Professional Documents
Culture Documents
School of Distance Education
Universiti Sains Malaysia
Lab Practical Module of
JIB 431/4
Biosystematics
Animal Systematics
Edited by
Lee‐Sim Lim
(leesim.lim@usm.my)
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Contents
Laboratory practical 3:Wet specimen preparation for animals 2
Laboratory practical 4:Phylogenetic construction of species 10
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Laboratory practical 1
Wet specimen preparation for animals
Su Yin Cheea, Lee‐Sim Limb
a
Centre for Marine and Coastal Studies (CEMACS), Universiti Sains Malaysia, 11800 Penang, Malaysia
b
School of Distance Education, Universiti Sains Malaysia, 11800 Penang, Malaysia
Objectives
At the end of this practical, students should be able to:
‐ know two types of wet specimen preparation methods, that are preservation using
formalin and preservation using alcohol.
‐ compare the advantages and disadvantages between preservation using formalin and
alcohol.
‐ pre‐process the collected dead animal before preserve the animal sample.
‐ handle the two types of wet preparation methods based on the guidelines provided.
‐ explain some other animal specimen preservation methods based on reading and group
discussions.
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Introduction
In animal taxonomic studies, voucher specimens are needed as models and references. In the
field, when a voucher sample is collected, it should be immediately bring to the lab or, if the
field is far away from the lab, it needs to be processed and preserved immediately on the spot.
Voucher specimens need to be preserved forever and keep in the museums or institutions for
future references too. Therefore, preservation of the voucher specimens is a key stage to
ensure the quality and the shelf life of the voucher specimens. Well preserved specimens also
generate more accurate morphological information to the taxonomists. In prominent museums
in the world several voucher specimens last more than a century!
There are a few types of specimen preservations for animal specimens, depend on the
purpose of the specimens and the species of the animal specimens. In some occasions, one
voucher specimen can be preserve in a few ways, bats for instance, the whole specimen can be
preserve in alcohol but its skull can be extracted from the body and preserved it as dried
specimen separately. For fish, whole specimens or tissue samples are fixed in formalin for 24‐
48 hours before they are transferred to 80% ethanol for long term preservation. In general,
there are two main types of specimens: the wet specimens and the dry specimens. Dry
preservation of animal specimens are usually more complicated and time consuming thus well
trained skills are needed. Hence in this practical,we will just focus on learning the wet
preservation. Students will be explored the dry preservation through literature searches at the
end of this practical.
Animal specimens can be preserving in liquid preservatives in two ways: preserve using
formalin 10% or either alcohol 70%. There are advantages and disadvantages for both of the
preservative liquids on animal specimens. For long term preservation in the museums, a
fixation process using formalin 10% before permanent preservation in alcohol 70% is needed.
Suggested extra reading materials
Inger, R.F. & Chin, P.K. (1962). The fresh‐water fishes of North Borneo. Chicago Natural History
Museum, Chicago.
K. P. Lim & N. Sivasothi. A guide to methods of preserving animal specimens in liquid
preservatives. http://preserve.sivasothi.com/
Kotellat, M. (2013). The fishes of the inland waters of Southeast Asia : a catalogue and core
bibliography of the fishes known to occur in freshwaters, mangroves and estuaries.
Kotellat, M. & Whitten, T. (1996). Freshwater biodiversity in Asia : with special reference to fish.
Singapore : National University of Singapore.
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Kotellat, M. & Yeo, D.C.J. (2005). Southeast Asian freshwater fish diversity. Dept. of Biological
Sciences, National University of Singapore. Washington, D.C.
Sampling and preservation for reference collections. http://www.loris‐
conservation.org/database/wild_survey/necropsy/Collections.html#fluid_preservation
The body worlds. http://www.bodyworlds.com/en.html (plastination preservation)
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Materials
*Formalin 100%
*Alcohol absolute
Water/ distilled water
Small mammal/fish voucher specimen
Forceps
Fish tagger and tags
Thread
Air‐tight jar
Digital caliper
Weighing scale
Surgical mask
Surgical glove
Surgical tray
Surgical scissors
Camera
Measuring cylinder
A few sheets of blank papers for measurement records
Fishes (bought by students from the market)
Caution!!
