Introduction
At a global level, our research project focuses on the evolution and history of human settlement. It is a historic question which has experienced substantial development during the 20th century, but especially during the last decades, due to genetic and palaeogenetic data which has been an important base for studies concerning humans from the last 12000 years in our laboratory, and due to paleontological data (anatomical, histological and ontogenetic) which remains the base to study older fossils. The beginning of the 21st century will add another question related to the causes and/or phenomena, or even genetic determinism, of the evolution and adaptation of populations to new environments (uterine, physical, biological, cultural and social). Consequently, it is probable that as questions regarding settlement history are answered, questions regarding the adaptations of populations will arise.
To define the data on which our research will be based, we have to define the geographic area, the populations and the specific environments. This is one of the strongest points of our laboratory but which requires a strong investment in time (three to twelve years per field) and planning.
To define what we hope to show, we depend on the methods available to analyse the data, but also on the interesting discoveries in anthropobiology in general. Within five years, the complete nuclear genome from hundreds of contemporary humans across the five continents will have been sequenced, and from this genetic data, detailed biological scenarios of human dispersal and contact between populations will be deduced. The conclusion will evidently be the comparison of these modern scenarios with those scenarios drawn using data from ancient populations.
This has several outcomes :
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To test the scenarios drawn from modern genetic data by comparing those from ancient genetic data. Data from ancient populations will always be less numerous than those from contemporary populations. Consequently contemporary data are taken as reference data. From this there are two hypothesis : either data from ancient populations confirm scenarios established by contemporary populations, so the scenarios remain undisputed; or data do not confirm these scenarios which must then be re-examined and/or modified according to new parameters.
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To appreciate the modifications in human variability over time, due to on one hand the loss of some lineages, and on the other the prevalence of other lineages due to the influence of selective pressures (genetic drift, selection, migration, bottleneck, marriage rules.)
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To assess these biological scenarios along with those of microevolution, cultural history and the environment (in particular the evolution of infectious diseases) in order to determine the factors that could have potentially played a major role.
The study of hominid evolution can be placed in a similar perspective, only if we consider that the term « lineage » is not referring to individuals but to species. Thus, the study of fossil variability is important, in order to assess whether some or all of the variation in certain characteristics overlap with human variability. When a fossil has a key characteristic included in the contemporary variability, the fossils (or its direct ancestor or any sub-species) inclusion in the Homo genus, is a key element of the diagnosis and discussion.
From this perspective, the exploration of the variability in ancient populations and the factors that affected them (especially infectious diseases) are major issues for the discipline in the coming years.
By 2014, it is hoped that :
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A large sample of Holocene individuals will have been sequenced for the complete mitochondrial genome and Y chromosome, so the laboratory will have to its disposal a large dataset from hundreds of ancient individuals. At the same time, the study of infectious diseases, especially large epidemics and/or pandemics of the past, will be crucial to understand their role in the evolution of populations.
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Regarding fossils, the study of their superstructure variability, development and microstructure characteristics (e.g. the interface between enamel and dentine) by scanner, will allow a robust investigation and consequently a better evaluation of the scenarios regarding human evolution.
Out data available are substantial because of the fieldwork we control and the ancient samples we have access to. The methods of data processing will be essential in the coming years. This requires both top researchers able to be leaders in their field, on specific equipments and on the means to function these equipments (funding and personnel). Our team is one of the international leaders in the field of ancient DNA of Holocene human populations. Likewise in the field of modern genetics and imagery we are very well positioned. We also have to our benefit an international network and the last generation of laboratory equipments. Our future in the next six years (c.f. self-analysis) depends on our aptitude to make these factors function efficiency. One of the means will be to develop a closer link between applied and fundamental research.
Laboratory Self-Analysis
When accomplishments at the international level are taken as reference, the strongest point is the link between the field and the laboratory, from the time a project is conceived. The monographs and/or articles with our laboratory as first or last author, in journals with a high impact factor, are all related to this point.
