*The following text was written a few years ago, but much of it never got published. So, I thought that this might be a good opportunity to make it available, since what it says is still true today.*

Since a phylogenetic tree is interpreted in terms of the monophyletic groups that it hypothesizes, it is important to quantitatively assess the robustness of all of these groups (i.e. the degree of support for each branch in the tree) — is the support for a particular group any better than would be expected from a random data set? This issue of clade robustness is the same as assessing branch support on the tree, since each branch represents a clade. Many different techniques have been developed, including:

**analytical procedures**, such as interior-branch tests (Nei et al. 1985; Sneath 1986), likelihood-ratio tests (Felsenstein 1988; Huelsenbeck et al. 1996b), and clade significance (Lee 2000);**resampling procedures**, such as the bootstrap (Felsenstein 1985), the jackknife (Lanyon 1985), topology-dependent permutation (Faith 1991), and clade credibility or posterior probability (Larget and Simon 1999); and**non-statistical procedures**, such as the decay index (Bremer 1988), clade stability (Davis 1993), and spectral signals (Hendy and Penny 1993).

**The bootstrap**

This method was first introduced by Efron (1979) as an alternative method to jackknifing for producing standard errors on estimates of central location other than the mean (e.g. the median), but it has since been expanded to cover probabilistic confidence intervals as well (Efron and Tibshirani 1993; Davison and Hinkley 1997). It was introduced into phylogenetic studies by Penny et al. (1982) and then formalized by Felsenstein (1985), who suggested that it could be implemented by holding the taxa constant and resampling the characters randomly with replacement, the tree-building analysis then being applied to each of the bootstrap resamples.

Bootstrapping is a monte carlo procedure that generates "pseudo" data sets from the original data, and then uses these new data sets for its inferences. That is, it tries to derive the population inferences (i.e the "true" answer) from repeated generation of new samples, each sample being constrained by the characteristics of the original data sample. It thus relies on an explicit analogy between the sample and the appropriate population: that sampling from the sample is the same as sampling from the population. Clearly, the strongest requirement for bootstrapping to work is that the sample be a reasonable representation of the population.

Bootstrap confidence intervals are only ever approximate, especially for complex data structures, as they are a fundamentally more ambitious measure of accuracy than is a simple standard error (SE). For example, the usual formula for calculating a confidence interval (CI) when the population frequency distribution is assumed to be normal is: CI = t * SE, where t is the Student t-value associated with the particular sample size and confidence percentage required. However, the main use of bootstrapping is in situations where the population frequency distribution is either indeterminate or is difficult to obtain empirically, and so this simple formula cannot be applied. Getting from the standard error to a confidence interval is then not straightforward. As a result, there are actually several quite distinct procedures for performing bootstrapping (Carpenter and Bithell 2000), with varying degrees of expected success.

**Types of bootstrap**

The original technique is called the

**percentile bootstrap**. It is based on the principle of using the minimum number of ad hoc assumptions, and so it merely counts the percentage of bootstrap resamples that meet the specified criteria. F§or example, to estimate the standard error of a median, the median can be calculated for each bootstrap resample and then the standard deviation of the resulting frequency distribution will be the estimated standard error of the original median. The method is thus rather simplistic, and is often referred to as the

**naïve bootstrap**, because it assumes no knowledge of how to calculate population estimates. It is a widespread method, as it can be applied even when the other methods cannot. However, it is known to have certain problems associated with the estimates produced, particularly for confidence intervals, such as bias and skewness (especially when the parent frequency distribution is not symmetrical). These were pointed out right from the start (Efron 1979), and efforts have subsequently been made to deal with them. Nevertheless, this is the form of bootstrap introduced by Felsenstein (1985), and it is the one used by most phylogeny computer programs. It is therefore the one that will be discussed in more detail below.

These known problems with the naïve bootstrap can be overcome by using bias-corrected (BC) bootstrap estimates — that is, the bias is estimated and removed from the calculation of the confidence interval. Possible dependence of the standard error on the parameter being estimated, which creates skewness, can be dealt with by using

**bias-corrected and accelerated (BCa) bootstrap**estimates, so that the bias and skewness are both estimated and removed from the calculation of the confidence interval. The BCa method is the one usually recommended for use (Carpenter and Bithell 2000), because it corrects for both bias and skewness. This method is much slower to calculate than the simple percentile bootstrap, because it requires an extra parameter to be estimated for each of the bias and skewness corrections, and the latter correction is actually estimated by performing a separate jackknife analysis on each bootstrap resample (which means that the analysis can take 100 times as long as a naïve analysis). There have been several attempts to apply this form of correction methodology to bootstrapping in a phylogenetic context (Rodrigo 1993; Zharkikh and Li 1995; Efron et al. 1996; Shimodaira 2002), but while these can be successful at correcting bias and skewness (Sanderson and Wojciechowski 2000) these have not caught on, possibly because of the time factor involved.

