SBB3023 BIODIVERSITY AND EVOLUTION OF PROTISTA AND ANIMALIA

LABORATORY SESSION
BIODIVERSITY AND EVOLUTION: GENETIC VARIATIONS

Introduction
One way of constructing evolutionary tree is by using similarities and differences in
organism genetic characteristics. During this lab session, you are going to use genetic
sequences from GenBank, which is an online database for genetic sequences of
various organisms, to construct phylogenetic tree. The method you are going to
learn during this session can be used to investigate differences between individuals
within species, identify species, and study the relatedness between species. The
phylogenetic tree that you will construct today is for hominoid species by using
cytochrome b gene sequence. Figure 1 shows the working area in constructing a
phylogenetic tree.

Own computer Online

BioEdit BLAST (GenBank)
(collect and handle sequences (compare own sequence to
in xxx.fas format) GenBank sequence)

MEGA GenBank
(construct phylogenetic tree in (find DNA sequence)
in xxx.meg format)

Figure 1. Working areas in constructing phylogenetic tree.

In general, you will find DNA sequences of cytochrome b gene of several hominoid
species (Table 1), align the sequences, and then construct the phylogenetic tree.

Table 1. List of hominoid species

Common name Species

Human Homo sapiens
Gorilla Gorilla gorilla
Sumatran orangutan Pongo abelii
Common chimpanzee Pan troglodytes

Objectives
1. To compare different hominoid species based on their DNA sequence.
2. To construct hominoid species phylogenetic tree.
Methodology

1. Download BioEdit and MEGA into computer

BioEdit download: http://www.mbio.ncsu.edu/BioEdit/bioedit.html

Download Mega: http://www.megasoftware.net/

2. Get cytochrome b sequences of different hominoid species

Go to GenBank (https://www.ncbi.nlm.nih.gov/nuccore). Type in “gorilla cytb” in the
search string box, chose “Nucleotide” and run a search in Genbank by clicking on
Search. Matching sequences will then be displayed. Choose for example “Gorilla
gorilla mitochondrion, complete genome”. The next page that comes up will show
the whole nucleotide sequence of the gorilla mitochondria at the end of the web
page). You have to find where the cytochrome b sequence begins. Scroll down until
you find the following text in the margin:

Click on “gene” so that you get the cytochrome b sequence only i.e. from nucleotide
14169 to 15309.

3. Copy sequence from GenBank to BioEdit

The next step is to transfer the sequences from GenBank to BioEdit (a programme
for sequence analysis). Before you can paste the sequence into BioEdit, you have to
obtain the sequence in FASTA format. Go to the bottom right of the GenBank page
and click on FASTA. Then highlight and copy (ctrl+C) the sequence.

Open BioEdit, go to “File” and choose “New alignment”. A new untitled window will
open. On the window, make sure that “Mode:” is set as “Edit” and “Insert” in the
upper left corner. Click on “File” and the “Import from clipboard”. BioEdit will then
automatically convert the file to a continuous nucleotide sequence and put it in your
alignment file. To rename the sequence (~out), click on “Sequence” --> “Rename” -->
“Edit Title”. No space should be put in the name. Save the file.

4. Add cytochrome b gene sequences for the same species in the BioEdit
alignment file
Add another 4-5 sequences for gorilla by using BLAST search based on the gorilla
sequence you have in the alignment file. First, copy your gorilla sequence in BioEdit
by highlighting the sequence name, and choose “Edit” and “Copy sequences to
clipboard (FASTA format)”.

Then go to BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and click on “Nucleotide

BLAST”. Now you see an open field where you can paste the copied sequence from
your alignment file.
Under “Choose set search”, select “Others” and “Nucleotide collection (nr/nt)”.

You can also try “Optimization for” with “HIghly similar sequence (megablast)” or
“More dissimilar sequences (discontiguous megablast)”. Click on the “BLAST” button.
The search will take a few seconds.

You will get the following result for the BLAST search. First an illustration of the
“Distribution of the top 100 Blast Hits on 100 subject sequence”, followed by a long
list of hits (sequences that show similarity to your sequence). Hits are ranked
according to similarity with the most similar sequence on the top of the list.
Click on the first hit.

You will be directed to the sequence. Click on GenBank. To copy the sequence, click
“FASTA” at the top left of the webpage, highlight the sequence and copy. Transfer
the sequence into BioEdit as in Step 3. Add other 4-5 sequences of the same species
in the BioEdit by re-running the BLAST search.

5. Add cytochrome b gene sequence from other species in the alignment file.
Repeat the GenBank search for other hominoid species as listed in Table 1. Perform
the BLAST search for each species as is Step 4.

6. Align sequence in ClustalW

Click “Accessory Application”, select “ClustalW Multiple alignment” in BioEdit, and
click “Run ClustalW”. A new file will open showing your sequences that have been
aligned. Save the alignment in the xxx.fas format.

Questions:
i. Examine your aligned sequences. Compare sequences from different species. Are
there any differences shown? If yes, give an example.
ii. Now compare sequences from same species. Are there any differences shown? If
yes, give an example.
Iii. Compare sequences from Gibbon and Orangutan. Are there more differences
within the same species or among the species?
7. Import file containing aligned sequences into MEGA
Open MEGA. Click on “File”, select “Convert file to MEGA format”. In the box, select
your BioEdit file containing aligned sequences, select FASTA format, and click “OK”.
Give a name to your MEGA file and save. The file is now converted from FASTA
format into MEGA format. Check that the file looks like example below:

8. Activating file in MEGA

Click on “File”, select “Open a file/session”, and choose your xxx.meg file. An “Input
data” window will pop up, click “Nucleotide Sequence” and then “OK”. MEGA will
ask “protein-coding sequence?”, so click “Yes”. Then for “Select Genetic Code”,
choose “Vertebrate Mitochondrial” and click “OK”.

9. Construct a phylogenetic tree in MEGA

Under “Analysis”, select “Phylogeny”, and go to “Neighbour-Joining (NJ)”. Click
“Compute”.

On the screen there will now appear a figure showing a phylogenetic tree. Such a
phylogenetic tree shows the similarities between sequences - the more similar two
sequences are, the closer they are in the tree i.e. the shorter brances link them
together. This tree also provides a picture of how these sequences have evolved, i.e.
how they changed over time. Thus, at least to some extent, it also shows how your
investigated species are related.

Construct another tree. Under “Analysis”, select “Phylogeny”, and go to

“Neighbour-Joining (NJ)”. Under “Test of Phylogeny”, select “Bootstrap” and
replications 1000. Click “Compute”.
You will then get a similar tree but with figures at each node in the tree. This figure
provides statistics for how likely it is that a species further out on a branch is
separated from the rest of the species in the tree. The maximum bootstraping value
is 100 (it is then very safe to say that the species to the right of this node is
separated from the rest of the tree), and the same is included for all the values
between 80 to 100. Values below 50 has no validity at all where in such nodes that
tree can take many other possible forms besides the one suggested in your figure
(what you see is just one suggestion out of many possibilities).

Question:
i. Are there any nodes showing bootstrap values less than 50? Which species are
they?
Ii. Include another Gibbon species called Hylobates lar among your sequence in
BioEdit. How does the tree look like now? Explain your answer in relation to the
variations in the sequences.

The evolutionary change of mitochondrial DNA genes (such as cytochrome b) is

estimated at ca 2% nucleotide substitution per 1 million years (refer to the value
0.02 under the Neighbour-Joining tree you have just made in MEGA). You could then,
from your phylogenetic tree, estimate as approximation time of when two species
split up.

Question:
i. When do you think the Sumatran Orangutan has split up from the Gibbon?