CARTA: Ancient DNA – Archaic Ancestry; Prehistoric Biology from Dental Calculus; The Oldest DNA

this ucsd-tv program is presented by university of california television like what you learn visit our website or follow us on Facebook and Twitter to keep up with the latest programs we are the paradoxical ape bipedal naked large-brained long the master of fire tools and language but still trying to understand ourselves aware that death is inevitable yet filled with optimism we grow up slowly we hand down knowledge we empathize and deceive we shape the future from our shared understanding of the past Carta brings together experts from diverse disciplines to exchange insights on who we are and how we got here an exploration made possible by the generosity of humans like you you so thanks so much for the invitation the great pleasure to be here and today I'm going to be talking about the legacy of archaic admixture in present-day humans and so to set the stage here based on genetic analyses of presently populations we've built up a fairly good broad outline of the history of modern humans so for example we know that modern humans evolved in Africa and about a hundred thousand years ago there was a dispersal outside of Africa to the rest of the world we also know based on archaeological evidence that during this dispersal modern humans overlapped with another archaic human population the Neanderthal and so this makes the question whether these two populations that overlapped potentially met and interbred and so several studies have tried to answer this this hypothesis but in the last five years there have been some major breakthroughs that allow us to get whole modern whole genomes from ancient human populations in particular we have genomes from the Neanderthals and the Pfister group the Denisovans and we'll hear a little bit more about the technical challenges that enable to break through in the later talks but what I like to focus on today is the fact that once we have these ancient whole genome sequences we can pretty much definitely answer the question of whether there was interbreeding between these two groups and so a number of studies have shown that there was indeed interbreeding or gene flow for example we now know that non-african populations trace about two percent of their genetic ancestry back to the Neanderthals we also know that there was Denisovan gene flow into some present-day human populations for example populations from Island Oceania have about three to six percent of their genetic ancestry tracing back to the Denisovans this is over and above the 2% that they inherit from the Neanderthals so the the big picture is all non-africans today carry a small amount of archaic ancestry and at first blush this might seem like a small contribution but when we examined this further one thing that we need to keep in mind is the Denisovans in the Neanderthals were highly diverged from modern human populations at the time of admixture so we think that they were separated by at least a couple of hundreds of thousands of years and as a result these populations accumulated novel mutations that were never seen in the modern human population and these novel mutations entered modern humans through the admixture process who we estimate that about 10% of the snips at the time of admixture in non-africans could have been of Neanderthal origin so our hypothesis is that these archaic add mixtures were having a potentially large impact on human biology so as one concrete example of the potential impact of archaic admixture we were involved in a genome-wide Association study a study that aims to determine genetic variants associated with type 2 diabetes in mexican-americans so in this study we determined a novel variant found in this gene called SLC 1611 and we found that this gene had a unique geographic distribution but when we look at the variant in this gene that confers increased risk for type 2 diabetes we found that the risk increasing variant is found at appreciable frequencies in the Americas is essentially absent in Africa and is present at low frequency everywhere else and what we found is that this risk increasing variant at this gene matches the genomic sequence found in one of the Neanderthal genomes that had been sequence so what we determined was this was a variant that had integrates into modern human populations from Neanderthals now a number of other studies have looked at specific genetic positions genetic loci and have documented substantial contributions of archaic ancestries at the slow site so here is a not very representative list but what we wanted to do is go beyond single Llosa analyses and ask what does the distribution of Neanderthal ancestry look if he were to look across the genome so we wanted to go from single OSA to a genome-wide assessment and to do this we need to build a map of archaic local ancestry so what do we mean by that basically what we want to do is to go along an individual person's genome and label the positions where they carry archaic ancestry and so here is a cartoon that depict what's going on and why we need to do this but we have a modern human genome ad mixing with an archaic human genome and if you look at the descendants of this interbreeding because of the way the genome is transmitted in every generation it gets broken down by a process called recombination and as a result if you look at the descendant of this interbreeding event this descendants genome is going to have this mosaic pattern where some portions of the genome trace their ancestry to the modern human and others trace their ancestry to the archaic human population and so what we'd like to be able to do is look at an present-day individual's genome and figure out which are the portions that are bred and which are the portions that are blue so to be able to do this we came up with a statistical model and what this model allows us to do is to infer these locations of archaic ancestry specifically we're going to look at a target genome this could be any genome that we are interested in and we compare this target genome to the genome of the Neanderthal the Denisovan as well as to a reference panel of African individuals we are looking at Africans because this is a population that we assume has little archaic ancestry it's a reasonable assumption but not entirely true but we work with that and our goal is to look at the star get 0 and for every position which we call a snip here along this target genome we like to label the ancestry or more precisely the local ancestry of this individual so we like to be able to say that this snip at the target individual is Neanderthal in ancestry whereas this other snip is modern human in ancestry so I'm going to give you a little bit of intuition for what goes under the layers of the statistical