The Complete Mitochondrial DNA Sequence of the Green Alga
Pseudendoclonium akinetum (Ulvophyceae) Highlights Distinctive
Evolutionary Trends in the Chlorophyta and Suggests a Sister-Group
Relationship Between the Ulvophyceae and Chlorophyceae
Jean-François Pombert, Christian Otis, Claude Lemieux, and Monique Turmel
Département de Biochimie et de Microbiologie, Université Laval, Québec, Québec, Canada
The mitochondrial genome has undergone radical changes in both the Chlorophyta and Streptophyta, yet little is known
about the dynamics of mtDNA evolution in either of these lineages. In the Chlorophyta, which comprises four of the five
recognized classes of green algae (Prasinophyceae, Trebouxiophyceae, Ulvophyceae, and Chlorophyceae), the
mitochondrial genome varies from 16 to 55 kb. This genome has retained a compact gene organization and a relatively
complex gene repertoire (‘‘ancestral’’ pattern) in the basal lineages represented by the Trebouxiophyceae and
Prasinophyceae, whereas it has been reduced in size and gene complement and tends to evolve much more rapidly at the
sequence level (‘‘reduced-derived’’ pattern of evolution) in the Chlorophyceae and the lineage leading to the enigmatic
chlorophyte Pedinomonas. To gain information about the evolutionary trends of mtDNA in the Ulvophyceae and also to
gain insights into the phylogenetic relationships between ulvophytes and other chlorophytes, we have determined the
mtDNA sequence of Pseudendoclonium akinetum. At 95,880 bp, Pseudendoclonium mtDNA is the largest green-algal
mitochondrial genome sequenced to date and has the lowest gene density. These derived features are reminiscent of the
‘‘expanded’’ pattern exhibited by embryophyte mtDNAs, indicating that convergent evolution towards genome
expansion has occurred independently in the Chlorophyta and Streptophyta. With 57 conserved genes, the gene
repertoire of Pseudendoclonium mtDNA is slightly smaller than those of the prasinophyte Nephroselmis olivacea and the
trebouxiophyte Prototheca wickerhamii. This ulvophyte mtDNA contains seven group I introns, four of which have
homologs in green-algal mtDNAs displaying an ‘‘ancestral’’ or a ‘‘reduced-derived’’ pattern of evolution. Like its
counterpart in the chlorophycean green alga Scenedesmus obliquus, it features numerous small, dispersed repeats in
intergenic regions and introns. Its overall rate of sequence evolution appears to be accelerated to an intermediary level as
compared with the rates observed in ‘‘ancestral’’ and ‘‘reduced-derived’’ mtDNAs. In agreement with the finding that
Pseudendoclonium mtDNA exhibits features typical of both the ‘‘ancestral’’ and ‘‘reduced-derived’’ patterns of evolution,
phylogenetic analyses of seven mtDNA-encoded proteins revealed a sister-group relationship between this ulvophyte and
chlorophytes displaying ‘‘reduced-derived’’ mtDNAs.
Introduction
The mitochondrial genomes of green plants are highly
variable in size, gene content, and organization and show
divergent evolutionary trends in some lineages, as revealed
by the complete mtDNA sequences that have been reported
thus far for 13 members of this group. Representatives of
the two main phyla of green plants, the Streptophyta and
Chlorophyta, have been examined in these genome
analyses. The Streptophyta (Bremer 1985) comprises all
embryophytes (land plants) and their closest green-algal
relatives, the members of the class Charophyceae sensu
Mattox and Stewart (1984). In this phylum, the mitochondrial genomes of the bryophyte Marchantia polymorpha
(Oda et al. 1992), of three angiosperms (Arabidopsis
thaliana [Unseld et al. 1997], Beta vulgaris [Kubo et al.
2000], and Oryza sativa [Notsu et al. 2002]), and of the
charophycean green alga Chaetosphaeridium globosum
(Turmel, Otis, and Lemieux 2002b) have been entirely
sequenced. The Chlorophyta (Sluiman 1985) comprises
the four other classes of green algae: the Prasinophyceae,
Ulvophyceae, Trebouxiophyceae, and Chlorophyceae. The
seven chlorophyte mitochondrial DNA (mtDNA) sequences that are currently available include those of four
Key words: Green algae, Ulvophyceae, Pseudendoclonium akinetum, mitochondrial DNA, group I introns, repeated sequences.
E-mail: [email protected].
Mol. Biol. Evol. 21(5):922–935. 2004
DOI:10.1093/molbev/msh099
Advance Access publication March 10, 2004
chlorophycean green algae (Chlamydomonas reinhardtii
[Michaelis, Vahrenholz, and Pratje 1990], Chlamydomonas eugametos [Denovan-Wright, Nedelcu, and Lee 1998],
Chlorogonium elongatum [Kroymann and Zetsche 1998],
and Scenedesmus obliquus [Kück, Jekosch, and Holzamer
2000; Nedelcu et al. 2000]), the nonphotosynthetic
trebouxiophyte Prototheca wickerhamii (Wolff et al.
1994), the prasinophyte Nephroselmis olivacea (Turmel
et al. 1999), and a chlorophyte of uncertain phylogenetic
affinity, Pedinomonas minor (Turmel et al. 1999). The
remaining green alga whose mitochondrial genome has
been scrutinized, Mesostigma viride (Turmel, Otis, and
Lemieux 2002a), belongs to the Streptophyta (Bhattacharya et al. 1998; Marin and Melkonian 1999; Karol et al.
2001) or to a lineage that emerged before the divergence of
the Streptophyta and Chlorophyta (Lemieux, Otis, and
Turmel 2000; Turmel et al. 2002; Turmel, Otis, and
Lemieux 2002a).
Because of their large size and their tendency to
incorporate foreign DNA (from the nucleus and the
chloroplast), land-plant mitochondrial genomes have been
reported to feature an ‘‘expanded’’ pattern of evolution
(Turmel et al. 1999). These mitochondrial genomes are the
largest (187 kb in Marchantia to 2,000 kb in muskmelon)
among green plants, and they also show the greatest
structural complexity. Most of the increased size of landplant mtDNAs relative to their green-algal homologs is
accounted for by noncoding sequences that reside either in
intergenic regions or introns (Oda et al. 1992; Unseld et al.
Molecular Biology and Evolution vol. 21 no. 5 Ó Society for Molecular Biology and Evolution 2004; all rights reserved.
