BACKGROUND
the exponentially accumulating sequence data in genbank have allowed for the publication of hundreds of nucleotide substitution rate estimates for the rapidly evolving rna viruses.
within a given viral species, published tip-calibrated bayesian substitution rate estimates are often highly consistent .
however, for some viruses, such as rabbit hemorrhagic disease virus , there is a significant discrepancy among published substitution rates  <cit> .
rhdv is a positive-sense, single-stranded rna virus of the family caliciviridae  and the causal agent of the highly lethal rabbit hemorrhagic disease .
since the emergence of rhd in china in  <dig>  <cit> , rhdv has spread worldwide and continues to be a growing concern for rabbit meat and fur industries  <cit> , as well as a threat to european ecosystems  <cit> .
heightened surveillance for rhdv has resulted in the identification of rabbit calicivirus , a nonpathogenic relative of rhdv, in australia, the united states, and europe  <cit> .
rhdv has mean published substitution rate estimates for the outer capsid  gene ranging from  <dig> x10- <dig> nucleotide substitutions per site per year   <cit>  to  <dig> x10-3 ns/s/y  <cit> , with non-overlapping 95% highest posterior density  intervals for these lowest and highest estimates.
not surprisingly, there is also significant variation among the time to most recent common ancestor  estimates for rhdv, with estimated mean coalescent ranging from  <dig>  <cit>  to  <dig>  <cit> .
as rhd was first described in  <dig>  <cit> , the oldest tmrca implies that the coalescent of virulent rhdv antedates the emergence of rhd by almost seven decades.
in the absence of an intermediate reservoir host, it is uncommon for the emergence of an acute, virulent virus to be so extensively decoupled from the appearance of its associated disease  <cit> .
as a result, there has been significant debate over the timing, location, and mechanisms of rhdv ’s emergence  <cit> .
it is possible that the discrepancy among evolutionary rate estimates for rhdv is partially attributable to variation among datasets  and/or subtle methodological variations  <cit> .
as this range of evolutionary rates is atypical for a gene of a single viral species, a systematic investigation was undertaken to explain variation among published nucleotide substitution rate and tmrca estimates for rhdv.
a combination of jackknifing controls was used to produce robust rate estimates for the vp <dig> gene and the first estimated substitution rate for rhdv ’s rna-dependent rna polymerase  gene.
these controls reveal that using one misdated taxon significantly slows the estimated rates, unnecessarily lengthening rhdv ’s tmrca.
we demonstrate the fragility of tip-calibrated evolutionary analyses and propose jackknife control beast runs as a way to identify potential misdated taxa.
RESULTS
complete dataset analyses
the best-fitting nucleotide substitution model for the complete vp <dig> datasets, regardless of whether or not rcv isolates were included, was gtr + i + Γ .
for the rdrp, the trnef + Γ model  was selected for the rhdv dataset, while the sym + i + Γ model  was selected for the rhdv + rcv dataset.
the uncorrelated lognormal clock model was determined to be a significantly better fit than the strict clock model for each of the datasets .
additionally, the 95% hpds of the standard deviation for the uncorrelated lognormal relaxed molecular clock rate estimates excluded zero for all of the demographic models, further rejecting a strict molecular clock for these alignments.
the bayesian skyline demographic model was best-fitting for each of the datasets, though it was not significantly better than the constant or exponentially growing population models.
however, there was no significant variation in nucleotide substitution rate or tmrca estimates among demographic models in any of the full datasets.
there was no significant substitution rate variation between rhdv and rhdv + rcv datasets of either gene , and the inclusion of the divergent rcv isolates had no significant effects on age estimates for the virulent rhdv clade or its individual lineages .
the subsequent analyses were performed only on the rhdv datasets.
