213 J. Phycol. 39, 213–220 (2003) MITOCHONDRIAL GENOME CONFORMATION AMONG CW-GROUP CHLOROPHYCEAN ALGAE 1 Mark Laflamme and Robert W. Lee 2 Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada Most green algal taxa have circular-mapping mito- chondrial genomes, whereas some have linear genome- or subgenomic-sized mitochondrial DNAs (mtDNA). It is not clear, however, if the circular-mapping ge- nomes represent genome-sized circular molecules, if such circular molecules and the linear forms are the predominant in vivo mtDNA structures, or if the lin- ear forms arose only once or multiple times among extant green algal lineages. We therefore examined the DNA components detected with homologous mtDNA probes after pulsed-field gel electrophoresis of total cellular DNA from the chlorophycean basal bodies displaced clockwise(CW)-group taxa Chlamy- domonas reinhardtii and Chlamydomonas moewusii. For C. reinhardtii, the 15.8-kb linear mtDNA was the only DNA component detected, and there was no evidence of circular or large linear precursors of this DNA. In the case of C. moewusii, which is known to have a cir- cular-mapping 22.9-kb mitochondrial genome, three DNA components were detected; these appeared to be circular (relaxed and supercoiled) and genome- sized linear DNA molecules, the latter of which likely resulted from random double-strand breaks in the circular forms during DNA isolation. In further stud- ies, DNA from additional CW-group taxa was exam- ined using conventional gel electrophoresis and DNA- filter blot analysis with C. reinhardtii and C. moewusii mtDNA probes. We conclude that all taxa from the “Volvox clade” (sensu Nakayama et al. 1996) of the CW-group have genome- or subgenomic-sized linear mtDNAs as their predominant mtDNA form and that these arose from a genome-sized circular form in an ancestor that existed near the base of this clade. Key index words: Chlamydomonas; Chlorophyceae; gel electrophoresis; mitochondrial DNA conformation; phylogeny Abbreviations: CW, basal bodies displaced clockwise; mt, mitochondrial; PFGE, pulsed field gel electro- phoresis Mitochondrial genomes vary in size and structure both among and within kingdoms of the Eukarya (Gray 1999, Gray et al. 1999, Lang et al. 1999b). In terms of size, at one extreme are the 6-kb mitochon- drial genomes of the apicomplexa that encode five genes (Feagin 1995), whereas at the other extreme are 200 kb and larger land plant counterparts that en- code 50–70 genes (Ward et al. 1981, Lang et al. 1999b) and the 100-kb mitochondrial genome of the jakobid protozoan Reclinomonas americana that en- codes 91 genes (Lang et al. 1999a). The conformation of mitochondrial DNA (mtDNA) can be a genome- sized circular molecule as in most animals, although genome-sized linear forms and subgenomic-sized cir- cular or linear forms have been reported in some ani- mal taxa (reviewed in Moritz et al. 1987, Bridge et al. 1992, Armstrong et al. 2000). Linear genome-sized mtDNAs with distinct telomeres have been identified in diverse taxa, most notably in some yeasts (reviewed in Nosek et al. 1998) and ciliates (Burger et al. 2000). In the apicomplexa (Wilson and Williamson 1997) and many fungi (Maleszka et al. 1991, Maleszka and Clark- Walker 1992, Maleszka 1993), most mtDNAs exist as larger than genome-sized linear molecules, with a small fraction of genome-sized circular molecules that are thought to give rise to the linear forms by a roll- ing-circle replication mechanism. Finally, land plant mtDNAs, the most structurally complex of these DNAs, exist primarily as linear molecules or “rosette-like” struc- tures that can be larger than the genome size, and it has been proposed that these use a recombination- dependent DNA replication mechanism similar to that of phage T4 (Backert et al. 1997, Oldenburg and Bendich 2001). Information pertinent to mitochondrial genome size and conformation in green algae (Chlorophyta, sensu Sluiman 1985) is summarized in Table 1. Those mitochondrial genomes that have been sequenced to completion vary in size from 16 to 55 kb, encode 12 to 60 genes, and are most commonly circular-mapping, although linear genome structures are found in some taxa. The smallest mitochondrial genomes in the green algae are found in the CW-group (Lewis et al. 1992, Friedl 1997) of the Chlorophyceae (sensu Mattox and Stewart 1984); the CW-group is characterized by the clockwise displacement of basal bodies in flagellated cells and includes all species of the artificial taxon Chlamydomonas and related genera (see Pröschold et al. 2001, and references therein). Essentially the same 12 genes are recognized in the mitochondrial ge- nomes of this group, and the size variation of these genomes (Table 1) is largely a result of optional in- tron content (reviewed by Nedelcu and Lee 1998a). The almost completely sequenced mitochondrial ge- nome of the CW-group taxon Polytomella parva exists in at least two subgenomic-sized linear pieces of 13.5 1 Received 28 March 2002. Accepted 10 October 2002. 2 Author for correspondence: e-mail Robert.Lee@dal.ca.