The impact of primer choice on results of metabarcoding studies was experimentally evaluated by analyzing fungal communities associated with leaves of four plant species. Significant differences in target specificity of primers were highlighted by a percentage of plant reads ranging from almost nothing to 30 to 35% of the total detected sequences. Overall, primer sets targeting the internal transcribed spacer 1 (ITS1) region proved to be more specific than those targeting the ITS2 region. A comparable taxa coverage was revealed for all investigated primer sets. However, each primer set detected only around 50% of the overall detected taxa highlighting that a consistent part of the actual fungal diversity remains undetected in studies conducted using a single couple of primers. The coverage was increased to 70 to 80% by combining results from two different primer sets. Some fungal taxa were preferentially or exclusively detected by certain primer sets and this association between primers and taxa was generally recurrent on several plant hosts. Data highlighted that a perfect set of primers to investigate the whole fungal diversity does not exist and that whatever the choice, only a fraction of the actual microbial diversity will be investigated. However, provided information may be valuable to select the best primers according to the objective of the analysis.
Chamaerops humilis is decreasing in abundance in Mediterranean Europe, which has induced the European community to call for its protection in Special Areas of Conservation. However, information about its genetic and morphological variability, which is crucial to the development of any conservation strategies, is insufficient. The present study aimed to investigate the genetic and morphological variability of C. humilis in Sicily, which was selected as a model because of the high number of dense populations. The relationships between morphological traits and climatic variables were studied to highlight patterns of adaptation to the environment, along with the genetic similarity among the populations. Ten natural populations were sampled, analyzed using 28 specifically designed SSR primers, and evaluated based on 29 morphological traits. The populations were clustered similarly based on genetic and morphological traits. Heterozygosity was high and inbreeding coefficients were low. These results, along with higher intra- than inter-population differentiation, suggest that C. humilis populations in Sicily differentiated from a common ancestor and that inter-population variation arose from secondary evolution processes induced by ecological adaptation. The correlations between climatic variables and morphological traits suggest that the morphological adaptation to arid environments depends more on summer temperatures than on evapotranspiration or rainfall and that autumn and winter temperatures are determinants of the species establishment at new sites. Considering the response of C. humilis to seasonal temperatures, the present results indicate this species as a candidate for tracking climatic changes in Europe. Further studies are needed to highlight the adaptation of C. humilis to cold environments. Palaeo-climatological and -ecological studies could help clarify its strategies for the colonization of new sites
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