*mean substitution rate, tmrca, and corresponding year estimates are shown with upper and lower 95% hpd bounds.
the maximum clade credibility  and maximum likelihood  trees for each complete gene dataset were highly congruent .
further, the trees generated for the two different genes were also congruent, with the exception of one taxon, genbank accession number ef <dig>  which switched from lineage b in the vp <dig> tree to lineage d in the rdrp tree .
while it has been previously suggested that this taxon has undergone a crossover event at the junction between the rdrp and vp <dig> genes  <cit> , it was not detected as a potential recombinant in these single gene analyses.
despite the congruence of tree topology, however, there was a significant difference  between the substitution rate estimates from the complete vp <dig> and rdrp datasets , with the vp <dig> rate estimate paralleling the lower vp <dig> rates from the literature  <cit> , and the rdrp rate paralleling the higher published vp <dig> rate  <cit> .
to assess the extent of temporal structure in the mcc trees, tip-date randomized controls were run.
for both genes, the upper 95% hpd interval from the tip-date randomized datasets occasionally overlapped the lower 95% interval of the real dataset substitution rates .
a post-hoc permutation test verified that estimates from the tip-date randomized data sets were different from the substitution rates estimated from the actual dataset .
root-to-tip regressions showed moderate correlations .
there is statistical support for temporal structure in each gene’s dataset.
control analyses
the jackknifed  <dig> taxa control analysis for the vp <dig> gene resulted in two distinct groups , roughly corresponding to the two divisions of rhdv rate and tmrca estimates in literature.
by comparing the compositions of the two groups, it was found there was only one taxon  that was present in every dataset from one group and absent from every dataset in the other group.
the statistical significance of grouping datasets by the presence or absence of ay <dig> is shown in figure  <dig>  in contrast to the vp <dig> dataset, the jackknifed  <dig> taxa control analysis for the rdrp dataset, which did not contain ay <dig>  yielded one continuous group of substitution rate estimates .
the n- <dig> jackknife control analysis for the vp <dig> gene further implicated ay <dig> as having a significant effect on the estimated substitution rate.
only the removal of ay <dig> resulted in a substitution rate estimate that was significantly higher than the other n- <dig> jackknife datasets  and the complete vp <dig> dataset .
removal of ay <dig> also resulted in a tmrca estimate that was substantially more recent than that from the complete dataset .
for the rdrp, the substitution rates estimated from the  <dig> n- <dig> jackknife datasets were nearly identical to each other and to the complete dataset .
ay <dig>  more formally “nj/china/ <dig> ” has been used in previous analyses with  <dig> as the year of isolation  <cit> .
by the leaf-dating method, it was estimated that a more appropriate year would have been  <dig> .
a root-to-tip regression of the vp <dig> ml tree, including ay <dig>  revealed that both ay <dig> and fr <dig> were potential outliers .
running a fresh beast analysis for the complete vp <dig> dataset without ay <dig> revealed that there was no overlap between the hpds of these substitution rate estimates and those of the dataset with ay <dig>  regardless of the demographic priors used.
by comparing the estimated years of emergence of virulent rhdv and its individual lineages from analyses with and without ay <dig> , it was revealed that while the inclusion of this taxon inflated tmrca estimates for all lineages, the most conspicuous effect was on the lineage in which it grouped .
removal of fr <dig>  however, had little effect on the estimated vp <dig> substitution rate and coalescent .
removal of ay <dig> from the complete vp <dig> dataset did not change the mcc or ml tree topology .
however, the tip-date randomization analysis performed on the vp <dig> dataset without ay <dig> revealed stronger evidence of a temporal signal .
similarly, excluding ay <dig> from a root-to-tip regression improved the correlation between genetic divergence and time .
discussion and
CONCLUSIONS
the notion that inaccurate specification of dates used for molecular rate calibration could produce misleading results is not a novel one  <cit> .
in the context of rhdv in particular, seven partial vp <dig> sequences were identified as misdated modern contaminants by maximum likelihood analysis  <cit> , but were nonetheless included in one beast analysis  <cit> , resulting in the slowest substitution rate published for rhdv.
it has also been suspected for some time that certain taxa could have a strong influence over inferred phylogenies, and a number of methods have been developed to identify weak clades in phylogenetic trees  <cit>  or the highly influential taxa responsible for weakening phylogenetic relationships  <cit> .
