The following exercises will help familiarize you with the basic tasks Notung can perform on a gene tree. The tree files used in these exercises are included in the Notung distribution, in the `sampleTrees` folder. If the program window becomes too cluttered, you may close trees that are no longer being used by selecting the tree and clicking on “File → Close.”

E.1 Exercise 1 - Reconciling a gene tree with a species tree

In this exercise, you will reconcile the gene tree genetree_NOTCH with the species tree speciestree_mega. You will also generate a pruned species tree, and use Notung to determine the upper and lower bounds on the time when a duplication occurred.

Open the tree files

Click “File → Open Gene Tree” and open genetree_NOTCH.
The gene tree is located in the sampleTrees folder, which is included in the downloaded zip file. Once loaded, the gene tree is displayed in the tree panel.
Click “File → Open Species Tree” and open speciestree_mega.
The species tree is located in the sampleTrees folder. Once loaded, the species tree appears in the tree panel. Because it is the most recent tree opened, it is now selected.

Note that the options that Notung offers differ depending on whether a species tree or a gene tree is selected. For example, because speciestree_mega is now selected, the box showing parameter values in the lower right corner has disappeared, and the task panel includes only two task modes, History and Annotation.

Reconcile the gene tree with the species tree

Click on the genetree_NOTCH tab to select the gene tree.
Click the “Reconciliation” tab.
The Reconciliation task panel opens below. From here you can reconcile a gene tree with a species tree, display a pruned species tree, show duplication bounds, and hide duplication marks and loss nodes.
Click “Reconcile/Rereconcile.”
The Reconciliation dialog appears. In this dialog box, Notung asks you to specify which species tree to use for the reconciliation and what naming convention is used in the gene tree to specify the species associated with each gene.
Select speciestree_mega in the drop-down menu labeled “Please select a species tree to reconcile with.”
Currently, the only selection available is speciestree_mega. However, if you have more than one species tree open in Notung, you must specify here which species tree to use.
Under the section labeled “Specify Species Label” select “Prefix of the gene label.”
This section in the dialog box asks you to specify the naming convention used in the gene tree to indicate from which species the genes originated. Notung tries to guess the naming convention, but it does not always guess correctly. Notung should have guessed correctly in this case. In general, remember to check the leaf node names in your gene tree during this step to make sure that they agree with the naming convention you choose.
For more details about the species label naming conventions, see Appendix A.4 - Specifying the Species Associated with Each Gene.
In the dialog box, click “Reconcile.”
Click on image to see larger version

Figure E.1: The gene tree should now look like this.

The reconciled gene tree now appears in the tree panel. The D/L Score of the reconciled tree, displayed in the bottom-left corner of the program window, is 20.5 - five duplications and thirteen losses. Five red D’s in the tree mark the inferred duplications. At the right end of the tree (at the leaves), thirteen loss nodes appear in light gray type.

Display the pruned species tree

The leaves of speciestree_mega include more species than are relevant to genetree_NOTCH. After reconciliation, you can view the species tree pruned of all species that are not represented by genes in the gene tree.

Click the “Show Pruned Species Tree” button.
A dialog box appears asking you to give a title for the pruned species tree. The default title is “Pruned Species Tree.”
In the dialog box text field, enter “Mega_Pruned” (or any other name you like), then click “OK.”
The pruned species tree appears in the tree panel. It contains only seven leaf nodes, all of which are species represented in the reconciled gene tree. The pruned species tree has a tab above the tree panel, labeled “Mega_Pruned.” You can now select and use this tree as you would any other species tree.

Click on image to see larger version

Figure E.2: The pruned species tree should look like this.

Check the duplication bounds

The duplication bounds provide information regarding when gene duplications occurred in the course of species evolution.