model that allows us to make these influences the basic idea is we are going to be looking at patterns of genetic variation that are informative of archaic ancestry so here is an example where we are going to be looking at features of genetic variation that are informative of Neanderthal ancestry so here we are looking at a single position in the genome where we are comparing the target genome to the Neanderthal the Denisovan and the Africans and what we see here is a genealogy or a tree that relates these genomes at this position and what we see at this position is that there is a mutation that clusters the target and the Neanderthal genome to the exclusion of all the other genomes so when we see this pattern we are likely to think that this is a position that has come into the target genome from Neanderthal admixture on the other hand here is another position where there is a mutation that clusters the target with the Denisovan and the Africans to the exclusion of the Neanderthals and when we see this we conclude the opposite that this is a position that is unlikely to carry me and Ethel ancestry so these kinds of features go into the statistical model which figures out what is an optimal way of combining these features to get the best possible predictions and similarly we can also do a statistical model that allows us to predict Denyce of an ancestry so we applied an initial version of this statistical model to a data set that comes from what is called the thousand genomes project and this already gave us some very interesting insights but a limitation of that data set was that this data set had a small sampling of populations from outside of Africa and a second limitation was that it lacked any individuals who carried any seven ancestry so we can make no inferences about the Denisovan contributions to modern human populations so instead we applied this data set with this method to a new data set this is a rich genomic data set that's called the Simon's genome diversity project it has genome sequences from over a hundred non-african populations and most importantly we have in this data set 20 genomes from Oceania from individuals who have substantial Denisovan ancestries so we applied our matter to this data set and the first thing we'd like to be able to conclude is that the method is giving us accurate inferences about archaic ancestries so one way to do this is to make sure that the influences are consistent with everything we know about human history so the way we do this is we take the influences that come out of the statistical model we average it across an individual's genome and compute what proportion of an individual's genome is archaic and ancestry in this particular case we are asking what proportion of an individual's genome is Neanderthal in ancestry and so what we have here is a circle for every population and the color is telling us whether you have low levels of the and ethyle ancestry to high levels of Neanderthal ancestry the first thing we observed is in general non-africans carries substantially more Neanderthal ancestry compared to South African hunter-gatherers which are not included here but this is what we expect based on a previous demographic models which tell us that there was Neanderthal admixture into non-africans after they split from Africans we also observe that when you look at eastern non-african populations they carried more Neanderthal ancestry compared to restorations which has also been a previous observation consistent with the literature now we can do the analogous thing for the niece of an ancestry and what we see here is that Oceanian populations have substantially more Denisovan ancestry compared to mainland Eurasian populations further amongst the mainland Asians there seems to be more Denisovan ancestry in East Asians compared to restorations again all of these are consistent with previous results now when we looked at the data further there was one element of surprise and a novel result and the novel result is that several populations in South and East Asia tend to have an excess of Denisovan ancestry that had not been observed before so these are populations like the Chaka which is a population from Nepal the Tibetans and Bengali were a population from East India it turns out that this is a trace amount of the niece of an ancestry that we carry we estimate that it's about five parts in a thousand which is why you mean these sensitive statistical methods to see these Denisovan contributions now one question we were interested in can this excess of Denise of an ancestry in South Asian populations be explained by a mixture between Eastern on Africans who have more Denisovan ancestry with restorations who have less than e7 ancestry to see this we plotted the DNI's of an ancestry as a function of what proportion of your genome is related to non West durations and what we see is generally as you get closer to the non West Eurasians you have more than e7 ancestry but when you look at South Asian populations they have systematically more that can be explained by this model so what this is telling us is one of several things a model that is consistent with this observation though not the only model is that there were actually three Denisovan integration events in the history of modern human populations one in the history of the pop ones for the Oceania pne's the second in the history of the East Asians and the third in the history of South Asian populations now we decide to zoom in and instead of looking at how archaic ancestry changes across individuals we'd like to look at how archaic ancestry changes as we move along the genomes so here as we move along the circle we are moving along the genome looking at different chromosomes and the outermost circle is telling us what is the DNI's of an ancestry proportion in the Oceanian and each of the inner circles are telling us what is the Neanderthal ancestry proportion in different continental populations and the key observation is the colors along these circles tell us which are positions in the genome where there is detectable proportions of archaic ancestry so the key takeaway from this figure is the archaic ancestry doesn't seem to be randomly scattered along an individual's genome there are certain hot spots where there is an elevated proportion of archaic ancestry so we call them Peaks and then there are certain other positions in the genome where nobody seems to be carrying archaic ancestry which we term deserts so this was another surprise and we'd like to figure out what is going on in these peaks and deserts of archaic ancestry so we looked at one or the most extreme example of a peak so this is a locus that overlaps a gene called based on you cling to and in the thousand genomes European population we find that about 60% of