Chloroplast DNA variation in Chlamydomonas and its potential application to the systematics of this genus

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BioSystems, 18 (1985) 293--298 Elsevier Scientific Publishers Ireland Ltd.

CHLOROPLAST DNA VARIATION IN CHLAMYDOMONAS AND ITS POTENTIAL APPLICATION TO THE SYSTEMATICS OF THIS GENUS

BERTRAND LEMIEUX, MONIQUE TURMEL and CLAUDE LEMIEUX* D~partement de Biochimie, Facultd des Sciences et de Gdnie, Universitd Laval, Quebec, Quebec, G I K 7P4 (Canada)

We have estimated the extent of chloroplast DNA (cpDNA) variation in three species of green algae belonging to the genus Chlamydomonas to determine if this variation could be used for taxonomic studies. The overall arrangement of sequences in the chloroplast genome of Chlamydomonas eugametos was compared with that of the closely related C. moewusii and that of the more distantly related C. reinhardtii. The results show that the chloroplast genomes of C. eugametos and C. moewusii are essentially co-linear and are highly homologous in sequence while those of C. eugametos and C. reinhardtii have been extensively rearranged and share a relatively low overall sequence homology. This wide range of chloroplast genome organization suggests that the analysis of cpDNA variation will be useful for the classification of algae belonging to the Chlamydomonas genus. Keywords: Heterologous fragment hybridizations; Alignment of physical maps; Chloroplast genes; Chloroplast genome evolution; Sequence rearrangements.

1. Introduction and objectives During the last 5 years, the chloroplast genome has proven to be a very useful molecular marker to trace evolutionary changes in the plant kingdom (Giannasi and Crawford, 1984; Palmer, 1984; Palmer et al., 1984, 1985). The main properties that make the chloroplast genome of higher plants well. suited for the determination of phylogenetic relationships are its relatively low complexity and its high degree of sequence conservation. Studies on the chloroplast DNAs (cpDNAs) of over 200 species of angiosperms have revealed that such conservation exists both at the fine level of nucleotide sequence and at the gross level of arrangement of large sequence elements (Palmer, 1984). Variations at the level of sequence arrangement have occurred very rarely during the evolution of the angiosperm chloroplast genome. However, sufficient variations have occurred at the nucleotide sequence level to allow the study of relationships between plants within the same genus or closely related genera. These *To whom all correspondence should be addressed.

fine variations are easily detected by comparing positions of restriction endonuclease sites on cpDNAs; base-pair substitutions are identified as restriction site polymorphisms and small deletions/additions as restriction fragment length polymorphisms. In many cases, such comparisons of cpDNA restriction sites have proven to be a needed alternative to other classification methodologies. In this study, we have addressed the two following questions: (1) Is the chloroplast genome of green algae as conserved as that of angiosperms? (2) Can the chloroplast genome be used as a marker to draw evolutionary relationships between green algae? In an attempt to answer these questions, we analyzed the extent of cpDNA variation among three species of green algae belonging to the genus Chlamydomonas. Chlamydomonas reinhardtii, C. eugametos and C. moewusii were chosen because they remain the only green algae for which cpDNA restriction maps are available (Rochaix, 1978; Lemieux et al., 1985a; our unpublished results). The genus Chlamydom. onas is one of the largest genera of the green algae (Ettl, 1976). In this genus, the classification is limited to the grouping of most

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294 species into clusters of related algae (subgenera and sections), according to morphological characteristics of the vegetative cell (Gerloff, 1940). The interfertile algae C. eugametos and C. moewusii are so similar with regard to physiological and biochemical properties that they have been considered varieties of the same species (Gowans, 1976). C. reinhardtii, however, is believed to be distantly related to these algae. Indeed, C. reinhardtii differs from C. eugametos and C. rnoewusii in many respects, including physiological and reproductive characteristics (Gowans, 1976), cross-reactivity of the cell-walldissolving enzyme (sporangium autolysin) (SchlSsser, 1984), G + C content of nuclear and cpDNAs (Lemieux et al., 1980) and restriction patterns of cpDNA (Lemieux et al., 1980). ~Okb.2

2. Structure and organization of the chloroplast DNAs of C. reinbardtii, C. eugametos and C. moewusii The chloroplast genomes of the three Chlamydomonas species examined here are structurally similar to those of most angiosperms (Whitfeld and Bottomley, 1983). They consist of single circular DNA molecules which are divided into two singie~opy, regions by a large inverted repeat sequence (Fig. 1). The size of this inverted repeat, as well as that of the entire chloroplast genome appears to be less conserved in Chlamydomonas than in most angiosperms (Palmer, 1984). The C. reinhardtii cpDNA, with its size of 190 kilobase-pairs (kbp) (Rochaix, 1978), is the smallest of the three algal cpDNAs. At 243 kbp, the C. eugametos cpDNA is 53 kbp larger than the C. reinhardtii genome, but the size of the two inverted repeats (20 kbp) is comparable (Lemieux et al., 1985a). Although C. moewusii is very closely related to C. eugametos, the cpDNAs of these two species differ by 50 kbp and most of this difference is accounted for by the enlarged C. moewusii repeat (Lemieux et al., 1985b). Physical mapping of a limited number of

