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, Robin R. Gutell, Jamie J. Cannone, Robert W. Rumpf and C. William Birky Jr

Department of Molecular Genetics, Ohio State University;
Institute of Cellular and Molecular Biology, University of Texas at Austin;
Department of Ecology and Evolutionary Biology and Graduate Interdisciplinary Program in Genetics, University of Arizona


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
Nonphotosynthetic land plants and algae serve as a basis for interesting natural experiments on the evolutionary consequence of the loss of a significant cell function. After losing the ability to do photosynthesis, nonphotosynthetic 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 ("leucoplast") 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 relatives. The majority of the genes in the chloroplasts of photosynthetic green algae and land plants encode proteins required for photosynthesis or gene expression (transcription and translation [Gillham 1994Citation ]; for recent data from complete chloroplast genome sequences, see Turmel, Otis, and Lemieux [1999]Citation , Lemieux, Otis, and Turmel [2000]Citation , 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, 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 indirect evidence that at least one of these genes, yet to be identified, codes for a protein that has an essential nonphotosynthetic (ENP) function (Gillham 1994Citation , 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 1990Citation ; Wimpee, Morgan, and Wrobel 1992a, 1992bCitation ; Nickrent, Duff, and Konings 1997Citation ; Wolfe and dePamphilis 1998Citation ; Young and dePamphilis 2000Citation ) and the euglenoid alga Astasia (Siemeister, Buchholz, and Hachtel 1990Citation ; Siemeister and Hachtel 1990a, 1990bCitation ). The leucoplasts of these nonphotosynthetic species differ from the chloroplasts of their close green relatives in numerous features. Morphologically, they are characterized 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 consequently are no longer subject to selection; they are expected to become pseudogenes and can be lost entirely. In contrast, the leucoplast genes needed for transcription 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 (Orobanchaceae), the oak parasite Conopholis americana (Orobanchaceae), 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 demonstrated (Siemeister, Buchholz, and Hachtel 1990Citation ; Siemeister and Hachtel 1990a, 1990bCitation ; Wimpee, Morgan, and Wrobel 1992Citation ; Wolfe, Morden, and Palmer 1992Citation ). Conversely, many leucoplast genes that coded for photosynthetic proteins appear nonfunctional in key domains, are grossly truncated, or are absent from these leucoplast genomes. Although most expression genes are selectively retained, the leucoplast genome in Epifagus is missing more than a dozen expression genes (tRNA genes, ribosomal protein genes, and all four RNA polymerase subunit genes; Morden et al. 1991Citation ), all four RNA polymerase genes are pseudogenes in Lathraea (Lusson, Delavault, and Thalouarn 1998Citation ), and the leucoplast genome in Conopholis is apparently missing several leucoplast tRNA genes (Wimpee, Morgan, and Wrobel 1992bCitation ). Presumably, these tRNAs and proteins are imported from the cytoplasm at a rate that may be too low for detection but is sufficient for protein synthesis 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 nucleotide 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 differences in physiology and habitats. Phylogenetic analyses of Rrn18 sequences show that the members of the nonphotosynthetic genus Polytoma belong to two different lineages within the clade that includes all Chlamydomonas species as well as a number of other photosynthetic genera and another nonphotosynthetic clade, Polytomella (Rumpf et al. 1996Citation ; unpublished data). Many species of Chlamydomonas are facultative auxotrophs, capable of utilizing acetate as their sole carbon and energy source, and nonphotosynthetic mutants are readily isolated in C. reinhardtii (Harris 1989Citation ). It is assumed 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 1963Citation ; Scherbel, Behn, and Arnold 1974Citation ; Siu, Chiang, and Swift 1976Citation ; Vernon-Kipp, Kuhl, and Birky 1989Citation ). Polytoma is sensitive to inhibitors of chloroplast protein synthesis, which is additional evidence that the leucoplast is synthesizing at least one protein that is essential for auxotrophic growth and reproduction (Scherbel, Behn, and Arnold 1974Citation ).

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 member of the second Polytoma lineage. For comparison, these genes were also sequenced from two closely related photosynthetic relatives (Chlamydomonas humicola 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 analyses of the rrn16 and tufA genes (figs. 2 and 3 ). Relative-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. 1992Citation ), but alternative explanations were not ruled out. We observed no increase in the substitution rate in the branch leading to P. oviforme, suggesting that photosynthesis was lost more recently in this lineage. This is the first analysis of the molecular evolutionary consequences of the loss of photosynthesis in a chlorophyte alga.



