Evolutionary relationships and finding new drugs in Daffodils




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Evolutionary relationships and finding new drugs in Daffodils


Students’ Sheet


  1. Constructing phylogenies (working out evolutionary relationships)




    1. Why constructing phylogenies (or understanding evolutionary relationships) is important.




    1. How phylogenies are constructed.


  1. Using phylogenies to help in the search for new drugs




    1. Creating a useful phylogeny.




    1. Choosing species to investigate.




  1. Constructing phylogenies (working out evolutionary relationships)




    1. Why constructing phylogenies (or understanding evolutionary relationships) is important.




    1. How phylogenies are constructed.



Sheet 1




a) Narcissus is the genus of plants containing daffodils. The articles on sheet 1 have investigated evolutionary relationships, evolution and useful drugs within this genus. From the information on sheet 1 and from your own ideas explain why scientists may be interested in understanding evolutionary relationships.


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b) A phylogeny, phylogenetic tree, or evolutionary tree is a diagram showing the evolutionary relationships between a group of organisms. Usually these diagrams show evolutionary relationships only, however they can be modified to include evolutionary time or how much a character differs between species.


The basic diagrams represent evolutionary relationships through their branching patterns. Nodes are points in the diagram where one line branches out from another. Phylogenies can be rotated about nodes without changing the evolutionary relationships they represent.
i) How many different possible evolutionary trees are there?
How many different ways can 3 species (A, B and C) be mapped onto the phylogeny below to show different evolutionary relationships? (remember that phylogenies can be rotated without changing the evolutionary relationship they represent)


Phylogenies can also look like this:






How many different ways can 4 different species be related evolutionarily? Draw all out all the phylogenies below. (Hint: the phylogenies come in 2 different shapes).

ii) Choosing the most likely evolutionary tree
So even with just 4 species there are quite a few different ways they could be related. The number of possible phylogenies increases very rapidly as the number of different species increases. For instance for 10 different species there are over 34 million ways they could be related.
So how do we choose which of the possible evolutionary relationships is the most likely?
The simplest way is to choose the evolutionary tree that requires the least number of evolutionary events (character changes) in order to match the current character states of the species being investigated.
To do this we need to have an idea of what the ancestral character states are by looking at one or several species called the “out-group”. The out-group is selected such that the species under investigation are definitely more closely related to each other than any of them are to the out-group. We then look for shared new character states within the species being studied.

Let’s try to do this for 3 species of Narcissus using the common snowdrop (Galanthus nivalis) as the out-group.


Any suitable characters can be used to construct phylogenies. Molecular data (either nucleotide sequences in DNA or amino acid sequences in proteins) is often used to construct phylogenies. Here we are going to use a sequence of 162 amino acids from a fragment of the protein NADH dehydrogenase subunit F (data freely obtainable from www.uniprot.com). Each letter in the sequence represents a different amino acid.
First we need to look at the sequences to see which amino acids (positions 1-162) might be informative. So on sheet 2 use 4 different colours to highlight the amino acid position according to the key below:

      1. If all amino acids at the position are the same for all 4 species then highlight all 4 letters with a vertical stripe in green.

      2. If all 3 Narcissus species share the same letter and that letter is different to the letter of Galanthus nivalis at the position then highlight all 4 letters with a vertical stripe in orange.

      3. If one of the Narcissus species is different but the other 3 species are the same at the position then highlight all 4 letters with a vertical stripe in yellow.

      4. If Galanthus nivalis shares a letter with one of the Narcissus species but the other two species share a different letter then highlight all 4 letters with a vertical stripe in blue.

      5. There should be one position that doesn’t meet any of the conditions above (position 93). This is because it looks like there have been two (or more) evolutionary changes at this position.

T
Sheet 2
he sequence of 162 amino acids from a fragment of the protein NADH dehydrogenase subunit F for 4 different species.
Lines 1a, 2a, 3a, and 4a contain amino acids number 1-100 and lines 1b, 2b, 3b, and 4b contain amino acids number 101-162
1. Galanthus nivalis (Common snowdrop)

