So, for today's lecture as you can see up there is molecular --
evolution, and ecology.
And what I mean by this,
it's basically the study or what we try to figure out in molecular
evolution and ecology is what genes or gene sequences can tell us about
the evolution and ultimately also the ecology of organisms
in the environment. And it's particularly relevant for
thinking about microorganisms, prokaryotes and the environment.
And I hope I can actually convince you today of that.
This is interesting. The topics that I want to cover
today is, first of all, I want to review a little bit what
we know about life on Earth, sort of give an overview of the
evolution of life on Earth. Then, I want to go into specific
topic that's of particular relevance for the evolution of eukaryotes.
That's the endosymbiosis theory. And then I'll explain how we can
use gene sequences to actually reconstruct events that have
happened a very, very long time ago.
OK, so we'll look at what we call molecular phylogenies,
with the use of gene sequences to reconstruct the evolutionary history
of organisms on Earth. Derived from that, we'll look at
what we call the tree of life. That's sort of the big picture
overview of the evolutionary relationships of all organisms on
the planet. And then finally, I'll introduce you to a topic called
molecular ecology. Again, that's how we can use gene
sequences to learn something about the diversity of microorganisms in
the environment that lead us then, next time, when I come back on
Monday, into this big topic of environmental genomics,
how we can actually expand this analysis to learn much more about
organisms in the environment. So, first of all, let's look at
life on Earth. Does anybody know how old we think
Earth is? Say again? Yeah, 4.5 to 4.6, I haven't my
notes 4.6. So, Earth's thought to have originated
about 4.6 billion years ago. When did the first solid rocks
appear on earth? So, when was the surface kind of
solidified? Anybody know? About 3.9 billion years ago, OK?
And when do we think life started to develop on the planet?
Any ideas? Take a guess. Two? One? 3.5 billion years ago,
OK? So, this is really remarkable.
We think it didn't, I mean, of course it took a long
time because were talking about millions of years and hundreds of
millions of years, but still, if you look at the big
picture, it didn't actually take life that long to evolve on the
planet. So, why do we think that is the case? What's the evidence for
that? Well, we look into sedimentary rocks,
so old rocks that arose from sediments, what you find around this
time, you find that chemicals start to appear, organic molecules that
really resemble organic molecules in modern life.
So, we have sort of chemical tracers, or chemical fossils.
So, tracers that indicate the
presence of organisms. But what we also find is so-called
micro-fossils, and I have a picture of that here
where when you actually take rocks and actually slice them into very,
very then slices, you can put them under specific microscopes.
And what you then find is that many rocks that are very,
very old, have those kinds of inclusions in them.
And these things really resemble very much modern prokaryotic cells,
modern bacterial cells, for example. And so, those micro-fossils are
generally taken as an indication, also, that life is already present
during those times. Now, when we take a quick sort of
overlook of the evolution of life on the planet, again this graph here
summarizes sort of the last 4. billion years or so when life
originated. We see that there was a period of chemical evolution,
and then somewhere here that region, it's, of course, not really well
understood when that exactly happens, the origin of life is placed.
But I want to alert you to a couple of really, really critical steps
here that are shown on this graph which we'll actually talk more about.
It is thought that life very early on is split into three major
lineages: the bacteria, the archaea, in what is called here
nuclear line. And I'll come back to that in a minute or so.
Then, a further major event which you may remember is oxygenic
photosynthesis actually evolved -- -- which means that cyanobacteria
evolved that started to produce oxygen as a byproduct of
photosynthesis. And that really fundamentally
changed the chemistry of the Earth. It actually became an oxidizing
atmosphere. And what you see here is, once the oxygen concentration
goes over a certain level, it allowed the development of an
ozone shield. Now, what does that mean?
What was the critical significance of the presence of an ozone shield?
Does anybody know? What does it block out? Anybody remember that?
What's the big significance of the ozone hole over Antarctica for
example? It allows UV radiation to heat the Earth's surface,
and in fact if there were no ozone, the UV radiation would be so strong
that there would be no life possible on land.
So, once the ozone shield actually developed, organisms could conquer,
basically, the land's surface and settle on the land surface.