individuals European individuals today carry the Neanderthal variant of the allele this needs to be contrasted with the 2% who would have carried it 50,000 years ago essentially the Neanderthal variant has increased from 2% to 60 percent over the last 50,000 years this is not an isolated example and we find out the order of 200 low-side with elevated Neanderthal ancestry in the different non-african populations and about 50 loss I with elevated ne7 ancestry in the Oceanian populations so these are all potential candidates for what we call archaic adaptive introgression putting it simply these are places in the genome where the archaic allele conferred an adaptive benefit in the modern human population which is why it rose up in frequencies so we'd like to understand what might be driving this increase in frequencies add these positions and that turns out to be a really challenging problem one way we try to address that is by looking at sets of genes that are known to be associated with certain functions or certain biological processes and we asked are these sets of genes harboring an excess of archaic ancestry much more than we'd expect and so we find several sets of genes that show an elevated proportion of archaic ancestry much more than we'd expect so for example genes that are involved in keratin filaments or mayshen so keratin is a protein that's found in hair and skin are enriched for Neanderthal ancestry across all the non African populations similarly we find that genes involved in trace immune receptors these are genes that are involved or important for olfaction tend to have elevated proportions of Denisovan ancestry so this again allows us to narrow down what the selection pressures might be but still we are quite some ways away from figuring out what the exact sequence of processes were that drove these archaic variants to high frequencies next we turn our attention to deserts so these are large regions tens of millions of bases long where we cannot detect either Neanderthal or dead of ancestry and even more impressively there are four such regions in the genome that are deserts for both Neanderthal and Denis seven ancestry so here is one example this is a desert on chromosome seven and it contains a number of genes within this region but one gene that caught our eye because of the prior work associated with it is a gene called Fox p2 so this is a gene that's shown to be important for speech and language so a possibility here is that these deserts of archaic ancestry places in the genome that are resistant to our kick introgression and a reason for that is that these are places that are important for the modern human phenotype the challenge again is that these are large regions of the genome and trying to localize what might the changes be that make them resistant to introgression is actually hurt quite challenging so we decided to look at this in a slightly more quantitative manner and the way we did this is we chopped up the genome according to a measure of the Selective strength in that feature of the genome and we asked how does the archaic ancestry change in different portions of the genome the x-axis as we go from right to left is going in directions of stronger selective constraint and what we find is the archaic ancestry whether we are looking at Neanderthals or Denisovans decreases as we move towards the strongly selectively constrained regions of the genome so this is consistent with the observation that there's been purifying selection to remove archaic alleles and there are several models that have been proposed one of these is that these archaic alleles are deleterious and they're being purged from the human population another one that we've also proposed is one of hybrid sterility where these populations have diverged and have accumulated genetic incompatibilities that are not tolerated on each other's genetic background so I'm just going to conclude very quickly I've talked about statistical models for inferring maps of archaic ancestry and so by combining these sensitive statistical models with the rich ancient and modern genomic data sets we can make some very fine scale inferences and the kinds of inferences lead us to conclude that there is a lot of complexity in the demographic histories of these populations we'll learn more about it in later talks and when we look at the variation along the genome clearly this is affected by selection but of there's also demography at play here and a major challenge for us is to separate out demography and selection and different kinds of selection from each other with that I'd like to acknowledge my colleagues at Harvard colleagues at the Max Planck and members of the Neanderthal genome consortium for comments and criticism different stages work so as I'd mentioned I'm sort of a bio chemist by training and I've been wondering for many years of how it is possible actually that ancient DNA is preserved and fossils over tens of thousands of years and I'm also wondering how much further can we go in time so how will it be in five four fifty years perhaps if I can still see that if someone giving a talk here of what material are they going to talk about is it a million years so in order to address the question of how long DNA can be preserved in bones I think it's worthwhile to look a bit into bone structure the bone consists of two major components one is an organic component collagen it's a protein that gives some level of elasticity to bones so that they don't break easily and then there's another very interesting component which makes up up to 50% of the bone mass which is hydroxyapatite it's of the form of calcium phosphate that has a very interesting biochemical property which is shown in this experiment here if you take a mixture of DNA of different sizes sort of similar to what you might find in an ancient bone and you dissolve this DNA in water and you add a suspension of pure hydroxyapatite DNA will instantly bind to the hydroxyapatite and then you can wash the hydroxyapatite powder often with water or with all sorts of low salt buffers and you will not release this DNA from hydroxyapatite it will only come down if you give it a harsh chemical treatment just using a decal see fire to break up the hydroxyapatite structure or using a strong phosphate buffer and you can repeat this experiment using ancient bone and so this is shown here so you the DNA pool of DNA fragments immediately binds to ancient bone that is also known to contain and dodging his ancient DNA and as of age forty to a hundred thousand years and it only gets comes down after intense treatment with chemicals and that even works if you have bones that are much older and bones had actually known at least that current level of resolution not to carry any ancient DNA like this one from a site in Germany