MrBayes lab
The following lab exercise is based on a tutorial written by Paul Lewis. Any mistakes in the version below were introduced in this adaptation. 
Contents
 1 Introduction
 2 Download the data files
 3 A typical analysis using MrBayes
 4 Other output files produced by MrBayes
 5 Using other programs to summarize MCMC results
 6 Running MrBayes with no data
 7 Binaries of MrBayes 3.2 for Windows and Mac OSX
 8 Compiling MrBayes 3.2 on Mac and Linux
 9 More resources
 10 References
Introduction
MrBayes^{[1]}^{[2]} is a program for Bayesian phylogenetic inference. There are currently two versions of of the program: the release version 3.1.2 and one that is under active development, version 3.2. The latter has been described in a book chapter (Ronquist et. al 2008) ^{[3]} which can be found here. On grendel, the executables for the two versions are called mb (version 3.1.2) and mb32 (version 3.2). For the first exercise of this lab we will run version 3.2 on grendel (windows and Mac binaries can be found in the shared folder's subdirectory mrbayes but we recommend that you first try it on grendel).
If you prefer to run a very similar tutorial to this one in version 3.1, please visit this page and work through the instructions there. You will need to download the data file from here.
Download the data files
The data files for today's lab can be found here.
A typical analysis using MrBayes
Log on to grendel and transfer the data files to a directory for today's lab. Start the program by typing
mb32
A typical analysis requires the following steps:
 Reading in the data (command execute)
 Specifying the substitution model (lset) and setting up the priors (prset)
 Specifying the MCMC settings (mcmcp)
 Running the analysis (mcmc, the command mcmcp is identical to mcmc except that it does not actually start a run.)
 Summarizing the results (sumt and sump)
Reading in the data
Type
execute algaemb.nex
Help!
You can obtain information (including the current settings) by typing help followed by the command name: for example,
help lset
Commands in MrBayes are (intentionally) similar to those in PAUP*, but the differences can be frustrating. For instance, lset ? in PAUP* gives you information about the current likelihood settings, but this does not work in MrBayes. Also, the lset command in MrBayes has many options not present in PAUP*, and vice versa. Get used to typing help followed by the name of a command in MrBayes to see what options are allowed for a particular command.
Specifying the model
The command
showmodel
prints the current model setting to the screen. In my case this looks like this
MrBayes screen output:
Model settings: Data not partitioned  Datatype = RNA Nucmodel = 4by4 Nst = 1 Covarion = No # States = 4 State frequencies have a Dirichlet prior (1.00,1.00,1.00,1.00) Rates = Equal Active parameters: Parameters  Statefreq 1 Ratemultiplier 2 Topology 3 Brlens 4  1  Parameter = Pi Type = Stationary state frequencies Prior = Dirichlet 2  Parameter = Ratemultiplier Type = Partitionspecific rate multiplier Prior = Fixed(1.0) 3  Parameter = Tau Type = Topology Prior = All topologies equally probable a priori Subparam. = V 4  Parameter = V Type = Branch lengths Prior = Unconstrained:Exponential(10.0) 
Note that Nst (number of substitution types) is fixed to one. The State frequencies are listed as active parameters and, if we started an analysis with the current settings, would be updated in the MCMC analysis. Other active parameters are the tree topology and the associated branch lengths. We can ignore the Ratemultiplier parameter. It is listed here but since we only have one data partition it has no effect here.

Let's assume we would like a 2parameter substitution matrix (i.e. the rate matrix has only two substitution rates, the transition rate and the transversion rate), and rates to vary across sites according to a gamma distribution with 4 categories.
lset nst=2 rates=gamma ngammacat=4
Type
showmodel
and compare the screen output to before. It should reflect the changes we have just made to the model.

Specifying the priors
Now that MrBayes knows which parameters we want in our model, we have to specify priors for all of them. Type
help prset
to see a list of the current settings (the list also includes priors that do not apply to the current analysis).
Specifying the prior on tree topologies
We can see from the previous output that the default prior on topologies is uniform. This means that the prior probability of any tree topology is 1/(# possible topologies for this data set). MrBayes only recognizes strictly bifurcating topologies.
Specifying the prior on branch lengths
prset brlenspr=unconstrained:exp(10.0)
The above command specifies that branch lengths are to be unconstrained (i.e. a molecular clock is not assumed) and the prior distribution to be applied to each branch length is an exponential distribution with mean 1/10. Note that the value you specify for unconstrained:exp is the inverse of the mean. (This is also the default setting so the last command didn't actually change anything.)