In this, then, is thought to be at least correlated with the
development of endosymbiosis. And I'll explain what I mean by
that. But it basically led to the origin of modern eukaryotes,
so your ancestors essentially. But there was still a long time,
obviously, until humans appeared. We have here the origin of animals
and metazoans, and then the age of the dinosaurs is
already a very small blip here on this graph. And humans don't even
get featured on that because we are so recent. So,
but what I want to show you here is that three major lineages
evolved early on. These are the bacteria,
archaea, and what we call a nuclear lineage. And the significance of
those nuclear lineages is that it basically combined with bacteria to
form the modern eukaryotic cell. So, the eukarya, or eukaryotes
they're also called. And it was this combination that we
called the endosymbiosis event. I want to explain this a little bit
more, and then I'll show you finally why we actually know that those
things are very likely to have occurred a long time ago.
Yes? It means the bacteria and the nuclear lineages combine to form a
eukaryote, OK? And I'm actually going to explain
this on the slide here. So, if you have any more questions
after that, please let me know. So, again, this shows you this early
evolution, this early split in two archaea, bacteria,
and this sort of nuclear line. It is thought that this nuclear
line, this was single celled organisms that increased in cell
size, and then developed or partitioned the DNA into a nucleus,
basically. So exactly how you find it in modern eukaryotic cells.
But then what happened is the cell took up a bacterial cell,
and over time this bacterial cell became symbiont.
In fact it became the mitochondria. And so what this mitochondria now
does in the moderate eukaryotic cell as you all know is it really took
over the energy metabolism. So, the proto-eukaryotic cell took
up a heterotrophic bacteria that form the mitochondria.
And this ultimately then gave rise to protozoa and to modern-day
animals. But there was a secondary symbiotic event.
This cell, once it had taken up a heterotrophic bacterium,
it took up an autotrophic bacterium, a cyanobacterium, an oxygenic
photosynthesizer. And this actually that led to the
development of modern algae and modern plants.
So what we can say is that mitochondria our ancient
heterotrophic bacteria --
And the chloroplasts are ancient cyanobacteria,
so, oxygenic, photosynthetic bacteria. And these obviously have
coevolved to then form animals and finally your plants.
So now, obviously we are talking here about events that happened a
very, very long time ago. And so, the big question is really
how do we really know this? But this takes me to the third
topic, which is that of molecular evolution. So, we can state
the problem again, And that is very simply put,
evolution is incredibly slow, OK? And therefore, its processes
are not directly observable.
And we need to actually use inference techniques to reconstruct
evolutionary processes. Now, what do we use when we want to
reconstruct the evolutionary history of animals and plants usually?
Anybody? Fossils. Exactly. So you take a shovel,
essentially, and dig down into the different layers.
And there's different techniques that you can actually determine the
age of different sedentary rocks. For example, and then you can
construct, if you're lucky, you'll find enough fossils of a
particular lineage. You can reconstruct the evolution
of the lineage. I'm sure you all have seen the
example of the horse, for example, where we have actually
quite good evidence when ancient horses look like.
And we can reconstruct the sequence of events that led to the evolution
of modern-day horses. Now, you can imagine,
though, that when we talk about such ancient events like these there really is no fossil record. OK, so what people have figured out, then, is that that was really a stroke of genius that came about in the late 60s, that DNA molecules can act as evolutionary chronometers.
OK, now what do I mean by that?
I mean that you can take DNA sequences or gene sequences from
different kinds of organisms. Based on those gene sequences you
can reconstruct the relationships to each other. You can determine
whether two organisms are closely related or whether they are only
very distantly related. And the underlying mechanism of that,
is that mutations happen with a certain probability all the time.
So, the idea is that as time passed on, DNA molecules will change.
So they will accumulate, actually, mutations, and so this will lead to,
and that the idea is that the amount of change in a particular DNA
sequence is proportional to the time of separate evolution of two
different lineages or two different organisms.
So, the amount is more or less proportional --
-- to time since the last
common ancestry.
So, let me explain how this is actually done.
What you really need in order to do this, is you need genes that are
related to each other, OK? So, genes, they need to be
universally distributed. That meets all organisms that you
want to compare need to have this type of gene. And,
those genes need to have conserved function.
In these genes,
we can then compare to each other, and I will explain how this is
actually done. Any questions so far?
OK, so the example that I actually want to bring is the 16S
ribosomal RNA genes.
We oftentimes abbreviate this rRNA. Now, does anybody remember what the
ribosomal RNAs are and do? What's the ribosome? Yes?
Right, and what does it do? Exactly, it's the location where
messenger RNA is translated into protein.
Now, the ribosomal RNAs are an integral part of the ribosome.
