BACKGROUND
the influence of genotype on phenotype is not limited to the coding of peptides and functional rnas by nucleotide sequences.
an organism's phenotype is also affected by the chromosomal arrangement of genes and the interaction of gene products.
comparative genomics has revealed a variety of gene clusters and chromosomal segments that have remained intact over hundreds of millions of years  <cit> .
selection for clustering of co-transcribed genes has been hypothesized to influence gene order within bacterial and organelle genomes, where gene clusters typically encode multiple components of a functional pathway  <cit> .
for example, the ribosomal proteins are encoded by similar operons in archaebacteria, eubacteria and plastids  <cit> .
in eukaryotic genomes, co-expression of neighboring genes is significantly associated with the functional roles of the genes   <cit> .
one way that those genes become clustered is through tandem duplication, which usually results in functionally related genes being adjacent.
on the other hand, unrelated genes may also be brought together through chromosome rearrangements .
unless selection is acting to maintain or promote gene clusters, gene orders in genomes subjected to rearrangements should become randomized with respect to function or co-expression profiles.
significant clustering has been inferred using permutation tests that compare observed physical distances between pairs or blocks of co-expressed or functionally related genes to a null distribution constructed from randomized gene orders  <cit> .
however, this approach is limited since the evolutionary history of the genome was not considered.
when comparing gene orders among related species, it is possible to estimate the ancestral genome and to simulate a null distribution for changes in gene order using a model.
this evolutionary approach can be used to test directly the influence of selection on genome structure, that is, whether present-day genome structure has been influenced by directional selection for clustering of functionally related genes.
small genomes, especially those of organelles and bacteria, are well suited to global comparisons of gene order.
like eukaryotic genomes, they are subject to structural changes such as inversion, transposition or translocation, as well as gene loss and  gene gain.
chloroplast dnas in most land plants share a highly conserved gene content and similar gene orders  <cit> .
most cpdnas include two identical regions in opposite orientations called the inverted repeat , flanked by large single copy  and small single copy  regions.
the irs generally contain the bacterial-like rrna gene clusters, and the genes involved in photosynthesis  are arranged similarly in chloroplast and cyanobacterial genomes  <cit> .
despite these well-characterized patterns, it is unknown to what extent the conserved gene order reflects a slow intrinsic rate of neutral chromosomal rearrangements, rather than selection against alternative gene orders.
a model of neutral rearrangement of gene order is required to test formally whether gene orders evolve under selection which prefers some gene arrangements over others.
nadeau and taylor first proposed a model for the neutral evolution of gene order in comparisons of mouse and human chromosomes  <cit> .
this "random breakage model" provides a null hypothesis for the evolution of gene order.
it assumes a random distribution of break points and allows all possible gene orders without restrictions.
the random breakage model has been used to infer organismal phylogenies from gene order data  <cit> .
the gene order difference can be measured using the inversion distance, which is the minimal number of inversions necessary to transform one gene order to another.
currently, the most accurate heuristic approach is implemented in the grappa software  <cit> , which is generally suitable for small taxon sets because the algorithm scores inversion medians for all nodes iteratively across all possible phylogenies.
algorithms for genomes with arbitrary rearrangements, a few deletions and duplications have been developed  <cit> , and the capacity of grappa can be scaled up with the discovering method  to potentially very large data sets  <cit> .
the random breakage model does not account for recombination hotspots, which have been reported from human-mouse genome comparisons  <cit> .
however, at this time it may be difficult to model these hotspots, because the precise locations of reused breakpoints are unknown due to insufficient resolution of gene orders and potential errors in homology assessment given the scale of eukaryotic chromosomes  <cit> .
thus, the fragile breakage model  <cit> , as an alternative to the random breakage model, has not been well established.
whereas gene order is generally conserved among land plant cpdnas, very little synteny is observed between this group and cpdnas of the chlorophytic green algae c. reinhardtii  <cit>  and chlorella vulgaris  <cit> .
the apparently increased rearrangement rate is associated with invasion by a large number of short dispersed repeats , for which the evolutionary distribution is still poorly defined.
the large number of rearrangements provides an excellent opportunity to test whether natural selection has preferred some changes in gene order.
here we present novel statistics and parametric tests that lead us to reject the models of random rearrangement in favor of directional selection for clustering of functionally related genes in c. reinhardtii.
we also present experimental evidence that adaptive evolution of chloroplast genome structure could be driven by the advantage of concerted regulation conferred by polycistronic transcription.
RESULTS
functional clusters are not randomly distributed
we compared gene orders of representative cpdnas from land plants, including tobacco   <cit>  and liverwort   <cit> , a charophytic green alga   <cit> , chlorophytic green algae , a green flagellate alga with uncertain affinities   <cit> , and the plastid of cyanophora paradoxa   <cit>   .
to measure the genome structure in terms of clustering by chromosome locations and gene functions, we defined "sided blocks" as contiguous genes coded on the same strand of the plastid chromosome, and "functional clusters" as blocks of functionally related genes .
the randomness in the observed distribution of shared genes in chloroplast genomes with respect to gene function was assessed using a kolmogorov-smirnov test.
the null hypothesis was rejected in all seven cpdnas investigated for genes in functional categories such as atp synthases and electron transport .
while this test suggests some degree of functional clustering in all chloroplast genomes, it does not take into account the phylogenetic relationship of these organisms, so it is unclear whether functional clustering in chloroplast genomes is a legacy of genome organization in a cyanobacteria-like ancestor, or the product of selection on gene order in the face of genome rearrangements.
