BACKGROUND
all variation found in natural populations is ultimately derived from evolutionary forces acting upon mutation and recombination.
by studying genome-wide patterns of variation among closely related species, it is possible to make inferences about the evolutionary process of that organism.
although the model organism caenorhabditis elegans has been well studied in biology, our knowledge of the evolution of natural populations of c. elegans is limited  <cit> .
in the past, molecular variation in c. elegans has been studied using assays involving single mitochondrial and nuclear genes  <cit> , transposons  <cit> , amplified fragment length polymorphisms , and microsatellites  <cit> .
in this study, we investigate genetic variation in three strains of c. elegans: n <dig>  cb <dig>  and cb <dig> using large-scale dna sequence datasets  <cit> .
n <dig>  is the most commonly used lab strain.
it is the strain for which the genome sequence has been completely determined.
cb <dig> is a strain of c. elegans from hawaii while cb <dig> is a strain of c. elegans from pasadena, ca.
strains cb <dig> and cb <dig> were isolated in  <dig> and  <dig>  respectively.
since then, they have been cultured in the lab.
in their c. elegans population genomics study, rockman and kruglyak  <cit>  distinguished  <dig> haplotypes among  <dig> wild isolates in their genotype data from  <dig> n2-cb <dig> snps.
they indicated that cb <dig> shares haplotype  <dig> with strains from other localities, including ab2- <dig>  from adelaide, australia, and cb <dig>  from palo alto, california.
the genotypic similarity among cb <dig>  cb <dig>  and ab2- <dig> suggests they may share an ancestor in a laboratory.
previous analysis of multiple caenorhabditis lines shows extensive variation in nuclear single-nucleotide polymorphism  density  <cit> , with the hawaiian strain cb <dig> having the highest snp density and most unique snps  <cit> .
additional polymorphisms in cb <dig> were found by swan et al.
<cit> , and analysis of the distribution of these polymorphisms by cutter et al.
<cit>  revealed that the polymorphisms occurred more frequently in the arms than in the core in chromosomes one to five, but are randomly distributed in chromosome x.
the patterns of natural variation found in a genome are the result of mutational events that are influenced by both drift and selection.
under neutral theory, a majority of mutations that arise spontaneously are neutral, and their fate  is primarily determined by drift.
in rare cases, a spontaneous mutation will confer a selective advantage  or disadvantage .
if by chance this mutation is not lost by drift, in consequent generations the mutation will spread throughout the population, and variation at linked sites will either accumulate or be purged.
the combination of neutral mutations fixed through drift and the mutations that are accumulated or purged determine the genomic landscape of the organism  <cit> .
by surveying the patterns of natural variation between the closely related caenorhabditis strains cb <dig> and cb <dig>  it is possible to investigate the evolutionary processes of drift and selection occurring in this lineage.
although previous studies provide insights into c. elegans snp patterns and densities in the natural isolates, a large-scale genomic analysis of the c. elegans polymorphic spectrum has not been accomplished until now.
other studies of c. elegans have not focused on examining the patterns of variation that we present here, but have instead focused on other important patterns.
for example, weber et al.
<cit>  reported the genome sequence of the lsj <dig>  strain and compared it to the reference wild type n <dig> .
lsj <dig> is a sibling of the reference wild type n <dig>  the authors indicated that comparing the genomes of n <dig> and lsj <dig> highlight genetic changes associated with lab domestication and errors in the c. elegans reference genome.
they confirmed that lsj <dig> and n <dig> are derived from the same bristol isolate.
in addition, they identified  <dig> predicted differences between the lsj <dig> and n <dig> genome sequences.
finally, by sequencing two recently isolated wild c. elegans strains, cb <dig>  and ed <dig> , they inferred the ancestral state of the original bristol isolate.
all of this makes cb <dig> an extremely valuable resource not only for c. elegans gene mapping  <cit>  but also for the study of the distribution and spectrum of caenorhabditis snps.
graustein et al.
<cit>  compared the levels of dna sequence polymorphism among three closely related species in the genus caenorhabditis: two self-fertilizing species, c. elegans and c. briggsae, and one cross-fertilizing species, c. remanei.
jovelin et al.
<cit>  analyzed nucleotide variation within and between species across two olfactory pathways in caenorhabditis to examine the relationship between evolutionary rate and gene function and their position within the pathway.
the authors found that transcription factors specifying chemosensory neuron subtype identity exhibit higher levels of nucleotide divergence and polymorphism than the structural genes in the signaling pathways.
they found much higher levels of polymorphism within c. remanei than within the related species c. elegans and c. briggsae.
their findings suggest behavioral differences between species in response to olfactory stimuli are likely to involve differences in gene regulation due to the divergence of regulatory genes.
we report the entire spectrum of events critical for understanding the fundamental divergence patterns of these natural isolates.
in addition, we compare our results to the n <dig> ma line results from denver et al.
<cit>  because we wish to compare the differences in mutation patterns when selection is present  versus when it is nearly absent .
RESULTS
overview of snp variation
we identified a total of  <dig> putative snps in our analysis of the cb <dig> genome compared to n <dig>  direct re-sequencing of  <dig> of these putative snps confirmed  <dig>  suggesting our rate of confirmation is 93%.
the  <dig> snps chosen for resequencing were representative of all the snps in this study.
the level of snp differences between n <dig> and cb <dig> is  <dig>  × 10- <dig> snp per base with insertion deletion events  contributing slightly more of the variation than base substitutions and inversions .
this is consistent with the known mutation spectrum described in denver et al.
