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Adapting to Global Change in the Mediterranean hotspot: from genes to ecosystems

Final Report Summary - ECOGENES (Adapting to Global Change in the Mediterranean hotspot: from genes to ecosystems)

Executive Summary:

Global change is introducing pervasive threats to the preservation of natural resources, with long-term consequences often difficult to measure. Concerned citizens demand answers from administrations; however, designing strategies that simultaneously improve the quality of life and incorporate sustainable development is an arduous effort due to the inertia that slows down any initiative aimed at limiting the socioeconomic growth. It is therefore imperative to support with sound and reliable scientific criteria the decisions that must be taken when incorporating the principles of sustainable development to the social and economic drivers that lead our society.
The Doñana Biological Station (EBD-CSIC) is a public research institute belonging to the Spanish National Research Council (CSIC) in the area of Natural Resources. Our major tasks are the understanding of the processes that threaten biodiversity in the face of global change, the development of tools for the analysis and prediction of social and environmental consequences, and the assessment of feasible solutions for society.
The objective of this project was to strengthen the capacities of EBD-CSIC in order to maintain these abilities and play a leading role in global change studies focused especially in the Mediterranean. To do so, a research institute requires the facilities to work at all the levels nature resources function, from molecules to ecosystems. Thus, we selected analytical tools highly demanded by our stakeholders and managed by all our research lines: Genetics and Genomics, Ecological Modelling, and Ecophysiology. Eight recruited experienced researchers introduced new approaches into running projects, and new collaborations promoted by secondments prompted the conceptual frame-theory of the actions. Training exchange activities and three international meetings encouraged discussion of issues of utmost relevance in the context of global change. At the same time, the upgrading of existing laboratories and the creation of a new stable isotopes laboratory at the EBD-CSIC was a milestone to achieve the propounded goals.
The results of the research carried out at EBD-CSIC have been constantly transferred to society through seminars, press releases, and EBD-CSIC and EcoGenes websites. One of the most important event in terms of disseminating the outcomes of the research carried out at the EBD-CSIC has been the final photographic outdoor exhibition on global change effects that has been on display during the last summer. This exhibition could be seen in September 2013 by thousands of people in one of the busiest pedestrian streets of Seville and will tour several Spanish cities in the coming months. Besides this immediate abidance, the international projection and collaborative approach of EBD-CSIC research staff will guarantee the continuity of the actions proposed beyond the duration of EcoGenes.

Project Context and Objectives:

The objective of this project has been to strengthen the potential of the EBD-CSIC research center. Providing facilities, new knowledge, and support, EcoGenes boosted the international projection of EBD-CSIC as a leading research centre in understanding the processes that threatened biodiversity in the face of Global Change. The location of this institute in the Mediterranean region, whose vulnerability to climate change makes it an important model system for conservation biology studies, adds strategic value to the project. The EBD-CSIC was founded in 1964 as a small research institute to provide scientific advice for the management of the newly created Doñana Natural Reserve, currently part of one of the most important national parks in Spain. Over the last 50 years, it has grown into an internationally respected academic institution in the field of biodiversity conservation at a global scale. Its fundamental mission is to carry out multidisciplinary research at the highest standard directed to understanding, from an evolutionary viewpoint, the way in which biodiversity is generated, maintained, and how it deteriorates, as well as the consequences of its loss and the possibilities of its conservation and restoration. An inseparable derivative is also to promote the transfer of the generated knowledge to society. However, the achievement of these objectives has been hindered because its analytical capabilities and technical expertise became outdated in some areas.
With the aid of outstanding research partnering organisations such as the University of Uppsala, the University of Groningen, the Helmholtz Centre for Environmental Research-UFZ, and the Institut National de la Recherche Agronomique (INRA), EcoGenes has helped us to build the frameworks that led us to the fulfillment of the major tasks: i) deepening the knowledge of the processes that threaten biodiversity in the face of global change; ii) the development of tools for the analysis and prediction of social and environmental consequences; iii) and the assessment of feasible solutions for society. Key objectives included: upgrading of the existing laboratories of Ecophysiology (LEF-EBD), Aquatic Ecology (LEA-EBD) and Molecular Ecology (LEM-EBD); the creation of a new laboratory of Stable Isotopes (LIE-EBD); recruitment of eight experienced researchers to increase the capacity for understanding the three focal areas (genetics, ecophysiology and ecological modeling); organization of three international conferences. Two-way secondments increased quality through collaboration with collaborative partners and other institutions included at a later stage. The loss of biodiversity due to global change, focused especially in the Mediterranean hotspot, was the common thread of the training exchange activities, seminars, and dissemination activities such as the photographic exhibition.
To enhance its prominence as a reference centre in Ecology and Environment in the Mediterranean, specific actions were meant to be consolidated in our institution. The lecture on Global Change has been converted in an annual event, focusing scientists attention on the dramatic changes that will affect Mediterranean areas in a global change scenario. EBD-CSIC is leading national and international research communities created with the goal of exploring specific aspects of global change effects in the Mediterranean, whose achievement requires pooling of database and sharing of information.

Project Results:

We choose analytical tools managed by all our research lines, such as genetics and genomics, ecological modelling, and ecophysiology. Since understanding how the responses of pair-wise interactions of species scale to entire assemblages remains one of the big challenges that must be met as society faces global ecosystem change, we aim at strengthening our capabilities for analysis and interpretation at all these levels: at the molecular level (genetics/genomics), at the physiological level (pathogens, stable isotopes) and at the population and higher levels of biological organization (modelling of populations, food webs, and ecosystems).
To understand the potential adaptability of ecological systems in light of global change, the EBD-CSIC required man power and research equipment. Thus, eight experienced researchers in the three focal areas (genetics, ecophysiology and ecological modelling) were recruited, and the existing laboratories of Molecular Ecology (LEM-EBD), Ecophysiology (LEF-EBD), and Aquatic Ecology (LEA-EBD) were provided with specific scientific equipment to attend the increasing demand for new analytical approaches. Besides, a new laboratory of Stable Isotopes (LIE-EBD) was created, and LEF-EBD was upgraded to bio-security level II.

Genetics and Genomics

The genomic department was equipped with a GS Junior Sequencer, a next-generation complete automated sequencing system, consisting of a DNA-sequencing instrument, a suite of point-and-click data analysis software, and a high-performance desktop computing station with 12 GB of RAM, 160 GB reserved for the application and 500 GB for data storage. GS Junior can greatly facilitate the study of genetic variation in non-model organisms, through the sequencing of large numbers of genomic regions in many individuals at once. With its excellent performance (a throughput higher than 35 Mb per run, comprehensive genome coverage with long 400 bp sequencing reads with an accuracy of over 99%, more than 100,000 reads for shotgun libraries, and over 70,000 sequences for amplicon libraries), this has brought a new type of technology into the lab, and triggered a series of new projects made uniquely possible through this different way of sequencing. These new projects range from getting similar types of results as can be gotten with other technology, but faster and much more, to different types of data.
To explore and understand the biases we introduce into experiments with particular experimental design decisions, experiments have been run to test the effects of different amplification protocols, the impact of different enzymes and also the impact of different analyses protocols. These will increase the quality of future experiments, and are of general interest and so are being prepared for publication in international scientific journals. For example, in the past sequencing loci from multigene families required bacterial cloning before sequencing, thus limiting the number of individuals and alleles per individual that could be characterized. Now with the chemical cloning incorporated in the pyrosequencing technology upon which the Junior is based, hundreds of ‘reads’ (analogous to clones) can be generated for each individual in populations in a single experiment. This type of experiment on the Major Histocompatiblity Complex (MHC), a gene family which plays an important role in the immune system, has already been set up with multiple taxa, including the Iberian lynx (Lynx pardinus) and domestic horse. Similarly, before we had access to next generation sequencing technology, the identification of highly polymorphic nuclear loci called microsatellites, which are useful for looking at within-family and within and between population genetic patterns, required extensive bacterial cloning and then sequencing of clones. We have now modified protocols and used them with Junior to identify many more loci, faster and more economically for four species of mosquitoes, and further experiments with other taxa are already in development.
We have also used this next generation sequencing technology to sequence fragments of DNA which are too long to traditional Sanger sequencing. This involves amplifying a gene or series of genes through long range polymerase chain reaction, and then fragmenting that DNA and sequencing the fragments on Junior. With the aid of the EcoGenes recruited researcher Eaaswarkhanth Muthukrishnan, we have tried both to sequence a candidate gene (IGF1) in dogs, and to sequence whole mitochondrial genomes in a variety of species, including tree squirrels of the subfamily Callosciurinae. Because of the additional power associated with the larger amount of data in the whole mitochondrial genome as opposed to small fragments which have been used historically in our field of study due to logistical reasons which don’t apply to Junior, this is likely to become a much more popular application of this technology in our lab.
The examples of applications of Junior explained above all depend on high quality DNA which can be obtained only from high quality, modern samples. However, multiple researchers in our institution also work on degraded, ancient DNA. This material ranges from historic skins that may be only decades or centuries old, to teeth and bones from archaeological and paleontological sites that are thousands to tens of thousands of years old. This material presents special problems, some of which can be addressed particularly well with next generation sequencing. We have been modifying and developing protocols to avoid amplification (and thus the errors associated with amplification) of ancient DNA through enriching extracts for target loci, and then using the enriched pool to develop libraries to run on Junior. We have been using these techniques on material from multiple mammalian species using both historic and more ancient material. So far we have been targeting mitochondrial DNA, but once these protocols are worked out, they will be useful for other loci as well.
So far experiments from six different principal investigators and many more PhD students have been run, and many more are in progress. As laboratory technician, Anna Cornellas was enrolled on 1/12/2012. She has been and is continuing to support the genomics line at EBD-CSIC through assisting in the preparation of various types of libraries from different types of starting material (i.e. high quality DNA, low quality/ancient DNA, regular PCR products, and long-range PCR products) to test and optimize the next-generation sequencer, 454 Junior.