Mitochondrial DNA Sequence of Pseudendoclonium 923
1997; Kubo et al. 2000; Notsu et al. 2002). Sixty-nine
mitochondrial genes have been identified in Marchantia
(Oda et al. 1992; Gray et al. 1998), whereas about 50 have
been found in angiosperms (Unseld et al. 1997; Kubo et al.
2000; Notsu et al. 2002). This substantial difference in
coding capacity is attributed to gene transfer to the
nucleus, a widespread and ongoing phenomenon (Schuster
and Brennicke 1994; Palmer et al. 2000; Adams et al.
2002). In embryophyte mitochondria, unicircular genomesized molecules coexist in a dynamic equilibrium with
subgenomic circles (Palmer and Shields 1984; Mackenzie,
He, and Lyznik 1994). In Marchantia mitochondria,
unicircular-genome sized molecules apparently coexist
with linear molecules and complex branched structures of
multigenomic sizes (Oldenburg and Bendich 2001). In
contrast to their fluid structure, land-plant mitochondrial
genomes evolve extremely slowly at the sequence level; in
angiosperm mitochondria, point mutations can occur at
a frequency up to 100 times lower than in vertebrate
mitochondria (Wolfe, Li, and Sharp 1987; Palmer and
Herbon 1988).
The more compact green-algal mitochondrial genomes display distinctive evolutionary patterns. They
range in size from 16 kb (in C. reinhardtii) to 67.8 kb
(in Mesostigma) and encode 12 (in C. reinhardtii, C.
eugametos, and Chlorogonium) to 67 (in Chaetosphaeridium) genes. An ‘‘ancestral’’ (minimally derived) evolutionary pattern (Turmel et al. 1999) has been ascribed to
the circular-mapping mtDNAs of Mesostigma, Chaetosphaeridium, Nephroselmis, and Prototheca, because of
their large number of conserved genes (.60), their high
gene density, and their important sequence conservation.
Not only fewer genes but also a greater variability in gene
content (12 to 42 genes) and structural organization (linear
or circular-mapping molecules, or even multimeric
molecules, as reported for Polytomella [Fan and Lee
2002]) have been found in chlorophycean green-algal
mtDNAs. The coding sequences of these genomes are
highly divergent from those of other green plants and
feature numerous deletions/additions; moreover, the rRNA
gene-coding regions are fragmented into pieces that are
dispersed throughout the genomes. As a consequence of
this high sequence divergence, chlorophycean taxa exhibit
very long branches in mitochondrial trees, and, most
probably because of long-branch attraction artifacts,
usually lie outside the green-plant clade when other green
plants and non–green-plant taxa are included in the
analyses. A ‘‘reduced-derived pattern’’ (Turmel et al.
1999) of evolution has been assigned to the three smallest
and gene-poorest chlorophycean mtDNAs (i.e., to C.
reinhardtii, C. eugametos, and Chlorogonium mtDNAs).
Because of its less-derived characters, the Scenedesmus
mtDNA sequence has been considered to display an
‘‘intermediate’’ pattern of evolution (Nedelcu et al. 2000).
The present study was undertaken to gather information about the evolutionary trends of the mitochondrial
genome in the Ulvophyceae and also to gain insights into
the phylogenetic relationships between ulvophytes and
other chlorophytes. Various hypotheses have been proposed concerning the phylogenetic position of the Ulvophyceae within the Chlorophyta, but none is strongly
supported by statistical analyses. On the basis of ultrastructural studies (Mattox and Stewart 1984; O’Kelly and Floyd
1984) and some phylogenetic analyses of nuclear small
subunit (SSU) rDNA sequences (Friedl 1995; Bhattacharya, Friedl, and Damberger 1996; Nakayama, Watanabe, and Inouye 1996; Chapman et al. 1998; Watanabe et
al. 2000; Wolf et al. 2002), it has been proposed that the
Ulvophyceae are a monophyletic assemblage that emerged
before the divergence of the Trebouxiophyceae and
Chlorophyceae. Independent inferences from nuclear SSU
rDNA sequences (Friedl 1997; Marin and Melkonian 1999)
and from concatenated chloroplast SSU and large subunit
(LSU) (Turmel et al. 2002) rDNA sequences suggest
a possible sister-group relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae
occupying a basal position. On the other hand, separate
nuclear SSU rDNA trees (Bhattacharya and Medlin 1998)
favor the hypothesis that the Chlorophyceae appeared
before the divergence of the Ulvophyceae and Trebouxiophyceae, whereas recent trees, including a wider diversity
of ulvophytes (Friedl and O’Kelly 2002) failed to revolve
the branching order of the Trebouxiophyceae, Chlorophyceae, and Ulvophyceae. Moreover, other nuclear SSU
rDNA trees, including several ulvophytes (Watanabe,
Kuroda, and Maiwa 2001) are in agreement with an earlier
report (Zechman et al. 1990) and with the concept of
the Ulvophyceae sensu Sluiman (1989) in supporting the
notion that the ulvophytes are nonmonophyletic. In the
Ulvophyceae sensu Sluiman, ulvophytes and trebouxiophytes are merged to form a green-algal group with
a counterclockwise arrangement of kinetid components.
The analyses supporting the monophyly of ulvophytes
suffer from a relatively poor and/or biased taxon sampling
(all five orders recognized in this class were not represented), whereas those supporting their nonmonophyly
may be plagued with long-branch attraction artifacts.
In this study, we report the complete mtDNA
sequence of Pseudendoclonium akinetum, a unicellular
member of the Ulvophyceae that belongs to a putatively
deep-branching lineage (Floyd and O’Kelly 1990). At
95,880 bp, this ulvophyte mtDNA is the largest greenalgal mtDNA analyzed thus far. Our phylogenetic analyses
provide support for a sister-group relationship between the
Ulvophyceae and Chlorophyceae.
Materials and Methods
Strain and Culture Conditions
Pseudendoclonium akinetum (Tupa 1974) was obtained from the University of Texas Algal Culture
Collection (UTEX 1912) and grown in modified Volvox
medium (McCracken, Nadakavukaren, and Cain 1980)
under 12 h light/dark cycles.
Isolation and Sequencing of mtDNA
A1T-rich organellar DNA was separated from
nuclear DNA by CsCl-bisbenzimide isopycnic centrifugation (Turmel et al. 1999). After nebulization of this A1Trich fraction, a plasmid library of DNA fragments (1200 to
2500 bp) was prepared (Lemieux, Otis, and Turmel 2000).