<cit>  that reports slightly more indels than snps in the c. elegans ma lines.
in contrast, a recent genome wide comparison of n <dig> and the strain cb <dig>  <cit>  revealed a much lower genome wide snp level  and those differences were dominated by base substitutions.
the difference in the ratio of base substitutions to indels in the two comparisons could be attributed to the differences in sequencing approaches used to assay variation.
the cb <dig> data was based on longer sanger reads  while the cb <dig> data was based on short read sequencing.
reads with more than one snp are difficult to assemble when using short read technology.
consequently, in cb <dig>  reads with more than  <dig> differences are incompatible with this analysis  <cit> .
in that scenario, sequential or highly clustered snps may not be detected in the cb <dig> dataset.
the total number of bases assayed in cb <dig> was  <dig> .
the numbers of snps observed in cb <dig> was based on the assay of 77% of the c. elegans reference genome .
the total number of bases assayed in cb <dig> was  <dig>  while the number of snps observed in cb <dig> was based on the assay of 77% of the c. elegans reference genome  <cit> .
even though  <dig> % is a small portion of the c. elegans reference genome, we assume that the sequences used to obtain the polymorphisms are randomly selected from the entire genome.
it is therefore reasonable to compare the results of cb <dig> to the results of cb <dig>
the base substitution/indel ratio is  <dig> : <dig>  for cb <dig> and  <dig> : <dig>  for cb <dig> .
two observations suggest that the differences in the observed base substitution/indel ratios in cb <dig> vs cb <dig> do not stem from read length differences in the two assays.
first, clusters of snps  do not contribute to a large fraction of the total variation.
therefore, sequential indels which may be undetectable using short-read technology do not greatly influence the base substitution/indel ratio.
second, when we limit the comparisons to single base substitutions and indels which are fully assayable using short read technology in cb <dig>  we find that the discrepancy between these two natural isolates remains .
based on the above observations, the cb <dig> snps have a higher proportion of indels than the cb <dig> snps.
these observations are consistent with those reported by denver et al.
<cit>  in which c. elegans ma lines showed a base substitution/indel ratio of  <dig> : <dig> .
either the cb <dig> genome is getting smaller or the n <dig> genome is getting larger
among the snps predicted in the comparison of cb <dig> and n <dig>  we observed a strong and significant bias for deletions  over insertions .
this bias was reflected in single event indels as well as larger indels .
although the direction in which the indel occurred is unknown, this pattern differs from the cb <dig> ratio , suggesting two possibilities:  the cb <dig> genome is becoming smaller or  the n <dig> genome is getting larger because it fails to purge insertions.
this result differs from the baseline mutation spectrum in c. elegans ma lines in denver et al.
<cit> .
they observed a high frequency of indels where the mutations appeared to be biased toward insertions.
there are two possible explanations for this observation:  there is a change in the mutation spectrum between n <dig> and cb <dig> or  selection constraints between n <dig> and cb <dig> have changed.
sequential vs single snps
sequential snps can arise from single mutational events involving multiple base pairs or as a consequence of individual events at adjacent positions.
there is a larger proportion of sequential snps in cb <dig> compared to cb <dig> .
the high proportions of observed sequential base substitutions  and indels  in cb <dig> are not consistent with expectations based on a hypothesis of independent adjacent polymorphisms.
this suggests a large fraction of the adjacent snps result from mutations involving multiple adjacent base pairs or that the adjacent snps are not independent.
the distribution of the sizes for sequential snps, however, suggests that the occurrence of sequential snps is negatively correlated with the number of bases involved.
due to the limitations of short read sequence alignment, the analysis between n <dig> and cb <dig> is limited to sequential base substitutions, indels, and putative inversions with a length of  <dig> bp.
in particular, inversions are a special subclass of sequential changes that occur through a single event.
we identified all size classes of inversions in cb <dig> and in cb <dig> .
these account for only a small fraction  of all snps and do not affect the overall conclusions of our study.
we note that in cb <dig>  the inversions may be downwardly biased due to the cb <dig> sequencing method.
shifting patterns of base substitution
the base substitution pattern when comparing n <dig> against the cb <dig> isolate reveals a c. elegans transition over transversion bias of  <dig> : <dig> .
however, when comparing n <dig> against cb <dig>  there is no transition over transversion bias .
furthermore, in cb <dig>  there is a significant bias in the direction of base substitution.
when comparing n <dig> against cb <dig>  the frequency of a or t bases in n <dig> that are g or c bases in cb <dig> greatly outnumber the opposite frequencies for transitions as well as transversions.
at equilibrium, the numbers of g:c to a:t differences between two genomes should be equal to the number of a:t to g:c differences.
the shift in base substitution results in a significant detectable shift in base composition between n <dig>  and cb <dig> .
in particular, there is a significant difference between the following rates in the n2/cb <dig> comparison: g →a lower than a→g; c →t lower than t →c; c →a lower than a→c; g→t lower than t→g.
none of these differences were observed in the n2/cb <dig> comparison .
*number
the results shown here differ from the expectation of a significant mutation bias from g:c to a:t and g:c to t:a based on mutation accumulation lines  <cit> .
because the base substitution pattern is consistent with the n2/cb <dig> comparison, but different in cb <dig>  the change is likely either a change in the mutational mechanisms or shifting selective forces.
cb <dig> is a prime example of base compositional differences  that can occur within a species.
cb4856/cb <dig> snp rate of chromosomes
both cb <dig> and cb <dig> exhibit significant differences in snp rates among the chromosomes .
in particular, chromosomes i to x do not have a uniform snp rate in either cb <dig>  or cb <dig> .
in addition, the snp rate is higher in the autosomes than in chromosome x in cb <dig> .
in cb <dig>  chromosome iii has the smallest snp rate while chromosome v has the largest snp rate.
although previous observations of this disparity have been attributed to the mutagenic effect of recombination  <cit> , the random distribution of mutations across all chromosomes in mutation accumulation lines  <cit>  makes it more likely that selective sweeps are purging polymorphisms from different regions of the genome.
a comparison of snp rates between chromosomes in cb <dig> is not appropriate because of the stochastic nature of the snps found in the cb4858-n <dig> comparison.
there are large stretches of dna that are shared by n <dig> and cb <dig>
cb4856/cb <dig> arms versus core snp rate
we compared the snp rate in the arms vs the cores in different chromosomes in cb <dig> and cb <dig> using chi-squared tests.
in both strains, 1) the snp rate in the arms is significantly higher than in the core in all of the chromosomes; 2) the rates of snps in the arms are different across the chromosomes; 3) the rates of snps in the core are different across the chromosomes .