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Fig. 1.—Cladogram showing the relationships of the taxa used in this study based on Rrn18 sequences (Rumpf et al. 1996Citation ; unpublished data)

 


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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

 

    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
Organisms
Polytoma uvella (UTEX 964) and P. oviforme (SAG 62-27) were obtained from the University of Texas Culture Collection of Algae and from Sammlung von Algenkulturen Gottingen, respectively. P. obtusum (designated strain DH1 by us) was obtained from David Herrin at the University of Texas at Austin; it originally came from Luigi Provasoli's collection at Yale. All cultures were subcloned once or twice and grown in Polytomella medium.

Chlamydomonas humicola UTEX 225 and C. dysosmos UTEX 2399, from the University of Texas Culture Collection of Algae, were combined under the species name Chlamydomonas applanata Pringsheim based on morphology and autolysin cross-reactions (Ettl 1976Citation ) and identity of the nuclear Rrn18 gene sequences (Gordon et al. 1995Citation ). Consistent with this, we found no substitution 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)Citation as described in Vernon (1996)Citation . This DNA was fractionated in a CsCl+bisbenzimide 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 alkaline lysis plasmid minipreps (Sambrook, Fritsch, and Maniatis 1989Citation ) 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 purified 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 sequencing. The 5' and 3' primers for rrn16 were A-17 (5'-GTTTGATCCTGGCTCAC-3') and 5005-15 (3'-CATGTGTGGCGGGCA-5'). The 5' and 3' degenerate primers for all but one of the tufA genes were 1F (5'-GGDCAYGTTGAYCAYGG-3') and 5R (3'-TGACANCCRCGRCCRCA-5'). Primer 5R did not amplify tufA from P. obtusum, so the 3' ends of the tufA genes from the other chlamydomonad species were inspected for conserved areas, and an alternative 3' primer (1130R: 3'-CCRATACGGDCCACTRGC-5') was designed and used, located 100 bases farther 5' of the original 3' primer 5R. This amplified a tufA fragment from P. obtusum that was approximately 100 bases shorter than the other chlamydomonad sequences. The rrn16 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 internal 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 1992Citation ) to match the rrn16 primary structure alignment in the Ribosomal Database Project (Maidak et al. 1994Citation ). 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 available 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 sequential format (fig. 6 in the supplement) and in interleaved Pretty Print format (fig. 7 in the supplement).



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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

 
Alignment of tufA Sequences
To assist alignment of tufA sequences, C. Delwiche and J. Palmer at Indiana University provided their alignment, with 18 eubacterial, 8 cyanobacterial, 26 algal, and 4 land plant sequences (array described in Delwiche, Kuhsel, and Palmer 1995Citation ). The Polytoma and Chlamydomonas 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 5' positions and the last 96 3' 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* (Swofford 1998Citation ) and PHYLIP, version 3.56 (Felsenstein 1993Citation ). Sequence differences were corrected for multiple hits using the Jukes-Cantor one-parameter model (Jukes and Cantor 1969Citation ); otherwise, all analyses used default settings. Before a set of sequences was subjected to phylogenetic 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 1973Citation ; Wu and Li 1985Citation ) were performed to detect differences between rates of nucleotide (or amino acid) substitution in the three Polytoma species studied, compared with green species. Each relative-rate test involved a Polytoma isolate (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 obtained 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)Citation two-parameter method, which allows different rates of transition versus transversion, and the Tamura (1992)Citation method, which uses information about G+C content as well as separate transition 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 = EONT and KOG = 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 lineages and the nongreen lineages can be expressed as the difference between these two numbers of substitutions (KON - KOG = [EON - EOG]T). The significance of rate differences was evaluated as in Muse and Weir (1992)Citation .

A second method was also used to separate observed 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 substitutions on each green or nongreen branch. The apportioned 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 - KAG. The difference divided by KAG, i.e., (KAN - 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. oviforme, C. humicola, and C. reinhardtii. All gaps were removed 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 using 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 1987Citation ). 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 1993Citation ); 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 1987Citation ).