924 Pombert et al.
Plasmid DNA templates and PCR fragments spanning
uncloned regions of Pseudendoclonium mtDNA were
sequenced using ABI Prism 373XL and 377 (Applied
Biosystems, Foster City, Calif.) DNA sequencers and the
ABI Prism Big Dye terminator sequencing kit (Applied
Biosystems) as described previously (Lemieux, Otis, and
Turmel 2000). Templates that yielded poor sequences
under these conditions were subjected to sequencing using
the DYEnamic ET (Amersham Pharmacia Biotech, Baie
d’Urfé, Canada) and/or the ABI Prism dGTP Big Dye
(Applied Biosystems) dye terminator sequencing kits.
Sequences were edited and assembled with AUTOASSEMBLER version 2.1.1 (Applied Biosystems).
Genome Analyses
Gene content was determined by Blast homology
searches (Altschul et al. 1990) against the nonredundant
database of NCBI. Protein-coding genes and open reading
frames (ORFs) were localized precisely using ORFFINDER at NCBI and various programs of the GCG
Wisconsin version 10.2 package (Accelrys, Burlington,
Mass.), whereas sequences coding for tRNAs were
identified with tRNAscan-SE 1.23 (Lowe and Eddy
1997). Patterns of codon usage for protein-coding genes
and ORFs were compared using CORRESPOND and
CODONPREFERENCE from the Wisconsin package and
CAI from the EMBOSS version 2.6.0 package (http://
www.hgmp.mrc.ac.uk/Software/EMBOSS/). Repeated sequence elements were identified with PIPMAKER
(Schwartz et al. 2000) and REPUTER version 2.74 (Kurtz
et al. 2001) and classified with REPEATFINDER
(Volfovsky, Haas, and Salzberg 2001).
trees were computed with PROTML in MOLPHY version
2.3b3 (Adachi and Hasegawa 1996) and CODEML in
PAML version 3.11 (Yang 1997) using the amino acid
substitution models JTT-F, mtREV24-F, and WAG-F
(Whelan and Goldman 2001). -distributed rates of
substitutions across sites (eight categories) and/or multiple
gene (Mgene option) corrections were applied in some
CODEML analyses. Local bootstrap probabilities were
estimated by resampling of the estimated log-likelihood
(RELL) (Adachi and Hasegawa 1996). Confidence assessments (P-values) of tree selections were evaluated by the
Approximately Unbiased, Kishino-Hasegawa, and Shimodaira-Hasegawa tests as implemented in CONSEL version
0.1d (Shimodaira and Hasegawa 2001). The effect of
invariable sites on topologies was determined by analysis
of a trimmed data set of 1,555 positions containing only
the variable sites.
-corrected ML distances were
calculated with TREE-PUZZLE version 5.0.2 (Strimmer
and von Haeseler 1996) under the WAG-F model, and
distance trees were computed by weighted neighborjoining as implemented in WEIGHBOR version 1.2
(Bruno, Socci, and Halpern 2000). Support for MLdistance trees was obtained by bootstrapping (100
replications) with PUZZLEBOOT version 1.03 (A. Roger
and M. Holder, http://www.tree-puzzle.de).
Comparisons of Amino Acid Substitution Rates in
Different Lineages
We used the data set of 2,107 amino acid positions
that was employed for the ML and ML-distance analyses.
Differences in the rates of amino acid substitutions among
lineages were assessed using the binomial test of Gu and
Li (1992).
Phylogenetic Analyses
Mitochondrial genome sequences were retrieved
from GenBank: Pseudendoclonium akinetum (accession
number AY359242 [this study]), Mesostigma viride
(accession number AF353999), Nephroselmis olivacea
(accession number AF110138), Prototheca wickerhamii (accession number NC_001613), Pedinomonas minor (accession number NC_000892), Scenedesmus obliquus
(accession number AF204057), Chlamydomonas eugametos (accession number NC_001872), Chlamydomonas
reinhardtii (accession number NC_001638), Chlorogonium elongatum (accession numbers Y13643 and
Y13644), Chaetosphaeridium globosum (accession number NC_004118), Marchantia polymorpha (accession
number NC_001660), Arabidopsis thaliana (accession
number NC_001284), Beta vulgaris (accession number
NC_002511), Chondrus crispus (accession number
NC_001677), and Porphyra purpurea (accession
number NC_002007). Deduced amino acid sequences
from individual genes were aligned using ClustalW
version 1.81 (Thompson, Higgins, and Gibson 1994).
Data sets were prepared by concatenating the alignments
of individual proteins and removing the ambiguously
aligned regions with GBLOCKS version 0.73b
(Castresana 2000). Phylogenetic trees were inferred using
maximum-likelihood (ML) and ML-distance methods. ML
Results
General Features
Pseudendoclonium mtDNA is a circular DNA
molecule of 95,880 bp (fig. 1). Its 57 conserved genes
and 36 free-standing ORFs (. 60 codons) represent 47.4%
and 11.3% of the genome sequence, respectively. A total
of seven introns interrupt four genes (atp1, cob, cox1, and
rnl). Intergenic sequences range from 5 to 2,663 bp in size,
with an average of about 600 bp, and feature similar proportions of A1T (61.4%) as compared with coding
regions (60.2%).
In terms of coding capacity, the mitochondrial genome
of Pseudendoclonium closely resembles those of Nephroselmis and Prototheca. Pseudendoclonium mtDNA lacks
six of the protein-coding genes encoded by its Nephroselmis
counterpart and only three of the protein-coding genes
encoded by Prototheca mtDNA (table 1). It encodes two
rRNAs, 25 tRNAs, 12 ribosomal proteins, 17 ATP synthase
and respiratory chain components, and also a protein
involved in the Sec-independent translocation pathway
(MttB). The encoded tRNAs each feature a conventional
cloverleaf secondary structure and together are sufficient to
translate all of the codons in Pseudendoclonium mtDNA
using the standard genetic code (table 2). In contrast to
Mitochondrial DNA Sequence of Pseudendoclonium 925
FIG. 1.—Gene map of Pseudendoclonium mtDNA. Genes on the outside of the map are transcribed in a clockwise direction; those on the inside of
the map are transcribed counterclockwise. tRNA genes are indicated by the one-letter amino acid code followed by the anticodon in parentheses (Me,
elongator methionine; Mf, initiator methionine). Seven group I introns (open boxes) interrupt conserved genes; six of these introns contain an internal
ORF (gray boxes). Only the free-standing ORFs larger than 60 codons are shown.
Nephroselmis and Prototheca mtDNAs, no gene for 5S
rRNA (rrn5) is present in Pseudendoclonium mtDNA.
