BACKGROUND
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  hypothesis  <cit>  asserts that much of this observed variation in genome compactness can be explained by the product of the effective genetic population size  and the mutation rate .
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  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 ng is large; thus, genomes with a high ngμ are predicted to be more compact than those with a small ngμ.
population-genetic theory tells us that at mutation-drift equilibrium the nucleotide diversity at neutral sites  is equal to 2ng μ .
estimates of πneutral can be acquired by measuring the nucleotide diversity at silent sites , which include noncoding sites and the synonymous sites of protein-coding dna.
because there are many factors that can cause ng to deviate from these neutral expectations, such as the influence of natural selection on linked variation, the only way to gain insight into 2ngμ 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 >  <dig> × 10-3; for the more noncoding-dense nuclear dna  of land plants πsilent is in the range of  <dig> × 10- <dig> to  <dig> × 10-3; and for the nuclear genomes of vertebrates, which abound with noncoding dna, πsilent tends to be ~ <dig> × 10- <dig>  <cit> .
similar trends are also observed for mitochondrial genomes: in the streamlined mitochondrial dna  of mammals πsilent is ~ <dig> × 10- <dig>  whereas that for land-plant mtdna, which is predominantly noncoding, is predicted to be <  <dig>  × 10- <dig>  <cit> .
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 ng, the mitochondrial and nuclear genomes of mammals have opposite coding densities – in mammals estimates of μ for mtdna are roughly  <dig> times those for nucdna  <cit> .
it is speculated that πsilent for plastid dna  also correlates positively with genome compactness  <cit> ; 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 .
of the  <dig> complete plastid-genome sequences available at the national center for biotechnology information  as of november  <dig>  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 s <dig> .
intriguingly, four of the five most bloated ptdna sequences come from the chlorophyta , 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  and expanded plastid genome, and it is also a model organism for studying plastids and their photosynthetic processes  <cit> .
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  <cit>  measured nucleotide diversity in its mitochondrial and nuclear genomes, which are respectively streamlined  and bloated .
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  <dig> times smaller than that of the nucdna .
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  <cit> .
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 .
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  <cit> .
thus, to directly confront these issues, we measured πsilent from the ptdna of various geographical isolates of c. reinhardtii.
RESULTS
strains and their genetic loci
for our analysis we employed seven geographical isolates of c. reinhardtii, which are listed in table  <dig>  these are the same isolates that were previously used for calculating πsilent of the mtdna and nucdna.
from each isolate,  <dig> distinct ptdna regions were sequenced, amounting to  <dig>  kb,  <dig>  kb, and  <dig>  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  <dig>
a ptdna sequences were data mined from this strain and compared only to those of cc-503
we also produced a complete plastid-genome sequence for c. reinhardtii strain cc- <dig>  by assembling ptdna trace files generated by the c. reinhardtii nuclear-genome sequencing project  <cit> .
the earlier complete c. reinhardtii ptdna sequence deposited at genbank  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  <cit>  – it is ideal to avoid using nc_ <dig> when calculating πsilent because sequence differences have been found between the ptdna of some laboratory strains  <cit> .
a comparison of our cc- <dig> ptdna sequence with nc_ <dig> reveals  <dig> single-nucleotide differences and  <dig> single-site indels; moreover, when the  <dig> ptdna regions sequenced from the geographical isolates were sequenced from two additional laboratory strains belonging to the "ebersold-levine" wild-type background  the resulting data were identical to our cc-503-generated sequence but showed differences with nc_ <dig>  suggesting that at least some of the discrepancies between cc- <dig> and nc_ <dig> 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  was obtained by data mining plastid sequences from genbank ; because very little of these sequence data overlap with the  <dig> regions described above they were only compared to the ptdna of cc- <dig>
nucleotide diversity
nucleotide-diversity measurements for the three genetic compartments of c. reinhardtii are summarized in table  <dig>  net values of πsilent for the plastid genome are  <dig>  × 10- <dig> when indels are removed from the alignment and  <dig>  × 10- <dig> when indels are included and counted as polymorphisms ; 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,  <dig>  and  <dig> times those of the mtdna, and  <dig>  and  <dig>  times those of the nucdna.
the nucleotide diversity values for the individual intergenic regions that were analyzed  range from  <dig> to  <dig>  × 10- <dig> , and the πintergenic+ measurements for these same regions span from  <dig> to  <dig>  × 10- <dig> .
the synonymous-site nucleotide diversity of the different protein-coding genes that were sequenced varies from  <dig> to  <dig>  × 10- <dig> .