§the test statistic dn measures whether the distribution of functionally related genes is random in gene clusters.
total  <dig> shared genes between seven cpdnas were included.
extensive rearrangements from the ancestral chloroplast genome to c. reinhardtii
in order to investigate evolutionary changes of gene order, we constructed a phylogeny of seven representative cpdnas and rooted with the sequence of c. paradoxa  <cit> .
maximum parsimony, neighbor joining and maximum likelihood analyses of an alignment of  <dig> concatenated protein sequences including a total of  <dig>  aligned sites , all yielded identical fully resolved topologies with high bootstrap support .
mesostigma was placed as a basal charophyte lineage in one previous analysis  <cit> .
the unrooted phylogeny of seven cpdnas  is congruent with the alternative placement of mesostigma either as a basal charophyte  <cit>  or basal to both charophyte and chlorophyte lineages  <cit> .
this tree was used as the reference phylogeny for gene order inference.
we scored the orders of  <dig> genes shared in the seven genomes .
then we used modified versions of grappa  <cit>  to compute the inversion distance between ancestral nodes and each terminal node .
the branches leading to two chlorophytic green algae, c. reinhardtii and c. vulgaris, are much longer than the branches leading to the other taxa, and that many more steps were inferred on the c. reinhardtii lineage relative to the c. vulgaris lineage.
gene duplications or deletions were mapped before scoring the ancestral genomes with inversions, and were not counted as rearrangements.
irs were present in all inferred ancestral nodes, and one copy was lost in c. vulgaris.
ancestral gene orders were scored on all the phylogenies using a two-step approach .
due to the computational time limit , we stopped scoring all possible ancestral gene orders with the data set after  <dig> days and took the best scored ancestral gene orders at that time .
the cpdnas of two land plants, n. tabacum and m. polymorpha, were separated by an estimated  <dig> inversions based on the data set.
one large inversion  in the lsc region has long been recognized to separate the two genomes  <cit> .
additional rearrangements are directly observable through comparison of gene order files for the two species .
using grappa, all rearrangements were inferred as inversions, but the total number of inversion events estimated by grappa may be greater than the true  mixture of inversions and transpositions because one transposition could result in the same change in gene order as two or three inversions.
increased order in the genome structure after rearrangements
two genomic structural characteristics were measured: the propensity of adjacent genes to be clustered on the same strand  and the clustering of functionally related genes  .
both indices were calculated for the inferred ancestral gene orders and extant daughter lineages.
among land plants and charophytes, the inferred sidedness among ancestral genomes was similar to extant lineages, however, among the chlorophytes an opposite trend was observed, especially in the c. reinhardtii lineage .
the large number of rearrangements in the c. reinhardtii cpdna lineage resulted in dramatically increased sidedness relative to the inferred most recent common ancestor of c. reinhardtii and c. vulgaris .
a small increase of cs was found in the n. olivacea lineage and there was almost no change in the lineage leading to c. vulgaris.
a large increase was also observed in the functional clustering index, cf, for c. reinhardti , whereas the trend was less profound in other lineages .
thus, even if the ancestral genome already had a "sided" structure, sidedness increased with genome rearrangements as c. reinhardtii cpdna evolved.
the inferred increase in sidedness and functional clustering in the face of the large number of rearrangements on the lineage leading to c. reinhardtii might be adaptive, if such increases were not expected under random rearrangements.
to test the null hypothesis that the changes in cs and cf were consequence of random genome rearrangements rather than a consequence of directional selection , we simulated random rearrangements starting with the inferred ancestral genome along the branch leading to c. reinhardtii.
although inversions are the most abundant type of rearrangement in cpdnas  <cit> , we also considered the contribution of transpositions under three inversion to transposition ratios, while the total number of rearrangements was fixed according to the branch length inferred using grappa .
three simulations with  <dig>  replicates were conducted with inversion to transposition ratios of 1: <dig>  10: <dig> and 1: <dig> under the random breakage model.
the mean cs values for the three sets were  <dig> ,  <dig>  and  <dig> , respectively, and the 95% confidence intervals were ,  and , respectively.
all datasets simulated under the random breakage model showed a significant decrease of sidedness from the ancestral level .
in contrast, the cs value calculated for c. reinhardtii increased significantly to  <dig>  , greatly exceeding the sidedness that would be expected in a genome that had undergone this much evolutionary change relative to its ancestor.
simulations using inferred ancestral genomes for land plant lineages  also strongly reject the null hypothesis of random rearrangements .
given the large number of rearrangements observed in the c. reinhardtii lineage, cf was also predicted to decrease significantly under the random breakage model, but cf did not decrease as observed in c. reinhardtii .
the simulations with three models described above  all yielded a large decrease in clustering as expected .
when transposition was included in simulations, decreases of cf were on a similar scale to the inversion-only simulations.
taken together, these results indicate that the remarkable increase in sidedness and functional clustering observed in c. reinhardtii cpdna has not been the outcome of solely chance events.
instead, the strong deviation from the range of outcomes expected under various random breakage models implies that the genome structure is the outcome of a directional selective process.
the increased level of organization in c. reinhardtii cpdna was associated with both maintenance of ancestral clusters and growth of new clusters.
there were six conserved blocks containing  <dig> of the  <dig> genes shared between the c. reinhardtii and the c. vulgaris cpdnas.
these blocks include concentrations of genes from a single functional category, such as ribosomal proteins , photosystem ii , translation apparatus , and atp synthase subunits .