Six intron ORFs in Pseudendoclonium mtDNA
encode proteins that are homologous to endonucleases/
maturases of the LAGLIDADG family (table 3); each of
these intron-encoded proteins contains two copies of the
LAGLIDADG motif. Two of the 36 free-standing ORFs,
orf289 and orf307, also code for putative LAGLIDADG
endonucleases/maturases with two copies of the LAGLIDADG motif. The endonuclease potentially encoded by
orf289 shows 48% sequence identity over 282 aligned
amino acid positions with the protein specified by orf298
in the fourth intron of cox1 (Pacox1.4), whereas the
endonuclease potentially encoded by orf307 has no
substantial homology with any of the Pseudendoclonium
intron-encoded endonucleases/maturases. Of the 34 remaining free-standing ORFs, only the four harboring more
than 300 codons, as well as orf61 and orf90, feature
a pattern of codon usage that is similar to that of conserved
genes (table 2). This observation suggests that these few
ORFs may be expressed at the protein level; however, we
have not been able to detect any similarity with known
gene sequences.
Pseudendoclonium mtDNA shares only two small
gene clusters (rps12-rps10 and trnE(uuc)-trnW(cca) with
Nephroselmis mtDNA) and a single cluster (atp1-nad1)
with Prototheca mtDNA. The large cluster of ribosomal
protein genes found in Nephroselmis mtDNA has been
segmented into several clusters in Pseudendoclonium
mtDNA, and as observed for the Nephroselmis and
Prototheca mtDNAs, the genes for the SSU and LSU
rRNAs have retained their continuous structure.
Introns
All seven introns in Pseudendoclonium mtDNA
belong to the group I family (table 3). All of these introns,
with the exception of Paatp1.1, show similarities with
group I introns inserted at equivalent positions in other
green-plant and/or fungal mtDNAs (table 4). The latter
green-plant mtDNAs include those of the streptophyte
Marchantia and of various chlorophytes exhibiting an
926 Pombert et al.
Table 1
Distribution of Conserved Genes Coding for Proteins and rRNAs in the mtDNAs of
Pseudendoclonium and Selected Chlorophytes
Genea
Nephroselmis
Prototheca
Pseudendoclonium
atp1
atp4b
atp8
atp9
cox2
cox3
nad7
nad9
nad10
rnpB
rpl5
rpl6
rpl14
rpl16
rps2
rps3
rps4
rps7
rps8
rps10
rps11
rps12
rps13
rps14
rps19
rrn5
s
s
s
Pedinomonas
Scenedesmus
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
a
Only the mitochondrial genes that are missing in one or more genomes are indicated. Genes shared between all green-algal
mtDNAs are atp6, cob, cox1, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, rnl, and rns. A filled/open circle denotes the presence/
absence of a gene in mtDNA.
b
Gene designated previously as ymf39 (Burger et al. 2003).
‘‘ancestral’’ or a ‘‘reduced-derived’’ pattern of mtDNA
evolution. Sequence conservation between the core
structures of four Pseudendoclonium introns (Pacox1.1,
Pacox1.2, Pacox1.4, and Parnl.1) and their green-algal
homologs is substantial (fig. 2).
Homologs of the Parnl.1 intron have also been
identified in the chloroplast rrl genes of several green
algae (Turmel et al. 1993). One of these chloroplast introns
(Chrnl.1) contains an ORF in L9.1 (as in Parnl.1) that
codes for the DNA-homing endonuclease I-ChuI (Côté et
Table 2
Codon Usage in the 30 Conserved Protein-Coding Genes of Pseudendoclonium mtDNA
Codon
%a
acb
aac
Codon
%
ac
aa
Codon
%
ac
aa
Codon
%
ac
aa
GCA
GCC
GCG
GCU
AGA
AGG
CGA
CGC
CGG
CGU
AAC
AAU
GAC
GAU
UGC
UGU
28
19
12
41
32
10
25
6
8
19
36
64
33
67
32
68
ugc
ugc
ugc
ugc
ucu
ucu
ucg
ucg
ucg
ucg
guu
guu
guc
guc
gca
gca
A
A
A
A
R
R
R
R
R
R
N
N
D
D
C
C
UAA
UAG
UGA
CAA
CAG
GAA
GAG
GGA
GGC
GGG
GGU
CAC
CAU
AUA
AUC
AUU
69
28
3
78
22
75
25
25
18
18
39
34
66
36
11
53
—
—
—
uug
uug
uuc
uuc
ucc
ucc
ucc
ucc
gug
gug
gau
gau
gau
*
*
*
Q
Q
E
E
G
G
G
G
H
H
I
I
I
CUA
CUC
CUG
CUU
UUA
UUG
AAA
AAG
AUG
UUC
UUU
CCA
CCC
CCG
CCU
AGC
14
3
4
20
47
12
78
22
100
22
78
27
14
15
44
19
uag
uag
uag
uag
uaa
uaa
uuu
uuu
cau
gaa
gaa
ugg
ugg
ugg
ugg
gcu
L
L
L
L
L
L
K
K
Me
F
F
P
P
P
P
S
AGU
UCA
UCC
UCG
UCU
ACA
ACC
ACG
ACU
UGG
UAC
UAU
GUA
GUC
GUG
GUU
26
19
8
6
22
37
16
10
37
100
34
66
38
8
12
42
gcu
uga
uga
uga
uga
ugu
ugu
ugu
ugu
cca
gua
gua
uac
uac
uac
uac
S
S
S
S
S
Td
Td
Td
Td
W
Y
Y
V
V
V
V
a
Percentage of each amino acid (aa) specified by a given codon.
