Tutorial 3: Identifying signatures of rapid radiations

Objectives

  • Complete the identifying signatures of rapid radiations workflow.

  • Interpret the reported values and generated artifacts in their scientific context.

  • Identify the canonical command references for each analysis step.

Prerequisites and working directory

Install the current PhyKIT release and create a dedicated working directory. Download the data linked in this tutorial into that directory before running the commands. All paths below are relative to this directory.

mkdir phykit-tutorial-03
cd phykit-tutorial-03

Workflow

Signatures of rapid radiations or diversification events can be identified by pinpointing polytomies in a putative species tree (Sayyari and Mirarab 2018; One Thousand Plant Transcriptomes Initiative 2019; Li et al. 2020).

PhyKIT uses a gene-based approach to evaluate polytomies. In other words, PhyKIT will determine what topology each gene supports. Thereafter, PhyKIT will conduct a chi-squared test to determine if there is equal support among gene trees for the various topologies. In the chi-squared test, the null hypothesis is that there is equal support among gene trees for the various topologies and the alternative hypothesis is that there is unequal support for the various topologies. Thus, failing to reject the null hypothesis would indicate that there is a polytomy whereas rejecting the null hypothesis would indicate there is no polytomy. The various topologies examined by PhyKIT are determined by the groups file. Formatting this file will be explained later.

To demonstrate how to identify polytomies, we will use a subset of 250 gene phylogenies from Steenwyk et al. 2019.

Download test data: polytomy_tutorial.tar.gz


Step 0: Prepare data

For this tutorial, the data has already been formatted for the user. There are two input files for the polytomy testing function:

  1. a file that specifies the location of gene trees

  2. a file that specifies the groups to test

Thus, this tutorial assumes that gene phylogenies have already been inferred and the area of the phylogeny that the user wishes to test for a polytomy has already been identified.

Examination of the first file reveals that it is a single column file that specifies the pathing of gene phylogenies to use during polytomy testing. Examination of the second file reveals that groups are specified using a tab-separated five column file.

column 1: an identifier for the test, which is not used by PhyKIT. Instead, this column is intended to be for the user to write any keywords or notes that can help remind them of what they were testing.

column 2-4: the tip names in the groups. Each column represents a single group to conduct polytomy testing for. If a group has multiple taxa, separate each tip name using a semi-colon ';'. For example, in groups_file0.txt there is one group with Aspergillus_persii;Aspergillus_sclerotiorum wherein this group has two taxa, Aspergillus_persii and Aspergillus_sclerotiorum.

column 5: the outgroup taxa. This column specifies the name of outgroup taxa, which are used to root the gene trees prior to determining what topology they support.

Step 1: Conduct polytomy test

Among the groups that have already been predetermined for the user, we will first conduct a polytomy test for groups_file0.txt. To execute the polytomy test, use the following command:

phykit ptt -t filamentous_fungi_250_trees.txt -g groups_file0.txt

Gene Support Frequency Results
==============================
chi-squared: 19.425
p-value: 6.1e-05
total genes: 240
0-1: 103
0-2: 49
1-2: 88

Note, if you are getting an error, it may be due to improper pathing in filamentous_fungi_250_trees.txt. Please check this file and modify it accordingly.

We will now go over the output of PhyKIT. PhyKIT will report the chi-squared value, the p value, the total number of genes used, followed by the support of sister relationships examined. Here, the chi-squared value is very high and the p value is very low indicating that the null hypothesis was rejected and that there is no evidence of a polytomy. The total number of genes used during the polytomy test was 240. However, you may have noticed that there were 250 genes used as input. This discrepancy is not an error but may be caused by two different reasons. (1) 10 genes were unable to be used due to incomplete taxon representation in the groups and (2) PhyKIT can account for gene tree uncertainty (i.e., gene phylogenies with collapsed bipartitions), which may render the support of a given gene tree to be uncertain and therefore not be used during polytomy testing.

Next, the section 0-1, 0-2, and 1-2 refers to the sister relationships between the groups. Group 0 is specified in column 2 of the groups file while group 1 and group 2 are specified in columns 3 and 4, respectively. Thus, 0-1 refers to the following topology (((0,1),2),outgroup); whereas 0-2 and 1-2 refers to the following topologies (((0,2),1),outgroup); and (((1,2),0),outgroup);, respectively. PhyKIT identified that 103 gene phylogenies support (((0,1),2),outgroup); whereas 49 and 88 gene phylogenies support the topologies (((0,2),1),outgroup); and (((1,2),0),outgroup);, respectively.


Next, conduct a polytomy test using the other group file using the following command:

phykit ptt -t filamentous_fungi_250_trees.txt -g groups_file1.txt

Gene Support Frequency Results
==============================
chi-squared: 0.129
p-value: 0.937521
total genes: 248
0-1: 84
0-2: 84
1-2: 80

In contrast to the previous test, the chi-squared value is very low and the p value is very high indicating a failure to reject the null hypothesis. Thus, there is a signature of rapid radiation or diversification event for these groups. Additional details provided by PhyKIT reveal 248 genes were used during the polytomy test and that there is nearly equal support for the various topologies.

Taken together, this tutorial reveals how to identify signatures of rapid radiation or diversification events in phylogenomic data.


Expected artifacts

Each step identifies its expected terminal output or generated files. Confirm that those artifacts exist before continuing to the next step; filenames are relative to the tutorial working directory unless an absolute path is shown.

Troubleshooting

  • Run phykit <command> --help to compare an invocation with the live interface.

  • Confirm that downloaded files are in the current working directory and retain the filenames shown in the tutorial.

  • For parsing errors, compare taxon names exactly across alignments, trees, and trait tables, including capitalization and underscores.

  • See Troubleshooting for installation, format, and error-reporting guidance.