Specifying the prior on the gamma shape parameter
prset shapepr=exp(1.0)
This command specifies an exponential distribution with mean 1.0 for the shape parameter of the gamma distribution we will use to model rate heterogeneity.
Specifying the prior on kappa
prset tratiopr=beta(1.0,1.0)
The command above says to use a Beta(1,1) distribution as the prior for the transition/transversion rate ratio. You may be thinking that it is a little strange to use a Beta distribution for a parameter that ranges from 0 to infinity, and if so you would be right! Allow me to explain as best I can. Recall that the kappa parameter is the ratio α / β, where α is the rate of transitions and β is the rate of transversions. Rather than allowing you to place a prior directly on the ratio α / β, which ranges from 0 to infinity, MrBayes asks you to instead place a joint (Beta) prior on α and β. Here, α and β act like p and 1 − p in the familiar coin flipping experiment. The reasoning behind this is esoteric, but is the same as the reasoning behind the (now commonplace) use of Dirichlet priors for the GTR relative rates, which is explained nicely in Zwickl and Holder (2004)^{[4]}.
You might wonder what the Beta(1,1) distribution (figure on the left) implies about kappa. Transforming the Beta density into the density of α / β results in a density for kappa that is very close, but not identical, to an exponential(1) distribution. This is known as the beta prime distribution, and has support (0, infinity), which is appropriate for a ratio such as kappa. The beta prime distribution is somewhat peculiar, however, when both parameters are 1 (as they are in this case): in this case, the mean is not defined, which is to say that we cannot predict the mean of a sample of kappa values drawn from this distribution. It is not necessary for a prior distribution to have a welldefined mean, so this is OK.
Specifying a prior on base frequencies
prset statefreqpr=dirichlet(1.0,1.0,1.0,1.0)
The above command states that a flat dirichlet distribution is to be used for base frequencies. The Dirichlet distribution is like the Beta distribution, except that it is applicable to combinations of parameters. Like the Beta distribution, the distribution is symmetrical if all the parameters of the distribution are equal, and the distribution is flat if all the parameters of the distribution are equal to 1.0. Using the command above specifies a flat Dirichlet prior, which says that any combination of base frequencies will be given equal prior weight. This means that (0.01, 0.99, 0.0, 0.0) is just as probable, a priori, as (0.25, 0.25, 0.25, 0.25). If you wanted base frequencies to not stray much from (0.25, 0.25, 0.25, 0.25), you could specify, say, statefreqpr=dirichlet(10.0,10.0,10.0,10.0)
instead.
Specifying MCMC options
Type
help mcmc
to view the current MCMC settings. The following options have to do with calculating a convergence diagnostic during the run. We will modify these below.
MrBayes screen output:
Mcmcdiagn Yes/No Yes Diagnfreq <number> 5000 Diagnstat Avgstddev/Maxstddev Avgstddev Minpartfreq <number> 0.10 Allchains Yes/No No Allcomps Yes/No No Relburnin Yes/No Yes Burnin <number> 0 Burninfrac <number> 0.25 Stoprule Yes/No No Stopval <number> 0.05 
Type
mcmcp ngen=100000 samplefreq=100 printfreq=1000 nruns=2 nchains=2 stoprule=yes stopval=0.01
to specify most of the remaining details of the analysis:
ngen=100000
tells MrBayes that its robots should each take 100,000 steps. You should ordinarily use much larger values for ngen than this! We're keeping it small here because we do not have a lot of time and the purpose of this lab is to learn how to use MrBayes, not produce a publishable result. samplefreq=100
says to only save parameter values and the tree topology every 100 steps. printfreq=100
says that we would like a progress report to the screen every 1000 steps.
nruns=2
says to do two independent runs (MrBayes performs two separate analyses by default).
nchains=2
says that we would like to have 2 heated chains running in addition to the cold chain. The default is one cold chain and three heated chains but this would take to long right now, because we are all sharing a few processors on the cluster. This way, MrBayes will run two independent analyses, each with a cold chain and a single hot chain.
stoprule=yes
tells MrBayes to stop the run when the average standard deviation of split frequencies has reached a certain threshold (stopval=0.01
).
Specifying an outgroup
outgroup Anacystis_nidulans
This merely affects the display of trees. It says we want trees to be rooted between the taxon Anacystis_nidulans and everything else.
