Research article | Open | Published:
Nucleotide diversity of the Chlamydomonas reinhardtii plastid genome: addressing the mutational-hazard hypothesis
BMC Evolutionary Biologyvolume 9, Article number: 120 (2009)
The mutational-hazard hypothesis argues that the noncoding-DNA content of a genome is a consequence of the mutation rate (μ) and the effective number of genes per locus in the population (N g ). The hypothesis predicts that genomes with a high N g μ will be more compact than those with a small N g μ. Approximations of N g μ can be gained by measuring the nucleotide diversity at silent sites (πsilent). We addressed the mutation-hazard hypothesis apropos plastid-genome evolution by measuring πsilent of the Chlamydomonas reinhardtii plastid DNA (ptDNA), the most noncoding-DNA-dense plastid genome observed to date. The data presented here in conjunction with previously published values of πsilent for the C. reinhardtii mitochondrial and nuclear genomes, which are respectively compact and bloated, allow for a complete analysis of nucleotide diversity and genome compactness in all three genetic compartments of this model organism.
In C. reinhardtii, the mean estimate of πsilent forthe ptDNA (14.5 × 10-3) is less than that of the nuclear DNA (32 × 10-3) and greater than that of the mitochondrial DNA (8.5 × 10-3). On average, C. reinhardtii has ~4 times more silent-site ptDNA diversity than the mean value reported for land plants, which have more compact plastid genomes. The silent-site nucleotide diversity of the different ptDNA loci that were studied varied significantly: from 0 to 71 × 10-3 for synonymous sites and from 0 to 42 × 10-3 for intergenic regions.
Our findings on silent-site ptDNA diversity are inconsistent with what would be expected under the mutational-hazard hypothesis and go against the documented trend in other systems of πsilent positively correlating with genome compactness. Overall, we highlight the lack of reliable nucleotide-diversity measurements for ptDNA and hope that the values presented here will act as sound data for future research concerning the mutational-hazard hypothesis and plastid evolution in general.
The magnitude of noncoding DNA in genomes can differ dramatically both among and within evolutionary lineages. This statement holds true for prokaryotic genomes and for the nuclear, mitochondrial, and plastid genomes of eukaryotes. The mutational-hazard (or mutational-burden) hypothesis  asserts that much of this observed variation in genome compactness can be explained by the product of the effective genetic population size (represented in this study as the effective number of gene copies at a locus [N g ], not individuals) and the mutation rate (μ). The hypothesis maintains that an allele with more noncoding nucleotides than an alternative allele will be selectively disadvantageous because the excess noncoding DNA can accumulate hazardous mutations that may negatively impact gene function; the burden (or selective disadvantage) of the allele containing the surplus of noncoding DNA is determined by μ and the number of additional noncoding nucleotides in the larger allele that can affect gene function. The hypothesis proposes that natural selection is more efficient at perceiving the burden of the expanded allele when N g is large; thus, genomes with a high N g μ are predicted to be more compact than those with a small N g μ.
Population-genetic theory tells us that at mutation-drift equilibrium the nucleotide diversity at neutral sites (πneutral) is equal to 2N g μ (where N g of uniparentally inherited organelle genes is thought to be about half that of haploid nuclear genes ). Estimates of πneutral can be acquired by measuring the nucleotide diversity at silent sites (πsilent), which include noncoding sites and the synonymous sites of protein-coding DNA. Because there are many factors that can cause N g to deviate from these neutral expectations, such as the influence of natural selection on linked variation, the only way to gain insight into 2N g μ is through empirical observation, i.e., by measuring πsilent.
