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
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Abstract |
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Introduction |
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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 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 1990
; Siemeister and Hachtel 1990a, 1990b
; Wimpee, Morgan, and Wrobel 1992
; Wolfe, Morden, and Palmer 1992
). 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. 1991
), all four RNA polymerase genes are pseudogenes in Lathraea (Lusson, Delavault, and Thalouarn 1998
), and the leucoplast genome in Conopholis is apparently missing several leucoplast tRNA genes (Wimpee, Morgan, and Wrobel 1992b
). 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. 1996
; 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 1989
). 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 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 evidence that the leucoplast is synthesizing at least one protein that is essential for auxotrophic growth and reproduction (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 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. 1992
), 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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Materials and Methods |
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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 1976
) and identity of the nuclear Rrn18 gene sequences (Gordon et al. 1995
). 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)
as described in Vernon (1996)
. 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 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 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 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 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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Phylogenetic Analyses
Gene trees for were produced with PAUP* (Swofford 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 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 1973
; Wu and Li 1985
) 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)
two-parameter method, which allows different rates of transition versus transversion, and the Tamura (1992)
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)
.
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 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
).
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Results and Discussion |
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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)
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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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)
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 1989
). 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/.
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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:
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, 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
) 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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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 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 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 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)
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 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. 1992
). 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. 1991
). Wolfe et al. (1992)
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)
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)
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.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Present address: National Institute of Standards and Technology, DNA Technologies Group, Gaithersburg, Maryland.
2 Present address: LabBook.com, Inc., Columbus, Ohio.
1 Keywords: Polytoma, Chlamydomonas
chloroplast rRNA gene
chloroplast elongation factor gene
substitution rate
codon bias
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
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References |
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Baldauf S. L., J. D. Palmer, 1990 Evolutionary transfer of the chloroplast tufA gene to the nucleus Nature 344:262-265[ISI][Medline]
