we applied the blastp program ( 5 ) to identify , for each predicted protein sequence in the h37rv genome ( genbank accession no .
ae000516 ) . following genbank annotations , we compared 3,972 predicted proteins in h37rv with 4,187 predicted proteins in cdc1551 .
we used a strict search criterion ( e = 10 - 50 ) to identify truly orthologous gene pairs .
we aligned ( 6 ) the putative orthologous pairs of amino acid sequences ( n = 3,428 ) , then imposed this alignment on the dna sequences .
visual inspection of amino acid alignments showed that certain alignments , usually near the n - terminus or c - terminus , had regions of very low sequence identity .
examination of the dna sequences of the corresponding genes showed that these regions of low identity were typically caused by a frameshift in one of the two genomes relative to the other .
whether these frameshifts are biologically real or result from sequencing error was uncertain ; therefore , we eliminated 119 such gene pairs from our data set .
for the remaining gene pairs ( n = 3,309 ) , we computed the proportion of synonymous substitutions per synonymous site ( ps ) and the proportion of nonsynonymous substitutions per site ( pn ) by using nei and gojobori s method ( 7 ) . because values of ps and pn were very low in most cases , we did not correct for multiple hits . because ps values appeared to fall into two groups ( see results ) , we used a simple probabilistic model to separate these two sets of gene pairs .
we assumed that the probability of synonymous substitution followed two separate binomial distributions , designated models a and b , with probabilities of  success 
( i.e. , of a synonymous difference ) designated pa and pb , respectively . using the bayes equation , for each gene pair with a given ps value ,
we computed the probability that model a applies , given the observed ps : p(a|ps ) = ( psa ) fa /[(psa ) fa + ( psb ) fb ] , where fa is the frequency of cases to which model a applies , fb the frequency of cases to which model b applies , psa is the binomial probability of obtaining the observed ps , given the number of synonymous sites in the gene and a probability of a synonymous difference equal to pa ; and psb is the binomial probability of obtaining the observed ps , given the number of synonymous sites in the gene and a probability of a synonymous difference equal to pb .
of 3,309 pairs of putatively homologous protein - coding genes in the h37rv and cdc1551 genomes of m. tuberculosis , 2,662 ( 80.5% ) showed no synonymous or nonsynonymous nucleotide differences between the two genomes , and 3,010 ( 91.0% ) showed no synonymous differences between the two genomes .
however , in a small number of gene pairs , the proportion of synonymous differences per synonymous site ( ps ) was surprisingly high . in 13 ( 0.4% )
gene pairs , ps was > 0.01 , and in 3 gene pairs ps exceeded 4% .
these extreme ps values seen in a small number of gene pairs are much higher than generally observed between alleles at neutrally evolving loci in eukaryotes ( 8) .
thus , the comparison of protein - coding genes between the two m. tuberculosis genomes suggested the existence of two distinct groups of gene pairs : a large group having few or no synonymous differences and a much smaller group with a substantial degree of synonymous divergence .
we used a simple probabilistic model ( see methods ) to separate these two sets of gene pairs , designated group a and group b , respectively ( figure ) .
the application of this method showed 11 loci with unusually high ps values and probabilities of assignment to group a of < 50% ( figure ) .
a plot of p(a|ps ) , the probability of assignment of a locus to group a given the observed ps value , as a function of ps at 3,309 loci compared between the h37rv and cdc1551 genotypes of mycobacterium tuberculosis .
the plot shows the bimodal nature of the distribution of ps values , with overall higher values of ps at the 11 loci having p(a|ps ) > 50% .
we assumed that group a members are truly orthologous gene pairs that diverged at the time of the common ancestor of the h37rv and cdc1551 genomes .
group a included 3,298 pairs , with mean ps for all genes of 0.000328  0.000022 standard error .
when ps was estimated for the 3,298 genes concatenated together ( a total of 934,413 synonymous sites ) , an estimate of ps = 0.000348  0.00019 was obtained .
the range of ps values in group a was between zero and 0.012 ; a total of 288 loci in group a had ps values other than zero .
these results show a substantial level of nucleotide diversity , approximately half the level of nucleotide diversity in humans ( 9 ) .