moreover, a number of small clusters of functionally related genes inferred in the ancestral genome were brought together in c. reinhardtii .
these include transcription/translation genes , electron transport genes , and photosynthetic genes  .
the new clusters contributed to the increase of cf in the c. reinhardtii chloroplast genome.
coordinated expression of genes in functional clusters
co-transcription of several clusters shown in figure 4b has been previously documented, including psbd-psaa exon 2-psbj-atpi  <cit> , psbf-psbl  <cit> , peta-petd  <cit> , and psbm-psbz  <cit> .
co-transcription of rpl and rps genes has been found in land plant chloroplasts  <cit> .
we documented co-transcription for an additional novel functional cluster, shown in figure 4a.
using rna gel blots, tricistronic transcripts of rpl36-rpl23-rpl <dig> and possibly dicistronic rpl2-rps <dig> species could be detected.
taken together, it appears that the clusters of functionally related genes observed in c. reinhardtii cpdna may be frequently co-transcribed.
discussion
by reconstructing the possible ancestral gene order in chloroplast genomes and simulating rearrangements, we have been able to formally test and reject the null hypothesis that c. reinhardtii cpdna has evolved through random rearrangements.
instead, we found that its observed gene order deviates strongly from the degree of sidedness and clustering expected under a random breakage model.
euglena gracilis cpdna also has a high degree of sidedness  <cit> , however, the asymmetry of its coding strand is concentrated in one half of the genome and associated with gc content, which could be influenced by asymmetrical replication of the chromosome  <cit> .
in c. reinhardtii, the sidedness is not associated with gc content and we hypothesize that it is driven by co-transcription of genes in a functional cluster.
whereas some clusters of co-transcribed genes  were maintained in both c. reinhardtii and c. vulgaris, novel clusters clearly formed in the c. reinhardtii lineage .
co-transcription of neighboring genes in the c. reinhardtii chloroplast is a widely documented phenomenon.
we demonstrated that in addition to the ribosomal protein clusters, global analyses support the elevated level of clustering of other functionally related genes.
the aggregate of genes in clusters include most essential genes involved in translation and transcription, and some photosynthetic genes.
coordinated transcription may play a crucial role in the regulation of plastid gene expression in response to light or circadian rhythms  <cit> .
it is also possible that some clusters contain cis-elements, similar to the artificial polydeoxyadenosine sequences  <cit> , which enhance transcription efficiency.
moreover, most of the putative co-transcription units are not conserved across chlorophytes.
therefore, the majority of functional clusters observed in c. reinhardtii represent new gene arrangements.
in the chloroplast gene order phylogeny , the c. reinhardtii lineage resides on a long branch compared to the c. vulgaris lineage, and both genomes are more rearranged than that of n. olivacea, relative to the common ancestral genome of the three chlorophyte lineages.
the elevated rate of chloroplast genome rearrangement in c. reinhardtii is associated with invasion of sdrs, which heavily populate the non-coding regions, increasing the total length of the intergenic regions compared to c. vulgaris cpdna by one-third  <cit> .
although simple sequence repeats are common to microbial genomes  <cit> , such elements are rare in most sequenced chloroplast genomes.
within the chlamydomonas genus , c. reinhardtii and c. gelatinosa cpdnas exhibit a prevalence of repetitive dna and a high degree of gene order variation compared to the c. moewusii/c.
pitschmannii lineage  <cit> .
the sister lineage to c. reinhardtii in our study, c. vulgaris , contains numerous cpdna repeat sequences.
besides chlorophyte algae, members of angiosperm families, including campanulaceae  <cit> , fabaceae  <cit>  and geraniaceae  <cit> , also contain repeat elements in rearranged cpdnas, albeit of a much lower copy number  <cit> .
these repeat elements may act as molecular "grease" that facilitates non-homologous recombination and creates a pool of diverse genome structures subject to selective retention.
future investigations will test whether the increased rates of rearrangement in plastid genomes with dispersed repeats typically lead to increased sidedness and functional clustering as we infer for c. reinhardtii.
gene order changes reflect relatively rare evolutionary events and are expected to result in much less homoplasy than substitution events in nucleotide or protein sequences over a deep time scale  <cit> .
phylogeny reconstruction using grappa is highly accurate even for divergent genomes  <cit> , and thus the ancestral gene orders inferred in our study contained sufficient phylogenetic information.
the only other software for genome rearrangement phylogeny, badger  <cit> , performed poorly on this data set .
grappa usually inferred unique ancestral gene orders on many data sets we tested.
furthermore, analyses on simulated data have shown that the inferred gene orders scored almost as well as true ancestral gene orders  <cit> .
in our simulation tests of three genomes with  <dig> genes each, and branch lengths of  <dig>   <dig> and  <dig> , the average score for ancestral gene orders computed by grappa was only about 7% less than the true scores.
in practice, we observed that the less optimal gene orders generally required more rearrangements.
therefore, it is quite likely that any error in our estimation of ancestral gene order has resulted in a downward bias in the inferred number of rearrangements on the branch leading to c. reinhardtii.
increasing the number of rearrangements on this branch would only lead to a more certain rejection of the neutrality of rearrangements.
the accuracy of ancestral genome reconstruction also depends on the degree of divergence among extant taxa and taxon sampling.
for example, accurate reconstruction of ancestral genomes at the mammalian cftr locus was achieved at the dna level  <cit> .