They play both a catalytic role as well as a structural role in the
ribosome. And so, fundamentally, because this is such
a fundamental organelle, all living organisms possess it.
So, all organisms have it. So this allows us to use these genes to
really compare all living organisms to each other.
OK, so this is a very important point.
I wanted to show you a, OK, if it wakes up. There we go.
An example of these ribosomal RNA genes, now this is actually,
what you see here is a secondary structure of the actual RNA,
the ribosomal RNA. Now, these molecules have a secondary structure
because they play a catalytic and structural role.
And so, the really amazing thing is when you look at the structure,
the structure determines really the function of those molecules in
different organisms. And then look at this.
We have here a bacterium, and here are an archaea. Now,
if you think back to the first couple of slides,
what I showed you is that those organisms have not shared a common
evolutionary history for about four, or so, billion years, or 3 billion
years, excuse me. But, if you just glance very
quickly at the structures, you see that they look very similar
to each other. So, there's an indication that the
function is really very highly conserved of those molecules.
However, when you actually look at the sequences in detail,
what you'll find is that there's different regions.
And I'd given some examples here denoted by A, B,
C in those molecules. And these different regions of the
molecules are really the key to its usefulness in figuring out the
evolution and ecology of many organisms.
The region number A here, or denoted by A, a sequence
stretches that are the same in all living organisms.
So they are universally conserved, which means that if you get a
mutation in a gene in that particular region,
you are dead. OK, that's why it's conserved essentially.
Then we have those regions B where the length is conserved,
but the sequence is not. So, there are sequence change
allowed, but the length needs to be conserved. And then there's the
region C were neither length nor sequence is actually conserved,
and where we get a lot of variation. So, let me write this down. We
have three types of sequence stretches.
We have A, what I called the
universally conserved sequences. We have B where length, but not
sequence is conserved. And, we have C where neither length
nor sequence is actually conserved.
And the first two stretches, the first two types of sequence
stretches, are very important in figuring out the phylogeny or the
evolutionary relationships amongst organisms. Whereas the sequence
stretches number C because they vary so dramatically,
are very important in identifying organisms.
And we'll talk more about this actually next time.
So what can we actually know do with those sequences?
Well, the first step is we need to generate an alignment.
OK, and this is actually shown here,
where each row denotes a gene from a particular organism.
OK, so these are all abbreviated here.
These actually aren't ribosomal RNA genes, but other genes.
And that what you will see here is we can recognize those three
different regions that I've pointed out before. You have the regions A
which tell you which nucleotides line up with each other,
so you use this sort of as an anchor because the sequences never vary
amongst organisms. And that the sequence region B
where you light up sequences that vary or stretches that vary in
sequence but not in length. Now, why is this important?
It's important because you have in each column that nucleotides that
have originated from a common ancestral nucleotide,
and whose variation over time you can actually monitor.
Is everybody with that? Any questions? OK, great.
The second step, then, is the calculation of a
similarity.
And this is shown here. Again, we have a very simplified
alignment now of four different organisms. Here,
we have the sequences that we want to compare. And what you'll see is
that they're overall very similar, but there are different sort of
nucleotides. And so, what we simply do is for
each pair of sequence combinations, we calculate the sequence similarity
value. So, what you see is that you have 12 nucleotides,
and the first pair differs in three nucleotides. OK,
so that tells us, or it's called actually a distance
here, I'm sorry. Let me write this down here.
It's simply one minus the similarity,
of course, but so basically a quarter of the nucleotides differ
where it's between A and C, a third of the nucleotides
difference on. OK, so you do this for each pair of
sequences, excuse me. The third step,
then, is to calculate the correction for multiple mutations affecting the
same nucleotides.
Now, you can imagine that over time there's a probability that a
particular nucleotide mutates, say, twice. So, in the first
instance it may change from A to a G, , but then it changes to a C.
But when you look at the modern-day sequences, you don't know that this
actually happened. And so there's ways to
statistically estimate what the likelihood is that a sequence
actually contains such multiple events.
OK, and this, we called, a corrective evolutionary distance
then. And what you will note is that the corrected evolutionary
distance is invariably larger than the actual observed one.
Now, what can we can do with those distances? We can constrain them
into a best fit tree of relationships.
So, we can draw what we call is a best fit tree.
That's shown here. We have our four organisms,
but when you look at those branches of the tree what you'll see is that
they add up roughly to the correct evolutionary distance here.
