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[Music]

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[Applause]

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I've had problems with this Absalom's in

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the past which I was just telling Nadia

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about so in the interest of

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interdisciplinarity I tried to go with a

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simpler more general title and I'm going

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to give rather than the talk I

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originally originally planned a more of

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an overview of the research program that

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we're trying to develop at Georgia Tech

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and so my new title will be stabilizing

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the evolutionary transition to

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multicellularity against reversion and

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so the evolution of multicellularity is

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one of a very small number of events in

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the history of life in which the

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hierarchical complexity of biological

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systems has increased and so the others

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in this very simplified diagram are so

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the origin of life may be considered as

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one or the origin of chromosomes the

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origin of the prokaryotic cell eukaryote

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Genesis then the evolution of

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multicellularity and later in some cases

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the evolution of eusociality and so

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multicellularity is quite unique in that

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unlike many of the other transitions

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like eukaryote genesis which we know has

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which we only know has occurred at least

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once multicellularity has evolved

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independently across multiple lineages

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here in the eukaryotes it's thought to

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have occurred at least 25 times and I

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think the big question for us in the

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field of multicellularity research now

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that Frank rosensweig set up very nicely

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is how do you go from a unicellular

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ancestor of say metazoan all metazoans

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to something like a kangaroo and so I've

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simplified that process in this

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three-part diagram so our understanding

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of multicellularity is sort of in its

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infancy in terms of the process that

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generates something like a kangaroo from

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something that may resemble a coin of

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flageolet so first we think there are

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it's necessary that you have external

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drivers that create the benefit to you

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forming a simple social group of cells

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so that that drives the increased group

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size from one to many

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and then later there are subsequent

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social changes associated with the

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transformation of simple groups to more

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complex ones and this could be anything

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and this is the part that we really

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don't quite understand so I would argue

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that our our understanding of these

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external drivers and their role is

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fairly well-established we have good

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theoretical and empirical results to

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support things like increased stress

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tolerance protection from predation and

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improved utilization of public goods as

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good drivers that might favor the

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evolution of simple social groups but

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what we really don't understand are

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these social changes but are associated

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with increases in the complexity of

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those simple groups and so as I may not

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have mentioned I work in the field of

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experimental evolution and actually

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there have been research groups evolving

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simple multicellular organisms in the

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laboratory for about 25 years and this

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is just a small sampling maybe I've

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captured about half of the instances of

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this happening in the field there have

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been multiple works with algae both this

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chlorella vulgaris in the middle and two

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experiments with different selective

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pressures with commit aluminium bonus

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there's also been work done in bacterial

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systems as well as different yeast model

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organisms and so there's just two

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lessons I want to summarize across these

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25 years of history of this emerging

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discipline so first simple

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multicellularity seems to evolve quite

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rapidly under the right conditions so

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all the experiments that I summarized

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here in the slide saw the evolution of

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multicellular genotypes over the course

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of a few hundred generations or less and

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this suggests that the genetic barriers

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to making this transition this first

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step of the transition at least are

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quite minimal the second lesson from

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experimental evolution is that simple

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multicellularity seems to be costly in

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the absence of these

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kernel drivers so just to pull out two

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examples in the yeast in the yeast

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picture on top right there was a ten

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percent cost measured by Ratcliffe a

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Talon twenty twelve when their snowflake

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yeast system was grown in liquid medium

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without the external driver in their

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case which was selection for rapid

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sedimentation and in the bacteria

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example there was a twenty percent cost

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measured in Pseudomonas fluorescens

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wrinkly spreaders when there was colony

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colonization of the air water interface

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was not necessary and so together these

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two lessons point towards a major

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problem for the increase subsequent

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increases in complexity for new

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multicellular forms which is that if you

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have something that's genetically labile

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and has high fitness costs when the

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external drivers are not present you

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should see the the reversion of back to

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unicellular t should the environment

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change and actually maria replicated

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gomez and mike travisano had a pair of

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great papers come out in the past year

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where they measured exactly this

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so using that snowflake yeast system

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they first show in a growth in liquid

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without settling selection that ten

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percent fitness costs they measure that

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i mentioned and they also demonstrated

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that when you do your selections on agar

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plates there's an even greater fitness

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cost for the multicellular forms so what

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they found really interestingly here we

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have a simple histograms of the area

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have these multicellular types that form

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a distribution of clump sizes over the

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course of 30 days when they're doing

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selection on plates you see rapidly

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going from the black distribution to the

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lighter ones you see size decrease quite

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rapidly but when you do your selections

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in liquid that size decreases both

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slower and less pronounced

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and so the cost associated with the

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simple multicellularity actually is a

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good predictor of how rapidly multi cell

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we'll be lost as well as the extent to

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which the phenotype will change and so

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we need at least one more arrow in my

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simple diagram which is that before you

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get locked into this positive feedback

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loop that produces things like kangaroos

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you might actually experience an

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environmental change that favors you

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exiting this simple this simple system

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and so the vicious cycle can't ever

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occur

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you'll never get increases in complexity

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and so this is this is just say that

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reversion may in fact be a major problem

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that we should consider in the evolution

