Systematics - Bio 615
Confidence - Assessment of the Strength of
Derek S. Sikes University of Alaska
Multiple optimal trees
•  Many methods can yield multiple equally
optimal trees
•  We can further select among these trees
with additional criteria, but
•  Typically, relationships common to all the
optimal trees are summarized with
consensus trees
Consensus methods
•  A consensus tree is a summary of the agreement
among a set of fundamental trees
•  There are many consensus methods that differ in:
  1. the kind of agreement
  2. the level of agreement
•  Consensus methods can be used with multiple
trees from a single analysis or from multiple
analyses
the Phylogenetic Signal - part 2
1. Consistency Index
2. g1 statistic, PTP - test
3. Consensus trees
4. Decay index (Bremer Support)
5. Bootstrapping / Jackknifing
6. Statistical hypothesis testing (frequentist)
7. Posterior probability (see lecture on Bayesian)
Multiple optimal trees
•  If multiple optimal trees are found we know
that all of them are wrong except, possibly,
(hopefully) one (as species tree, not gene trees)
•  Some have argued against consensus tree
methods for this reason
•  Debate over quest for true tree (point
estimate) versus quantification of uncertainty
Strict consensus methods
•  Strict consensus methods require agreement
across all the fundamental trees
•  They show only those relationships that are
unambiguously supported by the data
•  The commonest method (strict component
consensus) focuses on clades/components/full
splits
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Strict consensus methods Strict consensus methods

TWO FUNDAMENTAL TREES"
•  This method produces a consensus tree that A! B! C! D! E! F! G! A! B! C! E! D! F! G!

includes all and only those full splits found in all the
fundamental trees

•  Other relationships (those in which the

fundamental trees disagree) are shown as A! B! C! D! E! F! G!
unresolved polytomies

•  Can be less optimal than any of the optimal trees Simplest to interpret

STRICT CONSENSUS TREE!

Majority rule consensus Majority rule consensus

•  Majority-rule consensus methods require •  This method produces a consensus tree that
agreement across a majority of the fundamental includes all and only those full splits found in a
trees majority (>50%) of the fundamental trees

•  May include relationships that are not supported by •  Other relationships are shown as unresolved
the most parsimonious interpretation of the data polytomies

•  The commonest method focuses on clades/ •  Of particular use in bootstrapping and Bayesian
components/full splits Inference (best not to use for single searches)

•  Implemented in PAUP* and MrBayes

Majority rule consensus Majority rule consensus

THREE FUNDAMENTAL TREES Majority Rule Consensus trees are used for

A B C D E F G A B C E F D G A B C E D F G
1. Summarizing multiple equally optimal trees
from one search (but they shouldn’t be!)
2. Summarizing the results of a bootstrapping
analysis (multiple searches)

Numbers indicate A B C E D F G
3. Summarizing the results of a Bayesian
frequency of analysis
100 66
clades in the 66 66

fundamental trees 66 Don’t confuse these! The numbers on the branches

mean very different things in each case
MAJORITY-RULE CONSENSUS TREE

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Reduced consensus methods Consensus methods

Three
TWO FUNDAMENTAL TREES! fundamental
A! B! C! D! E! F! G! A! G! B! C! D! E! F! trees strict consensus agreement subtree
Ochromonas Ochromonas Euplotes excluded
Symbiodinium Symbiodinium
Prorocentrum Symbiodinium
Loxodes Prorocentrum
Loxodes Prorocentrum
Tetrahymena
Spirostomumum Tetrahymena Loxodes
Tracheloraphis Tracheloraphis Tetrahymena
Euplotes
Gruberia Spirostomum Spirostomum
Ochromonas
Euplotes Tracheloraphis
Symbiodinium Gruberia
A B!C! D!E! F! G!
Prorocentrum
Gruberia
A! B! C! D! E! F! Loxodes Ochromonas
Tetrahymena
Spirostomumum
majority-rule
Euplotes Ochromonas
Tracheloraphis Symbiodinium
Strict component consensus! Gruberia 100
Ochromonas
Prorocentrum
100
completely unresolved! Symbiodinium
66 100 Loxodes
Prorocentrum
Loxodes Tetrahymena
66
Tetrahymena Spirostomum
AGREEMENT SUBTREE - PAUP*! Euplotes
Spirostomumum Euplotes
100
Taxon G is excluded! Tracheloraphis Tracheloraphis
Gruberia
Gruberia