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  <dig> to  <dig>  × 10- <dig>  and from  <dig>  × 10- <dig> to  <dig>  × 10- <dig> , respectively; and for the nucdna, πintergenic varies from  <dig>  × 10- <dig> to  <dig>  × 10- <dig>  and πsyn extends from  <dig>  × 10- <dig> to  <dig>  × 10- <dig> .
the ptdna diversity of the rrna-coding regions that were analyzed is  <dig>  × 10- <dig>  which is slightly lower than that of the mtdna rrna-coding regions  – at present there are no nucleotide diversity data for rrna-coding nucdna.
# of indelsb
πc × 10-3
π+d × 10-3
note: s, number of segregating  sites; indels, insertion-deletion events; π, nucleotide diversity; π+, nucleotide diversity including both polymorphic sites and insertion-deletion events; πsyn, nucleotide diversity at synonymous sites; πnsyn, nucleotide diversity at nonsynonymous sites; sd, standard deviation.
mitochondrial- and nuclear-genome data come from  <cit> .
a comprises all sites in the nucleotide alignment, including those with indels.
b indels involving more than  <dig> nucleotide are counted as a single event.
indel length includes the sum of all indels and includes consecutive indel events.
c only includes sites in the alignment without indels.
d considers all sites, including those with indels.
consecutive indels are counted as a single polymorphic event.
indel states were measured using a multiallelic approach.
e for the ptdna and mtdna these values were generated from intergenic nucleotide sites, whereas for the nucdna they came from intronic sites.
f includes noncoding and synonymous sites; π+ values for the mtdna and nucdna differ slightly from those previously reported  <cit>  due to a different interpretation of indel sites in nucleotide-diversity calculations.
the silent-site ptdna diversity between cc- <dig>  and cc- <dig> is  <dig>  × 10- <dig> and πsilent+ is  <dig>  × 10-3; these values indicate that in the regions compared between cc- <dig> and cc- <dig>  single-site substitution differences are less frequent and indels are more frequent per site than in the regions compared in the group including cc- <dig> and the other six geographical isolates.
the various plastid-dna loci were examined for traces of selection using tajima's d-test , which compares the average number of nucleotide differences between pairs of sequences  to the total number of segregating sites   <cit> .
tajima's d is positive for the protein-coding genes atpa, cema, psba, rpoc <dig>  and rpl <dig> and negative for atpi, orf <dig>  rps <dig>  and ycf <dig>  all of the analyzed intergenic regions show positive values for tajima's d, with the exception of the atpf-rps <dig> 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 rpoc <dig>  and the region between the rrna-coding genes 23s- <dig> and 23- <dig> .
note: s, number of segregating  sites; indels, insertion-deletion events; π, nucleotide diversity; π+, nucleotide diversity including both polymorphic sites and insertion-deletion events; πsyn, nucleotide diversity at synonymous sites; πnsyn, nucleotide diversity at nonsynonymous sites; sd, standard deviation.
a comprises all sites in the nucleotide alignment, including those with indels.
b indels involving more than  <dig> nucleotide are counted as a single event.
indel length includes the sum of all indels and includes consecutive indel events.
c only includes sites without indels.
d considers all sites, including those with indels.
consecutive indels are counted as a single polymorphic event.
indel states were measured using a multiallelic approach.
discussion
accounting for the observed values of π
at mutation-drift equilibrium, the nucleotide diversity at neutral sites should approximate 2ngμ  <cit> ; 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 ~ <dig>  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  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  <cit> .
on the other hand, studies indicate that ptdna substitution rates at both synonymous and intergenic sites can vary considerably among loci within a genome  <cit> .
it would be ideal if we could interpret our ptdna nucleotide-diversity measurements in relation to μ and ng, 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  <cit> , and consequently the disparity of πsilent between these genomes can be explained by differences in ng .
other things being equal, in c. reinhardtii we would expect ng 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  <cit> , meaning organelle genomes may be more prone to the influences of natural selection on linked variation , which can cause ng to deviate from neutral expectations .