Anticodon (ac) of Pseudendoclonium mtDNA-encoded tRNA recognizing the corresponding codon. Some tRNAs with U in the wobble position of the anticodon
are assumed to decode all four members of four-codon families (i.e., leucine, CUN; valine, GUN; serine UCN; proline, CCN; alanine, GCN; arginine, CGN; glycine,
GGN; and threonine, ACN).
c
Amino acids are given in the one-letter code. Asterisks denote termination codons.
d
Among green-plant mtDNAs, the gene for tRNAThr (ugu) has been identified only in Prototheca and Pseudendoclonium mtDNAs. There is 57% sequence
identity between the two tRNAs.
e
Separate genes encode the elongator and initiator tRNAMet.
b
Mitochondrial DNA Sequence of Pseudendoclonium 927
Table 3
Main Features of Pseudendoclonium Mitochondrial Introns
Intron
Paatp1.1
Pacob.1
Pacox1.1
Pacox1.2
Pacox1.3
Pacox1.4
Parnl.1
Classa
Size (bp)
ORF Size (bp)
ORF Locationb
IB4
IB
IB4
ID
IB4
IB3
IB4
1425
911
2049
2148
1661
1732
1524
993
—
1335
789
534
897
777
L8
—
L8
L1
L8
L1
L9.1
Table 4
Group I Introns at Identical Gene Locations in Pseudendoclonium and Other mtDNAs
Pseudendoclonium
Intron
Pacob.1
Pacox1.1
a
Classification of group I introns was according to Michel and Westhof
(1990). Pacob.1 could not be assigned unambiguously to a subclass of IB introns.
al. 1993). The Chrnl.1 and Parnl.1 introns share 69%
sequence identity over 169 aligned nucleotides in their
core structures, and their encoded proteins show 65.3%
amino acid similarities over 219 aligned amino acid
positions. This important level of sequence conservation is
consistent with our previous finding of closely related
group I introns in the chloroplast and mitochondrial
genomes of green algae (Turmel et al. 1995; Turmel et al.
1999; Lucas et al. 2001; Turmel, Otis, and Lemieux
2002a) and further supports the idea that lateral transfers
of introns have occurred between different organellar
compartments during the evolution of green plants.
Pacox1.2
Pacox1.3
Pacox1.4
Repeated Sequence Elements
A comparison of the Pseudendoclonium mtDNA
sequence against itself using PIPMAKER (Schwartz et al.
2000) revealed the presence of many degenerated
repeated elements within intergenic regions and introns
(fig. 3). We identified these repeated sequences using the
program REPUTER and found that they are up to 350 nt
in size and do not differ substantially from the rest of the
Pseudendoclonium genome in terms of nucleotide
composition. The repeats that were 50 nt or more were
classified using REPEATFINDER (Volfovsky, Haas, and
Salzberg 2001); they represent about 10 kb of the
genome and form four distinct classes, designated C1 to
C4 (fig. 3 and table 5). The members of a given class
share no sequence similarity with those of other classes.
The first class of repeats (C1) is the largest. Its members
map to at least 16 different loci (fig. 3) and can be further
classified into 14 subclasses, designated C1R1 through
C1R14. The longest repeat within each subclass, defined
as the prototype, is made up of repeated units
(subrepeats) that also constitute the shorter repeated
elements found in the same subclass. Distinct subclasses
feature different arrangements of repeated units. The
coordinates of the prototypes and number of repeated
elements within each subclass are given in table 5. The
abundance of subclasses in class 1 suggests that
numerous recombination and shuffling events between
repeated units took place during the evolution of the
mitochondrial genome. On the other hand, classes 2, 3,
and 4 are much less complex than class 1. Each exhibits
only one subclass of repeats, with two identical copies of
the prototype sequence that are far apart from one
Parnl.1
Homologous Introna
Podospora anserina cob i3a (L9.1)
Podospora curvicolla cob i2 (L9.1)
Prototheca wickerhamii cox1 i1 (L8)
Chaetosphaeridium globosum cox1
i2 (L8)
Marchantia polymorpha cox1
i4 (L8)
Agrocybe aegerita cox1 i1 (L8)
Allomyces macrogynus cox1 i5
Saccharomyces douglasii cox1 i1
Schizosaccharomyces pombe cox1
i1 (L8)
Prototheca wickerhamii cox1 i2
Scenedesmus quadricauda cox1
i1 (L1)
Kluyveromyces lactis cox1 i2
Saccharomyces douglasii cox1 i2
Podospora anserina cox1 i8 (L1)
Agrocybe aegerita cox1 i2 (L8)
Emericella nidulans cox1 i3
Neurospora crassa cox1 i4
Schizosaccharomyces pombe cox1
i2 (L8)
Chlamydomonas eugametos cox1
i1 (L9.1)
Marchantia polymorpha cox1
i8 (L1)
Mesostigma viride cox1 i2 (L1)
Allomyces macrogynus cox1 i10
Dictyostelium discoideum cox1/2
i3 (L1)
Kluyveromyces lactis cox1 i4 (L1)
Podospora anserina cox1 i15 (L1)
Saccharomyces cerevisiae cox1
i5b (L9.1)
Saccharomyces douglasii cox1
i4 (L9.1)
Schizosaccharomyces pombe cox1 i4
Chlamydomonas eugametos rnl
i2 (L9.1)
Nephroselmis olivacea rnl i2 (L9.1)
Accession
Number
NC_001329
Z69894
NC_001613
NC_004118
NC_001660
AF010257
NC_001715
M97514
X00886
NC_001613
AB011524
X57546
M97514
NC_001329
AF010257
X00790
X14669
X00886
NC_001872
NC_001660
AF353999
NC_001715
NC_000895
X57546
NC_001329
NC_001224
M97514
M15671
NC_001872
AF110138
a
When an ORF is present in the homologous intron, its position is indicated in
parentheses. L followed by a number refers to the loop extending the base-paired
region identified by the number.
another. It is likely that these elements arose from single
duplication events. In addition to the repeats described
above, Pseudendoclonium mtDNA features numerous
repeated elements less than 50 nt that span about 4 kb
of this genome (fig. 3).
Phylogenetic Analyses
The amino acid sequences derived from the seven
protein-coding genes (cob, cox1, nad1, nad2, nad4, nad5,
and nad6) that are common to the mtDNAs of Pseudendoclonium and 11 other green plants were concatenated
and analyzed with ML and ML-distance methods of
phylogenetic inference, using as outgroup the homologous
sequences from Mesostigma. Note that when the red algae
Porphyra purpurea and Chondrus crispus were used as
outgroup, we observed no change in the position of
928 Pombert et al.
FIG. 2.—Structural similarities between group I introns in Pseudendoclonium mtDNA and their closest homologs in other green algal mtDNAs.
Pacox1.1 and Pacox1.2 were compared with the homologous introns in Prototheca mtDNA, Pacox1.4 was aligned with the homologous intron in
Mesostigma mtDNA, and Parnl.1 was compared with the homologous intron in Nephroselmis mtDNA. Introns were modeled according to the
nomenclature proposed by Burke et al. (1987). Splice sites between exon and intron residues are denoted by arrows. Identical residues found at the same
positions in the compared introns are shown in uppercase characters, whereas positions displaying different nucleotides are denoted by dots. Conserved
base-pairings are denoted by dashes. The numbers inside the variable loops indicate the sizes of these loops in the compared introns, with the upper
number referring to the Pseudendoclonium sequence.