Running the analysis
We are now ready to start the run by typing
mcmc
While MrBayes runs, it shows oneline progress reports. The first column is the step number. The next two columns show the loglikelihoods of the separate chains that are running, with the cold chain indicated by square brackets rather than parentheses. The asterisk separates the screen output for the two independent analyses. The last complete column is a prediction of the time remaining until the run completes. The columns consisting of only  are simply separators, they have no meaning.
When I tried this, the analysis hit the stop value after 40,000 generations:
MrBayes screen output:
36000  [3180.322] (3180.202) * [3179.141] (3180.397)  0:00:21 37000  [3180.560] (3181.537) * (3182.427) [3178.440]  0:00:20 38000  (3185.505) [3176.655] * (3177.682) [3182.832]  0:00:19 39000  (3180.690) [3179.084] * [3181.647] (3185.312)  0:00:20 40000  [3178.379] (3179.759) * [3177.612] (3183.339)  0:00:19 Average standard deviation of split frequencies: 0.007048 Analysis stopped because convergence diagnostic hit stop value. Analysis completed in 13 seconds Analysis used 12.78 seconds of CPU time 
MrBayes will then report various statistics about the run, such as the percentage of time it was able to accept proposed changes of various sorts. These percentages should, ideally, all be between about 20% and 40%, but as long as they are not extreme (e.g. 1% or 99%) then things went well.
Note the section of the output labeled Acceptance rates for moves in the "cold" chain. This gives the proportion of the time that proposals for various parameters were accepted:
In my case this was
MrBayes screen output:
Acceptance rates for the moves in the "cold" chain of run 1: With prob. (last 100) chain accepted proposals by move 33.7 % ( 35 %) Dirichlet(Tratio) 24.8 % ( 21 %) Dirichlet(Pi) 30.3 % ( 31 %) Slider(Pi) 46.1 % ( 44 %) Multiplier(Alpha) 7.6 % ( 12 %) ExtSPR(Tau,V) 2.5 % ( 1 %) ExtTBR(Tau,V) 10.1 % ( 10 %) NNI(Tau,V) 10.5 % ( 16 %) ParsSPR(Tau,V) 33.9 % ( 24 %) Multiplier(V) 28.5 % ( 34 %) Nodeslider(V) Acceptance rates for the moves in the "cold" chain of run 2: With prob. (last 100) chain accepted proposals by move 32.6 % ( 32 %) Dirichlet(Tratio) 25.3 % ( 25 %) Dirichlet(Pi) 31.8 % ( 26 %) Slider(Pi) 48.1 % ( 50 %) Multiplier(Alpha) 8.0 % ( 13 %) ExtSPR(Tau,V) 2.3 % ( 2 %) ExtTBR(Tau,V) 9.2 % ( 13 %) NNI(Tau,V) 10.2 % ( 10 %) ParsSPR(Tau,V) 33.7 % ( 22 %) Multiplier(V) 27.4 % ( 18 %) Nodeslider(V) 

In the above table, 46.1% (in run 1, in run 2 it was 48.1%) of proposals to change the gamma shape parameter were accepted. This makes it sounds as if the gamma shape parameter was changed quite often, but to get the full picture, you need to scroll up to the beginning of the output and examine this section:
MrBayes screen output:
The MCMC sampler will use the following moves: With prob. Chain will use move 2.08 % Dirichlet(Tratio) 1.04 % Dirichlet(Pi) 1.04 % Slider(Pi) 2.08 % Multiplier(Alpha) 10.42 % ExtSPR(Tau,V) 10.42 % ExtTBR(Tau,V) 10.42 % NNI(Tau,V) 10.42 % ParsSPR(Tau,V) 41.67 % Multiplier(V) 10.42 % Nodeslider(V) 
This says that an attempt to change the gamma shape parameter will only be made in 2.08% of the iterations. That means, out of 40,000 iterations (called generations by MrBayes), only about 832 attempts were made to change the gamma shape parameter, and in run one 46.1% of those, or about 383, were accepted. If you were keenly interested in the posterior distribution of the gamma shape parameter, you would probably want to base this on more values (you could also check the ESS with Tracer).
