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Bioinformatics and Systematics of the Beetles
Student Handout
Objectives

Use different types of characters to construct phylogenetic trees.

Use multiple trees to model the most likely evolutionary relationship between taxa.
Introduction
Scientists researching systematics use several types of evidence to construct evolutionary histories that
can be displayed as phylogenetic trees. Morphology has long been used to determine shared ancestry and
construct these trees; however, convergent evolution may lead to strikingly similar morphologies between
distantly related species. Modern genetic analyses, in particular nucleic acid and amino acid sequencing,
have allowed scientists to infer phylogenies based on genetic similarity, which is less prone to
convergence than morphology. In this exercise, you will create phylogenetic trees based on
morphological and molecular data.
There are more than 400,000 species of beetles, insects belonging to the order Coleoptera, known to
science, making them the most diverse taxon on the planet (Triplehorn and Johnson, 2005). You are
going to classify 5 beetle species using both their observable morphology and their cytochrome c oxidase
subunit I (COI) protein sequences.
COI is the catalytic portion of a large transmembrane protein complex found in the mitochondrial
membrane (Lunt et al., 2006). The COI gene is expressed as the COI protein. Review your textbook to
recall where this protein is located in the mitochondrial membrane and its critical role in energy
metabolism. This gene mutates very quickly and is variable among species, making it one of the most
reliable genes used to classify closely related animals.
Materials
Morphological observations will be collected using both live specimens and photographs of beetle species
available on the Bug Guide1 website.
Genetic information of all living things is published and openly available on the website of the National
Center of Biotechnology Information (NCBI). NCBI has a taxonomy browser2 that you will use to
choose beetles to classify.
Complete URLs are provided in the numbered list at the end of this handout.
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Methods
1. Your phylogeny will include the bean beetle Callosobruchus maculatus. Several COI proteins
have been sequenced for this species; you can choose whichever you prefer. Use this link to visit
the C. maculatus NCBI taxonomy page.3
On the upper right you will see a box of entry records. Click on the number beside the box
indicating “protein”. Depending on the species, you may see as few as one or several thousand
amino acid sequences. For some species, there
may not be protein sequences at all.
2. Find the cytochrome oxidase C subunit I
protein (for example, it is #14 on the protein list
for C. maculatus as of March 2013). Click on
the “FASTA” link underneath to bring up the
amino acid sequence written in letters (for a key
of the amino acid represented by each letter,
click here)4. Copy the entire FASTA sequence
and the associated heading, which includes the protein name, the species name in brackets, and
the GENBANK accession number, and paste this information into a blank MS Word document.
You will need to repeat this for 4 other beetle species.
3. Use the NCBI taxonomy browser to come up with 4 other beetle species to compare to C.
maculatus. Click this link to directly access the beetle page5.
Alternatively, you can browse all the way to beetles. Taxonomy browser2 > Eukaryota >
Eumetazoa > Protostomia > Mandibulata > Insecta > Neoptera > Coleoptera
Browse down to the level of species for each beetle – it will have a binomial scientific name in
italics. For best results, choose species that also have a common name (in parentheses). Pick
some beetles with fun names! Locate and paste the COI FASTA sequences into your MS Word
document, save a copy of the document, and be sure to include your name and any partner’s
names on the document. Be sure the scientific names of all beetles are indicated. You will print it
later.
4. Search online for images of your beetles that clearly show their complete morphology. Some will
look crazy and hopefully you will agree that some are beautiful. Choose images from
www.bugguide.net1 for best results, and copy the image(s) and the link(s) into your document as
a record. You may want to keep open pages or tabs for all of them for the next part of the
exercise. If you do not easily find an image for your exact species, you may either select a
different species and FASTA sequence, or select an alternate image of a closely related species.
5. Identify at least 4 morphological characters that you can use to classify your five species, and
record them in the table below. Choose carefully. You’ll want to choose characters that allow
you to compare traits between species. For instance, you cannot use “green color” as one
character and “blue color” as another – those are just two traits associated with one character,
color. The best characters to choose have two distinct traits, such as present (+) or absent (-).
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Use a beetle field guide or website6 for beetle morphology jargon.
Table 1: Beetle Morphological Characteristics. Write the scientific names of 5 beetle species, choose 4
characters, and fill the table.
Beetle 1
Beetle 2
Beetle 3
Beetle 4
Beetle 5
____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________
Character 1
_____________
Character 2
_____________
Character 3
_____________
Character 4
_____________
6. Next, use the character table to draw a phylogenetic tree. Groups that share characters share a
clade (branched section of the tree), while groups that lack them will typically be classified on a
branch outside the clade.
Figure 1. Draw here your phylogenetic tree of the five beetles using morphological characters.
7. Now, use your FASTA sequences to produce a tree based on COI sequences. This is easily done
online using the phylogentic key online tree builder website linked here7. Scroll down to
“Phylogeny Analysis” and select “One Click”.
8. Enter a “Name of the Analysis”. Paste your 5 FASTA sequences into the large box, preceded by
“> Genus_species” for each of the five sequences. You may click the example link to see the
example sequence entry. Select “submit”. The program will run for a minute, then produce a tree
similar to our example tree below.
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Figure 2. Example phylogenetic tree using the online tree builder.
9. Save your tree as a .PNG or .PDF file, then insert (or copy and paste) that into your MS Word file
of FASTA sequences.
Literature Cited
Lunt, D.H., Zhang, D.X., Szymura, J.M. and Hewitt, O.M. 1996. The insect cytochrome oxidase I gene:
evolutionary patterns and conserved primers for phylogenetic studies. Insect Molecular Biology 5(3):
153-165.
Triplehorn, C.A. and Johnson, N.F. 2005. Borror and DeLong’s Introduction to the Study of Insects. 7th
ed. Brooks/Cole. Belmont, CA.
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Questions
1. Did your two trees dispay the exact same phylogeny? If not, how different were they? Describe the
differences.
2. Look up the taxonomic information of your beetles (suborder, family, subfamily). Does the taxonomy
of these beetles match the phylogenetic trees you produced? If it matches one tree more closely, which is
it? Speculate why that is.
3. List at least 2 alternative morphological characters that would have changed your tree. Would these
build a tree more like the molecular tree or less, and why?
4. Do you predict the molecular tree would have looked different if you had selected a different molecular
marker (e.g., a nuclear gene or marker that is not an enzyme)?
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URLs for Website Resources
1. Bug Guide home page: http://bugguide.net/node/view/15740
2. NCBI Taxonomy Browser: http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/
3. Callosobruchus maculatus NCBI page:
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=64391&lvl=3&lin=f&kee
p=1&srchmode=1&unlock
4. Amino acid letter key: http://www.ddbj.nig.ac.jp/sub/ref2-e.html
5. NCBI Taxonomy Browser Coleoptera Homepage:
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Undef&id=7041&lvl=3&keep=1&
srchmode=1&unlock
6. Beetle Wikipedia entry: http://en.wikipedia.org/wiki/Beetle
7. Phylogenetic Keys for the Non-Specialist: http://www.phylogeny.fr/version2_cgi/index.cgi
This experiment was written by Evan Lampert and Jennifer L. Mook, 2014 (www.beanbeetles.org).
Copyright © by Evan Lampert and Jennifer L. Mook, 2014. All rights reserved. The content of this site may be
freely used for non-profit educational purposes, with proper acknowledgement of the source. All other uses are
prohibited without prior written permission from the copyright holders.
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