Consensus methods Recall

•  Use strict methods to identify those relationships
unambiguously supported by parsimonious •  Stochastic error vs Systematic error
interpretation of the data

•  Use reduced methods where consensus trees are

•  These assessment methods help identify
poorly resolved stochastic error
–  How repeatable are the results?
•  Avoid methods which have ambiguous –  How strongly do the data support them?
interpretations. Prevent possible confusion between –  This is a measure of precision (which is
MR consensus for an optimal tree search and a MR
hopefully related to accuracy)
consensus for a bootstrapping search

Confidence - Assessment of the Strength of

Accuracy and Precision the Phylogenetic Signal - part 2

•  Accuracy 1. Consistency Index

–  Accuracy is correctness. How close a
2. g1 statistic, PTP - test
measurement is to the true value. ""
"(unless we know the “true tree” in "" 3. Consensus trees
"advance we cannot measure this)"
4. Decay index (Bremer Support)

•  Precision 5. Bootstrapping / Jackknifing

–  Precision is reproducibility. How closely
6. Statistical hypothesis testing (frequentist)
two or more measurements agree with one
another. (this we can measure!) 7. Posterior probability (see lecture on Bayesian)

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Branch Support Decay analysis

•  Several methods have been proposed that attach
numerical values to internal branches in trees that
are intended to provide some measure of the
strength of support for those branches and the
corresponding groups
•  In parsimony analysis, a way to assess support for a
group is to see if the group occurs in slightly less
parsimonious trees also
•  The length difference between:
the shortest trees including the group and

•  These methods include:

the shortest trees that exclude the group
  - The Bootstrap (BS) and jackknife
  - Decay analyses (aka Bremer Support)
(the extra steps required to collapse a group)
  - Bayesian Posterior Probabilities (PP or BPP)

is the decay index or Bremer support

Decay analyses - in practice

Decay analysis -example •  Decay indices for each clade can be determined by:
Ciliate SSUrDNA data Randomly permuted data -  Using PAUP* to search for the shortest tree that
Ochromonas Ochromonas lacks the branch of interest using reverse
+27 Symbiodinium Symbiodinium topological constraints
+1
Prorocentrum Prorocentrum
+1 -  with the Autodecay or TreeRot programs (in
+45 Loxodes +3 Loxodes
Tracheloraphis Tetrahymena conjunction with PAUP*) - MacClade 4 will also
Spirostomum Tracheloraphis help prepare for a Decay analysis
Gruberia +8
+15 Spirostomum
+10 Euplotes Euplotes
Tetrahymena
-  An excellent use for the Parsimony Ratchet -
+7 Gruberia
because finding the shortest tree length is all that
matters (not finding multiple shortest trees)

Decay indices - interpretation Decay indices - interpretation

•  Generally, the higher the decay index the better the
relative support for a group
•  Like Bootstrap values (BS), decay indices may be
misleading if the data are misleading
•  Magnitude of decay indices and BS generally
correlated (i.e. they tend to agree)
•  Only groups found in all most parsimonious trees
have decay indices > zero
•  Unlike BS decay indices are not scaled (0-100)
–  This has the advantage that the value can exceed 100
whereas BS “tops - out” at 100 meaning that we cannot
distinguish between the support of two branches with BS
values of 100 although one might have a far greater
decay index than the other
•  It is even less clear what is an acceptable decay
index than a BS value…
–  Unlike the BS value very little work has examined the
properties and behavior of decay indices