Berthtold H., L. Reshetnikova, C. O. A. Reiser, N. K. Schirmer, M. Slprinzl, R. Hilgenfeld, 1993 Crystal structure of active elongation factor Tu reveals major domain rearrangements Nature 365:126-132[ISI][Medline]
Birky C. W. Jr.,, J. B. Walsh, 1988 Effects of linkage on rates of molecular evolution Proc. Natl. Acad. Sci. USA 85:6414-6418[Abstract]
Delwiche C., M. Kuhsel, J. D. Palmer, 1995 Phylogenetic analysis of tufA sequences indicates a cyanobacterial origin of all plastids Mol. Phylogenet. Evol 4:110-128[ISI][Medline]
dePamphilis C. W., J. D. Palmer, 1990 Loss of photosynthetic and chlororespiratory genes from the plastid genome of a parasitic flowering plant Nature 348:337-339[ISI][Medline]
dePamphilis C. W., N. D. Young, A. D. Wolfe, 1997 Evolution of plastid gene rps2 in a lineage of hemiparasitic and holoparasitic plants: many losses of photosynthesis and complex patterns of rate variation Proc. Natl. Acad. Sci. USA 94:7367-7372
Ettl H., 1976 Die Gattung Chlamydomonas Ehrenberg Beih. Nova Hedwigia 49:1-1122
Felsenstein J., 1993 PHYLIP (phylogeny inference package) Version 3.56. Distributed by the author, Department of Genetics, University of Washington, Seattle
Gilbert D. G., 1992 SeqApp, a biological sequence editor and analysis program for Macintosh computers Available via gopher or anonymous ftp to ftp.biol.indiana.edu
Gillham N. W., 1994 Organelle genes and genomes Oxford University Press, New York
Gordon J., R. Rumpf, S. L. Shank, D. Vernon, C. W. Birky Jr., 1995 Sequences of the rrn18 genes of Chlamydomonas humicola and C. dysosmos are identical, in agreement with their combination in the species C. applanata (Chlorophyta) J. Phycol 31:312-313[ISI]
Grant D. M., N. W. Gillham, J. E. Boynton, 1980 Inheritance of chloroplast DNA in Chlamydomonas reinhardtii Proc. Natl. Acad. Sci. USA 77:6067-6071[Abstract]
Gutell R. R., 1994 Collection of small subunit (16S- and 16S-like) ribosomal RNA structures: 1994 Nucleic Acids Res 22:3502-3507[Abstract]
. 1996 Comparative sequence analysis and the structure of 16S and 23S rRNA Pp. 111128 in R. A. Zimmerman and A. E. Dahlberg, eds. Ribosomal RNA. Structure, evolution, processing, and function in protein biosynthesis. CRC Press, New York
Harris E. H., 1989 The Chlamydomonas sourcebook Academic Press, New York
Jukes T. H., C. R. Cantor, 1969 Evolution of protein molecules Pp. 21123 in H. N. Munro, eds. Mammalian protein metabolism. Academic Press, New York
Kawashima T., C. Berthet-Colominas, M. Wulff, S. Cusack, R. Leberman, 1996 The structure of the E. coli EF-Tu/EF-Ts complex at 2.5 A resolution Nature 379:511-518[ISI][Medline]
Kimura M., 1957 Some problems of stochastic processes in genetics Ann. Math. Stat 28:882-901[ISI]
. 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences J. Mol. Evol 16:111-120[ISI][Medline]
Lang N. J., 1963 Electron-microscopic demonstration of plastids in Polytoma J. Protozool 10:333-339[ISI][Medline]
Lemieux C., C. Otis, M. Turmel, 2000 Ancestral chloroplast genome in Mesostigma viride reveals an early branch of green plant evolution Nature 249:649-652
Lusson N. A., P. M. Delavault, P. A. Thalouarn, 1998 The rbcL gene from the non-photosynthetic parasite Lathraea clandestina is not transcribed by a plastid-encoded RNA polymerase Curr. Genet 34:212-215[ISI][Medline]
Maidak B. L., N. Larsen, M. J. McCaughey, R. Overbeek, G. J. Olsen, K. Fogel, J. Blandy, C. R. Woese, 1994 The Ribosomal Database Project Nucleic Acids Res 22:3485-3487[Abstract]
Morden C. W., K. H. Wolfe, C. W. dePamphilis, J. W. Palmer, 1991 Plastid translation and transcription genes in a non-photosynthetic plant: intact, missing and pseudo genes EMBO J 10:3281-3288[Abstract]
Morton B. R., 1993 Chloroplast DNA codon use: evidence for selection at the psbA locus based on tRNA availability J. Mol. Evol 37:273-280[ISI][Medline]
. 1996 Selection on the codon bias of Chlamydomonas reinhardtii chloroplast genes and the plant psbA gene J. Mol. Evol 43:28-31[ISI][Medline]
Muse S. V., B. S. Weir, 1992 Testing for equality of evolutionary rates Genetics 132:269-276
Nedelcu A. M., 2001 Complex patterns of plastid 16S rRNA gene evolution in nonphotosynthetic green algae J. Mol. Evol. (in press)
Nickrent D. L., R. J. Duff, D. A. M. Konings, 1997 Structural analyses of plastid-derived 16S rRNAs in holoparasitic angiosperms Plant Mol. Biol 34:731-743[ISI][Medline]
Nickrent D. L., E. M. Starr, 1994 High rates of nucleotide substitution in nuclear small-subunit (18S) rDNA from holoparasitic flowering plants J. Mol. Evol 39:62-70[ISI][Medline]