rates of nucleotide substitution per unit time are difficult to estimate in bacteria given the lack of calibration from the fossil record ( 10 ) . to obtain an estimate of the rate of synonymous nucleotide substitution ,
we used published data on comparisons of escherichia coli and salmonella typhimurium ( 11,12 ) , which are believed to have diverged approximately 100 million years ago ( 13,14 ) ( table 1 ) .
this procedure yielded estimates for the last common ancestor of h37rv and cdc1551 in the range of 34,00038,000 years ( table 1 ) .
these estimates are approximately twice previous estimates of the age of the common ancestor of worldwide m. tuberculosis ( 2,3 ) . to obtain the observed mean ps value between h37rv and cdc1551 within 15,00020,000 years would require a rate of synonymous substitution approximately twice that observed in enterobacteria . based on synonymous substitutions between escherichia coli and salmonella typhimurium , assumed to have diverged 100 million years ago ( 13,14 ) . 
estimates are shown  standard error , based on standard error of mean ps . 
group b consisted of 11 gene pairs with mean ps of 0.0286  0.0050 ( table 2 ) . paired sample t - test of the hypothesis that ps = pn , p<0.01 .
the quantities ps and pn are the proportion of nucleotide difference per synonymous site and per nonsynonymous site , respectively . in enterobacteria , a negative correlation exists between observed proportions of synonymous difference and codon bias ( 11 ) . in the case of mycobacterium ,
codon bias results mainly from the very high third position g+c content of most genes ( 15 ) . in our data , however , we observed no correlation between ps and proportion g+c at third codon positions ( r = -0.010 ; not significant ) .
a number of additional possibilities may explain the occurrence of gene pairs with higher than expected ps values : 1 ) balanced polymorphism .
selectively maintained polymorphisms are expected to be much older than neutral polymorphisms and may even predate speciation events ( 16 ) . in the case of haploid organisms such as bacteria
, balancing selection would take the form of frequency - dependent selection rather than overdominant selection .
2 ) differential deletion . in a multi - gene family , if one member of an orthologous pair of genes were deleted in one genotype , the gene pairs would involve paralogous , not orthologous comparisons .
3 ) horizontal gene transfer . a gene obtained by one of the two genotypes from another bacterial species
one indication of a balanced polymorphism is a higher rate of nonsynonymous than synonymous substitution ( 8) . there was no strong evidence of such selection in the present case ; ps was greater than pn at 10 of the 11 loci , and pn exceeded ps only slightly at one locus ( table 2 ) .
in addition , we compared ps and pn in sliding windows of 30 codons along the length of these genes .
no regions were observed in which pn was greater than ps ( data not shown ) .
thus , there was no evidence of positive selection acting on specific regions of these genes .
on the other hand , differential deletion can probably explain some cases , most notably members of the pe multi - gene family ( 11 ) ( table 2 ) .
the remaining gene pairs are possibly cases of horizontal gene transfer ( table 2 ) , for which there is some recent evidence in m. tuberculosis ( 17 ) .
our results did not support the hypothesis that the common ancestor of m. tuberculosis was relatively recent ( 2 ) .
rather , the pattern of nucleotide substitution at synonymous sites suggested a divergence time for the two available genotypes of this species approximately 35,000 years ago .
since h37rv and cdc1551 represent two genotypes sampled from within the species , they are probably not the most divergent genotypes possible .
thus , the last common ancestor of m. tuberculosis likely occurred considerably earlier than 35,000 years ago .
while the difference between an estimate of 15,00020,000 years and one of 35,000 years is not large on an evolutionary time scale , such a difference is substantial on the scale of human history .
for example , the existence of two genotypes in the current population of m. tuberculosis with a common ancestor at 35,000 years is evidence against the hypothesis that m. tuberculosis arose , presumably from m. bovis , at the time of human domestication of cattle ( 18 ) .
our result is thus consistent with phylogenetic analyses based on insertion - deletion events , which suggest that the m. tuberculosis lineage was a human pathogen well before the origin of m. bovis ( 19 ) .
thus , along with recent evidence of an ancient origin and extensive polymorphism in the malaria parasite plasmodium falciparum ( 20,21 ) , our study provides evidence against the long - held view that virulent pathogens are invariably evolutionarily recent ( 22 ) .
our estimate is conservative because the rate of synonymous substitution may actually be lower in mycobacterium than in enterobacteriaceae , given the highly skewed g+c content in the former .
