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hello everyone and good morning I'm

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Abigail Karen and I'm going to be

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presenting some work I did with David

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gold Greg fornia and Roger summons

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entitled molecular data suggests sterile

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biosynthesis evolved around the great

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oxidation event and first I'm going to

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give you some backgrounds you can

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understand why you should care about

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this first thing you need to know is

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what is a sterile sterols are some lipid

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molecules they're organic they have

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these four rings and a sidechain group

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that varies depending on what specific

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sterile it is this one here ergosterol

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is a component of the cell membrane and

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fungi sterile you might know is

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cholesterol oftenly talked about sterols

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in general are created by most eukaryote

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lineages and only a very small number of

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bacteria as such they're indicative

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usually of presence of eukaryotes

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furthermore sterols require oxygen to be

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synthesized in this first step and also

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further downstream depending on what

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specific sterile they're going to end up

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making so the presence of sterols is

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indicative of aerobic metabolism because

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it requires oxygen and of eukaryotes so

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trying to date when sterile synthesis

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first evolved is an interesting has an

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interesting implications for early life

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so trying to date it so far people can

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look at sterols in the rock record their

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preserved as staring keeping their

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carbon backbone but losing key details

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like functional groups however the

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record is currently controversial back

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in 1999 sterols were reported at 2.7 and

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sorry 2.7 billion year old rocks in

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australia which was super interesting

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because as you might know the great

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oxidation event didn't occur until 2.4

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billion years ago and that's the first

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accumulation of molecular oxygen in the

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atmosphere so having me is earlier than

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the great oxidation event was a big deal

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because oxidative metabolism before we

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have appreciable oxygen however that was

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recently in the last few years refuted

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those rocks don't actually have stair

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is preserved in them and the next

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closest ones that were sure of in the

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rock record are at one point six for

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simple 23 to 24 carbon cells and 800

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million years ago for complex steering's

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with 26 to 30 carbons which are

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generally the ones that all modern

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eukaryotes create so there's some debate

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as to when this pathway evolved and

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looking at the rock record isn't

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currently helping so we decided to take

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an alternative approach and look at the

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genes of modern organisms to try and

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figure out when this approach when this

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adds we evolved just from the genetic

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record as such the first thing we had to

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do was pick two genes to study and we

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decided to pick the first two genes in

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the sterile synthesis pathway simply

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because these are conserved across all

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sterile synthesis pathways and some of

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the later genes are not on the first

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that I'm going to be talking about as

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squalene monooxygenase that converts

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squalene to squalene epoxide and the

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second gene I'll be talking about is

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oxido salt squalene cyclase which turns

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this epoxide into a proto sterile such

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as cyclo ordinal and now I'm going to

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briefly and quickly go through molecular

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clocks and how they work just because

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I'm not sure how familiar you guys are

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with them so the first thing we do is we

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acquire amino acid sequences from modern

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organisms a whole bunch of them and we

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run them through a program that deals

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with like insertions and deletions and

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make sure that similar parts are lined

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up for each gene just so that they're

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comparable we run that through some

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other programs to create phylogenetic

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trees using asian or maximum likelihood

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methods and the trees that are produced

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have similar sequences close together

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and very different sequences just

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further apart in the tree with longer

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branch lengths however that still

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doesn't give us any dates to add dates

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we need to add paleontological beta

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fossils to do that you can constrain

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specific nodes with specific fossils for

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example the mammal reptile split had to

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happen before the first solid reptile

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fossil so we can calibrate all of these

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amniotes with highly nomis and give it a

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date

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that is earliest and like some

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probability for when that node occurred

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we do this for a whole bunch of nodes

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and it creates a molecular clock which

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is basically just a phylogenetic tree

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with dates associated with each node and

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some probability there so we started

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doing this process for sqm oh and 40 SC

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and these are two maximum likelihood

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trees sqm oh and osc this is a

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representative sampling of eukaryotes

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and every single bacterial sequence we

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could find so the bacteria are this dark

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blue we only found 27 different species

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across six different phyla so it was

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like it's real weird bacteria that had

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these jeans and they correlate both in

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both Jean trees to these two groups one

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basil to eukaryotes and one like inside

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the eukaryotes so when you look at this

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what I want you to get from this is that

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all of these non dark blue things are

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eukaryotes and they generally reflect

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the species tree so we can say that

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these genes were probably present here

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in the stem eukaryote and we're just

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vertically inherited throughout Eukarya

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however the bacteria are weird here like

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this group here bacterial group to bec

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your ad in nabol from algae so they got

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this gene through horizontal gene

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transfer probably from some algae at

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some point same with this this basal

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group there's horizontal gene transfer

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here between these bacteria and the stem

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eukaryote we can't polarize which

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direction it is I can't say oh it

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evolved in eukaryotes but there was some

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sort of gene sharing here so we can say

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by this node we had functional sqm oh

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and osc also some of these bacteria been

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proven in the lab to actually create

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functional sterols so that was like a

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lot but basically you just need to know

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there's two horizontal gene transfer

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into or out of bacteria and we think

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since they're this they're in the same

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place that they were being transferred

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together so we looked at the genome of

