The evolution of mammoths and their living relatives

STUDENT’S GUIDE
Case Study
The evolution of
mammoths and their
living relatives
Anna Lorenc
Max Planck Institute for
Evolutionary Biology, Plön
Dean Madden [Ed.]
NCBE, University of Reading
Version 2.0
Case Stu
Case Stud
evolution of mammoths
Introduction
How are Asiatic and African elephants
related to Woolly mammoths?
IMAGE FROM: Sedwick, C. PLoS Biology 6 (4): e99
Woolly mammoths (Mammuthus primigenius) were a very successful species
that are thought to have existed in huge numbers. They ranged from Spain
to North America. The oldest fossils of woolly mammoths are 150 000 years
old. Most woolly mammoths died out at the end of the Pleistocene (10–12 000
years ago), while the most recent remains date from just 3 700 years ago.
Mammoths are closely related to present-day elephants, but until very
recently the exact relationship between these species was unknown. Did
mammoths share a common ancestor of today’s elephants (B)? Or were they
more closely related to one of the modern elephant species (A or C)?
A
B
C
African
elephant
Asiatic
elephant
Asiatic
elephant
Woolly
mammoth
African
elephant
Woolly
mammoth
Asiatic
elephant
Woolly
mammoth
African
elephant
Three possible evolutionary
relationships between Woolly
mammoths and modern elephants.
Scientists have disputed this relationship for a long time. Comparison
of teeth, the shape of the trunk tip, hair structure and immunological
reactions have all failed to resolve the dispute. Fortunately, DNA can be
Copyright © Anna Lorenc and Dean Madden, 2011
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used to assess this relationship — DNA from living elephants and DNA
extracted from a ~33 000-year-old mammoth leg found frozen in Siberia in
1986. (The frozen mammoth was named Enmyn after the Enmynveem river
valley in which it was found.)
DNA is the ultimate ‘forensic’ record of evolution. The DNA sequences of
the two living elephants can be compared with that from the mammoth
and an evolutionary tree based on differences in the sequences can be built.
The sequence data provided comes from mitochondrial DNA (mtDNA).
Mitochondria are the organelles within cells that enable energy to
be utilised. Mitochondria have their own small genome and each
mitochondrion contains several copies of it. Cells can contain tens of
thousands of mitochondria, so mtDNA is plentiful and it is easier to extract
from cells than nuclear DNA. This explains why mtDNA was, for many
years, the main source of ancient DNA.
20 cm
Part of the right back leg of a Woolly
mammoth used to obtain mtDNA
sequence data for this exercise. The
DNA was extracted from the very wellpreserved muscle tissue.
ATP synthase particles
Intermembrane
space Matrix
Cristae
Ribosome
Granules
DNA analysis
As the mtDNA used here is quite a long stretch of DNA (16 842 bases),
aligning three sequences would take a considerable time, even with
computer software. Therefore alignments have been prepared in advance
in the document: Elephants_and_mammoth.geneious.
DNA
Inner membrane
Outer membrane
Structure of a mitochondrion, showing
the location of the DNA.
1. Double click on the Elephants_and_mammoth.geneious document.
This will start the Geneious software and load the file of DNA sequence
data into the programme. Hint: if a box appears over the Geneious startup screen, saying that your trial of the ‘Pro’ version has ended, click on
‘Use Geneious Basic’.
2. The main Geneious window will now show the mtDNA sequences from
the Woolly mammoth, Asiatic and African elephants:
Copyright © Anna Lorenc and Dean Madden, 2011
IMAGE FROM: Rogaev, et al. PLoS Biology, 4(3): e73
evolution of mammoths
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evolution of mammoths
Zoom buttons
The ‘identity’ graph over the
sequences shows you how similar they
are: green= identical; mustard = some
differences; red=many differences.
3. Use the magnifying glass buttons to zoom in on the DNA data.
4. Scroll through the data, looking for any differences between the three
sequences of mtDNA (Hint: just look for different colours, or look out
for changes in the green bar at the top of the sequences). Keep a rough
note of the types of changes, if any, that you find e.g., A changing to C,
G changing to C etc. Don’t spend too long on this: the idea is simply to get
a first impression of the similarities and differences between the three
species’ sequences.
Questions
1. Which types of nucleotide substitutions are the most common?
2. Can you suggest why certain types of nucleotide mutations are more
common than others? Hint: think about the chemical structure of the
different bases in DNA, shown below.