S & T results
In a conservation context, genomics provides the opportunity of obtaining a detailed picture of the impact of genetic drift on genetic variation, including the so-far unexplored adaptive component. The collaboration between two of the recruited researchers, Fernando Cruz and Begoña Martínez, the Uppsala University, and EBD-CSIC researchers shed some light into the genetic consequences of species decline, eventually helping to improve management strategies to ameliorate genetic risks for species persistence. As part of the Iberian Lynx Genome Project ( the genome of the Iberian lynx, one of the most endangered feline in the world, has been sequenced. Eleven Iberian and one Eurasian male lynx (Lynx lynx) have been sequenced to medium coverage (~26X). The importance of this achievement is that it represents one of the few endangered animal species to be genome-sequenced in the world, and the first complex genome sequenced in Spain.
Another study centered on the domestication of dogs. The dog genome is at the basis of a genomics study in which the molecular evolution of insertions of foreign (mitochondrial) DNA into the nuclear chromosomes is studied. The first, and most complicated step, in this project was to identify all instances of insertions in the genome, and the next steps were to characterize the regions where the foreign DNA inserted, and lastly to characterize the foreign DNA. These data will then be used to test various hypotheses regarding how foreign DNA is incorporated into genomes.
Also, a project about using SNPs to study the back-crossing of Iberian wolves and domestic dogs is being carried out, with the idea to genotype wolves, hybrids, and dogs to estimate the fitness of the hybrids and the actual geneflow between wild and domestic populations.
The ecological genomics of natural Arabidopsis thaliana (thale cress) population in the Mediterranean region has been another core project in the area of genetics and genomics. Studying the genetic structure of thale cress is allowing better understanding the genetic basis of local adaptation and fitness, and to what extent adaptation can cope with rapid global change. The main objective of this project, carried out by Adrian Brennan, the EcoGenes recruited researcher, in collaboration with the EBD-CSIC researcher Xavier Picó, was to better understand the genetic basis of local adaptation in A. thaliana using a combination of high-throughput single nucleotide polymorphism (SNP) genotyping and phenotypic measures from reciprocal transplant experiments within the native Iberian range. Other objectives were to determine the biogeographic history of A. thaliana in the Western Mediterranean region using single nucleotide polymorphism genotypes, flowering gene sequence data, quantitative trait measures data. Finally we were interested in the influences of altitude and latitude on the population genetics of A. thaliana across its distribution range using a combination of the genotypic and phenotypic data described above. Phenotype data were monitored and collected from reciprocal transplant experiments at two locations, Grazalema and Sierra Nevada, through a complete lifecycle from seed to fruiting during the autumn-spring growing season of 2011-2012 and have commenced a replicate experiment for the coming growing season. Multi-year field studies are necessary to take account of considerable year to year variation in field conditions. The phenotypic characterization and reaction norms of Arabidopsis thaliana ecotypes is the aim of the project “Phenotypic characterization and reaction norms of Arabidopsis thaliana ecotypes by means of transplant experiments between contrasting environments (REACT)”. Funded by the Spanish Ministry of Economy and Competitiveness (Plan Nacional 2011), it will ensure the continuity of the collaboration between Adrian Brennan and EBD-CSIC researchers beyond the duration of the project.
A second line of research followed by Adrian Brennan focused on the ecological genetics of mating system diversity in Linum. Species in the genus Linum, closely related to the important fibre and oilseed crops flax and linseed, show extensive mating system variation from selfing to outcrossing by heterostyly (morphological selfing barriers), self-incompatibility (biochemical selfing barriers), or both systems together. Researchers plan to initiate an ambitious ecological genomics research program in wild Mediterranean Linum species to investigate the evolution and impacts of mating system diversity.
Finally, we highlight the study on the distribution range of an oak species involving the INRA-BIOGECO contact person Arndt Hampe. A set of 19 low-latitude marginal populations of Quercus robur (Pedunculate oak), one of the most important European forest trees, in order to elucidate how these have been able to sustain their viability and genetic diversity through multiple glacial cycles, and which are their perspectives under modern climate change. For this purpose, we are investigating how genetic diversity is distributed and transmitted across populations. Because gene dispersal plays a central role in this process, we examine patterns of historical and contemporary gene flow at three levels: within populations, among populations, and between species (i.e. hybridising with the closely related and much more abundant Quercus pyrenaica). Finally, germination and growth experiments under controlled conditions will be used to assess whether 1) low levels of genetic variation associated to small population size actually result in reduced fitness and decreased drought tolerance and 2) hybridisation with the more stress-tolerant Q. pyrenaica results in better performances of Q. robur under marginal conditions. The ultimate objective of this study is to better understand how glacial relict populations persist under adverse conditions and to provide valuable background information for the development of appropriate strategies for their conservation and management in a changing, and presumably drier, climate.

Scientific Publication
Recruited researchers of the genetics and genomics area published 5 peer reviewed papers during EcoGenes life, and five more have been submitted.

Exploitable foregrounds
As part of genomic analysis carried out at the LEM-EBD, Fernando Cruz with the support of researchers at the University of Uppsala has been collaborating with the EBD-CSIC IT Services to set up a server for genomic analyses, which is visibly facilitating analytical processes developed at the institute. Although current characteristics are quite modest, the machine is flexible enough to be upgraded to a high-end server with full capacities. At this moment, the server allows running processes of moderate computational demand, such as cleaning and pre-processing of sequence reads, mapping, alignment, blast, variant calling on small genomic regions or handling .sam, .bam and .vcf files. The operative system is CentOS release 5.8 (Final) compatible with the software of the 454 Sequencer GS Junior.
The expertise of recruited researchers in molecular ecology and bioinformatics has enabled a detailed characterization of the genomic patterns of variation in Iberian lynx, the identification of over 1 million polymorphic markers and the development of a SNP chip specific for the Iberian lynx. This new and powerful tool will allow building up exhaustive pedigrees and to study the genetic variation of the two remaining populations in the wild (Sierra Morena and Doñana) and the captive one to a previously unreachable detail, what will serve for the optimal genetic management of the populations.

The University of Uppsala has strictly collaborated with EBD-CSIC organizing the lecture on NGS analysis (March 2013), supporting the set up of the genomic server, and advising on multiple aspects of genetics issues. In particular, the person contact, Dr. Matthew T. Webster, visited the EBD-CSIC in four occasions for activities related with EcoGenes (First Conference; Steering Board meeting; Secondments). He leaded the “Limits of genomics to address adaptation” discussion round-table at the “Day 0” workshops (September 2013), and gave one seminar (March 2013). The two trips made by EBD-CSIC staff to the Uppsala University, contributed to the strengthening of genomics in multiple ways: assisting in the experimental design and analysis of ongoing projects, discussing laboratory work strategies for a collaborative grant proposal, meeting with PHD students, setting up potential future collaborations.
The BioGeco/INRA institute carries an intense collaboration with EBD-CSIC through their senior researcher Arndt Hampe. Dr. Hampe visited the EBD-CSIC in several occasions for activities related with EcoGenes (Steering Board Meeting; Second Conference; Secondments). He took advantage of his stay for carrying out meetings with his two PhD students hosted at EBD-CSIC and their other co-supervisor, Prof. Pedro Jordano (Ecological Synthesis. Ecology and Molecular Evolution. Molecular Ecology and Evolutionary Genetics Dept.). In one case, they discussed matters of field sampling and subsequent molecular analyses of the obtained material, a critical issue at the end of the student's first field season. In the other case, they discussed on an advanced manuscript entitled "Male fecundity and pollen dispersal in populations of a riparian tree species", that represents the first chapter of the student's PhD thesis. Moreover, Dr. Hampe gave a total of 12 hours of theory lectures in the fourth edition of the Master "Biodiversidad y Biologia de la Conservacion", a programme coordinated by the EBD-CSIC and the Pablo de Olavide University (Seville). He coordinated the Fourth International Course “Ecological Consequences of Climate Changes: Integrating Research Approaches” (September-October 2013). As a consequence, two meetings were organized to discuss the results of the course (evaluate students, exchange feedbacks about the teachers), and to advance the planning for the 2014.
A collaborative approach led to the creation of the Retuertas’ horse group (EBD-CSIC and University of Uppsala, with the participation of Gabriella Lindgren, University of Copenhagen, University of Potsdam, Laboratory of Applied Research- Military Horse Breeding, and University of the South Pacific). The aim of this group is to explore the Retuertas’ horse as a system to study the Genetics, Evolution and Domestication of this free-ranging horse breeding found in Doñana. The first step of this group is to write a proposal for the first genome to be sequenced to about 20x coverage, so it can be compared to other genomes from 31 other breeds, and also to compare the SNPS identified with those included in the 700K SNP chip expected to become available in a few months, in order to characterize potential bias.
In line with the advances in quality through collaborations, the creation of the Genomics Discussion Group arise from the incorporation of EcoGenes recruited researchers (September 2011 to date). These are internal meetings to support the genomics community within EBD-CSIC, for discussion of current genomics research, perspectives, collaborative projects, software, new methods or approaches. Driven by the participants from this internal group, the regional genomic network “SevinOmics” was created, as a research community interested in exploring Next Generation Sequencing technologies and constituted by scientists from different research centres located in Seville, such as the University of Seville, IBVF-CSIC and CABIMER. SevinOmics, who met at EBD-CSIC headquarter in February 2012 and June 2012, has set up an e-mail listserv and a wiki webpage.
The analysis of the ancient genomic data is fostering the collaboration between EBD-CSIC and the Center for Conservation and Evolutionary Genetics of the Smithsonian Institution (Washington D.C.). The application and development of genomics techniques to conservation and evolutionary questions, with special emphasis on the incorporation of low quality samples are currently discussed with LEM-EBD committee.
The project "Genetics of mating system variation in the genus Linum" is being carried out with the collaboration of Professor Juan Arroyo of the University of Seville. The project “Molecular characterization and evolution of a novel self-incompatibility system in the Asteraceae” foresees collaboration with Simon Hiscock, professor at the School of Biological Sciences of the University of Bristol. Future collaboration is expected with the University of Durham (UK), in order to develop SNP markers to unveil the genetic basis of heterostily in Linum tenue. The project “Evaluation, monitoring and genomic management of the Iberian lynx populations. Doñana National Park” was born from the collaboration with the Spanish institute INIA.