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all of these

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bacteria that have both genes and if you

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look OSC is orange eskimos blue and

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they're always right about next to each

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other on the genome of these bacteria

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which is pretty good evidence that the

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two genes were being transferred

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together in both of these events and

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that when we make our clocks if we're

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just looking at the transfers we can add

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data from sqm o to data from OSC to

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constrain those two nodes we ended up

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doing ten different analyses looking at

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two different topologies simply because

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there's some debate if excavates are

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basil to the other eukaryotes or if

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they're sister to the by cons and just

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to cover all our bases we did both

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topologies we also looked at five

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different datasets the SQ MO and osc

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genes concatenated like I just mentioned

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because they were being moved together

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also as qmo by itself as qmo constrained

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by an out group of genes from the Oba

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quinone biosynthesis pathway OSC by

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itself and osc constrained by an out

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group of squalene hoping cyclase it's

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sort of bacterial analogue each analysis

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had between 14 and 18 fossil

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calibrations added and I have to mention

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that we did exclude the SQ mo alone data

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set from our numerical analyses that

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we'll get into later simply because it

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was broadly inconsistent with our other

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analyses broadly and consistent with

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previous molecular clock work and very

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very old but when we added a

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constraining out group or the data from

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OSC it fixed the problem so here's an

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example of one of our molecular clocks

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this is the concatenated one so that I

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could particularly look at the two

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bacterial transfers first I want to talk

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about this green star that's

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representing crown Eukarya all of the

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eukaryotes here do make modern at least

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the modern versions create 26 to 30

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sterols 30 carbon sterols as such we can

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infer that way back here we had the

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genetic machinery to do so so we would

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expect to see 26 to 30 carbon sterols in

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the rock record around here but the

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average date we get for that node is one

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point

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six billion years ago which is much

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earlier than that 800 million i

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mentioned earlier furthermore if you

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look at this red node that represents

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the first transfer between the bacteria

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and the stem eukaryote so the pathway

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existed by then and we should expect to

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see at least proto sterols by this node

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as you can probably tell this

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ninety-five percent confidence interval

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is really big it's like 800 million

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years so I can't really say that much

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about when this occurred but the

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confidence interval in none of our

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analyses enters me so proterozoic so we

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can at least conclude that from this

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work these two genes should exist before

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the mesoproterozoic and if we look more

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closely at that node which is here the

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maximum probability in all of the

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analyses excluding that s qm o alone one

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the light blue occur like right during

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or right after the great oxidation event

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which is reasonable because the pathway

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requires oxygen so sharing it right

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about then would be useful I guess so

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that's about it in conclusion the

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pathway the the sterile synthesis

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pathway seems to have evolved much

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earlier than the rock record suggests if

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you look at this little chart the

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horizontal lines are our predictions and

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the vertical lines are the first thing

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we found in the rock record other things

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of note the evolution of the pathway

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correlates with the great oxidation

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event so as soon as we have oxygen we're

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making these sterols and a sterile

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biosynthesis predates the evolution of

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crown group Eukarya and might have been

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an important pre adaptation for

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subsequent you carry already ation and

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that's about it I'd like to acknowledge

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specifically David gold who was my

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mentor on this project and the summons

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questions for Abigail great talk so it

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seems reasonable that the you know by

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Owens biosynthetic pathway involving

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oxygen kind of came about around the

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great oxidation event but do you think

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there might have been like a different

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pathway that was still making you know

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sterols prior to oxidation that would

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not necessarily look very similar um I'm

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not entirely sure I sort of doubt it

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because at least the the cyclase

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involved is very similar to squalene

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Hopi and cyclase and hoping Tsar serve a

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very similar function but in bacteria

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and so at least when I do the trees it

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looks pretty clear that that gene sort

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of branched off around then from this

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hoping gene I guess there could have

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been an entirely different pathway but I

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have no way to tell yeah its retail with

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this method I guess I don't think it's

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very likely that some other pathway also

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um so being unfamiliar with the

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molecular clock method what's the

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uncertainty in this sort of analysis in

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this analysis I mean so we have a

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different amount of uncertainty on each

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of the nodes which are these it's kind

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of hard to see but like these bars so we

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have particularly large uncertainties

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because this clock is only being run

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with one gene or in this case two genes

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of information and like most published

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clocks that are just trying to determine

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the species tree are run with like tens

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of jeans all concatenated together so we

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do have more uncertainty than a clock

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that was just looking at the species

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tree but like I said our conclusions are

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so broad that I think it's fine like

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we're not saying this definitely

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occurred at this date

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our conclusion is like this occurred you

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know 600 million years earlier than the

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rocks so I think our conclusions are

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solid but there's still a lot of

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uncertainty in the exact dates okay

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thanks does that answer it okay I think

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we have time for one more quick question

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how conserved are these genes so how

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many mutations are you trying to make

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this molecular clock with they're fairly

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well conserved um i don't think i have

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like a good quantitative answer for you

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ah looking for eugene the tree in

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general hasn't like the genes have

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enough variety that we do just get the

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eukaryote species tree pretty reliably

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so I think we can be fairly confident

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that what we're seeing is real but they