1:00 - 1:30 pm
Bioprospecting & Benefits Sharing: What They Mean for Yellowstone
John Varley
Yellowstone Center for Resources

Transcript

John Varley:  This, however, is right off the griddle.  And I thought that those of you who came out from in the Park would be interested in the preliminary results of the Molecular All Taxa Biodiversity Inventory that we started with last August and now have its initial results.

The all taxa biodiversity inventory was attempted on Yellowstone Lake.  Yellowstone Lake was chosen for this test case because it had had for 133 years periodic and very good science associated with the life that lived in there.

That began with Yellowstone’s real pioneers, the Hayden Expedition and so forth.  In the 1890’s, a whole series of some of the most imminent scientists in the world came through and stopped at Yellowstone Lake and did some really extraordinary work.

That’s happened episodically ever since, right up to today.  During that time, they, and I’m calling them the Linnaen taxonomists because if you went to the same schools I did, at the same time, then you should be retired.

 (laughter)

But if you went more recently and took biology you became familiar with Linnaen taxonomy.  Linnaen taxonomy is a giant, elaborate way of classifying life, phylogenetically based upon the phylogeny, the evolution of different characteristics.  Then they were given a taxonomic name.  Homo Sapiens, for example or Canus domesticus, my dog.  For roughly 400 years that had been the accepted form of classifying life.

Since about 1990, roughly, we’ve seen a new cast of characters that came in and are upending that 400 years of classic taxonomy.  That’s the molecular geneticist.

They didn’t come from the tradition or culture of taxonomy, but their classifications of life are better because they’re looking at some portion of an organism’s genome instead of external characteristics, like eye color or earlobes or any number of other morphological characteristics.  

So, what we’ve got here is an alpine lake and the Linnaen taxonomists and the ecologists concluded over that 133 years of  study, that Yellowstone Lake, from a standpoint of the diversity of life, is a pretty unremarkable place.

I’ve said that myself in books and papers.  It has an extraordinarily simple ecosystem.  Seven common species in the plankton, for example.  One fish species.  Five or six or seven snails.  Those are just tiny numbers.  It was thought that was the state of things because it’s not far from distilled water.  It flows out of all those drainages…., that volcanic rock is relatively insoluble so it doesn’t have a lot of production capability.

Most places that have very little productive capability, called ligotrophy as it relates to water, are like that and we concluded… many ecologists concluded that was the case for Yellowstone Lake.

And you say, “Well, it has hydrothermal features; it can’t be that simple.  The hydrothermal features, which are primordial soups geochemically must give it a boost or something”  The ecologists considered that  and said, “Well, there’re some hot springs around both in the contributory streams and in the Lake itself but their input is not great enough to argue against the Lakes basic ligotrophy and its simple biodiversity.”

Our mission is to preserve and protect biodiversity and the premise is that it is difficult to protect what you don’t know exists.  So the existing knowledge is key in…is part of this assumption. 

The second bullet is just what I told you; the traditional assumption of low biodiversity is based upon Linnaen taxonomy and a ligotrophic lake environment.  The rationale for questioning that, is that most ecosystems, most biotopes, have recently shown, meaning that it’s molecular geneticists that showed it, surprisingly high biodiversity when using molecular approaches.  The hypothesis is that Yellowstone Lake may  resemble a deep sea ocean vent more than a typical alpine lake. 

As you know, in the last dozen or so years, that people are finding that the geothermal fields at the bottom of the lake are huge.  It might be the greatest collection of geysers, hot springs and fumeroles in the Park.  Any time you have that in the sea, you have some very novel life associated with it.

That’s the premise, therefore we came up with this proof of concept molecular all taxa biodiversity inventory.  The initial research plan was to analyze the Lake’s prokaryotic and eukaryotic diversity by ribosomal gene sequences. 