Associated with EcoGenes, we have hosted or presented several informational seminars on experimental design data processing and data analysis in order to encourage and support more research groups to embrace this new technology.
The acquisition of the GS Junior prompted the seminars “to Ecological and Conservation Genomics… with Junior” (February 2011), and “Junior: what goes in & what comes out” (July 2011). These seminars went through some of the applications of genomics in general to evolutionary and conservation studies. In particular, they focused on the steps of emulsion PCR, and the actual sequencing reaction- how it works in general, and how to do it with this machine. The kind of results one could expect with this machine given different types of libraries was summarized, and the actual results from the training run on our machine were presented. Lastly, some of the resources available to everyone to help them set up their own experiment were highlighted.
On February 2011, Paulo Celio Alves, from CIBIO, Portugal, presented the results of his research on the hybridization due to species distribution changes forced by climatic oscillations. Fernando Cruz, EcoGenes recruited researcher, gave a seminar on July 2013 on the population genomics of Iberian lynx. The video is available at the EcoGenes web site. Matthew Webster, contact person at the University of Uppsala, explained in the seminar “A worldwide survey of genome sequence variation reveals the evolutionary history of the honeybee Apis mellifera” the results of his studies on honeybee biology and genetics.
In November 2012, the LEM-EBD organized a seminar to get to know what’s going on in the laboratory. In December 2013, the second seminar took place at EBD-CSIC. Given the enthusiasm that the “LEM day” has arisen among EBD-CSIC staff and users of other centers using next generation sequencing, continuity is expected on a long term basis.

Training lectures
The trigger that led to starting up the genomic server was the organization of the course: “Introduction to the analysis of next generation sequencing data”. The course took place 5 – 8 March, 2013 at the EBD-CSIC facilities in Seville. Drs. Matthew Webster, Alvaro Martinez Barrio, and Marc Höppner from the collaborative partner Uppsala University, organized and run the classes. The course combined lectures on the theory of analysis algorithms with practical computational exercises demonstrating the use of common tools for analyzing data from next-generation sequencing projects. It was open for PhD students, postdocs, researchers, and other employees in need of bioinformatic skills within Spanish and international universities. Familiarity with basic linux command line was beneficial but not expected; no programming knowledge was required. To get the maximum benefit from the lectures, relevant previous experience in sequencing or analysis, and/or a current research project using next generation sequencing, was valuable. Beside, this course was most useful if participants were directly performing analyses or had a support role and were able to participate in a wide range of projects and transfer knowledge to others. Nineteen people attended the course. These were senior researchers, post and under graduate students, and laboratory technicians, coming mainly from EBD-CSIC but also from international research centres. The course combines lectures on the theory of analysis algorithms with practical computational exercises demonstrating the use of common tools for analyzing data from next-generation sequencing projects. The course extended to 4 days in order to include an all-day practical session, where people were offered the opportunity to work with their own data or -for those with no data yet- with an additional subset provided by the professors with a specific question in mind. This turned out to be a great opportunity for students to consolidate what they learned during the course.

The conference “Genetics and Genomics in Wildlife Studies. Implications for Ecology, Evolution and Conservation Biology” (Seville, 20-21 October 2011) was meant to help expanding genomics as a research line in the short-term at the EBD-CSIC. Given the existing expertise, infrastructures, and ongoing collaborations with other research centres, our institute pursued to take advantage of the current expansion of genomics to make substantial progress in fields such as ecology, evolution, behaviour and conservation, which have been the objects of intense research for decades within the EBD-CSIC. The expansion of the genomics approach in the projects of our researcher was perceived of great interest especially to face the biodiversity crisis in the Mediterranean region. However, the development of genomics is vertiginous, and keeping up with all the advances in this discipline is of paramount importance. This increased the importance of such an event, in which the main figures worldwide in the practice of genomics applied in wildlife studies provided an up-to-date account of the situation. Twelve leading researchers coming from 8 cutting-edge institutions worldwide agreed to come to Seville to speak about the application of genomic tools to address evolutionary, ecological and conservation questions. Participation was restricted to 65 assistants. The meeting highlighted the potential applications of cutting-edge genomic approaches, and the current revolution in next generation sequencing (NGS) technologies, for ecological, evolutionary and conservation genetics research. Talks covered work currently being done using genomic approaches, either in species with a reference genome or in non-model organisms, to tackle a wide array of evolutionary questions as diverse as phylogenomics, comparative genomics, population genomics, detection of adaptation and selection using genomic tools and genome-wide association analyzes. The meeting ended identifying a range of important needs: cost-effective genotyping methods at the population level, sequencing methods for low quantity or poor quality DNA and the assembly of duplicated genomes. Data storage is becoming a demanding issue as well because of the huge amount of NGS information becoming available. However, discussion at the end of the meeting cautioned against overuse of NGS. The conference’s participants agreed that genomics will definitely increase our understanding of adaptation and fitness components in a conservation context, but this might sometimes be overshadowed by the priority of preserving processes and habitats in the immediate short term for highly threatened species. This was a very successful event, with a great deal of fruitful and constructive discussion, and the key outcome as relevant as the publication of the findings in a specialized journal (Fernando Cruz et al. BioEssays, Vol. 34, Issue 3, 2012). Also, it provided a platform to start new collaborations, and to strengthen existing partnerships. The conference was chaired by Xavier Picó, Associate Professor of Research at the Department of Integrative Ecology (EBD-CSIC). The Organizing Committee was composed by other 9 researchers of EBD-CSIC, including three EcoGenes hired researchers (Adrian Brennan, Fernando Cruz and Eaaswarkhanth Muthukrishnan). Giulia Crema, the EcoGenes technical assistant was in charge of logistics.


The upgrading of existing laboratories has significantly improved the analytical capabilities of the ecophysiology department. The LEA-EBD has been provided with a state-of-the-art water quality auto-analyzer, which allows studies dealing with the understanding of ecological processes in freshwater ecosystems. Since September 2011, when commissioning was completed, the number of analysis of dissolved elements shifted from 32 to 892. Until then, only two projects could be attended by the services of this laboratory; after the purchase of the auto-analyzer 13 projects coordinated by at least 5 staff researchers are currently commissioning analytical samples.
The acquisition of the Roche LightCycler 480 by LEF-EBD allowed the implementation of the technique to measure relative telomere length in birds. This analysis has been offered to researchers at EBD-CSIC and external users. At this stage the laboratory have completed analyses for studies in Vultur gryphus, Parus major, Ficedula hypoleuca and Corvus monedula. Additionally there are several requests for information for new analyses of samples from observational and experimental studies. This equipment has also enabled the implementation of three different techniques to quantifying blood or feces parasites by far. The ability to quantify using RT-PCR has been implemented for the most common species from genus Plasmodium, Salmonella and Chlamydia. Each of these techniques is in different development process, although all of them are very advanced.
The stove/incubator incorporation has enabled the development of test to measure the antimicrobial capacity of serum in birds and reptiles. First we develop the technique in a conventional spectrophotometer, but finally to increase analysis speed the analyses are read in a micro plate reader Victor 3, getting a significance increase (20 to 48) in the number of samples analyzed per day. 1200 samples from Anas platyrhynchos and Fulica atra, and other 400 samples from Podarcis lilfordi have been analyzed. The technique will be applied to samples of mammals in the next months. The implementation of this technique has also served as the formation practices of a student of the program in “Experts in Environmental Health”.
In relation to HSP 70 technique, we analyzed 80 samples from Charadrius alexandrinus using ELISA technique. There are 120 samples actually waiting to analyze by this technique. We got ISO 9001:2008 and ISO 1400 certificates and we got level II on Biological Security too, and as much permanent workers as discontinuous ones respect rules without any problem. Since its creation in September 2009, LEF-EBD has analyzed a large number of samples. We are currently developing new techniques to offer to EBD-CSIC researchers. Through the participation in the EcoGenes project, the development of techniques and the number of analyzed samples have increased together with projects and users who make use of these facilities. Eighteen projects received support from LEF-EBD in 2013, and 30 users needed its service. Prominent results of the analysis carried out in this laboratory have been reported in international congresses by the lab coordinator, who has also received additional training thanks to secondments activities. The number of analyzed samples at LEF-EBD increased as well as projects and users who make use of their facilities: Telomere lengths from 0 (2011) to 2468 (2012-13); Immunochemical techniques from 4300 to 13940; Antimicrobial capacity from 0 to 4600.
The setting up of the LIE-EBD occurred throughout 2011. Leonard Wassenaar and Keith Hobson (Environment Canada) helped in the launch, and Gabriel Bowen (University of Utah) gave hints on the analysis of results. Lab coordinator has also been fully trained through workshops and seminars funded by secondments activities. The system of mass spectrometry consists of the following equipments: IRMS Delta V Advantage; Elemental Analyzer (EA) Flash HT Plus; ConFlo IV Universal Interface. Delta V analyzes stable isotopes, whose variation in temporal and spatial scales play a fundamental role in addressing climate change issues. The purchase of Delta V means strong savings of time and money, and gives a boost to the disentanglement of complex processes taking place in ecosystems, such as food-webs and migration. In addition, the complementary equipment purchased at a later stage is also enabling the preparation of the samples in the same laboratory, which is further increasing the savings, it allows analyzing a greater number of samples within each sequence, and it improves the accuracy in weighting, which is directly related with the accuracy of obtained results. As a whole, the Mass Spectrometer System has proven to be an effective tool for supporting many research projects, some of them directly related with the global change. The involvement of the hired researchers in the development and operation of laboratories has been particularly evident in the LIE-EBD. The work of Francisco Ramirez as a scientific advisor of isotope lab staff personnel consisted primarily in the development and validation of internal standards that allow the daily activity of the laboratory. Simultaneously, he gave the widest possible dissemination to the laboratory by providing scientific advice to all researchers interested in incorporating isotopic approaches in their research.