that six hundred thousand years old or this bone of a dinosaur that's 80 million years old so why this is sort of a very interesting proof that you can recover DNA from dinosaurs it's not yet proof that this can also be done for authentic dinosaur DNA but an important take-home message here is that the bone structure itself sort of provides a very good carrier for for DNA and there's basically no limit in the DNA binding capacity of bone over time so what is it then that sort of limits us in our ability to study ancient DNA through time and obviously there are also other processes processes taking place that will degrade DNA over time there are a lot of reactions some hydrolytic some oxidative reactions and the most the ones that are thought to damage DNA most are those ones with the red arrow here which is hydrolysis of purine bases or a's the letters s and g is coming off the DNA and then over time your DNA breaks down into shorter and shorter molecules and you can model this for example here assuming a strand break every 50 base pairs in your DNA then you will see that you have a very few long molecules but a lot of very short molecules and the more you damage your DNA s the more this distribution gets shifted to the left end until all your DNA has disappeared or broken down to fragments that are too short to make sense of so for that and it's important to know that this these chemical processes are temperature dependent so they occur much slower at lower temperature which is also why if you look for the world record of sort of ancient DNA genome sequencing you have to have to go to the permafrost so there's a two years ago I think there was a very exciting paper published an almost full genome of the seven hundred thousand years old horse from permafrost it gets more complicated if you leave permafrost and if you are then sort of if your faucets are subject to these temperature cycles that occur during the Pleistocene so for the last roughly hundred thousand years temperature mostly colder than they are now but then you enter these interglacials where you have elevated temperature and these are very detrimental to DNA preservation nonetheless sort of being limited to the last hundred thousand years and temperate climate zones is not too bad after all there are some very interesting events taking place in human evolution and one of them has been mentioned before by Shriram is the human dispersal out of Africa which may have occurred with the major wave around fifty thousand years ago and so humans went they when they left Africa they were still in the and that has living in the western and central Eurasia and we have generated genome sequences from several Indiana tiles also one to very high quality and we could make inferences from these genome sequences that show that Neanderthals are as expected also by morphological evidence a sister group to modern humans so they're different evolutionary lineage that we can or could also see and the analysis of these genomes was first time shown by a green in 2010 that there was actually admixture going on so that some that there was some contribution of the Neanderthal genome into modern humans and we've seen more recently sort of a weak signal also for the other way around so that some Neanderthals may have picked up modern human DNA when they interacted so if the analysis of genomes from the last hundred thousand years has also brought us the Denisovans so they were discovered based on just a few small fossils a tiny finger bone and a few teeth that were found in the knees of a cave and based on the analysis of their genome we could show that they are that they are indeed a sister group to Neanderthals so they are like the eastern brothers or sisters of Neanderthals and they have contributed also their DNA to modern humans but unlike Neanderthals not to all humans outside of Africa but to to humans in eastern Asia and mostly like in the highest proportion of ancestry of the nisman ancestry you find in Oceania here which makes us believe that the nice ovens were once much more widespread than just look in the mists of our cave now the problem is still we are looking sort of at the tips of these evolutionary branches so we are looking at the last sort of Denisovans the last Neanderthals sort of relatively late modern humans and would be tremendously exciting to look further down in the past and try to understand sort of what are the genomes like of these ancestors of the archaic ancestor and what form all for me would be very exciting is to sort of find the genome of the sort of last ancestor between humans and the arcades now as I mentioned before so the chemical reactions make DNA break down very short pieces so if you want to address that problem from a biochemical perspective what you have to do is you have to go for this extremely short fragments and hoping that you would find some surviving DNA in this size range so you the question then is where are we currently in doing this and this is sort of a typical fragment size size distribution of DNA sequences that we recover from fossils so from a few years ago from the world record holder the host genome the 700,000 year host genome that was sequenced by a group in Copenhagen and as you can see the DNA sequence is coming from this horse are indeed quite short sort of centering on about 70 base pairs but even even so there we could we could in principle make use also of sequences turn this up to maybe 30 35 base pairs so the question is are these sequences lost in sample preparation or or is it that truly there's no longer than a preserved in these bones so I I and a several graduate students in my group have sort of intensely looked at this over the past years and we've basically gone through every step of the sample preparation process and trying to refine it so that we maximize the number of molecules that we keep in each step and that we also extract the shortest possible DNA fragments and this starts obviously with DNA extractions or the process of isolating the DNA fragments from the bones or teeth and purifying them so that they can be used in downstream reactions there's a second very important process involved in DNA sequencing these days which is library preparation and in this process you attach short artificial pieces of DNA to the ends of each DNA fragment and this short adapter so-called adapters can then be used to make thousands of copies of each molecule and then also to prime the sequencing reaction read the molecule out as a last step of this process you're taking the sequences coming out of the sequence and trying to match them to a known reference genome in our case it's the human reference genome which has been in sequence to very high quality in 2001 and even this process actually has some issues and we are also