As predicted by the mutational-hazard hypothesis, studies have found a positive correlation between πsilent and genome compactness: for the coding-rich DNA of prokaryotes πsilent is generally > 50 × 10-3; for the more noncoding-dense nuclear DNA (nucDNA) of land plants πsilent is in the range of 3 × 10-3 to 15 × 10-3; and for the nuclear genomes of vertebrates, which abound with noncoding DNA, πsilent tends to be ~3 × 10-3 . Similar trends are also observed for mitochondrial genomes: in the streamlined mitochondrial DNA (mtDNA) of mammals πsilent is ~40 × 10-3, whereas that for land-plant mtDNA, which is predominantly noncoding, is predicted to be < 0.4 × 10-3 . The contrast in πsilent between mammalian and land-plant mtDNA is thought to be a consequence of the high mutation rate in the former and the low mutation rate in the latter. Mutation rate has also been invoked to explain why, despite similar proposed values of N g , the mitochondrial and nuclear genomes of mammals have opposite coding densities – in mammals estimates of μ for mtDNA are roughly 30 times those for nucDNA .
It is speculated that πsilent for plastid DNA (ptDNA) also correlates positively with genome compactness [1, 4]; however, this issue has not been formally addressed because there are very few ptDNA sequences for which both πsilent and genome-compactness data are available — we are aware of only two plastid genomes for which these two statistics are published: those of Arabidopsis thaliana and Cycas taitungensis; moreover, the silent-site diversities for these two genomes were derived in each case from only a single locus and, therefore, may have been unrepresentative because of a low sampling bias (see the supplementary material of Lynch et al. ).
Of the 146 complete plastid-genome sequences available at the National Center for Biotechnology Information (NCBI; ) as of November 2008, the noncoding-DNA content ranges from 5%, in the apicomplexan Eimeria tenella, to 56%, in the unicellular green alga Chlamydomonas reinhardtii – a complete compilation is shown in Supplementary Table S1 [see Additional file 1]. Intriguingly, four of the five most bloated ptDNA sequences come from the Chlorophyta (a phylum containing most of the green-algal diversification), suggesting that this lineage is ideal for evaluating the mutational-hazard hypothesis vis-à-vis ptDNA. However, no studies as of yet have measured silent-site ptDNA diversity from the Chlorophyta. C. reinhardtii, a unicellular haploid alga, is a good candidate for investigating ptDNA diversity because it has a large (204 kilobases [kb]) and expanded plastid genome, and it is also a model organism for studying plastids and their photosynthetic processes . From the viewpoint of the mutational-hazard hypothesis, πsilent in the C. reinhardtii ptDNA should be less than that of more compact organelle genomes.
A previous study on C. reinhardtii  measured nucleotide diversity in its mitochondrial and nuclear genomes, which are respectively streamlined (~16–20 kb and ~20–30% noncoding, depending on the presence of optional introns) and bloated (~121 Megabases and ~83% noncoding). The mutational-hazard hypothesis would have forecasted πsilent for the mitochondrial genome to be greater than that of the nuclear genome, but instead πsilent for the mtDNA was found to be 4 times smaller than that of the nucDNA (8.5 × 10-3 vs. 32 × 10-3). Although these findings were in opposition to the mutational-hazard hypothesis, it was suggested that introns in the mtDNA impose a greater burden than those in the nuclear DNA and predicted that the same may be true for the mitochondrial intergenic regions .
It would be interesting to see for C. reinhardtii how values of πsilent for the plastid genome compare to those of the mitochondrial and nuclear genomes. When considering the fraction of noncoding DNA in each of these genomes, the mutational-hazard hypothesis would predict πsilent for the ptDNA to be smaller than that of the mtDNA and larger than that of the nucDNA. But it is already known, as discussed above, that this is not the case: in C. reinhardtii the mtDNA has less silent-site diversity than the nucDNA. If the noncoding regions in the plastid genome carry an inflated burden, as suggested for those in the mtDNA, then we would expect a very low value of πsilent for the ptDNA, much smaller than that of the mtDNA (i.e., << 8.5 × 10-3). However, if πsilent for the ptDNA is significantly larger than that of the mtDNA but still smaller than that of the nucDNA, it will be difficult to find any support in our data for the mutational-hazard hypothesis. In addition, silent-site ptDNA diversity data from C. reinhardtii will allow for a comparison of the πsilent values for the three genetic compartments of this species with those of Arabidopsis lyrata, the only other species for which reliable πsilent estimates from ptDNA, mtDNA, and nucDNA are published . Thus, to directly confront these issues, we measured πsilent from the ptDNA of various geographical isolates of C. reinhardtii.