*based on chi-squared tests
there are many possible reasons why the snp rates differ between the arms and the core in both cb <dig> and cb <dig>  the first possibility is that there are more genes in the core.
since purifying selection removes mutations from coding regions, if the gene density is higher in the core, fewer snps will occur there  <cit> .
the second possibility is that there may be more recombination away from the core.
if recombination is mutagenic  <cit> , higher rates of recombination will increase snp density.
on the other hand, recombination interrupts linkage disequilibrium reducing the efficacy of selection.
as stated in cutter et al.
<cit> , chromosome arms have moderate to high rates of crossover recombination and low gene density and cores have strikingly little recombination despite comprising nearly half of each autosome.
rockman and kruglyak  <cit>  and barnes et al.
<cit>  provided evidence of these observations in their studies.
finally, there may be an inherent difference in mutation rates in the arms and the core.
as observed in this paper and by stein et al.
<cit> , there is a difference in the rate of snps in the arms vs the core.
observations by denver et al  <cit>  have suggested that the baseline rate of mutation in ma lines is not increased in the arms of autosomes.
this suggests that selection, not mutation, plays a larger role in the biased physical distribution of polymorphisms.
we then conclude that the reason the snp rate differs in the arms and core regions is primarily due to natural selection.
recombination and snp rate
in both cb <dig> and cb <dig> , the distribution of snp variation within the c. elegans chromosomes shows there is a significant relationship between snp frequency and recombination rate on the autosomes.
however, there is no significant linear relationship between snp frequency and recombination rate on the x chromosome in either isolate .
*p-values found doing spearman's rho test for correlation
the discrepancy with the x chromosome can be explained by the x chromosomes' many functional differences from the autosomes  <cit> .
recombination is almost non-existent in the right terminal arm of the x chromosome  <cit> .
furthermore, sex is determined by x chromosome dosage and this dosage is compensated in hermaphrodites  <cit> .
observations of correlation between snp rate and recombination on the autosomes in both cb <dig> and cb <dig> have been proposed to either reflect an inherent mutagenic effect of recombination or conditions where selection results in the loss  or fixation  of new mutations along with all linked polymorphisms.
the data in this study shows us that in the autosomes, the observed levels of polymorphisms are positively correlated with levels of recombination in both isolates.
using the chromosomal domains in rockman and kruglyak  <cit> , we found that 1) in the arms, the recombination rate is negatively correlated with chromosome size, although the correlation was not significant ; 2) the two tips of all autosomes and the x chromosome have an effectively zero recombination; 3) the c. elegans autosomes and the x chromosome have nearly constant recombination rates within each chromosomal domain .
findings 2) and 3) agree with the findings in rockman and kruglyak  <cit>  but finding 1) is different from the finding in rockman and kruglyak  <cit> .
cb <dig> hotspot analysis
the level of snp variation across the genome in any comparison reflects two possible mechanisms.
a high frequency of local polymorphisms can be a consequence of high localized mutation rate or a mutational hotspot.
alternatively, localized patterns of divergence can reflect the age of the allele and historic patterns of recombination.
as a consequence of recombination and interbreeding of the ancestors that gave rise to these isolates, each allele can have a unique history of coalescence.
to further investigate the pattern of variation across the c. elegans genome and specifically identify regions of significantly high localized polymorphisms, we identified clusters of snps with a significant number of polymorphisms .
in general, these hotspots are dna segments that have an unusual number of polymorphisms considering the length of the dna .
a more detailed description of the hotspots is given in the methods section.
these hotspots of polymorphisms may be mutational hotspots or may indicate old alleles.
we excluded the cb <dig> datasets due to the inherent inability of short reads to represent loci with highly clustered polymorphisms.
the summary statistics of the hotspots per chromosome are shown in table  <dig>  the optimal window size for the initial cluster analysis was constrained by the average single read length in the cb <dig> comparisons .
statistically significant hotspot clusters ranged from  <dig> bp to  <dig> bp in length .
there were  <dig> clusters when comparing n <dig> against cb <dig> and the average number of events per hotspot was  <dig> .
in general, the proportion of base substitutions was the largest in chromosome v, the proportion of nonsequential indels was the largest in chromosome x, and the proportion of sequential indels was the largest in chromosome iv.
there was a significant positive relationship between hotspot length  and the number of events in the hotspot .
chromosome v has the largest number of hotspots while chromosome x had the lowest number.
we did a linear regression with dummy variables to test if the location of snps  was a significant predictor of the number of hotspots.
we saw no significant hotspot distributional biases with respect to chromosomal arm/core boundaries .
the sizes of the hotspots vary per chromosome.
we categorized the hotspots into small and large hotspots based on a cutoff of  <dig> bp in length.
the number of small vs large hotspots is in table  <dig>  the hotspots with lengths less than  <dig> base pairs have on average  <dig>  of events and 15% of the events are base substitutions while 79% of the events are nonsequential indels.
on the other hand, the hotspots with lengths more than  <dig> base pairs have on average  <dig>  events and 58% of the events are base substitutions while 30% of the events are nonsequential indels.
in some taxa, it has been shown that the rate of mutation at specific loci is strongly correlated with gc content  <cit> .
to test this possibility, we assayed the gc content in all clusters and compared the %gc content in the clusters to the n <dig> genome as a whole .
there is sufficient evidence to indicate the %gc content in the statistical hotspots differs from the %gc content in the n <dig> genome as a whole .
the %gc content in different statistical hotspots is displayed in additional file  <dig>  some of the hotspots have a higher %gc content than n <dig> and some of them have a lower %gc content than n <dig>
we also compared these hotspots of polymorphism to mutations identified in a genome wide analysis of n <dig> mutation accumulation lines  <cit> .
denver et al  <cit>  found a random distribution of mutations in their ma lines.
the  <dig> mutations identified among ten n <dig> derived ma lines did not fall within any of the hotspots identified here.