Adenine
Thymine
Guanine
Cytosine
Copyright © Anna Lorenc and Dean Madden, 2011
An outline of the structure of DNA
and the base-pairing mechanism.
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evolution of mammoths
5. You should find that most of the sequences are identical in the three
species. Comparing sequences by eye is obviously very laborious
and prone to error (especially if you are colour blind). Fortunately,
Geneious has another method of allowing you to compare sequences:
the Statistics panel.
Statistics panel
(use the ‘%’ tab to
display this)
6. Select any two sequences by clicking on the name on the left and
holding down the � or � key (on a Mac) or the Ctrl key (on a PC) as you
select them, then examine the data in the Statistics panel. Look at the
‘Pairwise % Identity’ value. By comparing pairs of species’ sequences,
can you predict which of the three evolutionary trees shown in the
introduction might be correct?
7. You can build an evolutionary (phylogenetic) tree to check your
prediction. To make a reliable tree, we need an ‘outgroup’ — a
species that is only distantly-related to the elephants, which will
give the tree a root. For fun, we have chosen a species that was alive
at the time of the mammoths — the Neanderthal. Like the mammoth,
we have mtDNA recovered from fossil remains of this species.
Double-click the Geneious
Neanderthal.geneious
document:
Elephants_mammoth_
Select the mtDNA
sequences here by
clicking on the name.
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evolution of mammoths
8. Ensure that the alignment that includes the Neanderthal is sleected
in the uppermost window, then click the Tree button at the top of the
Geneious main window. A dialogue box will appear. Choose the options
shown below, then click OK.
Choose Tamura-Nei as the Genetic
Distance Model and NeighborJoining as the Tree build Method.
Select Neanderthal as the
outgroup from the drop-down list.
Select Resample tree, then set
the Number of replicates to 1,000
and the support threshold to 5%.
Ensure that Create Consensus
Tree is selected.
Tree build
button
9. A phylogenetic tree will be generated. Ensure you select Show Tip Labels
(Names) in the panel on the right hand side.
Select ‘Display names’ here.
Questions
1. Can you suggest why the relationship between modern elephants
and the Woolly mammoth was disputed for so long?
2. Which of the three trees shown on page 2 (A, B or C) is correct?
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evolution of mammoths
Extension activities
Living relatives of elephants
IMAGE FROM: Hans Hillewaert. Wikipedia.org
The Dugong (Dugong dugon) and Rock hyrax (Procavia capensis) are thought
to be close relatives of modern elephants. Their mitochondrial DNA
(mtDNA) has also been isolated and sequenced and can be used to generate
an evolutionary tree (phylogeny).
IMAGE FROM: Louise Murray. Science Photo Library
Rock hyrax (Procavia capensis).
Dugong (Dugong dugon).
If your computer is connected to the internet, you can download the mtDNA
sequences for these two animals. Alternatively, because it can take a long
time to align the sequences, you may be given the data ready to analyse.
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evolution of mammoths
To download the data from the internet
1. Start Geneious and click on Nucleotide under NCBI in the column on the
left-hand side:
2. Enter an Accession code in the search box and click the Search button:
3. The sequence will be downloaded from the internet and displayed in
the main Geneious window:
GenBank database
accession codes
The accession codes for the complete
mtDNA sequences from the following
species are:
African elephant
DQ316069
Asiatic elephant
DQ316068
Woolly mammoth
DQ316067
N. American mastodon
EF632344
Rock hyrax
NC_004919
DugongNC_003314
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evolution of mammoths
4. Drag the dowloaded sequence into the same folder as the two elephant,
mammoth and Neanderthal sequences, then search for and download
the second mtDNA sequence from the NCBI database. Drag the second
downloaded sequence in the same folder as the others.
5. Select all three sequences at the same time (by holding down the Shift
key as you click on the names), then click the Alignment button at the
top of the Geneious window.
Alignment
button
Select all of the
sequences by holding
down the ‘Shift’ key as
you click on them.
6. When the dialogue box appears, ensure that Genious Alignment is
selected, then click the OK button.
IMPORTANT
Alignment, even of a small number of
DNA sequences, can take several hours
on a slow computer. You may therefore
wish to use the ready-aligned sequences
provided instead (see next page).
7. The aligned sequences can be used to generate an evolutionary tree as
described on the following page.
Copyright © Anna Lorenc and Dean Madden, 2011
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evolution of mammoths
Using ready-aligned sequences to generate
an evolutionary tree
Either:
Double click on the document called: Dugong_Hyrax_Mammoth_
Elephants.geneious. This will start Geneious if the programme is not
already running.