Our success in ancient DNA from human remains is due to the fact that Eric Crubézy, head of the laboratory, anthropologist and archaeologist, and Bertrand Ludes, specialist in degraded DNA, work together to develop projects, for example the Siberian mission where DNA is especially well preserved. Likewise for palaeontology, where the excellent team is directed by J. Braga, who is in charge of both the French missions to South Africa and of the imagery laboratory. In the framework of our studies of contemporary genetics, under the direction of J.-M. Dugoujon, we have access to both current populations and to thousands of samples collected during the 1970s by current and/or previous researchers of our laboratory.
Our second strong point is the relationship between fundamental and applied research. The research our laboratory undertakes is « nourished » by the latest discoveries in the field of forensic sciences and by imagery techniques developed by Kodak, or by the expertise of private companies such as the Biopole. The transfer between fundamental and applied research is therefore immediate, and is also due to positive feedback between these two disciplines.
Our third strong point is our incorporation of important technical platforms: the Forensic Science Institute of Strasbourg (for degraded DNA), Génopole, microscanners of MEDES and/or Kodak.
The head of the laboratory is Professor Crubézy, who is advised by a strategic board on deciding the orientation and development of the laboratory, and by the teams who work in close relationship with the CNRS and the Universities of Toulouse and Strasbourg. The strategic board is formed of two co-directors : Professors D. Rougé and B. Ludes, Directors of the Forensic Science Institute in Toulouse and Strasbourg respectively.
The Emergence of Innovative Topics : Policy Incentives
One of our strong points since 2002 is our fieldwork in Southern and Eastern Siberia. We are also trying to develop a research program focusing on Holocene population adaptation and settlement, in the region of Verkhoïansk (2010). Our objective is to explore problematic in common with Siberian laboratories in Yakutia and Krasnoyarsk, with the goal to create to an international laboratory which has been accepted by the CNRS (October 2010).
The work made in this laboratory will benefit greatly from the results of the anthropobiological missions in Eastern Siberia, organised under the Minister of Foreign Affairs. The missions planned in the region of Verkoïansk (2010-2011) are in the framework of those supported by the Institut Polaire Paul Emile Victor (IPEV) in the Arctic, and are since august 2010 one of the research highlights of this project.
The status of the laboratory in 2014 will depend on our capacity to recruit young researchers. We will either continue on the same pattern of the last three years (recruiting one-two new researchers per year including one research engineer). Currently the laboratory is expanding and is in a position to play a key role at the regional, national and International level in certain fields, especially those of imagery, palegenetics and geomics from present day’s populations.
Projects of the « Settlement and Coevolution of Man-Environment » Team
Through our relationship with the forensic sciences, the analysis of degraded DNA, and our fieldwork (South and East Siberia where DNA is relatively well preserved), this team is a leader in the field of paleogenetics of Holocene human populations. A strong point will be the comparison of data from those obtained in Strasbourg and in Toulouse. Regarding the genetics of contemporary populations our strong points are: the samples collection, our new programs with genopole about genomics, our relationship with the Génopole
Since 1992 our team has participated in a significant way to the development of research in ancient DNA through work recognised at the international level in applied and fundamental research (publications of the last 15 years), regarding the settlement history of human Holocene populations and the most ancient mycobacteria.
This research confirms our position among the top laboratories of reference, in the field of degraded DNA from human origin. This is due to our pluri-disciplinary approach (traceability of samples from the field to the laboratory) which has allowed major methodological advances (diversity of molecular markers/techniques) for samples mainly coming from favourable environments (cold environments, caves etc). At the same time, we try to couple our studies on contemporary populations with ancient DNA studies in the same geographical area, as with the Siberian project, which will be used as a model for other projects.
Our project has several aims : (i) To work in parallel in the same geographic areas with both genetics and paleogenetics, in order to study settlement history and evolution; (ii) To explore the interaction among man-bacteria-parasite in ancient and contemporary populations, as a factor in the relationships between man and the environment. Particular attention is given to tuberculosis and the important pandemics responsible for the disappearance of indigenous populations in different parts of the world, especially Siberia. Our relationships with industry and the health sector for this latter research, will also allow us to plan a larger interpretative framework for the study of disease emergence, starting from the principle that during evolution all the current infectious diseases have been emergent.
The project takes place in areas where the DNA is well preserved (such as the Siberian project), but also in areas where the DNA is more degraded. In this context, we work in various areas of the world where the laboratory is well connected; Mediterranean Europe and South America, and in the Indian Ocean where important work has been in progress for two years.