however, there is currently no direct method for identifying the presence of a taxon, such as ay <dig>  that has significant influence over evolutionary rate and tmrca estimates, while not altering phylogenetic relationships.
further, the influence of misdated taxa on estimates of evolutionary parameters has yet to be extensively examined or quantified.
the comprehensive jackknifing controls used here demonstrate that the chinese rhdv isolate nj/china/ <dig>  genbank accession number ay <dig>  was responsible for dragging down the substitution rate estimate for the vp <dig> gene by 65%.
the chinese-language paper that described this taxon  <cit>  contained important details about the isolation and handling of this strain.
the first paragraph of the methods section  revealed that, though it was isolated from nature in  <dig>  it was maintained in the laboratory for vaccine preparation and was likely not sequenced until much later.
the  <dig> submission date of ay <dig> to genbank is concordant with the lower bound of the age estimated by the leaf-dating method, as well as its grouping with isolates from 2006– <dig> in the mcc and ml trees .
"“rhdv nj <dig> strain isolate was discovered and characterized byinstitute of veterinary medicine, jiangsu academy of agricultural sciences , from rabbits raised in an unknown farm in nanjing city, china, in  <dig>  since the discovery, this strain hasbeen maintained in lab rabbits until now.
this strain has been usedto prepare potent rabbit hemorrhagic disease vaccine for years our lab has cloned the gene vp <dig> in e. coli jm <dig> and bl <dig> ”"
the grouping with much more recent isolates could be explained by ay269825’s use in rhdv vaccine production.
whether attenuated or improperly inactivated, the strain could have been released into china, and now this lineage can be isolated from other regions of china and russia .
this is similar to the lab-escape strain that complicated substitution rate estimation for influenza a virus  <cit> .
instead of changing many dates of isolation, however, only ay <dig> would have to be assigned a different date .
previous studies have shown that the long-term rate of viral evolution in the lab can mimic the rate in nature  <cit> , so unlike the 20 years of frozen stasis that the influenza isolate experienced, ay <dig> was changing at a rate similar to its wild relatives.
however, consistent results were obtained by excluding this isolate from analyses altogether.
without ay <dig>  the tmrca for the entire virulent rhdv complex and each of its lineages is substantially lower, resolving the much of the debate over its puzzling evolutionary history.
indeed, without this misdated taxon, the ancestor of the entire complex is estimated to have existed between 1955- <dig> , as few as six years before the  <dig> appearance of rhd.
these results cannot address whether virulence was a shared trait of the most recent common ancestor of rhdv, or if it evolved independently in multiple lineages.
whenever virulence did evolve, it did not have to go undocumented for several decades  <cit> .
while the root-to-tip regression analysis identified ay <dig> as potentially deviating from the molecular clock, fr <dig>  a taxon that did not affect vp60’s evolutionary dynamics , also had a similarly high residual value .
this underscores the problem of using congruence with a strict molecular clock as the sole means of assessing the validity of dates of isolation.
deviation from the root-to-tip regression line is expected for viruses with variable rates of evolution, which would be accurately modeled with a relaxed molecular clock beast analysis  <cit> .
indeed, correlation between genetic distance and time was stronger for the vp <dig> dataset including the misdated ay <dig> taxon than the rdrp dataset, which produced a more trustworthy substitution rate .
the decision to include or exclude taxa based on residuals from a best-fitting line is largely subjective, as there are no guidelines for how common large residuals are in tip-dated viral datasets.
in fact, one of the ay269825-containing rhdv datasets had been subjected to this control prior to rate analysis, and the authors did not reject it as an outlier  <cit> .
finally, root-to-tip regression provides no insight into the magnitude of effect of any taxon on evolutionary estimates.
the jackknife controls proposed here focus on detecting taxa that have had a disproportionate effect on the beast results, and, in the case of rhdv, offered strong quantitative evidence against including ay <dig>
another interesting finding is that without ay <dig>  the estimated substitution rate for the vp <dig> gene is almost identical to that of the rdrp gene, despite the fact that the latter dataset had fewer than half as many taxa .