Select genetree_NOTCH.
Click “Display Options → Display Internal Node Names.”
Node name labels appear in red type next to each internal node. You can now identify each duplication by name. If internal node names are not provided in the gene tree file, Notung will assign the node an alphanumeric name (e.g. n132).
Select the Mega_Pruned species tree.
Click “Display Options → Display Internal Node Names.”
Node name labels appear in red type next to each internal node.
Select genetree_NOTCH.
Click the “Reconciliation” tab.
Click on “About This Tree → Duplication Bounds and Loss Counts” menu item.
A new window appears. Inferred duplications are listed in the left column, expressed as node names in the gene tree. The lower and upper bounds are listed in the middle and right columns, respectively, and are expressed as internal node names in the species tree. Information on losses is displayed below duplication bounds. The left column lists the species nodes in the species tree. The right column provides the number of losses that occurred in each species.
Find the duplication node in the bottom-right area of the tree, from which the XENLAnotch1 gene extends. Find its node name and duplication bounds in the “Duplications and Losses” window.
The node name may vary, depending on how many internal nodes Notung has counted in your current session.
Close the window and select the pruned species tree Mega_Pruned.
With Mega_Pruned selected, you can see internal nodes representing euteleostomi and coelom. The duplication occurred somewhere on the edge between those nodes.

E.2 Exercise 2 - Rooting an unrooted tree

The gene tree genetree_ANK is unrooted. In this exercise, you will select a root based on duplication loss parsimony.

Open the tree files

Click “File → Open Gene Tree” and open genetree_ANK.
The gene tree is located in the sampleTrees folder.
Since this tree is unrooted, it has a trifurcation (a node with 3 children) at the top of the tree, but is otherwise binary.
Click “File → Open Species Tree” and open speciestree_mega. If speciestree_mega is already opened, you may skip this step.
Be sure that the genetree_ANK tab is selected before proceeding.

Run the Rooting Analysis

Click the “Rooting” tab.
The Rooting task panel is displayed. Notung is now in Rooting mode.
Click “Run Rooting Analysis.”
A diagnostic message appears warning you that this tree contains a trifurcation at its root and may be unrooted. Click “OK.”
You will be asked to reconcile the tree. Select speciestree_mega and “Prefix,” click “Reconcile”. The edge at the top of the tree panel, leading to caeel*unc-44, is colored red. This means it has the minimum root score.
Optional: Click the “Display root score” checkbox.
Each edge is labeled with its root score. Notice that the red edge leading to caeel*unc-44 has a root score of 4.0. The next lowest score is 8.5.

Select a root

Click on the red edge in the tree panel.
The tree is now rooted on the edge leading to the caeel*unc-44 gene. The D/L Score of the tree is now 4.0, with two duplications and one loss.

Click on image to see larger version

Figure E.3: The gene tree should now look like this.

E.3 Exercise 3 - Rearranging a gene tree

In this exercise, you will reconcile the gene tree genetree_SMALL with the species tree speciestree_small and use Notung’s rearrangement tasks to investigate alternate gene trees with minimum D/L Score. Both input trees are located in the sampleTrees folder.

Reconcile the gene tree with the species tree

Click “File → Open Species Tree” and open speciestree_small.
Click “File → Open Gene Tree” and open genetree_SMALL.
This is an artificial tree made up for this exercise. The edge weights in this tree represent bootstrap values. Note that two internal edges have a bootstrap value of 100, one has a bootstrap value of 73, and several have not been assigned a weight. (Note that edges adjacent to leaves are usually not assigned bootstrap values since those edges are present in all trees.) Notung sets the default edge weight threshold to 90% of the maximum edge weight in the tree. Since the maximum edge weight in this tree is 100, the edge weight threshold is set to 90.0.
Click the “Reconciliation” tab.
Click “Reconcile/Rereconcile.”
In the “Reconciliation Options” dialog box, select speciestree_small and “Postfix” and click “Reconcile.”
The reconciled tree appears in the tree panel. Note that it has a D/L Score of 10.0, with four duplications and four losses.