Degraded DNA -the aim is to develop a multi-marker approach by overcoming the problems of DNA quantity and quality, and to access nearly all the genomic information (nuclear and/or mitochondrial) using the next generation of genotyping and sequencing. These approaches have been used in ancient DNA studies for the last two years and their development will bring us to the top of the field.
Development of a multiplex adapted to degraded DNA for the complete sequencing of the human mitochondrial DNA.
Such a methodology is actually in development in our lab.
MALDI-TOF. The mass spectrometer MALDI-TOF (Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight) is an analytical methodology actually used for DNA analysis. It is robust, sensitive, direct, quick and runs at little cost. It can therefore be used for SNP typing of ancient DNA samples in a very efficient manner. The sensitivity makes it possible to obtain spectrums with a lower quantity of DNA than those used with other technologies (classical sequencing or SNaPshot minisequencing).
Paleogenomic 454 sequencing. We are using the latest generation of the 454 Genome Sequencer FLX (www.454.com), through a partnership started with the Génopole Toulouse (www.genotoul.fr). This technology will allow us to generate quickly and in one run, about 500 million nucleotides and has many advantages when working with ancient and degraded DNA, in terms of the quality and quantity of molecular data obtained. In addition to obtaining an important quantity of DNA and to rebuild complete small-sized genomes, e.g. the mitochondrial genome, it’s simultaneously possible to clone every DNA fragment to estimate the rate of degradation and sequencing errors.
One of the strong points of the team is the availability of the two sites in Toulouse and Strasbourg, to analyse degraded DNA. This allows a unique efficiency of data processing at the European level, as it allows results to be replicated intra-lab and MALDI-TOF and 454 sequencing techniques to be compared, as each of these will be developed on each site.
Modern DNA - This will be investigated using mitochondrial DNA, Y chromosome and autosomal DNA. A more accurate and efficient approach (in both time and finance) will also be followed through routinely sequencing complete mitochondrial DNA. In collaboration with the Toulouse Génopole ( www.genotoul.fr) the use of a new methodology based on the analysis of large number of SNPs (illumina bead express) and the development of DNA chips to obtain, through a single analysis, all the polymorphic positions under study, is also in development. Micro RNA studies will be developed in the next two years.
Health and Environment: Detection of Bacteria - this research field is developed within the AMIS laboratory in close collaboration with the BioPole laboratory (PME Toulouse): Detection of bacterial DNA in specific environments (related to industry and/or archaeology) to develop original research protocols with technical links between ancient DNA analysis and rare DNA in the current environment (or visa versa). The goal is the efficient detection and quick identification of bacterial strains in the industrial environment, and in the micro-biological diagnosis of current infectious diseases. This can be later applied to the diagnosis of ancient infectious diseases from ancient human samples, or in the determination of bacterial ecology in the archaeological environment.
First-time experiments on ancient environments: teeth from archaeological graves where difficulties relate to the amplification of degraded DNA at weak concentrations. In parallel, in the air conditioning environment and in lake and river water, identification of bacteria using real time PCR and development of chip DNA technology. The project will also focus on the molecular analysis of bacterial communities found in confined environments, and for the first time to space flights which represent a confined environment with controlled environmental conditions for a long duration. Samples (from surfaces and the air) will be collected at regular intervals for significant statistical representation of the environment and will occur in two situations: during manned and non-manned space flights. DNA will be extracted from samples and the gene 16S rDNA will be amplified, cloned and sequenced. Negative controls will be carried out throughout the procedure. From the cloned sequences, strains will be identified and compared by phylogenetic analysis. This will allow the characterisation of bacterial populations within the confined environment at certain times, and to establish the pattern of microbial evolution before, during and after the space flight.
Finally we will assess the population dynamics and changes of equilibrium between the different strains, by specific statistical analyses. These studies will create a database reflecting the bacterial diversity and evolution present in the confined environments associated to the space flight, but which will also be transferable to other types of containment (industrial, research or medical). An important application is the archaeological environment; the study of graves and/or their associated objects, and the consequences of the bacterial community present. The analysis of different ecological environments may also be planned, and data on the origin and vectors of ancient bacterial pathogens may allow us to better understand their evolution. These aspects will be essential in understanding the emergence of infectious pathogens.