even while the rdrp dataset did not have as strong a temporal signal , probably due to the lower number of taxa, it still produced a significantly more accurate substitution rate estimate than datasets with two to three times as many taxa that included just one misdated taxon .
further, the estimated substitution rate from our vp <dig> rhdv + rcv dataset was nearly identical to that from another dataset which contained just  <dig> taxa , including one distantly related european brown hare syndrome virus taxon as an outgroup  <cit> .
this pattern of different sized datasets producing very similar substitution rates is not unique to rhdv.
for example, two beast analyses of dengue virus type  <dig> from datasets of  <dig> taxa and  <dig> taxa yielded nearly identical substitution rates  <cit> .
further, in the case of human parechovirus, three beast analyses of three different genomic regions based on datasets with a range of 29– <dig> taxa also produced nearly identical substitution rates  <cit> .
it is evident that assigning years of isolation to taxa should be done with great caution in tip-calibrated rate analyses.
these results support favoring data sets with fewer taxa with verifiable dates of isolation over larger data sets with less quality control: additional good data do not swamp out the effects of one badly dated taxon.
when researchers are including any ambiguously dated taxa, or when they want to be certain about the effects of each taxon on the rate analysis, jackknife controls provide a clear way to see these effects.
as many sequences are added to genbank without easily accessible papers describing in detail the isolation, passaging and sequencing of each isolate, it is necessary to verify if one or more of the sequences is having a disproportionate influence on the results.
we propose n- <dig> jackknifing as one method for researchers using tip-calibrated analyses in beast to ensure that a small number of taxa are not spoiling their estimates.

 the exponentially accumulating sequence data in genbank have allowed for the publication of hundreds of nucleotide substitution rate estimates for the rapidly evolving rna viruses. within a given viral species, published tip-calibrated bayesian substitution rate estimates are often highly consistent. however, for some viruses, such as rabbit hemorrhagic disease virus, there is a significant discrepancy among published substitution rates. rhdv is a positive-sense, single-stranded rna virus of the family caliciviridae and the causal agent of the highly lethal rabbit hemorrhagic disease. since the emergence of rhd in china in, rhdv has spread worldwide and continues to be a growing concern for rabbit meat and fur industries, as well as a threat to european ecosystems. heightened surveillance for rhdv has resulted in the identification of rabbit calicivirus, a nonpathogenic relative of rhdv, in australia, the united states, and europe. rhdv has mean published substitution rate estimates for the outer capsid gene ranging from x10- nucleotide substitutions per site per year to x10-3 ns/s/y, with non-overlapping 95% highest posterior density intervals for these lowest and highest estimates. not surprisingly, there is also significant variation among the time to most recent common ancestor estimates for rhdv, with estimated mean coalescent ranging from to. as rhd was first described in, the oldest tmrca implies that the coalescent of virulent rhdv antedates the emergence of rhd by almost seven decades. in the absence of an intermediate reservoir host, it is uncommon for the emergence of an acute, virulent virus to be so extensively decoupled from the appearance of its associated disease. as a result, there has been significant debate over the timing, location, and mechanisms of rhdv ’s emergence. it is possible that the discrepancy among evolutionary rate estimates for rhdv is partially attributable to variation among datasets and/or subtle methodological variations. as this range of evolutionary rates is atypical for a gene of a single viral species, a systematic investigation was undertaken to explain variation among published nucleotide substitution rate and tmrca estimates for rhdv. a combination of jackknifing controls was used to produce robust rate estimates for the vp gene and the first estimated substitution rate for rhdv ’s rna-dependent rna polymerase gene. these controls reveal that using one misdated taxon significantly slows the estimated rates, unnecessarily lengthening rhdv ’s tmrca. we demonstrate the fragility of tip-calibrated evolutionary analyses and propose jackknife control beast runs as a way to identify potential misdated taxa.  complete dataset analyses the best-fitting nucleotide substitution model for the complete vp datasets, regardless of whether or not rcv isolates were included, was gtr + i + Γ. for the rdrp, the trnef + Γ model was selected for the rhdv dataset, while the sym + i + Γ model was selected for the rhdv + rcv dataset. the uncorrelated lognormal clock model was determined to be a significantly better