1. 15
Chloroplast Gene Organization and
Phylogenetic Relationships in Green Algae
M. Turmel", E. Boudreau', R.R. Gutell', C. Otis' and C. Lemieux'
Dcpartemenl de biochimie, Faculle des sciences el de genie, Universite Laval. Quebec (Quebec) GIK 7P4.
Canada
2 Departments ~r Molecular, Cellular and Developmental Biology. Campus Box 347. University oj Colorado.
Boulder. CO 80309, USA
INTRODUCTION
Although the.chloroplasts of green algae and land plants share a common endosymbiotic
origin, their genomes appear to have followed very different evolutionary pathways.
It is well known that the land plant chloroplast genome evolves very conservatively and
• All correspondence should be sent to Monique Tunnel at the above address.
147

2. • .0..1." .."".... .h' .... ' ....... o..IlIU I.'''U'''''II
is under strong constraints to retain a compact gene organization (reviewed by Palmer 1991).•
Indeed studies of over 1000 rhotosynthetic plant srecies have indicated that this circular
~~
genome is remarkably conserved in structure, size (120-160 kb), gene content, primary~, j
sequence and overall gene order. Most of the 110-1 18 chloroplast genes encoded are grouped ,
into multicistronic operons, several of which are highly similar to those found in cyano- I
bacteria, the ancestors of chloroplasts. IIn contrast, the limited data available on green algal chloroplast DNAs (cpDNAs) have
revealed great variability in structure, size (89- >400 kb) and gene organization (reviewed by •
Palmer 199 I). Low-resolution chloroplast gene 'maps have been reported for only eight I
green algae representing three of the five major classes that have been proposed by Mattox, I
and Stewart (1984) on the basis of ultrastructural characters (see Palmer 1991). These three I
classes are the Charophyceae, whose members are the closest relatives of I~nd .plants,. the :m. 'I'
Ulvophyceae and the Chlorophyceae. Although all green algal cpDNAs studied In detail so 'Jfar consist of circular DNA molecules, some of which feature the large inverted repeat ,.;;;. ,
characteristic of the land plant genome, none of them shows strong similarity with the'·' I
consensus gene order found in land plants. Extensive gene rearrangements have been Iobserved even within the genus Chlamydomonas. In this large and highly diversified group
ofgreen algae, two divergent pairs of interfertile taxa have been examined: one pair consists
of C. reinhardtii and C. smithii and the other of C. eugametos and C. moewusii. On the basis I
of heterologous hybridizations with cloned cpDNA fragments spanning the compared I
chloroplast genomes, the cpDNAs of C. reinhardtii (196 kb) and C. eugametos (243 kb) i
have been found to be extremely scrambled in their gene order (Lemieux and Lemieux 1985), I
whereas the cpDNAs within each pair of interfertile algae have been found to be essentially Icolinear (Turmel et af. 1987; Boynton et af. 1992).
To gain insight into the tempo and, mode of evolution of the chloroplast genome in i
Chlamydomonas, we have recently examined the phylogenetic relationships among representa- I
tives ofthis genus as well as of other green algal genera, and have also begun to investigate
the organization ofabau! 75 chloroplast genes in representatives ofthe various Chlamydomonas
lineages identified. We report here the preliminary results of our phylogenetic analysis
based on the chloroplast large subunit rRNA gene (rrnL) and briefly review our most recent·
studies on the structure and organization of chloroplast genes in C. eugametoslC. moewusii
and C. reinhardtii, i.e. in members of the two major lineages found in Chlamydomonas. In
the presentation ofour results, we have placed a special emphasis on the differences between
these green algal cpDNAs and their land plant counterparts.
MATERIALS AND METHODS
Sequencing ojthe chloroplast rmL gene and phylogenetic analysis
The chloroplast rrnL gene from 28 green algae (see legend ofFig.15.1) and from the
chlorarachniophyte-like species designated Pedinomonas minutissima (see Chapman and
Buchheim 1992) was partially sequenced. For each organism, three overlapping segments of
the gene were PCR-amplified from total cellular DNA preparations with pairs of primers that
are complementary to highly conserved regions (Turmel et af. 1993a). PCR-fragments were
sequenced using the dsDNA cycle sequencing system from Life Technologies, Inc. (Gaithers-
burg, MD). Alignment of all chloroplast rrnL sequences and their analysis with the neighbor-
148
joining method ofSaitou and Nei (1987) were carried Ollt as described previollsly (Tunnel ('(
til. 1993n).