the high-quality reconstruction was attributed to a dense sampling of syntenic genome sequences from eutherian mammals, and the lack of gene order rearrangement at the locus.
because the c. reinhardtii cpdna is one of the most rearranged chloroplast genomes sequenced to date, we included all available chlorophyte chloroplast genomes for evolutionary distance estimation and ancestral gene order reconstruction.
the accuracy of our ancestral gene order estimation may improve with inclusion of additional chlorophyte plastid gene orders as they become available, but we do not foresee a substantial reduction in the inferred number of rearrangements separating c. reinhardtii and c. vulgaris from their common ancestor.
inversions are thought to be much more common than transpositions in chloroplast genome evolution  <cit> , and our estimation of ancestral genome order was made with the assumption that all rearrangements were inversions.
however, we did consider the contribution of inversions and transpositions under different scenarios in the simulation from the ancestral genome.
it should be noted that there is not a unique phylogeny distance measure using transposition only, because computationally one transposition is equivalent to two or three inversions  <cit> .
for this reason, we designed our simulations to allow for various ratios of inversion and transposition events.
the results of our simulation study did not vary significantly under these scenarios.
the grappa-ir algorithm was developed to account for the inverted repeat  region found in most plastid genomes the ir region seems to evolve at a slower rate in both nucleotide sequence and gene order than the single copy regions  <cit> , and frequent intra-molecular recombination homogenizes the two copies  <cit> .
the most conserved gene set in the ir region is the rrna operon.
in ir-containing green plastids, the order of rrna genes is conserved, but the ir boundaries can vary greatly even within one genus  <cit> .
the ir may restrict rearrangements that cross the boundary of single copy regions, and thus concentrate gene order changes within single copy regions.
however, this hypothetical constraint of the ir on genome rearrangements seems to have been lost in the c. reinhardtii/c.
vulgaris lineage.
notably, both lineages have undergone extensive rearrangements since their divergence from a common ancestor, and they contain only a few conserved clusters encoding rrna or ribosomal proteins.
in either genome, genes that typically reside together in the lsc region have often been scrambled and scattered.
when comparing the ancestral genome to the c. vulgaris gene order, there was no distinction of lsc and ssc regions although many large clusters were still shared .
if there were constraints on the breakpoint locations, as experimentally identified in bacterial inversion mutants  <cit> , it would limit the possible paths of evolution, and these constraints on the ancestral gene orders would increase the number of rearrangements relative to the estimations derived from grappa.
therefore, as discussed above, our approach of detecting strong deviation from expectation is conservative in that the number of rearrangements may be underestimated.
recent studies of plant, animal and fungal genomes have shown that genes involved in the same pathways or genes sharing similar expression patterns are often spatially clustered  <cit> .
in eukaryotes, the operon structure has only been demonstrated in the nematode caenorhabditis  <cit> .
comparative analyses of yeast genomes indicate that rearrangements brought together duplicate genes forming the dal cluster involved in allantoin metabolism  <cit> .
in this study, we demonstrated that positive selection for increased clustering has influenced gene order in the chloroplast.
gene clusters, as opposed to separated genes, permit polycistronic transcription and thus fewer transcriptional regulation units.
co-transcription may be facilitated by close spacing of genes in cpdna because transcription termination is inefficient  <cit> .
although post-transcriptional rna processing often creates multiple single-gene transcripts, co-transcription foments an initial stoichiometric accumulation of rna corresponding to each gene in a cluster.
thus, large clusters can be advantageous in coordinating gene expression on this level.
experimental approaches are necessary to understand whether these gene clusters function as operons.
because chloroplast primary transcripts are heavily processed – as just one example, the psbb cluster in maize accumulates as at least  <dig> distinct mrna species with varying translational capacities  <cit>  – direct analysis of the functional advantages of clustering in chloroplasts is challenging.
indeed, chlamydomonas may be a special case, since unlike land plants it has a single rather than multiple rna polymerases  <cit> .
this situation does not allow differential expression by promoter selectivity, and may therefore serve as a selective force that favors physical grouping of genes rather than evolution of promoter sequences of dispersed genes.
CONCLUSIONS
in conclusion, we infer that gene order in the c. reinhardtii plastid evolved in a non-random fashion, and hypothesize that genome structure has been influenced by directional selection acting on variation generated by an increased rate of rearrangement.
our results provide strong evidence that genetic responses to natural selection occur at the level of genome organization.
by estimating the ancestral gene order and simulating rearrangements under a null model, we provide a formal demonstration that the chloroplast genome of c. reinhardtii has been shaped by natural selection.
although the model of natural selection on gene order remains to be developed, application of our methods to sequences of additional chlorophyte plastid genomes would help to improve the accuracy of the ancestral genome reconstruction and inferred branch lengths.
the complex process of gene duplication and loss in bacterial and eukaryotic nuclear genomes presents challenges to reconstruction of ancestral gene orders.
still, the development of new comparative tools  <cit>  gives us hope that the type of analysis presented in this paper will soon be applicable to eukaryotic genomes.

