1. 1810
Mol. Biol. Evol. 18(9):1810–1822. 2001
᭧ 2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
Accelerated Evolution of Functional Plastid rRNA and Elongation Factor
Genes Due to Reduced Protein Synthetic Load After the Loss of
Photosynthesis in the Chlorophyte Alga Polytoma
Dawne Vernon,*1 Robin R. Gutell,† Jamie J. Cannone,† Robert W. Rumpf,*2
and C. William Birky Jr.*‡
*Department of Molecular Genetics, Ohio State University; †Institute of Cellular and Molecular Biology, University of Texas
at Austin; and ‡Department of Ecology and Evolutionary Biology and Graduate Interdisciplinary Program in Genetics,
University of Arizona
Polytoma obtusum and Polytoma uvella are members of a clade of nonphotosynthetic chlorophyte algae closely
related to Chlamydomonas humicola and other photosynthetic members of the Chlamydomonadaceae. Descended
from a nonphotosynthetic mutant, these obligate heterotrophs retain a plastid (leucoplast) with a functional protein
synthetic system, and a plastid genome (lpDNA) with functional genes encoding proteins required for transcription
and translation. Comparative studies of the evolution of genes in chloroplasts and leucoplasts can identify modes
of selection acting on the plastid genome. Two plastid genes—rrn16, encoding the plastid small-subunit rRNA, and
tufA, encoding elongation factor Tu—retain their functions in protein synthesis after the loss of photosynthesis in
two nonphotosynthetic Polytoma clades but show a substantially accelerated rate of base substitution in the P. uvella
clade. The accelerated evolution of tufA is due, at least partly, to relaxed codon bias favoring codons that can be
read without wobble, mainly in three amino acids. Selection for these codons may be relaxed because leucoplasts
are required to synthesize fewer protein molecules per unit time than are chloroplasts (reduced protein synthetic
load) and thus require a lower rate of synthesis of elongation factor Tu. Relaxed selection due to a lower protein
synthetic load is also a plausible explanation for the accelerated rate of evolution of rrn16, but the available data
are insufficient to test the hypothesis for this gene. The tufA and rrn16 genes in Polytoma oviforme, the sole member
of a second nonphotosynthetic clade, are also functional but show no sign of relaxed selection.
Introduction
Nonphotosynthetic land plants and algae serve as a
basis for interesting natural experiments on the evolution-
ary consequence of the loss of a significant cell function.
After losing the ability to do photosynthesis, nonphoto-
synthetic species use various alternative carbon sources,
with the plants becoming parasitic on other plants, while
the algae take up complex organic molecules from their
environment. Recognized by their lack of chlorophyll,
these nongreen organisms have unique plastid (‘‘leuco-
plast’’) genomes. The evolutionary consequences of the
loss of photosynthesis can be studied by comparing the
leucoplast genomes of nonphotosynthetic species with the
chloroplast genomes of their closest photosynthetic rela-
tives. The majority of the genes in the chloroplasts of pho-
tosynthetic green algae and land plants encode proteins
required for photosynthesis or gene expression (transcrip-
tion and translation [Gillham 1994]; for recent data from
complete chloroplast genome sequences, see Turmel, Otis,
and Lemieux [1999], Lemieux, Otis, and Turmel [2000],
and the NCBI chloroplast genome page at http://www.
ncbi.nlm.nih.gov:80/PMGifs/Genomes/plastids࿞tax.
html). These two functions account for 32 and 16 genes,
1 Present address: National Institute of Standards and Technology,
DNA Technologies Group, Gaithersburg, Maryland.
2 Present address: LabBook.com, Inc., Columbus, Ohio.
Key words: Polytoma, Chlamydomonas, chloroplast rRNA gene,
chloroplast elongation factor gene, substitution rate, codon bias.
Address for correspondence and reprints: C. William Birky Jr.,
Department of Ecology and Evolutionary Biology, Biological Sciences
West, University of Arizona, Tucson, Arizona 85721. E-mail:
birky@u.arizona.edu.
respectively, in Chlamydomonas reinhardtii, which also
encodes a minimal set of rRNA and tRNA genes (http:
//www.biology.duke.edu/chlamy࿞genome/chloro.html).
Genes encoding proteins with other functions, as well
as unidentified open reading frames, are found in some
taxa; C. reinhardtii has 15 of these. There is strong in-
direct evidence that at least one of these genes, yet to
be identified, codes for a protein that has an essential
nonphotosynthetic (ENP) function (Gillham 1994, pp.
83–86).
Investigations of leucoplast genome structure and
gene sequences of nonphotosynthetic organisms have
been limited to several parasitic angiosperm families
(Scrophulareae and Orobanchaceae; e.g., dePamphilis
and Palmer 1990; Wimpee, Morgan, and Wrobel 1992a,
1992b; Nickrent, Duff, and Konings 1997; Wolfe and
dePamphilis 1998; Young and dePamphilis 2000) and
the euglenoid alga Astasia (Siemeister, Buchholz, and
Hachtel 1990; Siemeister and Hachtel 1990a, 1990b).
The leucoplasts of these nonphotosynthetic species dif-
fer from the chloroplasts of their close green relatives
in numerous features. Morphologically, they are char-
acterized by reduction or elimination of the thylakoid
membranes. They all retain leucoplast ribosomes and
leucoplast DNA. However, their leucoplast genomes are
often reduced in size and complexity compared with
chloroplast genomes in photosynthetic relatives.
These leucoplast genomes allow investigation of
the effects of selection on rates of evolution. When the
ability to do photosynthesis is lost, the photosynthetic
and photorespiration genes lose their function and con-
sequently are no longer subject to selection; they are
expected to become pseudogenes and can be lost entire-
ly. In contrast, the leucoplast genes needed for transcrip-

2. Accelerated Evolution of Leucoplast Genes 1811
FIG. 1.—Cladogram showing the relationships of the taxa used in
this study based on Rrn18 sequences (Rumpf et al. 1996; unpublished
data).
tion and translation of the leucoplast genome probably
remain functional and subject to at least some degree of
selection because they are needed to transcribe and
translate the ENP genes. The leucoplast genomes of the
beech root parasite Epifagus virginiana (Orobancha-
ceae), the oak parasite Conopholis americana (Oroban-
chaceae), and the euglenoid Astasia longa display these
characteristics. Most expression genes are still present
in the leucoplast genomes and appear intact, and some
leucoplast RNA and protein products have been dem-
onstrated (Siemeister, Buchholz, and Hachtel 1990; Sie-
meister and Hachtel 1990a, 1990b; Wimpee, Morgan,
and Wrobel 1992; Wolfe, Morden, and Palmer 1992).
Conversely, many leucoplast genes that coded for pho-
tosynthetic proteins appear nonfunctional in key do-
mains, are grossly truncated, or are absent from these
leucoplast genomes. Although most expression genes
are selectively retained, the leucoplast genome in Epi-
fagus is missing more than a dozen expression genes
(tRNA genes, ribosomal protein genes, and all four
RNA polymerase subunit genes; Morden et al. 1991),
all four RNA polymerase genes are pseudogenes in
Lathraea (Lusson, Delavault, and Thalouarn 1998), and
the leucoplast genome in Conopholis is apparently miss-
ing several leucoplast tRNA genes (Wimpee, Morgan,
and Wrobel 1992b). Presumably, these tRNAs and pro-
teins are imported from the cytoplasm at a rate that may
be too low for detection but is sufficient for protein syn-
thesis in leucoplasts.