fit than the strict clock model for each of the datasets. additionally, the 95% hpds of the standard deviation for the uncorrelated lognormal relaxed molecular clock rate estimates excluded zero for all of the demographic models, further rejecting a strict molecular clock for these alignments. the bayesian skyline demographic model was best-fitting for each of the datasets, though it was not significantly better than the constant or exponentially growing population models. however, there was no significant variation in nucleotide substitution rate or tmrca estimates among demographic models in any of the full datasets. there was no significant substitution rate variation between rhdv and rhdv + rcv datasets of either gene, and the inclusion of the divergent rcv isolates had no significant effects on age estimates for the virulent rhdv clade or its individual lineages. the subsequent analyses were performed only on the rhdv datasets. *mean substitution rate, tmrca, and corresponding year estimates are shown with upper and lower 95% hpd bounds. the maximum clade credibility and maximum likelihood trees for each complete gene dataset were highly congruent. further, the trees generated for the two different genes were also congruent, with the exception of one taxon, genbank accession number ef which switched from lineage b in the vp tree to lineage d in the rdrp tree. while it has been previously suggested that this taxon has undergone a crossover event at the junction between the rdrp and vp genes, it was not detected as a potential recombinant in these single gene analyses. despite the congruence of tree topology, however, there was a significant difference between the substitution rate estimates from the complete vp and rdrp datasets, with the vp rate estimate paralleling the lower vp rates from the literature, and the rdrp rate paralleling the higher published vp rate. to assess the extent of temporal structure in the mcc trees, tip-date randomized controls were run. for both genes, the upper 95% hpd interval from the tip-date randomized datasets occasionally overlapped the lower 95% interval of the real dataset substitution rates. a post-hoc permutation test verified that estimates from the tip-date randomized data sets were different from the substitution rates estimated from the actual dataset. root-to-tip regressions showed moderate correlations. there is statistical support for temporal structure in each gene’s dataset. control analyses the jackknifed taxa control analysis for the vp gene resulted in two distinct groups, roughly corresponding to the two divisions of rhdv rate and tmrca estimates in literature. by comparing the compositions of the two groups, it was found there was only one taxon that was present in every dataset from one group and absent from every dataset in the other group. the statistical significance of grouping datasets by the presence or absence of ay is shown in figure  in contrast to the vp dataset, the jackknifed taxa control analysis for the rdrp dataset, which did not contain ay yielded one continuous group of substitution rate estimates. the n- jackknife control analysis for the vp gene further implicated ay as having a significant effect on the estimated substitution rate. only the removal of ay resulted in a substitution rate estimate that was significantly higher than the other n- jackknife datasets and the complete vp dataset. removal of ay also resulted in a tmrca estimate that was substantially more recent than that from the complete dataset. for the rdrp, the substitution rates estimated from the n- jackknife datasets were nearly identical to each other and to the complete dataset. ay more formally “nj/china/ ” has been used in previous analyses with as the year of isolation. by the leaf-dating method, it was estimated that a more appropriate year would have been. a root-to-tip regression of the vp ml tree, including ay revealed that both ay and fr were potential outliers. running a fresh beast analysis for the complete vp dataset without ay revealed that there was no overlap between the hpds of these substitution rate estimates and those of the dataset with ay regardless of the demographic priors used. by comparing the estimated years of emergence of virulent rhdv and its individual lineages from analyses with and without ay, it was revealed that while the inclusion of this taxon inflated tmrca estimates for all lineages, the most conspicuous effect was on the lineage in which it grouped. removal of fr however, had little effect on the estimated vp substitution rate and coalescent. removal of ay from the complete vp dataset did not change the mcc or ml tree topology. however, the tip-date randomization analysis performed on the vp dataset without ay revealed stronger evidence of a temporal signal. similarly, excluding ay from a root-to-tip regression improved the correlation between genetic divergence and time. discussion and  the notion that inaccurate specification of dates used for molecular rate calibration could produce misleading results is not