Fieldwork
Settlement and Adaptations of Siberian Populations.
To define and detail the origin and evolution of Siberian populations including their adaptations to particular environments, through the use of new molecular technology and acquisition of new samples from South Siberia, the Republics Altaï and Touva, the Peri-Baïkal area and the region Verkhoïansk. This research is done in the framework of MAE and the international laboratory of the CNRS. The partners for research are in progress, notably industries working in Siberian.
Andes/Amazonian Interface.
To evaluate the evolution of the genetic structure of indigenous populations in South America during the last millennia, and to test relationships between the first Paleo-Amerindian settlement (around 100 samples presently under analysis) and the origin of the current indigenous settlement. A specific focus will be made in Guyana and at the Andes/Amazonian interface with new fieldwork planned in Bolivia. Through the collaboration with Prof. A. Ruiz-Linares (UCL, United Kingdom) we will study the variability of several thousands of autosomal microsatellites and 550 000 SNPs, to fully understand the population of this area and to establish new scenarios of population dispersal and evolution. Social anthropology will bring information on group dynamics and their inscription to social time, at the local scale in the region of High-Maroni. Kinship patterns and their recent evolution will be taken into consideration in the genetic model. Projects are funded through our participation (Principal Investigator) to Amazonia II, specific funds to the Institute of Forensic Science in Strasbourg.
Mesolithic/Neolithic Transition in Mediterranean Europe and Ancient Populations in the Pyrenees.
To study the impact of the Mesolithic/Neolithic transition, from the main archaeological sites in the northern Mediterranean basin (from Spain to Turkey), by a sampling campaign and the analysis of human remains, Due to the post-glacial re-expansion, often hypothesised, a research thematic has been initiated on the settlement of the Pyrenees. In this context we have important genealogic, historic and genetic data. All this research will be conducted in partnership with our Spanish colleague (ancient DNA and Pyrenees), Italians (ancient DNA) and our English collaborators who are specialised in comparing cultural and biological factors (Y chromosome). Projects are funded in part through PRES, the Institute of Forensic Science in Strasbourg and regional funding.
Indian Ocean
Our current work underlines the ancient human settlement of Madagascar (southern dispersal) and contradicts aspects of current human dispersal models. The data are based on the analysis of the complete mitochondrial genome from samples of the last hunter-gather populations. For the coming years, we have expanded our partnership with Malagasy researchers (Archeologists, Historians, etc) in order to increase the number of populations sampled (particularly among different royal descent groups of the highlands and to the south-east, where the Arabian Muslims that are supposed to have settled in this region would have influenced the biological history). Contemporary and ancient population sampling in the eastern part of the Indian Ocean are also planned, especially focusing to the south-east of Borneo, the hypothesised origin of the Austronesian language spoken in Madagascar.
Conclusions
The pluri-disciplinary approach that we engage in, together with our background and the projects planned for the forthcoming four years, will allow us to continue the qualitative and quantitative jump necessary to move from paleogenetics to paleogenomics, from the mitochondria DNA to the nuclear DNA, from the study of populations to research of evolutionary factors.
Projects of the « Imagery and Anthropobiology » Team
We have setup on one side very efficient equipments (microscanner SCANCO at 41 microns, « Cone Beam » scanner, portable Kodak microscanner at six microns - under construction), and on the other side, several research programmes to analyse micro-tomographic resources or synchrotron imagery (in the European Marie-Curie Program in 6° PCRD). These equipments allow the development of innovative methodology, which is only available in a maximum of five other laboratories world-wide. After the period 2007-2009, with the development of new academic collaborations (in image processing/analysing to produce an anatomical atlas) and collaborations with industry (for the acquisition of high resolution data), we should be, in 2011-2014, among the top laboratories in Europe through the level of publications.
The activities in the laboratory are associated to fieldwork in Africa (South Africa, Mozambique) and in Asia (Mongolia), to look for new ancient fossils. Fieldwork in South Africa and Mongolia is already funded for the period 2011-2014, and funding is in process for fieldwork Mozambique. The partnerships underway with industries that finance our activities are : Kodak Dental Imaging/Trophy (medical imagery for the laboratory) and Telespazio (satellite imagery for fieldwork).