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Confidence - Assessment of the Strength of

Decay indices - interpretation the Phylogenetic Signal - part 2
One key study is that of DeBry (2001)
–  He showed that decay indices should be interpreted in 1. Consistency Index
light of branch lengths
2. g1 statistic, PTP - test
–  That the same values, even within the same tree, do not
represent the same support if the branch lengths differ 3. Consensus trees

4. Decay index (Bremer Support)

-  ie Decay Indices are not easily comparable as measures
of branch support
5. Bootstrapping / Jackknifing
-  Values < 4 should be considered weak regardless of
branch length 6. Statistical hypothesis testing (frequentist)
DeBry, R.W. (2001) Improving interpretation of the Decay Index for DNA sequence data. Systematic
Biology 50: 742-752. 7. Posterior probability (see lecture on Bayesian)

Bootstrapping (non-parametric)

•  Bootstrapping is a statistical
technique that uses computer
intensive random resampling
of data to determine sampling
error or confidence intervals
for some estimated parameter
•  Introduced to phylogenetics by
Decay values versus Bootstrap and Jacknife values Felsenstein in 1985
from one empirical study •  Based on idea of Efron (1979)
Norén, M. & U. Jondelius. 1999. Phylogeny of the Prolecithophora
(Platyhelminthes) inferred from 18S rDNA sequences. Cladistics 15: 103-112.

Bootstrapping (non-parametric)

1. Characters are sampled with replacement to create

many (100-1000) bootstrap replicate data sets
(think shuffle vs random play of music)

2. Each bootstrap replicate data set is analysed (e.g.

with parsimony, distance, ML)

3. Agreement among the resulting trees is

summarized with a majority-rule consensus tree

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Bootstrapping (non-parametric) Bootstrapping

•  Frequency of occurrence of groups, bootstrap Original data matrix! Resampled data matrix!

support (BS), is a measure of support for those Characters! Characters!

Summarize the results of
Taxa 1 2 3 4 5 6 7 8! Taxa 1 2 2 5 5 6 6 8!
groups A R R Y Y Y Y Y Y! A
B
R
R
R R Y Y Y Y Y!
R R Y Y Y Y Y!
multiple analyses with a
B R R Y Y Y Y Y Y! majority-rule consensus tree
C Y Y Y Y Y R R R! C Y Y Y Y Y R R R!
D Y Y R R R R R R! D Y Y Y R R R R R! Bootstrap values (BS) are the
Outgp R R R R R R R R! Outgp R R R R R R R R! frequencies with which
•  Additional information is given in partition tables (for groups are encountered in
analyses of replicate data
Randomly resample characters from the original data with
groups below 50% support) replacement to build many bootstrap replicate data sets of the sets
same size as the original - analyse each replicate data set
A! B! C! D!
A! B! C! D! A! B! C! D!
•  Can ask PAUP* to create MR con-tree of higher 1!
5!
2! 1! 5!
cut-off, eg 80% - all weaker branches collapse 8!
7!
2!
8!
2!
96%!
6! 6!
5! 6! 2! 66%!
4! 1!
3!
Outgroup!
Outgroup! Outgroup!

Bootstrapping - an example Bootstrapping - random data

Ciliate SSUrDNA - parsimony bootstrap Partition Table Partition Table

Randomly permuted data - parsimony bootstrap 123456789 Freq!

