1. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Phylogenetic Reconstruction: Distance Matrix Methods Anders Gorm Pedersen Molecular Evolution Group Center for Biological Sequence Analysis Technical University of Denmark gorm@cbs.dtu.dk

2. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Trees: terminology

3. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Trees: terminology

4. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Trees: representations Three different representations of the same tree

5. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Trees: rooted vs. unrooted A rooted tree has a single node (the root) that represents a point in time that is earlier than any other node in the tree. A rooted tree has directionality (nodes can be ordered in terms of “earlier” or “later”). In the rooted tree, distance between two nodes is represented along the time-axis only (the second axis just helps spread out the leafs) EarlyLate

6. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Trees: rooted vs. unrooted EarlyLate A rooted tree has a single node (the root) that represents a point in time that is earlier than any other node in the tree. A rooted tree has directionality (nodes can be ordered in terms of “earlier” or “later”). In the rooted tree, distance between two nodes is represented along the time-axis only (the second axis just helps spread out the leafs)

7. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Trees: rooted vs. unrooted EarlyLate A rooted tree has a single node (the root) that represents a point in time that is earlier than any other node in the tree. A rooted tree has directionality (nodes can be ordered in terms of “earlier” or “later”). In the rooted tree, distance between two nodes is represented along the time-axis only (the second axis just helps spread out the leafs)

8. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Trees: rooted vs. unrooted In unrooted trees there is no directionality: we do not know if a node is earlier or later than another nodeIn unrooted trees there is no directionality: we do not know if a node is earlier or later than another node Distance along branches directly represents node distanceDistance along branches directly represents node distance

9. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Trees: rooted vs. unrooted In unrooted trees there is no directionality: we do not know if a node is earlier or later than another nodeIn unrooted trees there is no directionality: we do not know if a node is earlier or later than another node Distance along branches directly represents node distanceDistance along branches directly represents node distance

10. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Reconstructing a tree using non- contemporaneous data

11. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Reconstructing a tree using present-day data

12. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Data: molecular phylogeny DNA sequencesDNA sequences –genomic DNA –mitochondrial DNA –chloroplast DNA Protein sequencesProtein sequences Restriction site polymorphismsRestriction site polymorphisms DNA/DNA hybridizationDNA/DNA hybridization Immunological cross-reactionImmunological cross-reaction

13. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Morphology vs. molecular data African white-backed vulture (old world vulture) Andean condor (new world vulture) New and old world vultures seem to be closely related based on morphology. Molecular data indicates that old world vultures are related to birds of prey (falcons, hawks, etc.) while new world vultures are more closely related to storks Similar features presumably the result of convergent evolution

14. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Molecular data: single-celled organisms Molecular data useful for analyzing single-celled organisms (which have only few prominent morphological features).

15. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Distance Matrix Methods 1. Construct multiple alignment of sequences 2. Construct table listing all pairwise differences (distance matrix) 3. Construct tree from pairwise distances Gorilla : ACGTCGTA Human : ACGTTCCT Chimpanzee: ACGTTTCG GoHuCh Go-44 Hu-2 Ch- Go Hu Ch 2 1 1 1

16. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Finding Optimal Branch Lengths S1S1S1S1 S2S2S2S2 S3S3S3S3 S4S4S4S4 S1S1S1S1-121314 S2S2S2S2-2324 S3S3S3S3-34 S4S4S4S4- Observed distance S1 S3 S2 S4 a b c d e Distance along tree D 12 ≈  d 12 = a + b + c D 13 ≈ d 13 = a + d D 14 ≈ d 14 = a + b + e D 23 ≈ d 23 = d + b + c D 24 ≈ d 24 = c + e D 34 ≈ d 34 = d + b + e Goal:

17. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Superimposed Substitutions Actual number ofActual number of evolutionary events:5 Observed number ofObserved number of differences:2 Distance is (almost) always underestimatedDistance is (almost) always underestimated Real distance can be estimated from observed distance using models of how evolution occursReal distance can be estimated from observed distance using models of how evolution occurs ACGGTGC C T GCGGTGA

18. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Optimal Branch Lengths: Least Squares Fit between given tree and observed distances can be expressed as “sum of squared differences”:Fit between given tree and observed distances can be expressed as “sum of squared differences”: Q = ∑ (D ij - d ij ) 2 Q = ∑ (D ij - d ij ) 2 Find branch lengths that minimize Q - this is the optimal set of branch lengths for this tree.Find branch lengths that minimize Q - this is the optimal set of branch lengths for this tree. S1 S3 S2 S4 a b c d e Distance along tree D 12 ≈ d 12 = a + b + c D 13 ≈ d 13 = a + d D 14 ≈ d 14 = a + b + e D 23 ≈ d 23 = d + b + c D 24 ≈ d 24 = c + e D 34 ≈ d 34 = d + b + e Goal: j>i

19. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Least Squares Optimality Criterion Search through all (or many) tree topologiesSearch through all (or many) tree topologies For each investigated tree, find best branch lengths using least squares criterionFor each investigated tree, find best branch lengths using least squares criterion Among all investigated trees, the best tree is the one with the smallest sum of squared errors.Among all investigated trees, the best tree is the one with the smallest sum of squared errors.

20. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Exhaustive search impossible for large data sets No. taxa No. trees 31 43 515 6105 7945 810,395 9135,135 102,027,025 1134,459,425 12654,729,075 1313,749,310,575 14316,234,143,225 157,905,853,580,625

21. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Heuristic search 1. Construct initial tree; determine sum of squares 2. Construct set of “neighboring trees” by making small rearrangements of initial tree; determine sum of squares for each neighbor 3. If any of the neighboring trees are better than the initial tree, then select it/them and use as starting point for new round of rearrangements. (Possibly several neighbors are equally good) 4. Repeat steps 2+3 until you have found a tree that is better than all of its neighbors. 5. This tree is a “local optimum” (not necessarily a global optimum!)

22. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Clustering Algorithms Starting point: Distance matrixStarting point: Distance matrix Cluster least different pair of sequences:Cluster least different pair of sequences: Repeat until all nodes are linkedRepeat until all nodes are linked Results in only one tree, there is no measure of tree-goodness.Results in only one tree, there is no measure of tree-goodness.

23. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Neighbor Joining Algorithm For each tip compute u i = Σ j D ij /(n-2)For each tip compute u i = Σ j D ij /(n-2) (this is essentially the average distance to all other tips, except the denominator is n-2 instead of n) Find the pair of tips, i and j, where D ij -u i -u j is smallestFind the pair of tips, i and j, where D ij -u i -u j is smallest Connect the tips i and j, forming a new ancestral node. The branch lengths from the ancestral node to i and j are:Connect the tips i and j, forming a new ancestral node. The branch lengths from the ancestral node to i and j are: v i = 0.5 D ij + 0.5 (u i -u j ) v j = 0.5 D ij + 0.5 (u j -u i ) Update the distance matrix: Compute distance between new node and each remaining tip as follows:Update the distance matrix: Compute distance between new node and each remaining tip as follows: D ij,k = (D ik +D jk -D ij )/2 Replace tips i and j by the new node which is now treated as a tipReplace tips i and j by the new node which is now treated as a tip Repeat until only two nodes remain.Repeat until only two nodes remain.

24. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Bootstrapping ratGAGGCTTATC humanGTGGCTTATC turtleGTGCCCTATG fruitflyCTCGCCTTTG oakATCGCTCTTG duckweedATCCCTCCGG 0123456789 Original alignment Many more replicates (between 100 - 1000) ratGGAAGGGGCTTTTTA humanGGTTGGGGCTTTTTA turtleGGTTGGGCCCCTTTA fruitflyCCTTCCCGCCCTTTT oakAATTCCCGCTTCCCT duckweedAATTCCCCCTTCCCC 001122234556667 Boostrapped alignment 1 ratCCTTTTAAATTTTCC humanCCTTTTAAATTTTCC turtleCCCCCTAAATTTTGG fruitflyCCCCCTTTTTTTTGG oakCCTTTCTTTTTTTGG duckweedCCTTTCCCCGGGGGG 445556777888899 Boostrapped alignment 2...... rat human turtle fruit fly oak duckweed Inferred tree rat human turtle fruit fly oak duckweed...... rat human turtle fruit fly oak duckweed

25. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Bootstrapping values rat human turtle fruit fly oak duckweed 100 65 55