Strains and their genetic loci
For our analysis we employed seven geographical isolates of C. reinhardtii, which are listed in Table 1. These are the same isolates that were previously used for calculating πsilent of the mtDNA and nucDNA. From each isolate, 14 distinct ptDNA regions were sequenced, amounting to 9.5 kb, 7.2 kb, and 2.7 kb of intergenic, protein-coding, and rRNA-coding ptDNA, respectively. A genetic map of the C. reinhardtii plastid genome highlighting these regions is shown in Figure 1.
We also produced a complete plastid-genome sequence for C. reinhardtii strain CC-503 (one of the isolates described in Table 1) by assembling ptDNA trace files generated by the C. reinhardtii nuclear-genome sequencing project [9, 10]. The earlier complete C. reinhardtii ptDNA sequence deposited at GenBank (accession# NC_005353) is a mosaic derived by linking the sequence data of various laboratory strains, most of which came from the "Ebersold-Levine" wild-type background of C. reinhardtii  – it is ideal to avoid using NC_005353 when calculating πsilent because sequence differences have been found between the ptDNA of some laboratory strains . A comparison of our CC-503 ptDNA sequence with NC_005353 reveals 471 single-nucleotide differences and 955 single-site indels; moreover, when the 14 ptDNA regions sequenced from the geographical isolates were sequenced from two additional laboratory strains belonging to the "Ebersold-Levine" wild-type background (CC-277 and CC-2454) the resulting data were identical to our CC-503-generated sequence but showed differences with NC_005353, suggesting that at least some of the discrepancies between CC-503 and NC_005353 are the result of sequencing errors in the latter. Thus, at present, the C. reinhardtii plastid-genome sequence presented here appears to the most accurate.
Twenty kilobases of intergenic ptDNA-sequence data from an additional geographical isolate of C. reinhardtii (CC-2290) was obtained by data mining plastid sequences from GenBank (Figure 1 and Supplementary Table S2 [see Additional file 2]); because very little of these sequence data overlap with the 14 regions described above they were only compared to the ptDNA of CC-503.
Nucleotide-diversity measurements for the three genetic compartments of C. reinhardtii are summarized in Table 2. Net values of πsilent for the plastid genome are 14.5 × 10-3 when indels are removed from the alignment and 18.4 × 10-3 when indels are included and counted as polymorphisms (πsilent+); note, indels involving more than one nucleotide are considered to be a single polymorphic site. These values of πsilent and πsilent+ for the ptDNA are, respectively, 1.7 and 2 times those of the mtDNA, and 0.45 and 0.5 times those of the nucDNA. The nucleotide diversity values for the individual intergenic regions that were analyzed (outlined in Table 3) range from 0 to 41.6 × 10-3 (average πintergenic = 11.3 × 10-3), and the πintergenic+ measurements for these same regions span from 0 to 53.2 × 10-3 (average πintergenic+ = 14.4 × 10-3). The synonymous-site nucleotide diversity of the different protein-coding genes that were sequenced varies from 0 to 71.1 × 10-3 (average πsyn = 7.8 × 10-3; Table 3). Relative to the mitochondrial and nuclear genomes, the ptDNA shows more variance in nucleotide diversity among different regions: πintergenic and πsyn of the various mtDNA loci range from 0 to 17.3 × 10-3 (average = 11.4 × 10-3) and from 1.6 × 10-3 to 15.3 × 10-3 (average = 8.1 × 10-3), respectively; and for the nucDNA, πintergenic varies from 21.6 × 10-3 to 58.3 × 10-3 (average = 36.1 × 10-3) and πsyn extends from 2.8 × 10-3 to 41.1 × 10-3 (average = 20.9 × 10-3). The ptDNA diversity of the rRNA-coding regions that were analyzed is 1.8 × 10-3, which is slightly lower than that of the mtDNA rRNA-coding regions (2.4 × 10-3) – at present there are no nucleotide diversity data for rRNA-coding nucDNA.