 the influence of genotype on phenotype is not limited to the coding of peptides and functional rnas by nucleotide sequences. an organism's phenotype is also affected by the chromosomal arrangement of genes and the interaction of gene products. comparative genomics has revealed a variety of gene clusters and chromosomal segments that have remained intact over hundreds of millions of years. selection for clustering of co-transcribed genes has been hypothesized to influence gene order within bacterial and organelle genomes, where gene clusters typically encode multiple components of a functional pathway. for example, the ribosomal proteins are encoded by similar operons in archaebacteria, eubacteria and plastids. in eukaryotic genomes, co-expression of neighboring genes is significantly associated with the functional roles of the genes. one way that those genes become clustered is through tandem duplication, which usually results in functionally related genes being adjacent. on the other hand, unrelated genes may also be brought together through chromosome rearrangements. unless selection is acting to maintain or promote gene clusters, gene orders in genomes subjected to rearrangements should become randomized with respect to function or co-expression profiles. significant clustering has been inferred using permutation tests that compare observed physical distances between pairs or blocks of co-expressed or functionally related genes to a null distribution constructed from randomized gene orders. however, this approach is limited since the evolutionary history of the genome was not considered. when comparing gene orders among related species, it is possible to estimate the ancestral genome and to simulate a null distribution for changes in gene order using a model. this evolutionary approach can be used to test directly the influence of selection on genome structure, that is, whether present-day genome structure has been influenced by directional selection for clustering of functionally related genes. small genomes, especially those of organelles and bacteria, are well suited to global comparisons of gene order. like eukaryotic genomes, they are subject to structural changes such as inversion, transposition or translocation, as well as gene loss and gene gain. chloroplast dnas in most land plants share a highly conserved gene content and similar gene orders. most cpdnas include two identical regions in opposite orientations called the inverted repeat, flanked by large single copy and small single copy regions. the irs generally contain the bacterial-like rrna gene clusters, and the genes involved in photosynthesis are arranged similarly in chloroplast and cyanobacterial genomes. despite these well-characterized patterns, it is unknown to what extent the conserved gene order reflects a slow intrinsic rate of neutral chromosomal rearrangements, rather than selection against alternative gene orders. a model of neutral rearrangement of gene order is required to test formally whether gene orders evolve under selection which prefers some gene arrangements over others. nadeau and taylor first proposed a model for the neutral evolution of gene order in comparisons of mouse and human chromosomes. this "random breakage model" provides a null hypothesis for the evolution of gene order. it assumes a random distribution of break points and allows all possible gene orders without restrictions. the random breakage model has been used to infer organismal phylogenies from gene order data. the gene order difference can be measured using the inversion distance, which is the minimal number of inversions necessary to transform one gene order to another. currently, the most accurate heuristic approach is implemented in the grappa software, which is generally suitable for small taxon sets because the algorithm scores inversion medians for all nodes iteratively across all possible phylogenies. algorithms for genomes with arbitrary rearrangements, a few deletions and duplications have been developed, and the capacity of grappa can be scaled up with the discovering method to potentially very large data sets. the random breakage model does not account for recombination hotspots, which have been reported from human-mouse genome comparisons. however, at this time it may be difficult to model these hotspots, because the precise locations of reused breakpoints are unknown due to insufficient resolution of gene orders and potential errors in homology assessment given the scale of eukaryotic chromosomes. thus, the fragile breakage model, as an alternative to the random breakage model, has not been well established. whereas gene order is generally conserved among land plant cpdnas, very little synteny is observed between this group and cpdnas of the chlorophytic green algae c. reinhardtii and chlorella vulgaris. the apparently increased rearrangement rate is associated with invasion by a large number of short dispersed repeats, for which the evolutionary distribution is still poorly defined. the large number of rearrangements provides an excellent opportunity to test whether natural selection has preferred some changes in gene order. here we present novel statistics and parametric tests that lead us to reject the models of random rearrangement in favor of directional selection for clustering of functionally related genes in c. reinhardtii. we also present experimental evidence that adaptive evolution of chloroplast genome structure could be driven by the advantage of concerted regulation conferred by polycistronic transcription.  functional clusters are not randomly distributed we compared gene orders of representative cpdnas from land plants, including tobacco and liverwort, a charophytic green alga, chlorophytic green algae, a green flagellate alga with uncertain affinities, and the plastid of cyanophora paradoxa. to measure the genome structure in terms of clustering by chromosome locations and gene functions, we defined "sided blocks" as contiguous genes coded on the same strand of the plastid chromosome, and "functional clusters" as blocks of functionally related genes. the randomness in the observed distribution of shared genes in chloroplast genomes with respect to gene function was assessed using a kolmogorov-smirnov test. the null hypothesis was rejected in all seven cpdnas investigated for genes in functional categories such as atp synthases and electron transport. while this test suggests some degree of functional clustering in all chloroplast genomes, it does not take into account the phylogenetic relationship of these organisms, so it is unclear whether functional clustering in chloroplast genomes is a legacy of genome organization in a cyanobacteria-like ancestor, or the product of selection on gene order in the face of genome rearrangements. §the test statistic dn measures whether the distribution of functionally related genes is random in gene clusters. total shared genes between seven cpdnas were included. extensive rearrangements from the ancestral chloroplast genome to c. reinhardtii in order to investigate evolutionary changes of gene order, we constructed a phylogeny of seven representative cpdnas