So, between A and B we have 0. 3 and 0.08, which roughly gives you
0.3 here, OK, whereas between A and C the tree is constrain such that we
have 0.31, and here 0. 5, and so overall you roughly get
the distance here that we have calculated. And so what this means
is that you ordered the organisms by their calculated evolutionary
distance. And so you have now obtained, actually,
a very intuitive picture of the relationship of organisms to each
other where A and B are obviously the most closely related ones,
and A and D are the most distantly related.
Is everybody with it? Any questions? OK, now,
this best fit tree is what we call a phylogeny.
Now, excuse me,
these techniques really revolutionized the study of
evolutionary relationships, and one of the things that it
allowed us to do is to construct universal phylogenetic trees or what
we can also call the tree of life. And I will show you this on the next
slide, and that I want to make a few general statements about this.
So first of all, when you analyze all known organisms,
and obviously that would be a big task, but representative of all
known organisms, what you'll find is that,
indeed, we have three major lineages: the bacteria,
the archaea, and the eukarya. OK, so we have what we call three
domains of life: the archaea, bacteria, and the eukarya.
So, this really is the evidence that life really split very,
very early on into those three lineages that I showed you before.
Interestingly, two of those major domains here are
prokaryotic, OK? So, two of the domains are
prokaryotes. Moreover, if you actually look at the types of
organisms that are on here, you'll notice that even on the
eukaryotic side of the tree, most of the organisms here are
actually microbial. So, the single celled organisms: and
that means that most of the life on the planet is microbial.
The vast diversity of organisms on the planet are microorganisms.
So, we can say that most life is microbial.
And when you, then, look at analysis of mitochondria,
and chloroplasts which all have their own genetic machinery,
and therefore also their own ribosomes you'll see that the
mitochondrion, OK, and the chloroplasts both tree
within the bacteria. So, we really have an amazing
confirmation of this endosymbiont theory which actually developed in
the absence of gene sequences by some Russian scientists in the early
20th century. So, we have that mitochondria and
chloroplasts tree within bacteria, and this really supports the
endosymbiont theory. So really, you could say eukaryotes
are really just walking, and swimming, and flying incubators
for bacteria, right? So, just hosts for microorganisms.
OK, so basically you can, what you should take home from this is the
three domains of life. Two are prokaryotic, and even more
so most of the diversity that we find is actually microbial,
and then finally the endosymbiont theory is actually confirmed by
those phylogenies. Now, what I want to cover in the
remaining time, is how we can actually use now those
sequences to learn something about organisms in the environment.
That's the topic of molecular ecology.
To introduce this,
I just want to show you a couple slides that really sort of capture
what the big problem is that we're facing here. Now,
when we look at the abundance of prokaryotic cells in different types
of environments, what we see is that there is an
enormous number of different prokaryotes out there.
This summarizes, here, different types of
environments. We have the marine environment, freshwater environment,
sediment and soils, subsurface sentiments and animal guts.
And that this number here gives you the average number of prokaryotic
cells either per milliliter or per gram. And it here we have the total
number of cells obtained by multiplying the average number with
the total volume of the particular environment.
So what you can see is that in the marine environment,
we have an average half a million cells per milliliter of water,
OK? It freshwater, we have about a million cells.
What is that telling you? There's a ton of prokaryotes out
there. What you go swimming, you take a little gulp of water:
you've probably eaten several million prokaryotes,
that it's nothing to worry about because what this also tells us is
that very, very few prokaryotes out there are really pathogens because
otherwise you'd be sick all the time.
Now, in sediments and soils, in as little as a gram you have five
times 10^9 prokaryotic cells almost. 5 billion prokaryotic cells are out
there, and even in very, very deep sediments that reach down
to 3,000 m, you have a substantial number of prokaryotic cells.
Well, and here's your guts, 10^5 times 10^6 gives you 10^11 per
gram. So again, you're just a walking incubator for
a very complex microbial community. Here's the global abundance. You
see that steeps of surface sediments and the marine environment,
probably in terms of numbers at least, the most important
microbial environments. Now, faced with this enormous
abundance of prokaryotes out there, very important question is how many
of them are out there? Or, how diverse our prokaryotes in
the environment? That's important if you want to
figure out their function and the environment, and want to understand
also their evolution. And what I want to show you here is
that we've gone through an amazing development in our understanding of
prokaryotic diversity in the environment over the last
10 to 15 years or so. Who knows about E.