This brings up a couple of important points. First, in each iteration, MrBayes chooses a move at random to try. Each move is associated with a "proposal rate" (Rel. prob). The proposal rates can be listed using the
showmoves
command (note: this command is called props in version 3.1.2, where it shows a list of all possible moves). The output will be something like this:
MrBayes screen output:
Moves that will be used by MCMC sampler (rel. proposal prob. > 0.0): 1  Move = Dirichlet(Tratio) Type = Dirichlet proposal Parameter = Tratio [param. 1] (Transition and transversion rates) Tuningparam = alpha (Dirichlet parameter) alpha = 47.088 [run 1, chain 1] 46.620 [run 1, chain 2] 47.088 [run 2, chain 1] 46.156 [run 2, chain 2] Targetrate = 0.250 Rel. prob. = 1.0 2  Move = Dirichlet(Pi) Type = Dirichlet proposal Parameter = Pi [param. 2] (Stationary state frequencies) Tuningparam = alpha (Dirichlet parameter) alpha = 102.020 [run 1, chain 1] 100.000 [run 1, chain 2] 102.020 [run 2, chain 1] 104.081 [run 2, chain 2] Targetrate = 0.250 Rel. prob. = 0.5 3  Move = Slider(Pi) Type = Sliding window Parameter = Pi [param. 2] (Stationary state frequencies) Tuningparam = delta (Sliding window size) delta = 0.208 Targetrate = 0.250 Rel. prob. = 0.5 4  Move = Multiplier(Alpha) Type = Multiplier Parameter = Alpha [param. 3] (Shape of scaled gamma distribution of site rates) Tuningparam = lambda (Multiplier tuning parameter) lambda = 0.878 Targetrate = 0.250 Rel. prob. = 1.0 5  Move = ExtSPR(Tau,V) Type = Extending SPR Parameters = Tau [param. 5] (Topology) V [param. 6] (Branch lengths) Tuningparam = p_ext (Extension probability) lambda (Multiplier tuning parameter) p_ext = 0.500 lambda = 0.098 Rel. prob. = 5.0 6  Move = ExtTBR(Tau,V) Type = Extending TBR Parameters = Tau [param. 5] (Topology) V [param. 6] (Branch lengths) Tuningparam = p_ext (Extension probability) lambda (Multiplier tuning parameter) p_ext = 0.500 lambda = 0.098 Rel. prob. = 5.0 7  Move = NNI(Tau,V) Type = NNI move Parameters = Tau [param. 5] (Topology) V [param. 6] (Branch lengths) Rel. prob. = 5.0 8  Move = ParsSPR(Tau,V) Type = Parsimonybiased SPR Parameters = Tau [param. 5] (Topology) V [param. 6] (Branch lengths) Tuningparam = warp (parsimony warp factor) lambda (multiplier tuning parameter) r (reweighting probability) warp = 0.100 lambda = 0.098 r = 0.050 Rel. prob. = 5.0 9  Move = Multiplier(V) Type = Random brlen hit with multiplier Parameter = V [param. 6] (Branch lengths) Tuningparam = lambda (Multiplier tuning parameter) lambda = 3.657 [run 1, chain 1] 4.042 [run 1, chain 2] 3.549 [run 2, chain 1] 3.883 [run 2, chain 2] Targetrate = 0.250 Rel. prob. = 20.0 10  Move = Nodeslider(V) Type = Node slider (uniform on possible positions) Parameter = V [param. 6] (Branch lengths) Tuningparam = lambda (Multiplier tuning parameter) lambda = 0.191 Rel. prob. = 5.0 Use 'Showmoves allavailable=yes' to see a list of all available moves 
Summing the five proposal rates yields 1+0.5+0.5+1+5+5+5+5+20+5=48. To get the probability of using one of these moves in any particular iteration, MrBayes divides the proposal rate for the move by this sum. Thus, the gamma shape parameter will be chosen with probability 1/48 = 0.0208, i.e. it will be updated in about 2.08% of the iterations.
Second, note that MrBayes places a lot of emphasis on modifying the tree topology and branch lengths (in this case 97% of proposals), but puts little effort (3%) into updating other model parameters. You can change the percent effort for a particular move using the props command. (Note: this is easier to do in version 3.1.2 but version 3.2 gives you more control, in version 3.2 you can also use the propset command in batch mode).
The section entitled "Chain swap information" reports the number of times the cold chain attempted to swap with the heated chain (lower left) and the proportion of time such attempts were successful (upper right). The total number of attempted swaps should be the same as the number of generations, i.e. in this case 40,000.
In my case this was
MrBayes screen output:
Chain swap information for run 1: 1 2  1  0.80 2  40000 Chain swap information for run 2: 1 2  1  0.79 2  40000 Upper diagonal: Proportion of successful state exchanges between chains Lower diagonal: Number of attempted state exchanges between chains Chain information: ID  Heat  1  1.00 (cold chain) 2  0.91 Heat = 1 / (1 + T * (ID  1)) (where T = 0.10 is the temperature and ID is the chain number) 

Running MrBayes in batch mode
MrBayes does not have to be run interactively. Instead, the commands can be written in a separate command block in a Nexus file and they will be executed sequentially. In fact, this is the preferred method of running an analysis once you are familiar with the commands. A minimal MrBayes block would be
A minimal MrBayes block:
begin mrbayes; < YOUR COMMANDS HERE >; end; 
Note that there are semicolons at the end of each command!