The tempo of evolution has also changed in the
leucoplast genomes of these nonphotosynthetic species.
Most, but not all, of the apparently still-functional genes
analyzed in leucoplasts show an increased rate of nu-
cleotide substitution compared with rates in their green
relatives. Most of the rate increases found in functional
leucoplast genes in Astasia and Epifagus are in the range
of 1.5-fold to 8-fold, while rrn16 genes in Epifagus and
Conopholis show 40-fold rate increases (references in
Results and Discussion). This suggests that selection on
these genes has been relaxed, even though it has not
been eliminated completely.
To test the generality of these results, we extended
the analysis of the sequence and evolution of leucoplast
translation genes to the nonphotosynthetic chlorophyte
algae, separated from the euglenoid plastid and from
land plants by over 400 Myr of evolution and large dif-
ferences in physiology and habitats. Phylogenetic anal-
yses of Rrn18 sequences show that the members of the
nonphotosynthetic genus Polytoma belong to two dif-
ferent lineages within the clade that includes all Chla-
mydomonas species as well as a number of other pho-
tosynthetic genera and another nonphotosynthetic clade,
Polytomella (Rumpf et al. 1996; unpublished data).
Many species of Chlamydomonas are facultative auxo-
trophs, capable of utilizing acetate as their sole carbon
and energy source, and nonphotosynthetic mutants are
readily isolated in C. reinhardtii (Harris 1989). It is as-
sumed that Polytoma species arose as nonphotosynthetic
mutants of facultative auxotrophs similar to the extant
Chlamydomonas. The single large cup-shaped leucoplast
in Polytoma does not have thylakoid membranes but still
contains ribosomes, DNA (lpDNA), rRNA, and stored
starch granules (Lang 1963; Scherbel, Behn, and Arnold
1974; Siu, Chiang, and Swift 1976; Vernon-Kipp, Kuhl,
and Birky 1989). Polytoma is sensitive to inhibitors of
chloroplast protein synthesis, which is additional evi-
dence that the leucoplast is synthesizing at least one
protein that is essential for auxotrophic growth and re-
production (Scherbel, Behn, and Arnold 1974).
Although these data strongly suggest that Polytoma
retains a functional leucoplast expression system, the
leucoplast genes involved have not been identified and
demonstrated to be functional. We sequenced the rrn16
gene and the tufA gene (encoding the plastid elongation
factor Tu) from two representatives of the Polytoma
uvella clade (P. uvella 964 and Polytoma obtusum
DH1), plus Polytoma oviforme, which is the sole mem-
ber of the second Polytoma lineage. For comparison,
these genes were also sequenced from two closely re-
lated photosynthetic relatives (Chlamydomonas humi-
cola SAG 11-9 and Chlamydomonas dysosmos UTEX
2399). The evolutionary relationships of these strains
and some others involved in the analysis are shown in
figure 1. This cladogram agrees with phylogenetic anal-
yses of the rrn16 and tufA genes (figs. 2 and 3). Rela-
tive-rate tests showed increased substitution rates in
rrn16 and tufA, compared with green relatives, in the
two P. uvella species. However, sequence analyses
showed that the genes were subject to selection and
therefore functional. The increase in substitution rate
was greater at sites subject to less stringent selection,
implicating a partial relaxation of selection. The tufA
gene of P. obtusum showed a large reduction in codon
preference, suggesting that the relaxed selection is due
at least partly to a reduced load of protein synthesis.
This was proposed earlier for the increased substitution
rates in Epifagus (Wolfe et al. 1992), but alternative ex-
planations were not ruled out. We observed no increase
in the substitution rate in the branch leading to P. ovi-
forme, suggesting that photosynthesis was lost more re-
cently in this lineage. This is the first analysis of the
molecular evolutionary consequences of the loss of pho-
tosynthesis in a chlorophyte alga.
Materials and Methods
Organisms
Polytoma uvella (UTEX 964) and P. oviforme
(SAG 62-27) were obtained from the University of Tex-

3. 1812 Vernon et al.
FIG. 2.—Neighbor-joining tree of rrn16 sequences. Branch lengths in percentages of substitutions are shown above the branches; below
the branches are the lengths in the most parsimonious tree, which had an identical topology.
FIG. 3.—Neighbor-joining tree of tufA sequences. Branch lengths in percentages of substitutions are shown above the branches; below the
branches are the lengths in the most parsimonious tree and the maximum-likelihood tree, which had identical topologies.
as Culture Collection of Algae and from Sammlung von
Algenkulturen Gottingen, respectively. P. obtusum (des-
ignated strain DH1 by us) was obtained from David Her-
rin at the University of Texas at Austin; it originally
came from Luigi Provasoli’s collection at Yale. All cul-
tures were subcloned once or twice and grown in Po-
lytomella medium.
Chlamydomonas humicola UTEX 225 and C. dy-
sosmos UTEX 2399, from the University of Texas Cul-
ture Collection of Algae, were combined under the spe-
cies name Chlamydomonas applanata Pringsheim based
on morphology and autolysin cross-reactions (Ettl 1976)
and identity of the nuclear Rrn18 gene sequences (Gor-
don et al. 1995). Consistent with this, we found no sub-
stitution differences and only one insertion or deletion
difference in their chloroplast rrn16 sequences, while
the tufA sequences showed two synonymous differences
and one nonsynonymous differences and no insertions
or deletions. Consequently, we included only the C.
humicola sequences in the analyses described here.
DNA Preparation
The rrn16 gene of P. uvella was cloned. Whole-
cell DNA was isolated with a lysis method designed to
yield high-molecular-weight chloroplast DNA, modified
from Grant, Gillham, and Boynton (1980) as described
in Vernon (1996). This DNA was fractionated in a
CsClϩbisbenzimide equilibrium gradient. The top band
in the gradient was identified as leucoplast DNA by
Southern hybridization with an rrn16 probe and dot blot
hybridization with a tufA probe. The C. reinhardtii
cpDNA probes were provided by Elizabeth Harris (Duke
University) and Jeffrey Palmer (Indiana University). The
top band was used to prepare a HindIII library cloned
in pBluescript. DNA obtained from these clones by al-
kaline lysis plasmid minipreps (Sambrook, Fritsch, and
Maniatis 1989) was electrophoresed, and Southern blots
on GeneScreen Plus were hybridized with the cpDNA
probes. A clone containing the rrn16 gene in a 6.2-kb
insert was identified and purified for sequencing with a
GeneClean kit (Bio 101). All other new sequences used
in this study were of genes amplified from partially pu-
rified whole-cell DNA isolated from CTAB lysates of
1L algal cultures.
Polymerase Chain Reaction Amplifications
Primers located near the ends of the rrn16 and tufA
genes were used to obtain DNA templates for sequenc-
ing. The 5Ј and 3Ј primers for rrn16 were A-17 (5Ј-
GTTTGATCCTGGCTCAC-3Ј) and 5005-15 (3Ј-CA-
TGTGTGGCGGGCA-5Ј). The 5Ј and 3Ј degenerate
primers for all but one of the tufA genes were 1F (5Ј-
GGDCAYGTTGAYCAYGG-3Ј) and 5R (3Ј-TGA-
CANCCRCGRCCRCA-5Ј). Primer 5R did not amplify
tufA from P. obtusum, so the 3Ј ends of the tufA genes
from the other chlamydomonad species were inspected
for conserved areas, and an alternative 3Ј primer
(1130R: 3Ј-CCRATACGGDCCACTRGC-5Ј) was de-
signed and used, located 100 bases farther 5Ј of the orig-
inal 3Ј primer 5R. This amplified a tufA fragment from
P. obtusum that was approximately 100 bases shorter
than the other chlamydomonad sequences. The rrn16

4. Accelerated Evolution of Leucoplast Genes 1813
amplification products sequenced were about 1.3 kb
long, except for P. uvella, which was about 1.6 kb long;
the tufA amplification products were about 1.1 kb long,
except for P. obtusum, which was about 1.0 kb long.