In the beginning of 2007, the Computer Aided Palaeoanthropology and Anatomy (C@PA) Group (within the framework of the « AMIS » Unit, FRE 2960 CNRS) was created after the appointment of Professor José Braga. This Group associates research in palaeoanthropology, biological anthropology, anatomy and computational sciences, but also develops biomedical imaging and clinical applications. Since 2007, the primary research of the C@PA Group centres on the emergence of the genus Homo (first humans) and of the species Homo sapiens, but also on fundamental nature of dental/skeletal microstructure, growth, development and biomechanics, including its variation in hominoid non-human primates, fossil and extant humans, as well as applications for understanding human anatomical variability, ontogeny and phylogeny.
Overall Research, Training Objectives and Strategy
This project is both multidisciplinary and intersectorial. Based on computer sciences, it focuses on the use of up-to-date methods and the development of new tools for both field and laboratory investigations dedicated to palaeoanthropological studies : (i) Computer assisted anatomy will be used to interpret anatomical variability in humans, the search for their ancestors and relatives, and to develop medical applications; (ii) Computer aided satellite imagery (mainly radar) will be used for field explorations in Africa (Southern Africa) and Asia (Mongolia) and the discovery of new fossil hominids.
Field explorations
Palaeoanthropological research in South Africa has brought many significant fossil discoveries to the world’s attention and increased our understanding of human origins. Over the last decade, members of the C@PA Group conducted research in the « Cradle of Humankind », a World Heritage Site first named by UNESCO [3]http://www.anthropobiologie.fr/ - _ftn3, producing a large collection of new fossil hominids (from the Drimolen and Kromdraai B sites) and producing a large virtual anatomical database with high-resolution 3D reconstructions. Continued exploration of the fossil deposits primarily on the sites of Kromdraai B and Sterkfontein will provide a key opportunity to resolve some major questions on human evolution in the interval between three and two million years ago.
Our new intensive field research initiative which is planned over the next eight years (2008-2016), will address specific questions relevant to the emergence of Homo and the spread of this genus out of Southern Africa. It is planned to run continuous fieldwork throughout the year, rather than to concentrate the annual field expeditions solely in a limited number of months. At the same time, using the latest technology and methods of analysis, we will incorporate new approaches and we will continue to build our unique virtual anatomical database of fossil hominids.
In 2009, after the discovery of an Homo sapiens skullcap in Northeast Mongolia (Palaeolithic superior), the C@PA Group will organise its first palaeoanthropological expedition to this country. In light of the promising discovery of this fossil human, the C@PA Group plans field explorations in Northeast Mongolia over at least the next three years (2009-2012).
Laboratory investigations – The C@PA Group currently develops a number of projects investigating coordinated development and evolution of the dentition and craniofacial skeleton, the interaction between maxilla-facial biomechanics and dentition during development and evolution. Currently the world’s largest research group in this field (the « European Virtual Anthropology Network » or EVAN; www.evan.at) includes 15 partners from different sectors (academic, clinical, industrial research sectors) and six European countries. EVAN is an EU funded network over the 2006-2009 period within the framework of the FP6 (Marie Curie Actions, Human Resource and Mobility Activity). The C@PA Group is part of this EVAN consortium and therefore develops up-to-date and leading edge tools for digital image processing. Since opening, the C@PA laboratory has processed terabytes of micro-computed tomography (microCT) data and third generation synchrotron microCT in order to accurately visualise and measure internal dental and skeletal anatomical structures. The C@PA Group is currently building a multi-scale anatomical atlas of virtual data which, upon publication, will be hosted free of charge in a public open-access database at the University of Toulouse (Paul Sabatier).
We aim to reach two fundamental goals in order to have a significant impact on biomedical research topics ranging from craniofacial growth and development, to craniofacial biomechanics: (i) We first aim to formalise a strengthened and long-lasting new international collaborative research network which will lead to the promotion of the C@PA Group as one of the major European centres for our research activities. This network will implement recent advances in separate digital-image domains of expertise in order to provide the best possible multi-centre research and training facilities; (ii) Within the next five years, the access to the best possible anatomical virtual database will be pivotal in the success of any research project in our field. Therefore, we aim to a multi-scale anatomical atlas of high-resolution virtual database.