Prokaryotic is the bacteria and archea, all microscopic organisms.  The eukaryotes have a number a number of microscopic organisms, as well, but it also includes trees and human beings.  So, life now is divided into those two giant categories. 

The pitch was, that we can find and cultivate, actually grow these hidden things using traditional and novel technologies developed by a San Diego biotech company called Diversa. Then we can compare the molecular findings of a 133 years of Linnaen taxonomy vs 4 days of molecular…four days versus a 133 years. 

The beautiful picture is a stereonella(?) formosa which is the most common diatom in Yellowstone Lake.  I’ve seen a lot of pictures of it but that’s the most gorgeous and it’s because they were able to cultivate this in a laboratory.  I’ll show you how in a minute.

We put together an interesting team to do this.  The Diversa Corporation, a for profit corporation, Park Service, the Yellowstone Park Foundation, Eastern Oceanatics, Dave La (?)and his wonderful robot, several USGS scientists including Lisa Morgan and we sort of designed…balled all this up and came to the Lake last August to do this work.

 The Diversa Corporation was key to this because they have fundamentally automated what is in a traditional university laboratory. (They have) The ability to look at genes and bring order to them.  They also hold a patent and have the ability to take community DNA, and that would be if I came through and took a mouth swab of all of you in this room and put it into a common vial and shook it up.  That would be community DNA. My DNA would be mixed up with Paul’s and right on down the line and how do you sort that out?

Well, they have a very interesting way of sorting it out.  It’s a process that’s patented by them and allows them to look at this community DNA.  It just takes us light years ahead of the traditional way of looking at genetics. 

Well, we know  our goals, our objectives, are to conduct species inventories, demonstrate the value of protecting biodiversity, strengthening and protection and conservation of biodiversity, developing tools for monitoring biodiversity, studying extreme environments, sharing information with the public, serving the public, maintaining our long standing tradition of being first, conserving and preserving, and maintaining over a 100 year tradition of being an environmental laboratory.

Now, let’s look at Diversa’s objectives.  They want to identify what novel diversity but for new products.  They want to demonstrate the value of protecting biodiversity.  They demonstrate the ability to access biodiversity without harming it.  Being non-invasive is always good,  advancing technology for detecting and assessing novel biodiversity,  accessing new organisms at the extremes of life,  discovering novel enzymes and gene pathways,  developing long term mutual beneficial collaborations, serving their shareholders, most of which are average citizens, and maintaining leadership and novelty in …..(?) in other words, their shockingly similar mission. 

Now, the preferred method of surveying biodiversity is the micro and macro morphological inventory.  That’s the way it’s been done since Linnaeus, 400 years ago.  It’s all based on eye color, and ear lobes and hair color and so forth.  We’re still doing this and there’s a place for it; I’m not putting it down at all. 

Phylogenetic profile takes a section, called the 16S gene, it’s really a section of the gene. Some of the genes and gene sequences in 16S are very conservative, meaning they don’t change much, you don’t often see them morphing into something different.  What you get with those is these primers that go and identify that gene sequence.

Then culturing organisms using standard laboratory microbial methodologies.  Accessing genomes of organisms that can not be cultured and that would be missed due to their having unusual 16S ribosomal genes.  For example, the brand new group of organisms only known for the last four years called Nanoarchaeum.

Accessing organisms where DNA can not be purified and would be missed by traditional culturing methods.  This high throughput culturing just means they’re doing it on a massive scale.  I’ll show you some of that in a minute. 

Collating and comparing our data to the Linnaen data to test the hypothesis and, ideally, establish a program.  Now how this works, is you go out and get an environmental sample, in this case it’s a soil sample.  You extract the DNA from the entire sample, in other words, community DNA.  You (?) it using  polyermase which came from Yellowstone originally and make these genome libraries.  Then it’s screened to determine which biomolecules they are.  Then Diversa takes it and attempts to match it to these industries: agriculture, chemical, industrial and pharmaceuticals. 