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 ng of the organelle dna and nucdna did not depart significantly from what was expected under neutrality  <cit> .
thus, the fact that silent-site nucleotide diversity in c. reinhardtii ptdna is only within a factor of  <dig> from that of the mtdna and nucdna can easily be accounted for by slight differences in μ and/or ng.
plastid dna diversity for the c. reinhardtii ptdna 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.
<cit>  who compiled a summary of silent-site ptdna diversity values from  <dig> land-plant species and found that on average πsilent is  <dig>  × 10- <dig>  with a standard error of  <dig>  × 10- <dig> – most of these diversity data were calculated using an indels-out approach but some were generated with the indels-in method .
more recently published πsilent estimates from the ptdna of land plants are concordant with these values: 0– <dig>  × 10- <dig> , ~ <dig> × 10- <dig> , and ~ <dig> × 10- <dig>   <cit> .
in comparison, the silent-site ptdna diversity of c. reinhardtii is  <dig> times the mean estimate for land plants .
the average πsilent estimates from the mtdna and nucdna of land plants are, respectively,  <dig>  × 10- <dig> and  <dig>  × 10- <dig>  <cit> .
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  <cit> : values of πsilent for the ptdna, mtdna, and nucdna are  <dig>  × 10- <dig>   <dig>  × 10- <dig>  and  <dig> × 10- <dig>  respectively.
therefore, silent-site diversity in the a. lyrata ptdna is  <dig> times that of the mtdna and  <dig>  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 , greatest for the bloated nucdna , and intermediary for the plastid genome , 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  =  <dig>  × 10-3; 41% noncoding) and cycas taitungensis  =  <dig>  × 10-3; 37% noncoding)  <cit> .
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 , and, therefore, may be biased because of insufficient sampling.
if we assume that the mean πsilent estimate of land-plant ptdna , derived by lynch et al.
<cit> , is representative of the silent-site ptdna diversity in land plants for which plastid-genome-compactness values are available , then, based on the noncoding-dna densities  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  <dig> 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 ~ <dig> × 10- <dig> , those for arthropods , birds , echinoderms , and mollusks  are  <dig> – <dig>  times the πsilent value reported here for the c. reinhardtii ptdna, which is reasonably close considering the stark contrast in genome architectures.
of the  <dig> kb of noncoding nucleotides in the c. reinhardtii plastid genome, < <dig> 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 ; 2) per-nucleotide mutation rate ; and 3) effective number of genes per locus in the population  – where the overall population-genetic barrier to noncoding-dna colonization is defined by ngμn.
by measuring nucleotide diversity we were able to approximate 2ngμ; however, n is more difficult to estimate.
for organelle introns n is believed to be relatively large, perhaps as high as  <dig> per intron  <cit> , 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 ?
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  <cit> .
in c. reinhardtii, however, most chloroplast genes appear to be transcribed into monocistronic  transcripts  <cit> .
although speculative, it is possible that the intergenic dna in the c. reinhardtii plastid genome carries a reduced burden  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  <cit>  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.
CONCLUSIONS
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 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 hypothesis asserts that much of this observed variation in genome compactness can be explained by the product of the effective genetic population size and the mutation rate. 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 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 ng is large; thus, genomes with a high ngμ are predicted to be more compact than those with a small ngμ. population-genetic theory tells us that at mutation-drift equilibrium the nucleotide diversity at neutral sites is equal to 2ng μ. estimates of πneutral can be acquired by measuring the nucleotide diversity at silent sites, which include noncoding sites and the synonymous sites of protein-coding dna. because there are many factors that can cause ng to deviate from these neutral expectations, such as the influence of natural selection on linked variation, the only way to gain insight into 2ngμ 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 > × 10-3; for the more noncoding-dense nuclear dna of land plants πsilent is in the range of × 10- to × 10-3; and for the nuclear genomes of vertebrates, which abound with noncoding dna, πsilent tends to be ~ × 10-. similar trends are also observed for mitochondrial genomes: in the streamlined mitochondrial dna of mammals πsilent is ~ × 10- whereas that for land-plant mtdna, which is predominantly noncoding, is predicted to be < × 10-. 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 ng, the mitochondrial and nuclear genomes of mammals have opposite coding densities – in mammals estimates of μ for mtdna are roughly times those for nucdna. it is speculated that πsilent for plastid dna also correlates positively with genome compactness; 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. of the complete plastid-genome sequences available at the national center for biotechnology information as of november 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 s. intriguingly, four of the five most bloated ptdna sequences come from the chlorophyta, 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 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 and bloated. 