Rearrange the reconciled tree

Click the “Rearrange” tab.
The Rearrange task panel is now displayed.
Click the “Highlight weak edges” checkbox.
Several edges in the reconciled tree are highlighted in yellow. These are edges with weights below the Edge Weight Threshold and are considered “weak.” Weak edges may be rearranged to reduce the number of duplications and losses in the tree. Edges with weights above the threshold will not be rearranged.
Note that in addition to the edge with weight 73.0, the internal edges with no edge weight are also highlighted in yellow. Notung assumes that any internal edge that is not explicitly assigned a weight is considered weak.

Click on image to see larger version

Figure E.4: The gene tree with weak edges highlighted.
Click “Perform Rearrangement.”
The rearranged tree appears in the tree panel. It now has a D/L Score of 4.0, with two duplications and only one loss.

Click on image to see larger version

Figure E.5: The gene tree should now look like this.

Change the parameter values and rearrange again

In the previous steps, we rearranged the tree using the default parameter values (c_D=1.5 and c_L=1.0). For the default values, there is only one minimum cost tree. We now explore what happens when we rearrange the tree when duplications and losses are weighted equally.

Click the “Edit Values” button in the bottom-right corner of the program window.
In the dialog box, change the Duplication Cost to 1.0.
Click “Apply Changes.”
A message appears to warn us that although we have changed the parameter values, this has had no effect on the tree. We must rearrange the tree again to see the effect of rearrangement with this choice of parameter values. Click “OK.”
Duplications and losses are now weighted equally in Notung’s reconciliation algorithm.
Click the “Rearrange” tab, if it is not already selected.
Click “Perform Rearrangement.” The tree is rearranged with the new parameter values. The newly rearranged tree appears in the tree panel. The D/L Score of this tree is 3.0, with three duplications and no losses.
Click on image to see larger version

Figure E.6: The gene tree should now look like this.

View a different alternate event history

With the new parameter values, there is more than one alternate gene tree with minimal D/L Score. You are currently viewing history 0.

In the Rearrange task panel, click on the drop-down menu labeled “Select an optimal event history.”
This opens a list of available alternate event histories. You should see history 0 and history 1.
Select history 1.
A different tree appears. This tree also has a D/L Score of 3.0, but has two duplications and one loss instead of three duplications and no losses.

Swap nodes in the rearranged tree

Note that this tree groups gB_human with gA_mouse and gA_human with gB_mouse. However, the tree that groups gA_human with gA_mouse and gB_human with gB_mouse has the same score.

Click the “Examine same cost swaps" button.
Nodes that can be interchanged without changing the D/L Score are marked with enlarged light blue boxes.
Click the gB_human node.
To select the node, you must click on the enlarged blue box. When you are able to click and select a node, a blue triangle will mark the node(s). Once selected, the node is marked with a light blue triangle. Each node it can be swapped with is marked with a pink triangle. In this case, there is just one: gA_human.
Click the gA_human node.
The nodes gB_human and gA_human are swapped. Once they have been swapped, they are temporarily highlighted with yellow triangles, so that you can see the results of the most recent action. Note that the gA genes are now grouped together, and the gB genes are together in the same subtree, along with the g_gorilla gene.

Click on image to see larger version

Figure E.7: The gene tree should now look like this.

Try performing additional swaps to see how many alternate, minimum cost trees you can find.

E.4 Exercise 4 - Binary gene tree with a non-binary species tree

In this exercise, you will perform Notung’s main tasks on the gene tree exercise4_genetree with the non-binary species tree exercise4_speciestree. You will reconcile and root the gene tree, and use Notung to determine the upper and lower bounds on the time when a duplication occurred.

Open the tree files

Click “File → Open Gene Tree” and open exercise4_genetree.
This is an artificial tree made up for this exercise.
Click “File → Open Species Tree” and open exercise4_speciestree.
As you will notice, this is a non-binary species tree with a polytomy representing the common ancestor of the marsupials.

Reconcile the gene tree with the species tree

Select exercise4_genetree.
Click the “Reconciliation” tab.
Click “Reconcile/Rereconcile.”
In the “Reconciliation Options” dialog box, select exercise4_speciestree and “Prefix” and click “Reconcile.”
The reconciled tree appears in the tree panel. Note that it has a D/L Score of 8.0, with two duplications, one conditional duplication, and five losses. Two red D’s in the tree mark the required duplications, while the one pink cD marks the conditional duplication. At the leaves of the tree, five loss nodes appear in light gray type.