Even though the mrbayes block can simply be added below the data matrix in the nexus file, we recommond keeping the data and the commands separate. This way you can easily use the same data set with different settings or programs. Write the following batch file and call it algaemb_batch.nex:
algaemb_batch.nex:
#NEXUS [ Text in square brackets is interpreted as a comment and will not be read by the program ] begin mrbayes; set autoclose=yes nowarn=yes; [ data file ] execute algaemb.nex; outgroup Anacystis_nidulans; [ substitution model ] lset nst=2 rates=gamma ngammacat=4; [ priors ] prset brlenspr=unconstrained:exp(10.0) shapepr=exp(1.0) tratiopr=beta(1.0,1.0) statefreqpr=dirichlet(1.0,1.0,1.0,1.0); [ mcmc settings ] mcmcp ngen=100000 samplefreq=100 printfreq=100 nruns=2 nchains=2 stoprule=yes stopval=0.01; mcmc; sump burninfrac=0.25; sumt burninfrac=0.25; quit; end; 
Now save the file, and  although we are not doing this now because we do not want to overwrite our results  you could rerun the analysis by providing MrBayes with the filename as a command line argument: mb32 algaemb_batch.nex
. In this case you could also redirect the screen output to a file, or you can use the log command in MrBayes. Note: alternatively you could also just start MrBayes and execute algaemb_batch.nex from the program's command prompt.
Summarizing and interpreting the results
The sump command
MrBayes saves information in several files. Only two types of these will concern us today. One of them will be called algaemb.nex.run1.p. This is the file in which the sampled parameter values from run 1 were saved (there is also a corresponding file for run 2, both will be summarized). This file is saved in tabdelimited format so that it is easy to import into a spreadsheet program such as Excel. We will examine this file graphically in a moment, but first let's get MrBayes to summarize its contents for us.
At the MrBayes prompt, type the command sump. This will generate a crude graph showing the loglikelihood as a function of time. Note that the loglikelihood starts out low on the left (you started from a random tree, remember), then quickly climbs to a constant value.
Below the graph, MrBayes provides the arithmetic mean and harmonic mean of the marginal likelihood. The harmonic mean is used in estimating Bayes factors, which are in turn useful for deciding which among different models fits the data best on average. We will talk about how to use this value in lecture, where you will also get some warnings about Bayes factors calculated in this way.
The table at the end is quite useful. It shows the posterior mean, median, variance and 95% credible interval for each parameter in your model based on the samples taken during the run. The credible interval shows the range of values of a parameter that account for the middle 95% of its marginal posterior distribution. If the credible interval for kappa is 3.8 to 6.8, then you can say that there is a 95% chance that kappa is between 3.8 and 6.8 given your data (and of course the model). The parameter TL represents the sum of all the branch lengths. Rather than reported every branch length individually, MrBayes just keeps track of their sum.
The sumt command
Now type the command sumt. This will summarize the trees that have been saved in the files algaemb.nex.run1.t and algaemb.nex.run2.t.
The output of this command includes a bipartition (=split) table, showing posterior probabilities for every split found in any tree sampled during the run. After the bipartition table is shown a majorityrule consensus tree containing all splits that had posterior probability 0.5 or above.