Optimal amplification conditions were determined for
each gene empirically; multiple separate amplifications
were performed and pooled, then purified using
GeneClean.
Sequencing
Both strands of all genes were sequenced manually
using a modified dsDNA Cycle Sequencing kit (Life
Technologies). Most internal primers for sequencing
were obtained from Paul Fuerst for the rrn16 gene and
from Jeffrey Palmer for the tufA gene; additional inter-
nal primers in conserved regions were designed to fill
gaps in sequence coverage.
Alignment of rrn16 Sequences
The five new sequences for the study reported here
(P. uvella, P. obtusum, P. oviforme, C. humicola, and
C. dysosmos) were initially aligned using CLUSTAL W
in SeqApp (Gilbert 1992) to match the rrn16 primary
structure alignment in the Ribosomal Database Project
(Maidak et al. 1994). The alignment was further refined
by comparison with 70 publicly available plastid rrn16
sequences using a SUN Microsystems workstation with
the alignment editor AE2 (developed by T. Macke,
Scripps Research Institute, San Diego, Calif., and avail-
able at http://www.cme.msu.edu/RDP/html/index.html).
Sequences were initially aligned for maximum primary
structure similarity; then, all positions associated with
the comparatively inferred base pairs were checked to
assure that these base-paired positions were properly
aligned. The final alignment (with a complete list of
species and numerous chloroplast and Polytoma SSU
rRNA secondary-structure diagrams) is available in the
supplement (on the MBE web site) as GenBank files
(fig. 3c in the supplement); a subset of sequences used
for phylogenetic analysis is shown in less detail in se-
quential format (fig. 6 in the supplement) and in inter-
leaved Pretty Print format (fig. 7 in the supplement).
Alignment of tufA Sequences
To assist alignment of tufA sequences, C. Delwiche
and J. Palmer at Indiana University provided their align-
ment, with 18 eubacterial, 8 cyanobacterial, 26 algal,
and 4 land plant sequences (array described in Delwiche,
Kuhsel, and Palmer 1995). The Polytoma and Chlamy-
domonas sequences were aligned to various subsets of
this array using DNA sequences but were influenced by
the resulting amino acid alignment. One thousand fifty-
three base pairs of the tufA gene were aligned (85% of
the coding region), leaving out the first 72 5Ј positions
and the last 96 3Ј positions for lack of data in some or
all species. The complete alignment is available in the
supplement (fig. 5); a subset of sequences is shown in
less detail in sequential format (fig. 6 in the supplement)
and in interleaved Pretty Print format (fig. 8 in the
supplement).
Phylogenetic Analyses
Gene trees for were produced with PAUP* (Swof-
ford 1998) and PHYLIP, version 3.56 (Felsenstein
1993). Sequence differences were corrected for multiple
hits using the Jukes-Cantor one-parameter model (Jukes
and Cantor 1969); otherwise, all analyses used default
settings. Before a set of sequences was subjected to phy-
logenetic analysis or relative-rate tests, sites that were
missing in one or more species were removed from all
sequences.
Relative-Rate Tests
Relative-rate tests (Sarich and Wilson 1973; Wu
and Li 1985) were performed to detect differences be-
tween rates of nucleotide (or amino acid) substitution in
the three Polytoma species studied, compared with green
species. Each relative-rate test involved a Polytoma iso-
late (nongreen, N), its closest photosynthetic relative
(green, G), and a photosynthetic outgroup species (O).
The test parameters were KON and KOG, the estimated
numbers of base substitutions per site occurring along
the lineages leading from the outgroup to the nongreen
Polytoma and to the green ingroup, respectively. The
estimated numbers of substitutions per site (K) were ob-
tained by correcting the observed sequence differences
per site for multiple hits with the Jukes-Cantor model
implemented in the MEGA sequence analysis package,
PHYLIP, or PAUP*. Two other correction methods were
used for comparison: the Kimura (1980) two-parameter
method, which allows different rates of transition versus
transversion, and the Tamura (1992) method, which uses
information about GϩC content as well as separate tran-
sition and transversion rates, again using MEGA. All
three correction methods added approximately the same
number of unobserved substitutions (data not shown),
so the Jukes-Cantor method was used for the relative-
rate test because it had the smallest variance. KON and
KOG were related to the evolutionary rates EON and EOG
along the nongreen and green lineages by KON ϭ EONT
and KOG ϭ EOGT, where T is the time since divergence
of the two lineages and was, of course, the same for
both lineages. Any rate differences between green lin-
eages and the nongreen lineages can be expressed as the
difference between these two numbers of substitutions
(KON Ϫ KOG ϭ [EON Ϫ EOG]T). The significance of rate
differences was evaluated as in Muse and Weir (1992).
A second method was also used to separate ob-
served sequence differences into rates along different
green or nongreen lineages, employing phylogenetic
software. Gene trees in which the observed substitutions
were apportioned to the various branches of the tree by
phylogenetic algorithms provided the inferred substitu-
tions on each green or nongreen branch. The appor-
tioned substitutions from a nongreen Polytoma species
and from its nearest green relative (ingroup) to their
nearest ancestral node, KAN and KAG, respectively, were
used to calculate the ratio KAN/KAG or the difference KAN

5. 1814 Vernon et al.
Table 1
Pairwise Numbers of Substitutions per Site Among rrn16 Genes
Chlamydomonas
reinhardtii
Chlamydomonas
moewusii
Polytoma
oviforme
Chlamydomonas
humicola
Polytoma
uvella
Polytoma
obtusum
Chlorella . . . . . . . . . . . . . . . . . . . . . .
Chlamydomonas reinhardtii. . . . . . .
Chlamydomonas moewusii . . . . . . . .
Polytoma oviforme . . . . . . . . . . . . . .
Chlamydomonas humicola . . . . . . . .
Polytoma uvella . . . . . . . . . . . . . . . .
0.162 0.185
0.138
0.171
0.123
0.106
0.170
0.119
0.125
0.095
0.227
0.212
0.213
0.194
0.152
0.220
0.193
0.200
0.166
0.142
0.054
NOTE.—Estimated number of substitutions per site (sequence divergence) ϭ sequence differences per site corrected for multiple hits by the Jukes-Cantor method.
Table 2
Relative-Rate Tests on rrn16 Based on Neighbor-Joining, Maximum-Parsimony, and Maximum-Likelihood Tree
Branch Lengths and on Pairwise Numbers of Substitutions
NONGREEN
SPECIES
GREEN
SPECIES
KAN/KAG
Neighbor
Joining
Maximum
Parsimony
Maximum
Likelihood
OUTGROUP
SPECIES KON ϪKOG
Polytoma uvella 964
Polytoma obtusum
Polytoma oviforme
Chlamydomonas humicola
C. humicola
Chlamydomonas moewusii
3.32
2.89
0.67
4.34
4.03
0.50
3.05
2.68
0.65
Chlamydomonas moewusii
Chlamydomonas reinhardtii
C. moewusii
C. reinhardtii
Chlamydomonas humicola
C. reinhardtii
0.088*
0.093**
0.075**
0.074**
Ϫ0.030
Ϫ0.015
* Significant at the 1% level.