Click on image to see larger version

Figure E.8: The gene tree should now look like this.

Check the duplication bounds

The duplication bounds provide information regarding when gene duplications occurred in the course of species evolution.

Click “Display Options → Display Internal Node Names.”
Select exercise4_speciestree.
Click “Display Options → Display Internal Node Names.”
Select exercise4_genetree.
Click the “About This Tree → Duplication Bounds and Loss Counts” from the menu.
In the new window, required duplications are described first. Conditional duplications are described below the required duplications. For both types of duplications, the duplication nodes are listed in the left column, expressed as node names in the gene tree. The lower and upper bounds are listed in the middle and right columns, respectively, and are expressed as internal node names in the species tree. Information on losses is provided below the conditional duplication bounds.
Close the window by clicking the “Close this window” button.

Run the Rooting Analysis

Click the “Rooting” tab.
Click “Run Rooting Analysis.”
The edge leading to genes from placental mammals (cow, mouse, and human) is colored red. This means it has the lowest root score.
Optional: Deselect “Display Internal Node Names” (under “Display Options“”) and click the “Display root score” checkbox.
Notice that the red edge has a root score of 7.0. The next lowest root score is 8.0.
Click “Display Options → Display Internal Node Species Names.”
The name of the species to which the node is mapped appears in italics next to each internal node.
Select the optimal root by clicking on the red edge in the tree panel.
The tree is rooted on the edge which splits the tree between placental mammals (Eutheria) and marsupials (Metatheria). The D/L Score of the tree is now 7.0, with two duplications, one conditional duplication, and four losses.
Do not close these trees yet - they will be used in upcoming steps.

Click on image to see larger version

Figure E.9: The gene tree should now look like this.

Reconcile the Tree using the Combined Polytomy Losses algorithm

This step uses the command line interface and can be skipped, if desired. You will use the command line interface to reconcile the gene tree exercise4_genetree with the species tree exercise4_speciestree using the combined losses algorithm.

On the command line, navigate to the Notung directory.
For instructions on using Notung from the command line, see Chapter 12.2 - Running Notung from the command line.
Type the following in the command window/terminal and hit enter:
java -jar Notung-2.6.jar sampleTrees/exercise4_genetree -s sampleTrees/exercise4_speciestree --reconcile --exact-losses --outputdir sampleTrees --report-heuristic-losses
Notung will print information to the screen as it reconciles the tree for both combined and explicit losses. Notice that the first unrooted gene tree has a D/L Score of 8.0, with two duplications, one conditional duplication and five heuristic losses as compared to the second unrooted gene tree, which has a D/L Score of 7.0, with two duplications, one conditional duplication, and four exact losses. The tree, reconciled and with exact losses, will be saved to the sampleTrees folder (as specified by --outputdir) as exercise4_genetree.reconciled.

Root the tree reconciled with the Combined Polytomy Losses algorithm

In the previous step, you reconciled the gene tree while using the combined polytomy losses algorithm. In this step you are will find the optimal root for this gene tree. If you skipped the previous step, you will need to use the gene tree exercise4_genetree-exactLosses.ntg instead of exercise4_genetree.reconciled.

In Notung’s graphical user interface, click “File → Open Gene Tree” and open exercise4_genetree.reconciled.
If you skipped the last step, use exercise4_genetree-exactLosses.ntg instead.
A warning will appear stating that the tree was reconciled using --exact-losses. Click the “OK” button.
Click the “Rooting” tab.
Click “Run Rooting Analysis.”
Select the optimal root by clicking on the red edge in the tree panel.
The tree is rooted on the edge leading to placental mammals. The D/L Score of the tree is now 6.0, with two duplications, one conditional duplication, and three losses.

Click on image to see larger version

Figure E.10: The gene tree should now look like this.