MrBayes also calculates the maximum and average standard deviation of split frequencies (should approach 0.0), as well as the potential scale reduction factor (PSRF, should approach 1.0)^{[5]}. For my tree sample the output looked like this:
MrBayes screen output:
Summary statistics for informative taxon bipartitions (saved to file "algaemb.nex.tstat"): ID #obs Probab. Sd(s)+ Min(s) Max(s) Nruns  9 1202 1.000000 0.000000 1.000000 1.000000 2 10 1202 1.000000 0.000000 1.000000 1.000000 2 11 1075 0.894343 0.017648 0.881864 0.906822 2 12 887 0.737937 0.003530 0.735441 0.740433 2 13 651 0.541597 0.003530 0.539101 0.544093 2 14 478 0.397671 0.011766 0.389351 0.405990 2 15 129 0.107321 0.008236 0.101498 0.113145 2 16 122 0.101498 0.016472 0.089850 0.113145 2  + Convergence diagnostic (standard deviation of split frequencies) should approach 0.0 as runs converge. Summary statistics for branch and node parameters (saved to file "algaemb.nex.vstat"): 95% HPD Interval  Parameter Mean Variance Lower Upper Median PSRF+ Nruns  length[1] 0.008367 0.000013 0.001950 0.015120 0.007852 1.001 2 length[2] 0.022923 0.000033 0.012460 0.034131 0.022379 1.002 2 length[3] 0.007468 0.000012 0.001151 0.014112 0.006961 0.999 2 length[4] 0.100903 0.000248 0.070782 0.132716 0.098969 1.000 2 length[5] 0.027781 0.000096 0.010363 0.047241 0.027005 0.999 2 length[6] 0.132494 0.000459 0.090394 0.172768 0.130331 1.002 2 length[7] 0.115070 0.000380 0.078286 0.152808 0.113921 0.999 2 length[8] 0.109665 0.000377 0.076268 0.152571 0.108245 1.005 2 length[9] 0.020557 0.000031 0.010935 0.031613 0.019942 0.999 2 length[10] 0.031238 0.000077 0.015405 0.048266 0.030336 1.002 2 length[11] 0.033435 0.000176 0.009834 0.060309 0.032323 0.999 2 length[12] 0.014773 0.000062 0.000372 0.029653 0.014318 1.001 2 length[13] 0.021518 0.000096 0.004489 0.041987 0.020622 0.999 2 length[14] 0.017349 0.000068 0.003676 0.033102 0.016950 0.998 2 length[15] 0.006425 0.000026 0.000008 0.014339 0.005335 1.029 2 length[16] 0.028166 0.000208 0.004058 0.052153 0.026935 1.029 2  + Convergence diagnostic (PSRF = Potential Scale Reduction Factor; Gelman and Rubin, 1992) should approach 1.0 as runs converge. NA is reported when deviation of parameter values within all runs is 0 or when a parameter value (a branch length, for instance) is not sampled in all runs. Summary statistics for partitions with frequency >= 0.10 in at least one run: Average standard deviation of split frequencies = 0.007648 Maximum standard deviation of split frequencies = 0.017648 Average PSRF for parameter values ( excluding NA and >10.0 ) = 1.004 Maximum PSRF for parameter values = 1.029 
If you chose to save branch lengths (and we did), MrBayes shows a second tree in which each branch is displayed in such a way that branch lengths are proportional to their posterior mean. MrBayes keeps a running sum of the branch lengths for particular splits it finds in trees as it reads the file algaemb.nex.t. Before displaying this tree, it divides the sum for each split by the total number of times it encountered the split to get a simple average branch length for each split. It then draws the tree so that branch lengths are proportional to these mean branch lengths.
Finally, the last thing the sumt command does is tell you how many tree topologies are in credible sets of various sizes. For example, in my run, it said that the 99% credible set contained 18 trees. What does this tell us? MrBayes orders tree topologies from most frequent to least frequent (where frequency refers to the number of times they appear in algaemb.nex.t). To construct the 99% credible set of trees, it begins by adding the most frequent tree to the set. If that tree accounts for 99% or more of the posterior probability (i.e. at least 99% of all the trees in the algaemb.nex.t file have this topology), then MrBayes would say that the 99% credible set contains 1 tree. If the most frequent tree topology was not that frequent, then MrBayes would add the next most frequent tree topology to the set. If the combined posterior probability of both trees was at least 0.99, it would say that the 99% credible set contains 2 trees. In our case, it had to add the top 18 trees to get the total posterior probability up to 99%.
Type
quit
(or just q
), to quit MrBayes now.
Other output files produced by MrBayes
That's it for the lab today. You can look at plots of the other parameters if you like. You should also spend some time opening the other output files MrBayes produces in a text editor to make sure you understand what information is saved in these files. Note that some of MrBayes' output files are actually NEXUS tree files, which you can open in FigTree. For example, algaemb.nex.con.tre contains the consensus tree from the Bayesian analysis. The file algaemb.nex.trprobs contains all distinct tree topologies, sorted from highest to lowest posterior probability. The file algaemb.nex.mcmc contains useful statistics about the MCMC run (e.g. proposal acceptance rates, etc.).
Using other programs to summarize MCMC results
Tracer
The Java program Tracer is very useful for summarizing the results of Bayesian phylogenetic analyses. Tracer was written to accompany the program Beast, but it works well with the output file produced by MrBayes as well.