** Significant at the 0.1% level.
Ϫ KAG. The difference divided by KAG, i.e., (KAN Ϫ
KAG)/KAG, can be used to compare the magnitudes of
the rate increases along two different nonphotosynthetic
lineages with different ingroups.
Codon usage and the amount of codon usage bias
in tufA were also investigated in P. obtusum, P. ovifor-
me, C. humicola, and C. reinhardtii. All gaps were re-
moved from the aligned sequences of these four species,
leaving 349 codons. DNA Strider was used to calculate
codon usage in these sequences. Relative synonymous
codon usage (RSCU) was calculated for each codon us-
ing MEGA. RSCU is the ratio of the observed frequency
of a particular codon to the expected frequency of that
codon calculated on the assumption that all codons are
used equally frequently; an RSCU value significantly
different from 1 is evidence of biased codon usage
(Sharp and Li 1987). A Pascal program provided by
Brian Morton was used to calculate the codon bias index
(CBI) and the codon adaptation index (CAI). The CBI
is an overall measure of codon bias for the entire gene
(Morton 1993); the CBI ranges from 0 (no codon bias
in the gene) to 1 (maximum codon bias). The CAI is
measure of bias in the use of a codon relative to its use
in a reference set of highly expressed genes (Sharp and
Li 1987).
Results and Discussion
The Evolutionary Rate of rrn16 is Accelerated in P.
uvella and P. obtusum but not in P. oviforme
Figure 2 shows the neighbor-joining tree of rrn16
sequences; the topology of the most parsimonious tree
from an exhaustive maximum parsimony search is iden-
tical. This tree is compatible with the trees of the nuclear
Rrn18 gene (fig. 1). Above each line in figure 2 is the
length of the branch in the Neighbor-Joining tree in per-
centage of substitutions; below each line is the length
of the same branch in the parsimony tree. The tree
shows a strong acceleration of substitution rate along
the branches leading to the nonphotosynthetic P. uvella
lineage.
We used the distances on the tree in figure 2 to
calculate the ratio of substitution rates on nonphotosyn-
thetic and photosynthetic lineages, as well as the differ-
ence between nonphotosynthetic and photosynthetic
rates. We also used the method of Wu and Li (1985) for
relative-rate tests based on corrected frequencies of pair-
wise substitutions (table 1). Table 2 shows the results of
these relative-rate calculations. The tests for P. uvella
and P. obtusum used C. humicola as their closest relative
and Chlamydomonas moewusii or C. reinhardtii as the
outgroup; the test for Polytoma oviforme used C. moe-
wusii as the closest green relative and C. humicola or
C. reinhardtii as the outgroup. All relative-rates tests
showed significantly increased substitution rates in the
branch leading to P. obtusum versus the branch leading
to the ingroup (C. humicola), and an even greater rate
increase was seen in P. uvella. Figure 2 shows that the
branch leading from the common ancestor of the P.
uvella clade and C. humicola to the common ancestor
of P. uvella and P. obtusum is longer, i.e., has more
substitutions, than the branch leading to C. humicola.
This shows that the acceleration began in the common
ancestor of the P. uvella clade, as expected. As a con-
trol, we performed a relative-rate test on the nuclear
Rrn18 gene of P. uvella (not shown). The test showed
a small increase in this nongreen species, but it was not

6. Accelerated Evolution of Leucoplast Genes 1815
Table 3
Pairwise Numbers of Substitutions per Site Among tufA Genes
ORGANISMS
SEQUENCE DIVERGENCE
All Sites 3rd Position
1st ϩ 2nd
Positions
Polytoma obtusum–Chlamydomonas reinhardtii. . . .
Chlamydomonas humicola–C. reinhardtii . . . . . . . . .
P. obtusum–C. humicola . . . . . . . . . . . . . . . . . . . . . . .
Polytoma oviforme–C. humicola. . . . . . . . . . . . . . . . .
P. oviforme–C. reinhardtii . . . . . . . . . . . . . . . . . . . . .
0.25334
0.13349
0.22619
0.15501
0.15988
0.74805
0.31078
0.62276
0.43202
0.40108
0.09112
0.05843
0.08612
0.04732
0.06324
NOTE.—Estimated number of substitutions per site (sequence divergence) ϭ sequence differences per site corrected for multiple hits by the Jukes-Cantor method.
Table 4
Relative-Rate Tests on tufA Based on Tree Branch Lengths and Pairwise Distance Matrices
NONGREEN
SPECIES
GREEN
SPECIES
KAN/KAG
Parsimony
Neighbor
Joining
Maximum
Likelihood OUTGROUP
KON Ϫ KOG
All Sites
3rd
Position
1st ϩ 2nd
Positions
Polytoma obtusum
Polytoma oviforme
Chlamydomonas humicola
Chlamydomonas reinhardtii
3.23
1.26
2.7
1.18
4.39
1.34
C. reinhardtii
C. humicola
0.1998**
0.0215
0.4408*
0.1167
0.0278*
Ϫ0.00951
* Significant at the 1% level.
** Significant at the 0.1% level.
statistically significant. No rate increase was seen in the
branch leading to P. oviforme.
The Evolutionary Rate of tufA is Accelerated in P.
obtusum but not in P. oviforme
Sequences of tufA are available for C. humicola, P.
obtusum, C. reinhardtii, P. oviforme, and a number of
green algae outside of the Chlamydomonadaceae. Of
these, Codium is the closest relative, but when we used
it as an outgroup, all three tree-making algorithms
grouped Codium with P. obtusum, presumably due to
long-branch attraction. We therefore used only the four
Chlamydomonadaceae, with C. reinhardtii serving as
the outgroup for C. humicola and P. obtusum, and C.
humicola serving as the outgroup for C. reinhardtii and
P. oviforme. The tufA sequences of these four species
have 1,014 sites in common. The topology of the neigh-
bor-joining tree of these genes (fig. 2) is consistent with
the Rrn18 tree (fig. 1). However, long-branch attraction
was still a problem with the parsimony and maximum-
likelihood algorithms, which favored the tree that placed
P. obtusum with C. reinhardtii. The correct parsimony
tree (the one with the same topology as the Neighbor-
Joining tree and all trees involving rrn16 or Rrn18) was
the least parsimonious and had the lowest likelihood
scores, although not by much. The branch lengths for
the correct trees from all three algorithms are shown in
figure 2. In every case, the branch leading to P. obtusum
is much longer than that leading to C. humicola, while
P. oviforme shows no acceleration. Table 3 shows the
estimated pairwise numbers of substitutions among
these species; the branch leading to P. obtusum is ac-
celerated in all three trees (parsimony, Neighbor-Join-
ing, and maximum likelihood).
We performed relative-rate tests of the evolution of
tufA in P. obtusum and P. oviforme, using the pairwise
differences with the Jukes-Cantor correction for multiple
hits (table 3). In addition to calculating relative rates of
nucleotide substitutions for all aligned sites, we com-
pared first ϩ second codon positions with third codon
positions. The results are shown in table 4; all tests
showed a significantly higher substitution rate in the
branch leading to the nonphotosynthetic P. obtusum than
in the branch leading to the photosynthetic ingroup, C.
humicola. The increase was greater in the third codon
positions than in the first and second positions. No sig-
nificant difference was found between the branches lead-
ing to P. oviforme and C. reinhardtii, in agreement with
the data from rrn16.
