1. First & Last Name
August X, 2000
Evolution
“To study history one must know in advance that one is attempting something fundamentally impossible, yet necessary and highly important.”
Father Jacobus (Hesse's Magister Ludi)
(c) 2000 CGDN

2. Some history is known Bacterial evolution observed
First & Last Name
Some history is known
August X, 2000
Bacterial evolution observed
Manchester Moths – light to dark
(c) 2000 CGDN

3. Key Concepts Fundamentals of Systematics
First & Last Name
August X, 2000
Fundamentals of Systematics
Appreciate that phylogenetic analysis allows you to estimate or infer the evolutionary relationships between sequences/organisms
Learn how to better interpret trees
Gain insight into the different phylogenetic methods
Appreciate the need for new algorithms
DNA and protein analysis - benefits and pitfalls of each
(c) 2000 CGDN

4. First, some terminology…
Systematics, an attempt to understand the interrelationships of living things
Taxonomy, the science of naming and classifying organisms (evolutionary theory not necessarily involved)
Phylogenetics is the field of systematics that focuses on evolutionary relationships between organisms or genes/proteins (phylogeny).
Cladistics is a particular method of hypothesizing relationships among organisms/genes/proteins.

5. Three basic assumptions of cladistics
Any group of organisms/ genes/proteins are related by descent from a common ancestor (fundamental tenant of evolutionary theory)
There is a bifurcating pattern of cladogenesis
(most controversial assumption)
Change in characteristics occurs in lineages over time
(necessary for cladistics to work!)

6. A phylogenetic tree Human Mouse Fly
First & Last Name
August X, 2000
A phylogenetic tree
A node
Human
Mouse
Fly
A clade
You have bunches of taxa, where groups of taxa proposed to have a common ancestral origin is called a clade- the base of these clades are nodes
Any group of organisms/ genes/proteins are related by descent from a common ancestor (fundamental tenant of evolutionary theory)
There is a bifurcating pattern of cladogenesis (most controversial assumption)
Change in characteristics occurs in lineages over time (necessary for cladistics to work!)
taxon -- Any named group of organisms – evolutionary theory not necessarily involved.
clade -- A monophyletic taxon (evolutionary theory utilized)
(c) 2000 CGDN

7. A phylogenetic tree with branch lengths
First & Last Name
August X, 2000
A phylogenetic tree with branch lengths
A node
Human
Mouse
Fly 4
A clade 2 3 1
Can also view this way – note that in this case horizontal branch lengths are meaningful – mouse is more related to fly than human is related to fly
Branch length can be significant…
In this case it is and mouse is slightly more similar to fly than human is to fly
(sum of branches is less than sum of 1+2+4)
(c) 2000 CGDN

8. Phylogenetic analysis
First & Last Name
August X, 2000
Phylogenetic analysis
Organismal relationships
Gene/Protein relationships
However most of the evolutionary analyses being done in genomics involves more ancient relationships- anciently duplicated genes, comparing human and mouse sequences (organisms which are thought to have diverged x million years ago)
(c) 2000 CGDN

9. Organismal relationships

12. Improving our understanding of organismal relationships
Realization that rates of change are not constant

13. Improving our understanding of organismal relationships
Better appreciation for what sequences may be suitable for analysis of different degrees of divergence
For the tree of life:
rRNA genes
Multiple genes
“Whole genome” datasets of genes
rRNA genes!

14. Improving our understanding of organismal relationships
Better sampling of all the species in our world
Amazing but true!
More bacteria in our bodies than human cells!
More different types of bacterial genes in our body then there are human genes!
“The second human genome project”
(David Relman)

15. Gene/Protein Relationships
Homolog, ortholog, paralog??

16. Homologs Have common origins but may or may not have common activity.
Homologous or not?: Often determined by arbitrary threshold level of similarity determined by alignment

17. Homologs
…have common ancestry, but the way they are related can vary (i.e. the reasons they have diverged into different sequences can vary)
orthologs - Homologs produced by speciation. They tend to have similar function.
paralogs - Homologs produced by gene duplication. They tend to have differing functions.
xenologs -- Homologs resulting from horizontal gene transfer between two organisms.