S & T results
The phenotypic flexibility of the Black-tailed Godwit Limosa limosa limosa, a significant species in terms of global change effects, is one of the core projects at the ecophysiology area, and results can shed light on some facets of global change impact on wildlife. The main objective of this research conducted by the EcoGenes hired researcher Rocío Marquez in collaboration with EBD-CSIC staff and the University of Groningen, is to identify the costs and benefits of wintering site selection in long distance migratory birds. Doñana Wetlands, in South-West of Spain, is an important key site for many waterbirds during the whole year, especially during winter. The study species is the endangered Black-tailed Godwit, a waterbird that breed in continental Europe and winters in southern Spain or in the southern part of the Sahara desert. This study tries to answer specific questions at the light of climate change models predicting environmental changes in the Mediterranean due to increases in mean monthly temperature and decreases in precipitation, and considering that the degradation of the Mediterranean wetlands occurred in the last century makes highly vulnerable these ecosystems: (i) Is the species population trend related with environmental variables and changes in the land use occurred in the last years in Doñana? (ii) Do individuals with different migration strategies have different survival, breeding success or habitat choice during the breeding season? (iii) Do the pathogens found in shorebirds that use different habitat in Doñana, differs depending of the habitat use and the migration strategy? To answer these questions, intensive field work is required. Actually, our main work during the last year focused in data collection, which will presumably be concluded in winter 2013. Analyses up to now have focused on the first question. As a summary, in contrast to the considerably decline of the population of the nominate subspecies L. limosa limosa in Europe and to the decline found in the main wintering areas in West-Africa, the number of individuals in Doñana Wetlands is increasing, mainly in the artificial wetlands (i.e. cultivated areas and fish ponds) created during the last decades. This could be a response of the species to global change, since land use changes have occurred along the distribution range. The species could have found in the protected Doñana Natural Area predictable wetlands where to stay during the non-breeding season. Fundamental aspects of the study on the Black-tailed godwit study lies in the analysis conducted in the LEF-EBD and LIE-EBD. The analysis of blood samples for the detection of blood parasites, antibodies against different virus and characterization of different components of the immune system have been conducted at EBD-CSIC thanks to the new methodologies supported by the upgraded LEF-EBD. On the other hand, the isotope laboratory analyzed the primary feather samples of individuals breeding in the Netherlands and samples of individuals using Doñana. With these analyses we aim to identify the moulting places from the birds breeding in the Netherlands in order to determine moulting strategies related with different migration strategies and analyze the potential fitness consequences. The first results show that the individuals start to moult the primary feathers in their breeding grounds and finish their moult in Doñana or in Africa. At the moment, we try to identify if Doñana signature could be different from those signals found in Doñana based on Deuterium. This result in combination with our observations of the individuals in Doñana could support our findings.
The complex interactions between the individuals and the environment are the objective of several studies by the EcoGenes recruited researcher Laura Gangoso, centered on the Eleonora’s falcon (Falco eleonorae). What mechanisms can individuals use to adjust their physiology, including metabolism, thermal relationships, endocrine responses and immune parameters, to changing habitat conditions, is one of the questions addressed on a study based on the genetic colour polymorphism of Eleonora’s falcons. This species exhibits a striking melanin-based colour polymorphism with individuals displaying a pale or dark morph with little variation within these two morphs. We studied possible covariations between colouration and immunity. Results confirmed the ones previously found regarding cell-mediated immunity, reinforcing the idea that the capacity to mount a cellular response was morph-specific, which was genetically determined. But environmental effects played also a role. In sum, this study highlights that the importance of MHC diversity cannot be generalized among vertebrates and hints the evolution of compensatory immune mechanisms in falcons to cope with emerging and continuously evolving pathogens. Another study under preparation will determine whether there are differences in population dynamics between morphs. To do this, we will use a long-term database (2006-2012) on breeding performance and survival (using a capture-recapture approach). The analysis of oxidative stress, biochemistry, humoral immunity (antibodies against New Castle virus), oxidative stress, heat shock proteins (Hsp) as well as detection and quantification of the blood parasites (Plasmodium, Haemoproteus and Leucozytozoon) were carried out in the LEF-EBD. In the coming months, we will continue the detection and quantification of the blood parasites and we will carry out the telomeres length measurement at LEF-EBD.
One of the possible variations associated to climate change is the availability of food resources. Another study investigates the ecological specialization of the Eleonora's falcons towards a fluctuating food resource. The ecological specialization of this species towards a fluctuating food resource has several associated trade-offs. This specialization, on the one hand, has allowed the widespread colonization of the Mediterranean basin, where they can exploit almost exclusively a temporally superabundant food resource (i.e. small migratory birds) to raise offspring, while avoiding inter-specific competition during spring (the season during which most other bird species breed in the Northern Hemisphere). On the other hand, and precisely due to its high specialization, Eleonora’s falcons are forced to travel long distances (up to 10.000 km) thus facing its implicit high costs in terms of potential mortality twice a year. Moreover, this specialist may be particularly susceptible to incidental variations associated to climate change in the phenology of their prey populations, such as advances or delays in their migration schedules (Jenny and Kéry 2003), as well as potential changes in wind regimes. To investigate feeding ecology and hunting behaviour in this species and to what extent they may be influenced by variations associated to climate change, we tagged six individuals from the Canarian population with GPS/accelerometers data-loggers. These data are currently being processed.
The use of stable isotopes, remote sensing and Geographic Information Systems (GIS) to investigate the impact of global change on the trophic structure and dynamic of natural communities underlie a number of studies developed by hired EcoGenes researcher Francisco Ramirez, in collaboration with Phillip Island (Australia), Environment Canada and the University of Utah.
The first deal with the natural and anthropogenic factors affecting the feeding ecology of a top marine predator, the Magellanic penguin. We used a multidisciplinary approach to explore the mechanisms through which a typical central-place forager, the Magellanic penguin (Spheniscus magellanicus) from the Chubut province of Argentina, responds to variations in oceanic conditions and prey resources. We combined habitat and species distribution modelling with isotopic dietary reconstructions based on blood δ13C and δ15N values to quantitatively evaluate the role of bathymetry, sea-surface temperature and chlorophyll-a concentration, abundance of conspecifics, and extent of fisheries activities in explaining the foraging and feeding preferences of individuals breeding at different colonies. The at-sea distribution of penguins was tightly coupled with the spatial distribution of their staple prey species, anchovies (Engraulis anchoita), especially in areas over the continental shelf (>200 m depth), with relatively warm water (from 16 to 21ºC), and moderate abundances of conspecifics (from 50 to 250 individuals). Competition with conspecifics and human fisheries were also identified as important factors explaining penguin diet with decreasing relative contributions of anchovies with increasing abundance of conspecifics and fishing activity. Our multifactorial approach allowed us to simultaneously explore different physical, biological and anthropogenic features likely affecting marine resource availability, and, consequently, driving the feeding and foraging ecology of this central-place forager. Our approach can be extended to a large suite of central-place foragers, thus providing important advances in the way we tackle investigations into the conservation and management of these species.
Another study being develop in this area is entitled “Evaluating the role of man-made habitats as alternative feeding areas for Slender-billed gulls (Chroicocephalus genei) breeding at the Natural Area of Doñana”. The methodology and results derived from this study could be extended to a large suite of threatened natural communities worldwide, thus providing a useful framework for management and conservation. Current rates of wildlife habitat loss has placed increasing demands on managers to develop, validate and implement tools aimed at improving our ability to evaluate such impacts on wildlife. In this project we combined remote sensing and stable isotope (δ13C, δ15N) analyses of individuals to unravel (1) the effect of variations in availability of natural food resources on reproductive performance of a Slender-billed Gull population breeding at the Natural Area of Doñana (SW Spain), and (2) the role of two adjacent, artificial systems (a fish farm and saltmines) as alternate anthropogenic feeding areas. The estimated flooded areas (ranging from extreme drought [ca. 151 ha, 1995] to high moisture [15,049 ha, 2004]) were positively related to reproductive success of gulls, suggesting that habitat availability played a role in determining their reproductive performance. Based on blood δ13C and δ15N values of fledglings, 2001-2004, and a Bayesian isotopic mixing model, we conclude that saltmines acted as the main alternative foraging habitat for gulls, with relative contributions increasing as the extent of marshland decreased. Although adjacent, anthropogenic systems have been established as the preferred breeding sites for this gull population, dietary switches towards exploitation of alternative (anthropogenic) food resources negatively affected the reproductive output of this species (Figure 3), thus challenging the perception that these man-made systems are necessarily a reliable buffer against loss of natural feeding habitats. Analyses of this study were conducted at the LIE-EBD.
The demography of the same species, the Slender Billed gull, is the objective of another study (Living on the edge: Demography of the in the Western Mediterranean) aimed at investigating the role of peripheral populations, being typically vulnerable to local extinction processes but important for the metapopulation dynamics of species. Using long-term data on annual breeding success and capture-resights of marked individuals (14 years), we estimated and compared the vital rates and evaluated the connectivity of two Spanish local populations (Ebro Delta and Doñana) varying in their local environmental conditions. At a metapopulation scale, we analyzed 22 years of data on breeding numbers to predict their future prospects by means of population demographic models. Local survival and breeding success of gulls from the Ebro Delta was lower than those from Doñana, which is likely the result of higher permanent emigration and/or winter mortality in the former. Gulls from the Ebro Delta wintered mostly in Mediterranean areas whereas those from Doñana did so in Atlantic coasts, where food availability is higher. Whereas adult local survival was constant, juvenile local survival showed temporal parallel variations between colonies, probably related to natal dispersal to other breeding colonies. Our results suggested that dispersal was higher at the Ebro Delta where gulls preferentially emigrate to close patches with higher breeding expectances. We found large fluctuations in breeding numbers among local populations probably related to an evolutionary load typical of a species evolved in unstable habitats and the ability to disperse between patches depending on environmental stochastic conditions during breeding.