Compare this tree with the previously rooted gene tree (exercise4_genetree). Can you find the difference between the trees? In exercise4_genetree, the loss node, tasmanian_devil*LOST, above the subtree containing genes gene3 and gene2, has been moved below the duplication node and combined with opossum*LOST and bandicoot*LOST in the gene3 and gene2 subtrees, respectively, in exercise4_genetree.reconciled. This resulted in a reduction of the total number of losses.

View polytomy losses without species names included

There are two display options for polytomy losses. In this step, you will see the other way to display these losses.

Select the exercise4_genetree.reconciled gene tree. (Use
exercise4_genetree-exactLosses.ntg if you skipped the step for reconciling the tree using the exact losses algorithm.)
Deselect the “Display Options → Use Species Names in Polytomy Losses” option. The gene tree no longer shows the species names for polytomy losses. For example the loss that was previously displayed as “[tasmanian_devil, bandicoot] of Metatheria*LOST” is now displayed as “2/4 of Metatheria*LOST.”
Click on image to see larger version

Figure E.11: The gene tree should now look like this.

E.5 Exercise 5 - Non-binary gene tree with a binary species tree

In this exercise, you will perform Notung’s main tasks on the non-binary gene tree exercise5_genetree with the species tree exercise5_speciestree. You will reconcile, root, resolve, and rearrange the gene tree, and use Notung to determine some general statistics about the trees.

Open the tree files

Click “File → Open Gene Tree” and open exercise5_genetree.
This is an artificial tree made up for this exercise. Notice that this gene tree is non-binary and contains multiple polytomies.
Click “File → Open Species Tree” and open exercise5_speciestree.
Select exercise5_genetree.
Click “Display Options → Highlight Polytomies.”
The polytomies in the gene tree are circled and highlighted in cyan.

Click on image to see larger version

Figure E.12: The gene tree with polytomies highlighted.

Reconcile the gene tree with the species tree

Click the “Reconciliation” tab.
Click “Reconcile/Rereconcile.”
In the “Reconciliation Options” dialog box, select exercise5_speciestree and “Prefix” and click “Reconcile.”
The reconciled tree appears in the tree panel. Note that it has a D/L Score of 20.0, with ten duplications and five losses. Also note that some of the polytomies have more than one duplication associated with the node (ex: the polytomy with eight children has two duplications).

Click on image to see larger version

Figure E.13: The gene tree should now look like this.

Get general tree statistics for the gene tree

In this step you will gather some general statistics about the reconciled gene tree and the species tree.

Click on “About This Tree → General Tree Statistics”
The General Tree Statistics window appears. In this window is information on both the gene tree, the reconciled gene tree, and the species tree. You may have to scroll down to view all the information.
The General Tree Statistics Window should look like this.

Click on image to see larger version

Figure E.14: The General Tree Statistics Window should look like this.
After reviewing this information, click “Close this window” to close the window and continue.
For more information on the data in the General Tree Statistics window, see Chapter 3.4 - General Tree Statistics.

Resolve the polytomies in the gene tree

In this step, you will resolve all the polytomies in the gene tree, thus creating a binary gene tree.

Click the “Resolve” tab.
The Resolve task panel opens below.
Make sure that the “Highlight polytomies” checkbox is selected.
The polytomies in the gene tree are circled and highlighted in cyan.
Click “Resolve Polytomies.”
The resolved tree appears in the tree panel. Edges associated with the resolved polytomies are now colored cyan. This is the same tree as before, only now the polytomies have been resolved. The number of duplications and losses are identical to the reconciled tree, and even the duplication bounds are the same.

Click on image to see larger version

Figure E.15: The gene tree should now look like this.

Change the parameter values and view alternate event histories

In the previous steps, we reconciled and resolved the tree using the default parameter values (CD=1.5 and CL=1.0). For the default values, there is only one minimum cost tree. We now explore what happens when we reconcile the tree when duplications and losses are weighted equally.