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 times smaller than that of the nucdna. 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. 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 these are the same isolates that were previously used for calculating πsilent of the mtdna and nucdna. from each isolate, distinct ptdna regions were sequenced, amounting to kb, kb, and 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 a ptdna sequences were data mined from this strain and compared only to those of cc-503 we also produced a complete plastid-genome sequence for c. reinhardtii strain cc- by assembling ptdna trace files generated by the c. reinhardtii nuclear-genome sequencing project. the earlier complete c. reinhardtii ptdna sequence deposited at genbank 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_ when calculating πsilent because sequence differences have been found between the ptdna of some laboratory strains. a comparison of our cc- ptdna sequence with nc_ reveals single-nucleotide differences and single-site indels; moreover, when the ptdna regions sequenced from the geographical isolates were sequenced from two additional laboratory strains belonging to the "ebersold-levine" wild-type background the resulting data were identical to our cc-503-generated sequence but showed differences with nc_ suggesting that at least some of the discrepancies between cc- and nc_ 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 was obtained by data mining plastid sequences from genbank; because very little of these sequence data overlap with the regions described above they were only compared to the ptdna of cc- nucleotide diversity nucleotide-diversity measurements for the three genetic compartments of c. reinhardtii are summarized in table net values of πsilent for the plastid genome are × 10- when indels are removed from the alignment and × 10- when indels are included and counted as polymorphisms; 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, and times those of the mtdna, and and times those of the nucdna. the nucleotide diversity values for the individual intergenic regions that were analyzed range from to × 10-, and the πintergenic+ measurements for these same regions span from to × 10-. the synonymous-site nucleotide diversity of the different protein-coding genes that were sequenced varies from to × 10-. 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 to × 10- and from × 10- to × 10-, respectively; and for the nucdna, πintergenic varies from × 10- to × 10- and πsyn extends from × 10- to × 10-. the ptdna diversity of the rrna-coding regions that were analyzed is × 10- which is slightly lower than that of the mtdna rrna-coding regions – at present there are no nucleotide diversity data for rrna-coding nucdna. # of indelsb πc × 10-3 π+d × 10-3 note: s, number of segregating sites; indels, insertion-deletion events; π, nucleotide diversity; π+, nucleotide diversity including both polymorphic sites and insertion-deletion events; πsyn, nucleotide diversity at synonymous sites; πnsyn, nucleotide diversity at nonsynonymous sites; sd, standard deviation. mitochondrial- and nuclear-genome data come from. a comprises all sites in the nucleotide alignment, including those with indels. b indels involving more than nucleotide are counted as a single event. indel length includes the sum of all indels and includes consecutive indel events. c only includes sites in the alignment without indels. d considers all sites, including those with indels. consecutive indels are counted as a single polymorphic event. indel states were measured using a multiallelic approach. e for the ptdna and mtdna these values were generated from intergenic nucleotide sites, whereas for the nucdna they came from intronic sites. f includes noncoding and synonymous sites; π+ values for the mtdna and nucdna differ slightly from those previously reported due to a different interpretation of indel sites in nucleotide-diversity calculations. the silent-site ptdna diversity between cc- and cc- is × 10- and πsilent+ is × 10-3; these values indicate that in the regions compared between cc- and cc- single-site substitution differences are less frequent and indels are more frequent per site than in the regions compared in the group including cc- and the other six geographical isolates. the various plastid-dna loci were examined for traces of selection using tajima's d-test, which compares the average number of nucleotide differences between pairs of sequences to the total number of segregating sites. tajima's d is positive for the protein-coding genes atpa, cema, psba, rpoc and rpl and negative for atpi, orf rps and ycf all of the analyzed intergenic regions show positive values for tajima's d, with the exception of the atpf-rps 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 rpoc and the region between the rrna-coding genes 23s- and 23-. note: s, number of segregating sites; indels, insertion-deletion events; π, nucleotide diversity; π+, nucleotide diversity including both polymorphic sites and insertion-deletion events; πsyn, nucleotide diversity at synonymous sites; πnsyn, nucleotide diversity at nonsynonymous sites; sd, standard deviation. a comprises all sites in the nucleotide alignment, including those with indels. b indels involving more than nucleotide are counted as a single event. indel length includes the sum of all indels and includes consecutive indel events. c only includes sites without indels. d