Another study being develop in this area is entitled “Spatial movements of two sympatric seabirds: influence of the breeding status, environment and fishing activity”. As central place foragers, pelagic seabirds are constrained by spatiotemporal heterogeneity to find productive marine areas and compete for prey. We analyzed 97 foraging trips to study the movement and oceanographic characteristics of foraging habitats of two different - yet closely related - species of shearwaters (Scopoli's shearwater Calonectris diomedea and Cory's shearwater C. borealis) breeding in sympatry in the Mediterranean. We combined various methodological approaches (GPS tracking, species distribution modelling and stable isotope analysis) to explore the foraging strategies of these two species. Isotopic results suggested that trophic habits of both shearwater species were similar, mainly based on pelagic fish consumption. Foraging areas of both species were characterized by shallow waters near the colony. Both shearwater species exploited persistent productive marine areas. The foraging areas of the two species broadly overlapped during the incubation period, but during chick-rearing period Scopoli's shearwaters apparently foraged in different areas than Cory's shearwaters.
Reconstructing the diet of top marine predators is of great significance in several key areas of applied ecology, requiring accurate estimation of their true diet. However, from conventional stomach content analysis to recent stable isotope and DNA analyses, no one method is bias or error free. Here, we evaluated the accuracy of recent methods to estimate the actual proportion of a controlled diet fed to a top-predator seabird, the Eudyptula minor (Little penguin). Within the research entitled “Searching for the true diet of marine predators: Incorporating Bayesian priors into stable isotope mixing models”, we combined published DNA data of penguins scats with blood plasma δ15N and δ13C values to reconstruct the diet of individuals fed experimentally. Mismatch between controlled (true) ingested diet and dietary estimates obtained through the separately use of stable isotope and DNA data suggested some degree of differences in prey assimilation (stable isotope) and digestion rates (DNA analysis). In contrast, combined posterior isotope mixing model with DNA Bayesian priors provided the closest match to the true diet. We provided the first evidence suggesting that the combined use of these complementary techniques may provide better estimates of the actual diet of top marine predators- a powerful tool in applied ecology in the search for the true consumed diet.
Deepening in the studies on the effects of climate change on the trophic ecology and population dynamics, the Little Penguin is also the subject of a study currently under development. This project aims to evaluate the effect of inter- and intra-annual variations in both local (meso-scale) and global (macro-scale) climate-change related oceanographic variables on the diet and reproductive output of Little penguins at Phillip Island (Victoria, Australia). This objective will be accomplished by combining remote sensing and GIS with isotopic approaches. In particular, we will use an already existing long-term (2003-2011) isotopic dataset (that will be enhanced with additional analyses at the LIE-EBD), which include the stable isotope composition of penguin blood from Penguin Parade colony and their main preys, as well as dietary estimates obtained through conventional methods. So far, we have analyzed the spatiotemporal component environmental variables likely driving prey abundance and distribution and, therefore, the diet and foraging distribution of Little penguins. Among different oceanographic features commonly used for identifying highly productive hotspots, chlorophyll-a concentrations (hereafter CHL, mg/m3), sea surface temperature (SST, ºC) and bathymetry (BAT, m) are widely considered as the most biologically relevant. In particular, CHL can be considered a reliable surrogate of marine productivity and, consequently, of prey abundance, whereas SST and BAT may provide relevant information on physical processes or oceanographic features driving prey distribution.
The study “Older female Little penguins adjust nutrient allocations to both eggs and chicks” investigated an important but poorly quantified aspect of avian life-history theory such as the differential allocation of nutrients by females to their eggs and subsequent effort expended in raising chicks. Here, we study the nutrient allocation to eggs of Little penguins using body-mass recording and δ13C and δ15N values for incubating females and their chicks. Overall, endogenous contribution to eggs was relatively low (~20%). However, older females accumulated larger amounts of reserves before laying (up to 40% body mass increases) and relied more on endogenous resources (up to 45%) for clutch production. Females were able to adjust their endogenous investment between their two chicks, with somatic reserve inputs to heavier siblings showing a bimodal distribution (mean±SD; 16.7±5.9% and 36.5±4.4%) that contrasted with the unimodal distribution observed for lighter siblings (17.8±8.7%). In addition to differential nutrient investment in eggs, females apparently adjust provisioning effort to favour siblings with greater chances to fledge.
Understanding the resource allocation to egg formation is also the aim of the study “The role of Calcium in constraining egg synthesis in the Audouin's Gull Ichthyaetus audouinii”, a crucial step for our fully comprehension of avian reproductive strategies. However, little is known about how egg synthesis in wild birds might be constrained by the availability of specific micronutrients. Through this project we aimed to investigate the role of Calcium (Ca) in constraining egg synthesis in the Audouin's Gull. In particular, we evaluated the relationship between plasma Ca levels in incubating females (considered as a reliable surrogate of Ca available for females during the clutch production period) and several fitness-related traits regarding egg size (i.e. egg volume and surface area), egg shape (i.e. egg shape index) and eggshell thickness from three-egg clutches. Preliminary results indicate that, remarkably, egg volume and surface area were positively related with female plasma Ca levels, being this relationship significantly higher for latter-laid eggs. Although we cannot prove for the causality of such relationship, these results pointed to the constraining role of Ca for egg synthesis and suggested that Ca may also have a role in modulating the intra-clutch pattern of egg size variation typical of this gull species.
The ISOSCAPE project (ISOSCAPE of the wetland at the Natural Area of Doñana: Long-term monitoring of water dynamic) is framed within the EcoGenes Project. In the context of the global change on the structure and functioning of ecosystems, mainly those linked to Mediterranean areas, isotopic approaches have revealed as useful tools to identify potential environmental changes associated to natural and/or anthropogenic processes. As a consequence, there is a growing interest in implementing such isotopic approaches within programs of ecological monitoring. The utility of stable isotopes is based in the fact that any environmental change (natural or induced by human activity) is reflected in changes in the isotopic composition of the different compartments of the ecosystems. Therefore, the characterization of spatio-temporal patterns of isotopic variability in the different components of the ecosystems provides very valuable information when studying perturbations and their effects. The aim of the ISOSCAPES project is to generate a long term monitoring protocol, based on the use of stable isotopes, and focused on the study of water dynamic and quality at the wetlands of the Natural Area of Doñana (Espacio Natural de Doñana, END). To address this goal, we will evaluate temporal and spatial variability in δ2H and δ18O values, along with additional chemical (NO3, NH4, etc) and physical parameters (conductivity, etc) of the water from this Natural Area. In addition, we will measure δ15N and δ13C in blood of cattle that is spatially segregated in this area along the year. We are currently obtaining and analyzing preliminary results. So far, we have typified the isotopic composition of δ2H, δ18O, δ15N and δ13C from first samples of water and cattle blood from the END. Figure 1 shows preliminary ISOSCAPES, based on δ2H and δ18O values for water samples from the END and showing the influence of isotopically depleted water from the aquifer at the North-west of the marshland.
The suitability of sentinel species to monitor environmental pollution is often hampered by an insufficient knowledge on pollutant trophodynamics. In the project “Investigating the trophodynamic of inorganic pollutants in wide-range feeder, the Yellow-legged Gull (Larus michahellis)” we used an isotope approach to evaluated simultaneously the influence of individuals' trophic position and dietary exploitation of particular systems on inorganic pollutant body-burden (mercury Hg, Selenium of a wide-range feeder, the Yellow-legged Gull -Larus michahellis). Concentrations of total Hg and Se in fledgling feathers were under the threshold points for deleterious effects on seabirds. On the contrary, alarming Pb concentrations were found in one colony. With the exception of Pb, pollutant concentrations were positively influenced by consumption of marine resources, whereas trophic position played a minor role in determining pollutant body burdens. A paper was published.
The study “Stable isotope analyses to unravel an ecological gradient depending on altitude” is being developed in collaboration with the University of Barcelona and Girona (Spain). The Atlas day gecko Quedenfeldtia trachyblepharus is a diurnal and social gecko endemic from High Atlas Mountains in Morocco, and the dominant species of the alpine assemblage. It appears from 1,200 m altitude, being more abundant from 2,500 m, and becoming gradually became scarce at lower altitudes, likely as a result of increasing species richness of lizards at lower altitudes that might compete for the same trophic resources. Here, we used an isotopic approach, based on nitrogen and carbon isotope determinations, to investigate how variations in the degree of inter-specific competence affect the trophic niche of Atlas day gecko. The isotopic composition of the Atlas day gecko significantly differed throughout the altitudinal gradient with depleted isotopic values for those individuals inhabiting at lower altitudes. Moreover, isotopic variability increased with altitude likely reflecting wider trophic niches as a result from a lower inter-specific competence for food resources. A scientific publication is expected.
The study “A new spatiotemporal scale to integrate oceanographic variables into marine animals” is currently in preparation. Its goal is to test a novel way to identify water masses flowing to the seabirds foraging grounds using connectivity analysis based on circuit theory which we applied to marine currents.
Within the study “Ecological consequences of central-place foraging behavior: a ten-years case study on Little penguins, we try to provide a suitable methods informing on individuals' feeding strategies throughout the breeding cycle, in order to understand the ecological response of seabirds to the various constraints and requirements imposed by a central-place foraging behavior.