Fig. 1. Structure and organization of the chloroplast genome in C. reinhardtii (Cr), C. eugametos (Ce)and C. moewusii (Cm). The inverted repeat sequence k represented by the thick line. The positions of the 16 S and 23 S ribosomal RNA genes, psbA and rbcL are indicated. The ~ m s of the C. reinhardtii, C. eugametos and C. moewusii cpDNAs are 190, 243 and 292 kbp, respectively.

genes on the three algal cpDNAs provided the first indication that the organization of these chloroplast genomes is not conserved. Figure 1 shows that all three algal inverted repeats encode the genes for the 16 S and 23 S ribosomal R N A s as well as the gene for the "32 kilodalton" thylakoid membrane protein (psbA) (Erickson et al.,1984; Lemieux et al., 1985b). The C. eugametos and C. moewusii inverted repeats also contain the gene for the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase (rbcL) (Lemieux et al., 1985b). In C. reinhardtii, however, rbcL is located in a single-copy region (Malnoe et al., 1979). Obviously, a more detailed comparison of the arrangement of sequence elements common to the three algal cpDNAs was needed to determine the extent of sequence rearrangements between these cpDNAs.

295 3. Overall sequence homology between the chloroplast DNAs of C. reinbardtii, C. eugam e w s and C. moev;usii We tested if the cpDNAs of the three Chlamydomonas species share enough overall sequence homology to allow us to compare the arrangement of their common sequence elements. EcoRI digests of the three algal cpDNAs were electrophoresed in an agarose gel, the resolved fragments were transferred to a nitrocellulose filter and this filter was hybridized with 3:P-labelled whole cpDNA from C. eugametos. Comparison of the intensities of the heterologous versus homologous hybridization signals indicated that the cpDNA of C. eugametos is more closely related to the C. moewusii cpDNA than to the C. reinhardtii cpDNA (Fig. 2). The amount of conserved sequences and degree of sequence homology between the C. reinhardtii and C. eugametos cpDNAs, however, are sufficiently high to permit a detailed comparison of the arrangement of sequence elements common to these genomes.

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-1 Fig. 2. Overall sequence homologies between the cpDNA of C. eugametos and each of the C. reinhardtii and C. moewusii cpDNAs. A nitrocellulose filter containing EcoRI cpDNA digests from C. eugametos (Ce), C. reinhardtii (Cr) and C. moewusii (Cm) was hybridized with nick-translated whole cpDNA from C. eugametce as described in Lemieux and Lemieux (1985). Alongside the 0.8% agarose gel electrophoretic pattern are shown the corresponding eutoradiogranm obtained after 3-h and 48-h exposures. The C. eugametos hybridization pattern matches the corresponding restriction pattern, thus indicating that the cpDNA probe was evenly labelled.

4. Comparison of the arrangement of sequence elements common to the chloroplast DNAs Df C. reinbardtii, C. eugametos and C. raoewusii

Heterologous fragment hybridizationswere used to compare the arrangement of sequence elements shared between the C. eugametos c p D N A and each of the C. reinhardtii and C. moewusii cpDNAs. Individual cloned c p D N A restriction fragments from one of the algae being compared were hybridized to filterbound c p D N A restrictionfragments from the other alga. The resultsof these hybridizations are summarized in Fig. 3. Clearly, there is a greater variation in the organization of the chloroplastgenome within the genus Chlamydomonas than within most angiosperms. Numerous sequence rearrangements are observed between the C. eugametos and C. reinhardtii c p D N A s and these are so extensive that they cannot be explained in terms of single events, such as inversionsand deletions. Based on these results,we suggest that the C. eugametos and C. reinhardtii algal lineages were established earlier than the angiosperm lineages. Nonetheless we cannot exclude the possibility that the cpDNAs of these algae have evolved fasterthan those of angiosperms. An independent measure of the time of divergence separatingthe C. eugametos and C. reinhardtii lineagesis needed to decide between these hypotheses. In contrast, the cpDNAs of C. eugametos and C. moewusii share essentially the same arrangement of c o m m o n sequences. Virtually every restrictionfragment from C. moewusii has its homologous counterpart(s) in C. eugametos. T w o separate regions of the C. moewusii chloroplast genome, however, contain sequences unique to thisalga.The firstconsists of a 5.9 kbp sequence located in the singlecopy region bordering the 16 S ribosomal R N A genes, and the second of a 21 kbp sequence located in the invertedrepeat region between psbA and rbcL. The lattersequence accounts for the largersizeof the C. moewusii repeat relativeto that of C. eugametos (Lemi-