Alternatively, we can decide not to be naïve when calculating confidence intervals, and to therefore calculate them in the traditional manner, using the standard error and the t-distribution. However, we then need to overcome any non-normal distribution problems of these two estimates by estimating both of them using bootstrapping. That is,

**bootstrapped-t confidence intervals**are derived by calculating both the standard error and the t-value using bootstrapping, and then calculating the confidence interval as ±t * SE. To many people, this is the most natural way to calculate confidence intervals, since it matches the usual parametric procedure, and thus it is frequently recommended (Carpenter and Bithell 2000). Once again, this method is much slower to calculate than the percentile bootstrap, because the t-value is actually estimated by performing a separate bootstrap analysis on each bootstrap resample (which means that the analysis can take 100 times as long as a naïve analysis). This methodology seems not to have yet been suggested in a phylogenetic context, and in any case the time factor may be restrictive.

It is also possible to calculate

**test-inversion confidence intervals**. This idea is based on the reciprocal relationship of statistical tests and confidence intervals, where (for example) non-overlapping 95% confidence intervals indicate statistically significant patterns at p<0.05 and vice versa. Thus, if we work out the situation where the pattern has a probability of p=0.05 of occurring by chance, then this defines the 95% confidence limit of the pattern. Clearly, this can be a complex process, especially for two-sided tests (which double the required number of calculations), as it can only be done by iteratively modifying the pattern and re-calculating the probability until the solution is reached. Once again, no-one yet seems to have suggested this methodology in a phylogenetic context, which is not unexpected given the general problems in deciding how to test branches statistically.

The above methods all count as non-parametric bootstrap methods. More recently,

**parametric bootstrapping**methods have also been developed, which make the more restrictive assumption that a parametric model can be applied to the data (e.g. that the standard deviation of the parameter can be reliably estimated). In parametric bootstrapping we generate simulated datasets based on the assumed frequency distribution of the data, rather than by resampling from the data set itself. That is, instead of sampling from the sample, we sample from the assumed theoretical distribution to generate the set of bootstrap samples. We can then apply the percentile, BCa or bootstrap-t methods, described above, in the usual way. Clearly this method assumes that we know the appropriate frequency distribution; and the method will only be appropriate if this assumption is true, but not otherwise. However, if the assumption is correct, then this can be the most powerful method (Huelsenbeck et al. 1996a; Newton 1996) because it is not dependent on the representativeness of the data sample. The method has been introduced into phylogenetics in several contexts (Goldman 1993; Adell and Dopazo 1994; Huelsenbeck et al. 1996a), but the appropriate frequency distribution for branch support is not obvious (i.e. a phylogeny is a complex structure and cannot be represented by a single number but rather requires a model of sequence evolution and a model tree) and so it is not used for this purpose.

**Issues with the bootstrap**

Thus, for several reasons, all of the best bootstrapping methods are not likely to be available when assessing the robustness of a phylogenetic tree, and we are left with the naïve percentile bootstrap, which can be expected a priori to provide biased and skewed estimates of confidence intervals (because the frequency distribution associated with tree branches will not be symmetrical). Sadly, these problems have been repeatedly confirmed for the assessment of branch support in phylogenetic tree-building, both theoretically (Zharkikh and Li 1992a, 1992b; Felsenstein and Kishino 1993; Li and Zharkikh 1994; Sitnikova et al. 1995; Berry and Gascuel 1996; Efron et al. 1996; Huelsenbeck et al. 1996a; Newton 1996; Sanderson and Wojciechowski 2000; Suzuki et al. 2002; Alfaro et al. 2003; Erixon et al. 2003; Galtier 2004; Huelsenbeck and Rannala 2004) and empirically (Sanderson 1989; Hillis and Bull 1993; Buckley et al. 2001; Buckley and Cunningham 2002; Wilcox et al. 2002; Taylor and Piel 2004).

