This example shows how to generate a timetree in MEGA. For this analysis, MEGA uses a Timetree Wizard window which will walk you through the necessary steps. The data files used in this example can be found in the MEGA/Examples folder (The default location for Windows users is C:\Users\UserName\Documents\MEGA7\Examples\. The default location for Mac users is $HOME/MEGA/Examples, where $HOME is the user’s home directory).
Setting up the analysis
From the main MEGA window, select Clocks | Compute Time Tree (RelTime-ML). The Timetree Wizard window, which outlines the 6 steps for creating a timetree in MEGA will be displayed.
Step1: First, we will load a sequence alignment file. In the Timetree Wizard window, click the Browse... button and then using the file open dialog, find and select the “mtCDNA.meg” sequence alignment file. After the alignment file is parsed by MEGA, the Load Tree File action in step 2 will become enabled.
Step 2: Second, we will load the newick tree file which gives the topology for our timetree. Click the Browse … button and using the file open dialog that is displayed, find and select the “mtCDNA.nwk” tree file. After this file is parsed and validated against the sequence alignment begin used, step 3 will become enabled.
Step 3: Next, we need to specify an outgroup taxon (we will specify one but multiple taxa can be in the outgroup). Click the Select Taxa… button and the Taxa/Groups window will be displayed with all taxa in our data listed in the Ungrouped Taxa list box (alternatively you can click the Select Branch… button and use the Tree Explorer to specify the outgroup). Select the gibbon taxon and move it from the Ungrouped Taxa list box to the Taxa in Outgroup list box by clicking the left-pointing arrow. Click the Close button to save your changes and exit the Taxa/Groups dialog.
Step 4: Now, an option to specify divergence time calibrations constraints will become available (if this step is skipped, then only relative times of divergence will be calculated). Click the Add Constraints… button. MEGA will display the Calibration Editor window that is used for specifying divergence time constraints in the timetree.
First, we will create a divergence time calibration constraint by specifying two taxa whose most recent common ancestor is the node for which the time constraint applies. In the Calibration Editor window, select the Calibration | Calibrate MRCA menu item (or click the add new constraint button on the upper left toolbar [it looks like a clock with a plus sign on the bottom right]). This will create a new calibration constraint with a default name. From the Taxon A and Taxon B dropdown lists select chimpanzee and bonobo. The Calibration Name edit box and the MRCA Node Label edit box are populated with default names but you can edit these if you like. The MRCA node label is especially useful for interpreting the tabular Timetree output produced by MEGA’s Timetree system so that you can quickly identify calibrated nodes by name instead of by node number. In the Min Divergence Time edit box enter 1.2. In the Max Divergence Time edit box enter 5.0.
Next, we will create another calibration constraint by selecting a node in the tree display. In the tree display, select the node whose descendents are orangutan and sumatran (click this node to select it. It will then have a red diamond around it when it is selected). Select Calibration | Calibrate Selected Node menu item (or on the upper-right toolbar, click the new divergence time constraint button [it also looks like a clock but has a plus sign on its lower-left instead of lower-right]). This will create a new calibration. Now type 13.0 in the Max Divergence Time edit box. Leave the Min Divergence Time Edit box blank. Click the Finished button to complete step 4.
Step 5: Next, we can set several analysis settings such as substitution model, treatment of missing data, etc… Back in the Timetree Wizard window, click the Set Analysis Options… button in order to open the Analysis Preferences dialog. Click the Save button to use the default settings.
Step 6: Finally, in the Timetree Wizard window, click the Execute button. Progress will be displayed as the analysis runs. When the analysis completes, the Tree Explorer window will return and display the time tree.
Viewing the results
In the Tree Explorer window, the calculated timetree will be displayed with absolute times of divergence for all branching points in the tree shown. Blue diamonds indicate those nodes which were used to calibrate the tree. To display node height error bars, click View | Show/ Hide | Node Height Error Bars (if branch lengths are also shown, you can hide them by clicking View | Show/Hide | Branch Lengths).
Select File | Export Current Tree (Timetree) and MEGA’s text editor will be displayed with a description of the tree in tabular format.
Go back to the Tree Explorer window and select View | Show/Hide | Node Ids from the main menu. Now the divergence times are no longer shown but node Ids are shown. These correspond to the Node Ids in the tabular description of the timetree in the text editor.
The first column in the text editor has node labels. The one specified in the Calibration Editor is there and there is another one which was contained in the mtCDNA.nwk file. Open the mtCDNA.nwk file using the text editor and find this node label.
Select View | Show/Hide | Data Coverage and the data coverage for each internal node in the tree will be displayed.