We’re interested in this part of the process but especially this loop which includes the phylogenetics of the organisms.  How one organism relates to another and so forth and the cultivation of those organisms in addition to that. 

How they cultivate these is really interesting.  They collect the environmental samples, they prepare the samples and purify the cells of the mixed population, then it goes through this machine that has a laser beam.  Every time a single cell comes through, that laser beam bounces it out of the stream.  They have single cells and they are put into microcapsules with a nutrient solution.  You need a microscope to see these microcapsules.  It’s all robotic.  They pack these things into a column, and they have many of these, so they can have them at different temperatures, and have different nutrients, and so forth.  They grow in these microcapsules and then the microcapsules are put into regular agar plates and start growing there in these wells.  Each of the plates has  100,000 holes and they fundamentally can treat, in this manner, about a billion samples a day.

It’s just staggering what this does.  Of course, what you get out of it is some pretty interesting stuff. 

Now you’re wondering what the Douc Langurs are doing here.  A Douc Langur is this cute little fellow up here that is an extremely endangered animal and they’re trying propagation in the San Diego Zoo.  They’re just not having a lot of luck so Diversa volunteered to come in and follow these things.

 The colors in this phylogenetic chart each represent an organism of life from their guts.  From the Douc Langur gut.  The green are healthy and normal Langurs and the greens are mostly found over here.  I think you can see that, you probably can see the scale.  The red are early sick Langurs.  The yellow are advanced sick and the blue is deceased.

A Veterinarian can go through here and see what’s going on in the Langur’s gut when it’s healthy, when it’s sick, when it’s really sick in a big way and finally, when it’s ready to expire.  That’s part of their pro bono program for the San Diego Zoo. 

This paper was published…it’s talking about the comparative metogenomics of microbial communities and was published about a week ago so it’s right hot off the presses.  It gets to the new and emerging field of taking this community DNA and looking at different ecosystems.  Again, trying to go where we want to go and that is to find one ecosystem and try to describe all of the life in it. 

Now, in their example here, they took three different samples, one from the Sargasso Sea, one from Minnesota farm soil, and in this corner, a deep sea whale fall.  Does everybody know what a whale fall is?  That’s when a whale dies, goes down on the bottom and starts to decay.  They’re called whale falls and they’re rather extraordinary ecosystems by the time it’s all gone, just because of their mass. 

They are not only able to sort all three ecosystems out, using this metogenomic, using a functional analysis.  This is going beyond just naming species, as we saw in the Langur a minute ago, but instead, going to the functional processes that are inferred by the gene sequence observed. 

What would be an example?  The gene sequences in our acid loving microorganism, sulfalobasol (?), common out around Norris, live at a pH of 1 or less….boiling water, now there’s a physiology, and they’ve got a particular array of gene sequences that allows them to live in there but would kill you or I in seconds if we fell in it. 

So they are able to separate out these three different physiologies from these three very different physiologies, from Minnesota farm soil to the Sargasso Sea, to that which is associated with a deep sea whale fall. 

They’re all very unique and they put them into a family trees and build these massive gene libraries that is of great interest.  The way these Operons are scattered in the triangle, shows those things that can process sodium very well and those that process potassium very well. 

That’s all foundational stuff to get to the results of what we found on Yellowstone Lake.  In the Eukaryotes, we use a different gene, it’s 18S gene because that’s a better gene to separate Eukaryotes, which are insects, crustaceans, eublyna(?), fungi, those kinds of things. … Cultivation of library…  and the Prokaryotes, which are bacteria and the Archaea, all microscopic. 

Here is the phylogenetic analysis for Eukaryotes and less than 16% of the gene sequences they found had any appreciable identity with gene sequences that are deposited in the giant depository for all this of information.  That’s called GeneBank.  Most of the sequences represent really novel lineages of those that they found. 