Pseudendoclonium. The WAG-F model of amino acid
replacement was selected for all phylogenetic analyses,
because it gave higher likelihood values than the JTT-F
and mtREV24-F models (table 6). The ML and MLdistance trees inferred by assuming -distributed rates of
substitutions across sites revealed a strong affiliation of
Pseudendoclonium with the highly supported clade
containing Pedinomonas and the four chlorophycean
green algae whose mtDNA sequences has been determined
to date (fig. 4). As shown by their very long branches, the
members of this clade, designated here ‘‘reduced-derived’’
clade, display a higher rate of mtDNA sequence evolution
compared to the other green plants examined. Phylogenetic relationships among members of the ‘‘reducedderived’’ clade were found to be well resolved in all
analyses, and the affiliation of Pseudendoclonium with this
clade remained strongly supported when we excluded
invariable sites and/or when we included an additional
parameter (Mgene option in CODEML) to take into
account the different evolutionary rates of the selected
mitochondrial proteins.
To our surprise, we found that under certain
conditions of phylogenetic analyses the ‘‘reduced-derived’’
clade groups with the rest of green algae and land plants.
For instance, in the best ML tree shown in figure 4, the
cluster formed by Pseudendoclonium and the ‘‘reducedderived’’ clade occupies the Chlorophyta lineage. This
result contrasts with previously reported phylogenies in
which members of the ‘‘reduced-derived’’ clade branch
consistently outside the monophyletic group formed by the
rest of green plants, most certainly as a result of longbranch attraction artifacts (Turmel, Otis, and Lemieux
1999; Nedelcu et al. 2000). We explored the phylogenetic
conditions that contribute to the recovery of the correct
branching pattern in our best ML tree and found that the
selected inference method, evolutionary model, and taxon
sampling all play an important role in the analyses.
Analyses based on ML-distances and performed under the
Mitochondrial DNA Sequence of Pseudendoclonium 929
FIG. 3.—Positions of repeated sequence elements in Pseudendoclonium mtDNA as revealed by PIPMAKER. Repeats of 50 nt or more were sorted
into four classes by REPEATFINDER. Repeats less than 50 nt were not classified. Genes and their polarities are denoted by horizontal arrows, and
exons are represented by filled boxes. Similarities between repeated sequence elements are shown as average percentage identity (between 50% to
100% identity).
JTT-F and mtREV24-F models failed to recover the
correct topology as the best tree. Moreover, the inclusion
of the Pseudendoclonium taxon in the concatenated data
set and the addition of a
correction for amino acid
substitutions across sites also represent important factors in
the ML analyses. However, even under the most favorable
ML conditions for recovering the correct tree, alternate
topologies in which the ‘‘reduced-derived’’ cluster
branches outside the clade formed by the rest of green
plants were detected relatively frequently among the
RELL bootstrap replicates. These alternate topologies
proved to be not significantly different (P , 0.1) from the
best tree in the Kishino-Hasegawa and the ShimodairaHasegawa tests.
Rates of Amino Acid Substitutions
Considering the branch lengths of the best ML tree
(fig. 4), it appears that Pseudendoclonium mtDNA evolves
at a slower rate than the mtDNAs of taxa from the
‘‘reduced-derived’’ clade. To determine whether this
interpretation is correct, we assessed the relative rates of
930 Pombert et al.
Table 5
Prototypes of Repeated Sequence Elements of 50 nt or
More in Pseudendoclonium mtDNA
Class/
Subclass
Starta
Enda
Size (nt)
Number of
Occurrencesb
C1R1
C1R2
C1R3
C1R4
C1R5
C1R6
C1R7
C1R8
C1R9
C1R10
C1R11
C1R12
C1R13
C1R14
C2R1
C3R1
C4R1
7829
22728
27871
29398
35292
41072
71594
80073
83421
83605
84050
86491
93473
94722
11683
30366
74522
8178
22833
28104
29461
35372
41233
71764
80204
83505
83742
84176
86566
93685
94839
11792
30429
74606
350
106
234
64
81
162
171
132
85
138
127
76
213
118
110
64
85
8
3
9
2
3
4
5
4
6
4
3
3
7
4
2
2
2
a
Coordinates in accession number AY359242.
Number of occurrences of the prototype and subsets of this sequence within the genome.
b
amino acid substitutions of mtDNA-encoded proteins
among the different lineages using the binomial test of
Gu and Li (1992) (table 7). The substitution rates of
Pseudendoclonium proteins were found to be significantly
smaller (P , 0.001) than those observed for Scenedesmus
and other green algae from the ‘‘reduced-derived’’ clade.
Pseudendoclonium mtDNA–encoded proteins, however,
evolve significantly faster (P , 0.001) than their
Nephroselmis and Prototheca counterparts.
Discussion
Distinctive Features of Pseudendoclonium mtDNA
Of all the green-algal mtDNAs sequenced to date,
Pseudendoclonium mtDNA is the genome with the largest
size and the lowest gene density (table 8). At 95,880 bp,
the size of this genome is almost twofold larger than those
of the two chlorophytes displaying an ancestral pattern of
evolution (Nephroselmis and Prototheca). Despite this
substantial expansion in size, Pseudendoclonium mtDNA
displays a smaller repertoire of conserved genes compared
with its Nephroselmis and Prototheca counterparts (table
8). Most of its increased size is accounted for by dispersed
repeats and sequences of unknown nature/origin that reside
mainly within the intergenic regions.
The unusually large size and low gene density of
Pseudendoclonium mtDNA, together with the presence of
dispersed repeats, is reminiscent of the ‘‘expanded’’ pattern
of evolution exhibited by embryophyte mtDNAs. Both the
expanded Pseudendoclonium and the embryophyte
mtDNAs encode a similar number of conserved genes,
although they differ in their gene repertoire; moreover,
most of their extraneous DNA sequences are of unknown
origin and/or function (Unseld et al. 1997; Kubo et al.