RESULTS AND DISCUSSION
ChlamydomonasJorms a highly diversified group ofgreen algae within the
Chlorophyceae
To understand the evolutionary basis for the extensively rearranged cpDNAs in
Chlamydomonas, we have inferred the phylogenetic positions of C. reinhardtii and C.
moewlisii relative to those of other green algae from this genus as well as from other genera
by comparing sequences for the chloroplast rrnL gene. The phylogeny presented in
Fig. 15. I features 45 green algae from the classes Chlorophyceae, Pleurastrophyceae and
Micromonadophyceae sensu Mattox and Stewart (1984), eight land plants, two euglenophytes,
one chlorarachniophyte-like species, one brown alga and one red alga. The 25 Chlamydomonas
taxa examined represent all of the groups that were distinguished in this genus on the basis
of morphological and biochemical characters (Ettl 1976; Schiisser 1984). These taxa cluster
into two major lineages, one of which comprises C. reinhardtii and the other C. moewlisii.
Within each of these major lineages, there are additional lineages that form sister groups. As
reported previously (Buchheim et af. 1990), Chlamydomonas is clearly polyphyletic as some
taxa classified in this genus (e.g. C. applanata ) are more closely related to green algae
belonging to other genera (e.g. Chlorogonium and Dunaniella) than to other Chlamydomonas
taxa. Altogether, the Chlamydomonas lineages show at least twice the range of sequence
divergence seen in all land' plants. This molecular diversity is supported by the greater
variability in chloroplast gene organization among Chlamydomonas taxa as compared to that
observed among land plants. In the inferred phylogeny, the micromonadophycean green
algae and land plants clearly occupy basal positions relative to the pleurastrophycean and
chlorophycean green algae, a result that is consistent with a recently reported phylogenetic
analysis based on partial sequences of the nuclear small subunit rRNA (Kantz et af. 1990).
.The highly rearranged cpDNAs oJe. moewusii and C. reinhardtiifeature conserved
gene clusters
We have extended to 74 and 75 the number of genes mapped on the C. reinhardtii and
C. moewusii cpDNAs, respectively (see Fig. 15.2) (Boudreau et af. 1994). To map additional
genes on these cpDNAs, we first attempted, but with little success, to carry out Southern blot
hybridizations with gene-specific fragments from the tobacco cpDNA. Since, most of the
probes proved ineffective, we undertook the partial sequencing ofthe colinear C. eugametos
and C. moewusii cpDNAs. We have sequenced thus far about 140 kb of the genome using
both random and directed sequencing approaches. The resulting sequences along with those
reported for the C. reinhardtii cpDNA allowed us to generate gene-specific probes that proved
useful in heterologous hybridizations., The results of these hybridizations indicated that the
C. moewusii and C. reinhardtii cpDNAs share a similar gene complement. Only four of the
genes mapped, tseA and three open reading frames (ORF715, ORFA and ORFB), have not
been reported in any cpDNAs and may thus be specific to Chlamydomonas or a larger
taxonomic group. Interestingly, rpl5 resides on both the C. reinhardtii and C. moewusii
cpDNAs, but is absent from the cpDNAs ofland plants (Ohyama et al. 1986; Shinozaki et af.
1986; Hiratsuka et af. 1989). From our results, it appears that a very small fraction of the
149

3. Fig. 15.1
C. mexicana
C. peterf,;
C. gigantea
C lrankii
- C. palidostigmata
~--- C.nivalis
C. mdiata
C.mutabilis
Chlorogonvm dmglltum
C.humicola
C.lIpplanala
~ DunalitJIa paIVa
C. lIgtul(ormis
Stephanosptz.tua pllviafs
' - - - - - HaematoCDCDlS lacustris
C. pischmlJnni
C. ~ugllm.tos
C. mOflwusi
O'lbrococcum ttchnozygotum
C. species 66.72
C.geifleri
C. pstJudapettusa
C. monadna
~
Catteriacrudera
camuiatildiosa
Garteria luzsnsis
~S::::/:::,s:~~~'::tus
L-_____ Uonema btlDcae
Chlomlfa vulgaris
Prototheca widuuhami
o:T
o
a1:J
:T
'<
o
(!)
III
m
"U
m-e
OJ
!!!.
a1:J
~
o
(!)
III
m
Chlamlla elipsoidea
Tmbouxia aggre9a,a
{=====-TetJaselmiscatteriilormis
Pleur-utrum tenestra
L-___ Pedinomonas mnor
;;::
fi'
o
3
o
=>
::jJ
~
=-!OI)'U sativa
aa mays
Nicotiaflll labacum
Anusncanus
Eplagus rig'iniana
- Conopho& americm..