Click the “History” tab.
We must go back in the history before we change parameter values, as the tree has already been resolved and the change in values might effect the current resolution of the tree.
Go back to the pre-resolved step in the history by clicking “Reconciled with exercise5_speciestree.”
The tree panel shows the state of the tree before the polytomies were resolved.
Click the “Edit Values” button in the bottom-right corner of the program.
In the dialog box, change the Duplication Cost to 1.0.
Click “Apply Changes.”
Duplications and losses are now weighted equally, and the gene tree is automatically rereconciled with the new parameter values.
Click the “Reconciliation” tab.
The reconciled tree appears in the tree panel. There is now more than one alternate gene tree with the minimal D/L Score. You are currently viewing history 0.
Click on the drop-down menu labeled “Select an optimal event history.”
Select history 1.
A different tree appears. This tree has a D/L Score of 15.0, with ten duplications and five losses. This tree has the same duplications and losses as the tree reconciled with a duplication cost of 1.5 and a loss cost of 1.0 (see Figure E.14).
Click on the drop-down menu labeled “Select an optimal event history” and select history 0.
A different tree appears. This tree also has a D/L Score of 15.0, but has eleven duplications and four losses rather than the ten duplications and five losses in history 1. The large polytomy with seven children now has three duplications and one loss, whereas in history 1 it had two duplications and two losses.

Click on image to see larger version

Figure E.16: The gene tree should now look like this.

Run the Rooting Analysis

Click the “Rooting” tab.
Click “Run Rooting Analysis.”
Many edges and one polytomy are colored red, which indicates that all of these components of the tree have the lowest root score.
Notice that the large polytomy is circled in red. Placing a root at a polytomy indicates that at least one edge in the binary resolution of the polytomy has the lowest root score.

Click on image to see larger version

Figure E.17: The gene tree should now look like this.
Optional: Click the “display root score” checkbox.
Each edge and polytomy is labeled with its root score.
Select an optimal root by clicking on the polytomy with the red circle.
The tree is rooted on the polytomy and the D/L Score of the tree is still 15.0, with eleven duplications and four losses.

Click on image to see larger version

Figure E.18: The gene tree should now look like this.

Resolve the polytomies in the gene tree

In this step, you will resolve all the polytomies in the gene tree, thus creating a binary gene tree.

Click the “Resolve” tab.
The Resolve task panel opens below.
Make sure that the “Highlight polytomies” checkbox is selected.
The polytomies in the gene tree are circled and highlighted in cyan.
Click “Resolve Polytomies.”
The resolved tree appears in the tree panel. Edges associated with the resolved polytomies are now colored cyan.

Click on image to see larger version

Figure E.19: The gene tree should now look like this.

View a different alternate event history

With these parameter values, there is more than one alternate gene tree with minimal D/L Score. You are currently viewing history 0.

Click on the drop-down menu labeled “Select an optimal event history.”
This displays a list of available alternate event histories. You should see history 0 and history 1.
Select history 1.
A different tree appears. This tree also has a D/L Score of 15.0, but has ten duplications and five losses instead of eleven duplications and four losses.
Note that these alternate histories correspond to the same alternate histories that were presented after reconciliation.

Swap nodes in the resolved tree

Note that this tree groups human-gene-BB1 with mac-gene-BB2 and human-gene-BB2 with mac-geneBB1. However, the tree that groups human-gene-BB1 with mac-geneBB1 and human-gene-BB2 with mac-gene-BB2 has the same score.

Click the “Examine same cost swaps” button.
Nodes that can be interchanged without changing the D/L Score or history implied by the polytomies are marked with enlarged light blue boxes.
Click the node for mac-gene-BB1.
The node is now marked with a light blue triangle. Each node it can be swapped with is marked with a pink triangle. In this case, there is just one: the node leading to mac-gene-BB2.
Click the node for mac-gene-BB2.
The nodes mac-gene-BB1 and mac-gene-BB2 are swapped. Once they have been swapped, they are temporarily highlighted with yellow triangles, so that you can see the results of the most recent action. Note that the BB1 genes are now grouped together, and the BB2 genes are together in the same subtree.