Or:
Use the sequences you have downloaded and aligned.
1. Generate a phylogeny (evolutionary tree) by selecting the data in the
upper Geneious window and clicking the Tree button at the top of the
Geneious main window. A dialogue box will appear. Choose the options
shown below, then click OK.
Choose Tamura-Nei as the Genetic
Distance Model and NeighborJoining as the Tree build Method.
Select Neanderthal as the
outgroup from the drop-down list.
Select Resample tree, then set
the Number of replicates to 1,000
and the support threshold to 5%.
Ensure that Create Consensus
Tree is selected.
Tree build
button
Questions
1. Describe where the Rock hyrax and Dugong are positioned on the
evolutionary tree.
2. What does the tree tell you about how closely related these two animals
are to modern elephants and mammoths?
3. If you placed humans on the same tree, where would you put them?
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evolution of mammoths
Mammoths and mastodons
The extinct North American Mastodon (Mammut americanum) had thick
body hair, like a Woolly mammoth but unlike modern elephants. It had
straighter tusks than the Woolly mammoth and unlike the mammoth, its
back did not slope and it had a larger, flatter head. The mastodon was about
the same size as a modern Asian elephant (~3 m tall). The mastodon’s teeth
suggest that it was a browser (that is, it fed on high-growing vegetation),
not a grazer (unlike modern elephants and the Woolly mammoth).
Question
IMAGE FROM: Dantheman9758. Wikipedia.org
1. Based on its physical appearence and habitat, where would you
place the North American Mastodon on the evolutionary tree of the
mammoth and living elephants?
North American mammoth or
Mastodon (Mammut americanum).
Mitochondrial DNA has been recovered and sequenced from a Mastodon
tooth found in Alaska. This has been aligned with mtDNA from modern
elephants and the Woolly mammoth.
1. Double click on the document called: Plus_Mastodon.geneious. This
will start Geneious if the programme is not already running, and load
the mtDNA sequence data into the software.
Copyright © Anna Lorenc and Dean Madden, 2011
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evolution of mammoths
1. Generate a phylogeny (evolutionary tree) by selecting the data in the
upper Geneious window and clicking the Tree button at the top of the
Geneious main window. A dialogue box will appear. Choose the options
shown below, then click OK.
Choose Tamura-Nei as the Genetic
Distance Model and NeighborJoining as the Tree build Method.
Select Neanderthal as the
outgroup from the drop-down list.
Select Resample tree, then set
the Number of replicates to 1,000
and the support threshold to 5%.
Ensure that Create Consensus
Tree is selected.
Tree build
button
Questions
1. Does the tree generated by the mastodon mtDNA data agree with your
prediction?
2. How can you explain the pattern of evolutionary relationships
shown in the tree? (You may wish to research the Bering Strait
landbridge theory and to find out more about Mammoths and
Mastodons, and the evolutionary history of the elephant family.)
Further reading
Cooper, A. (2006) The year of the mammoth. PLoS Biology, 4(3): e78.
doi: 10.1371/journal.pbio.0040078.
This is an easy-to-follow article that places the sequencing of mammoth
mitochondrial genomes in context and outlines the methods used to date.
Gross, L. (2006) Reading the evolutionary history of the woolly mammoth
in its genome. PLoS Biology, 4(3): e74. doi: 10.1371/journal.pbio.0040074.
A simple report which explains the findings of the Rogaev et al. paper.
Rogaev, E.I. et al (2006) Complete mitochondrial genome and phylogeny of
pleistocene mammoth Mammuthus primigenius. PLoS Biology, 4(3): e73. doi:
10.1371/journal.pbio.0040073.
This is one of the scientific papers that this exercise is based on.
Rohland, N. et al (2007) Proboscidean mitogenomics: Chronology
and mode of elephant evolution using mastodon as outgroup.
PLoS Biology, 5(8): e207. doi: 10.1371/journal.pbio.0050207.
The mastodon sequence data was obtained from this research.
Rohland, N. et al (2010) Genomic DNA sequences from mastodon and woolly
mammoth reveal deep speciation of forest and savanna elephants. PLoS
Biology, 8(12): e1000564. doi: 10.1371/journal.pbio.1000564.
Nuclear DNA shows that African elephants are two different species: Forest
and the Savanna elephants, which diverged 2.6–5.6 million years ago.
Copyright © Anna Lorenc and Dean Madden, 2011
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Note
All of these papers are available freeof-charge at: www.plosbiology.org
Additional references are listed in the
teacher’s notes.
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