furthermore , our estimate of the mean proportion of synonymous difference was conservative because we excluded 119 loci with potential frameshifts between the two genotypes as well as a set of 12 loci with unusually high ps values .
in addition to the 12 loci assigned to our group b , certain other loci might also have originated from horizontal gene transfer . however , even if horizontal gene transfer has occurred at other loci besides those in group b , eliminating further loci with relatively high ps values from group a will not affect the results greatly .
for example , if we eliminate the 10 loci with highest ps values from group a , mean ps will be reduced only to 0.000299 , and the estimated age of the common ancestor will be barely affected .
the degree of polymorphism observed in this study is unlikely to have been substantially influenced by sequencing errors .
the error rate for finished sequences from the institute for genomic research ( where cdc1551 was sequenced ) has been independently estimated at < 1 in 88,000 bases ( 23 ) . assuming a similar error rate for both 4.4 mega - bp m. tuberculosis genomes
, we would expect to see approximately 100 differences between them due to sequencing errors .
approximately 21 such differences would be expected in the 938,778 synonymous sites in group a and group b genes .
in fact , 411 synonymous differences were observed at these sites ; thus , even if present , sequencing errors are likely to have made up only a small fraction ( approximately 5% ) of the total synonymous polymorphism . at such a rate ,
sequencing errors would have little effect on our estimates of nucleotide diversity at synonymous sites or the age of the common ancestor of the two genomes .
in addition , the hypothesis that the single nucleotide polymorphisms ( snps ) observed between these genotypes are real received strong support from a recent study that observed a number of the same snps in clinical isolates ( 24 ) . moreover , since sequencing errors are expected to occur at random with respect to the reading frame of coding sequences , the fact that mean ps exceeded mean pn in both group a and group b was strong evidence against the hypothesis that a substantial proportion of the observed polymorphism was due to sequencing error .
simple considerations of probability can explain why earlier studies produced relatively low estimates of this species age .
if we assume that the per - site probability of a synonymous difference between two m. tuberculosis genomes is equal to the mean ps observed between h37rv and cdc1551 ( 0.000328 ) , then the probability is approximately 95% that no synonymous differences will be seen in a gene with 150 synonymous sites .
the probability that no synonymous differences will be seen in 10 such loci chosen at random is approximately 60% , and the probability that no synonymous differences will be observed at 20 such loci is approximately 37% . on the other hand , the probability that no synonymous differences will be seen at 100
these calculations emphasize the need to examine a very large number of nucleotide sites to obtain a reliable estimate of nucleotide diversity and thus of the age of the most recent common ancestor in cases where the frequency of substitution is less than one in a thousand .
even when the frequency of substitution is between one in a thousand and one in a hundred , substantial stochastic error is possible if the number of loci examined is small .
thus , any study that estimates population parameters from nucleotide sequence data needs to survey a substantial number of loci .
these considerations are particularly important in the case of pathogenic microorganisms , where a number of factors ( including both natural selection and horizontal gene transfer ) may lead to substantial differences among loci with respect to the level of nucleotide diversity .
comparison of two complete genomes of m. tuberculosis showed a greater extent of sequence polymorphism than would be expected on the basis of previous studies , in turn suggesting that analysis of additional genomes will likely show further polymorphism .
polymorphism in any species of pathogen may complicate therapeutic strategies because it implies the existence of variation on which selection can act , including selection imposed by human vaccines and pharmacologic agents ( 20 ) .
on the other hand , known polymorphisms may prove useful to investigators in reconstructing the evolutionary relationships among clinical isolates and in providing markers for understanding the genetic basis of complex phenotypic traits .
   comparison of the pattern of synonymous nucleotide substitution between two complete genomes of mycobacterium tuberculosis at 3,298 putatively orthologous loci showed a mean percent difference per synonymous site of 0.000328  0.000022 . 
 although 80.5% of loci showed no synonymous or nonsynonymous nucleotide differences , the level of polymorphism observed at other loci was greater than suggested by previous studies of a small number of loci . 
 this level of nucleotide difference leads to the conservative estimate that the common ancestor of these two genotypes occurred approximately 35,000 ago , which is twice as high as some recent estimates of the time of origin of this species . 
 our results suggest that a large number of loci should be examined for an accurate assessment of the level of nucleotide diversity in natural populations of pathogenic microorganisms . 