a novel one. in the context of rhdv in particular, seven partial vp sequences were identified as misdated modern contaminants by maximum likelihood analysis, but were nonetheless included in one beast analysis, resulting in the slowest substitution rate published for rhdv. it has also been suspected for some time that certain taxa could have a strong influence over inferred phylogenies, and a number of methods have been developed to identify weak clades in phylogenetic trees or the highly influential taxa responsible for weakening phylogenetic relationships. however, there is currently no direct method for identifying the presence of a taxon, such as ay that has significant influence over evolutionary rate and tmrca estimates, while not altering phylogenetic relationships. further, the influence of misdated taxa on estimates of evolutionary parameters has yet to be extensively examined or quantified. the comprehensive jackknifing controls used here demonstrate that the chinese rhdv isolate nj/china/ genbank accession number ay was responsible for dragging down the substitution rate estimate for the vp gene by 65%. the chinese-language paper that described this taxon contained important details about the isolation and handling of this strain. the first paragraph of the methods section revealed that, though it was isolated from nature in it was maintained in the laboratory for vaccine preparation and was likely not sequenced until much later. the submission date of ay to genbank is concordant with the lower bound of the age estimated by the leaf-dating method, as well as its grouping with isolates from 2006– in the mcc and ml trees. "“rhdv nj strain isolate was discovered and characterized byinstitute of veterinary medicine, jiangsu academy of agricultural sciences, from rabbits raised in an unknown farm in nanjing city, china, in since the discovery, this strain hasbeen maintained in lab rabbits until now. this strain has been usedto prepare potent rabbit hemorrhagic disease vaccine for years our lab has cloned the gene vp in e. coli jm and bl ”" the grouping with much more recent isolates could be explained by ay269825’s use in rhdv vaccine production. whether attenuated or improperly inactivated, the strain could have been released into china, and now this lineage can be isolated from other regions of china and russia. this is similar to the lab-escape strain that complicated substitution rate estimation for influenza a virus. instead of changing many dates of isolation, however, only ay would have to be assigned a different date. previous studies have shown that the long-term rate of viral evolution in the lab can mimic the rate in nature, so unlike the 20 years of frozen stasis that the influenza isolate experienced, ay was changing at a rate similar to its wild relatives. however, consistent results were obtained by excluding this isolate from analyses altogether. without ay the tmrca for the entire virulent rhdv complex and each of its lineages is substantially lower, resolving the much of the debate over its puzzling evolutionary history. indeed, without this misdated taxon, the ancestor of the entire complex is estimated to have existed between 1955-, as few as six years before the appearance of rhd. these results cannot address whether virulence was a shared trait of the most recent common ancestor of rhdv, or if it evolved independently in multiple lineages. whenever virulence did evolve, it did not have to go undocumented for several decades. while the root-to-tip regression analysis identified ay as potentially deviating from the molecular clock, fr a taxon that did not affect vp60’s evolutionary dynamics, also had a similarly high residual value. this underscores the problem of using congruence with a strict molecular clock as the sole means of assessing the validity of dates of isolation. deviation from the root-to-tip regression line is expected for viruses with variable rates of evolution, which would be accurately modeled with a relaxed molecular clock beast analysis. indeed, correlation between genetic distance and time was stronger for the vp dataset including the misdated ay taxon than the rdrp dataset, which produced a more trustworthy substitution rate. the decision to include or exclude taxa based on residuals from a best-fitting line is largely subjective, as there are no guidelines for how common large residuals are in tip-dated viral datasets. in fact, one of the ay269825-containing rhdv datasets had been subjected to this control prior to rate analysis, and the authors did not reject it as an outlier. finally, root-to-tip regression provides no insight into the magnitude of effect of any taxon on evolutionary estimates. the jackknife controls proposed here focus on detecting taxa that have had a disproportionate effect on the beast results, and, in the case of rhdv, offered strong quantitative evidence against including ay another interesting finding is that without ay the estimated substitution rate for the vp gene is almost identical to that of the rdrp gene, despite