The silent-site ptDNA diversity between CC-2290 (the strain from which ptDNA sequences were data mined) and CC-503 is 6.5 × 10-3 and πsilent+ is 18.8 × 10-3; these values indicate that in the regions compared between CC-2290 and CC-503, single-site substitution differences are less frequent and indels are more frequent per site than in the regions compared in the group including CC-503 and the other six geographical isolates.
The various plastid-DNA loci were examined for traces of selection using Tajima's D-test (Table 3), which compares the average number of nucleotide differences between pairs of sequences (i.e., π) to the total number of segregating sites (S) . Tajima's D is positive for the protein-coding genes atpA, cemA, psbA, rpoC2, and rpl2 and negative for atpI, orf1995, rps9, and ycf3. All of the analyzed intergenic regions show positive values for Tajima's D, with the exception of the atpF-rps11 intergenic spacer, which has a negative D value. The only cases where Tajima's D-test is statistically significant are for the protein-coding gene rpoC2 (Tajima's D = 2.03, P value < 0.05) and the region between the rRNA-coding genes 23S-1 and 23-2 (Tajima's D = 2.10, P value < 0.05).
Accounting for the observed values of π
At mutation-drift equilibrium, the nucleotide diversity at neutral sites should approximate 2N g μ ; thus, an essential question of this study is: are the sites that we used to measure πsilent for the C. reinhardtii ptDNA neutrally evolving? We employed both noncoding sites and synonymous sites in our calculations of πsilent; these are generally considered to be among the more neutrally evolving positions in a genome. Indeed, the nucleotide diversity at these sites within the C. reinhardtii ptDNA exceeds that of the more functionally constrained positions, such as first and second codon positions and rRNA-coding sites. Among the different types of silent-sites, intergenic regions have ~1.8 times more nucleotide diversity than synonymous sites. Given that synonymous sites can be subject to selection for specific tRNA anticodons, one might expect them to be under more selective constraints than intergenic regions; therefore, it is not surprising that nucleotide diversity for the intergenic regions is greater than πsyn. Even so, because we sequenced more intergenic sites than synonymous sites, there is not a significant downward bias to our C. reinhardtii ptDNA-diversity measurements by including synonymous sites.
Another issue is the discrepancy in nucleotide diversity among the ptDNA loci that were studied. Factors that can result in inter-loci nucleotide-diversity discrepancy include selection (e.g., balancing-, purifying-, or positive-selection) and inconsistencies in the mutation rate across the plastid genome; however, without interspecific ptDNA-divergence data, it would be overly speculative to focus on any one of these factors. Tajima's D-test did yield statistically significantly positive values for two of the loci that were studied, which could be an indication of balancing selection. It is noteworthy that the magnitude of variation among the C. reinhardtii ptDNA loci is significantly more pronounced than what is typically observed for ptDNA: the nucleotide diversity of most plastid genomes appears to be relatively homogeneous across loci [8, 13]. On the other hand, studies indicate that ptDNA substitution rates at both synonymous and intergenic sites can vary considerably among loci within a genome [14–16].
It would be ideal if we could interpret our ptDNA nucleotide-diversity measurements in relation to μ and N g , but this is difficult because the mutation rate for the C. reinhardtii plastid genome is unknown. There is evidence that μ for the mtDNA and nucDNA of C. reinhardtii are approximately the same , and consequently the disparity of πsilent between these genomes can be explained by differences in N g (see  for a more detailed discussion). Other things being equal, in C. reinhardtii we would expect N g of the uniparentally-inherited plastid genome to be about the same as that of the mitochondrial genome, which is also uniparentally inherited, and about half that of the nuclear genome. Uniparental inheritance also implies that the organelle DNA has less opportunity for recombination during sexual reproduction compared with the nucDNA , meaning organelle genomes may be more prone to the influences of natural selection on linked variation (i.e., genetic hitch-hiking), which can cause Ng(organelle) to deviate from neutral expectations (e.g., Bazin et al. ). Nevertheless, the only study to seriously investigate this issue with respect to the ptDNA, mtDNA, and nucDNA from a single species, Arabidopsis lyrata, found that N g of the organelle DNA and nucDNA did not depart significantly from what was expected under neutrality . Thus, the fact that silent-site nucleotide diversity in C. reinhardtii ptDNA is only within a factor of 2 from that of the mtDNA and nucDNA can easily be accounted for by slight differences in μ and/or N g .