The Plastid tufA Genes in Polytoma Remain
Functional After the Loss of Photosynthesis
One possible explanation for the accelerated evo-
lution of rrn16 and tufA is that the genes became non-
functional in the nonphotosynthetic lineages. This is un-
likely, given the evidence that they remain functional in
nonphotosynthetic land plants. We found additional ev-
idence that tufA remained subject to selection, and hence
functional, in the Polytoma lineages:
1. There are no premature stop codons in the entire
gene. This could be because no stop mutations oc-
curred since the loss of photosynthesis or because
they were eliminated by selection. For the P. uvella
clade, we estimated the probability of no stop mu-
tations occurring as follows: First, we assumed that
a truncation would not inactivate the protein if it oc-
curred between the carboxyl terminal of the protein
and the 14th amino acid, since the first 13 amino
acids are not involved in intermolecular bonding in
Escherichia coli (Kawashima et al. 1996). In the re-
mainder of the protein, we found 108 codons that
were one substitution away from being stop codons.
As described above, we know that more synonymous

7. 1816 Vernon et al.
substitutions occurred along the branch leading to P.
obtusum than along the branch leading to C. humi-
cola from their common ancestor. There were 0.2235
extra substitutions per site in third codon positions,
which must have occurred after photosynthesis was
lost; this is also an estimate of the number of muta-
tions per site. We found 81 sense codons in the tufA
gene of P. obtusum that could have become a stop
codon as a result of one kind of substitution (e.g.,
UCG to UAG); the expected number of such substi-
tutions in the absence of selection was 81 ϫ 0.2235
ϫ 1/3 ϭ 6.034. We found 27 sense codons that could
have become stop codons as a result of either of two
kinds of substitutions (e.g., UAC to UAA or UAG);
the expected number of such substitutions was 27 ϫ
0.2235 ϫ 2/3 ϭ 4.023. Consequently, the expected
number of premature stop codons in the absence of
selection was 10.057, and from the Poisson distri-
bution the probability of finding no premature stop
codons was eϪ10.057 ϭ 4.3 ϫ 10Ϫ5. We conclude that
the tufA gene of P. obtusum must have been under
selection that eliminated genes with premature stop
codons most or all of the time since the loss of
photosynthesis.
Additional evidence was obtained using a tufA se-
quence obtained from P. uvella by Nedelcu (2001)
using the UTEX stock without subcloning. We
aligned 999 bp, or 333 complete codons, of P. uvella
and P. obtusum. The sequences of these species dif-
fered by 0.07892 synonymous substitutions per site,
all of which must have occurred since they diverged
from a common ancestor, after photosynthesis was
lost. We used parsimony to reconstruct 321 codons
of the sequence of their most recent common ances-
tor. This sequence contained 72 codons which could
have become stop codons if they had incurred single
specific mutations. The expected number of such
substitutions in the absence of selection was 72 ϫ
0.07892 ϫ 1/3 ϭ 1.894. The ancestral sequence also
contained 28 codons that could have become stop
codons as a result of either of two kinds of substi-
tutions; the expected number of such substitutions
was 28 ϫ 0.07892 ϫ 2/3 ϭ 1.473. Consequently, the
expected number of premature stop codons in the ab-
sence of selection is 3.367, and from the Poisson dis-
tribution the probability of finding no premature stop
codons was eϪ3.367 ϭ 0.0345.
2. All of the amino acid substitutions that occurred (out-
side of the hypervariable region discussed below) in
P. oviforme must be compatible with the normal
function of tufA, because each of the substituted ami-
no acids can be found at a comparable position in at
least one functional algal, cyanobacterial, or nonpho-
tosynthetic bacterial gene in the alignment array of
Delwiche, Kuhsel, and Palmer (1995). The same is
true of all but six amino acid substitutions seen in P.
obtusum. Both Polytoma sequences contain only con-
servative amino acid substitutions, except for some
nonconservative substitutions on the surface of the
EF-Tu protein of P. obtusum. None of these amino
acid substitutions are likely to change the folding of
the EF-Tu protein.
3. Nucleotide substitution rates in the tufA sequences at
first and second codon positions are much lower than
the rates at third positions (table 3), a difference that
can only be due to selection.
4. Relative to all other species, the tufA sequences from
Polytoma and Chlamydomonas contain numerous
amino acid substitutions, insertions, and deletions in
the hypervariable region. Despite the variability in
this region, we believe that it is compatible with
functionality of the protein in both Polytoma species
for the following reasons. First, the hypervariable re-
gion of P. oviforme is identical in length to that of
C. reinhardtii, and nearly identical in sequence, with
only two conserved amino acid differences between
the two species. The hypervariable region in P. ob-
tusum differs from that in C. reinhardtii in 13 sub-
stitutions and 3 gaps. However, this region is also
hypervariable and unusually long in functional EF-
Tu proteins from C. reinhardtii (Baldauf and Palmer
1990), C. humicola, and C. dysosmos, which are pho-
tosynthetic and therefore have functional EF-Tu pro-
teins. Second, the hypervariable region is on the out-
side surface of the protein in functional domain 3,
where amino acid changes or extra amino acids
would probably not affect the conformational chang-
es that occur during catalysis (Berthtold et al. 1993),
especially since the amino acid composition of the
hypervariable region is even more hydrophilic in P.
obtusum than in C. humicola and C. reinhardtii.
Moreover, the face of domain 3 that interacts with
other EF-Tu molecules (Kawashima et al. 1996) and
with the acceptor stem or T stem of the tRNA (Nis-
sen et al. 1995) is opposite the hypervariable region.
The Plastid rrn16 Genes in Polytoma Remain
Functional After the Loss of Photosynthesis
The growth of P. uvella and of another member of
the same clade, P. uvella 62-3 ϭ P. mirum, is inhibited
by the antibiotics erythromycin, streptomycin, and spec-
tinomycin at 800, 400, and 50 mg/ml, respectively (data
not shown). Scherbel, Behn, and Arnold (1974) previ-
ously found that growth of P. mirum is inhibited by
streptomycin. This antibiotic is known to inhibit chlo-
roplast protein synthesis in C. reinhardtii at similar or
lower (erythromycin) concentrations (Harris 1989).
Spectinomycin sensitivity is especially interesting: it has
no known side effects, and Chlamydomonas mutants re-
sistant to high concentrations have mutations only in the
rrn16 gene. These data suggest that Polytoma, like
Chlamydomonas, synthesizes at least one essential pro-
tein on plastid ribosomes which contain functional 16S
rRNA molecules.
Consistent with the antibiotic studies, an analysis of
the primary and secondary structures of the 16S rRNA
molecules, inferred from the rn16 sequences, strongly sup-
ports functionality of the molecules. Here we present only
a summary; the complete analysis is included with the
secondary-structure figures and sequence alignments in the

8. Accelerated Evolution of Leucoplast Genes 1817
FIG. 4.—Relative synonymous codon usage values of codons in
Polytoma obtusum (open bars) compared with Chlamydomonas hum-
icola (filled bars). Separate graphs are shown for amino acids that are
sixfold-, fourfold-, and twofold-degenerate. Stars represent codons for
which the complementary anticodons are found in tRNAs encoded in
plant chloroplast genomes.
supplement and at the web site http://www.rna.icmb.
utexas.edu/PUBLICATIONS/BIRKY/.