finally, in a comparison of cb <dig> and cb <dig>  there were  <dig> snps that were shared between cb <dig> and cb <dig> , which is larger than the expected number of snps shared between cb <dig> and cb <dig> .
we hypothesize that the hotspots of polymorphisms in this study are alleles that are shared between cb <dig> and cb <dig> but not by n <dig> and are not mutational hotspots because  the %gc content in the hotspots differs from the %gc content in n <dig> and  because the snps found in cb <dig> vs n <dig> were not in the ma lines.
distribution of snps by function
to investigate the distribution of snps across major functional partitions in the c. elegans genome, we identified the putative functional role of each snp position based on the annotation of the n <dig> reference genome .
the general variation in both cb <dig> and cb <dig> follows expected patterns.
the functional categories of snps are ordered by intergenic>introns>utr>exons in cb <dig> and intergenic>utr>exons>introns .
the functional categories of snps are ordered as expected for cb <dig> but for cb <dig>  where the frequency of snps in exons is higher than in introns.
cb <dig> may have relaxed selection constraints on coding mutations .
insert
the numbers represent isolate × 10- <dig> snps per base.
*
*for ease in comparison of frequencies across categories and strains, we divided the number of polymorphisms by the number of analyzed sites and multiplied that number by  <dig>  for example, the proportion of insertion polymorphisms in cb <dig> is 9942*10- <dig>
the numbers represent isolate × 10- <dig> snps per base.
*
*for ease in comparison of frequencies across categories and strains, we divided the number of polymorphisms by the number of analyzed sites and multiplied that number by  <dig>
cb4856/cb <dig> neutrality
the ka/ks ratio is the rate of nonsynonymous substitutions  to the rate of synonymous substitutions , which can be used as an indicator of selective pressure acting on a protein-coding gene.
a value of ka/ks> <dig> indicates positive selective pressure whereas a value of ka/ks =  <dig> indicates neutral evolution.
finally, a value of ka/ks < <dig> indicates pressures to conserve amino acid sequence  <cit> .
we test for neutrality by testing the null hypothesis of ka/ks =  <dig> in both cb <dig> and cb <dig>  <cit> .
analysis of genome-wide snp patterns reveal a ka/ks of less than  <dig> for all chromosomes in both cb <dig> and cb <dig> in table  <dig>  adjusting for multiple comparisons  <cit> .
this result is consistent with the role that purifying selection has on snps in most protein coding regions.
*all ka/ks values in both isolates are significantly smaller than 1
in cb <dig>  the autosomes have ka/ks values that are not significantly different from x in either direction .
in addition, when considering all of the chromosomes, the ka/ks values are not significantly different in the different chromosomes in either direction .
however, the snp rate is lower in chromosome x  compared to the autosomes  in cb <dig> .
differences in the length or the number of cell divisions in the male germline compared to the hermaphrodites in cb <dig> may explain the lower snp rate in the x chromosome  <cit> .
when the x chromosome is passed through the male germline, it is in a hemizygotic state and exposed to increased purifying selection  <cit> .
in addition, the mitotic cell cycle length has been shown to be shorter in males than hermaphrodite germ cells  <cit> .
taken together, both of these factors can contribute to a lower snp rate in the x chromosome than in the autosomes in cb <dig>  these observations are dependent upon how often males are involved in reproduction from generation to generation.
while direct observations of c. elegans populations show a low frequency of males and low levels of heterozygosity, it is possible that some lineages of caenorhabditis have historically undergone much more outbreeding involving males  <cit> .
in cb <dig>  the autosomes have ka/ks values  that are significantly smaller than x  on the average .
unlike cb <dig>  the different chromosomes have significantly different ka/ks values .
it is not appropriate to consider the differences in snp rates between chromosomes in cb <dig> due to the long stretches of dna shared by n <dig> and cb <dig>
because the ka/ks ratio in the x chromosome is higher than the autosomes on average in cb <dig>  purifying selection appears to be reduced on the x chromosome in this strain compared to cb <dig>  the relative difference in ka/ks ratios in cb <dig> could reflect differences in the life history of cb <dig>  we suggest that males  could contribute to a smaller degree in natural populations of cb <dig> than in cb <dig> populations, resulting in a lower rate of protein sequence evolution for x-linked mutations in the hawaiian isolate cb <dig>  previous studies have presented varying levels of the genetic contribution of males in different natural isolates of caenorhabditis elegans  <cit> .
CONCLUSIONS
this study provides a large scale genomic portrait of the c. elegans polymorphisms in cb <dig> and cb <dig>  the patterns observed here give us insight into the evolutionary spectrum of the caenorhabditis lineage.
there is a clear bias of deletions over insertions in cb <dig>  suggesting two possibilities.
the first one is that the cb <dig> genome is becoming smaller.
other studies have argued that possible reasons that genomes could become smaller in general are gene loss, natural selection, or the occurrence of long terminal repeat  sequences of transposable elements  <cit> .
the second possibility is that the n <dig> genome is getting larger because it fails to purge insertions.
interestingly, direct sequencing of c. elegans insertion deletion rates in mutation accumulation lines  <cit>  observe a bias towards insertions.
taken together, we conclude that cb <dig> is evolving under different mutation or selection pressures than n <dig> or cb <dig>
our results show that in both cb <dig> and cb <dig>  snp rates are higher in the chromosome arms than in the chromosome cores in agreement with previous studies  <cit> .
direct genome sequencing of c. elegans mutation accumulation lines  <cit>  show that the mutation rate is not higher in arm regions compared to the core regions.
we hypothesize snp rates differ in the core regions of n <dig>  cb <dig>  and cb <dig> in our study due to higher levels of purifying selection in the core regions.
we found several statistically significant hotspots of polymorphisms in our cb4856-n <dig> comparison.
some of the cb <dig> hotspots contained significantly higher levels of g/c when compared to the genome average, consistent with observations of higher rates of deamination at methylated cytosines  <cit> .
none of these hotspots coincided with c. elegans mutations identified in denver et al.