Plastid DNA diversity for the C. reinhardtiiptDNA relative to that of other taxa
There is a paucity of nucleotide-diversity data from ptDNA, and the estimates that are published are limited to a small number of model land-plant species. Most of these available estimates are listed in the supplementary material of Lynch et al.  who compiled a summary of silent-site ptDNA diversity values from 17 land-plant species and found that on average πsilent is 3.7 × 10-3, with a standard error of 1.1 × 10-3 – most of these diversity data were calculated using an indels-out approach but some were generated with the indels-in method (e.g., Huang et al. ). More recently published πsilent estimates from the ptDNA of land plants are concordant with these values: 0–1.2 × 10-3 (Rhododendron spp.), ~4 × 10-3 (Machilus spp.), and ~2 × 10-3 (Silene spp.) [13, 20, 21]. In comparison, the silent-site ptDNA diversity of C. reinhardtii is 4 times the mean estimate for land plants (14.5 × 10-3 vs. 3.7 × 10-3). The average πsilent estimates from the mtDNA and nucDNA of land plants are, respectively, 0.4 × 10-3 and 15.2 × 10-3 [3, 4]. Thus, when considering all three genetic compartments, the πsilent values from C. reinhardtii match the general trend observed in land plants, with silent-site nucleotide diversity being intermediate for the plastid genome, lowest for the mitochondrial genome, and highest for the nuclear genome; however, there is an overall increase of silent-site diversity for C. reinhardtii, in all three of its genomes, relative to that of land plants.
To the best of our knowledge, the only species, heretofore, for which nucleotide-diversity data are available from all three genetic compartments is A. lyrata : values of πsilent for the ptDNA, mtDNA, and nucDNA are 1.0 × 10-3, 0.35 × 10-3, and 20 × 10-3, respectively. Therefore, silent-site diversity in the A. lyrata ptDNA is 3 times that of the mtDNA and 0.05 times that of the nucDNA. Again, the same general trend is observed for C. reinhardtii but with a less dramatic difference between the silent-site diversity of the organelle DNA versus that of the nucDNA.
Addressing the mutational-hazard hypothesis
Contrary to what the mutational-hazard hypothesis forecasted, the πsilent data for the three genetic compartments of C. reinhardtii do not positively correlate with genome compactness. In fact, the opposite trend is observed, with silent-site diversity being lowest for the compact mitochondrial genome (8.5 × 10-3), greatest for the bloated nucDNA (32.3 × 10-3), and intermediary for the plastid genome (14.5 × 10-3), which has a noncoding-DNA density that is halfway between the mtDNA and nucDNA.
Due to a lack of available data, it is difficult for us to compare πsilent and genome-compactness values of the C. reinhardtii ptDNA with those of other plastid genomes; we are aware of only two ptDNA sequences for which both these data are published: those of Arabidopsis thaliana (πsilent(ptDNA) = 1.4 × 10-3; 41% noncoding) and Cycas taitungensis (πsilent(ptDNA) = 12.8 × 10-3; 37% noncoding) . Based on their relative fractions of noncoding ptDNA, the mutational-hazard hypothesis would forecast A. thaliana and C. taitungensis to have more silent-site ptDNA diversity than C. reinhardtii, but instead they have less. However, it is important to stress that the πsilent values for the A. thaliana and C. tatiungensis ptDNA are derived, in each case, from only a single locus (one protein-coding gene and one intergenic region, respectively), and, therefore, may be biased because of insufficient sampling.