1. The primary and secondary structures for the three
Polytoma rRNA sequences contain all of the struc-
tural elements present in the chloroplast rRNAs that
are functional, and, apart from a few insertions dis-
cussed below, all of the nucleotide positions in the
Polytoma sequences correspond to all of the positions
present in these 70 functional 16S rRNA chloroplast
sequences (figs. 1, 2, 3a, and 3d–f in the supplement
and at our web site mentioned above; see also Gutell
1994).
2. Differences between the Polytoma sequences occur
at positions that also vary in the functional SSU
rRNAs in the nuclear genes of the Eucarya, Bacteria,
and Archaea and in chloroplast and mitochondrial
genes (http://www.rna.icmb.utexas.edu/RDBMS/,
http://www.rna.icmb.utexas.edu/CSI/BPFREQ/
16S-MODEL-BP/, and figs. 2, 3a, and 3c in the sup-
plement and at our web site mentioned above). Con-
versely, positions that are conserved in the three phy-
logenetic domains plus chloroplasts and mitochon-
dria are also conserved in the Polytoma sequences.
3. Base pairs were predicted with comparative sequence
analysis (Gutell 1996; http://www.rna.icmb.utexas.
edu/METHODS/); two aligned positions that change
coordinately are considered possible base pairs.
These base pairs are highly conserved in the Poly-
toma SSU rRNA and are consistent with function-
ality. There are 70 base-paired positions at which the
Polytoma sequences differ from the chloroplast con-
sensus: of these, 24 are compensatory changes; 10
involve an A·U or G·C interchange to a G·U base
pair; and only 12 change an A·U, G·C, or G·U pair
to a noncanonical pair (figs. 2 and 3 at our web site
mentioned above). The number of noncanonical base
pairs in the Polytoma SSU rRNA is approximately
the same as in other rRNAs that are known to be
active in protein synthesis (unpublished data).
4. A comparison of the sequences of P. obtusum and P.
uvella reveals functional and structural constraints
acting on these 16S rRNA sequences since their com-
mon ancestor; because this ancestor was nonphoto-
synthetic, all of the differences between the two Po-
lytoma isolates arose in nonphotosynthetic lineages.
Among the 55 variable paired positions, 30 are in-
volved in a covariation at base pairs predicted with
comparative sequence analysis (fig. 4 in the supple-
ment and at our web site mentioned above). Of these
positions, 12 involve an exchange between Watson-
Crick base pairs, while three involve a Watson-Crick
base pair with something else. Of the 25 paired po-
sitions with a single change, 14 exchanged between
AU or GC and GU. Since our covariation algorithms
search for simultaneous changes regardless of the
base pair types, the fact that AU and GC are the most
frequent pairs identified at positions that covary in-
dicates that these genes are still undergoing positive
selection. In addition, several of the noncanonical co-
variation base pairs have been experimentally veri-
fied (reviewed in Gutell 1996), suggesting that all of
these covarying bases could be base-paired in the
secondary structure.
5. Substitution rates were higher at paired sites than at
unpaired sites, presumably because single substitu-
tions at paired sites are detrimental but pairs of com-
pensating changes are neutral (data not shown).
There are three large uncharacteristic insertions in
the Polytoma sequences. The P. oviforme gene has an
incompletely sequenced insertion larger than 438 bp be-
tween positions 427 and 428 (E. coli numbering). Po-
lytoma obtusum and P. uvella have insertions of 223 bp
and Ͼ596 bp, respectively, between positions 746 and
747. Finally, P. uvella has a second insertion of 85 nt
between (E. coli) positions 138 and 140. All of these
insertions occur in regions of the 16S rRNA that are
variable in sequence and not considered to be function-
ally important. The first, between positions 138 and 140
(E. coli numbering) contains 85 extra nucleotides in P.

9. 1818 Vernon et al.
uvella; 75 of these are A or U. We believe that this
insertion may be an internal transcribed spacer that is
excised after transcription for two reasons. First, this
extra sequence has a very high AU content, a charac-
teristic of internal transcribed spacer (ITS) sequences.
Second, an ITS has been found in the bacterium Cam-
pylobacter sputorum 16S rRNA at position 220 (E. coli
numbering; VanCamp et al. 1993; accession number
X67775), which is directly across the helix from our
presumed ITS. The two other insertions occur at the
ends of helices and may be either introns or ITSs.
Relaxed Selection Is at Least Partly Responsible for
the Accelerated Evolution
The preceding sections show that the rrn16 and
tufA genes in Polytoma remained subject to selection,
and hence functional, long after the loss of photosyn-
thesis. Although we cannot exclude the possibility that
these genes lost their function recently, they were prob-
ably still functional when we tested their antibiotic sen-
sitivity. Consequently the increased rate of base pair
substitution in the tufA and rrn16 genes in the P. uvella
lineage is not due to complete loss of function. We
therefore considered four other possible explanations for
their accelerated evolution:
1. An increased mutation rate would increase the rate
of substitution, provided, of course, that the fixation
probability was not reduced by the same factor. How-
ever, an increase in mutation rate cannot explain our
data because it would affect different functional re-
gions of a gene to the same extent; in contrast, we
found that the substitution rate in the rrn16 gene was
greater in regions coding for the stems of the rRNA
than in regions coding for the loops, and in tufA there
was a much greater increase in the synonymous rate
than in the nonsynonymous rate. These observations
show that an increased mutation rate is not the sole
cause of the accelerated evolution in rrn16 and tufA
but do not rule it out as a contributing factor.
2. There could be an increased level of adaptive or pos-
itive selection by fixation of mutations that were for-
merly neutral or detrimental but are now advanta-
geous. This explanation is unlikely because of the
large number of such mutations that would be re-
quired to explain the data. For example, parsimony
analysis (data not shown) suggests that about 152
base pair substitutions occurred in the rrn16 gene
along the lineage leading to P. uvella from the most
recent common ancestor of P. uvella and C. humi-
cola, while only 35 occurred in the C. humicola lin-
eage. The nonphotosynthetic lineage thus incurred
152 Ϫ 35 ϭ 117 more substitutions that would have
to be adaptive; this is 77% of all of the substitutions
after photosynthesis was lost. Moreover, it is proba-
bly not a complete explanation because it is not likely
to increase the synonymous substitution rate. How-
ever, it is possible that positive selection contributed
in part to the increase in substitution rate.
3. A reduced effective population size (Ne) would make
natural selection against detrimental mutations less
effective. By making some mutations that were for-
merly detrimental effectively neutral, it would in-
crease the overall fixation probability of new muta-
tions. A decrease in the frequency of recombination
of plastid genes would reduce Ne as a result of the
Hill-Robertson effect (Birky and Walsh 1988), but
there is no reason to suspect this in nonphotosyn-
thetic lineages. A more plausible reason for a de-
crease in Ne is that an obligate heterotroph has fewer
niches available to it, which might reduce Ne by de-
creasing the total population size or increasing the
variance in offspring number. In any event, a reduced
Ne cannot explain our data by itself, because the ratio
KAN/KAG was greater for kinds of substitutions sub-
ject to weak selection (third positions in tufA; stems
in rrn16) than for those subject to relatively strong
selection (first and second positions in tufA; loops in
rrn16). A theoretical analysis of the combined effects
of directional selection and drift, using Kimura’s
(1957) equation for the fixation probability of a mu-
tation, showed that a decrease in effective population
size by itself would cause KAN/KAG to be larger, not
smaller, for strongly selected detrimental mutations
than for relatively weakly selected mutations (unpub-
lished data). In addition, a reduced Ne would affect
nuclear genes as well as organelle genes, whereas we
observed no significant increase in substitution rate
in the nuclear Rrn18 genes of P. obtusum and P.
uvella. We cannot rule out the possibility that some
of the observed acceleration in evolutionary rate is
due to a reduction in effective population size too
small to be detected in the nuclear gene, but this
cannot be a complete explanation.