and rooted with the sequence of c. paradoxa. maximum parsimony, neighbor joining and maximum likelihood analyses of an alignment of concatenated protein sequences including a total of aligned sites, all yielded identical fully resolved topologies with high bootstrap support. mesostigma was placed as a basal charophyte lineage in one previous analysis. the unrooted phylogeny of seven cpdnas is congruent with the alternative placement of mesostigma either as a basal charophyte or basal to both charophyte and chlorophyte lineages. this tree was used as the reference phylogeny for gene order inference. we scored the orders of genes shared in the seven genomes. then we used modified versions of grappa to compute the inversion distance between ancestral nodes and each terminal node. the branches leading to two chlorophytic green algae, c. reinhardtii and c. vulgaris, are much longer than the branches leading to the other taxa, and that many more steps were inferred on the c. reinhardtii lineage relative to the c. vulgaris lineage. gene duplications or deletions were mapped before scoring the ancestral genomes with inversions, and were not counted as rearrangements. irs were present in all inferred ancestral nodes, and one copy was lost in c. vulgaris. ancestral gene orders were scored on all the phylogenies using a two-step approach. due to the computational time limit, we stopped scoring all possible ancestral gene orders with the data set after days and took the best scored ancestral gene orders at that time. the cpdnas of two land plants, n. tabacum and m. polymorpha, were separated by an estimated inversions based on the data set. one large inversion in the lsc region has long been recognized to separate the two genomes. additional rearrangements are directly observable through comparison of gene order files for the two species. using grappa, all rearrangements were inferred as inversions, but the total number of inversion events estimated by grappa may be greater than the true mixture of inversions and transpositions because one transposition could result in the same change in gene order as two or three inversions. increased order in the genome structure after rearrangements two genomic structural characteristics were measured: the propensity of adjacent genes to be clustered on the same strand and the clustering of functionally related genes. both indices were calculated for the inferred ancestral gene orders and extant daughter lineages. among land plants and charophytes, the inferred sidedness among ancestral genomes was similar to extant lineages, however, among the chlorophytes an opposite trend was observed, especially in the c. reinhardtii lineage. the large number of rearrangements in the c. reinhardtii cpdna lineage resulted in dramatically increased sidedness relative to the inferred most recent common ancestor of c. reinhardtii and c. vulgaris. a small increase of cs was found in the n. olivacea lineage and there was almost no change in the lineage leading to c. vulgaris. a large increase was also observed in the functional clustering index, cf, for c. reinhardti, whereas the trend was less profound in other lineages. thus, even if the ancestral genome already had a "sided" structure, sidedness increased with genome rearrangements as c. reinhardtii cpdna evolved. the inferred increase in sidedness and functional clustering in the face of the large number of rearrangements on the lineage leading to c. reinhardtii might be adaptive, if such increases were not expected under random rearrangements. to test the null hypothesis that the changes in cs and cf were consequence of random genome rearrangements rather than a consequence of directional selection, we simulated random rearrangements starting with the inferred ancestral genome along the branch leading to c. reinhardtii. although inversions are the most abundant type of rearrangement in cpdnas, we also considered the contribution of transpositions under three inversion to transposition ratios, while the total number of rearrangements was fixed according to the branch length inferred using grappa. three simulations with replicates were conducted with inversion to transposition ratios of 1: 10: and 1: under the random breakage model. the mean cs values for the three sets were, and, respectively, and the 95% confidence intervals were, and, respectively. all datasets simulated under the random breakage model showed a significant decrease of sidedness from the ancestral level. in contrast, the cs value calculated for c. reinhardtii increased significantly to, greatly exceeding the sidedness that would be expected in a genome that had undergone this much evolutionary change relative to its ancestor. simulations using inferred ancestral genomes for land plant lineages also strongly reject the null hypothesis of random rearrangements. given the large number of rearrangements observed in the c. reinhardtii lineage, cf was also predicted to decrease significantly under the random breakage model, but cf did not decrease as observed in c. reinhardtii. the simulations with three models described above all yielded a large decrease in clustering as expected. when transposition was included in simulations, decreases of cf were on a similar scale to the inversion-only simulations. taken together, these results indicate that the remarkable increase in sidedness and functional clustering observed in c. reinhardtii cpdna has not been the outcome of solely chance events. instead, the strong deviation from the range of outcomes expected under various random breakage models implies that the genome structure is the outcome of a directional selective process. the increased level of organization in c. reinhardtii cpdna was associated with both maintenance of ancestral clusters and growth of new clusters. there were six conserved blocks containing of the genes shared between the c. reinhardtii and the c. vulgaris cpdnas. these blocks include concentrations of genes from a single functional category, such as ribosomal proteins, photosystem ii, translation apparatus, and atp synthase subunits. moreover, a number of small clusters of functionally related genes inferred in the ancestral genome were brought together in c. reinhardtii. these include transcription/translation genes, electron transport genes, and photosynthetic genes. the new clusters contributed to the increase of cf in the c. reinhardtii chloroplast genome. coordinated expression of genes in functional clusters co-transcription of several clusters shown in figure 4b has been previously documented, including psbd-psaa exon 2-psbj-atpi, psbf-psbl, peta-petd, and psbm-psbz. co-transcription of rpl and rps genes has been found in land plant chloroplasts. we documented co-transcription for an additional novel functional cluster, shown in figure 4a. using rna gel blots, tricistronic transcripts of rpl36-rpl23-rpl and possibly dicistronic rpl2-rps species could be detected. taken together, it appears that the clusters of functionally related genes observed in c. reinhardtii cpdna may be frequently co-transcribed. discussion by reconstructing the possible ancestral gene order in chloroplast genomes