<cit> , and are likely to be older alleles shared between cb <dig> and cb <dig> but not n <dig>
the ka/ks ratios in both cb <dig> and cb <dig> are consistent with the effect of purifying selection on snps in most protein coding regions.
the differences in ka/ks in the autosomes vs the x chromosome in cb <dig> suggest selection may be more efficient for x-linked genes.
in addition, these differences may be attributed to male-driven evolution of the x chromosomes.
we hypothesize that natural populations of cb <dig> strains have fewer males than cb <dig> populations, resulting in a lower rate of protein sequence evolution for x-linked mutations.
the finding in cb <dig> of a significant difference in selection on the x chromosome compared to the autosomes is consistent with a biological role for males in the lineage giving rise to cb <dig>  consistent with previous published results  <cit> .

 all variation found in natural populations is ultimately derived from evolutionary forces acting upon mutation and recombination. by studying genome-wide patterns of variation among closely related species, it is possible to make inferences about the evolutionary process of that organism. although the model organism caenorhabditis elegans has been well studied in biology, our knowledge of the evolution of natural populations of c. elegans is limited. in the past, molecular variation in c. elegans has been studied using assays involving single mitochondrial and nuclear genes, transposons, amplified fragment length polymorphisms, and microsatellites. in this study, we investigate genetic variation in three strains of c. elegans: n cb and cb using large-scale dna sequence datasets. n is the most commonly used lab strain. it is the strain for which the genome sequence has been completely determined. cb is a strain of c. elegans from hawaii while cb is a strain of c. elegans from pasadena, ca. strains cb and cb were isolated in and respectively. since then, they have been cultured in the lab. in their c. elegans population genomics study, rockman and kruglyak distinguished haplotypes among wild isolates in their genotype data from n2-cb snps. they indicated that cb shares haplotype with strains from other localities, including ab2- from adelaide, australia, and cb from palo alto, california. the genotypic similarity among cb cb and ab2- suggests they may share an ancestor in a laboratory. previous analysis of multiple caenorhabditis lines shows extensive variation in nuclear single-nucleotide polymorphism density, with the hawaiian strain cb having the highest snp density and most unique snps. additional polymorphisms in cb were found by swan et al., and analysis of the distribution of these polymorphisms by cutter et al. revealed that the polymorphisms occurred more frequently in the arms than in the core in chromosomes one to five, but are randomly distributed in chromosome x. the patterns of natural variation found in a genome are the result of mutational events that are influenced by both drift and selection. under neutral theory, a majority of mutations that arise spontaneously are neutral, and their fate is primarily determined by drift. in rare cases, a spontaneous mutation will confer a selective advantage or disadvantage. if by chance this mutation is not lost by drift, in consequent generations the mutation will spread throughout the population, and variation at linked sites will either accumulate or be purged. the combination of neutral mutations fixed through drift and the mutations that are accumulated or purged determine the genomic landscape of the organism. by surveying the patterns of natural variation between the closely related caenorhabditis strains cb and cb it is possible to investigate the evolutionary processes of drift and selection occurring in this lineage. although previous studies provide insights into c. elegans snp patterns and densities in the natural isolates, a large-scale genomic analysis of the c. elegans polymorphic spectrum has not been accomplished until now. other studies of c. elegans have not focused on examining the patterns of variation that we present here, but have instead focused on other important patterns. for example, weber et al. reported the genome sequence of the lsj strain and compared it to the reference wild type n. lsj is a sibling of the reference wild type n the authors indicated that comparing the genomes of n and lsj highlight genetic changes associated with lab domestication and errors in the c. elegans reference genome. they confirmed that lsj and n are derived from the same bristol isolate. in addition, they identified predicted differences between the lsj and n genome sequences. finally, by sequencing two recently isolated wild c. elegans strains, cb and ed, they inferred the ancestral state of the original bristol isolate. all of this makes cb an extremely valuable resource not only for c. elegans gene mapping but also for the study of the distribution and spectrum of caenorhabditis snps. graustein et al. compared the levels of dna sequence polymorphism among three closely related species in the genus caenorhabditis: two self-fertilizing species, c. elegans and c. briggsae, and one cross-fertilizing species, c. remanei. jovelin et al. analyzed nucleotide variation within and between species across two olfactory pathways in caenorhabditis to examine the relationship between evolutionary rate and gene function and their position within the pathway. the authors found that transcription factors specifying chemosensory neuron subtype identity exhibit higher levels of nucleotide divergence and polymorphism than the structural genes in the signaling pathways. they found much higher levels of polymorphism within c. remanei than within the related species c. elegans and c. briggsae. their findings suggest behavioral differences between species in response to olfactory stimuli are likely to involve differences in gene regulation due to the divergence of regulatory genes. we report the entire spectrum of events critical for understanding the fundamental divergence patterns of these natural isolates. in addition, we compare our results to the n ma line results from denver et al. because we wish to compare the differences in mutation patterns when selection is present versus when it is nearly absent.  overview of snp variation we identified a total of putative snps in our analysis of the cb genome compared to n direct re-sequencing of of these putative snps confirmed suggesting our rate of confirmation is 93%. the snps chosen for resequencing were representative of all the snps in this study. the level of snp differences between n and cb is × 10- snp per base with insertion deletion events contributing slightly more of the variation than base substitutions and inversions. this is consistent with the known mutation spectrum described in denver et al. that reports slightly more indels than snps in the c. elegans ma lines. in contrast, a recent genome wide comparison of n and the strain cb revealed a much lower genome wide snp level and those differences were dominated by base substitutions. the difference in the ratio of base substitutions to indels in the two comparisons could be attributed to the differences in sequencing approaches used to assay variation. the cb data was based on longer sanger reads while the cb data was based on short read sequencing. reads with more than one snp are difficult to assemble when using short read technology. consequently, in cb reads with more than differences are incompatible with this analysis. in that scenario, sequential or highly clustered snps may not be detected in the cb dataset. the total number of bases assayed in cb was. the numbers of snps observed in cb was based on the assay of 77% of the c. elegans reference genome. the total