working on sort of optimizing the bioinformatics algorithm that we use to identify human-like sequences now going through all this sort of in several cycles for several years we eventually sort of determine the fragment size distribution in ancient bones that looks more like this so this is what I've shown you before this was the distribution of fragments from the permafrost this now is a typical fragment size distribution that we see in in most bones from from caves and temperate climate zones and what you see indeed there are very very short fragments in the bones many fragments much shorter than we can currently use for analysis because sequences then so short you can't even identify it as a human sequence anymore but it's not that we are sort of losing these long ones and now we gain the short molecules indeed the sort of an extra gain of short molecules so this year did the small black stripes are the fragment size distribution that I've shown you before and sort of this gain of short molecules is on top of this now from the sort of methods perspective we are all ready to sequence order DNA than before but the problem of course still is you also need a site that allows an extraordinary level of DNA preservation and we were lucky enough to identify this site in northern Spain the site is called the Sima de los huesos the pit of bones and it carries the remains of at least 28 virtually complete hominid and skeleton separate dated to four hundred thirty thousand years ago and this is for the biggest assemblage of fossils from the Middle Pleistocene in Eurasia the time period preceding hundred thirty thousand years the site is very special and that it's sort of 30 meters below ground you can it's very difficult to reach it it doesn't it's only through a sort of very hidden cave entrance and you have to sort of crawl in there so there's very little air exchange with outside and very stable temperature in the cave around eleven degrees and this basically makes it a perfect fridge now we were indeed successful in recovering DNA fragments from that problem from several fossils from this site and then blue here I'm showing sort of the part of the size distribution where we find evidence for the presence of short highly damaged ancient DNA molecules the rest of these distributions mostly made up by microbial sequences and modern human contamination now the problem is these are there were very few sequences only coming out so we had to so in order to make some sense out of them we first had to look at one particular type of DNA and the mammalian cell which is mitochondrial DNA and this DNA is special and that it's not present and only two copies per cell as the 23 chromosomes that everyone knows about but it's indeed presence in in several copies in in hundreds of mitochondria so for each for each part of the nuclear genome you have hundreds of of copies of the mitochondrial genome and even even so we still needed around 2 grams of material which is a lot with current dates then a current a standard so just get one sort of nice assembly of the mitochondrial genome and if you want to compare this to what we typically get from late pleistocene sites from sort of moderately preserved Neanderthal remains there we get much more many more copies of the mitochondrial genome from much less material in summary it's about a thousand times less than what we've seen in other Neanderthal samples so having the mitochondrial genome we could build a tree from mitochondrial DNA and the first thing to note here is that if you look just at the size of the branches you will see that all of these all of these lineages here modern humans Neanderthals Denisovans they are relatively long and the Scimitar loss vessel sequences on a short branch indicating sister ordinary age right while the other individuals kept mutating this individual died a few hundred thousand years earlier the snakes mutations further than this this tree is actually very confusing and this is because mitochondrial DNA even though it is preferable for technical reasons only shows very limited gives very limited information about the ancestry it's only inherited from mothers to their children and it reflects only a small small part of the population history so instead of looking at mitochondrial DNA it's much better to look at nuclear DNA to determine how this individual was related to the nice events in the anitha's and sort of after screening more samples from the site we were eventually able to recover as much as a thousands of the genome of a often individual from Seema Dallas whistles and this extremely little data but still it's enough to do a very simple analysis now having so few sequencers one thing you cannot do is actually compare these sequences to high quality reference genome and then ask where does it differ and then you see this red dots here which indicate sequencing arrows because an average one in 100 base pairs is wrong so but what you can do instead is use a trick and use the data set a reference data set of high quality genomes including the genomes of many present-day humans but also the archaic genomes that we have sequenced to high-quality and identify positions in the genome where for example the Neanderthal differs from all the other humans where the Denisovans differ from Neanderthals humans in the chimpanzee and so on and then you can just count at positions overlapping these sites how does the Sima de los ways of sequence look like and we did identify a hundred sequences that overlap sites that are sort of diagnostic for Neanderthals and 42 of them actually shot the Neanderthal State in contrast to this we had a sort of roughly equal number of sequences matching at an incident specific site but only 10 share the Denisovan State so and this is highly significance difference and and there's a very simple inference from this is that indeed the Sima de los vasos hominins were closer to Neanderthals and Denisovans which also means that the undertows and Annisa ones must have diverged by the time these individuals lived so one sort of important message from this is that it gives us for the first time a relatively stable anchor point in the timeline of human evolution so both the geological dating methods as well as sort of the branch lengths of the mitochondrial DNA that we've looked at arrive at an age estimate about four hundred thousand years and so we can say that the common ancestor of these two archaic languages must have lived before now they have been in the past a number of attempts to to infer the population split times using using the genomes of Neanderthals and Denisovans and then depending on the parameters that you use you arrive at different ages if I use the most compatible estimates the estimates that are most compatible with this number then indeed I