2000; Notsu et al. 2002). The finding that Pseudendoclonium mtDNA is much larger than its chlorophyte
counterparts suggests that mitochondrial genome expan-
Table 6
Tree-Related Statistics and Log-Likelihood Values for the
Mitochondrial Data Set of 2,107 Amino Acid Positions
Model of Evolution
Tree length
Longest branch
Shortest branch
Median branch
Average branch
Log-likelihood
mtREV24-F
JTT-F
WAG-F
4.804
1.360
0.011
0.112
0.209
229224.44
4.084
1.126
0.010
0.101
0.178
229114.30
3.905
1.060
0.010
0.092
0.170
228984.66
sion occurred independently in the Chlorophyta and
Streptophyta. Analyses of additional ulvophyte mtDNAs
will be required to determine whether the Ulvophyceae
displays the pattern of progressive genome expansion
observed in the Streptophyta.
On one hand, the gene content (a relatively complex
gene repertoire) and gene structure (lack of fragmented and
scrambled rRNA genes) of Pseudendoclonium mtDNA are
characteristic of ‘‘ancestral’’ mtDNAs. On the other hand,
its low gene density, abundant repeats, and the absence of
certain genes (nad9, rpl6, rps7, and rrn5) are typical of
Scenedesmus and Pedinomonas mtDNAs. The finding of
genomic features that are typical of the ‘‘reduced-derived’’
pattern of mtDNA evolution, together with the presence of
ancestral features, suggests that Pseudendoclonium belongs to a lineage that appeared after the emergence of the
Trebouxiophyceae but before the divergence of the
Chlorophyceae. Further supporting this notion are three
independent observations. First, ML and ML-distance
trees inferred from mtDNA-encoded proteins always
cluster Pseudendoclonium with chlorophycean green algae
and consistently place the trebouxiophyte Prototheca at
the base of this clade (see next section). Second, the
overall rate of sequence evolution appears to be accelerated to an intermediary level in Pseudendoclonium
mtDNA as compared with the slow rates observed in
‘‘ancestral’’ mtDNAs and the very fast rates detected in
Pedinomonas, Scenedesmus and chlorophycean greenalgal mtDNAs (fig. 4). Third, close relatives of the
Pacox1.2, Pacox1.4, and Parnl.1 introns are present in
mtDNAs displaying both the ‘‘ancestral’’ and ‘‘reducedderived’’ patterns of evolution.
Phylogenetic Niche of the Ulvophyceae
Our phylogenetic analyses of concatenated mtDNAencoded protein sequences reveal a close relationship
between Pseudendoclonium and chlorophycean green
algae, with the trebouxiophyte Prototheca occupying
a basal position (fig. 4). These analyses included all the
green-algal mtDNA sequences available in public databases to minimize possible undesirable effects of small
taxon sampling and used Mesostigma viride as outgroup to
maximize the amount of phylogenetic information. The
position of Pseudendoclonium relative to the Chlorophyceae and Trebouxiophyceae may be the result of genuine
phylogenetic signal because it is consistent with our
Mitochondrial DNA Sequence of Pseudendoclonium 931
Table 7
Differences in the Number of Amino Acid Substitutions in
Seven Mitochondrial Proteins and Test for Equal Rates of
Substitutions in Selected Pairs of Green Algal Lineages
FIG. 4.—Phylogenetic position of Pseudendoclonium as inferred
from the deduced amino acid sequences of seven mitochondrial genes.
ML and ML-distances analyses of the data set (2,107 amino acid
positions) were carried out under the WAG-F model of amino acid
replacement, assuming -distributed rates of substitutions across sites.
The best-supported ML tree computed with CODEML is shown.
Numbers above the nodes indicate support values; upper and lower
values are from the ML and ML-distance analyses, respectively. The
chlorophyte lineages displaying a ‘‘reduced-derived’’ pattern of mtDNA
evolution are represented as thick lines.
finding that Pseudendoclonium mtDNA shares derived
structural features with its homologs in chlorophycean
green algae. However, the branching order of the
Chlorophyceae/Ulvophyceae/Trebouxiophyceae remains
an unresolved issue. The tree recovered in our analyses
may reflect variations in the evolutionary rates of mtDNA
sequences rather than the underlying phylogenetic signal,
and although we have included all currently available
green-algal mtDNAs in our analyses, taxon sampling is
still very low, with only one representative of the
Ulvophyceae and of the Trebouxiophyceae.
Pseudendoclonium appears to be closely related to
Pedinomonas, a green alga of uncertain affiliation. In
mitochondrial trees, Pedinomonas branches immediately
after the emergence of the Pseudendoclonium lineage.
Given the very long branch displayed by Pedinomonas,
the phylogenetic position of this chlorophyte might be
attributed to long-branch artifacts. However, the existence
of a close relationship between Pedinomonas and
ulvophytes is supported by the observation that the basal
bodies of Pedinomonas and ulvophytes display an absolute
counterclockwise orientation that contrasts with the directly opposed or clockwise orientation found in chlorophycean green algae (Melkonian 1990). Considering that
the 25-kb mtDNA of Pedinomonas is highly reduced in
size and gene content relative to its Pseudendoclonium and
Scenedesmus relatives and also considering the much
longer branch displayed by Pedinomonas mtDNA, it
appears that the mitochondrial genome evolved at a more
accelerated rate in the lineage leading to Pedinomonas
than in the Pseudendoclonium and Scenedesmus lineages.
Repeated Sequence Elements As an Evolutionary
Force in the Chlorophyta
Small repeats have been previously identified in
chlorophyte mtDNAs. The few that are found in Proto-
Compared Green Algae
(Taxon 1/Taxon 2)
N1a
N2b
N1/
( N1 1 N2)c
Pseudendoclonium/Nephroselmis
Pseudendoclonium/Prototheca
Pseudendoclonium/Scenedesmus
Pseudendoclonium/Pedinomonas
393
374
237
173
142
151
373
525
0.735
0.712
0.389
0.248
Pc
3.0
7.4
4.1
4.4
E-28
E-23
E-8
E-42
a
Number of informative substitutions attributable to taxon 1; that is, the
number of amino acid positions at which taxa 1 and 2 are different but taxa 2 and 3
are identical. Taxon 3, the outgroup, was Mesostigma.
b
Number of informative substitutions attributable to taxon 2; that is the
number of positions at which taxa 1 and 2 are different but taxa 1 and 3 are identical.
c
P values were obtained for the binomial tests of the null hypothesis
N1/(N1 1 N2) ¼ 0.5.
theca mtDNA vary from 30 to 200 nt, are mostly arranged
in tandem, and the recurring motifs are rich in A1T (Wolff
et al. 1994). Repeats in Scenedesmus mtDNA range from
16 to 118 nt, and, like their Pseudendoclonium counterparts, account for at least 15% of the genome, flank many
individual genes, are composed of various subrepeats, and
show a low bias in base composition (Nedelcu et al. 2000).
