Ivan Radosavljević

Development of new microsatellite markers for Salvia officinalis L. and its potential use in conservation-genetic studies of narrow endemic Salvia brachyodon Vandas.

Nine new microsatellite markers (SSR) were isolated from Salvia officinalis L. A total of 125 alleles, with 8 to 21 alleles per locus, were detected in a natural population from the east Adriatic coast. The observed heterozygosity, expected heterozygosity, and polymorphic information content ranged from 0.46 to 0.83, 0.73 to 0.93 and 0.70 to 0.92, respectively. New microsatellite markers, as well as previously published markers, were tested for cross-amplification in Salvia brachyodon Vandas, a narrow endemic species known to be present in only two localities on the Balkan Peninsula. Out of 30 microsatellite markers tested on the natural S. brachyodon population, 15 were successfully amplified. To obtain evidence of recent bottleneck events in the populations of both species, observed genetic diversity (HE) was compared to the expected genetic diversity at mutation-drift equilibrium (HEQ) and calculated from the observed number of alleles using a two-phased mutation model (TPM). Recent bottleneck events were detected only in the S. brachyodon population. This result suggests the need to reconsider the current threat category of this endemic species.

New microsatellite markers for Salvia officinalis (Lamiaceae) and cross-amplification in closely related species

PREMISE OF THE STUDY Microsatellite primers were identified to study the genetic diversity, population genetic structure, and relationships of common sage and closely related species. METHODS AND RESULTS Nine novel polymorphic trinucleotide microsatellite loci were identified for Salvia officinalis. A total of 127 alleles were observed. The observed and expected heterozygosity values ranged from 0.375 to 0.880 and from 0.624 to 0.931, respectively. Nine new and 11 previously published microsatellite primers were tested for cross-amplification. The species with the most successful cross-amplification was S. fruticosa. CONCLUSIONS The nine new microsatellite markers will be useful in genetic studies of wild and cultivated populations of common sage and are potentially useful in genetic studies of closely related species.

Genetic Diversity and Structure of Dalmatian Pyrethrum (Tanacetum cinerariifolium Trevir. /Sch./ Bip., Asteraceae) within the Balkan Refugium

Dalmatian pyrethrum (Tanacetum cinerariifolium Trevir. /Sch./ Bip.) is an outcrossing, perennial insecticidal plant, restricted to the eastern Adriatic coast (Mediterranean). Amplified fragment-length polymorphisms (AFLP) were used to investigate the genetic diversity and structure within and among 20 natural plant populations. The highest level of gene diversity, the number of private alleles and the frequency down-weighted marker values (DW) were found in northern Adriatic populations and gradually decreased towards the southern boundary of the species range. Genetic impoverishment of these southern populations is most likely the result of human-related activities. An analysis of molecular variance (AMOVA) indicated that most of the genetic diversity was attributed to differences among individuals within populations (85.78%), which are expected due to the outcrossing nature of the species. A Bayesian analysis of the population structure identified two dominant genetic clusters. A spatial analysis of the genetic diversity indicated that 5.6% of the genetic differentiation resulted from isolation by distance (IBD), while 12.3% of the genetic differentiation among populations followed the pattern of isolation by environmental distance (IBED). Knowledge of the genetic diversity patterns of the natural populations and the mechanism behind these patterns is required for the exploitation and possible conservation management of this endemic and economically important species.

Genetic Diversity and Demographic History of Wild and Cultivated/Naturalised Plant Populations: Evidence from Dalmatian Sage (Salvia officinalis L., Lamiaceae)

Dalmatian sage (Salvia officinalis L., Lamiaceae) is a well-known aromatic and medicinal Mediterranean plant that is native in coastal regions of the western Balkan and southern Apennine Peninsulas and is commonly cultivated worldwide. It is widely used in the food, pharmaceutical and cosmetic industries. Knowledge of its genetic diversity and spatiotemporal patterns is important for plant breeding programmes and conservation. We used eight microsatellite markers to investigate evolutionary history of indigenous populations as well as genetic diversity and structure within and among indigenous and cultivated/naturalised populations distributed across the Balkan Peninsula. The results showed a clear separation between the indigenous and cultivated/naturalised groups, with the cultivated material originating from one restricted geographical area. Most of the genetic diversity in both groups was attributable to differences among individuals within populations, although spatial genetic analysis of indigenous populations indicated the existence of isolation by distance. Geographical structuring of indigenous populations was found using clustering analysis, with three sub-clusters of indigenous populations. The highest level of gene diversity and the greatest number of private alleles were found in the central part of the eastern Adriatic coast, while decreases in gene diversity and number of private alleles were evident towards the northwestern Adriatic coast and southern and eastern regions of the Balkan Peninsula. The results of Ecological Niche Modelling during Last Glacial Maximum and Approximate Bayesian Computation suggested two plausible evolutionary trajectories: 1) the species survived in the glacial refugium in southern Adriatic coastal region with subsequent colonization events towards northern, eastern and southern Balkan Peninsula; 2) species survived in several refugia exhibiting concurrent divergence into three genetic groups. The insight into genetic diversity and structure also provide the baseline data for conservation of S. officinalis genetic resources valuable for future breeding programmes.

New microsatellite markers for Campanula pyramidalis (Campanulaceae) and cross-amplification in closely related species

Premise of the study: Microsatellite markers were identified and characterized to study the genetic diversity and structure, conservation status, taxonomy, and biogeography of subspecific taxa and populations of Campanula pyramidalis (Campanulaceae). Methods and Results: Eleven microsatellite markers were developed from genomic libraries enriched for di- and trinucleotide repeats. A total of 80 alleles were observed in the tested natural population. The number of alleles per locus, observed heterozygosity, and expected heterozygosity ranged from four to 13, 0.217 to 0.913, and 0.521 to 0.895, respectively. Conclusions: The new microsatellite markers will be useful for studying genetic diversity and structure as well as for better assessing the conservation status of subspecific taxa and populations of C. pyramidalis. Furthermore, a set of seven loci was successfully cross-amplified in C. secundiflora and C. versicolor and will be of great value for addressing unsolved taxonomic and biogeographic issues within the C. pyramidalis species complex.

Causes and consequences of contrasting genetic structure in sympatrically growing and closely related species

To understand the historical circumstances that shape populations of sympatric and closely related taxa, microsatellite markers were used, while populations of three Salvia species served as a study model. In the widespread S. officinalis no population genetic disturbances were detected, in the endemic S. brachyodon evidence for clonality and a genetic bottleneck were found while the results of the S. fruticosa population indicated high inbreeding levels and hybridization with S. officinalis. As many findings regarding demography of individual population or species can be reached only through their comparison with closely related taxa, this study demonstrates the importance of the multi-species approach.

Development and characterization of new polymorphic microsatellite markers for Degenia velebitica (Degen) Hayek (Brassicaceae)

Twelve novel polymorphic microsatellite markers were developed for Degenia velebitica. All 12 microsatellite markers showed single-locus amplification and reveal 88 alleles in 20 individuals from a natural population. The number of alleles per microsatellite locus varied from 3 to 19. The observed heterozygosity, the expected heterozygosity, and polymorphic information content ranged from 0.250 to 0.950, 0.234 to 0.938 and 0,220 to 0,908, respectively. The significant deviations from the Hardy–Weinberg expectations were detected in two microsatellite loci. These new microsatellite markers will be a useful tool for studying genetic diversity, genetic structure and phylogeography of D. velebitica, as well as for successful conservation and management of this endangered species.