Periodic Reporting for period 2 - CO-PHY-MED (Comparative Phylogeography of a Megadiverse Fauna across the Mediterranean) Periodo di rendicontazione: 2018-11-01 al 2019-10-31 Sintesi del contesto e degli obiettivi generali del progetto Comparative Phylogeography and Next Generation SequencingPhylogeography is an integrative scientific field that uses genetic information to study the geographic distribution of genealogical lineages. Its major goal is to infer the evolutionary and ecological processes that have left a genetic imprint on organisms. For these reasons, phylogeography provides a historical perspective, vital to biogeography, speciation, biodiversity research, taxonomy and conservation biology. Phylogeography is in the early stages of embracing modern massively-parallel methods of DNA sequencing (Next Generation Sequencing - NGS), to generate robust phylogenies and phylogeographical histories. Comparing phylogeographical patterns among multiple co-distributed species can elucidate the complex relationship between landscape and biota through time. The project CO-PHY-MED investigates the mechanisms and processes that drive divergence and speciation, and shape the biogeographical history of organisms, by using the Mediterranean as an empirical example and squamate reptiles (lizards and snakes) as models to conduct a large-scale multi-species comparative phylogeographic analysis.Model regionThe Mediterranean Basin is an area where biotic elements of the biogeographical regions of Europe, Asia and Africa converge and is recognized as a Global Biodiversity Hotspot. The Aegean region and the strait of Gibraltar, specifically, have become natural laboratories for phylogeographic investigations. Several factors are responsible for the rich biodiversity in these regions: (1) Position and complex geological history: The Aegean lies between Asia, Europe and Africa (through the Middle East), while the strait of Gibraltar, that now acts as a barrier to species’ dispersal, has served as a corridor between Europe and Africa. This resulted in an interesting mixture of faunal elements. (2) Geomorphology and islands: Islands provide important models for understanding drivers of diversification and the Mediterranean includes almost 12,000 islands, the majority of which are located in the Aegean. (3) Refugia: The Balkan and Iberian peninsulas have acted as refugia during the climatic changes of the past 2-3 million years. (4) Human activity has been recorded here for more than 10,000 years and has influenced the distribution of many organisms. Model organismsMore than 350 terrestrial reptile-species live in the Mediterranean region: most of them are squamates, about half of them are endemic, and the largest number of European species is found in Spain and Greece. Reptiles display a great diversity in their biology and offer outstanding models in ecology and evolution, mostly because of their temperature-dependence that renders them particularly sensitive to climatic changes and their diversity of dispersal and colonisation capacities, and ability to overcome barriers. Barriers are essential for speciation and biogeography, by isolating populations. Major research and training objectives - Conclusions of CO-PHY-MEDFollowing our research objectives, we collected genomic data for co-distributed species of squamate reptiles in the focal regions using NGS and applied sequence capture (seq-cap) and double-digest restriction-site associated DNA sequencing (ddRAD). We reconstructed the phylogenetic relationships among and within the studied species, inferred across-barrier diversification among co-distributed species and tested alternative hypotheses to explain patterns. Finally, we identified the major barriers that have influenced the formation and evolution of biodiversity. Our results show that squamate reptiles have presented a variety of responses to biogeographic barriers, mostly related to their dispersal capacities, size and biology. Sea barriers have differently affected the generation and maintenance of biodiversity, depending on their age and position.Accordingly, training included experimental design in phylogenomics, preparation of biological samples for genomic approaches, handing of genomic data and phylogenomic analyses. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti The outgoing phase of the project (2016-2018) acted mostly as a training phase regarding the technical aspects of the analyses (Leaché Lab, Biology Department, University of Washington-UW, Seattle, USA); the incoming phase (2018-2019) represented an application of the acquired knowledge in the European institution (Carranza Lab, Institute of Evolutionary Biology, Barcelona, Spain). The integration of the fellow within the hosting organisations and the initiation of the project’s activities took place at the early stages of the programme and the fellow was introduced to the goals, environment and style of work of the host institutes. During the same time, the list of species/samples to be used in the outgoing phase was finalized and the samples were transferred to the UW, with the respective permits and documentation. Samples were prepared for genomic analyses using two approaches (seq-cap and ddRAD). Smaller projects (subset of samples) were first analysed, to help the fellow acquaint himself with the procedures of the methodology. Subsequently, lab analyses were performed for a total of more than 750 samples included in the project. Genomic data were processed and further analyses included (1) data processing, filtering and generation of files, (2) phylogenetic-tree reconstruction, (3) estimation of divergence times, (4) biogeographical and species-delimitation analyses. So far, results were presented at the Second Joint Congress on Evolutionary Biology (Montpellier, August, 2018) and the XX European Congress of Herpetology (Milan, September 2019), and in four open-access publications in SCI scientific journals, with several others in preparation. Primary data are freely accessible in public repositories (GenBank and Dryad). Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) The fellow, through (1) the achievement of the planned training objectives, (2) the skills and experience acquired in project design and execution, and (3) working at an institution of international reputation, hosted by leading experts in their field, is in a very good position in becoming an expert in the field of phylogenomics and phylogeography in the NGS era. Additionally, this project represents an innovative and important work that contributes to the grand idea of the Tree of Life. The results from the first phase and the anticipated results from the second one, will provide important insights towards this goal. The training-through-research contributes to the enhancement of EU excellence in phylogenomics, promoting the European Research Area (ERA) as a highly innovative, productive and attractive centre for scientific research. Finally, the “protection and sustainable management of natural resources and ecosystems” is one of the objectives of environmental research under HORIZON 2020. Phylogeography provides a fine-scale documentation of species’ diversity in a region, identifies lineages within species and studies the drivers of biodiversity. The results from the current project will, therefore, assist with the effective management and conservation of biodiversity, a major current challenge, especially in the face of environmental change and will contribute to meeting the objectives of several EU and global initiatives.