would estimate that the human archaic die virgins are something like five hundred fifty-two seven eight hundred thousand years ago and probably even toward sort of the higher end of this so the sort of the good or the bad news is as you will but if you want a sequence that common ancestor between humans and Neanderthals we have to go much further back in time so there still challenges waiting for us here and I'm looking forward to hopefully being able to present this at some point so I want to thank all my colleagues in Leipzig especially since Fanta Payable who's of the mastermind behind all the ancient DNA work in Leipzig the Max Planck Society for the very good funding that we have and all the colleagues and in my lab and graduate students as well as importantly the our collaborators in space I've done brilliant work in excavating these samples very cleanly I must say and thank you for your attention thank you so much it's such a pleasure to be here I'm going to be talking about some other genomes that contribute to what makes us human so this may look like a photograph of outer space but these pinpoint points of light are not stars they're in fact the glowing genomes of millions of bacterial cells on the human surface of human teeth this is dental plaque now the human body contains an estimated 30 trillion human cells and by the latest estimate 40 trillion bacterial cells and so if you add these two numbers up together you will find that we are more than 50% bacterial now 40 trillion bacterial cells is an incredible number and at an average length of just over one micron if you were to line them up just the ones in your own body alone end-to-end they would actually wrap around the entire earth and span more than 20,000 miles 40 trillion bacterial cells is truly an astronomical number and even this fails to capture the immensity of this number because they are only about 300 billion stars in the Milky Way galaxy and so the number of bacteria in and on your body actually exceed the number of stars in more than a hundred galaxies so at this point you may be wondering how you appear human at all and the answer is that although numerous these bacteria are also very small an average there are a thousand times smaller than a human cell and so when you add them all up together they make up about 2% of your body weight or roughly 1.5 kilograms and that's a really interesting number because that's about the same weight as your brain and your liver and so some have argued that we should start to think of the microbiome as an additional organ system now most of these bacteria are concentrated in the gut and specifically in the distal colon and in feces they're actually concentrated to an extraordinary degree there are over a hundred billion viable bacterial cells per gram of feces and so if you follow the math through that means with each trip to the toilet you actually lose 20% of your total body cells but don't worry you regenerate them very quickly after your next meal now in addition to the gut a smaller but also very important fraction of your microbiome lives within your oral cavity and there they inhabit the buccal mucosa the surface of the tongue and the surfaces of your teeth where they are called done the plaque now the oral microbiome actually plays a very important role in the history of microbiology because the first undisputed description of bacteria comes from a letter written by Anthony Van Leeuwen hook to the Royal Society of London approximately 300 years ago in which he described very many small animals which moved themselves very extravagantly within his dental plaque he drew many of these organisms and he tried to count them but he eventually gave up and he wrote the number of these animals in the scurf of man's teeth are so numerous that I believe they exceed the number of men in a kingdom now if anything this is a gross understatement because we now know that there are nearly as many bacteria on the surface of your teeth as there are humans on earth and each day you swallow an average of eighty billion bacteria in your saliva so in addition to being numerous these bacteria also contain an immense diversity of genes and on average there are about a hundred and fifty times more genes in your microbiome than in venn your human genome this collector bacterial genome is so large that's often referred to as your accessory genome and in fact you require these genes in order to perform some of your most basic human life functions and this has led some to describe the relationship between humans and their microbes as that of a super organism like a colony of bees so many independent organisms contributing together or more recently as a hollow beyonds an ecosystem so tightly interdependent that it behaves as a single organism like a coral so rather than just being a leaf on the great tree of life in some ways humans are actually more like a tree house a home woven from many permanent and transient microbial inhabitants and yet we have only very recently come to even notice this large number of underexplored and mostly nameless microorganisms that inhabit the human body in fact it was only in 2001 that Joshua Lederberg coined the term microbiome in order to quote signify the ecological community of commensal symbiotic and pathogenic microorganisms that literally share a body space and have been all but ignored as a terminus of health and disease the is rather remarkable because these microbial communities perform essential major functions within their host bodies that include various aspects of digestion vitamin production and drug metabolism education of the immune system and defense against pathogens but the microbiome can also be a source of infectious agents for example the oral microbiome is the natural reservoir for numerous respiratory opportunistic pathogens that are responsible for infections ranging from pneumonia to meningitis and the oral and gut microbiomes have been implicated in several chronic inflammatory diseases including a cardiovascular disease where in a recent study was found that more than 80% of diseased valve and arterial tissues contain oral bacteria so the microbiome clearly plays a pivotal role in human biology and therefore it is critical to understand its evolution and changing ecology through time and one way we can we can do this is we can investigate the ancestral human microbiome by directly measuring and analyzing it from archaeological material now ancient DNA studies have long focused on the analysis of bones and teeth and we've made major advancements studying these tissues and we've been able to for example recover host and pathogen DNA and reconstruct the entire genomes of extinct animals archaic humans and even ancient pathogens but studying the microbiome has been very challenging the