 I would like to start my presentation saying that this was supposed to be a joint presentation with my colleague Professor Janet Montgomery. She was going to present the isotope part, but due to unforeseen circumstances, she cannot be here, so I will be presenting the isotope part myself. I'm not an isotope expert, so I will use Janet's note for that part of the presentation. So what I'm presenting here is a multidisciplinary project involving different people from different institutions. So we studied Musgrave burial, an hypogeum, dated back to the late Neolithic that was unearthed during works for the high speed train line in the north of Barcelona in an area called La Sagrera. So that you have here. So this hypogeum, this mass grave burial, contained more than 200 skeletons, and as I said they were dated back to the Neolithic and you have some of the radiocarbon dates here. Three phases of use could be identified within the hypogeum. We have a first collective burial phase which is at the bottom of the hypogeum followed by the simultaneous intimation of more than 100 skeletons, which means that we have more than 100 people that they were placed together or almost together at the same time in this mass grave. And then finally we have a single enumation of one individual which was placed on top. So here we have the dates from the three different phases of use of the hypogeum. We selected 13 individuals, I will explain why in the next slide, from this simultaneous intimation of more than 100 skeletons for anthropological genetic and isotopic analysis. The anthropological analysis was done by Pat Balaguer and in the anthropological analysis they found that they estimated the age and the sex of the individuals finding that in this second phase, the second episode, there were more juvenile individuals compared to the first episode. The first episode had a normal demographic profile and the second one it seems to be a bit anomalous with a high proportion of juvenile individuals. There were no significant signs of trauma, perimortem trauma that could be indicative of a kind of a violent episode that could explain this simultaneous deposition of these 100 plus individuals and we didn't find also great goods associated to the burials. The anthropologists, they studied the dynamics of the deposition and they could identify several population groups of clusters of individuals that they had been placed at the same time or almost at the same time. So we did a selection of 13 individuals based on these three different population groups. We have here population group A with two children and a young adult female. We have population group three also with three individuals, three of them adults and then you have here population group C including juvenile adult individuals and also children. So you can see just by looking at the pictures of the different skeletons that they seem to be they seem to be arranged in a quite intimate position. So you can see for example that individual 18 has his hand placed over the scalp of individual 56 and his spine is aligned with the spine of the young adult individual 68. In population group C you can see the scalp here of the adult individuals the children will be at the bottom and all of them are also oriented in the same position. So it seems to be indication of intentional deposition of these people at the same time. So we sample these individuals and we have three questions in mind. We wanted to know if the individuals from these population clusters, if the individuals from the second face of use of the ipodium were related in particular those from coming from the same population cluster. We also wanted to know which was the diet of the individuals buried at La Sagrada and finally just to try to see we could have an indication of the cause of death of the simultaneous death of this group of individuals that they were placed together in the second phase of the of the site. For that we sampled two samples per individual. We used a complete sample for ancient DNA analysis and the second sample we just extracted the dentin and we need replication in an independent laboratory in the Manchester Institute for Biotechnology with Professor Tavi Brown and for isotope analysis we use both dentin and enamel to measure strontium and carbon and nitrogen and oxygen ratios. In terms of DNA methods I'm not going to extend we applied initially classical PCR approach that was focusing on the maternal lines because the rate of success is higher and we wanted to know if we could extract DNA with quality out of these samples. So we target the most valuable region within the mitochondrial DNA as I said the results were reproduced in a second lab and we also cloned the PCR products to assess contamination and also molecular damage so artifacts. We assessed when we got the upload site so the sequences we assessed we are trying to prove kinship here so it's important to assess if these sequences that we retrieve are frequent in the population or not and for that we use to that databases one containing all the sequences from mitochondrial DNA published up today from different time periods and the other one is a modern forensic database. So here we have a summary of the DNA results so basically what we are looking at I have organized here the skeletons by cluster A, B or C in different colors so what we are looking at is at the coincidence of the positions that we have here. So we can see that we could get data out of 11 out of 13 skeletons which is quite good considering that these are late neolithic quite old and most of the upload types they seem to be different from each other with two exceptions. So we have this upload type that we search in the database and it wasn't represented which means that these are fairly quite rare upload type and it was present in two individuals but coming from two different clusters. We also have individuals 43 and 45 from the same cluster that they may share this mutation but for these individuals for one of them we couldn't retrieve the whole sequence so still in the air they could be related or not and these are the only maternal relationships that we can infer out of all the individuals that produce results. So moving into the isotope methods this is our methodology. I'm just going to say about this that the samples that were prepared at the preparation lab at Durham University and samples were analyzed also at Earth Sciences also within Durham University at the oxygen isotopes at NIGL keywords and the carbon and nitrogen isotopes at the University of dashboard. So these here you can you can see the strontium and oxygen isotope ranges that we estimated were characteristic of the area of people living in coastal Barcelona. So we estimated that for strontium the local ranges would be within this interval and also using modern rain and ground water studies we estimated that local individuals may fall within this range for oxygen isotopes. So these baselines they are indicating when we get the results of the individuals if they were local to the area from the Sagrada in this case the plain of Barcelona or if they were coming from somewhere else. So here you have the results for strontium and oxygen isotopes so the key thing to take home