considers all sites, including those with indels. consecutive indels are counted as a single polymorphic event. indel states were measured using a multiallelic approach. discussion accounting for the observed values of π at mutation-drift equilibrium, the nucleotide diversity at neutral sites should approximate 2ngμ; 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 ~ 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 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. on the other hand, studies indicate that ptdna substitution rates at both synonymous and intergenic sites can vary considerably among loci within a genome. it would be ideal if we could interpret our ptdna nucleotide-diversity measurements in relation to μ and ng, 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 ng. other things being equal, in c. reinhardtii we would expect ng 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, which can cause ng to deviate from neutral expectations. 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 ng 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 from that of the mtdna and nucdna can easily be accounted for by slight differences in μ and/or ng. plastid dna diversity for the c. reinhardtii ptdna 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 land-plant species and found that on average πsilent is × 10- with a standard error of × 10- – most of these diversity data were calculated using an indels-out approach but some were generated with the indels-in method. more recently published πsilent estimates from the ptdna of land plants are concordant with these values: 0– × 10-, ~ × 10-, and ~ × 10-. in comparison, the silent-site ptdna diversity of c. reinhardtii is times the mean estimate for land plants. the average πsilent estimates from the mtdna and nucdna of land plants are, respectively, × 10- and × 10-. 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 × 10- × 10- and × 10- respectively. therefore, silent-site diversity in the a. lyrata ptdna is times that of the mtdna and 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, greatest for the bloated nucdna, and intermediary for the plastid genome, 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 = × 10-3; 41% noncoding) and cycas taitungensis = × 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, and, therefore, may be biased because of insufficient sampling. if we assume that the mean πsilent estimate of land-plant ptdna, derived by lynch et al., is representative of the silent-site ptdna diversity in land plants for which plastid-genome-compactness values are available, then, based on the noncoding-dna densities 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 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 ~ × 10-, those for arthropods, birds, echinoderms, and mollusks are – times the πsilent value reported here for the c. reinhardtii ptdna, which is reasonably close considering the stark contrast in genome architectures. of the kb of noncoding nucleotides in the c. reinhardtii plastid genome, < 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; 2) per-nucleotide mutation rate; and 3) effective number of genes per locus in the population – where the overall population-genetic barrier to noncoding-dna colonization is defined by ngμn. by measuring nucleotide diversity we were able to approximate 2ngμ; however, n is more difficult to estimate. for organelle introns n is believed to be relatively large, perhaps as high as 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? 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. in c. reinhardtii, however, most chloroplast genes appear to be transcribed into monocistronic transcripts. although speculative, it is possible that the intergenic dna in the c. reinhardtii plastid genome carries a reduced burden 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 mutational-hazard hypothesis argues that the noncoding-dna content of a genome is a consequence of the mutation rate and the effective number of genes per locus in the population. the hypothesis predicts that genomes with a high ngμ will be more compact than those with a small ngμ. in c. reinhardtii, the mean estimate of πsilent forthe ptdna is less than that of the nuclear dna and greater than that of the mitochondrial dna. 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. on average, c. reinhardtii has ~ times more silent-site ptdna diversity than the mean value reported for land plants, which have more compact plastid genomes. approximations of ngμ can be gained by measuring the nucleotide diversity at silent sites. the silent-site nucleotide diversity of the different ptdna loci that were studied varied significantly: from to × 10- for synonymous sites and from to × 10- 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 mutational-hazard hypothesis argues that the noncoding-dna content of a genome is a consequence of the mutation rate and the effective number of genes per locus in the population. the hypothesis predicts that genomes with a high ngμ will be more compact than those with a small ngμ. approximations of ngμ can be gained by measuring the nucleotide diversity at silent sites. we addressed the mutation-hazard hypothesis apropos plastid-genome evolution by measuring πsilent of the chlamydomonas reinhardtii plastid dna, 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 is less than that of the nuclear dna and greater than that of the mitochondrial dna. on average, c. reinhardtii has ~ 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 to × 10- for synonymous sites and from to × 10- 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. 