human body decays rapidly after death in some cases among the fication can occur but in the vast majority of instances we are left with only a skeleton but there is one microbiome that routinely persists after death and that is something called dental calculus this is a it's what it essentially is as a dental plaque that has spontaneously calcified during life you know this by the name tartar which is what your dentist calls it it calcifies during life in a way it actually it's the only part of your body that fossilizes while you're still alive and therefore persist like your skeleton long after death this is just a close-up image of dental calculus so you can kind of see what it is if you're wondering what this looks like in a living person here's an image here this is calcified microbial matrix on the surface of teeth this particular example from a woman who died approximately a thousand years ago and we can take as we can zoom in on this particular tooth here and here I've just shown that same tooth now in cross-section using scanning electron microscopy and we can zoom in on this calculus deposit that you can see on the surface of the enamel there it is right there and we're gonna zoom in again on this part right here and one of the things that's very clear immediately is that it has structure it has a layered structure and that is because dental calculus forms incremental II dental pock undergo spontaneous calcification which kills the microorganisms inside but also in tombs them and then another layer of dental plaque forms in this process repeats and over time it builds up layers like tree rings or layers of an onion but what's really significant about this is what it means is that we have an ordered record of this person's life history from the earliest period closest to the surface of the teeth to to the latest period of life just before they died this structure never remodels and therefore is a remarkable record of this person's life history we can zoom in even further and actually see the individual microbial cells that have been calcified in situ we can also decalcify it and and use stains like Gram stain to visualize these bacteria remarkably when you remove the the mineral the the bacteria do not disintegrate and in fact you can see individual microbial cells here so these are individual gram-positive bacteria for example here still with cell wall intact and what to me was most remarkable as we tried kind of on a lark we didn't think it would work we decided to use another stain in this case hooks dye which is very similar to dot B if you've ever used that it's a DNA diet binds to double-stranded DNA and this is the image we have we got after that which we saw in the beginning this is the only archaeological material that I know to exist that has so much DNA inside that you can actually see it under a microscope this property turns out to be pretty remarkable for calculus if we're gonna use by comparison let's just sort of talk a little bit about bone and dentin those are the tissues that are most commonly studied they actually have very little DNA inside even when a person is alive so bone for example has fewer than a thousand cells per milligram it's almost a cellular we have typically in archaeological context we recover very little DNA typically less than an anagram of DNA per milligram that we study and even that is mostly environmental bacteria that have invaded post-mortem so here's a Justin D that we've generated in my own lab we looked at four teeth from different parts of the world different time periods and we look to see what the endogenous content was of dentin what we find is that only a tiny fraction of that DNA is actually human most of it the vast majority is a post-mortem environmental bacteria that are decomposing the teeth and on average we get something like 0.1 to 8% human DNA so very very low amounts of endogenous DNA calculus is completely different first off it starts off with far more cells so on average it has more than 200 million cells per milligram in our lab we've isolated in excess of 500 nanograms of DNA per milligram we've studied and what's also really remarkable is we have very low contamination from environmental sources so these are the same teeth but now we've analyzed the calculus from them and what you can see is the endogenous content the oral microbiome proportion is much much higher it's on the order of 60 to 80 percent we can look then it pairs again of calculus and dentin and we can see that the amount of DNA that we can recover this is a logarithmic scale is on average about two orders of magnitude higher so nearly a hundred times more DNA in calculus than in dentin and in some cases a thousand times higher we thought for in the beginning this might just because it starts off with more DNA and so we decided to test this we took a tooth that was very diseased it had a massive carious lesion and also a giant abscess so this would have had tons of bacterial DNA tons of bacteria at the time this person died and we expected we might see elevated levels of DNA from these samples and in fact we don't we find they're almost the same as what we see for healthy dentin and it seems to imply that there's something special about calculus that facilitates DNA preservation we can then actually go in and say well what what do we find in calculus is this is amazing structure it's full of DNA what sorts of information can we learn from it we can break it down into different categories and we can say well that 99% of it is bacterial that makes sense we know this is made of dental plaque dental plaque is primarily a microbial matrix but about 1% is is quite interesting we have a little bit more than a half a percent which is eukaryotic that's mostly hosts and dietary I'm able to find a tiny bit of archaea and a little bit of virus the virus is actually a bacteriophage which are viruses that infect bacteria we can also extract proteins from dental calculus and we can also classify them what we find is about 80% of the proteins are bacterial in origin and about 20% are human I'll come back to that in just a few minutes let's focus on the bacteria first we've identified more than a thousand species but the vast majority more than 85% of the sequences that bacterial sequences that we find actually belong to a hundred highly abundant taxa and some of them are very interesting so here's this as a selection of some of the bacteria that we've identified in dental calculus ancient dental calculus and I mentioned earlier that the oral microbiome is a major reservoir for opportunistic infections and we find many of these bacteria preserved in dental calculus now I want to caution here that finding them does not indicate this person had this disease while they were alive carriage of these organisms is