Nissen P., M. Kjeldgaard, S. Thirup, G. Polekhina, L. Reshetnikova, B. F. C. Clark, J. Nyborg, 1995 Crystal structure of the ternary complex of Phe-tRNA, EF-Tu, and a GTP analog Science 270:1464-1472[Abstract]
Rumpf R., D. Vernon, D. Schreiber, C. W. Birky Jr., 1996 Evolutionary consequences of the loss of photosynthesis in Chlamydomonadaceae: phylogenetic analysis of Rrn18 (18S rDNA) in 13 Polytoma strains (Chlorophyta) J. Phycol 32:119-126[ISI]
Sambrook J., E. F. Fritsch, T. Maniatis, 1989 Molecular cloning: a laboratory manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y
Sarich V. M., A. C. Wilson, 1973 Generation time and genomic evolution in primates Science 179:1144-1147[ISI][Medline]
Scherbel G., W. Behn, C. G. Arnold, 1974 Untersuchungen zur genetischen Funktion des farblosen Plastiden von Polytoma mirum Arch. Microbiol 96:205-222[ISI][Medline]
Sharp P. M., M. Averof, A. T. Lloyd, G. Matassi, J. F. Peden, 1995 DNA sequence evolution: the sounds of silence Philos. Trans. R. Soc. Lond. B Biol. Sci 349:241-247[ISI][Medline]
Sharp P. M., W.-H. Li, 1987 The codon adaptation indexa measure of directional synonymous codon usage bias, and its potential applications Nucleic Acids Res 15:1281-1295[Abstract]
Siemeister G., C. Buchholz, W. Hachtel, 1990 Genes for the plastid elongation factor Tu and ribosomal protein S7 and six tRNA genes on the 73 kb DNA from Astasia longa that resembles the chloroplast DNA of Euglena Mol. Gen. Genet 220:425-432[ISI][Medline]
Siemeister G., W. Hachtel, 1990a. Organization and nucleotide sequence of ribosomal RNA genes on a circular 73 kbp DNA from the colourless flagellate Astasia longa Curr. Genet 17:433-438[ISI][Medline]
. 1990b. Structure and expression of a gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) in the colourless euglenoid flagellate Astasia longa Plant Mol. Biol. 14:825-833[ISI][Medline]
Siu C.-H., K.-S. Chiang, H. Swift, 1976 Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. III. Ribosomal RNA cistrons of the nucleus and leucoplast J. Cell Biol 69:383-392[Abstract]
Swofford D. L., 1998 PAUP*: phylogenetic analysis using parsimony (*and many other methods) Version 4. Sinauer, Sunderland, Mass
Tamura K., 1992 Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C content biases Mol. Biol. Evol 9:678-687[Abstract]
Turmel M., C. Otis, C. Lemieux, 1999 The complete chloroplast DNA sequence of the green alga Nephroselmis olivacea: insights into the architecture of ancestral chloroplast genomes Proc. Natl. Acad. Sci. USA 96:10248-10253
VanCamp G., Y. Van de Peer, J. M. Neefs, P. Vandamme, R. DeWachter, 1993 Presence of internal transcribed spacers in the 16S and 23S ribosomal RNA genes of Campylobacter Syst. Appl. Microbiol 16:361-368[ISI]
Vernon D., 1996 Evolutionary consequences of the loss of photosynthesis in the nonphotosynthetic chlorophyte alga Polytoma Dissertation, Ohio State University, Columbus
Vernon-Kipp D., S. A. Kuhl, C. W. J. Birky, 1989 Molecular evolution of Polytoma, a non-green chlorophyte Pp. 284286 in C. T. Boyer, J. C. Shannon, and R. C. Hardison, eds. Physiology, biochemistry, and genetics of nongreen plastids. American Society of Plant Physiologists, Rockville, Md
Wimpee C. F., R. Morgan, R. L. Wrobel, 1992a. An aberrant plastid ribosomal RNA gene cluster in the root parasite Conopholis americana Plant Mol. Biol 18:275-285[ISI][Medline]
. 1992b. Loss of transfer RNA genes from the plastid 16S-23S ribosomal RNA gene spacer in a parasitic plant Curr. Genet 21:417-422[ISI][Medline]
Wolfe A. D., C. W. dePamphilis, 1998 The effect of relaxed functional constraints on the photosynthetic gene rbcL in photosynthetic and nonphotosynthetic parasitic plants Mol. Biol. Evol 15:1243-1258
Wolfe K. H., C. W. Morden, S. C. Ems, J. D. Palmer, 1992 Rapid evolution of the plastid translational apparatus in a nonphotosynthetic plant: loss or accelerated sequence evolution of tRNA and ribosomal protein genes J. Mol. Evol 35:304-317[ISI][Medline]
Wolfe K. H., C. W. Morden, J. D. Palmer, 1992 Function and evolution of a minimal plastid genome from a nonphotosynthetic parasitic plant Proc. Natl. Acad. Sci. USA 89:10648-10652[Abstract]
Wu C.-I., W.-H. Li, 1985 Evidence for higher rates of nucleotide substitution in rodents than in man Proc. Natl. Acad. Sci. USA 82:1741-1745[Abstract]
Young D. Y., C. W. dePamphilis, 2000 Purifying selection detected in the plastid gene matK and flanking ribozyme regions within a group II intron of nonphotosynthetic plants Mol. Biol. Evol 17:1933-1941