These studies have demonstrated that the probability of bootstrap resampling supporting the true tree may be either under- or overestimated, depending on the particular situation. For example, bootstrap values >75% tend to be underestimates of the amount of support, while they may be overestimates below this level, as shown in the first graph (above). That is, when the branch support is strong (i.e. the clade is part of the true tree) there will be an underestimation and when the support is weak (i.e. the clade is not part of the true tree) there will be an overestimation. This situation has been reported time and time again, with various theoretical explanations (e.g. Felsenstein and Kishino 1993; Efron et al. 1996; Newton 1996), although there are dissenting voices (e.g. Taylor and Piel 2004) as would be expected for a complex situation. Unfortunately, practitioners seem to ignore this fact, and to assume incorrectly that bootstrap values are

*always*underestimates.

Just as importantly, the theoretical studies show that the pattern of over- and underestimation depends on (i) the shape of the tree and the branch lengths, (ii) the number of taxa, (iii) the number of characters, (iv) the evolutionary model used, and (v) the number of bootstrap resamples. This was first reported by Zharkikh and Li (1992a), and has been reconfirmed since then. For example, with few characters the bootstrap index tends to overestimate the support for a clade and to underestimate it for more characters. This is particularly true if the number of phylogenetically informative characters is increased or the number of non-independent characters is increased; and the index becomes progressively more conservative (i.e. lower values) as the number of taxa is increased.

Moreover, these patterns of under- and overestimation are increased with an increasing number of bootstrap replications, as shown in the next graph — this called "being wrong, with confidence".

The following graph pair of graphs show the effect of varying the evolutionary model used to generate the data, where under-specification of the analysis model leads to a general over-estimate of the true probability (cross-over at p=0.8, as shown in the first graph of the pair), while matching the generating and analysis models leads to a general under-estimation (cross-over at p=0.3, as shown in the second graph of the pair).

These are serious issues, which seem to be often ignored by practitioners. We can't just assume that the "true" support value is larger than our observed bootstrap value. In particular, this means that bootstrap values are not directly comparable between trees, even for the same taxa, and thus there can be no "agreed" level of bootstrap support that can be considered to be "statistically significant". A bootstrap value of 90% on a branch on one tree may actually represent

*less*support than a bootstrap value of 85% on another tree, depending on the characteristics of the dataset concerned and the bootstrapping procedure used (although within a single tree the values should be comparable).

This complex situation means that we have to consider carefully how best to interpret bootstrap values in a phylogenetic context (Sanderson 1995). The bootstrap proportion (i.e. the proportion of resampled trees containing the branch/clade of interest) has variously been interpreted as (Berry and Gascuel 1996):

- a measure of reliability, telling us what would be expected to happen if we repeated our experiment;
- a measure of accuracy, telling us about the probability of our experimental result being true; and
- a measure of confidence, interpreted as a conditional probability similar to those in standard statistical hypothesis tests (i.e. measuring Type I errors or false positives).

The issue of support values depending on the number of bootstrap replicates is also of interest. It is usually recommended that at least 1,000–2,000 bootstrap resamples are taken for estimating confidence intervals, and this generality has been applied to phylogenetic trees (Hedges 1992). However, it is important to recognize that these suggestions relate to the precision of the confidence estimates not to their accuracy. Accuracy refers to how close the estimates are to the true value (i.e. correctness) while precision refers to how variable are the estimates (i.e. repeatability). Accuracy depends on a complex set of characteristics many of which have nothing to do with bootstrap replication. Precision, on the other hand, is entirely to do with the number of bootstrap replicates and the expected accuracy of the estimates. As shown in the next graph, 100 replicates at a conventional level of accuracy produces estimates that are expected to be within ±4% of the "true" values while 2,000 replicates produces estimates ±1%. This needs to be borne in mind when deciding whether to call a particular value "significant support" or not.

There have also been attempts to overcome some of the practical limitations of bootstrapping for large data sets by adopting heuristic procedures, including resampling estimated likelihoods for maximum-likelihood analyses (Waddell et al. 2002) and reduced tree-search effort for the bootstrap replicates. However, approaches using reduced tree-search effort produce even more conservative estimates of branch support, and the magnitude of the effect increases with decreasing bootstrap values (DeBry and Olmstead 2000; Mort et al. 2000; Sanderson and Wojciechowski 2000).

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