and simulating rearrangements, we have been able to formally test and reject the null hypothesis that c. reinhardtii cpdna has evolved through random rearrangements. instead, we found that its observed gene order deviates strongly from the degree of sidedness and clustering expected under a random breakage model. euglena gracilis cpdna also has a high degree of sidedness, however, the asymmetry of its coding strand is concentrated in one half of the genome and associated with gc content, which could be influenced by asymmetrical replication of the chromosome. in c. reinhardtii, the sidedness is not associated with gc content and we hypothesize that it is driven by co-transcription of genes in a functional cluster. whereas some clusters of co-transcribed genes were maintained in both c. reinhardtii and c. vulgaris, novel clusters clearly formed in the c. reinhardtii lineage. co-transcription of neighboring genes in the c. reinhardtii chloroplast is a widely documented phenomenon. we demonstrated that in addition to the ribosomal protein clusters, global analyses support the elevated level of clustering of other functionally related genes. the aggregate of genes in clusters include most essential genes involved in translation and transcription, and some photosynthetic genes. coordinated transcription may play a crucial role in the regulation of plastid gene expression in response to light or circadian rhythms. it is also possible that some clusters contain cis-elements, similar to the artificial polydeoxyadenosine sequences, which enhance transcription efficiency. moreover, most of the putative co-transcription units are not conserved across chlorophytes. therefore, the majority of functional clusters observed in c. reinhardtii represent new gene arrangements. in the chloroplast gene order phylogeny, the c. reinhardtii lineage resides on a long branch compared to the c. vulgaris lineage, and both genomes are more rearranged than that of n. olivacea, relative to the common ancestral genome of the three chlorophyte lineages. the elevated rate of chloroplast genome rearrangement in c. reinhardtii is associated with invasion of sdrs, which heavily populate the non-coding regions, increasing the total length of the intergenic regions compared to c. vulgaris cpdna by one-third. although simple sequence repeats are common to microbial genomes, such elements are rare in most sequenced chloroplast genomes. within the chlamydomonas genus, c. reinhardtii and c. gelatinosa cpdnas exhibit a prevalence of repetitive dna and a high degree of gene order variation compared to the c. moewusii/c. pitschmannii lineage. the sister lineage to c. reinhardtii in our study, c. vulgaris, contains numerous cpdna repeat sequences. besides chlorophyte algae, members of angiosperm families, including campanulaceae, fabaceae and geraniaceae, also contain repeat elements in rearranged cpdnas, albeit of a much lower copy number. these repeat elements may act as molecular "grease" that facilitates non-homologous recombination and creates a pool of diverse genome structures subject to selective retention. future investigations will test whether the increased rates of rearrangement in plastid genomes with dispersed repeats typically lead to increased sidedness and functional clustering as we infer for c. reinhardtii. gene order changes reflect relatively rare evolutionary events and are expected to result in much less homoplasy than substitution events in nucleotide or protein sequences over a deep time scale. phylogeny reconstruction using grappa is highly accurate even for divergent genomes, and thus the ancestral gene orders inferred in our study contained sufficient phylogenetic information. the only other software for genome rearrangement phylogeny, badger, performed poorly on this data set. grappa usually inferred unique ancestral gene orders on many data sets we tested. furthermore, analyses on simulated data have shown that the inferred gene orders scored almost as well as true ancestral gene orders. in our simulation tests of three genomes with genes each, and branch lengths of and, the average score for ancestral gene orders computed by grappa was only about 7% less than the true scores. in practice, we observed that the less optimal gene orders generally required more rearrangements. therefore, it is quite likely that any error in our estimation of ancestral gene order has resulted in a downward bias in the inferred number of rearrangements on the branch leading to c. reinhardtii. increasing the number of rearrangements on this branch would only lead to a more certain rejection of the neutrality of rearrangements. the accuracy of ancestral genome reconstruction also depends on the degree of divergence among extant taxa and taxon sampling. for example, accurate reconstruction of ancestral genomes at the mammalian cftr locus was achieved at the dna level. the high-quality reconstruction was attributed to a dense sampling of syntenic genome sequences from eutherian mammals, and the lack of gene order rearrangement at the locus. because the c. reinhardtii cpdna is one of the most rearranged chloroplast genomes sequenced to date, we included all available chlorophyte chloroplast genomes for evolutionary distance estimation and ancestral gene order reconstruction. the accuracy of our ancestral gene order estimation may improve with inclusion of additional chlorophyte plastid gene orders as they become available, but we do not foresee a substantial reduction in the inferred number of rearrangements separating c. reinhardtii and c. vulgaris from their common ancestor. inversions are thought to be much more common than transpositions in chloroplast genome evolution, and our estimation of ancestral genome order was made with the assumption that all rearrangements were inversions. however, we did consider the contribution of inversions and transpositions under different scenarios in the simulation from the ancestral genome. it should be noted that there is not a unique phylogeny distance measure using transposition only, because computationally one transposition is equivalent to two or three inversions. for this reason, we designed our simulations to allow for various ratios of inversion and transposition events. the results of our simulation study did not vary significantly under these scenarios. the grappa-ir algorithm was developed to account for the inverted repeat region found in most plastid genomes the ir region seems to evolve at a slower rate in both nucleotide sequence and gene order than the single copy regions, and frequent intra-molecular recombination homogenizes the two copies. the most conserved gene set in the ir region is the rrna operon. in ir-containing green plastids, the order of rrna genes is conserved, but the ir boundaries can vary greatly even within one genus. the ir may restrict rearrangements that cross the boundary of single copy regions, and thus concentrate gene order changes within single copy regions. however, this hypothetical constraint of the ir on genome rearrangements seems to have been lost in the c. reinhardtii/c. vulgaris lineage. notably, both lineages have undergone extensive