Ochromonas (1)! 123456789 Freq -----------------!
Symbiodinium (2)!
----------------- Ochromonas Ochromonas
.*****.** 71.17!
..**..... 58.87!
100! .**...... 100.00 Symbiodinium
16
Symbiodinium ....*..*. 26.43!
Prorocentrum (3)!
...**.... 100.00 59 Prorocentrum 59 Prorocentrum .*......* 25.67!
Loxodes Loxodes .***.*.** 23.83!
Euplotes (8)! .....**.. 100.00 71
Tracheloraphis
26
Spirostomumum ...*...*. 21.00!
84! 21
...****.. 100.00 Spirostomumum 71 16 Tetrahymena .*..**.** 18.50!
Tetrahymena (9)! .....*..* 16.00!
...****** 95.50 Euplotes Euplotes
.*...*..* 15.67!
96! Tetrahymena Tracheloraphis
100!
Loxodes (4)! .......** 84.33 .***....* 13.17!
Gruberia Gruberia ....**.** 12.67!
Tracheloraphis (5)!
...****.* 11.83 ....**.*. 12.00!
100! ...*****. 3.83 50% Majority-rule consensus (with minority components)
..*...*.. 12.00!
Spirostomum (6)! .**..*..* 11.00!
100! .*******. 2.50 .*...*... 10.80!
Gruberia (7)! .**....*. 1.00 .....*.** 10.50!
Majority-rule consensus .**.....* 1.00
.***..... 10.00!

Bootstrapping Bootstrap - interpretation

The probability of a character being omitted •  Bootstrapping was introduced as a way of
from a bootstrap sample ranges from establishing confidence intervals for
0-0.367 (depending on N, the number of phylogenies
characters)
Rule of thumb: a branch must be
•  This interpretation of bootstrap values
N P depends on the assumption that the
supported by 3 or more characters to be
1  0 recovered in >95% of bootstraps original data is a random sample from a
2  0.25 much larger set of independent and
3  0.29 identically distributed data (i.i.d.)
4  0.31
… 0.367

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Bootstrap - interpretation “…bootstrapping provides

•  However, several things complicate this interpretation us a confidence
interval within which is
contained not [necessarily]
-  These assumptions are often wrong - making any the true phylogeny but
strict statistical interpretation of BS invalid the phylogeny that
would be estimated
-  Some theoretical work indicates that BS are very on repeated sampling of
conservative (too low), and may underestimate many characters from the
confidence intervals - problem increases with underlying pool of
numbers of taxa characters.”

-  BS can be high for incongruent relationships in

separate analyses - and can therefore be misleading Joseph Felsenstein (1985)
(misleading data -> misleading BS) recall the
Mantra: The data are the things

Bootstrap - interpretation Bootstrap - interpretation

Huelsenbeck & Rannala (2004) list 3 common interpretations •  High BS (e.g. > 85%) is indicative of strong ‘signal’
in the data (some use 70% as the cutoff, there is no
1. Probability that a clade is correct (accuracy) consensus as to which value is best)

2. Robustness of the results to perturbation •  Provided we have no evidence of strong misleading

(repeatability / precision)
signal due to violation of assumptions (e.g. base
composition biases, great differences in branch
3. Probability of incorrectly rejecting a hypothesis of
monophyly (1-P) : probability of getting that
lengths) high BS values are likely to reflect strong
much evidence if, in fact, the group did not exist phylogenetic signal

Huelsenbeck, J.P. and Rannala, B. (2004) Frequentist properties of Bayesian posterior probabilities of •  In other words, although technically they are meant
phylogenetic trees under simple and complex substitution models. Systematic Biology 53: 904-913.
to be a measure precision, they are usually thought
to be at least strongly correlated with accuracy

Bootstrap - interpretation Bootstrap - interpretation

Be suspicious of
•  Low BS values, however, need not mean the
maximum bootstrap
relationship is false, only that it is poorly supported
values…
they might be due –  This is especially true of morphological data
to systematic error. –  Morphologists often use the Decay index instead

•  Bootstrapping can be viewed as a way of exploring

the robustness of phylogenetic inferences to
perturbations in the balance of supporting and
conflicting evidence for groups

Paul Lewis

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Bootstrap - interpretation Bootstrap - interpretation