PIsum sativum
Uan:/lantia polymotph..
' - - - - - i'kphlOsernis OMara
f----------- M.i:mmonas pusila
1-----:-----'- Pedinomonasmnutissina
G Pyfaiela iftorais
Pamatia pamata
Astasia Jonga
Euglena gracilis
Anacystis nidulans Escherichia. col
r'<
III 0
=> m
a. III
1:J m
iii
=>
u;
==:J-~o
g
=>
D>
a.
.g
~
g
D>
m
Neighbor-joining phylogeoetic analysis ofplastid rmL sequences ftum 45 green algal taxa (Chlorophyceae,
Pleurastrophyceae and Micromonadophyceae sensu Mattox and Stewart). eight land plants. two
euglenophytes (Astaria longa and Euglena gracilis), one chlorarachniophyte-like species (Pedinomonas
minutissimo), one brown alga (Pylaiella littoralis) and one red alga (Palmoria palmata). This tree, which
includes also a cyanobacterium (Anacystis nidu/ans), is a 50% majority-rule tree derived from a Kimura
dissimilarity matrix that was computed from a data set of 2,1 02 semiconserved positions. One thousand
bootstrap replications were done in. which the Escherichia coli rmL sequence was used as an outgroup.
Branch lengths on the horizontal axis represent the evolutionary distance between the nodes. The P.
minutissima chloroplast rrnL sequence and all green algal rrnL sequences, with the exception of that
from Chlorella elJipsoidea (accession no.: M36158), were determined in our laboratories. Of these
sequences, those from 17 Chlamydomonas taxa have been reported (see Tunnel et al. 1991a, 1993a).
Accession numbers for the published non-green algal sequences are as follows: Oryza sativa, X15901; Zea
mays, X01365; Nicotiana tabacum, JOl446 and Z00044; Alnus incana, M76448 and M75722; Epifagus
virginiana, M81884; Conopholis americana, X59768; Pisum salivum, M37430; Marchantia pO(l'morpha,
XOI647 and X04465; Astasia longa, X14386; Euglena gracilis, XI331O; Pylaiella littoralis, X61179
and 536159; and Palmaria palmata, ZI8289
150
.."..
f"i~'l:,1~1 "1
-)< 1 ,
;~ili
~ ':
II
I
" I
I,
Increased size of Chlamydomonas cpDNAs relative to their land plant countcrparIs is
explained by the presence of additional genes. Most of this extra size seems 10 be due to the
presence of enlarged spacers between coding regions and also to Ihe presence of unusually
long genes (see below) (Boudreau et al. 1994).
As shown in Fig. 15.2, all gene rearrangements between the C. moewlIsii and C.
reinhardtii cpDNAs, with the exception of those accounting for the relocations of rbcL and
the apiA and psbl gene pair, occurred within corresponding regions of the genome. Despite
these numerous rearrangements, 40 genes were found to define 13 conserved clusters of
closely linked loci (see Fig. 15.2). Ten of the 13 clusters (rpsIB-rps2-trnD-psbB-ycj8-psbH-
tmEI, ycJ3-ycJ4 and petB-chlL are the exceptions) have been partially or entirely sequenced,
and interestingly, each of them features contiguous genes that are encoded by the same DNA
strand. It is very likely that these ten gene clusters were present in the common ancestor of
all Chlamydomonas species. Only 16 of the genes mapped on the C. moewusii and C.
reinhardtii cpDNAs are organized similarly to ancestral operons found in other cpDNAs (see
Fig. 15.2); they reside within five of the 13 conserved clusters identified. The rRNA gene
cluster shows exactly the same gene content as its land plant homologue, whereas segments
ofland plant operons are represented by the four remaining clusters. These results suggest
that most of the ancestral operons that characterize the chloroplast genome organization of
land plants and early-diverging photosynthetic eukaryotes have been disrupted before the
emergence of Chlamydomonas. Of the multiple sequence rearrangements that marked the
evolution of the Chlamydomonas chloroplast genome, one seems to have led to the break-up
of the ancestral region containing rp123, rpl2, rpsl9, rpll6, rpll4, rpl5, rpsB and thepsaA
exon 1. This gene cluster, which differs from the corresponding land plant operon by the
absence ofrp122, rps3 and infA and also by the presence ofrpl5 and the psaA exon 1, is found
in C. reinhardtii, while it is divided into two separate fragments, rpI23-rpI2-rps19 and rplJ6-
plJ4-rpI5-rpsB-pasA exon I, in C. moewusii (see Fig. 15.2).