2. Narcissus cernuus

3. Narcissus tazetta

4. Narcissus asturiensis

1a. INRNLLLSTM NNKVSFFSKD IYRIDDNVRN RVRYFSTYFR NKYTCTYPHE SDNTMLFPLL VLGLFTLFIG AIGIHFDRGV IDFDLLSKWI TPYADFFHPN

2a. VNRNLLLSTM NNRVSFFSKD IYRIDDNVRN GVRDFSTYFR NKYTYTHPHE SDNTMLFPLL VLVLFPLFIG AIGIHFDLGV IDFDLLSKWL TPSADFFHPN

3a. VNRNLLLSTM NNKVSFFSKD IYRIDDNVRN GVRYFSTYFR NKYTYTYPHE SDNTMLFPLL VLVLFTLFIG AIGIHFDRGV IDFDLLSKWL TPSADFFHPN

4a. VNRNLLLSTM NNRVSFFSKD IYRIDDNVRN GVRYFSTYFR NKYTYTHPHE SDNTMLFPLL VLVLFTLFIG AIGIHFDRGV IDFDLLSKWL TPPADFFHPN

1b. SKDSSDWYEF LKNVVFSVSI ALFGLFVASI LYGSVYSSLQ NLGLVNSFVK KSPKRILLDQ VK

2b. AKDSSDWCEF LKNAVFSVSI ALFGLFVASI LYGSVYSSLQ NLGLVNSFVK KSPKRILLDQ AQ

3b. SKDSSDWYEF LKNAVFSVSI ALFGLFVASI FYGSVYSSLQ NLGLVNSFVK KSPKRILLDQ VK

4b. AKDSSDWCEF LKNAVFSVSI ALFGLFVASI LYGSVYSSLQ NLGLVNSFVK KNPKRILLDQ VQ

Seeing as there are only 3 different possible phylogenies when investigating 3 different species it is possible to map evolutionary changes onto every tree to see which one we would consider to be the most likely. Remember Galanthus nivalis is the out-group and so has a fixed position as the most distantly related species in the phylogeny – we are only investigating the evolutionary relationships between the 3 species of Narcissus.
The most likely phylogeny (i.e. the one that is our best estimate of the true evolutionary relationships) is often selected as the one with the least number of evolutionary changes required to match the current character states of the species being investigated.
We can now try mapping on all of the evolutionary changes that have been highlighted in the sequence of amino acids on to the 3 possible phylogenies on sheet 3:


  1. The amino acid positions highlighted for part 1 of the key previously show no evolutionary changes and so don’t need to be mapped onto the 3 possible phylogenies.




  1. As with selecting the most likely phylogeny, evolutionary changes are usually mapped onto phylogenies so that they require the minimum number of events to match the current character states of the species being investigated.




  1. One way of mapping position 93 onto the phylogenies is done for you (there are other, equally likely ways of mapping this position).




  1. Follow the method used for position 93 to map on the character changes for the amino acid positions you have highlighted for parts 2, 3 and 4 of the key.

Remember in this case:



    • We are assuming that the amino acid present at any particular location in Galanthus nivalis is the ancestral condition (i.e. the condition at the base of the tree).

    • Evolutionary changes are mapped onto the trees in a way that minimises the number of changes needed to produce the amino acids present in the species “at the tips of the branches of the trees”.

    • Sometimes the amino acids at one position in the 4 species can be explained by one evolutionary change but sometimes two evolutionary changes are needed to explain the variation between species (for some trees).


Sheet 3


A

B


C



By mapping all the evolutionary changes onto the 3 possible evolutionary trees you can count up how many evolutionary changes are needed to produce the variation seen in this fragment of a protein for each of the possible trees.
Which evolutionary tree do you think is most likely to represent the true evolutionary relationships between these 3 species and why?
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This method of choosing which phylogeny is best, works when you only have 3 or 4 species of interest and when the evolutionary changes being used turn out to be relatively simple. However when researchers study a large number of species they cannot possibly map evolutionary changes onto all possible trees. Even a computer would struggle to do this in a reasonable amount of time when the number of species of interest increases too far beyond 10. In this case researchers rely on complex statistical analysis and cutting of corners!
Sometimes it is possible to work out the most likely evolutionary tree from the evolutionary changes. This can only be done if there ends up being a reasonably simple course of evolutionary changes and if the researchers have only selected the informative characteristics.
When you mapped the evolutionary changes onto the 3 phylogenies did you notice that some of them always require the same number of evolutionary changes no matter which tree they are being mapped on to?
For example the one done for you, position 93, requires 2 changes on each of the 3 trees. This means that position 93 is an uninformative characteristic for determining the evolutionary relationships between these 3 species.
Complete the table below using your work on sheet 3 to work out which types of characteristic are informative for determining evolutionary relationships.


Number in the key

Description of amino acid variation

Informative characteristic?