*Handle these chemicals in a fume hood or with surgical mask and glove, and away from flame.
Formalin is carcinogenic and will trigger irritation and allergic on normal tissue when get
exposed. Alcohol is a flammable chemical and it will cause irritation after expose for a long
time and in high concentration.
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Procedures
1. Record the locality and sampling date and time for the voucher specimen.
2. Wash the specimen under running tap water to clean it off slime, blood, etc.
3. Take a digital photograph of your specimen. Note: Align your fish with its head facing
the left hand side of the image.
4. Take external measurements of the voucher specimen using a ruler or a digital caliper
(Figure 1).
5. Record the information from step 1 and 3 into a table as shown in Table 1. These
information can be used to form a Truss Network essential in morphometric studies:
Figure 1. Truss network measurements of a fish
6. Identify your fish based on the morphology of its mouth, eye, operculum, pectoral fin,
pelvic fin, dorsal fin, anal fin, caudle peduncle and caudal fin with the aid of an
identification key.
7. Record the sample code, species, sampling locality and date, as well as the person who
collected the specimen on a fish tag.
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8. By using a fish tagger, tag the tear‐proof card at the base of the dorsal fin of the fish as
such (Figure 2):
Figure 2. Tagging site on a fish
9. For specimens that needs to be preserved in 10% formalin, first dilute formalin 100% to
formalin 10% and pour the solution into an air‐tight jar. Then put the labeled specimen
into the jar. Make sure the entire specimen is totally soaked in the solution. Cover the
lid tightly to avoid evaporation.
10. Also prepare 70% ethanol by diluting absolute ethanol with distilled water until the
concentration reaches 70%. The fishes should be transferred to this medium 24‐48
hours (depending on the size of the fish) after fixation in formalin. Replace the ethanol
once a day, then once in two days, then once a week, then once a month or when the
colour of the ethanol in the jar changes. This is to make sure there is no further dilution
of the ethanol from bodily liquids of the fish. Again, ensure the specimen is totally
covered with ethanol.
11. Keep the jars in a dark space that is dry and room temperature that is cool, away from
light sources.
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Table 1. Truss network values of fish
Values for 1‐2, 1‐3, 1‐4, etc. to be measured based on Figure 1
TL = Total length (from the tip of the mouth to the tip of the tail)
SL = Standard length (from the tip of the mouth to the peduncle)
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Discussion
1. If many specimens were collected from the field at one time and limited jars and
chemicals are available in the lab, how can we organise the specimens during
preservation?
2. What are the advantages and disadvantages of preserving specimens in formalin 10%?
3. What are the advantages and disadvantages of preserving specimens in alcohol 70%?
4. How can we ensure the internal organs of a big volume specimens can be preserved as
well?
5. In your opinion, which animal species are not suitable to preserve in the solution?
6. Besides preserving voucher specimens in a solution, what other preservation methods
that we can consider? List of one or two and describe these methods with references.
7. What sort of characters are used for fish identification?
8. What is a Truss Network and why is it important?
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Laboratory practical 2
Phylogenetic analysis
Lee‐Sim Lim
School of Distance Education, Universiti Sains Malaysia, 11800 Penang, Malaysia
Objectives
At the end of this practical, students should be able to:
‐ explain the term phylogeny.
‐ explain the purpose for the construction of phylogenetic relationships on target species.
‐ describe the process of preparing the data for phylogenetic construction, via
morphology and genetic of the species.
‐ construct simple phylogenetic tree using phylogenetic and evolutionary softwares.
‐ analyse tree topology of the constructed phylogenetic tree.
‐ list and brief explain some latest published phylogenetic findings with the evidence of
the publication.
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Introduction
Systematic studies usually will lead to the study of evolutionary history of species. Therefore, in
this practical, students will be exposed to some basic skills in phylogenetic relationships
construction.