. Wilson here? One person? So, he wrote a very famous book on
biodiversity, which was published in 1988, where he tried to summarize,
really, how diverse the known organisms are on the planet it also
try to extrapolate to the total diversity.
And what you see is that he came up with about 1.4 million different
species here, mostly dominated by insects. That's the big section
here on this pie chart. The plants: very important.
And if you look, the prokaryotes feature with about 3,
00 different species. So, in 1988 we thought there were very
few prokaryotic species out there. If you look about 10 years into the
future and take the assessment here, and this just exemplifies how the
thinking has changed, you see that we think now that there
is about 11 million different species out there,
and that the vast majority of them are prokaryotic,
OK, 10 million. So, this big part of the pie chart is
really the prokaryotic diversity. Now, what really has changed is
that we've actually started to use molecular techniques to determine
the diversity of prokaryotes in the environment.
So molecular ecology is really the use of molecular gene sequences
obtained directly from the environment --
-- to learn about the diversity
prokaryotic --
-- diversity out there. Now, this slide just quickly
summarizes this. Basically, the idea is that you go
out into the environment and collect either water or soil samples that,
as I just showed you, invariably contain a lot of different
prokaryotic cells. You then lyse the cells and purify
their DNA. And so that you end up with a mixture of DNA that
represents the organisms out there, and then you can use universal PCR
primers to actually amplify ribosomal RNA genes from all the
organisms that are present in your samples.
Now, why can you use universal PCR primers? Well,
they target the regions number A that I showed you before.
Those regions in the genes are invariant amongst all organisms.
You guys all remember how the PCR works, right? We cover this.
OK? Yes? No? Who doesn't? You don't? All right,
come to the board. Just kidding. OK, you should look it up. I don't
have time to cover this, unfortunately, but basically it's a
technique that allows you to amplify specific types of genes millions to
billion fold. And once you have done this, what you can do is that
you can purify the genes on gels, and then separate them by cloning
them into individual plasmids. And those plasmids have been
inserted into E. coli cells, and the E.
coli cells are then individually grown up so that each culture
contains only a single plasmid, and you can then sequence these
ribosomal DNAs or ribosomal RNA genes from those clones.
And so, you have obtained a library of the ribosomal RNA genes
from the environment. So, we use environmental ribosomal
RNA gene libraries from which we then can actually compare how many
different types of genes are out there.
So let me show you an example of this. What we have done recently,
we've gone out in one of the first really comprehensive samplings of
coastal bacteria plankton, which means the bacteria that are
present free living in ocean water. And so, we've done this, we've
collected all those clones, and then basically we constructed
those phylogenetic trees that I showed you before that really allow
us see how many different types are out there, and how closely related
they are to one another. And what we found is that in this
environment that you think might be very simple because it just the
water column right? No, not much structure in there.
We found over 1500 bacterial 16S ribosomal RNA sequences to occur,
so an enormous diversity of prokaryotes of bacteria in that
particular environment. And the important point is that when
you actually look at a collection of such studies that I just showed you,
what you find is that the vast majority of microorganisms in the
environment have never been cultured. So traditionally what we do of
course to learn about microorganisms when you grow E.
coli, or so, you throw them onto culture plates.
You make lots of different cells, and that allows you to study some of
their properties. But when you look,
for example, at results from the ocean, this summarizes now coastal
and open ocean environments, again, the bacteria plankton is
those free-floating bacterial cells in the water.
And you compare this to what we've actually been able to culture from
those environments. What you see is that you have some
dominant groups here. They have all funny names,
most of them, because they're just clones and clone libraries.
But these are the dominant groups that show up in clone libraries.
Here's their relative representation in different clone
libraries from a variety of environments. And so here you have
one very important one, the SAR11 group, or this one,
the SAR86, that always show up in clone libraries.
But we've never see them in culture, so the important point to realize
here is that what is actually happening is that whenever we go out,
we find a great diversity of bacteria out there,
but we have no idea what they actually do.
And this is one of the big questions that we need to answer to understand,
really, how the planet actually works. What are those uncultured
microorganisms out in the environment really doing,
and what is their importance? And we'll talk about this next time.
We're going to talk about environmental genomics because
essentially what we can do now, is we have techniques available that
allow us to isolate and least large fragments of the genomes,
sequence those, and look at what kinds of genes they have present.
And that allows us, then, to infer some of their
function in the biogeochemical cycles in the environment.
OK, so with this I'm going to close today unless you have
any more questions.