Endosymbiosis -- Evolution -- Glaucocystophyta -- Glaucophyta -- Phylogeny -- Plastid -- 16S ribosomal RNA Introduction The number and tinting of endosymbiotic events com- prise two of the most interesting and controversial prob- lems in plastid evolution. The theory of endosymbiosis as an explanation for the origin of plastids from one or more cyanobacterial ancestors is now widely accepted (Mereschkowsky 1905, 1910; Margulis 1981; Gray 1989). The Glaucocystophyta (Kies 1979; Kies and Kremer 1986; synonym Glaucophyta, Sknja 1954), a group of algae with a cyanobacterial cell inclusion ("cy- anelle," Pascher 1929), is particularly interesting in the context of plastid evolution. Because cyauelles resemble cyanobacteria in ultrastructure (e.g., unstacked thyla-