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
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 identical. 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 percentage 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 nonphotosynthetic and photosynthetic lineages, as well as the difference between nonphotosynthetic and photosynthetic rates. We also used the method of Wu and Li (1985)Citation for relative-rate tests based on corrected frequencies of pairwise 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. moewusii 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 control, 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 statistically significant. No rate increase was seen in the branch leading to P. oviforme.


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Table 1 Pairwise Numbers of Substitutions per Site Among rrn16 Genes

 

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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

 
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 neighbor-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 accelerated in all three trees (parsimony, Neighbor-Joining, and maximum likelihood).


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Table 3 Pairwise Numbers of Substitutions per Site Among tufA Genes

 
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 compared first + 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 significant difference was found between the branches leading to P. oviforme and C. reinhardtii, in agreement with the data from rrn16.


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Table 4 Relative-Rate Tests on tufA Based on Tree Branch Lengths and Pairwise Distance Matrices

 
The Plastid tufA Genes in Polytoma Remain Functional After the Loss of Photosynthesis
One possible explanation for the accelerated evolution of rrn16 and tufA is that the genes became nonfunctional in the nonphotosynthetic lineages. This is unlikely, given the evidence that they remain functional in nonphotosynthetic land plants. We found additional evidence 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 occurred since the loss of photosynthesis or because they were eliminated by selection. For the P. uvella clade, we estimated the probability of no stop mutations occurring as follows: First, we assumed that a truncation would not inactivate the protein if it occurred 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. 1996Citation ). In the remainder of the protein, we found 108 codons that were one substitution away from being stop codons. As described above, we know that more synonymous substitutions occurred along the branch leading to P. obtusum than along the branch leading to C. humicola 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 mutations 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 substitutions in the absence of selection was 81 x 0.2235 x 1/3 = 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 x 0.2235 x 2/3 = 4.023. Consequently, the expected number of premature stop codons in the absence of selection was 10.057, and from the Poisson distribution the probability of finding no premature stop codons was e-10.057 = 4.3 x 10-5. 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 sequence obtained from P. uvella by Nedelcu (2001)Citation 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 differed 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 ancestor. 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 x 0.07892 x 1/3 = 1.894. The ancestral sequence also contained 28 codons that could have become stop codons as a result of either of two kinds of substitutions; the expected number of such substitutions was 28 x 0.07892 x 2/3 = 1.473. Consequently, the expected number of premature stop codons in the absence of selection is 3.367, and from the Poisson distribution the probability of finding no premature stop codons was e-3.367 = 0.0345.
  2. All of the amino acid substitutions that occurred (outside of the hypervariable region discussed below) in P. oviforme must be compatible with the normal function of tufA, because each of the substituted amino acids can be found at a comparable position in at least one functional algal, cyanobacterial, or nonphotosynthetic bacterial gene in the alignment array of Delwiche, Kuhsel, and Palmer (1995)Citation . The same is true of all but six amino acid substitutions seen in P. obtusum. Both Polytoma sequences contain only conservative 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 region 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. obtusum differs from that in C. reinhardtii in 13 substitutions and 3 gaps. However, this region is also hypervariable and unusually long in functional EF-Tu proteins from C. reinhardtii (Baldauf and Palmer 1990Citation ), C. humicola, and C. dysosmos, which are photosynthetic and therefore have functional EF-Tu proteins. Second, the hypervariable region is on the outside surface of the protein in functional domain 3, where amino acid changes or extra amino acids would probably not affect the conformational changes that occur during catalysis (Berthtold et al. 1993Citation ), 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. 1996Citation ) and with the acceptor stem or T stem of the tRNA (Nissen et al. 1995Citation ) 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 = P. mirum, is inhibited by the antibiotics erythromycin, streptomycin, and spectinomycin at 800, 400, and 50 mg/ml, respectively (data not shown). Scherbel, Behn, and Arnold (1974)Citation previously found that growth of P. mirum is inhibited by streptomycin. This antibiotic is known to inhibit chloroplast protein synthesis in C. reinhardtii at similar or lower (erythromycin) concentrations (Harris 1989Citation ). Spectinomycin sensitivity is especially interesting: it has no known side effects, and Chlamydomonas mutants resistant to high concentrations have mutations only in the rrn16 gene. These data suggest that Polytoma, like Chlamydomonas, synthesizes at least one essential protein 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 supports 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 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 structural elements present in the chloroplast rRNAs that are functional, and, apart from a few insertions discussed 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 3df 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 supplement and at our web site mentioned above). Conversely, positions that are conserved in the three phylogenetic domains plus chloroplasts and mitochondria are also conserved in the Polytoma sequences.
  3. Base pairs were predicted with comparative sequence analysis (Gutell 1996Citation ; 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 Polytoma SSU rRNA and are consistent with functionality. There are 70 base-paired positions at which the Polytoma sequences differ from the chloroplast consensus: 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 common ancestor; because this ancestor was nonphotosynthetic, all of the differences between the two Polytoma isolates arose in nonphotosynthetic lineages. Among the 55 variable paired positions, 30 are involved in a covariation at base pairs predicted with comparative sequence analysis (fig. 4 in the supplement 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 positions 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 indicates that these genes are still undergoing positive selection. In addition, several of the noncanonical covariation base pairs have been experimentally verified (reviewed in Gutell 1996Citation ), 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 substitutions at paired sites are detrimental but pairs of compensating changes are neutral (data not shown).