the fact that the latter dataset had fewer than half as many taxa. even while the rdrp dataset did not have as strong a temporal signal, probably due to the lower number of taxa, it still produced a significantly more accurate substitution rate estimate than datasets with two to three times as many taxa that included just one misdated taxon. further, the estimated substitution rate from our vp rhdv + rcv dataset was nearly identical to that from another dataset which contained just taxa, including one distantly related european brown hare syndrome virus taxon as an outgroup. this pattern of different sized datasets producing very similar substitution rates is not unique to rhdv. for example, two beast analyses of dengue virus type from datasets of taxa and taxa yielded nearly identical substitution rates. further, in the case of human parechovirus, three beast analyses of three different genomic regions based on datasets with a range of 29– taxa also produced nearly identical substitution rates. it is evident that assigning years of isolation to taxa should be done with great caution in tip-calibrated rate analyses. these results support favoring data sets with fewer taxa with verifiable dates of isolation over larger data sets with less quality control: additional good data do not swamp out the effects of one badly dated taxon. when researchers are including any ambiguously dated taxa, or when they want to be certain about the effects of each taxon on the rate analysis, jackknife controls provide a clear way to see these effects. as many sequences are added to genbank without easily accessible papers describing in detail the isolation, passaging and sequencing of each isolate, it is necessary to verify if one or more of the sequences is having a disproportionate influence on the results. we propose n- jackknifing as one method for researchers using tip-calibrated analyses in beast to ensure that a small number of taxa are not spoiling their estimates. 
the control methods show that using even one misdated taxon in a large dataset can significantly skew estimates of evolutionary parameters and suggest that it is better practice to use smaller datasets composed of taxa with unequivocal isolation dates. in an attempt to reconcile the conflicting data and further elucidate details of rhdv ’s evolutionary history, we undertook fresh bayesian analyses and employed jackknife control methods to produce robust substitution rate and time to most recent common ancestor estimates for rhdv based on the vp and rna-dependent rna polymerase genes. the literature is ripe with phylogenetic estimates of nucleotide substitution rates, especially of measurably evolving species such as rna viruses. after excluding the misdated taxon, both genes support a significantly higher substitution rate as well as a relatively recent emergence of rhdv, and obviate the need for previously hypothesized decades of unobserved diversification of the virus. these jackknife controls would be useful for future tip-calibrated rate analyses that include taxa with ambiguous dates of isolation.
 the literature is ripe with phylogenetic estimates of nucleotide substitution rates, especially of measurably evolving species such as rna viruses. however, it is not known how robust these rate estimates are to inaccuracies in the data, particularly in sampling dates that are used for molecular clock calibration. here we report on the rate of evolution of the emerging pathogen rabbit hemorrhagic disease virus, which has significantly different rates of evolution for the same outer capsid gene published in the literature. in an attempt to reconcile the conflicting data and further elucidate details of rhdv ’s evolutionary history, we undertook fresh bayesian analyses and employed jackknife control methods to produce robust substitution rate and time to most recent common ancestor estimates for rhdv based on the vp and rna-dependent rna polymerase genes.  through these control methods, we were able to identify a single misdated taxon, a passaged lab strain used for vaccine production, which was responsible for depressing the rhdv capsid gene’s rate of evolution by 65%. without this isolate, the polymerase and the capsid protein genes had nearly identical rates of evolution: x10- nucleotide substitutions/site/year, ns/s/y, x10-3- x10-3) and x10-3 ns/s/y, respectively.  after excluding the misdated taxon, both genes support a significantly higher substitution rate as well as a relatively recent emergence of rhdv, and obviate the need for previously hypothesized decades of unobserved diversification of the virus. the control methods show that using even one misdated taxon in a large dataset can significantly skew estimates of evolutionary parameters and suggest that it is better practice to use smaller datasets composed of taxa with unequivocal isolation dates. these jackknife controls would be useful for future tip-calibrated rate analyses that include taxa with ambiguous dates of isolation. rhdvsubstitution ratetip-calibratedbeastmisdated taxon 