The molecular mechanisms underlying the tremendous rearrangements that occurred
during the evolution of the Chlamydomonas chloroplast genome remain unknown. As
discussed previously (Boudreau et af. 1994), repeated sequences located in intergenic spacers
and/or tRNA genes might have been implicated in such rearrangements. To identify the
nature ofthe sequence elements involved ip gene reshuffling, it will be necessary to analyze
the endpoints of rearranged cpDNA segments from closely related green algae showing
specific mutations. In this regard, it would be interesting to undertake the molecular character-
ization of the few rearrangements we have uncovered between the cpDNAs of C. moewusii
and C. pitschmannii (Boudreau and Turmel 1995) and between those of C. reinhardtii and C.
gelatinosa (see Tunnel et af. 1991b).
As more cpDNAs from chlorophycean green algae are investigated, we expect that a
number of the gene clusters conserved between C. moewlIsii and C. reinhardtii will be
identified in taxa residing in basal lineages relative to the Chlamydomonas lineages.
At present, the petA-petI? gene cluster is the only one that has been reported outside
Chlamydomonas. This cluster has been identified in the chlorophycean green alga Scenedesmlls
obliqulls (Kiick 1989). Like its Chlamydomonas counterparts, the Scenedesmlls petA and petD
genes are contiguous and encoded by the same DNA strand, suggesting that these genes were
present in the most recent common ancestor ofChlamydomonas and Scenedesmus. We suspect
that the gene cluster from the latter ancestor also comprised the tRNAM. (UeU) gene, which
is present immediately downstream ofpetD in both C. eugametos and Scenedesmlls. In C.
151

4. C. reinhardtii
196 kb
atpE
a'pH
rpoBl
rpoB2
rpoC2b
rpoC2.
fefS
puB
S[GCUI
atpl,rp1J.4
I;~r-;P~___J
a.......,
,,~
p.bK, CjOCAI, WICCAI
I rplid Sru:;J.1 I
ctllB TIUGUI
AIUCU]
psbE-psbF-psbL-psbJ
psbB-ycfB-psbH-petB-petD
rmS-tmt-tmA-f77lL~ rONA
rpt23-rpt2-rpsI9-rpI22-rps3-rptl6-
rpI14-rpsB-intA-rpl36-rpsll-rpaA
rpt23-rpt2-rpsl9-rpI22-rps3-rptl6-
rp/14-rpsB-infA-rpl36-rpsll-rpaA
C. moewusii
292 kb
152
'1' i
i~:': . f'Hi:;. .:,
''; .'..
l.i~
.>~
.·:fizt
. '!!l
;;r"
:;~
I
I
III(}('I·U.III, petO and Imlll ul·U) arc separaled by an exira sequcncc (II' 5.9 kh, which alSo exISis
as a linear DNA in this alga (Turmel el al. 1986).
Chlamydomonas Gild lalld plalll cpDNAs differ ill their ;,lIron content
Different sets of genes are interrupted by g';oup I and group II inIrons in land plant and
Chlamydomonas cpDNAs, indicating that most ofthese introns have a recent and independent
origin. While group I introns are more abundant than group [[ introns in Chlamydomonas
cpDNAs, the opposite situation prevails in land plant cpDNAs. A total of 24 group I intron
insertion sites located within five Chlamydomonas chloroplast genes have been described; in
contrast, a single group I intron [in the tRNALEU (UAA) gene] has been identified in land plant
cpDNAs_ Twelve of the Chlamydomonas chloroplast group I insertion sites have been found
during a recent analysis of the rrnL gene from 17 Chlamydomonas species (Turmel el al.
1991a, 1993a), while the twelve remaining sites have been identified in the rrnS (four sites),
psbA (five sites),psbC (two sites) and psaB (one site) genes of C. reinhardlii, C. eugamelos
and/or C. moewusii (see Turrnel el al. 1993b; our unpublished data). Ofthese sites, only three
(in the rmL gene) have been reported outside the polyphyletic genus Chlamydomonas (see
Turrnel e/ al. 1993a; Turrnel el al. 1995a). Five rrnL insertion sites were observed in the two
major Chlamydomonas evolutionary lineage, whereas the seven remaining ones were found
to be restricted to one or the other lineages, a result suggesting that the origin ofthe latter sites
is more recent To gain insight into the mode ofinsertion and proliferation of group I introns
in cpDNAs, we have undertaken the sequencing of the Chlamydomonas chloroplast introns
inserted at common positions as well as at distinct sites. Analysis of the intron sequences
available so far strongly suggests that, in two pairs ofrelated introns (CmpsaB'I/CepsbC'2 and
CmLSU-4/CeLSU'2), one of the members arose from transposition mediated through reversal
of the self-splicing reaction (Turrnel et al. 1993b). Some Chlamydomonas introns may have
also arrived at their present locations through transposition events imd/or lateral transfers that
were initiated by double-strand DNA breaks caused by intron-encoded endonucleases, as
CeLSU·5 (Gauthier et al. 1991), CrLSU·I (Diirrenbergerand Rochaix 1991), ChLSU·I (Cote
et al. 1993) and CPLSU·2 (Turmel et al. 1995a) have been shown to encode distinct
endonucleases that cleave specifically the exon junction sequence in the corresponding
intronless genes..