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Fig. 4.—Relative synonymous codon usage values of codons in Polytoma obtusum (open bars) compared with Chlamydomonas humicola (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

 
There are three large uncharacteristic insertions in the Polytoma sequences. The P. oviforme gene has an incompletely sequenced insertion larger than 438 bp between positions 427 and 428 (E. coli numbering). Polytoma obtusum and P. uvella have insertions of 223 bp and >596 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 functionally important. The first, between positions 138 and 140 (E. coli numbering) contains 85 extra nucleotides in P. 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 characteristic of internal transcribed spacer (ITS) sequences. Second, an ITS has been found in the bacterium Campylobacter sputorum 16S rRNA at position 220 (E. coli numbering; VanCamp et al. 1993Citation ; 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 photosynthesis. Although we cannot exclude the possibility that these genes lost their function recently, they were probably still functional when we tested their antibiotic sensitivity. 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. However, an increase in mutation rate cannot explain our data because it would affect different functional regions 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 positive selection by fixation of mutations that were formerly neutral or detrimental but are now advantageous. This explanation is unlikely because of the large number of such mutations that would be required 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. humicola, while only 35 occurred in the C. humicola lineage. The nonphotosynthetic lineage thus incurred 152 - 35 = 117 more substitutions that would have to be adaptive; this is 77% of all of the substitutions after photosynthesis was lost. Moreover, it is probably not a complete explanation because it is not likely to increase the synonymous substitution rate. However, 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 formerly detrimental effectively neutral, it would increase the overall fixation probability of new mutations. A decrease in the frequency of recombination of plastid genes would reduce Ne as a result of the Hill-Robertson effect (Birky and Walsh 1988Citation ), but there is no reason to suspect this in nonphotosynthetic lineages. A more plausible reason for a decrease in Ne is that an obligate heterotroph has fewer niches available to it, which might reduce Ne by decreasing 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 subject 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)Citation equation for the fixation probability of a mutation, 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 (unpublished 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 require that some mutations that were detrimental in a chloroplast are neutral or effectively neutral in a leucoplast 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 proteins due to mistranslation. At least the first of these conditions is very likely to be met. Most of the chloroplast protein-coding genes code for proteins involved in photosynthesis, and all mutations affecting these genes are selectively neutral after photosynthesis 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 abundant proteins in photosynthetic organisms, ribulose bisphosphate carboxylase. A number of photosynthetic 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 elongation 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. obtusum 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, 1996Citation ; Sharp et al. 1995Citation ). 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 1987Citation ) measures codon bias for a gene relative to one or more highly expressed genes that show high codon bias. Two reference 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 paradoxa, and Odontella sinensis. The CBI and both CAI values were lower for P. obtusum than for the two Chlamydomonas species and P. oviforme (table 5 ).