from this slide is that it seems you have here these pink bands they indicate the local ranges for strontium here and for oxygen here. So you can see that while some individuals these individuals here according they have a geological pattern according to the isotopes that is compatible from with them being from the area of Barcelona most if not all of the oxygen ratios indicate well not only just with this individual it shows a little overlap with the estimated values of oxygen for the region. This is telling us that most if not all the individuals that they were buried at la Sagrada were from all of them were for someone else they were not local. When we we studied the carbon and nitrogen results for the children individuals we did bulk carbon and nitrogen analysis but for the adult individuals we did incremental analysis. So we used the first molars for all the individuals and basically what the incremental analysis is looking at you just slice the dentin like different layers and we measure the isotopes and this gives us a relationship between the diet of the individual at a particular time of his life. So here you can see the carbon and nitrogen results so one of the things that it's obvious from the plot is that the children and the adult individuals that we analyzed they seem to have quite different values for both carbon but especially nitrogen. We have the children here that it seems that it suggests that the diet of the children and the diet of the adults at the time of death was substantially different. So basically these values over here of the adults they are characteristic of a typical neolithic diet so predominant C3 terrestrial and plant protein diet. However the diet of the children it has a higher content of both carbon and nitrogen and this could be suggestive of a maritime diet more marine diet. So another alternative explanation for this could be that the data indicated that the children were breastfeeding however and therefore there are a trophic level that is higher than the adults however we are using first molars and we also did incremental analysis so basically we are looking at the same point and the same time in life both in adults and in children and moreover the carbon shift is too large to be explained solely by breastfeeding effect. So we looked at the incremental dentin analysis as well so I have just presented one of the plots of one of the adults but more most of them they look the same way. So this slide basically is adding further evidence to the interpretation than the children when eating a different diet of the adults. So here you have a representation of the nitrogen values and we have the bulk dentin data for the children as you can see that they have very high nitrogen content. So these values are outside the range of that will correspond to a diet typical of neolithic populations. They have a much higher nitrogen content and here we have the representation of the incremental data for the adults by age. So as I said this is average of all individuals at a particular moment in time so we can see that when the adults there were children the nitrogen values they were much lower. However as life progressed they consumed started consuming food sources with a higher and higher content of nitrogen especially prior to the death of the individuals. So just to summarize the objectives of the talk and the conclusions that we can draw from our analysis in terms of the individuals from the Sagrada we were wondering if these population clusters they were made out of individuals that they were related. So we have our data argues against this so there's in principle no maternal relationship that we can draw within each cluster but on top of everything when we look at the strontium and oxygen data we can see that there is no particular distinctive isotopic signature within each cluster. It seems that individuals within the same cluster they were coming from different locations so this is indicating that they are not related as well and as I said this variety within each cluster indicates that individuals within a cluster they have different places of origin. So if we look at the individuals cluster by cluster we can see this clearly so not in all the cases we could retrieve genetic data out of all the individuals but in this case we can use the isotope data so we didn't get a sample from individual two but we can see that individual four the strontium grade should suggest that they are not local and the same for individual eight. So still in the air why these individuals they were placed in such an intimate position at the same time but they seem not to be related the same situation we have for population group B so the strontium and oxygen ratios they suggest that they are not local and they come from different places and also the DNA at least the maternal lineage cannot be compared it's different suggesting no kinship between them. The third case we have the same situation non-local values and different quite different in some cases for the adult individuals and also with the children as I said before the only genetic link that we could establish was between one individual from cluster C and one individual from cluster A but not among individuals belonging to the same cluster. In terms of the diet of the individuals buried in the ipodium we had we just wanted to know what was the diet of the people but we got this result of differences between the adults and the sub-adults so our hypothesis is that we observe these values in other populations for example of the Shetland Islands a study by Montgomery 2013 so we propose that these people they shift towards a marine diet just right before they die so we see that when the adults were children they had that typical in Neolithic diet but before death these individuals had a much increased levels of nitrogen so this could be also an indication of why this shifting day it would be related also to the massive death of these individuals maybe perhaps. In terms of the most probable cause of the simultaneous deposition just looking at the Strontium values suggesting that these people this is indeed is not a population it seems like a conglomerate of individuals coming from different places around Catalonia probably in the north in the mountains if we look at the Strontium map and considering this change in death in in diet that we observed before the death of the individuals we could hypothesize that maybe this individual there was a sort of agricultural disaster that forced these individuals that they were living in the mainland in the north of Catalonia in the interior part of Catalonia to move towards the coast to make use of these marine protein resources so as I said this has been proposed for other populations in the Shetland Islands by Montgomery in 2013 so this could be explained maybe will indicate points towards the right direction of why these individuals they died and they were put together at the same time so finally just to have knowledge of Cerbega Arqueologia the Instituta Cultura de la Ligenda Barcelona who fully funded this study and the data presented here is the work of two master students from Liverpool University and from Durham University so thank you very much for your attention