4. Relaxed selection against detrimental mutations is a
plausible explanation for the increase in substitution
rate in nonphotosynthetic lineages. This would re-
quire that some mutations that were detrimental in a
chloroplast are neutral or effectively neutral in a leu-
coplast and thus more likely to be fixed. The rrn16
and tufA genes are involved in translation of plastid
proteins, and mutations in these genes are likely to
be detrimental if they directly or indirectly reduce the
rate or accuracy of protein synthesis. Selection would
be relaxed if heterotrophic algae could tolerate either
a lower rate of plastid protein synthesis or a higher
proportion of amino acid substitutions in plastid pro-
teins due to mistranslation. At least the first of these
conditions is very likely to be met. Most of the chlo-
roplast protein-coding genes code for proteins in-
volved in photosynthesis, and all mutations affecting
these genes are selectively neutral after photosynthe-
sis is lost in the ancestor of a nonphotosynthetic
clade; as a result, they will accumulate mutations that
block transcription or translation or will be deleted
entirely. Leucoplasts thus have to translate only about
two-thirds as many proteins. Among the genes that
do not have to be expressed in heterotrophic algae or
plants is rbcL, which encodes one of the most abun-
dant proteins in photosynthetic organisms, ribulose
bisphosphate carboxylase. A number of photosyn-

10. Accelerated Evolution of Leucoplast Genes 1819
Table 5
Codon Adaptation Index (CAI) and Codon Bias Index
(CBI) for tufA in Chlamydomonas and Polytoma Species
Organism CAI(Cre) CAI(Cum) CBI
Chlamydomonas reinhardtii. . . .
Polytoma oviforme . . . . . . . . . . .
Chlamydomonas humicola . . . . .
Polytoma obtusum. . . . . . . . . . . .
0.487
0.359
0.432
0.22
0.77
0.688
0.687
0.37
0.775
0.774
0.722
0.455
NOTE.—CAI(Cre) is calculated relative to the codon usage in C. reinhardtii
psbA gene; CAI(Cum) is calculated relative to the codon usage in the psbA genes
of C. reinhardtii, Porphyra purpurea, Odontella sinensis, and Cyanophora par-
adoxa.
thetic genes show high codon bias characteristic of
highly expressed genes.
We hypothesized that the loss of photosynthesis in
Polytoma permitted a lower rate of synthesis of elon-
gation factor Tu because these heterotrophic organisms
can tolerate a lower overall rate of protein synthesis than
their photosynthetic relatives. This hypothesis predicts
that the tufA gene will show less codon bias in P. ob-
tusum than in C. humicola, and probably less codon bias
than in other Chlamydomonas species. Codon bias is
generally greater in highly expressed genes, probably
because selection favors codons that are read by more
abundant tRNAs (e.g., Morton 1993, 1996; Sharp et al.
1995). We calculated two measures of codon bias for
the tufA genes of the Chlamydomonadaceae. The CBI
measures the overall level of codon bias in a gene and
ranges from 0 to 1; the CAI (Sharp and Li 1987) mea-
sures codon bias for a gene relative to one or more high-
ly expressed genes that show high codon bias. Two ref-
erence databases of codon use in a highly expressed
plastid gene were provided by Brian Morton: the psbA
gene of C. reinhardtii and the combined psbA genes of
C. reinhardtii, Porphyra purpurea, Cyanophora para-
doxa, and Odontella sinensis. The CBI and both CAI
values were lower for P. obtusum than for the two Chla-
mydomonas species and P. oviforme (table 5).
These data show that codon bias in tufA is reduced
in P. obtusum compared with its closest photosynthetic
relative, C. humicola. The change in codon bias might
be due to a change in relative mutation rates, e.g., to
favor AT pairs over GC. To test this possibility, we cal-
culated expected numbers of the four codons in all four-
fold-degenerate amino acids and in the fourfold-degen-
erate codon set of sixfold-degenerate amino acids, as-
suming that the expected proportions of A, T, G, and C
were the averages of those found in the third positions
of all codons. The deviation of observed numbers from
these expected numbers can be summarized by the chi-
square value, which was much greater for C. humicola
(64.3) than for P. obtusum (46.2). We also found that
the percentages of AϩT were not significantly different
in the first ϩ second codon positions in C. humicola and
P. obtusum (53.0% vs. 52.6% respectively; P k 0.05
by Fisher’s exact test) but were significantly different in
the third codon positions (60.5% vs. 75.9%, respective-
ly; P K 0.001). Since mutation rates do not differ sys-
tematically between codon positions, we conclude that
the third-position base composition difference between
Chlamydomonas and Polytoma is due to selection on
synonymous codons rather than to mutation pressure.
The reduced codon bias in Polytoma is presumably due
to relaxed selection against mutations that substitute
less-used codons for those that are most used in the C.
humicola lineage.
A more detailed comparison of codon bias in P.
obtusum and C. humicola suggests that the bias that is
relaxed in the nonphotosynthetic species is largely a
preference for codons that can be read without wobble
(a mismatched base pair between tRNA and the mRNA
third codon position). Several plant genomes have been
completely sequenced and found to have a set of 29
tRNAs. Gene sequences of 15 of these have been
mapped on the chloroplast genome of C. humicola (E.
Boudreau, personal communication), and no tRNA
genes have been identified in any green alga other than
those known from plant chloroplasts, so it is likely that
these algae have the same set of chloroplast tRNAs as
do plants. Many codons require wobble to be read by
this set of tRNAs, and several require ‘‘superwobble,’’
in which one tRNA reads A, G, C, or U in the third
codon position. We calculated the RSCU for all degen-
erate amino acids (those using more than one codon) in
P. obtusum and C. humicola. Figure 4 shows that much
of the difference in codon bias is in the sixfold-degen-
erate amino acids leucine, serine, and arginine, which
preferentially use codons that can be read without wob-
ble. This preference is much greater for C. humicola
than for P. obtusum (chi-square test; 0.01 Ͼ P Ͼ 0.001).
Similar analyses for fourfold- and twofold-degenerate
amino acids show no significant difference between
these species; both species show preference for no wob-
ble over wobble in the twofold-degenerate amino acids
(P ഠ 0.9) and for no wobble or normal wobble over
superwobble in those fourfold-degenerate amino acids
that require superwobble (0.8 Ͼ P Ͼ 0.7).