Fig. 15.2 Comparative gene organization ofthe C: moewusii and C. reinhardtii cpDNAs. DNAs are drawn to scale
and are linearized at one ofthe junctions ofthe inverted repeat (denoted by thick lines) and the single-copy
region bordering the rrnS genes. Gene loci are denoted by dark areas, with their size reflectiog the length
ofcoding regions. Note that the rrnL and psaA coding regions from both green algae as well as those of
the C. moewusii rmS, psaB and psbC are oversized in this figure, as the intraD sequences interrupting them
were not represented, All corresponding C. mo~ii and C. reinhardtii gene loci are connected by lines:
those that are part of conversed clusters (framed areas) are linked by solid lines, whereas the remaining
genes are connected by dashed lines. To,the right of five conversed clusters are indicated the land plant
chloroplast operons to which they share similarity, ForaH genes that are indicated on only one of the two
green algal cpDNAs, our heterologous hybridizations failed to identifY their counterparts in the compared
DNA- For each gene, the polarity ofthe DNA strand containing the coding region was denoted by an arrow
when this infonnation was available. Contiguous genes with the same polarity were assigned a common
arrow. This figure was modified from Boudreau et al. (1994). Note that the C. moewusii trnL (UAG) gene
was indicated at an incorrect position in the figure presented by Boudreau et ai, (1994); it was localized
by heterologous hybridization to the EcoRl fragment 10'" containing the 5' part of clpP and psaA exon 2,
but was inadvertently assigned to the adjacent fragment containing the 3' pan ofclpP, tm! (CAU) and trnjM
(CAU). The relative order of trnL (UAG) and psaA exon 2 has been recently determined by DNA
sequencing (our unpublished data)
153

5. In Chialllydolllollas, psa:l is lhe only gcne lhal is known 10 contain group II IIltrons.
Two trans-spliced psaA inlrons have been idenlified in C. reinhardlii (Klick el al. 1987;
Choquel el al. 1988). Our hybridizalions Wilh specific exon probes suggcSl lhallhe psaA
genes of six other Chiamydomonas species, including C. ellgametos and C. moewll.';il
resemble their C. reinhardtii counlerpart in consisting of three widely spaced exons
by 5'- and 3'-segments of group II introns (Turmel el al. 1995b; our unpublished data).
all lhe chloroplast psaA genes characterized so far in other algae and land plants do
feature any trans-spliced introns nor cis-spliced introns at the same locations as those
in C. reinhardtii, insertion and splitting of group II introns probably occurred
recently in a common ancestor of Chlamydomonas species.
Some Chlamydomonas chloroplast genes exhibit an unusual structure
Although the chloroplast rrnL gene is fragmented in Chlamydomonas and land plants,
corresponding gene products in these two groups oforganisms share no common
(see Turmel et al. 1993a). Four mature chloroplast large subunit rRNA fragments, called a,
f3, rand 8, have been identified in all 17 Chlamydomonas taxa examined thus far, whereas two
to three mature rRNA species have been observed in land plants. These differences reflect
variations in the location and number of short internal transcribed spacers that are excised
from the primary transcript to yield the mature rRNA species.
The Chlamydomonas chlB, clpP and rps3 are functional genes despite the presence of
long insertion sequences relative to their homologues in land plants and other organisms
(Fong and Surzycki 1992b; Li et al. 1993; Liu el al. 1993a,b; Huang et al. 1994; Turmel and IOtis 1994). Remarkably, these insertion sequences are in frame with the coding regions and
show no similarity with group I and group II introns and with known DNA sequences. They
are not excised at the RNA level; so if they are translated and not removed at the protein leve4=". ~
the corresponding proteins would be significantly larger than their land plant counterp~ :'~' ISimilar insertion sequences have been identified at the same positionS in the chlB, clpP and
rps3 genes ofspecies from the two major Chlamydomonas lineages, suggesting that they have
a common origin and that they were present in the comnion ancestor of Chlamydomonas
species. One ofthe two sites ofinsertion sequences in the C. eugametos clpP gene, howevCIj
appears to be of more recent origin, as it has not been found in all other ChlamydomonaS
species examined.