number of bases assayed in cb was while the number of snps observed in cb was based on the assay of 77% of the c. elegans reference genome. even though % is a small portion of the c. elegans reference genome, we assume that the sequences used to obtain the polymorphisms are randomly selected from the entire genome. it is therefore reasonable to compare the results of cb to the results of cb the base substitution/indel ratio is: for cb and: for cb. two observations suggest that the differences in the observed base substitution/indel ratios in cb vs cb do not stem from read length differences in the two assays. first, clusters of snps do not contribute to a large fraction of the total variation. therefore, sequential indels which may be undetectable using short-read technology do not greatly influence the base substitution/indel ratio. second, when we limit the comparisons to single base substitutions and indels which are fully assayable using short read technology in cb we find that the discrepancy between these two natural isolates remains. based on the above observations, the cb snps have a higher proportion of indels than the cb snps. these observations are consistent with those reported by denver et al. in which c. elegans ma lines showed a base substitution/indel ratio of:. either the cb genome is getting smaller or the n genome is getting larger among the snps predicted in the comparison of cb and n we observed a strong and significant bias for deletions over insertions. this bias was reflected in single event indels as well as larger indels. although the direction in which the indel occurred is unknown, this pattern differs from the cb ratio, suggesting two possibilities: the cb genome is becoming smaller or the n genome is getting larger because it fails to purge insertions. this result differs from the baseline mutation spectrum in c. elegans ma lines in denver et al.. they observed a high frequency of indels where the mutations appeared to be biased toward insertions. there are two possible explanations for this observation: there is a change in the mutation spectrum between n and cb or selection constraints between n and cb have changed. sequential vs single snps sequential snps can arise from single mutational events involving multiple base pairs or as a consequence of individual events at adjacent positions. there is a larger proportion of sequential snps in cb compared to cb. the high proportions of observed sequential base substitutions and indels in cb are not consistent with expectations based on a hypothesis of independent adjacent polymorphisms. this suggests a large fraction of the adjacent snps result from mutations involving multiple adjacent base pairs or that the adjacent snps are not independent. the distribution of the sizes for sequential snps, however, suggests that the occurrence of sequential snps is negatively correlated with the number of bases involved. due to the limitations of short read sequence alignment, the analysis between n and cb is limited to sequential base substitutions, indels, and putative inversions with a length of bp. in particular, inversions are a special subclass of sequential changes that occur through a single event. we identified all size classes of inversions in cb and in cb. these account for only a small fraction of all snps and do not affect the overall conclusions of our study. we note that in cb the inversions may be downwardly biased due to the cb sequencing method. shifting patterns of base substitution the base substitution pattern when comparing n against the cb isolate reveals a c. elegans transition over transversion bias of:. however, when comparing n against cb there is no transition over transversion bias. furthermore, in cb there is a significant bias in the direction of base substitution. when comparing n against cb the frequency of a or t bases in n that are g or c bases in cb greatly outnumber the opposite frequencies for transitions as well as transversions. at equilibrium, the numbers of g:c to a:t differences between two genomes should be equal to the number of a:t to g:c differences. the shift in base substitution results in a significant detectable shift in base composition between n and cb. in particular, there is a significant difference between the following rates in the n2/cb comparison: g →a lower than a→g; c →t lower than t →c; c →a lower than a→c; g→t lower than t→g. none of these differences were observed in the n2/cb comparison. *number the results shown here differ from the expectation of a significant mutation bias from g:c to a:t and g:c to t:a based on mutation accumulation lines. because the base substitution pattern is consistent with the n2/cb comparison, but different in cb the change is likely either a change in the mutational mechanisms or shifting selective forces. cb is a prime example of base compositional differences that can occur within a species. cb4856/cb snp rate of chromosomes both cb and cb exhibit significant differences in snp rates among the chromosomes. in particular, chromosomes i to x do not have a uniform snp rate in either cb or cb. in addition, the snp rate is higher in the autosomes than in chromosome x in cb. in cb chromosome iii has the smallest snp rate while chromosome v has the largest snp rate. although previous observations of this disparity have been attributed to the mutagenic effect of recombination, the random distribution of mutations across all chromosomes in mutation accumulation lines makes it more likely that selective sweeps are purging polymorphisms from different regions of the genome. a comparison of snp rates between chromosomes in cb is not appropriate because of the stochastic nature of the snps found in the cb4858-n comparison. there are large stretches of dna that are shared by n and cb cb4856/cb arms versus core snp rate we compared the snp rate in the arms vs the cores in different chromosomes in cb and cb using chi-squared tests. in both strains, 1) the snp rate in the arms is significantly higher than in the core in all of the chromosomes; 2) the rates of snps in the arms are different across the chromosomes; 3) the rates of snps in the core are different across the chromosomes. *based on chi-squared tests there are many possible reasons why the snp rates differ between the arms and the core in both cb and cb the first possibility is that there are more genes in the core. since purifying selection removes mutations from coding regions, if the gene density is higher in the core, fewer snps will occur there. the second possibility is that there may be more recombination away from the core. if recombination is mutagenic, higher rates of recombination will increase snp density. on the other hand, recombination interrupts linkage disequilibrium reducing the efficacy of selection. as stated in cutter et al., chromosome arms have moderate to high rates of crossover recombination and low gene density and cores have strikingly little recombination despite comprising nearly half of each autosome. rockman and kruglyak and barnes et al. provided evidence of these observations in their studies. finally, there may be an inherent difference in mutation rates in the arms and the core. as observed in this paper and by stein et al., there is a difference in the rate of snps in the arms vs the core. observations by denver et al have suggested that the baseline rate of mutation in ma lines is not increased in the arms of autosomes. this suggests that selection, not mutation, plays a larger role in the biased physical distribution of polymorphisms. we then conclude that the reason the snp rate differs in the arms and core regions is primarily due to natural selection. recombination and snp rate in both cb and cb, the distribution of snp variation within the c. elegans chromosomes shows