rearrangements since their divergence from a common ancestor, and they contain only a few conserved clusters encoding rrna or ribosomal proteins. in either genome, genes that typically reside together in the lsc region have often been scrambled and scattered. when comparing the ancestral genome to the c. vulgaris gene order, there was no distinction of lsc and ssc regions although many large clusters were still shared. if there were constraints on the breakpoint locations, as experimentally identified in bacterial inversion mutants, it would limit the possible paths of evolution, and these constraints on the ancestral gene orders would increase the number of rearrangements relative to the estimations derived from grappa. therefore, as discussed above, our approach of detecting strong deviation from expectation is conservative in that the number of rearrangements may be underestimated. recent studies of plant, animal and fungal genomes have shown that genes involved in the same pathways or genes sharing similar expression patterns are often spatially clustered. in eukaryotes, the operon structure has only been demonstrated in the nematode caenorhabditis. comparative analyses of yeast genomes indicate that rearrangements brought together duplicate genes forming the dal cluster involved in allantoin metabolism. in this study, we demonstrated that positive selection for increased clustering has influenced gene order in the chloroplast. gene clusters, as opposed to separated genes, permit polycistronic transcription and thus fewer transcriptional regulation units. co-transcription may be facilitated by close spacing of genes in cpdna because transcription termination is inefficient. although post-transcriptional rna processing often creates multiple single-gene transcripts, co-transcription foments an initial stoichiometric accumulation of rna corresponding to each gene in a cluster. thus, large clusters can be advantageous in coordinating gene expression on this level. experimental approaches are necessary to understand whether these gene clusters function as operons. because chloroplast primary transcripts are heavily processed – as just one example, the psbb cluster in maize accumulates as at least distinct mrna species with varying translational capacities – direct analysis of the functional advantages of clustering in chloroplasts is challenging. indeed, chlamydomonas may be a special case, since unlike land plants it has a single rather than multiple rna polymerases. this situation does not allow differential expression by promoter selectivity, and may therefore serve as a selective force that favors physical grouping of genes rather than evolution of promoter sequences of dispersed genes.  in conclusion, we infer that gene order in the c. reinhardtii plastid evolved in a non-random fashion, and hypothesize that genome structure has been influenced by directional selection acting on variation generated by an increased rate of rearrangement. our results provide strong evidence that genetic responses to natural selection occur at the level of genome organization. by estimating the ancestral gene order and simulating rearrangements under a null model, we provide a formal demonstration that the chloroplast genome of c. reinhardtii has been shaped by natural selection. although the model of natural selection on gene order remains to be developed, application of our methods to sequences of additional chlorophyte plastid genomes would help to improve the accuracy of the ancestral genome reconstruction and inferred branch lengths. the complex process of gene duplication and loss in bacterial and eukaryotic nuclear genomes presents challenges to reconstruction of ancestral gene orders. still, the development of new comparative tools gives us hope that the type of analysis presented in this paper will soon be applicable to eukaryotic genomes. 
genome rearrangements influence gene order and configuration of gene clusters in all genomes. simulations and experimental evidence suggest that both selective maintenance and directional selection for gene clusters are determinants of chloroplast gene order. ancestral gene orders were inferred and then subjected to simulated rearrangement events under the random breakage model with varying ratios of inversions and transpositions. we report evidence of co-transcription of neighboring genes, which may be responsible for the observed gene clusters in c. reinhardtii cpdna. conserved gene orders may reflect a slow intrinsic rate of neutral chromosomal rearrangements, or selective constraint. we found that adjacent chloroplast genes in c. reinhardtii were located on the same strand much more frequently than in simulated genomes that were generated under a random rearrangement processes. we use a novel parametric bootstrap approach to test if directional selection is responsible for the clustering of functionally related genes observed in the highly rearranged chloroplast genome of the unicellular green alga chlamydomonas reinhardtii, relative to ancestral chloroplast genomes. we investigate the influence of natural selection on gene order in association with increased rate of chromosomal rearrangement. most land plant chloroplast dnas share a highly conserved gene content and with notable exceptions, a largely co-linear gene order. it is unknown to what extent observed changes in gene order are random or adaptive. in addition, functionally related genes were found to be more clustered than those evolved under random rearrangements.
 genome rearrangements influence gene order and configuration of gene clusters in all genomes. most land plant chloroplast dnas share a highly conserved gene content and with notable exceptions, a largely co-linear gene order. conserved gene orders may reflect a slow intrinsic rate of neutral chromosomal rearrangements, or selective constraint. it is unknown to what extent observed changes in gene order are random or adaptive. we investigate the influence of natural selection on gene order in association with increased rate of chromosomal rearrangement. we use a novel parametric bootstrap approach to test if directional selection is responsible for the clustering of functionally related genes observed in the highly rearranged chloroplast genome of the unicellular green alga chlamydomonas reinhardtii, relative to ancestral chloroplast genomes.  ancestral gene orders were inferred and then subjected to simulated rearrangement events under the random breakage model with varying ratios of inversions and transpositions. we found that adjacent chloroplast genes in c. reinhardtii were located on the same strand much more frequently than in simulated genomes that were generated under a random rearrangement processes. in addition, functionally related genes were found to be more clustered than those evolved under random rearrangements. we report evidence of co-transcription of neighboring genes, which may be responsible for the observed gene clusters in c. reinhardtii cpdna.  simulations and experimental evidence suggest that both selective maintenance and directional selection for gene clusters are determinants of chloroplast gene order. 