In the case of rrn16, one can imagine that the re-
duced load of protein synthesis in the leucoplast has
resulted in less intense selection for translation rate or
fidelity. Alternatively, there may be a greater tolerance
for mutations that affect the rate of transcription of the
gene, of posttranscriptional processing, or of assembly
of the ribosome. Relaxed selection for translational rate
or fidelity might result in decreased stability of the sec-
ondary structure of the small-subunit rRNA. Compared
with C. humicola, both P. uvella and P. obtusum had
fewer GC pairs and more AU pairs, with GC/AU ratios
of 1.80, 1.40, and 1.34, respectively. These numbers are
compatible with the hypothesis that the loss of photo-
synthesis has been accompanied by a relaxation of se-
lection for helix stability in the leucoplast small-subunit
rRNA molecule. However, it is also compatible with a
decrease in the GϩC content of the genome as a whole.
A more definitive test of the hypothesis will require
comparisons of secondary-structure stability in a larger
sample of nonphotosynthetic species and their close
photosynthetic relatives, using the nuclear small-subunit
rRNA molecule as a control, together with data on the
GϩC contents of the genomes.

11. 1820 Vernon et al.
The bulk of the evidence suggests that the in-
creased rate of base pair substitution in the tufA and
rrn16 genes of Polytoma is due at least in part to relaxed
selection, such that mutations that reduce the rate or
fidelity of translation are less likely to be eliminated.
Rigorous tests of this hypothesis will require more de-
tailed analyses of a larger number of sequences of these
and other plastid expression genes.
Polytoma oviforme May Have Lost Photosynthesis
More Recently
The tufA gene of P. oviforme shows codon bias
similar to that of several close green relatives, and nei-
ther the rrn16 gene nor the tufA gene shows an increased
substitution rate in the lineage leading to P. oviforme
relative to green lineages. In contrast, the expression
gene sequences that we sampled from the P. uvella clade
all show significant rate increases compared with green
relatives, and tufA from P. obtusum (the only protein-
coding gene we obtained from the P. uvella clade)
shows minimal codon bias. These data are consistent
with the hypothesis that the P. uvella clade may have
resulted from a more ancient loss of photosynthesis than
the P. oviforme lineage, and only the P. uvella clade has
been evolving without photosynthesis long enough for
the consequences of relaxed selection to be evident.
Accelerated Evolution of Leucoplast Expression Genes
in Other Organisms
Smaller increases in evolutionary rates have been
demonstrated in the leucoplast expression genes rrn16,
rrn23, and tufA, as well as in the rbcL gene, of the
heterotrophic euglenoid alga Astasia longa (Siemeister,
Buchholz, and Hachtel 1990; Siemeister and Hachtel
1990a, 1990b). An increase in the substitution rate of
the rrn16 genes of some nonphotosynthetic angiosperms
was reported by Nickrent, Duff, and Konings (1997),
although it was not verified by relative-rate tests. The
increase was accompanied by an increase in AϩT con-
tent in the genes and consequently by an increase in
destabilizing AU pairs in the rRNA, such as we ob-
served. The authors did not compare the rates in stems
and loops. Nickrent and Starr (1994) also reported an
increased substitution rate in the nuclear Rrn18 genes of
a different set of species of holoparasitic angiosperms
and presented evidence that the increase could not be
explained by a decrease in generation time or effective
population size. In contrast, we observed no increase in
the evolutionary rate of Rrn18 in Polytoma (Rumpf et
al. 1996) or Polytomella (unpublished data). The holo-
parasitic angiosperm Epifagus virginiana was subjected
to an intensive analysis of evolutionary rates in leuco-
plast genes; rate increases were detected in rrn16 and
rrn23 and the pooled data for 17 tRNAs and 15 ribo-
somal proteins (Wolfe et al. 1992). In the ribosomal pro-
teins, accelerated evolution was seen in both nonsynon-
ymous and synonymous substitutions; in contrast to our
results, the increase was greater for nonsynonymous
substitutions. Epifagus also differed from Polytoma in
showing no change in codon bias (Morden et al. 1991).
Wolfe et al. (1992) proposed that the increase in non-
synonymous substitutions was probably due to relaxed
selection for both the rate and fidelity of protein syn-
thesis, while the increase in synonymous substitutions
probably resulted from an increased mutation rate. How-
ever, alternative explanations were not ruled out. In par-
ticular, both the synonymous and the nonsynonymous
rate increase might be entirely due to a decreased effec-
tive population size, which under some circumstances
can increase the rate of detrimental substitutions more
when they are under stronger selection (unpublished
data). dePamphilis, Young, and Wolfe (1997) found that
the substitution rate of the ribosomal protein gene rps2
was significantly accelerated in some, but not all, hol-
oparasitic (nonphotosynthetic) angiosperm lineages in
the Orobanchaceae and Sdrophulariaceae. In some cas-
es, there was a significant increase in synonymous but
not nonsynonymous substitutions, and in some other
cases, the reverse held. dePamphilis, Young, and Wolfe
(1997) proposed that increases in nonsynonymous sub-
stitution rates are probably due to reduced functional
constraint when abundant photosynthetic proteins do not
have to be synthesized. The increases in synonymous
substitutions were attributed to increased mutation rates.
However, no formal analyses of the data were given in
support of these conclusions.
The independent losses of photosynthesis in a num-
ber of different plants and algae provide biologists with
a series of natural experiments in which selection has
been reduced or eliminated to different extents in genes
with different functions. Future comparative studies of
leucoplast expression and photosynthetic gene sequenc-
es from Polytoma and Polytomella will help to unravel
the roles of mutation and selection in the molecular evo-
lution of plastid genes. The data to date suggest that
accelerated evolution of plastid expression genes is a
general consequence of the loss of photosynthesis and
that the rate of evolution of expression genes is strongly
influenced by the rate of protein synthesis that they must
sustain.
Supplementary Material
The DNA sequences have been deposited in
GenBank under accession numbers AF352839 (P. ob-
tusum tufA), AF352840 (P. oviforme tufA), AF352838
(C. humicola tufA), AF397587 (P. obtusum rrn16),
AF397589 (P. uvella rrn16), AF397588 (P. oviforme
rrn16), AF397590 (C. reinhardtii rrn16), and
AF397586 (C. humicola rrn16). Additional supplemen-
tary materials on the Molecular Biology and Evolution
web site include tufA and rrn16 sequence alignments
in sequential GenBank format and in interleaved Pretty
Print format, and the complete argument and additional
data showing that the rrn16 gene is functional in
Polytoma.
Acknowledgments
We are grateful to Brian Morton for supplying his
Macintosh Pascal program and reference databases for
calculating measures of codon bias, and to Aurora Ned-

12. Accelerated Evolution of Leucoplast Genes 1821
elcu for allowing us to analyze her tufA sequence from
P. uvella before it was published. Sally Otto analyzed
Kimura’s (1957) equation to verify that reducing Ne af-
fects strongly selected sites more than weakly selected
sites. Primers were kindly provided by Paul Fuerst and
Jeffrey Palmer. The large tufA database was kindly pro-
vided by Charles Delwiche. Other members of the Birky
lab at Ohio State University, notably Pamela Mackowski
and Stacy Seibert, assisted in numerous ways. We are
grateful to Jennifer Wernergreen, Aurora Nedelcu, and
two anonymous reviewers for helpful comments on ear-
lier manuscripts. D.V. submitted the data and a prelim-
inary analysis in a thesis in partial fulfillment of the
requirements for the Ph.D. degree at the Ohio State Uni-
versity. This work was supported in part by research
grants from NIH (GM34094) and NSF (BSR-9107069)
to C.W.B. and from NIH (GM48207) to R.R.G., and by
funds from the Ohio State University, the University of
Arizona, and the University of Texas.
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DAVID RAND, reviewing editor
Accepted May 30, 2001