Two types of precision (Hillis & Bull 1993): Hillis & Bull (1993) examined precision, repeatability, and
accuracy of the bootstrap
Precision of bootstrap value vs repeatability of
finding a branch: - Found that BS provide a very imprecise measure of
repeatability - so imprecise as to be worthless as a
measure of repeatability
- Precision of bootstrap values increases with the
number of bootstrap replicates (variance - Determined that in some cases a BS as low as 70%
among analyses decreases) was equivalent to a 95% probability of being true - Bias
confirmed by Newton (1996)
- Repeatability tells us how likely we are to find the
Hillis, D.M. and Bull, J.J. (1993) An empirical test of bootstrapping as a method for assessing confidence in
same results using a different but similar phylogenetic analysis. Systematic Biology, 42: 182-192.
dataset - Felsenstein’s original idea

Hillis & Bull (1993) examined

precision, repeatability, and Bootstrap - interpretation
accuracy of the bootstrap
BS values have been criticized for a variety of
a) 1,089 BS of 100 reps e from 1 reasons:
“real” dataset =1,089 pseudo datasets
Sanderson, M.J. (1995) Objections to Bootstrapping Phylogenies: A Critique. Systematic Biology, 44:
299-320.
b) 100 real datasets
But the top reason has been that they seem to
“Comparison of these two be too conservative - ie underestimates of the
distributions reveals that the probability of the branch being correct - ie
process of bootstrap resampling
is not the same as repeated, biased downward (erratically & unpredictably)
independent sampling of data.”
Newton, M.A. (1996) Bootstrapping phylogenies: Large deviations and dispersion effects. Biometrika,
Hillis, D.M. and Bull, J.J. (1993) An empirical test of bootstrapping 83: 315-328.
as a method for assessing confidence in phylogenetic analysis.
Systematic Biology, 42: 182-192.

Jackknifing Low Support

•  Jackknifing is very similar to bootstrapping and Low branch support can result from
differs only in the character resampling strategy
1. Conflicting data (homoplasy)

•  Some proportion of characters (e.g. 37%, 50%) are 2. Lack of data - even a dataset with no homoplasy can yield
randomly selected and deleted poorly resolved trees if there are branches without change

3. Use of a poorly fitting model (too complex or too simple)

•  Replicate data sets are analyzed and the results
summarized with a majority-rule consensus tree
•  Jackknifing and bootstrapping tend to produce
broadly similar results and have similar
interpretations - Jackknifing is preferred by cladists
4. Artifact of mid-sized clades? “This indicates that, for all support
measures on trees of a given size, the largest clades and the smallest clades are
supported most strongly, whereas medium sized clades receive lower support”
Picket, K.M. and Randle, C.P. (2005) Strange bayes indeed: uniform topological priors imply non-uniform
clade priors. Molecular Phylogenetics and Evolution 34: 203-211. SEE ALSO: Brandley, M. et al. (2006)
Are unequal clade priors problematic for Bayesian phylogenetics? Systematic Biology 55: 138-146.

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Confidence - Assessment of the Strength of Terms - from lecture & readings

the Phylogenetic Signal - part 2 consensus methods
consensus tree
1. Consistency Index strict consensus
splits
2. g1 statistic, PTP - test majority rule consensus
reduced consensus trees
3. Consensus trees agreement subtree
branch support
4. Decay index (Bremer Support) Decay analysis
Decay index (Bremer Support)
5. Bootstrapping / Jackknifing DeBry (2001)
Bootstrapping
6. Statistical hypothesis testing (frequentist) resampling with replacement
repeatability
7. Posterior probability (see lecture on Bayesian) jackknifing

Study questions
Describe the difference between a strict and majority rule
consensus tree."

What were the key findings of DeBry in his (2001) paper on Decay
Indices?"

What is the rule of thumb in bootstrapping for a branch to receive >

95% support?

What are two common but different interpretations of bootstrap

values? What did Hillis & Bull (1993) conclude regarding these
interpretations?"

What are two common explanations for low branch support?"