Click on image to see larger version

Figure E.20: The gene tree should now look like this.

Annotate the Gene Tree

This step will introduce you to Notung’s annotations capabilities.

Click the “Annotations” tab.
The Annotations task panel is displayed.
Click on the “New” button to add a new annotation.
A box will appear to edit the new annotation.
In the space labeled “Please enter a title for the annotation”, type in “-A” Select a color from the palate and click “OK”.
This will automatically annotate all the leaves that contain the string “-A” with the color you selected.
Click on the “New” button. In the space labeled “Please enter a title for the annotation,” type in “-BA” and select a color from the palate and click “OK.”
This will automatically annotate all the leaves that contain the string “-BA” with the color you selected.
Click on the “New” button. In the space labeled ‘Please enter a title for the annotation,” type in “BB1” and select a color from the palate and click “OK”.
This will automatically annotate all the leaves that contain the string “BB1” with the color you selected.
Click on the “New” button. In the space labeled “Please enter a title for the annotation,” type in “BB2” and select a different color from the palate and click “OK”.
This will automatically annotate all the leaves that contain the string “BB2” with the color you selected.
Click on the “New” button. In the space labeled “Please enter a title for the annotation,” type in “BA 4, 5, 6” and select a different color from the palate and select the button labeled “I want to manually select the nodes and subgroups to add.” Click “OK”.
This option lets you select the nodes to add to the annotation without searching for a substring.
Click on the node leading to the subtree with genes BA4, 5, and 6 in humans.
Notice that these leaves were previously in the color selected in step 3. The leaves are a new color now because the newer annotation takes precedence.
Click on the “New” button. In the space labeled “Please enter a title for the annotation,” type in “BA 1, 2, 3” and select a different color from the palate and select the button labeled “I want to manually select the nodes and subgroups to add.” Click “OK.”
Click on the node leading to the subtree with genes BA1, 2, and 3 in humans.
Click on image to see larger version

Figure E.21: The gene tree should now look something like this.

Rearrange the resolved tree

In this step, you will rearrange the gene tree to obtain the minimal D/L Score. In this exercise, you have resolved the polytomies in the gene tree before rearranging the weak areas of the tree. However, it is possible to do both task at the same time while in the rearrangement mode. Both Resolve and Rearrangement are available because these two functions have different purposes. If you want to obtain a hypothesis of the binary gene tree, but wish to retain all the information in the gene tree, use the Resolve task mode. However, if you wish to consider edges with an edge weight below a certain value as uninformative, use the Rearrangement task mode.

Click the “Rearrange” tab.
Click the “Highlight weak edges” checkbox.
Several edges in the reconciled tree are highlighted in yellow. These are edges with weights below the Edge Weight Threshold and are considered “weak.“” Weak edges may be rearranged to reduce the number of duplications and losses in the tree. Edges with weights above the threshold will not be rearranged.

Click on image to see larger version

Figure E.22: The gene tree with weak edges highlighted.
Click “Perform Rearrangement.”
The rearranged tree appears in the tree panel. It has a D/L Score of 15.0, with twelve duplications and only three losses. Note that the score did not change; the rearranged tree is not necessarily “better” than the original tree.

Click on image to see larger version

Figure E.23: The gene tree should now look like this.

View a different alternate event history

You are currently viewing history 0.

Click on the drop-down menu labeled “Select an optimal event history.”
This opens a list of available alternate event histories. You should see history 0, history 1, and history 2.
Select another history and examine the same cost swaps by clicking the “Examine same cost swaps” button.
Nodes that can be interchanged without changing the D/L Score are marked with enlarged light blue boxes. Try performing additional swaps to see how many alternate, minimum cost trees you can find.
See if you can find the original tree by changing the histories and examining same cost swaps.
HINT 1: Select the history with ten duplications and five losses.
HINT 2: Swap the subtree of BA1 and BA2 genes in “pan” with the LOST “pan” gene in the BA subtree.
HINT 3: Swap the subtree of BA4, BA5, and BA6 in human with the node for BA3 in human.