(My screwed up slide here; I don’t know why these things float around)

For example, , these are all diatoms right here, and there’s one, two, three, four, five, six genera that have never been described from Yellowstone Lake before.  At the genus level and at the species level and we also had one there that we had in common, (?) between the MATBI and the Linnaen taxonomy.  So we have one match in the diatoms.  The rest of them are novel for Yellowstone Lake.

Thalasseosyra (?) is a species that is almost totally marine, and it has only one known fresh water species and that only occurs in tropical areas.  You get these little individual stories that suggest a great deal of novelty.

This group here are the water molds and that was the first time they have ever been sampled and collected from Yellowstone Lake.  The fungi just literally knocked my socks off.  There were one, two, three, four species of fungi that are very likely new to science.  Two named species, Bulgaria is a mushroom that is most common in Europe and Asia and what that’s doing in Yellowstone Lake is anyone’s guess.  If that’s a bizarre story, then this cryptococcus is even more bizarre because that species has never been found outside the McMurdo Valley in Antarctica.  But we found it in Yellowstone Lake. 

There’s a bunch of ciliates here that are actually fairly boring as a group.  Very common things.  A species of paramecium we found in Yellowstone Lake, you can buy a little jar of it from Ward’s supply catalogue in Chicago, enough for 30 students to work on.  The same for Homolozoon, which is really a lot bigger than a paramecium and is easier to study.  Obertrumia is a ciliate that is most commonly found in polluted environments, so that’s pretty interesting.   

Down here, in the section you cannot see, but I can read it here, you’ve got the crustaceans that form the food pyramid for the trout in the Lake and nematodes and we’re still sorting those out.

Here are some of the cultivated eukaryotes, the one with the big circle here, are mostly diatoms, but these are two micro green algae species that were found.  They’re minute and they’re often solitary or found in groups of four or eight.  That was the first time we found green algae in Yellowstone Lake.

This little inset here is the nanoarchaem that would be, looking at these diatoms, if it were to scale, a little pinprick on this screen. 

Here’s the phylogenetic tree for bacteria.  The bacteria that was found in the Lake, and no bacteria have ever been described from Yellowstone Lake, less that 4% of their gene sequence have a high identity with anything in GenBank,   Possibly 85 new bacterial species; new to science in that sample and there were no sequences with high identity to GenBank and over 100 new archeal species were found.

This talks about nanoarchaem, those are the organisms I mentioned that have only been known to science as a group for about four years.  There are two species known from the world, one from the deep sea oceanic hot springs from very deep vents off the coast of Iceland and another from the deep sea off the coast of Indonesia.  So there were two species known going into this and they discovered twenty eight in the bottom of Yellowstone Lake.  That was pretty exciting.  At Diversa, they made the statement that a larger nanoarchael diversity was found, at the inflated plain, that’s the bubble that goes between Stevenson Island and Mary Bay, than any other site in the world.  

They’re a very minute thing and they’re either an endosymbiont (?) or a parasite of bacteria and archea. 

We should have brought the artist to describe this.  This is the family tree of the biodiversity in Yellowstone Lake.   I’m selling that poster for….just kidding…

(laughter)

It was done pro bono by the joint genome institute, which is located on the Berkeley campus of the University of California.  If we go into another phase of this study then they will be a full partner and will be able to help us immensely.  The long and short of it is that the Linnaens found 233 species, the molecular scientists in four days found about 250.  There were only two in common.  The mathematicians can take that number, and it’s way beyond my algebra ability I’ll tell you, but they can come up with an estimate of the total number of species in Yellowstone Lake.  The low estimate is 50,000 and the high estimate is two million. 

So that’s what we’re looking at from Yellowstone Lake, quite a bit more diverse than anybody every thought.  It looks like each one of those vents on the bottom of Yellowstone Lake is a different habitat to a different group of organisms.  And now we know, double the numbers, over 500 roughly, of the species and we are a long ways from 50,000, the low estimate where two million is the high estimate.  Thank you.

(applause)