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Table 5 Codon Adaptation Index (CAI) and Codon Bias Index (CBI) for tufA in Chlamydomonas and Polytoma Species

 
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 calculated expected numbers of the four codons in all fourfold-degenerate amino acids and in the fourfold-degenerate codon set of sixfold-degenerate amino acids, assuming 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 A+T were not significantly different in the first + second codon positions in C. humicola and P. obtusum (53.0% vs. 52.6% respectively; P >> 0.05 by Fisher's exact test) but were significantly different in the third codon positions (60.5% vs. 75.9%, respectively; P << 0.001). Since mutation rates do not differ systematically 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 degenerate 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-degenerate amino acids leucine, serine, and arginine, which preferentially use codons that can be read without wobble. This preference is much greater for C. humicola than for P. obtusum (chi-square test; 0.01 > P > 0.001). Similar analyses for fourfold- and twofold-degenerate amino acids show no significant difference between these species; both species show preference for no wobble over wobble in the twofold-degenerate amino acids (P {approx} 0.9) and for no wobble or normal wobble over superwobble in those fourfold-degenerate amino acids that require superwobble (0.8 > P > 0.7).

In the case of rrn16, one can imagine that the reduced 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 secondary 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 photosynthesis has been accompanied by a relaxation of selection for helix stability in the leucoplast small-subunit rRNA molecule. However, it is also compatible with a decrease in the G+C 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 G+C contents of the genomes.

The bulk of the evidence suggests that the increased 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 detailed 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 neither 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 1990Citation ; Siemeister and Hachtel 1990a, 1990bCitation ). An increase in the substitution rate of the rrn16 genes of some nonphotosynthetic angiosperms was reported by Nickrent, Duff, and Konings (1997)Citation , although it was not verified by relative-rate tests. The increase was accompanied by an increase in A+T content in the genes and consequently by an increase in destabilizing AU pairs in the rRNA, such as we observed. The authors did not compare the rates in stems and loops. Nickrent and Starr (1994)Citation 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. 1996Citation ) or Polytomella (unpublished data). The holoparasitic angiosperm Epifagus virginiana was subjected to an intensive analysis of evolutionary rates in leucoplast genes; rate increases were detected in rrn16 and rrn23 and the pooled data for 17 tRNAs and 15 ribosomal proteins (Wolfe et al. 1992Citation ). In the ribosomal proteins, accelerated evolution was seen in both nonsynonymous 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. 1991Citation ). Wolfe et al. (1992)Citation proposed that the increase in nonsynonymous substitutions was probably due to relaxed selection for both the rate and fidelity of protein synthesis, while the increase in synonymous substitutions probably resulted from an increased mutation rate. However, alternative explanations were not ruled out. In particular, both the synonymous and the nonsynonymous rate increase might be entirely due to a decreased effective 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)Citation found that the substitution rate of the ribosomal protein gene rps2 was significantly accelerated in some, but not all, holoparasitic (nonphotosynthetic) angiosperm lineages in the Orobanchaceae and Sdrophulariaceae. In some cases, there was a significant increase in synonymous but not nonsynonymous substitutions, and in some other cases, the reverse held. dePamphilis, Young, and Wolfe (1997)Citation proposed that increases in nonsynonymous substitution 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 number 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 sequences from Polytoma and Polytomella will help to unravel the roles of mutation and selection in the molecular evolution 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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
The DNA sequences have been deposited in GenBank under accession numbers AF352839 (P. obtusum 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 supplementary 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.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
We are grateful to Brian Morton for supplying his Macintosh Pascal program and reference databases for calculating measures of codon bias, and to Aurora Nedelcu for allowing us to analyze her tufA sequence from P. uvella before it was published. Sally Otto analyzed Kimura's (1957)Citation equation to verify that reducing Ne affects strongly selected sites more than weakly selected sites. Primers were kindly provided by Paul Fuerst and Jeffrey Palmer. The large tufA database was kindly provided 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 earlier manuscripts. D.V. submitted the data and a preliminary analysis in a thesis in partial fulfillment of the requirements for the Ph.D. degree at the Ohio State University. 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.


    Footnotes
 
David Rand, Reviewing Editor

1 Present address: National Institute of Standards and Technology, DNA Technologies Group, Gaithersburg, Maryland. Back

2 Present address: LabBook.com, Inc., Columbus, Ohio. Back

1 Keywords: Polytoma, Chlamydomonas chloroplast rRNA gene chloroplast elongation factor gene substitution rate codon bias Back

2 Address for correspondence and reprints: C. William Birky Jr., Department of Ecology and Evolutionary Biology, Biological Sciences West, University of Arizona, Tucson, Arizona 85721. birky{at}u.arizona.edu Back


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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
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 Acknowledgements
 References
 

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Accepted for publication May 30, 2001.