After starting Tracer, choose File > Import... to choose a parameter sample file to display. Select the algaemb.nex.run1.p in your working folder, then click the Open button to read it.
You should now see 8 rows of values in the table labeled Traces on the left side of the main window. The first row (LnL) is selected by default, and Tracer shows a histogram of loglikelihood values on the right, with summary statistics above the histogram.
A histogram is perhaps not the most useful plot to make with the LnL values. Click the Trace tab to see a trace plot (plot of the loglikelihood through time).
Tracer determines the burnin period using an undocumented algorithm. You may wish to be more conservative than Tracer. Opinions vary about burnin. Some Bayesians feel it is important to exclude the first few samples because it is obvious that the chains have not reached stationarity at this point. Other Bayesians feel that if you are worried about the effect of the earliest samples, then you definitely have not run your chains long enough! You might be interested in reading Charlie Geyer's rant on burnin some time.
Click the Estimates tab again at the top, then click the row labeled kappa on the left.

Click the row labeled alpha on the left. This is the shape parameter of the gamma distribution governing rates across sites.

Click on the row labeled TL on the left (the Tree Length).

AWTY
AWTY^{[6]}^{[7]}
Running MrBayes with no data
Why would you want to run MrBayes with no data? Here's a possible reason. You discover by reading the text that results from typing help prset that MrBayes assumes, by default, the following branch length prior: exp(10). What does the 10 mean here? Is this an exponential distribution with mean 10 or is 10 the socalled "rate" parameter (a common way to parameterize the exponential distribution)? If 10 is correctly interpreted as the rate parameter, then the mean of the distribution is 1/rate, or 0.1. Even good documentation such as that provided for MrBayes does not explicitly spell out everything you might want to know, but running MrBayes without data can provide answers, at least to questions concerning prior distributions.
Also, it is not possible to place prior distributions directly on some quantities of interest. For example, while you can specify a flat prior on topologies, it is not possible to place a prior on a particular split you are interested in. This is because the prior distribution of splits is induced by the prior you place on topologies. Running a Bayesian MCMC program without data is a good way to make sure you know what priors you are actually placing on the quantities of interest. Even if you think you know, running without data provides a good sanity check.
If there is no information in the data, the posterior distribution equals the prior distribution. An MCMC analysis in such cases provides an approximation of the prior.
To do this in version 3.2 you need to make a simple modification to the batch file algeamb_batch.nex above. Change the line
mcmcp ngen=100000 samplefreq=100 printfreq=100 nruns=2 nchains=2 stoprule=yes stopval=0.01;
to
mcmcp ngen=100000 samplefreq=100 printfreq=100 nruns=2 nchains=2 stoprule=yes stopval=0.01 data=no;
For version 3.1.2 execute the file blank.nex (see below) in MrBayes (NOTE: do not use this file with version 3.2. For some reason the program becomes unresponsive when you try to do it.). Here is what this file looks like (use your favorite text editor to create this file in the MrBayes directory):
blank.nex
#NEXUS begin data; dimensions ntax=4 nchar=1; format datatype=dna missing=? gap=; matrix A ? B ? C ? D ? ; end; begin mrbayes; set autoclose=yes; prset brlenspr=unconstrained:exp(10.0); prset tratiopr=beta(1.0,1.0); prset statefreqpr=Dirichlet(1.0,1.0,1.0,1.0); prset shapepr=exp(1.0); lset nst=2 rates=gamma ngammacat=4; mcmcp ngen=1000000 samplefreq=100 printfreq=100 nruns=1 nchains=1; mcmc; end; 
Note that the data matrix consists of just one character, and that character declares that data are missing for all taxa!
Type
mcmc
to perform the analysis. Because calculation of the likelihood is the most timeconsuming part of a Bayesian analysis, this analysis will go quickly because the likelihood for just one site takes almost no time to compute.
Consulting Bayes' formula, what value of the likelihood would cause the posterior to equal the prior? Is this the value that MrBayes reports for the loglikelihood in this case?
Checking the shape parameter prior
Import the output file blank.nex.p in Tracer. Look first at the histogram of alpha, the shape parameter of the gamma distribution.
What is the mean you expected for alpha based on the prset shapepr=exp(1.0) command in the blank.nex file? What is the posterior mean actually estimated by MrBayes (and presented by Tracer)? An exponential distribution always starts high and approaches zero as you move to the right along the xaxis. The highest point of the exponential density function is 1/mean. If you look at the approximated density plot (click on the Marginal Density tab), does it appear to approach 1/mean at the value alpha=0.0?
Checking the branch length prior
Now look at the histogram of TL, the tree length.