Scientific Publication
Eleven peer reviewed papers have been published by EcoGenes recruited researchers in the ecophysiology area, and at least 7 articles have been submitted.

Exploitable foregrounds
For the genus Salmonella we have designed specific primers for several species which affect most avian species. Amplifications have been very satisfactory, and sequencing has also confirmed our success with the designed primers. Currently we are working on deleting some non specific peaks in the melting curve that may affect our analysis which we will get adjusting our coupling temperature and / or adding BSA.
For Chlamydia situation the technique is ready to be used having a high level of quantification and a quite good efficiency. We are waiting the first samples from analyses that come from wild parakeets’ populations from Barcelona.
The ISO 9001:2008 and ISO 1400 certificates provide a guarantee of the standard of quality of the analyses done at the laboratory. This is relevant both for researchers at the institute but also for other institutes or enterprises requesting the services offered by the laboratory.
The Level II on Biological Security allows working on a wide range of pathogens under the necessary measures of biological control.
Measurement of relative telomere lengths. Telomeres protect the extreme of the chromosomes during replication. The length of the telomeres is affected by different processes related with organisms well being. This is an intense area of research in oncology but little information is available on wildlife. This service is being requested by a large number of local researchers and from researchers of other institutes. A biomedical enterprise has also showed interest on the results of the laboratory and possible ways of collaboration.
Measurement of antimicrobial capacity of serum. This technique aimed at measuring the capacity of serum to control different potential pathogens. It is applicable to evolutionary ecology studies and researchers at the institute and in Spanish and USA universities are using this service.
Detection and quantification of Salmonella sp. in birds using RT-PCR. Salmonella is an important pathogen transmitted with vertebrate faeces. This technique has already been applied in the laboratory to reptile and avian samples. We anticipate the interest of different researchers groups to use this service.
Detection and quantification of Chlamydia sp. in birds using RT-PCR. This is an important zoonotic pathogen that may have implications in human health. In addition to local researchers, health services from a large Spanish city plan to use this service during the next year.
HSP 70 technique. Heat shock proteins are affected by environmental stress. Researchers working with thermal stress are currently using this service.
Measurement of isotope stable of d15N, d13C, d18O, d34S. The mass spectrometer and the elemental analyser services allow the EBD-CSIC to deal with a variety of studies involving migration, paleoclimate, trophic ecology, and the distribution of marine species population to assess conservation measures
Measurement of stable isotope of d2H, d18O in water and vapour water by laser spectrometry. Laser spectrometry extended the capabilities of the mass spectrometer, allowing simultaneous analysis of solids and liquids in the LIE-EBD. Besides, parameters that can be analyzed with this equipment are particularly relevant when applied to the Mediterranean location of the EBD-CSIC, where monitoring the quality and dynamics of water has direct implications on habitat conservation.
Quality and nutrient composition in water: nitrate, nitrite, phosphate, ammonium analysis. Water is the main limiting factor in the Mediterranean ecosystem; Determining nutrient composition and quality over the annual cycle is of paramount importance to understand numerous ecological processes. The results of the analysis are relevant for the EU Water Framework Directive as they have permitted a huge step in understanding of the water quality in Doñana, and the problems of eutrophication caused by nutrient input from surrounding agricultural and urban areas.

The strict collaboration with the University of Groningen is highlighted by the frequent use of the secondments activities made by the staff members at the Department of Animal Ecology of this centre. In particular, Prof. Theunis Piersma visited in October 2011 the EBD-CSIC, where he had the opportunity to discuss with EBD-CSIC people of the Wetland Ecology Department and Rocío Marquez the importance of Doñana for the wintering of the Black-tailed Godwits. In order to assess the potential impacts of the recent change in the wintering distribution range of this species on the breeding performance and survival of the individuals, Prof. Piersma discussed the field work approach and the sampling protocols to adopt in the future, along with the analysis for papers derived from EcoGenes project. Collaboration with the University of Groningen will continue through the 7FP ExpeER project. In this UE funded project (Habitat use by a threatened long-distance migrant the Black-tailed Godwit Limosa l. limosa at Doñana Wetland during the non-breeding period) our collaborators from the Groningen University are going to visit Doñana wetlands to collect more data of the marked Black-tailed Godwits present in this area. The project started in September 2013 until the end of March 2014.
The Fedération Nationale des Chasseurs from France has expressed its interest in funding a follow-up of the Black-tailed Godwit project in order to answer important questions rising from the evidence that the continental population of Black-tailed Godwit that breeds in The Netherlands had increased the number of wintering birds in Spain.
The EBD-CSIC participates in the EDENext, Biology and Control of Vector-Borne Infections in Europe, a research project bringing together 46 international partners dedicated to investigating the biological, ecological and epidemiological components of vector-borne disease introduction, emergence and spread, and the creation of new tools to control them.
Collaboration with Andre Chiaradia at the Research Department at the Phillip Island Nature Park, Australia, has been strengthened through the Marie Curie action fellowship granted to EBD-CSIC (Phillip Island as host institution) under the FP7-PEOPLE-2012-IOF ECOLIGHTSFORSEABIRDS project. EcoGenes hired researchers Francisco Ramirez has also submitted a proposal to the European Marie Curie Actions, (IOF; Call: FP7-PEOPLE-2013-IOF): Global-Warming and Marine Ecosystems: seabirds as integrators of Environmental Information (Phillip Island as host institution).
The new collaboration with Gabriel Bowen (University of Utah) is meant to improve the implementation of stable isotope analysis techniques, and further investigate important but poorly quantified aspect of avian life-history theory related with global change issues.
The setting up of the LIE-EBD has fostered contacts with Environment Canada through Leonard Wassenaar and Keith Hobson. In particular, a project in collaboration with Hobson on the study of migration and wintering areas of European Birds by applying stable isotopes is ready for submission to international or national calls for funding: “Preliminary Study on European – African connectivity in trans-Saharan avian migrants using stable isotope (δ2H, δ13C, δ15N) analysis of feathers”. Several papers are currently under submission.
Several institutes from the Mediterranean area have been involved in the numerous isotope studies carried out within EcoGenes: Institut Mediterrani d’Estudis Avançats, IMEDEA (CSIC-UIB), Islas Baleares, Spain; Institut des Sciences de l’Evolution, Université de Montpellier, France; Unitat d’ecosistemes aquatics, Institut de Reserca i Tecnologie Agroalimentaries, IRTA, Tarragona, Spain. This have led to the creation of the Group of Applied Marine Ecology of the Biological Station of Doñana (EBD-GEMA) promoted by hired researchers.
Studies on the genus Falco were carried out in collaboration with the University of Lausanne (Prof. Alexandre Roulin); University of British Columbia (Dr. Miguel Alcaide); U. S. Geological Survey, Alaska Science Center (Prof. Sandra Talbot); Computational Geo-Ecology Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam (Prof. Willem Bouten); College of Science King Saud University, Riyadh, Saudi Arabia (Prof. Boudjema Samraoui).
The collaboration with the Eawag, Switzerland, was meant to improve the use of novel techniques to study key taxa such as the amphibians in understanding the effects of global change on the Mediterranean region. Katja Raesaenen visited EBD-CSIC in November 2012 to help in the laboratory of the Wetland Ecology group at EBD-CSIC, as part of her study on the evolution of tolerance to water acidity in Mediterranean amphibian species through the proteomic analysis of embryonic capsules. She has trained EBD-CSIC staff in a procedure to extract and preserve the jelly and fertilization envelopes of amphibian clutches, as a starting point for a joint collaboration. Exposure to acid water constitutes a high risk of mortality for amphibian embryos, a risk that is often increasing in different regions as a consequence of human activities.
The Black-tailed Godwit has generated two master projects related with Black-tailed Godwit and waders in Doñana Wetlands: “A needle in a haystack. Is the behaviour of the Dutch breeding Black-tailed Godwit Limosa l. limosa wintering in Southern Iberia based on condition?” Master Forest and Nature Conservation at the Wageningen University and Research (WUR), the Netherlands. 2013-2014. “Old questions, new approaches: effects of salinity on the prevalence of avian malaria parasites in waders”. Master in Biodiversity and Conservation Biology. Pablo de Olavide University–EBD-CSIC, Spain. 2012-2013.
Laura Gangoso, involved in the Eleonora’s falcon studies, co-supervised the master project “Patrones de transmisión de protozoos sanguíneos por hipobóscidos en el Halcón de Eleonor: ¿la historia de un salto de hospedador fallido?” (Master in Biodiversity and Conservation Biology. Pablo de Olavide University, EBD-CSIC).

The seminar of Leonard Wassenaar “Applications of isoscapes to global wildlife migration studies: a decade of progress and the road ahead” (October 2011) was framed within the creation of the LIE-EBD, whose inauguration was celebrated in October 2011 with the special seminar “Stable Isotope Lab” that brought together both guests researchers and EBD-CSIC staff in an initiative to unveil the potential of LIE-EBD. Keith Hobson gave another seminar related with LIE-EBD: “Recent advances in the use of stable isotopes to track animal movements”. Andre Chiaradia (Phillip Island) during his stay at EBD-CSIC in 2013 was in charge of explaining some effects of the increase of ocean temperature in marine species distribution. His seminar “News from the Antipodes: the smallest penguin in hot waters” took place on April 2013.
Results from a study with direct implications in stable isotope analysis were presented by Renaud de Stephanis (EBD-CSIC) in the seminar “Long term monitoring of cetaceans in the Strait of Gibraltar: from research to conservation” (March 2011). The technician in charge of the LEF-EBD was commissioned to disseminate the new services that the upgrading of the existing LEF-EBD would provide to EBD-CSIC. The seminar “Ecophysiology Lab” took place in April 2011.
The contact person at the University of Groningen, Theunis Piersma, gave the seminar “Habitat selection in shorebirds”, whose video is available at the EcoGenes web site. The results of Jonathan Jeschke (Technische Universität München) studies involving invasive species were presented at the seminar “What we know and don’t know about invasive species and other novel organisms” (April 2013), an issue with many implications for global change studies. On the same subjects fits the seminar of Alena Sonjia Gsel (Leibniz Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany) carried out in November 2013.