there is a significant relationship between snp frequency and recombination rate on the autosomes. however, there is no significant linear relationship between snp frequency and recombination rate on the x chromosome in either isolate. *p-values found doing spearman's rho test for correlation the discrepancy with the x chromosome can be explained by the x chromosomes' many functional differences from the autosomes. recombination is almost non-existent in the right terminal arm of the x chromosome. furthermore, sex is determined by x chromosome dosage and this dosage is compensated in hermaphrodites. observations of correlation between snp rate and recombination on the autosomes in both cb and cb have been proposed to either reflect an inherent mutagenic effect of recombination or conditions where selection results in the loss or fixation of new mutations along with all linked polymorphisms. the data in this study shows us that in the autosomes, the observed levels of polymorphisms are positively correlated with levels of recombination in both isolates. using the chromosomal domains in rockman and kruglyak, we found that 1) in the arms, the recombination rate is negatively correlated with chromosome size, although the correlation was not significant; 2) the two tips of all autosomes and the x chromosome have an effectively zero recombination; 3) the c. elegans autosomes and the x chromosome have nearly constant recombination rates within each chromosomal domain. findings 2) and 3) agree with the findings in rockman and kruglyak but finding 1) is different from the finding in rockman and kruglyak. cb hotspot analysis the level of snp variation across the genome in any comparison reflects two possible mechanisms. a high frequency of local polymorphisms can be a consequence of high localized mutation rate or a mutational hotspot. alternatively, localized patterns of divergence can reflect the age of the allele and historic patterns of recombination. as a consequence of recombination and interbreeding of the ancestors that gave rise to these isolates, each allele can have a unique history of coalescence. to further investigate the pattern of variation across the c. elegans genome and specifically identify regions of significantly high localized polymorphisms, we identified clusters of snps with a significant number of polymorphisms. in general, these hotspots are dna segments that have an unusual number of polymorphisms considering the length of the dna. a more detailed description of the hotspots is given in the methods section. these hotspots of polymorphisms may be mutational hotspots or may indicate old alleles. we excluded the cb datasets due to the inherent inability of short reads to represent loci with highly clustered polymorphisms. the summary statistics of the hotspots per chromosome are shown in table the optimal window size for the initial cluster analysis was constrained by the average single read length in the cb comparisons. statistically significant hotspot clusters ranged from bp to bp in length. there were clusters when comparing n against cb and the average number of events per hotspot was. in general, the proportion of base substitutions was the largest in chromosome v, the proportion of nonsequential indels was the largest in chromosome x, and the proportion of sequential indels was the largest in chromosome iv. there was a significant positive relationship between hotspot length and the number of events in the hotspot. chromosome v has the largest number of hotspots while chromosome x had the lowest number. we did a linear regression with dummy variables to test if the location of snps was a significant predictor of the number of hotspots. we saw no significant hotspot distributional biases with respect to chromosomal arm/core boundaries. the sizes of the hotspots vary per chromosome. we categorized the hotspots into small and large hotspots based on a cutoff of bp in length. the number of small vs large hotspots is in table the hotspots with lengths less than base pairs have on average of events and 15% of the events are base substitutions while 79% of the events are nonsequential indels. on the other hand, the hotspots with lengths more than base pairs have on average events and 58% of the events are base substitutions while 30% of the events are nonsequential indels. in some taxa, it has been shown that the rate of mutation at specific loci is strongly correlated with gc content. to test this possibility, we assayed the gc content in all clusters and compared the %gc content in the clusters to the n genome as a whole. there is sufficient evidence to indicate the %gc content in the statistical hotspots differs from the %gc content in the n genome as a whole. the %gc content in different statistical hotspots is displayed in additional file some of the hotspots have a higher %gc content than n and some of them have a lower %gc content than n we also compared these hotspots of polymorphism to mutations identified in a genome wide analysis of n mutation accumulation lines. denver et al found a random distribution of mutations in their ma lines. the mutations identified among ten n derived ma lines did not fall within any of the hotspots identified here. finally, in a comparison of cb and cb there were snps that were shared between cb and cb, which is larger than the expected number of snps shared between cb and cb. we hypothesize that the hotspots of polymorphisms in this study are alleles that are shared between cb and cb but not by n and are not mutational hotspots because the %gc content in the hotspots differs from the %gc content in n and because the snps found in cb vs n were not in the ma lines. distribution of snps by function to investigate the distribution of snps across major functional partitions in the c. elegans genome, we identified the putative functional role of each snp position based on the annotation of the n reference genome. the general variation in both cb and cb follows expected patterns. the functional categories of snps are ordered by intergenic>introns>utr>exons in cb and intergenic>utr>exons>introns. the functional categories of snps are ordered as expected for cb but for cb where the frequency of snps in exons is higher than in introns. cb may have relaxed selection constraints on coding mutations.  insert the numbers represent isolate × 10- snps per base. * *for ease in comparison of frequencies across categories and strains, we divided the number of polymorphisms by the number of analyzed sites and multiplied that number by for example, the proportion of insertion polymorphisms in cb is 9942*10- the numbers represent isolate × 10- snps per base.