The Virtual Database Project
The anatomy of fossil humans or modern skeletons of known age and sex that usually represent rare and precious museum material will no longer be studied on the specimens themselves but on their virtual copies at a high resolution scale. Gathering these digital anatomical data, building virtual databases and being top notch in their analysis will become a dominant axis of activity which will imply an increasing network of collaboration with museums and research laboratories at the international level. In the one year or so since the launch of the C@PA Group database project, we have already held the world’s largest maxillofacial virtual database through the high-resolution micro-computed tomographic imaging of one of the best preserved collection of known-age at death infant skeletons in Europe (housed in the Institut d’Anatomie Normale of Strasbourg, France). Therefore, we are currently developing (with the assistance of Kodak Dental Imaging/Trophy) a portable microCT system which will exhibit a higher spatial resolution than the laboratory (Scanco) microCT and which will allow us to collect data anywhere. Over the next eight years, this portable device will be used to gather a considerable amount on new high-resolution anatomical data from skeletal remains housed in several institutions, The Royal Museum for Central Africa in Belgium, the Pretoria Bone Collection, The Transvaal Museum in Pretoria (the world’s first largest collection of fossil hominids from Africa) and The Institute for Human Evolution at the University of the Witwatersrand (Johannesburg) (the world’s second largest collection of fossil hominids from Africa).
Virtual Comparative Anatomy
These virtual representations of anatomical objects have opened a number of new possibilities for the analysis of skeletal anatomy in a wide comparative context in order to understand the evolution of morphology and to decipher phylogenetic relationships. The precise and virtual (non-invasive) quantitative analysis of inaccessible internal structures represents a major axis of the C@PA Group research. This research can be used, for example, for the « virtual extraction » and reconstruction (with correction of plastic distortions) of fragile fossil remains which cannot be prepared physically. The pilot study of this research topic will be focused on the first adult « ape-man skull », the preliminary account of which was published in « Nature » of 19th September 1936. Since then, this 2.5 million years old complete skull is in three pieces and is « waiting » to be prepared in the collections of the Transvaal Museum (South Africa) because it cannot be removed from its current location (as for most of these precious fossil remains). The larger piece (comprising of the major part of the skull) is still embedded in a block of breccias (rock) with many parts of it still not visible. This project will last several years (starting from 2010) depending upon the technical difficulties. In this research topic, we will also continue to use 3D morphometric analysis (so called « Geometric Morphometric » methods) and to develop new mathematical tools, such as new computational methods for automated morphometric analyses, automated digital reconstruction of the skull from its fragments and automated segmentation of teeth and other parts of the skull. This work will be done within joint research projects with the Asclepios Team at INRIA Sophia Antipolis, Rennes (Unit/Project VisAGeS, IRISA) and the ICAR team at CNRS Montpellier.
Medical and Biological Engineering
We believe that the next step toward advancing 3D virtual tools is biological engineering for the modelling of bone, the understanding of its form and function; at all hierarchical levels. The C@PA Group has a special interest in the modelling of the maxillofacial skeleton to examine how the mechanical environment affects morphology. Models and computer simulations will serve many purposes in this biomechanical research. The key technologies that we aim to apply are finite element analysis and dynamics analysis, with complex image and data processing.
These solid modelling techniques have been used to determine interface stresses, deformations, forces, pressures and alignments in biomechanics systems, consisting of structural components like bones, teeth, muscles, joints and ligaments. A solid model is a mathematical and graphical description of some geometric object. Our project will be unique because the core input data will be provided for living patients by a cone beam CT (to provide the 3D virtual model of the maxillofacial anatomy), the EMG (surface only) of the masticatory muscles, before and after a TENS (transcutaneous electrical nervous stimulation), the recording of the dental contacts during usual occlusion (during 3 seconds of time) and the mandibular movements from its resting-place to the maximum of dental contact (maximum intercuspation). This project will last over 2010-2013. It will be also used to assist in the design of implants and the diagnosis of maxillofacial pathologies.
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