If we assume that the mean πsilent estimate of land-plant ptDNA (3.7 × 10-3), derived by Lynch et al. , is representative of the silent-site ptDNA diversity in land plants for which plastid-genome-compactness values are available (i.e., those with completely sequenced plastid genomes), then, based on the noncoding-DNA densities (Supplementary Table S1 [see Additional file 1]) the mutational-hazard hypothesis would predict less silent-site diversity for the C. reinhardtii ptDNA relative to the more coding-rich plastid genomes of land plants; however, C. reinhardtii appears to have 4 times more silent-site ptDNA diversity than the mean estimate for land plants.
Let us now compare the πsilent and genome-compactness measurements of the C. reinhardtii ptDNA to those of animal mtDNA – the only organelle genomes for which these data are readily available. As highlighted earlier, the size and non-coding-DNA density of the C. reinhardtii plastid genome is significantly larger than that of animal mitochondrial genomes, but contrary to what would be predicted under the mutational-hazard hypothesis, the silent-site diversity of animal mtDNA is not dramatically greater than that of the C. reinhardtii ptDNA. Although reported πsilent values for animal mitochondrial genomes can be as high as ~67 × 10-3 (nematodes), those for arthropods (~27 × 10-3), birds (~17 × 10-3), echinoderms (~11.7 × 10-3), and mollusks (~13.5 × 10-3) are 0.8–1.9 times the πsilent value reported here for the C. reinhardtii ptDNA, which is reasonably close considering the stark contrast in genome architectures.
Of the 114 kb of noncoding nucleotides in the C. reinhardtii plastid genome, <2 kb represent intronic DNA – the remainder are intergenic DNA. Why have intergenic nucleotides proliferated in the C. reinhardtii plastid genome when intronic DNA has been kept at bay? Recall, that under the mutational-hazard hypothesis the proliferation of noncoding DNA is dependent on the: 1) number of noncoding nucleotides associated with gene function (n); 2) per-nucleotide mutation rate (μ); and 3) effective number of genes per locus in the population (N g ) – where the overall population-genetic barrier to noncoding-DNA colonization is defined by N g μn. By measuring nucleotide diversity we were able to approximate 2N g μ; however, n is more difficult to estimate. For organelle introns n is believed to be relatively large, perhaps as high as 100 per intron , but n for organelle intergenic regions is generally unknown. One might ask, is there any reason to believe that intergenic DNA in the C. reinhardtii plastid genome carries a reduced burden (i.e., has fewer sites that are crucial for gene function relative to other plastid genomes)? In regards to this question, two observations are worth noting. In land plants, chloroplast genes are organized into operons, which are first transcribed into polycistronic primary transcripts and then subsequently processed into mature monocistronic units via endo- and exonucleolytic cleavage [23–25]. In C. reinhardtii, however, most chloroplast genes appear to be transcribed into monocistronic (or in some cases dicistronic) transcripts [26–28]. Although speculative, it is possible that the intergenic DNA in the C. reinhardtii plastid genome carries a reduced burden (because of a smaller n) relative to that of land plant ptDNA – a mutation in the intergenic DNA of land plant ptDNA could affect the expression of many genes by interfering with transcriptional or posttranscriptional steps, an outcome that seems less likely for the C. reinhardtii ptDNA, which has a preponderance of monocistronically expressed genes. A final comment is that in C. reinhardtii, genes in the mtDNA, unlike those in the ptDNA, show extensive transcriptional linkage  and although our estimates of 2Ngμ for the mitochondrial genome are low, the intergenic regions are reduced in size, which may imply that n for mitochondrial intergenic DNA is relatively large.
The primary goal of this study was to measure nucleotide diversity for the ptDNA of C. reinhardtii and by doing so investigate a novel theory regarding genome evolution – the mutational-hazard hypothesis. Ultimately, the results presented in this study go against the documented trend of πsilent positively correlating with genome compactness, and thus challenge the central premise of the mutational hazard hypothesis.