18. Orthologous or paralogous homologs
Early globin gene
Gene Duplication
-chain gene
ß-chain gene
mouse 
human 
cattle 
cattle ß
human ß
mouse ß
Orthologs ()
Orthologs (ß)
Paralogs (cattle)
Homologs
Orthologs – diverged after speciation – tend to have similar function
Paralogs – diverged after gene duplication – some functional divergence occurs
Therefore, for linking similar genes between species, or performing “annotation transfer”, identify orthologs

19. True or False?
A1x is the ortholog in species x of A1y?
A1x is a paralog of A2x?
A1x is a paralog of A2y?

20. Identifying Gene/Protein Relationships from Phylogenetic trees
orthologs - Homologs produced by speciation. Gene phylogeny matches organismal phylogeny.
paralogs - Homologs produced by gene duplication. Multiple copies of homologs in a given species or evidence that gene duplication involved through phylogenetic analysis and lack of match to organismal phylogeny
xenologs -- Homologs resulting from horizontal gene transfer between two organisms. Gene phylogeny does not match organismal phylogeny in a tree where most genes do match organismal phylogeny well.

21. Othologs and Paralogs of the fly1 gene?
Chimpanzee o
Human
Mouse
Fly1
Fly p
Known organismal phylogeny
Chimpanzee
Human
Mouse
Fly
Worm
Human
O?P?
O?
Mouse o
Fly1
Worm
Chimpanzee o
Human
Worm p
Fly1

22. Xenologs: Horizontal gene transfer
E. coli

23. Gene Orthology: How to detect?
Most common high throughput computational method: Identify reciprocal best BLAST hits (EGO, COGs,…)
Example Problem:
If making comparisons between human and bovine, for example, the bovine gene dataset is still quite incomplete
Therefore, current best hit may be a paralog now and the true ortholog not yet sequenced
human
cattle
mouse
cattle

24. Can we improve orthology analysis for linking functionally similar genes?
One solution: Phylogenetic analysis of all putative human-bovine orthologs, using mouse as an outgroup
Assumption: - Mouse and Human gene datasets are more complete, with more true orthologs identified
Expect (organismal phylogeny): Reject:
mouse
human
cattle
human
mouse
cattle

25. PaAlgU is a paralog of ? Blue genes are from the same species
PaAlgU is an ortholog of ?
PaAlgU is a paralog of ?

26. 2 Forms in 1 Species + + ++ +
Slides from Jonathan Eisen

27. 2 Forms in 1 Species - LGT + + + + + + Both forms maintained
Red and blue forms
diverge
Gene present in common ancestor +

28. 2 Forms in 1 Species - Gene Loss+ + ++ +
Loss
Loss
Gene duplicated in common ancestor ++

29. Unusual Distribution Pattern+ +

30. Unusual Distribution - LGT+ +
Acquires new
type of gene
Gene originates here

31. Unusual Distribution - Gene Loss+ +
Gene lost
here
Gene present in ancestor

32. Unusual Distribution - Evolutionary Rate Variation-?
Gene too diverged to be found + +

33. Unusual Distribution - Incomplete Data
+/-
+/- + +
Gene present in ancestor

34. Hope for the future Better sampling of all the species in our world
Amazing but true!
More bacteria in our bodies than human cells!
More different types of bacterial genes in our body then there are human genes!

35. “So….. how do we construct a phylogenetic tree??”

36. Most common methods
Parsimony
Neighbor-joining
Maximum Likelihood

37. Parsimony “Shortest-way-from-A-to-B” method
The tree implying the least number of changes in character states (most parsimonious) is the best.
Note:
May get more than one tree
No branch lengths
Uses all character data

38. Neighbor-joining (and other distance matrix methods)
“speedy-and-popular” method
distance matrix constructed
distance estimates the total branch length between a given two species/genes/proteins
Neighbor-joining approach: Pairing those sequences that are the most alike and using that pair to join to next closest sequence.