The Chlamydomonas rpoB and rpoC2 genes are very unusual in featuring more than on~
ORF. If transcription and translation occur and there is no edited RNA sites, each of these
genes would be expected to specifY separate polypeptides. Fong and Surzycki (1992a) have
reported that the C. reinhardtii rpoB consists of two ORFs (designated rpoB] and rpoB2 in
Fig. 15.2) that are closely linked to each other on the genome ofthis green alga. Their order
and polarity are those found in conventional, unfragmt;nted rpoB genes, suggesting that
insertion ofa sequence element might have led to splitting ofthe C. reinhardtii gene. In the
C. ellgametos and C. moewusii cpDNAs, the ';poB sequences corresponding to these ORFs
map also to closely linked loci (Boudreau et al. 1994). Results derived from partial
sequencing of the rpoC2 gene in both C. reinhardtii (Fong and Surzycki 1992a) and C.
eugametos (our unpublished results) and from Southern blot hybridizations (Boudreau et al.
1994) suggest that the C. reinhardtii gene displays at least two ORFs (designated rpoC2a and
rpoC2b) that are close to each other on the chloroplast genome. Disruption of one of these
ORFs has revealed that the C. reinhardtii rpoC2 gene is functional (Goldschmidt-Clermont
154
Il)l) I; ,ce Boudreau etal. I'N4). By Soulhern bioI hybridizalions. we have found lhal closely
linked loci correspond to Ihe identified C. reinhart/Iii ORFs in lhe C. C'ugolllelos and C.
1I/01!1I'lisii cpDNAs (Boudreau el al. 1994). Obviously, delerminalion of lhe complele
nucleolide scquence of the C. reinhardlii rpoC2 along wilh analyses at the RNA and protein
levels will be necessary 10 understand how this gene transcripl is translaled into a functional
protein.
In conclusion, the sequence data that have been collected from Chlamydomonas cpDNAs
indicale that a few genes have been invaded by foreign sequences, but have remained
functional. Survey of these genes for the presence of similar insertion elements among other
green algal lineages will be needed to determine when such events occurred during the
evolution of the green algal chloroplast genome.
Acknowledgment: This research was supported by grants from the National Sciences and
Engineering Research Council of Canada (GP0003293 to M.T. and GP0002830 to c.L.) and
"Le Fonds pour la Formation de Chercheurs et l'Aide ala Recherche" (93-ER-0350 to M.T.
and c.L.). M.T. and c.L. are Scholars in the Evolutionary Biology Program of the Canadian
Institute for Advanced Research
REFERENCES
Boudreau E, Tunnel M(1995): Gene rearrangements in Chlamydomonas chtoroplast DNAs are accounted for by
inversions and by the expansion/contraction of the inverted repeat. Plant Mol. BiD!.. 27: 351-364.
Boudreau E, Otis C, Tunnel M(1994): Conserved gene clusters in the highly rearranged chloroplast genomes of
Chlamydomonasmoewusii and Chlamydomonas reinhardtii. Plant Mol. Bioi. 24: 585-602.
Boynton lE, Gillham NW, Newman SM, Harris EH (1992): Organelle genetics and transformation of
Chlamydomonas. In: Hennann RG (ed) Plant Gene Research, Vol. VI. Springer-Verlag, Vienna. pp.3--{)4.
Buchheim MA, Tunnel M, Zimmer EA, Chapman RL (1990): Phylogeny of Chlamydomonas (Chlorophyta) based
on cladistic analysis ofnudear 18S rRNA sequence data. J. Phycol. 26: 689--{)99.
Chapman RL, Buchheim MA (1992): Green algae and the evolution ofland plants: inferences from nuclear-encoded
rRNA gene sequences. BioSystems 28: 127-137. ()
Choquet Y, Goldschmidt-Clennont M, Girard-Bascou 1, KUck U, Bennoun P, Rochaix lD (1988): Mutant phenotypes
support a trans-splicing mechanism for the expression of the tripartite psaA gene in the C. reinhardtii
chloroplast. Cel/52: 903-913.
Cote V, Mercier lP, Lemieux C, Tunnel M (1993) The single group-I intron in the chloroplast rrnL gene of
Chlamydomonas humicola encodes a site-specific DNA endonuclease (l-chu J). Gene 129: 69-76.