The C. reinhardtii strains used in this study were obtained from the Chlamydomonas Center at Duke University. DNA was extracted from the same clonal isolate of each strain as used previously by Smith and Lee  for studies on the nucleotide diversity of the C. reinhardtii mitochondrial and nuclear genomes. PtDNA was amplified by PCR using total genomic DNA as the template; the purified PCR products were sequenced on both strands. All of the ptDNA-sequence data presented here were blasted against the C. reinhardtii draft nuclear genome sequence (v3.0) to insure that they are not nuclear-encoded plastid sequences (NUPTS). Our blast results suggest that very few NUPTS are in the nuclear genome (<3 kb), and the few copies that are present are highly degenerate. Nucleotide diversity and its standard deviation were calculated with DnaSP 4.5 . Two different methods for calculating silent-site nucleotide diversity were employed: one that excludes indels (indels-out), which was employed for calculating πsilent, and another that considers indels as polymorphic sites (indels-in), which was used for measuring πsilent+. For our estimates of πsilent+, indels involving more than one nucleotide were considered to be a single polymorphic site.
We acquired the complete plastid-genome sequence of C. reinhardtii strain CC-503 by assembling ptDNA sequences collected from the C. reinhardtii Whole Genome Shotgun Reads Trace Archive Database at GenBank. Blast hits showing >99% similarity to C. reinhardtii ptDNA were downloaded and assembled; all of the downloaded ptDNA sequences were subsequently blasted against the C. reinhardtii draft nuclear genome sequence (v3.0) to insure that no NUPTS were collected. Our assembly of the ptDNA data gave a complete CC-503 plastid genome with >50-fold coverage.
GenBank accession numbers for the ptDNA sequences produced in this study are: FJ436944–FJ436977, FJ458164–FJ458275, and FJ423446; the latter number represents the CC-503 plastid-genome sequence.
Lynch M: The Origins of Genome Architecture. 2007, Sunderland: Sinauer Associates, Inc
Birky CW, Fuerst P, Maryama T: Organelle gene diversity under migration, mutation, and drift: equilibrium expectations, approach to equilibrium, effect of heteroplasmic cells, and comparison to nuclear genes. Genetics. 1989, 121: 613-627.
Lynch M, Conery JS: The origins of genome complexity. Science. 2003, 302: 1401-1404. 10.1126/science.1089370.
Lynch M, Koskella B, Schaack S: Mutation pressure and the evolution of organelle genomic architecture. Science. 2006, 311: 1727-1730. 10.1126/science.1118884.
National center for biotechnology information entrez organelle-genome database. [http://www.ncbi.nlm.nih.gov/]
Harris EH: Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol. 2001, 52: 363-406. 10.1146/annurev.arplant.52.1.363.
Smith DR, Lee RW: Nucleotide diversity in the mitochondrial and nuclear compartments of Chlamydomonas reinhardtii: investigating the origins of genome architecture. BMC Evol Biol. 2008, 8: 156-10.1186/1471-2148-8-156.
Wright SI, Nano N, Foxe JP, Dar V-uN: Effective population size and tests of neutrality at cytoplasmic genes in Arabidopsis. Genet Res. 2008, 90: 119-128. 10.1017/S0016672307008920.
Merchant SS, Prochnik SE, Vallon O, (117 co-authors), et al: The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science. 2007, 318: 245-250. 10.1126/science.1143609.
JGI Chlamydomonas reinhardtii v3.0. [http://genome.jgi-psf.org/Chlre3/Chlre3.home.html]
Maul JE, Lilly JW, Cui L, dePamphilis CW, Miller W, Harris EH, Stern DB: The Chlamydomonas reinhardtii plastid chromosome: islands of genes in a sea of repeats. Plant Cell. 2002, 14: 2659-2679. 10.1105/tpc.006155.
Tajima F: Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics. 1989, 123: 585-595.
Wu SH, Hwang CY, Lin TP, Chung JD, Cheng YP, Hwang SY: Contrasting phylogeographical patterns of two closely related species, Machilus thunbergi and Machilus kusanoi (Lauraceae), in Taiwan. J Biogeogr. 2006, 33: 936-947. 10.1111/j.1365-2699.2006.01431.x.
Wolfe KH, Li W-H, Sharp PM: Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proc Natl Acad Sci USA. 1987, 84: 9054-9058. 10.1073/pnas.84.24.9054.