1

All amino acids the same


No

2

Amino acid only common to all 3 Narcissus species




3

Amino acid different in only one Narcissus species




4

2 species have one amino acid and the other 2 have a different one




5

Position 93


No

The characteristics that you should have identified as being informative for determining evolutionary relationships are called synapomorphies. They are shared, derived characteristics.
Being “shared” is important because if only one species has the characteristic then it is not informative about evolutionary relationships (characteristic type number 3 in the key).
“Derived” means that something has evolved to be different from the state the characteristic was in for the ancestor of the group of interest. Any characteristic that the species share because that’s what the ancestor had is not informative either (Numbers 1 and 2 in the key).
Position 93 is uninformative because it is possible that the common ancestor of all the Narcissus species has an “S” at that location and only one species has evolved a “P” there instead. That means the “S” state for position 93 is not “derived” and the “P” state is not shared. Therefore there are no “shared, derived” characteristic states for position 93 for these species.
So when looking at lots of different characters, e.g. a whole sequence of 162 different amino acids, it’s quite easy to cut that down very quickly so that we only look at those characteristics which are informative (i.e. the shared derived ones). This is what has been done in the next activity.

2. Using phylogenies to help in the search for new drugs


    1. Creating a useful phylogeny.




    1. Choosing species to investigate.

There are between 65 and 150 known species (depending on your definition of a species) in the genus Narcissus. Some studies have been done on some of these species to look for drugs to treat Alzheimer’s disease or viral infections. Because there are so many different species and because it is time consuming and expensive to discover new drugs, and work out whether they will be effective, some way of targeting which species to investigate is useful.


Due to evolution by natural selection closely related species are likely to have similar characteristics with some differences. This means that there are two strategies to select which species it’s worth investigating first:


  1. Identify species that are closely related to species that have already been shown to have at least “quite useful” or “quite effective” chemicals. In doing this we are hoping that the new species that we investigate have “more useful” or “more effective” chemicals than the ones already known.




  1. Identify areas of the evolutionary tree that haven’t been investigated at all and pick species within those areas. In doing this we are hoping that the new species being investigated might have chemicals that are very different from those chemicals in species that have already been studied and that these chemicals may have useful properties.

You are going to have a go a constructing an evolutionary tree of 12 species of Narcissus in order to then use this to make some decisions about which species should be investigated to look for new drugs to treat Alzheimer’s and viral infections such as H5N1 (bird flu).


The table on sheet 4 shows the character states for amino acids at 13 different locations in the protein fragment studied earlier (NADH dehydrogenase subunit F) for 12 species from the genus Narcissus and the outgroup (Galanthus nivalis). These are the phylogenetically “informative” characters from this protein fragment for these species.
There are over 13.7 billion possible evolutionary trees for these 12 species so it would be impossible to write them all out, map on the character changes and see which tree requires the fewest changes.
Nevertheless you are going to find the most likely evolutionary tree for these species!

  1. Creating a useful phylogeny

It turns out that the character states in the table on sheet 4 suggest an evolutionary tree with the minimum possible changes for those characters. There are 13 characters (the different amino acid positions) and all of them have a single change in amino acid except the one at position 93 as we’ve already seen.


This means that the minimum possible number of changes is 14 for these character states and it turns out that there is one evolutionary tree that only requires these 14 steps.
Constructing the evolutionary tree
The information on sheet 4 is ordered alphabetically by species and so it is difficult to see patterns in which organisms should be grouped together and which species branch off from the rest of the species earlier than others.


  1. Highlight the shared, derived character states for each of the amino acid positions (e.g. the “R”s at position 13). As we’ve seen, position 93 has two changes from the out-group. You should assume that the “Y to S” changed occurred at the very base of the Narcissus genus and so is uninformative. Only the “P” at this location is a shared, derived character state.




  1. Arrange the species in the blank table below so that any species that share derived characteristics for any amino acid position are next to each other.

You might need to do this in rough first before filling in the table.


To save time, and to make it clearer, you could just write in the letters for the derived state at each position.
There are two ways to start building the evolutionary tree:


  1. Tip down




  1. Base up

Either or both of these may help you find the best evolutionary tree. “Tip down” construction involves finding the most closely related pairs of species and working backwards through evolutionary time from there. “Base up” construction involves working from the outgroup and progressively branching the tree into smaller and smaller groups until each species has its own branch. You need to identify which evolutionary changes occur earliest (i.e. those that most species share) for any subset of species and work out your branching pattern from that. We will focus on the “Tip down” construction.

Tip down construction:


  1. Any two species that share all the same letters must share a pair of branches of the tree. Any pairs of species like this should be next to each other in the table on sheet 4 that you completed. Join them up with a small square bracket (see →) on the right-hand side of the table.

  2. Some of these pairs of species will share one or more derived amino acid character states with one (but no more than one) other species. Link this third species to the pair with another square bracket (see →).