* *for ease in comparison of frequencies across categories and strains, we divided the number of polymorphisms by the number of analyzed sites and multiplied that number by cb4856/cb neutrality the ka/ks ratio is the rate of nonsynonymous substitutions to the rate of synonymous substitutions, which can be used as an indicator of selective pressure acting on a protein-coding gene. a value of ka/ks> indicates positive selective pressure whereas a value of ka/ks = indicates neutral evolution. finally, a value of ka/ks < indicates pressures to conserve amino acid sequence. we test for neutrality by testing the null hypothesis of ka/ks = in both cb and cb. analysis of genome-wide snp patterns reveal a ka/ks of less than for all chromosomes in both cb and cb in table adjusting for multiple comparisons. this result is consistent with the role that purifying selection has on snps in most protein coding regions. *all ka/ks values in both isolates are significantly smaller than 1 in cb the autosomes have ka/ks values that are not significantly different from x in either direction. in addition, when considering all of the chromosomes, the ka/ks values are not significantly different in the different chromosomes in either direction. however, the snp rate is lower in chromosome x compared to the autosomes in cb. differences in the length or the number of cell divisions in the male germline compared to the hermaphrodites in cb may explain the lower snp rate in the x chromosome. when the x chromosome is passed through the male germline, it is in a hemizygotic state and exposed to increased purifying selection. in addition, the mitotic cell cycle length has been shown to be shorter in males than hermaphrodite germ cells. taken together, both of these factors can contribute to a lower snp rate in the x chromosome than in the autosomes in cb these observations are dependent upon how often males are involved in reproduction from generation to generation. while direct observations of c. elegans populations show a low frequency of males and low levels of heterozygosity, it is possible that some lineages of caenorhabditis have historically undergone much more outbreeding involving males. in cb the autosomes have ka/ks values that are significantly smaller than x on the average. unlike cb the different chromosomes have significantly different ka/ks values. it is not appropriate to consider the differences in snp rates between chromosomes in cb due to the long stretches of dna shared by n and cb because the ka/ks ratio in the x chromosome is higher than the autosomes on average in cb purifying selection appears to be reduced on the x chromosome in this strain compared to cb the relative difference in ka/ks ratios in cb could reflect differences in the life history of cb we suggest that males could contribute to a smaller degree in natural populations of cb than in cb populations, resulting in a lower rate of protein sequence evolution for x-linked mutations in the hawaiian isolate cb previous studies have presented varying levels of the genetic contribution of males in different natural isolates of caenorhabditis elegans.  this study provides a large scale genomic portrait of the c. elegans polymorphisms in cb and cb the patterns observed here give us insight into the evolutionary spectrum of the caenorhabditis lineage. there is a clear bias of deletions over insertions in cb suggesting two possibilities. the first one is that the cb genome is becoming smaller. other studies have argued that possible reasons that genomes could become smaller in general are gene loss, natural selection, or the occurrence of long terminal repeat sequences of transposable elements. the second possibility is that the n genome is getting larger because it fails to purge insertions. interestingly, direct sequencing of c. elegans insertion deletion rates in mutation accumulation lines observe a bias towards insertions. taken together, we conclude that cb is evolving under different mutation or selection pressures than n or cb our results show that in both cb and cb snp rates are higher in the chromosome arms than in the chromosome cores in agreement with previous studies. direct genome sequencing of c. elegans mutation accumulation lines show that the mutation rate is not higher in arm regions compared to the core regions. we hypothesize snp rates differ in the core regions of n cb and cb in our study due to higher levels of purifying selection in the core regions. we found several statistically significant hotspots of polymorphisms in our cb4856-n comparison. some of the cb hotspots contained significantly higher levels of g/c when compared to the genome average, consistent with observations of higher rates of deamination at methylated cytosines. none of these hotspots coincided with c. elegans mutations identified in denver et al., and are likely to be older alleles shared between cb and cb but not n the ka/ks ratios in both cb and cb are consistent with the effect of purifying selection on snps in most protein coding regions. the differences in ka/ks in the autosomes vs the x chromosome in cb suggest selection may be more efficient for x-linked genes. in addition, these differences may be attributed to male-driven evolution of the x chromosomes. we hypothesize that natural populations of cb strains have fewer males than cb populations, resulting in a lower rate of protein sequence evolution for x-linked mutations. the finding in cb of a significant difference in selection on the x chromosome compared to the autosomes is consistent with a biological role for males in the lineage giving rise to cb consistent with previous published results. 
we hypothesize the hotspots found represent alleles that are shared between cb and cb but not n because the ka/ks ratio in the x chromosome is higher than the autosomes on average in cb purifying selection is reduced on the x chromosome. the frequency of a or t bases in n that are g or c bases in cb outnumber the opposite frequencies for transitions as well as transversions. the base substitution pattern when comparing n against cb reveals a transition over transversion bias that is not present in cb in cb there is a significant bias in the direction of base substitution. the clear bias of deletions over insertions in cb suggests that either the cb genome is becoming smaller or the n genome is getting larger. genome wide analysis of variation within a species can reveal the evolution of fundamental biological processes such as mutation, recombination, and natural selection. in both cb and cb based on a measure of the strength of selection, all the chromosomes are under negative selection and in cb there is no difference in the strength of natural selection in either the autosomes versus x or between any of the chromosomes. similarly, we observed a strong bias for deletions over insertions in cb that is not present in cb in both cb and cb there is a significant correlation between snp rate and recombination rate on the autosomes but not on the x chromosome. by contrast, in cb ka/ks values are smaller in the autosomes than in the x chromosome.
 genome wide analysis of variation within a species can reveal the evolution of fundamental biological processes such as mutation, recombination, and natural selection. we compare genome wide sequence differences between two independent isolates of the nematode caenorhabditis elegans and the reference genome.  the base substitution pattern when comparing n against cb reveals a transition over transversion bias that is not present in cb in cb there is a significant bias in the direction of base substitution. the frequency of a or t bases in n that are g or c bases in cb outnumber the opposite frequencies for transitions as well as transversions. these differences were not observed in the n2/cb comparison. similarly, we observed a strong bias for deletions over insertions in cb that is not present in cb in both cb and cb there is a significant correlation between snp rate and recombination rate on the autosomes but not on the x chromosome. furthermore, we identified numerous significant hotspots of variation in the cb4856-n comparison. in both cb and cb based on a measure of the strength of selection, all the chromosomes are under negative selection and in cb there is no difference in the strength of natural selection in either the autosomes versus x or between any of the chromosomes. by contrast, in cb ka/ks values are smaller in the autosomes than in the x chromosome. in addition, in cb ka/ks values differ between chromosomes.  the clear bias of deletions over insertions in cb suggests that either the cb genome is becoming smaller or the n genome is getting larger. we hypothesize the hotspots found represent alleles that are shared between cb and cb but not n because the ka/ks ratio in the x chromosome is higher than the autosomes on average in cb purifying selection is reduced on the x chromosome. 