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C. eugametos Fig. 3. Compared arrangements of c o m m o n sequence elements on the cpDNAs of C. eugametos and C. reinhardtii (upper) and on the cpDNAs of C. eu&ametos and C. moewusii (lower). The C. eugametos/C, reinhardtii comparison was taken from Lemieux and Lemieux (1985), while the C. eugametos/C, moewusii comparison was taken from our unpublkhed data. The cpDNAs of each pair of algae were linearized and the cro~-hybridizing regions were connected. The EcoRI restriction sites on the three algal cpDNAs are indicated by (I) and the BamHI sites on the C. refnhardtit cpDNA by (~). The gene nomenclature proposed by Hallick and Bottomley (1983) was used to denote the genes that have been mapped on the cpDNAs of the three algae. The positions of the C. reinhardfi/genes were taken from: Rochalx and Malnoe (1978) for the rRNA genes; Malnoe et al. (1979) for rbcL; Watson and Surzycki (1982) for tufA; Erickson et al. (1984) for pshA; Roehaix et al. (1984) for pshD; Woesner et al. (1984) for atpA, atpB and atpH. The position of the C. eugametos and C. moewulii genes were taken from: Lemieux et al. (1985b) for the 16 S and 23 S ribosomal RNA genes, pshA and rbeL; our unpublished data for atpA, atpB, atpH and psbD.

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eux et al.,1985b). Apart from these two large deletions/additions, the C. eugametos and C. moewusii c p D N A s display restriction siteand fragment length polymorphisms (unpublished results). Although such minor differences are c o m m o n in the c p D N A s of closely related higher plants, deletion/addition differences as important as the 21 kbp sequence have not been observed in these cpDNAs (Palmer, 1984; Palmer et al., 1984). 5. Conclusion The most important finding of this study is that there is a wider range of cpDNA variation in a given genus of green algae than within the vast majority of higher plants. We believe that the analysis of this variation will be useful to green algal systematics. The analysis of smallscale c p D N A variation islikelyto be applicable only to the classificationof veryclosely related green algae. Our results indicate that it is possible to align the c p D N A restriction maps of the closely related C. eugametos and C. moewusii and to analyse the minor differences in the position of restriction sites. However, this is impossible in the distantly related C. eugametos and C. reinhardtii because of the extensive variation in the arrangement of their cpDNA sequences. The mapping and comparison of chloroplast genomes from other Chlamydomonas species is needed to determine if intermediate cases can be found between these two extremes in the arrangement of common sequences. Sequence rearrangements may not always be as numerous as those detected between C. eugametos and C. reinhardtii and more importantly, they could be interpreted in terms of individual events. If this proves to be the case, the analysis of the rearrangement events will be applicable to the determination of phylogenetic relationships at the level of the entire Chlamydomonas genus. Acknowledgements We thank Robert W. Lee and Rose Ann Cattolico for critically reading the manuscript.

W e are also grateful to Guy Bellemare for providing the laboratory space and facilitiesfor this research. This investigation was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. References Erickson, J.M., M. Rahire, J.D. Rochaix, 1984, Chlamydomonas reinhardtii gene for the 32 000 tool. wt. protein of photosystem II contains four large introrts and is located entirely within the chloroplast inverted repeat. EMBO J. 3, 2753-2762. Ettl, H., 1976, Die Gattung Chlamydomonas Ehrenberg (Cramer, Varduz). Gerloff, J., 1940, Beitr~/ge zur Kenntnis der Variabilitiit und Systematik der Gattung Chlamydomonas. Arch. Protistenkd. 94, 311--502. Giannasi, D.E. and D.J. Crawford, 1984, Plant chemosystematics. Evol. Biol., in press, Gowans, C.S., 1976, Genetics of Chlarnydomonas moewusii and Chlamydomonas eugametos, in: The Genetics of Algae, R.A. Lewin (ed.) (Blackwell Scientific Publication, Oxford) p. 145. Hallick, R.B. and W. Bottomley, 1983, Proposals for the naming of chloroplast genes. Plant Mol. Biol. Reporter 1, 38--43. Lemieux, B. and C. Lemieux, 1985, Extensive sequence rearrangements in the chloroplast genomes of the green algae Chlamydomonas eugametos and Chlamydomonas reinhardtii. Curt. Genet., in press. Lemieux, C., M. Turmel and R.W. Lee, 1989, Characterisation of chloroplast DNA in Chlamydomonas eugametos and C. moewusii and its inheritance in hybrid progeny. Curr. Genet. 2,139---147. Lemieux, C., M. Turmel, V.L. Seligy and R.W. Lee, 1985a, The large subunit of ribulose-l,5-bisphosphate carboxylase-oxygenase is encoded in the inverted repeat sequence of the Chlamydomonas eugametos chloroplast genome. Curt. Genet. 9, 139--145. Lemieux, C., M. Tunnel, G. Bellemare and R.W. Lee, 1985b, A 21 kilobase-pair deletion/addition difference in the inverted repeat sequence of chloroplast DNA from Chlamydomonas eugametos and C. moewusii. Plant Mol. Biol., 5, 77--84. Malnoe, P., J.D. Rochaix, N.H. Chua and P.F. Spahr, 1979, Characterization of the gene and messenger RNA of the large subunit of ribulose-l,5-diphosphate carboxylase in Chlamydomonas reinhardtii. J. Mol. Biol. 133,417--434. Palmer, J.D., 1984, Evolution of chloroplast and mitochondrial DNA, in: Monographs in Evolutionary Biology: Molecular Evolutionary Genetics, R.J. MacIntryre (ed.) (Plenum, New York).

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