  3. Some of the groups of species that you have already identified will share one or more derived amino acid character states with one other species or with one other group of species. Link up these groups as before (e.g. see ↓)




  1. Keep going until all species are linked together. You have now drawn the evolutionary tree for this group of species.

  2. On a separate sheet of paper now draw out this tree to fill the whole sheet.

  3. Map the evolutionary changes onto the tree as you did before and check that the amino acid sequences for all species can be explained by only 14 changes (including the “Y to S” in position 93 at the very base of the Narcissus genus).

  4. Now check your evolutionary tree with the one on sheet 5. Remember that trees can be rotated about branch points without changing the evolutionary relationships that it shows. This means that your tree may not look exactly like the one on sheet 5 but may still show the same relationships so check carefully!

A
Sheet 4


mino acids at positions that are informative characteristics for working out the evolutionary relationships between these species.

Species

Amino acid at each position number

13

34

47

75

78

93

101

108

112

131

142

152

162

Galanthus nivalis (Common snowdrop) = outgroup

K

Y

Y

H

R

Y

S

Y

K

L

L

S

K

Narcissus asturiensis

R

Y

H

H

R

P

A

C

K

L

L

N

Q

Narcissus atlanticus

K

Y

Y

R

R

S

S

C

K

L

F

S

K

Narcissus calcicola

K

Y

Y

H

R

S

S

C

K

L

F

S

K

Narcissus cernuus

R

D

H

H

L

S

A

C

K

L

L

S

Q

Narcissus jacetanus

R

Y

H

H

R

P

A

C

K

L

L

N

Q

Narcissus longispathus

K

Y

Y

H

R

S

A

C

Q

L

L

S

Q

Narcissus nevadensis

K

Y

Y

H

R

S

A

C

Q

L

L

S

Q

Narcissus pseudonarcissus

R

Y

H

H

R

S

A

C

K

L

L

N

Q

Narcissus scaberulus

K

Y

Y

R

R

S

S

C

K

L

F

S

K

Narcissus serotinus

K

Y

Y

H

R

S

S

Y

K

F

L

S

K

Narcissus tazetta

K

Y

Y

H

R

S

S

Y

K

F

L

S

K

Narcissus triandrus

R

D

H

H

L

S

A

C

K

L

L

S

Q




Species

Amino acid at each position number

13

34

47

75

78

93

101

108

112

131

142

152

162

Galanthus nivalis (Common snowdrop) = outgroup

K

Y

Y

H

R

Y

S

Y

K

L

L

S

K



























































































































































































































































































































































































































































































































Sheet 5






















  1. Choosing species to investigate

Some Narcissus species have been investigated for finding drugs to treat Alzheimer’s disease. Some of the drugs used to treat Alzheimer’s disease are acetylcholine esterase (AChE) inhibitors. Acetylcholine esterase (AChE) inhibitory activity of a species can be assessed by finding out how much plant material is needed for 50% inhibition of a known quantity of the enzyme. This is called an IC50 value. Plants with better inhibitory activity require a lower mass of material for 50% inhibition and therefore have a lower IC50 value.


Only 3 of the species in this group of Narcissus species have been studied. Their IC50 values are in the table below.


Species

IC50 value

Narcissus asturiensis

?

Narcissus atlanticus

?

Narcissus calcicola

?

Narcissus cernuus

?

Narcissus jacetanus

1.75

Narcissus longispathus

?

Narcissus nevadensis

?

Narcissus pseudonarcissus

2.30

Narcissus scaberulus

?

Narcissus serotinus

?

Narcissus tazetta

?

Narcissus triandrus

>50

If we assume that closely related species will have similar IC50 values, use the evolutionary tree you’ve made (or the one on sheet 5) to work out:


i) Which species is most closely related to the species that has the lowest known IC50 value? Maybe this one will have an even lower IC50 value.
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ii) Which species is most closely related to the species that has the highest known IC50 value? This is probably the species least likely to possess a useful new drug.
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iii) Are there any groups of species on the evolutionary tree that haven’t been studied at all? Maybe there are very different chemicals in some of these groups that might have a much lower IC50 value than any that have been discovered before. Select 3 species that you think would be best to study first if we are aiming to make sure that we have investigated all groups of species.
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iv) The abstract from the research article below shows that one species of Narcissus has been found to possess chemicals that might be useful in the treatment or prevention of bird flu (H5N1).



Use your evolutionary tree to identify the species most closely related to the one from the research article. Maybe this species also has chemicals that will help in the fight against bird flu.


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Science & Plants for Schools: www.saps.org.uk



Evolutionary relationships and finding new drugs: p.



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