quite widespread even asymptomatic carriage but these bacteria have the capacity to cause disease under the right circumstances for example if the host is immuno compromised what's really significant about this is it gives us the opportunity to study the evolution of these opportunistic pathogens because they do not preserve in any other context and some of them are very interesting so for example streptococcus pneumoniae is the causative agent of pneumonia Streptococcus pyogenes that's one of the causes of strep throat Hamas Luis influenza causes respiratory infections and Neisseria meningitidis is a leading cause of bacterial meningitis now we have a reliable source to study the evolution of these opportunistic pathogens but a couple others that we found extremely interesting are these three a little bit lesser-known we have here poor Mona shindy Valis Tanner left for Sofia and Treponema denta Kola we have them at really high abundance and this is potentially clinically significant because these are the major causative agents of periodontal disease today we investigated this little further we wanted to know that our ancient samples have unusually high abundances of these three taxa and so what we did we compared to a reference set of healthy individuals from the human microbiome project we found that the frequency of these organisms within healthy individuals are vanishingly small very very few copies of these bacteria are present and healthy people but our ancient individuals have much higher frequencies of these bacteria this did make sense however because we'd actually selected these individuals for study because they showed osteological evidence for periodontal disease what was interesting about this is we were able to show that despite a century of intensive efforts to proactively treat periodontal disease and prevent it we still see the same organisms that are causing it over a span of more than a thousand years now one of these organisms Tanner alone for Sofia we actually found at such high abundance that we were able to reconstruct a full draft genome we're now investing in it to try to understand the evolution of this pathogen through time in addition to the bacteria that are present I mentioned before that we do find some really interesting other material there it turns out dental calculus like dental plaque acts as a kind of sink for other all the other things that you put into your mouth including host biomolecules as well as food and talk really quickly about the hosts early on we noticed that we were seeing human DNA within dental calculus and this was really interesting to us we just published a study last month we're using it to capture based approach we were able to enrich for human DNA and reconstruct full mitochondrial genomes in this case from a cemetery of Native American cemetery from Illinois this is potentially actually quite impactful especially in North America there are many tribes who do not allow genetic analysis of skeletal remains because it is a destructive process but if dental calculus can serve as a surrogate and may be a way of conducting ancient genetic analysis without disturbing the actual scale to remains themselves we also find a huge abundance of human proteins within dental calculus and this is where it gets extremely interesting most of these proteins I've color-coded them by sort of what their function is most of these proteins are colored red and that's because they are part of the innate immune system what's interesting about this is that by having proteins we get an additional level of information we are not only identifying that we have human proteins but many of these proteins are specifically expressed in different cell types in this case many of them are specific to neutrophils so we can identify the source of these proteins as a specific immune cell that's reacting to dental calculus and so what we're actually visualizing here is evidence of an active infection at the time of death now in terms of diet it's long been known that microphones plant microfossils and animal microfossils preserve within dental calculus so all that little bit of bits of food that gets stuck between your teeth they actually stick around for a really long time and we can see them under the microscope so what you're looking at here this is a little bit of connective tissue from some sort of animal tissue this person ate this is a phytolith it's a little piece of plant class these two here are actually intact starch granules this one the morphology is consistent with the plant tribe triticeae which includes things like wheat and barley and this one has characteristic structures of the plant family fabaceae which include things like peas and beans we can also extract DNA from this and get even higher species level resolution so this particular tooth came from a man who lived in Germany about a thousand years ago and in his teeth we found evidence of sheep pigs cabbage and wheat and therefore conclusively demonstrated that German diet has not changed much more than 900 years we can also isolate proteins from dental calculus to investigate diet one of the most interesting ones that we have found so far is this one called beta lactic lobule in' beta lactic 11 is specific to milk and as a result we've now been able to test more than a hundred individuals and identify the presence of dairy throughout Europe dating back to at least the Bronze Age we published these results last year we're building on them now and our goal and the next phase of this project is to try to understand the origins and spread of daring in the Middle East and Europe what's also amazing about proteins and using this approach is that because it's sequence space there are sequence variants that are species specific and we can actually distinguish cattle milk sheep milk and goat milk and have a very fine scale resolution of how how these dietary practices changed so if you'll pardon my pun I think we've only scratched the surface of where we can go with it analysis and one of the most exciting things that I find about it is that is its ubiquity so dental calculus is found in all living and all known living populations today and we find it ubiquitously in skeletal assemblages from the past it's also found on Neanderthal teeth and the teeth of Foster for the scenes it's actually quite abundant on chimpanzee teeth as well and I think by studying it it can provide a unique window a lens through which we can be get better begin to understand the evolution of our ancient microbial self so thank you very much you

Channel: University of California Television (UCTV)

Views: 39,945

Rating: 4.8193021 out of 5

Keywords: DNA, CARTA, evolution, Sankararaman, Warinner, Meyer

Id: pEPZI5DsUJs

Length: 57min 50sec (3470 seconds)

Published: Fri Aug 05 2016