What is the posterior mean of TL, as reported by Tracer? What value did you expect based on the prset brlenspr=unconstrained:exp(10) command? Does the approximated posterior distribution of TL appear to be an exponential distribution? The second and third questions are a bit tricky, so I'll just give you the explanation. Please make sure this explanation makes sense to you, however, and ask us to explain further if it doesn't make sense. We told MrBayes to place an exponential prior with mean 0.1 on each branch. There are five branches in a fourtaxon, unrooted tree (2N3, where N is the number of taxa). Thus, five times 0.1 equals 0.5, which should be close to the posterior mean you obtained for TL. That part is fairly straightforward.
The marginal distribution of TL does not look at all like an exponential distribution, despite the fact that TL should be the sum of 5 exponential distributions. It turns out that the sum of n independent Exponential(λ) distributions is a Gamma(n, 1 / λ) distribution. In our case the tree length distribution is a sum of 5 independent Exponential(10) distributions, which equals a Gamma(5, 0.1) distribution. Such a Gamma distribution would have a mean of 0.5 and a peak (mode) at 0.4. If you want to visualize this, fire up R and type the following commands:
x < seq(0, 2, .001) y < dgamma(x, shape=5, scale=0.1) plot(x, y, type="l")
How does the Gamma(5, 0.1) density compare to the distribution of TL as shown by Tracer? (Be sure to click the "Marginal Density" tab in Tracer)
Binaries of MrBayes 3.2 for Windows and Mac OSX
There are two precompiled binaries in the /Shared/mrbayes folder on the cluster for you to try on your own machine. There is no guarantee these will work on your computer.
The Windows version is: mb3.2svnWindows.zip
The MacOSX version is: mb32osx
Compiling MrBayes 3.2 on Mac and Linux
The following instructions are specific to the version we downloaded from the SVN repository for building the program without BEAGLE support. You'll need a C compiler and GNU autotools installed.
Download mb3207292011.tgz from /Shared/mrbayes folder on cluster to your own computer. (Do not move it from the Shared folder and do not compile it on the cluster).
Doubleclick on it to uncompress this file and move into the newly created folder (called "src")in a Terminal window.
At the prompt enter:
autoconf ./configure withbeagle=no
For this specific download, there are two files in the src folder that you will need to edit
Open the "config.h" file in TextWrangler (or other text editor)
Find the lines (~4344) that say
config.h (lines ~4344)
/* Define to enable beagle library */ /* #undef BEAGLE_ENABLED */ 
and uncomment the second line (remove /* and */) to look like:
/* Define to enable beagle library */ #undef BEAGLE_ENABLED
then... Open the "command.c" in TextWrangler and comment out the line (~56) that reads
command.c (line ~56)
#include "libhmsbeagle/beagle.h" 
to read
/*#include "libhmsbeagle/beagle.h"*/
Go back to the Terminal window (you should still be in the /src directory). Type
make
The computer will chug along. Check to see that there are no "errors". The executable file will be called mb.
More resources
 Fredrik Ronquist's MrBayes page: http://people.sc.fsu.edu/~fronquis/mrbayes/
 Tutorials on the MrBayes wiki site
References
 ↑ Huelsenbeck, J. P., and F. Ronquist. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754755
 ↑ Ronquist F., and J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:15721574
 ↑ Ronquist F., P. v. d. Mark, and J. P. Huelsenbeck. 2008. Bayesian phylogenetic analysis using MrBayes. In P. Lemey, Salemi M., and Vandamme A. (eds.) The Phylogenetic Handbook: a Practical Approach to Phylogenetic Analysis and Hypothesis Testing. Cambridge University Press.
 ↑ Zwickl, D., and Holder, M. T. 2004. Model parameterization, prior distributions, and the general timereversible model in Bayesian phylogenetics. Systematic Biology 53(6):877–888
 ↑ Gelman, A and Rubin, DB (1992) Inference from iterative simulation using multiple sequences, Statistical Science, 7, 457511.
 ↑ Wilgenbusch J.C., Warren D.L., Swofford D.L. 2004. AWTY: A system for graphical exploration of MCMC convergence in Bayesian phylogenetic inference: http://king2.scs.fsu.edu/CEBProjects/awty/awty_start.php
 ↑ Nylander, J. A., J.C. Wilgenbusch, D. L. Warren, D. L. Swofford. 2008. AWTY (Are We There Yet): a system for graphical exploration of MCMC convergence in Bayesian phylogenetics. Bioinformatics 24:581583.