39. Practical comparison of common distance matrix methods: Some PHYLIP and PAUP programs as an example
Neighbor-joining: fast – not so good for highly divergent sequences
Fitch: Better but slower and result not that different (seeks to maximize fit of pairwise distances)
Kitsch: Assumes equal rate of evolution – can greatly bias results so do not use!
Minimum Evolution (PAUP): Similar to Fitch but fixes location of internal verses external nodes when maximizing fits
Note: gap info not incorporated into analysis

40. Maximum Likelihood “Inside-out” approach
produces trees and then sees if the data could generate that tree.
gives an estimation of the likelihood of a particular tree, given a certain model of nucleotide substitution.
Notes:
All sequence info (including gaps) is used
Based on a specific model of evolution – gives probability
Verrrrrrrrrrrry slow (unless topology of tree is known)

41. How reliable is a result?
Non-parametric bootstrapping
analysis of a sample of (eg. 100 or 1000) randomly perturbed data sets.
perturbation: random resampling with replacement, (some characters are represented more than once, some appear once, and some are deleted)
perturbed data analysed like real data
number of times that each grouping of species/genes/proteins appears in the resulting profile of cladograms is taken as an index of relative support for that grouping

42. Bootstrapping
The number of times a particular branch is formed in the tree (out of the X times the analysis is done) can be used to estimate its probability, which can be indicated on a consensus tree
High bootstrap values don’t mean that your tree is the true tree!
Alignment and evolutionary assumptions are key

43. Parametric Bootstrapping
Data are simulated according to the hypothesis being tested.

44. Are we confident that the cow, mole and hedgehog has one ancestor?

45. Orthologs, paralogs and homologs – one more test!
Early globin gene
Gene Duplication
-chain gene
ß-chain gene
mouse 
human 
cattle 
cattle ß
human ß
mouse ß
Orthologs – diverged after speciation – tend to have similar function
Paralogs – diverged after gene duplication – some functional divergence occurs
Therefore, for linking similar genes between species, or performing “annotation transfer”, identify orthologs

46. Phylogenetics – More info
First & Last Name
August X, 2000
Li, Wen-Hsiung Molecular evolution Sunderland, Mass. Sinauer Associates. - a good starting book, clearly describing the basis of molecular evolution theory. It is a 1997 book, so is starting to get a bit out of date.
Nei, Masatoshi & Kumar, Sudhir Molecular evolution and phylogenetics Oxford ; New York. Oxford University Press. - a relatively new book, by two very well respected researchers in the field. A bit more in-depth than the previous book, but very useful.
(c) 2000 CGDN

47. Phylogenetic Tree Construction: Examples of Common Software
First & Last Name
August X, 2000
PHYLIP
PAUP
MEGA 2.1
TREEVIEW
Extensive list of software
(c) 2000 CGDN

48. PhyloBLAST – a tool for analysis

49. Challenges
How do we classify?

50. Computational Challenges
Need to incorporate more evolutionary theory into the multiple sequence alignment and phylogenetic algorithms used in phylogenetic analysis
Phylogenetic analyses are computationally intensive – great way to benchmark your CPU speed!

51. More Challenges Increasing the sampling of our genetic world
More accurately differentiating orthologs, paralogs, and horizontally acquired genes
How frequent is gene loss, gene duplication, and horizontal gene transfer in genome evolution?
To what degree can we predict protein/gene function using phylogenetic analysis?

52. First & Last Name
August X, 2000
Evolution
“To study history one must know in advance that one is attempting something fundamentally impossible, yet necessary and highly important.”
Father Jacobus (Hesse's Magister Ludi)
(c) 2000 CGDN

53. Evolutionary theory is evolving
First & Last Name
August X, 2000
Evolutionary theory is evolving
(c) 2000 CGDN