Research Methods in Evolutionary Biology – Assignment 2

Method

Figure 1: Above is the data set of the three sampled species of extant wombat

Uploading and Renaming

1. Download and save FASTA files from myuni
2. Import FASTA files into Geneious Prime, ensure the option to ‘Keep sequences
separated’ is selected.
3. Select all BRCA1 genes, select Edit -> Batch Rename and add the gene name into the
description column, ensure to include the outgroup
4. Complete step 3 for the cytb and ND1 genes
5. For concatenation purposes later remove the suffix from each gene name by going
Edit -> Batch Rename -> Delete and specifying the number of characters required to
delete the underscore and suffix
Aligning Gene Sequences
1. Select all the sequences of one gene (BRCA, ND1, cytb)
2. Select Align/Assemble
3. Select Multiple Align and opt for the MUSCLE option as it is a progressive aligner that
makes comparisons by Log expectations
4. Repeat for all genes

Forming Neighbour Joining Trees

1. Select Gene Sequence you wish to make a tree for and select Tree
2. Select Geneious Tree Builder tab at the top of the menu
3. Ensure Tree Build Method is set to Neighbour Joining
4. Change Outgroup to ‘Koala’
5. Increase number of replicates to 1000 to help differentiate between different
methods
6. Click OK to build the tree
7. Repeat for other gene sequences
Neighbour Joining trees are sometimes inaccurate as they make trees with the shortest
branches, however Maximum Likelihood Trees take into account branch length which assists
in modelling evolution. This is why I also chose to construct ML trees for all sequences and
the concatenated alignments.

Forming Maximum Likelihood Trees

1. Select Gene Sequence you wish to make a tree for and select Tree
2. Select RAxML tab at the top of the menu
3. Include these parameters
Nucleotide Model: GTR GAMMA
Algorithm: Rapid Bootstrapping and search for best scoring
Number of starting trees/b-strap replicates: 1000
Parsimony random seed 1
Partitioning: DNA, p1=1-X\3. 2-X\3
DNA, p2=3-X\3
Additional Command Line Options:-o Koala
4. Under Partitioning substitute X with the length of the sequence
5. Click Ok to create Tree
Concatenated Sequences and Trees
1. Concatenation is important as it allows you to combine multiple alignments to
determine if there is a more comprehensive alignment.
2. Begin by selecting the two (or three) alignments you wish to combine. I chose cytb
and ND1 as these are both mitochondrial genes
3. Select Tools - > Concatenate Sequences or Alignments
 Figure 2: Maximum Likelihood tree of the cytb gene sequence Figure 3: Maximum Likelihood tree of the BRCA1 gene sequence
taken from samples of three extant species of wombat with taken from samples of three extant species of wombat with
Koala as an outgroup Koala as an outgroup

Figure 4: Neighbour Joining tree of the cytb gene sequence taken Figure 5: Neighbour Joining tree of the BRCA1 gene sequence
from samples of three extant species of wombat with Koala as taken from samples of three extant species of wombat with
an outgroup Koala as an outgroup
Figure 6: Neighbour Joining tree of the ND1 gene sequence Figure 7: Maximum Likelihood tree of the ND1 gene sequence
taken from samples of three extant species of wombat with taken from samples of three extant species of wombat with Koala
Koala as an outgroup as an outgroup

Figure 8: Maximum Likelihood tree of the ND1 and cytb

concatenated gene sequence taken from samples of three Figure 9: Neighbour Joining tree of the ND1 and cytb
extant species of wombat with Koala as an outgroup concatenated gene sequence taken from samples of three
extant species of wombat with Koala as an outgroup