What Can Evolution REALLY Do? Part IV How many ways can a population head for an evolutionary dead end? March 12, 2009

1. What Can Evolution REALLY Do? Part IVHow many ways can a population head for an evolutionary dead end?March 12, 2009 Ralph Seelke, U. Wisconsin-Superior

2. Where We’re Going • Confessions of an experimentalist who loves making (bacterial) mutants • What evolution has been able to accomplish • What it has NOT been able to accomplish • Some conclusions

3. Two Warnings 1) Do not expect to be overwhelmed. 2) Some of this will get complicated!

4. Four Take-Homes Lessons • We really CAN test evolution • Two is an evolution stopper • Even doing one thing at a time can be a problem for evolution- a gene can “evolve to become unevolveable” • There are at least 7 ways our test gene can “evolve to become unevolveable”

5. Experimental Evolution? ??? Source: Ronald Pine, http://home.honolulu.hawaii.edu/~pine/book1-2.html

6. We Can Do This “Absurd” Experiment With Microbes! For Evolution to Occur You Need A LARGE Population and/or MANY Generations A Trait That Can Evolve !!!!BACTERIA!!!!

7. Up to 4 TRILLION in a 1 Gal Milk Jug! • Thousands of Generations in a Year! • COMPLEX Traits! • When they evolve, we can FIND THEM!

8. Each day= 6.64 generations of evolution!

9. We can FIND evolution BecauseWhen the microbe EVOLVESitGROWSor GROWS BETTER!!!!

10. Evolution Before your very eyes!

11. About 3 mm MORE Evolution Before your very eyes!

12. My Question: Can evolution do two things at once? • Can a microbe evolve when two mutational events BOTH have to happen for evolution to occur? • Not just my pet question! • Acknowledged by others as a problem • The fundamental problem of irreducible complexity- 2 or more components, all required for a function, and all required for any function.

13. Why Should Requiring Two Changes Make Evolution Difficult? • Mutation rates are typically one in 100 million; a teaspoonful of bacteria would have over about 50 mutants! • If you need TWO mutations, then the chances of BOTH mutations occurring is 1 in 10,000 trillion! • Now you would need a population of 10,000 liters to produce the mutant!

14. Is the Need for Two Independent Mutations REALLY an Evolution-Stopper?Studies with the trpA Gene of Escherichia coli

15. Testing the Two Mutation Rule • Find a well-studied gene, with known mutations that inactivate it. • Introduce 1,2,3, or four inactivating mutations • Let the gene evolve under highly selective conditions

16. 60 49 234 175 Source: Hyde, et. al, 1988 The Gene of Choice: trpA (tryptophan synthase A) Mutation 2 When trp is scarce, evolution to Trp+ is HIGHLY selective! Mutation 1

17. Results So Far:If Evolution Requires Two or More Independent MutationsNOTHING HAPPENS

18. Nothing Happens Means • Not when 2+ trillion cells are tested • Not when they are given >7,600 generations in which to evolve

19. The cultures have evolved to be able to grow better in the tryptophan-limited medium

20. CONCLUSION:TWO is an evolution stopperImportant, but somewhat boring

21. Warning: This Next Part May be Hard!

22. Then the story got more interesting- and more complicated DeadTrpA

23. We thought that BOTH mutations inactivated trpA, but only Mutation 1 did; Mutation 2 only weakened the gene When we made a version with just Mutation two- the gene was not completely dead! weakTrpA

24. Why was this a big deal? It meant that our gene with two mutations should have evolved! Selection because of fitness advantage Mutation M1, M2 Dead TrpA M1, M2 Weak TrpA THIS DOES NOT HAPPEN!! Evolution not only can’t do two things at a time, in this case it can’t do one thing at a time! M1, M2 Strong TrpA Population of fully Trp+ cells

25. So why didn’t our gene evolve?? Maybe, while it was evolving, the expression of the mutant trpA gene was lowered, so that now a trpA gene with just M2 would be DEAD. This would mean that EVOLUTION MADE IT UNEVOLVEABLE

26. How can we find out if it “evolved to become unevolveable”? We did “part swapping” experiments! We took the large part (red) from our original plasmid, combined it with trpA-M2; the gene was weakly trpA. When we took the same piece was from a plasmid that had evolved, the trpA-M2 gene was DEAD! Evolved piece- trpA-M2 gene DEAD Original piece- trpA-M2 gene WEAK

28. What did this mean? Most likely, a mutation had occurred, outside the trpA-M2 gene, that had turned the weakly trpA gene OFF. Mutation somewhere in red, not in the trpA gene! Evolution in red switched off the trpA gene in blue. That somewhere was a single base change at 1584. 1584

29. A case of evolution preventing evolution This path NEVER happens Selection because of fitness advantage Mutation M1, M2 DeadTrpA M1, M2 Weak TrpA Change at 1584 ALWAYS happens M1, M2 Dead TrpA M1, M2 Strong TrpA M1, M2 STILL Dead TrpA Mutation lowers trpA expression, increases fitness Population of fully Trp+ cells Reversion of M1 no longer selective; both must now revert for a Trp+ cell

30. The Concept of a “Fitness Peak”

31. It gets worse... • OK so it hit a dead end that one time- what if you “rewind the tape”- maybe a different time, when allowed to evolve, it will. • We did that in 2007- Jason Uviasah's research- Sorry, no evolution with 12 cultures in 500 generations. • Last summer I looked at 11 of them, to see if they had evolved to become unevolveable-

32. All 11 had evolved to become unevolveable • 6 of 12 were physically different from their ancestors- they had added DNA! • Laramie Rapp and Jory Fleischauer identified the location of the new DNA, and sequenced it.

33. Plasmid produces Trp+ phenotype when large SalI-HpaI fragment is from unevolved pRS202-5 Inserts at 1516, 1524,1531, 1544- IS1 & IS2 events A1584C transversion responsible for altered phenotype One IS1 at 2132 trpA M1,M2 mutant gene Plasmid produces Trp- phenotype when large SalI-HpaI fragment is from pRS202-5 after 2,000 generations of evolution

34. How is evolution like a guy? 1) It has trouble doing two things at once 2) Even when it only has one thing to do, it sometimes gets sidetracked 3) It has many ways to get sidetracked

35. Acknowledgements • Merck Foundation • Biologic Institute • UW-Superior • A.C. Matin Lab and Stanford University • NUMEROUS undergraduate students! • Lynn Meyers, Sarah Rahn, Stephanie Ebnet, Jason Uviasah, Benjamin Okemwa, Laramie Rapp, Jory Fleischauer