New techniques for the early identification of reduction in biodiversity were developed, and existing ones refined to meet the specific needs of the study sites and other similar sites in the Mediterranean Region. Key species were selected that were abundant and common at each study site. These species may be proposed as bioindicators, once demonstrated their links within the ecosystem, as estimated from the data base from the field studies, and the correlation of the polymorphism and/or behaviour with adaptability to changes.
The techniques involve measurement of variation of the key species (the littoral crustacean Talitrus saltator and the dune plant Cakile maritima were chosen for this purpose) at the level of the genome, by analysis of DNA, and iso-enzymes. Genetic heterozygosity and polymorphism were estimated at study sites and similar sites subjected to different human impacts, to gain indication of the adaptive potential of the populations under study. Electrophoretic analysis of iso-enzymes was applied. Preliminary analyses were carried out with RAPDs and with a PCR method where primers were designed from regions upstream of genes known to be relevant for the development and thereby for the fitness. Potentially hypervariable sequences were selected. The results from the different methods were merged to evaluate genetic structure and evolutionary trends of the populations analysed.
Easy to apply macro-molecular kits containing the best primers and non-radioactive techniques for their use, were developed and tuned to the specific needs, and their potential use for environmental impact evaluation directly tested at the study areas.