The evolution of mammoths and their living relatives

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Case Study

The evolution of mammoths and their living relatives

Anna LorencMax Planck Institute for Evolutionary Biology, Plön

Dean Madden [Ed.]NCBE, University of Reading

STUDENT’S GUIDE

Version 2.0

Copyright © Anna Lorenc and Dean Madden, 2011

evolution of mammoths

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IntroductionHow are Asiatic and African elephants related to Woolly mammoths?

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.

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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)?

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

Three possible evolutionary relationships between Woolly mammoths and modern elephants.

African elephant

African elephant

African elephant

Asiatic elephant

Asiatic elephant

Asiatic elephant

Woollymammoth

Woollymammoth

Woollymammoth

A B C



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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.

DNA analysisAs 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.

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 start-up 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:

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 well-preserved muscle tissue.

Structure of a mitochondrion, showing the location of the DNA.

DNAInner membrane

Outer membrane

ATP synthase particles

Intermembranespace Matrix

CristaeRibosome

Granules

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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.

Zoom buttons

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

Guanine

Thymine

CytosineAn outline of the structure of DNA and the base-pairing mechanism.

The ‘identity’ graph over the sequences shows you how similar they are: green= identical; mustard = some differences; red=many differences.



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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.

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 document: Elephants_mammoth_Neanderthal.geneious

Statistics panel(use the ‘%’ tab to

display this)

Select the mtDNAsequences here by clicking on the name.



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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.

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.

Tree build button

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?

Choose Tamura-Nei as the Genetic Distance Model and Neighbor-Joining 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.



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Extension activitiesLiving relatives of elephants

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).

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.

Rock hyrax (Procavia capensis).

Dugong (Dugong dugon).

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GenBank database accession codes

The accession codes for the complete mtDNA sequences from the following species are:

African elephant DQ316069Asiatic elephant DQ316068Woolly mammoth DQ316067N. American mastodon EF632344Rock hyrax NC_004919Dugong NC_003314

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:



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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.

Select all of the sequences by holding down the ‘Shift’ key as you click on them.

Alignment button

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.



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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.

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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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

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 orMastodon (Mammut americanum).

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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.



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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 readingCooper, 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.

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.

Note All of these papers are available free-of-charge at: www.plosbiology.org

Additional references are listed in the teacher’s notes.

Tree build button


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The evolution of mammoths and their living relatives

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