Durrenberger F. Rochaix 1D (1991): Chloroplast ribosomal intran of Cltlamydomonas reinhardtii: in vitro self-
splicing, DNA endonuclease activity and in vivo mobility. EMBOJ. 10: 3495-3501.
Ettl H(1976): Die Gattung Chlamydomonas Ehrenberg. Nova Hedwigia 49: 1-1122.
Fang SE, Surzycki S1 (1992a): Organization and structure of plastome psbF. psbL, petG and 0RF712 genes in
Chlamydomonas reinhardtii. Curro Genet. 21: 527-530.
Fong SE, Surzycki S1 (l992b): Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an
unusual structure and arrangement. Curro Genet. 21: 485-497.
Gauthier A, Tunnel M, Lemieux C (199I): A group 1intran in 'he chloroplast large subunit rRNA gene of
Chlamydomonas eugametos encodes a double-strand endonuclease that cleaves the homing site of this intran.
Curro Genet. 19: 43-47.
Goldschmidt-Clennont M(199.1): Transgenic expression of aminoglycoside adenine transferase in the chloroplast:
a selectable marker for site-specific directed transformation of Chlamydomonas. Nucleic Acids Res. 19:
4083-4089.
Hiratsuka 1, Shimada H, Whittier R, Ishibashi T, Sakamoto M, Mori M, Kondo C, Honji Y, Sun CR, Meng BY, Li
YQ, Kanno A, Nishizawa Y. Harai A, Shinozaki K, Sugiura M(1989): The complete sequence of the rice
(Or)'za sativa) chloroplast genome: Intermolecular recombination between distinct tRNA genes accounts for a
major plastid DNA inversion during the evolution of the cereals. Mol. Gen. Genet. 217: 185-194.
155

6. ,
c
;;
-~t
~
tl
.1j
i
~
j
ijj
J
I
f
J~
Huang C. Wang S. Chen L. Lemieux C. Olis C. Tunnel M. Liu XQ (1994): The CMaml'donwllas chloroplasl cJpp
gene contains translated large insertIOn sequences and is essential for cell growth. Mol. Gen. Genet.244i ~
151-159. .
Kanlz TS. Theriot EC, Zimmer EA. Chapman RL (1990): The Pleuraslrophyceae and Micromonadophyceae: a
cladistic analysis of nuclear rRNA sequence data. J. Phycol. 26: 711-721.
Klick U (1989): The intran of a plastid gene from a green alga contains an open rcading frame for a
• transcriptase·like enzyme. Mol. Gen. Genet. 218: 257-267.
Klick U, Choquet Y. Schneider M, Dron M, Bennaun P (1987): Structural and transcription analysis of twc'i
homologous genes for the P700 chlorophyll a-apoproteins in Chlamydomonas rdnhardtii: evidence for in
vivo trans-splicing. EMBO 1. 8: 2185-2195.
Lemieux B, Lemieux C (1985): Extensive sequence rearrangements in the chloroplast genomes of the green
Chlamydomonas eugametos and Chlamydomonas reinhardtii. Curro Genet. 10: 213-219.
Li J. Goldschmidt-Clennont M, Timko MP (1993): Chloroplast-encoded cMB is required for light-independent;
protochlorophyllide reductase activity in Chlamydomonas reinhardtii. Plant Cell 5: 1817-1829. :~
Liu XQ. Xu H, Huang C (1993a): Chloroplast ciliB gene is required for light-independent chlorophyll aecumulatio':'
in Chlamydomonas reinhardtii. Plant Mol. Bioi. 23, 297-308. .
Liu XQ, Huang C, Xu H (1993b): The unusual rps3-like orj712 is functionally essential and structurally conserved
in Chlamydomonas. FEBS Lell. 336: 225-230. .' .
Mattox K, Stewart K (1984): Classification ofthe green algae: a concept based on comparative cytology. In: Irvine
DEG, John DM (eds) Systematics oJthe Green Algae, Academic Press, London. pp.29-72.
Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sana T, Sana S, Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota
S, Inokuchi H, Ozeki H (1986): Chloroplast gene organization deduced from complete sequence of liverwort
Marchantia polymorpha. Nature 322: 572-574..
Palmer JD (1991): Plastid chromosomes: structure and evolution. In: Bogorad L, VasiliK (eds) Cell Culture and
Somatic Cell Genetics ojPlants. Vol. 7A: The Molecular Biology ojPlastids, Academic Press, San Diego.
pp.5-53.