The repeats harbored by chlamydomonad mtDNAs are
shorter (9 to 14 nt) and richer in G1C (Boer and Gray
1991; Nedelcu and Lee 1998). Unlike all their known
chlorophyte counterparts, the numerous repeated sequences
found in Pedinomonas mtDNA are densely packed within
a discrete, noncoding region of the genome (Turmel et al.
1999). The low abundance of repeats in Nephroselmis and
Prototheca mtDNAs, together with the increased occurrence of these elements in the more derived lineages
leading to Pseudendoclonium and Scenedesmus, raise the
question on their origin. Repeats might have been present
in the mitochondrial genome of the last common ancestor
of the ulvophytes and chlorophycean green algae, and have
diverged after the split of these lineages. Alternatively, they
might have arisen independently in the Ulvophyceae and
Chlorophyceae.
Considering that recombination between dispersed
repeats can lead to genome rearrangements (gene losses
or inversions) and that gene content became reduced as
families of dispersed repeats emerged and grew in size
during the evolution of chlorophyte mtDNAs (at least in
derived lineages), we speculate that repeated elements
have played an important role in generating the great
diversity of size and gene arrangement seen in these
mtDNAs. In chlamydomonad mtDNAs, where gene
content is the poorest of the Chlorophyta lineage,
excision of coding regions via recombination between
flanking short repeats has been invoked to explain their
reduced gene content (Nedelcu 1997; Nedelcu 1998).
Dispersed repeats are also thought to act as hot spots for
recombination in land-plant (Palmer and Herbon 1988;
Mackenzie, He, and Lyznik 1994), fungal (Jamet-Vierny,
Boulay, and Briand 1997), and animal (Lunt and Hyman
1997) mtDNAs. Aside from dispersed repeats, other
factors most probably determine the tempo of gene loss.
932 Pombert et al.
Table 8
Compared Features of Pseudendoclonium mtDNA and Other Green-Plant mtDNAs
Size
(bp)
A1T
Content (%)
Coding
Sequences (%)a
Conserved
Genesb
Group I
Introns
Group II
Introns
186,609
366,924
368,799
490,520
57.6
55.2
56.1
56.2
65.0
36.8
33.0
—
69
49
48
53
7
0
0
0
25
23
20
23
Charophyte
Chaetosphaeridium
56,574
65.6
76.3
67
9
2
Prasinophytes
Mesostigma
Nephroselmis
42,424
45,223
67.8
67.2
86.6
80.6
65
65
4
4
3
0
Trebouxiophyte
Prototheca
55,328
74.2
70.6
61
5
0
Ulvophyte
Pseudendoclonium
95,880
60.7
58.7
57
7
0
Chlorophycean algae
C. eugametos
C. reinhardtii
Chlorogonium
Scenedesmus
22,897
15,758
22,704
42,919
65.4
54.8
62.2
63.7
84.6
83.1
89.1
60.6
12
12
12
42
9
0
6
2
0
0
0
2
Uncertain affiliation
Pedinomonas
25,137
77.8
60.5
22
0
1
Green Plant
Land plants
Marchantia
Arabidopsis
Beta
Oryza
a
Conserved genes, unique ORFs, introns, and intron ORFs were considered as coding sequences. The values for
Marchantia and Arabidopsis mtDNAs were taken from Lang, Gray, and Burger (1999) and Marienfeld, Unseld, and Brennicke
(1999), respectively. The proportion of coding sequences in Oryza mtDNA could not be determined because coordinates for
unique ORFs that are less than 150 codons and duplicated coding regions are not reported in the genome descriptions (accession
numbers AB076665 and AB076666).
b
Unique ORFs and intron ORFs were not taken into account.
For angiosperms, it has been shown that the tempo of
mitochondrial gene loss (and probably gene transfer to
the nucleus) is remarkably punctuated. Certain lineages
have rapidly lost most or all of their 16 ribosomal protein
and sdh genes, whereas other lineages, mostly ancient
lineages, have maintained a constant set of mitochondrial
genes for hundreds of millions of years (Adams et al.
2002). This punctuated pattern appears to be driven by
major episodic rises in the rate of functional gene
transfer.
Interestingly, short, dispersed repeats have been
observed in the chloroplast DNAs of Chlamydomonas
taxa (Boudreau and Turmel 1996; Maul et al. 2002) and of
the trebouxiophyte Chlorella vulgaris (Maul et al. 2002)
but not in Nephroselmis chloroplast DNA (Maul et al.
2002). The phylogenetic distribution of small, repeated
elements within chlorophyte chloroplast DNAs thus
parallels that observed for the mitochondrial genome.
Given the evidence for lateral transfers of introns (genetic
elements often associated with short repeats) between the
mitochondrial and chloroplast compartments (Turmel et al.
1995; Turmel et al. 1999; Lucas et al. 2001; Turmel, Otis,
and Lemieux 2002a), it is possible that such events
contribute to the dispersal of short repeats and account for
the presence of these elements in both organelle genomes
of the same cell. In this context, it will be interesting to see
if short, repeated elements are found in ulvophyte
chloroplast DNAs.
Conclusion
Representing the first ulvophyte organelle genome
sequence publicly available, Pseudendoclonium mtDNA is
a valuable source of information to our understanding of
mitochondrial genome evolution in the Chlorophyta. It
will be important to examine mtDNAs from additional
representatives of the Ulvophyceae to determine if the
distinctive traits exhibited by Pseudendoclonium mtDNA
are conserved in other ulvophyte mtDNAs or are restricted
to the lineage leading to this ulvophyte. A broader study of
mitochondrial genomes, including more trebouxiophytes
and chlorophycean green algae, will be required to resolve
the question about the monophyly of the Ulvophyceae, to
clarify the interrelationships between the Chlorophyceae,
Trebouxiophyceae, and Ulvophyceae, and also to better
understand how the great diversity seen at the mitochondrial genome level arose during the evolution of
chlorophytes.
Supplementary Material
The genome sequence reported in this paper has been
deposited in the GenBank database (accession number
AY359242).
Acknowledgments
This work was supported by a grant from the Natural
Sciences and Engineering Research Council of Canada (to
Mitochondrial DNA Sequence of Pseudendoclonium 933
M.T. and C.L.). J.-F.P. gratefully acknowledges a scholarship from CREFSIP (Centre de Recherche sur la Fonction,
la Structure et l’Ingénierie des Protéines).
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Mark Ragan, Associate Editor
Accepted January 8, 2004