Phylogeny means the historical or evolutionary relationships among lineages of
organisms, or may be certain parts of the organisms. We can study the relationships among
targeted species (e.g. the divergence of cat, Felidae family) or targeted genes found in certain
groups of organisms (e.g. the evolutionary history of the hearing genes in echolocating bats).
These relationships are inferred by mathematical algorithms provided in softwares. Since the
calculations by the softwares are calibrated based on predicted evolutionary rates of certain
genes or age of certain fossils, we usually consider the phylogenetic results as inferences rather
than exact results. However, if the information such as substitution rate, fossil age and
molecular clock is accurate, the inferences can be very accurate and match with historical
events for evolution (e.g. sea level change, Last Glacial Maximum or major global biodiversity
turnover).
Since phylogenetic construction base on algorithms and calculations, morphological
data of target species need to be converted into numerical form before import into
phylogenetic programs. In the last few decades, molecular techniques has improved rapidly
and DNA sequences are widely used to construct phylogenetic inferences of target species. In
these cases, sequences with nucleotide bases of each individual, that are A (adenine),T
(thymine),G (guanine) and C (cytosine) can be directly used in the data for the analyses. For
example, one nucleotide represents one character in a sequence, which means a DNA sequence
of 1000 base pairs will have 1000 characters to be analysed for a single individual. This applies
to amino acid sequence data as well. Students will be exposed to some molecular techniques in
obtaining DNA sequences from eucaryotic cells in the lecture. Here, we only focus on how to
analyse the data we already have and expose to some technics in phylogenetic construction.
There are several phylogenetic analytical methods provided to date, included the
Neighbor‐joining, Maximum parsimony, Maximum likelihood and Bayesian methods. Neighbor‐
joining is a method derived from distance‐based approach. This method build phylogram based
on finding two closest operational taxonomic units (OTUs) and link them together at each time,
until the tree is formed. Phylogram generated from neighbor‐joining provides tree topology
and branch lengths. This is a quick method to generate a phylogeny tree and therefore we will
learn this technique in this practical. Unlike neighbor‐joining, maximum parsimony may
generate more than one tree based on one set of data and the best tree is the tree with the
shortest total branch lengths. Maximum likelihood is a method of model based approach and
aims to generate a tree with highest likelihood of producing observed data set. Bayesian
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approach of analysis is another model‐base method which applies Bayesian probability test in
the phylogenetic tree construction. This method assumes the tree topology is known and
calculate branch length of a tree with likelihood method.
Phylogenetic consider a new branch of knowledge which are developing fast. Almost
each year, new lab technics, new analytical methods and new analytical tools are developed
and contribute to this field. Therefore, if you are interested with this, you are encouraged to
explore and read a lot to get yourself updated and improved!
Suggested extra reading materials
Nei, M. and Kumar, S. 2000. Molecular Evolution and Phylogenetics. Oxford University Press:
New York.
Wiley, E.O., Lieberman, B.S. 2011. Phylogenetics: Theory and Practice of Phylogenetic
Systematics. 2nd ed. Wiley‐Blackwell: New Jersey.
Simpson, M.G. 2010. Chapter 14: Plant Molecular Systematics. Plant Systematics. Elsevier Inc.:
USA.
Materials
Computer with internet connection
BioEdit software
MEGA‐X software
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Procedures
1. By using a computer with good internet connection, visit GenBank
(http://www.ncbi.nlm.nih.gov/genbank/).
2. Type a species scientific name in the search space and click on “search” button. For
example, Rhinolophus luctus, cytochrome b. Remains “Nucleotide” for the space in the
left, as we are going to construct the phylogenetic tree using DNA sequences.
Type the species name and the target gene here
3. GenBank will show all the results related to the DNA/genome sequences of the species
you searched for as the printed screen showed below.
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4. Press on the FASTA link of the sequence you are interested. We need the sequence in
FASTA format for our following analysis.
5. FASTA format of the sequence you have selected will be displayed. Click on the “Send”
link to save the sequence into your computer. For the saving options, choose
“Complete record” and “File”. For the file format, choose “FASTA” then click on “Create
file” and save it to your destination.