The conference “Global Change in the Mediterranean. Learning from Experiences Worldwide” took place in Seville, 15-16 November 2012. The aim of this meeting was to provide a more general view to common problems in the Mediterranean, gathering some leading experts in different fields that could collectively provide a cutting-edge, multifaceted view of the manifold ecological and evolutionary implications of global change. Investigating phenotypic responses of individuals using a multidisciplinary approach at scales above and below the organism provided a suitable framework for predicting the consequences of Global Change on wildlife and understanding feedbacks with the biosphere. Fastening inter-institutional relationships by exchanging experiences and sharing standards and data to deal with the threats posed by the impact of Global Change was the main objective of the symposium. This meeting addressed the following four major research topics: plasticity and epigenetics; physiological response; use of stable isotopes; ecological modelling for tracing the effects of global change. The symposium was attended by 21 invited speakers, and we limited to 120 the number of attendees. The main conclusions of the symposium indicate that organisms have mechanisms to interact with changing environments. There is great plasticity within the organisms themselves which is not necessarily reflected in immediate changes in DNA sequence. The response to changes in the environment may come through genetic modification, but also through changes in the regulation of genes and the development of organisms. Organizers suggest that one of the main contributions of this meeting is that there are new research methods to document not only the current climate change processes, but also to determine the speed and direction of the changes that occurred in the past, particularly in the transition from cold to warm periods, dry to wet, and the response of organisms and populations. One of the most useful tools is stable isotopes. The conference was chaired by Jordi Figuerola, Research Scientist at the department of Wetland Ecology (EBD-CSIC). Five EBD-CSIC researchers joined the committee, which was completed by three EcoGenes hired researchers in charge of moderating three sessions. Giulia Crema took care of logistics.

Ecological Modelling

S & T results
The area of Ecological Modelling has made impressive progress thanks to the collaboration with the hired researchers, who have focused in improving the analysis of prediction of extinction according to the direction of change, in developing and applying the concept of structural stability to complex ecological networks, and in studying patterns of association during recruitment on community level.
One of the analyses conducted was aimed at studying how a systematic change in pair-wise interaction strength (increase or decrease) induces species extinction and more specifically if we can predict the most vulnerable species. We have found that predicting the most vulnerable species is very sensitive to the direction of change. This indicates that we first have to identify the direction of the change in interaction strength. i.e. increase or decrease, before being able to predict the most endanger species. This result has been published in Nature Communication.
Within the second research line, we have developed and applied the concept of structural stability to complex ecological networks. Structural stability is the study of the range of constraints on an ecosystem such that all species can coexist in a stable way. More specifically, we have suited how the specific architecture of ecological network are relaxing this set of constraints, in other words, how network architecture modulates the amplitude of perturbation that a system can tolerate before losing one of its constituting species. We have demonstrated that the empirical nested architecture is the one maximizing the range of conditions compatible with the stable coexistence of all species. This result indicates that the architecture of the observed networks is already optimized for facing higher environmental variability under global change.
In a third study, we have provided an historical overview of the twenty-two years over which spatial point pattern analysis have been used to infer ecological processes in several fields. Specifically, we aim to (i) assess which of the practical difficulties in the use of these techniques have been already overcome and (ii) which are still common, and (iii) detect these new methods that are still under-used but have a great potential to tackle certain issues. Firstly, we made a brief presentation of some basic concepts that may be unknown to those readers initially approaching to spatial point pattern analysis, and describe the methods and the main results of our systematic review. Secondly, we discussed on the major achievements and the unresolved pitfalls in the use of SPPA in ecology, and on the newest and most promising techniques. Finally, we provided a new list of recommendations to guide through the use of SPPA in ecological research. During the last two decades, spatial point pattern analysis has been increasingly used in ecology but it is unknown at which extent some of the critical points associated with the use of these techniques have been solved and many recently developed methods are still unknown for many authors. We conducted a systematic and integrative review of 259 articles published over the period 1992-2012 in order to assess which of the practical difficulties in the use of spatial point pattern analysis in ecology have been overcome and which are still common, and to detect new methods still under-used but with a great potential for ecological research. Our results clearly indicate that over the study period ecologists have used increasingly complex null models and a larger variety of summary statistics. They have also successfully modified the standard techniques according to their needs and problems associated with edge effects and data comparisons have been definitively overcome. We have also detected some potentially useful methods such as the methods to deal with isotropic and multivariate point patterns, the summary statistics adapted to objects of finite size and irregular shape, the use of remote sensing and aerial photographs, the extraction of point patterns from other types of datasets, and new null models such as independent labeling and pattern reconstruction. However, pattern-process inference and the analysis of heterogeneous point patterns are still a critical point in the use of spatial point pattern analysis in ecology. Confusing terminologies can be extremely discouraging for researchers initially approaching to these methods and there is still a lack of communication between the statisticians developing the methods and the ecologists using them. We offer a list of recommendations to assist in the correct use of spatial point pattern analysis in ecology.
In a fourth study, we have focused on community level patterns of association during recruitment. We have conducted a detailed analysis on the spatiotemporal patterns of seedling emergence and survival in a temperate forest in Northwestern Spain. We pursued the following specific research questions: (i) Is there any spatial relationship between seedlings and either conspecific or heterospecific adults? (ii) Do these relationships remain constant within and between seasons? (iii) Are spatial patterns of recruitment enough to explain the relationship between adults? We found that negative or null patterns of association dominated at intermediate to large scales in a temperate deciduous forest stand of NW Spain. Surprisingly, there was almost no attraction at small scales, except for some pairs of fleshy-fruited species. At the same time, the massive recruitment of F. sylvatica following a mast event was accompanied by positive associations at larger scales. Spatial changes in seedling abundance were demonstrated to depend not only on the distribution of conspecific adult trees, but to lay a spatial signature of the location of adults from other species. The temporal persistence of some of these patterns and changes associated to varying production highlight the need for a community approach to study tree recruitment.
Another analysis conducted in this area consist in combining experimental and observational studies, both in the field and in silico, using the dynamics of temperate and mountain forest ecosystems as a benchmark system, focusing on the spatial pattern of biotic interactions. In particular, two different subjects within this general area of interests are the centre of interest. A first study has dealt with the response of forest ecotones to climate change in mountain ecosystems, addressing the following questions: (i) Should we expect an upward migration of Pyrenean tree lines? (ii) Will tree line advance represent a real thread to Pyrenean alpine ecosystems? (iii) Are predictions based on empirical Species Distribution Models (SDM) dependent on spatial resolution and on dynamic processes like dispersal limitation?

Scientific Publication
Twelve peer reviewed papers have been published by EcoGenes researchers recruited in the ecological modelling area, and at least 10 articles have been submitted.

The partner organization in the area of Ecological Modelling, the Helmholtz Centre for Environmental Research-UFZ (Leipzig, Germany) offered his support to the EcoGenes recruited researcher Isabel Martinez during the whole duration of the project. She visited in several occasions this centre. At the same time, senior researchers at UFZ visited EBD-CSIC within secondments activity. Additionally, this centre organized and implanted a Training course in Ecological Modelling, in April 2012. The collaboration will continue Helmholtz Centre for Environmental Research-UFZ. Cooperation on Cantabrian brown bear population studies under climate change.
Rudolf Rohr, the second hired researcher in this area, is actively collaborating with the University of Fribourg, Switzerland for matching-centrality decomposition and the forecasting of new links in networks.
Other important contacts are: Ohio State University (Understanding demographic consequences of anthropogenic disturbance using an ecological network approach); Department of Applied Mathematics and Statistics of the Technical University of Madrid (Nestedness in mutualistic networks); University of Lausanne, Switzerland. (Incorporating trophic networks in spatial distribution models).

Vasileios Dakos (EBD-CSIC) in the seminar “Probing the resilience of complex systems” in November 2012 summarized the principles of early-warning signals using simple ecological models. In June 2013 Priyanga Amarasekare (UCLA) gave the seminar “A trait-based perspective of ecological and evolutionary dynamics” on the integration of theory and data to make testable predictions about the effects of climate warming on population viability, biodiversity and the control of invasive species.

Training lectures
The Ecological Modelling course took place in Seville 10-20 April 2012. This intensive training event provided an introduction to the modelling of ecological systems, a valuable tool for scientific practice and decision-making in conservation and environmental management. The course enables EBD-CSIC to increase the critical mass of researchers generating theory, improving their ability to create future scenarios in the frame of global change. The collaborative partner UFZ played a major role, providing their long experience in organizing this activity. EBD-CSIC staff and EcoGenes hired researchers I. Martinez and R. Rohr also contributed to this course that emphasized the importance of modelling in understanding ecological systems. Twenty people, mainly EBD-CSIC staff, attended the course.