Shaw J, Lickey EB, Beck JT, Farmer SB, Liu W, Miller J, Siripun KC, Winder CT, Schilling EE, Small RL: The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. Am J Bot. 2005, 9: 142-166. 10.3732/ajb.92.1.142.
Guisinger MM, Kuehl JV, Boore J, Jansen RK: Genome-wide analyses of Geraniaceae plastid DNA reveal unprecedented patterns of increased nucleotide substitutions. Proc Natl Acad Sci USA. 2008, 105: 18424-18429. 10.1073/pnas.0806759105.
Popescu CE, Lee RW: Mitochondrial genome sequence evolution in Chlamydomonas. Genetics. 2007, 175: 819-826. 10.1534/genetics.106.063156.
Bazin E, Glémin S, Galtier N: Population size does not influence mitochondrial genetic diversity in animals. Science. 2006, 312: 570-572. 10.1126/science.1122033.
Huang S, Chiang YC, Schaal BA, Chou CH, Chiang TY: Organelle DNA phylogeography of Cycas taitungensis, a relict species in Taiwan. Mol Ecol. 2001, 10: 2669-2681. 10.1046/j.0962-1083.2001.01395.x.
Chung JD, Lin TP, Chen YL, Cheng YP, Hwang SY: Phylogeographic study reveals the origin and evolutionary history of a Rhododendron species complex in Taiwan. Mol Phylogenet Evol. 2006, 42: 14-24. 10.1016/j.ympev.2006.06.027.
Muir G, Filatov D: A selective sweep in the chloroplast DNA of dioecieous Silene (section Élisanthe). Genetics. 2007, 177: 1239-1247. 10.1534/genetics.107.071969.
Lang BF, Laforest MJ, Burger G: Mitochondrial introns: a critical view. Trends Genet. 2007, 23: 119-125. 10.1016/j.tig.2007.01.006.
Hudson GS, Mason JG, Holton TA, Koller B, Cox GB, Whitfeld PR, Bottomley W: A gene cluster in the spinach and pea chloroplast genomes encoding one CF1 and three CF0 subuntis of the H+-ATP synthase complex and ribosomal protein S2. J Mol Biol. 1987, 196: 283-298. 10.1016/0022-2836(87)90690-5.
Barkan A: Proteins encoded by a complex chloroplast transcription unit are each translated from both monocistronic and polycistronic RNAs. EMBO J. 1988, 7: 2637-2644.
Haley J, Bogorad L: Alternative promoters are used for genes within maize chloroplast polycistronic transcription units. Plant Cell. 1990, 2: 323-333. 10.1105/tpc.2.4.323.
Sakamoto W, Sturm NR, Kindle KL, Stern DB: petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing. Mol Cell Biol. 1994, 14: 6180-6186.
Bruik RK, Mayfield SP: Processing of the psbA 5' untranslated region in Chlamydomonas reinhardtii depends upon factors mediating ribosome association. J Cell Biol. 1998, 143: 1145-1153. 10.1083/jcb.143.5.1145.
Jiao HS, Hicks A, Simpson C, Stern DB: Short dispersed repeats in the Chlamydomonas chloroplast genome are collocated with sites for mRNA 3' end formation. Curr Genet. 2004, 45: 311-322. 10.1007/s00294-004-0487-7.
Gray MW, Boer PH: Organization and expression of algal (Chlamydomonas reinhardtii) mitochondrial DNA. Philos Trans R Soc Lond B Biol Sci. 1988, 319: 135-147. 10.1098/rstb.1988.0038.
Rozas J, Sánchez-DelBarrio JC, Messeguer X, Rozas R: DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics. 2003, 19: 2496-2497. 10.1093/bioinformatics/btg359.
This work was supported by a grant to RWL from the Natural Sciences and Engineering Research Council (NSERC) of Canada. DRS is an Izaak Walton Killam Memorial Scholar and holds a Canada Graduate Scholarship from NSERC.
DRS carried out the molecular studies, data analyses, and wrote the manuscript. RWL helped in interpreting the data and revising the manuscript. Both DRS and RWL have read and approved the final version of this manuscript.