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6. Now you can open your file in BioEdit, a software installed in your computer.
7. Back to GenBank, try to search for the gene (Cytochrome b) you are looking for just now
for four other species within the same genus (e.g. Rhinolophus sinicus, Rhinolophus
pearsonii, Rhinolophus ferrumequinum, Rhinolophus arcuatus), by repeating step 4 and
step 5.
**Please note that if you plan to study a gene for a species, you need to choose the
same gene for the following species that you are interested in.
8. Choose an outgroup for the data you have to polarise the phylogenetic tree you are
going to construct. In my case, I chose Hipposideros armiger. We need an outgroup
from different genus but still close to the species here. So you may consider species
from the same family but different genus. However, target gene of study must be the
same as ingroup, which in this exercise, our target gene is Cytochrome b.
9. Next, try to copy every single sequences that you saved into another newly created file
in BioEdit. To do this, highlight the sequence you decided to copy, go to “Edit” pull down
menu, and click on “Copy sequence (s)” then paste into the new file using “Paste
sequence (s)”. Change the name of the sequence into abbreviation not more than 20
characters, no space, and only “_” symbol is allowed.
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10. Go to Accessory Aplication pull down menu, and choose for “ClustalW multiple
Alignment”.
11. A window will pop out, choose for Full Multiple Alignment. Then press “Run ClustalW”
button at the bottom of the window.
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12. The following screen will pop out, indicating the aligning process. It will take a while,
depend on your data size.
13. When the sequences were aligned, the black window will disappear and another
sequence file generated. Schroll the sequences towards right to compare the aligned
file with the unalign one, what have you notice?
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14. Saved the aligned outcome file in FASTA format, with file name “rhinolophus aligned
outcome”.
15. Start MEGA‐X software, go to File pull down menu and select “Open A File/ Session”.
16. Open the sequences aligned file that you just saved as “rhinolophus aligned
outcome.fas”.
17. Another new window will pop out within the MEGA to ask if you would like to align or
analyse your file. Since we are opening our aligned file, we choose the option “Analyze”.
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18. Another window will pop out after you choose “Analyze”, asking for input data options.
Here, we choose “Nucleotide Sequences” and click “OK”.
19. Third window to pop out is to ask if our data set a protein‐coding nucleotide data, click
“Yes”. Fourth window to come out needs us to select for a genetic code, we choose for
“Vertebrate Mitochondrial”.
20. The file “rhinolophus aligned outcome.fas” is now ready to be analysed by the program,
when you see the window below.
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21. Click on the button “PHYLOGENY”, then choose for “Construct/Test Neighbor‐Joining
Tree”.
22. Click “Yes” for “Would you like to use the currently ctive data (xxxx)?”, when a window
pops out after choosing to “Construct/Test Neighbor‐Joining Tree”.
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23. A Neighbor‐joining (NJ) analysis preferences window will be next displayed. Increase
the Bootstrap replications to 1000 times and keep the rest of the options as they are.
Press OK at the bottom after update the Bootstrap replication to 1000 times.
24. The analysis will start immediately by showing a progress window as below:
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25. A result window will pop out automatically when the analysis is complete, as below:
26. Go to “Image” menu to save your Neighbor‐Joining tree in any of the image format.
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27. Next, save the caption of the generated NJ tree as .txt file by clicking at the “txt” button
as below:
28. Now you can use your saved tree image for your practical reports!
Analysis fo the phylogram
Discuss with your instructor, facilitators and group members, and analyse the phylogram that
generated by you. Write down your analysis.
Discussion
1. What is phylogeny and what are the ultimate goals of phylogenetic studies?
2. By giving examples, show how the morphological characters can be used in computerise
phylogenetic analyses.
3. In phylogenetic analyses, how do we interpret one nucleotide/amino acid?
4. By using Google Scholar, search one latest finding on phylogenetic relationships of some
species on Earth. Summarise the key findings in this study within 50 words and attach
the publication as appendix of this practical report. REMINDER: You need to summarise
the study using your own words and ways of explaination, NO PLAGIARISE PLEASE!
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