The extent of two staple activities included in EcoGenes exceeds the limits set by the development of this report in the three focal areas. With this assumption, we would highlight those events that integrate the different research lines to provide those long-range results that endorse EBD-CSIC as a centre of reference on biodiversity loss studies in the light of global change.
The lecture on global change “Ecological consequences of climate changes: integrating research approaches” is one of these horizontal activities. This course took place at the Doñana Biological Reserve (RBD) (Huelva province, Spain). This was the fourth edition of a course that is being increasingly coveted among international postgraduate students and that has been converted in an annual event, focusing scientists’ attention on the dramatic changes that will affect Mediterranean areas. Juan José Negro (Director of EBD-CSIC and EcoGenes coordinator) and Arndt Hampe (INRA) organized and coordinated the course. The Ecological Consequences of Climate Changes course is meant to provide future generations with scientific tools to face questions related with climate (and other) changes, elucidate these responses across diverse spatio-temporal scales, and improve projections of future processes. Throughout the course, the global change issue, and in particular the climate change, has been studied under several points of view, reflecting the different research lines and expertise of teachers, from the impact on phenological aspects, to the complex interactions among biological invasions and climate change, through past climate change studies or species distribution consequences under climate change. Eight lecturers coming from international research centres gave lecturers and supervised practical works on global and climate change issues. Thirteen students met the requirements set by the organizers. They came mainly from European countries, some of them located in the Mediterranean area (France, Portugal, Italy, Greece and Croatia) which shows the interest on the global change topic in this region.
The second horizontal activity we have to mention here is the third and final conference. The congress “Adapting to Global Change in the Mediterranean” (Seville, 18-20 September 2013) successfully recruited 37 excellent speakers for all nine of the sessions held across three days, and also for the three plenary talks. There was a high level of participation in the poster session and participation in the congress was constantly near maximum capacity. Interactions between researchers, especially from different fields, were intense especially in response to the day 0 workshops. This situation generated novel points-of-view. These new perspectives are in the process of being written up for publication in the special issue of the scientific journal Evolutionary Ecology Research, associated with this congress. Many participants commented to the organizing committee that they were pleasantly surprised at the overall exceptional level of the talks. All of the plenary talks, and several of the sessions were recorded and made available on the internet. A request was received for some of them for use in a university level course. Thus, the congress was a great success in terms of scientific interactions at the time, but will also have more lasting results in the form of: i) talks that can be viewed in the future and incorporated into university level courses, ii) a special issue of a scientific journal including multi-disciplinary discourse and novel approaches to long-standing issues, and iii) new international collaborations. The conference was chaired by Jennifer Leonard, Associate Professor of Research at the Department of Evolutionary Ecology (EBD-CSIC). The Scientific Committee was composed by other 21 EBD-CSIC researchers, of which 6 EcoGenes recruited researchers. Giulia Crema was in charge of logistics.

Potential Impact:
EcoGenes had strong impacts at three main levels:
1) Impacts on our institute
2) Impacts on our scientific network
3) Impacts in the Mediterranean region
It is evident that the project had improved the scientific capabilities of the institute to focus on the global change effects in the Mediterranean. Upgrading the existing laboratories with the latest equipment and creating a new laboratory has enabled EBD-CSIC to attend the increasing demand for new analytical and innovative approaches, and had satisfied the need to open up new working areas related with global change. Reinforcing the activities in molecular ecology and ecophysiology had immediate benefits such as budgetary savings and reduction in wait time in typically saturated service laboratories. Day to day work at the centre had improved, evidence of this being the increase in number of users of lab services users. Recruited researchers acted as scientific advisors of lab staff personnel, and gave the widest possible dissemination to the laboratories services to all researchers interested in incorporating those approaches in their research. The incorporation of recruited researchers had also improved our capacity in ecological modeling, data processing and simulation experiments, developing new theoretical scenarios on tolerance of environmental variability facing biodiversity loss. Strengthening our capabilities for analysis and interpretation at all range of levels nature resources are functioning, reverted also in a substantial increase in scientific production in the form of papers published in high-impact multidisciplinary and ecological journals.
Thanks to the project, EBD-CSIC scientific network increased in quality through collaboration. EcoGenes helped strengthening and expanding the collaboration between our research groups and research centres located in other EU member states. Increasing the visibility of our centre through international conferences, workshops, secondments and training activities, EcoGenes enhanced EBD-CSIC international exposure and recognition. This prompted our participation in the European Research Area, and nowadays most of the research projects developed within EcoGenes is continuing through funding lines coming from the European Commission. Increasing our implication in key international projects entails a significant training not only of research staff, but also of technical and management teams, making EBD-CSIC a more competitive research centre. The role of partnering organizations had been paramount to help increasing our scientific network, either strengthening existing relationships, or expanding contacts through new research lines. Numerous working groups composed of researchers from international and national centers have been created as a result of the new research lines introduced by the recruited researchers, have been encouraged by the improvement of laboratories’ capacities.
The Mediterranean basin is experiencing some of the most critical experiences in terms of economy, society and politics. The situation from the environmental point of view is also the most critical, since theoretical scenarios predict greater warming, greater variability in rainfall and temperature and a higher frequency of extreme events such as droughts and floods. Great efforts are being devoted into measuring the extent of climate change and predicting its effects on human activities. However there is not a recognized leading centre for biodiversity research. EcoGenes has helped meet this need. Having assured high standard for research in one of the most critical theme for the European Union such as Environment, legitimize EBD-CSIC to lead studies on biodiversity loss in this area. All the activities carried out within EcoGenes, involved researchers working in Mediterranean research centres or dealing with global change issue affecting this region. These activities had both improved ongoing relationships and extended our contacts with other centres located in the area.
To summarize all the positive effects that EcoGenes has exerted over EBD-CSIC, it is worth mentioning the obtainment of the Severo Ochoa Excellence Program Award (Spanish Ministry). This three year-program award was granted in 2012 by 3 CSIC centres only. The scientific committee responsible for selecting emphasized that EBD-CSIC is developing a multidisciplinary research at the highest level, aimed at understanding, from an evolutionary point of view, how biodiversity generates, maintains and deteriorates, the consequences of its loss, and the chances of its restoration.
Dissemination is one of the pillars of EcoGenes. The EBD-CSIC is aware of the importance of transferring knowledge to society, particularly when global change menaces indicate that there should be limits to the current pattern of development. It is therefore imperative to provide society with accurate and concise information on global change evidence as derived from the results of research carried out in our centre. In this way we both reach our goals to serve the Society of Knowledge and to boost EBD-CSIC as a reference institution in biodiversity studies on global change in the Mediterranean hotspot.
The dissemination actions carried out within EcoGenes addressed the following target audiences: a) the general public, in Spain and target countries (exhibition, Science Week, blog, press releases, book, virtual visits to labs, radio and press coverage); b) policy makers. Regional and national public administrative involved in EcoGenes activities (exhibition, assessment committee, press releases); c) Scientific community. The results of research achieved in the project contribute to identify EBD-CSIC as the reference research centre in Ecology and Environment in the Mediterranean region (conferences, workshops, lectures, training courses, seminars, secondments, working groups).
Below, more details are provided on some of the most important events in terms of disseminating the outcomes of studies. The exhibition "What Next? Environmental Challenges in a Changing Planet" is certainly one of these. This photographic production that rounded off EcoGenes in September 2013 was on display in the centrally-located street of Seville Avenida de la Constitución. Through 36 panels showing impactful images and succinct text aimed at a broad spectrum of public people received precise information on some of the effects of biodiversity loss due to global change. A QR code provides access to a web log, inviting to learn more about the subject, and supplying further bibliographic references. Local and national press and radio well echoed the exhibition. As a consequence, What next? will travel to a few Spanish cities during summer 2014, and we expect to be able to extend the route to more locations.
After that, the exhibition was again on display at EBD-CSIC during the Science Week (November 2013), one of the biggest media events of science and technology held in Europe. During the Science Week, the CSIC centers, universities and other research institutes, propose activities of all sorts to citizenship, according to the characteristics of each institute. The EBD-CSIC opened the doors of its laboratories and adapted the outdoor panels of the EcoGenes exhibition to make it available inside the EBD-CSIC facilities.
Within the objective of increasing the visibility of EBD-CSIC among civil society and research institutions, we provided virtual visits to the upgraded and newly created laboratories of our institute. This service allows getting a real perspective of each laboratory, with access to specific information on equipment and accompanied by a brief description of the lab, both in English and Spanish, made by each responsible. To date, virtual tours are available on the EcoGenes webpage for the LIE and LEM laboratories only. However, they will be shortly extended to the remaining laboratories, and in the next months virtual tours can reach the whole EBD-CSIC facilities.
The publication of the book “Aerial Censuses of Waterfowls in the Guadalquivir Marshes. Forty Year of History” is further evidence of the commitment of our institute with society regarding global change issues in the Mediterranean. Doñana is a Natural Park located in south-western Spain, and one of the most significant areas for biodiversity in the Mediterranean basin. The core area (Doñana Biological Reserve –RBD) is owned and managed by the CSIC. A book on global change evidences in Doñana wetland is going to be published on autumn 2014. These evidences are inferred by long time series analysis of variables that characterize global change, such as the pattern of distribution and relative abundance of waterbird populations. Data have been collected by aerial censuses over the last 40 years, and the information provided is useful both in terms of academic knowledge and in the decision making related to global change management policies. Its conclusions can be useful to other wetland areas in the Mediterranean region. The book is aimed at researchers, but also at high-school teachers, environment technicians, ornithologists, and decision-makers.
The results of the project have also been spread in scientific journals, congresses contributions and seminars. The International Innovation Journal helped to disseminate EcoGenes research not only to the core scientific community but also to a wider range of potential stakeholders throughout Europe. The EBD-CSIC web page reported the scientific production by the EcoGenes researchers and EBD-CSIC staff as well, providing the abstracts of the principal findings, always in English and most of the time in Spanish too, and a link to the paper as well. The same information appears simultaneously on the EcoGenes project web page. Most of the seminars concerning the three focal areas were also reported in the EcoGenes webpage.
To guarantee EBD-CSIC prominence as a reference centre in ecology and environment in the Mediterranean, the lecture on Global Change has been converted in an annual event, drawing scientists’ attention to the dramatic changes that will affect Mediterranean areas in a global change scenario. The recent participation of EBD-CSIC in the EUBON and ExpeER networks, confirm the willingness of collaborating in the sharing of evidence-based database.

List of Websites:
EcoGenes coordinator: Juan José Negro
Uppsala University. Contact Person: Matthew Webster.
University of Groningen. Contact Person: Theunis Piersma.
Helmholtz Centre for Environmental Research- UFZ. Contact Person: Volker Grimm.
Institut National de la Recherche Agronomique (INRA) Contact Person: Arndt Hampe.