Saitou N, Nei N (1987): The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mal. Bioi.
Eva!. 4: 406-425.
Schlosser UG (1984): Species-specific sporangium autolysins (cell-wall-dissolving enzymes) in the genus
Chlamydomonas. In: Irvine DEG, John DM (eds) Systematics oJthe Green Algae, Academic Press, London.
pp.40~18.
Shinozaki N, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J,
Yamagochi-Shinozaki K, Ohto C, Torazawa K, Meng BY, Sugita M, Dena H, Komogashira T, Yamada Ki
Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M (1986): The complete nucleotide sequence
of the tobacco chloroplast genome: its gene organization and expression. EMBO 1. 5: 2043-2049. ;
Tunnel M, Otis C (1994): The chloroplast gene cluster containing psbF, psbL, petG and rps3 is conserved in .
Chlamydomonas. Curro Genet. 27: 54--{j I . . -'
Turmel M, Bellemase G, Lee RW, Lemieux C (1986): A linear DNA molecule of 5.9 kilobase-pairs is highly
homologoos to the chloroplast DNA in the green alga Chlamydomonas moewusii. Plant Mol. Bia!. 6: 313-319:
Turmel M, Bellemare G, Lemieux C (1987): Physical mapping ofdifferences between the chloroplast DNAs ofthe
interfertile algae Chlamydomonas eugametos and Chlamydomonas moewusii. Curro Genet. II: 543-552.
Tunnel M, Boulanger J, Schnare MN, Gray MW, Lemieux C (199Ia): Six group 1 introns and three internal
transcribed spacers in the chloroplast large subunit ribosomal RNA gene of the green alga Chlamydomonas
eugametos. 1. Mo!. Bioi. 218: 293-311.
Tunnel M, Boudreau E, Boulanger J, Mercier JP, Otis C, Lemieux C (199Ib): Chloroplast DNA evolution and
phylogenetic relationships in Chlamydomonas. In: Dudley DM (ed) The Unity ojEvolutionary Biology. The
Proc. 4th Int. Congr. System. & Evo!. Bio!., Dioscorides Press, Portland, Oregon. pp.816-827. .
Tunnel M, Choquet Y, Goldschmidt-Clermont M, Rochaix JD, Otis C, Lemieux C (1995b): The trans·spliced
intron 1 in the psaA gene of the Chlamydomonas chloroplast: a comparative analysis. Curro Genet. 27:
270-279.
Tunnel M, Cote V, Otis C, Mercier JP, Gray MW, Lonergan KM, Lemieux C (I 995a): Evolutionary transfer ofORF-
containing group I introns between different subcellular compartments (chloroplast and mitochondrion). Mol.
Bioi. Evol. 12: 533-545.
Tunnel M, Gutell RR, Mercier JP, Otis C, Lemieux C (1993a): Analysis of the chloroplast large subunit ribosomal
RNA gene from 17 Chlamydomonas taxa: three internal transcribed spacers and 12 group I intran insertion sites.
1. Mal. Bioi. 232: 446-467.
Tunnel M. Mercier JP, Cote MJ (1993b): Group I introns interrupt the chloroplast psaB and psbC and the
mitochondrial rrnL gene in Chlamydomonas. Nucleic Acids Res. 21: 5242-5250.
156
_..
III•
16
Structure and Evolution of the Chloroplast Genome
in Seedless Land Plants
L.A. Raubeson' , D.B. Steint• and D.S. Conant'
Department ojBiological Science. Mount Holyoke College. South Hadley. MA 01075. USA
Department ojNatural Sciences. Lyndon State College. Lyndonville. VT 05851. USA
INTRODUCTION
Gross aspects of chloroplast DNA (cpDNA) structure are shared widely in land plants
(Palmer 199I). The circular genome typically contains a large inverted repeat (IR) that
separates the remainder ofthe molecule into regions of unique DNA - the large and small
single copy regions (LSe, SSC). Early work suggested that the structure of the genome (its
basic organization, gene content and order) was highly conserved (Palmer and Stein 1986).
Now, many instances of structural change are known, with most of the investigated land
plant cpDNAs being from flowering plants (reviewed in Palmer et al. 1988; Downie and
Palmer 199I). Still structural changes, while not quite as rare as first thought, are uncommon.
Structural mutations"that have occurred in the chloroplast genome during land plant evolution
include gene duplication, gene loss, and inversions. Where these mutations occur, the
systematic distribution of the change often provides an important phylogenetic marker.
The gene order common to the fern, Osmunda, the gymnosperm, Ginkgo, and the
·Corresponding author
157