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of multicellularity and so in my view

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there's probably two possible routes to

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stabilizing the evolution of

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multicellularity the first being you can

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reduce the number of potential reversion

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mutations so here I've drawn just a

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simplest distribution of fitness effects

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of reversion mutations that I could

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imagine so here you have this greyed out

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dashed you know modal distribution that

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represents all the possible mutations

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that will cause a reversion and the

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arrow is saying that through

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evolutionary time while you're in this

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positive feedback loop you may lose some

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of those potential routes back to

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unicellular T that's just a question

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about the availability of mutations

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another route would be to reduce the

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selective advantage to fixing one of

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those reversion mutations so if by

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further genetic interactions and

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mutations reversion is no longer

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beneficial even when you have

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environmental change then that should

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also prevent the exiting of this

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cyclical this cycle here that I had on

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the previous slide or of course you can

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do both and so we saw we our aim was to

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look for evidence of whether either of

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these two processes were happening in

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our laboratory experiments so we also

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are working with the sacrum icy service

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see a snowflake yeast system

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and what will Ratcliffe in my crevice on

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Oh and others did in this original

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experiment is they subjected a

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unicellular strain of baker's yeast to

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selection for rapid settling in liquid

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media and over the course of 60 days or

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fewer they got things that grew in this

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beautiful fractal like pattern and being

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from Minnesota as I already spoiled they

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called it snowflake yeast because that

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was familiar so the thing that really

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enables our present study is that the

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genetics of multicellularity are

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excessively simple in this system at

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least in five of the 10 original

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isolates the mutation was a single loss

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of function in a transcription factor

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called ace 2 that produced the

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multicellular growth form and the

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restoration of a functional copy of ace

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2 can produce unicellular progeny so

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here's the original mutant here's a

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functional ace to put into that mutant

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and here if you knock out both copies of

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the ancestral type you recapitulate the

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snowflake yeast form and so we did this

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from the original 60 day evolution

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experiment and just at two-two intervals

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so and then we measured a competitive

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fitness of the unicellular riverton's

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that were derived from multicellular

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types against their ancestors and what

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we saw was really striking is that at

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the beginning their fitness is equal to

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their ancestor they haven't undergone

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any mutations but then by 30 transfers

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they were a bit lower which is the

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opposite of what you normally expect and

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then by 60 days there was about a 5%

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fitness cost associated with this

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reversion mutation so this is this is

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evidence of that second class of

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mutations potentially fixing and at

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least in the case of the transition the

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the difference between day 30 and day 60

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we had a few candidate traits that we

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thought might explain this decrease in

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unicellular Fitness so

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he's populate this particular population

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of all of dicks elevated rates of

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programmed cell death or apoptosis as

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well as increased cell size and we have

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these uh priori reasons to think that

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those traits might be costly in a

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unicellular background but beneficial

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for the multi cells so what we really

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want to know after getting this

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provocative first piece of data is what

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are the dynamics and the consequences of

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long-term selection on size and luckily

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for me and hopefully for other people we

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already had a fantastic postdoc that

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joined the lab years before me that was

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conducting a long term evolution

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experiment with settling selection and

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that is ozone buzz Doug who's here in

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the audience and later today he'll be

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giving a poster on this long term

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experiment and also talking a bit about

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the effects of high and low oxygen

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levels on the evolution of multicellular

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size so please check out his poster this

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evening but what we did is again take

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all of the isolates from this original

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experiment and perform these genetic

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reversions on them and so on the next

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slide and I promised Frank it'll

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actually happen on the next slide will

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either these dots that are representing

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day 200 day 400 and day 600 isolates

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from two parallel experiments that ozone

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had run will sit tell you whether or not

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the transform ins with a functional h2

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are unicellular or multicellular and so

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unsurprisingly based on the way I set

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this up we have some some some of the

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populations have actually lost the

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ability to revert to you know

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cellularity when we put a functional ace

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2 back into them this was particularly

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validating for us because the two

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populations are actually the three

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populations at the bottom there that

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were unable to revert also exhibited the

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most pronounced phenotypic changes over

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the course of these 600 days of

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evolution and one of those really

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dramatic changes was that the site the

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average size of the snowflake yeast

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clusters increased from 50 microns in

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diameter

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so we did some genome sequencing of

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these large 600 transfer isolates those

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suggest further genes of interest that

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might be costly in a unicellular context

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but beneficial in a multicellular

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context and many of them are associated

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with the phenotypes that we think are

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enabling this extremely large size

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namely very elongated cells and both

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polar and side budding and so if you're

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interested in some of these new

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candidate genes and the interaction

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between those genes we have another

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postdoc here also giving a poster this

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evening Toni Bernette II and his poster

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will be in the Evergreen Ballroom as

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well and so in summary our results

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suggest this third model where both the

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Fitness effects of reversion mutations

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as well as the availability of reversion

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mutations is changing over evolutionary

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time at least for some of our

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populations the failure of the

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functional ASA to to restore unicellular

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T is associated with more dramatic

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phenotypic changes and we'll continue

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working on this and be doing some direct

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pairwise competitions with these more

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derived strains in the future so thank