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AFLP (Amplified Fragment Length Polymorphism) and SSR (Single Sequence Repeats ; microsatellites) markers are widely used in plant genetic studies and it is fair to say that the fundamental insights into plant population dynamics and structure have been gained from the analysis of either AFLP or SSR data, or both. The AFLP technique requires no prior knowledge of the genetic make-up of a species and produces a large number of polymorphic fragments that are generally scored as dominant markers. SSRs, on the other hand, are species specific, codominant, multiallelic markers. In this study, we wanted to compare some basic population genetic parameters calculated on the basis of both marker systems.

Centaurea ragusina L. (Asteraceae) is a Dalmatian narrow endemic species that grows as typical chasmophyte mostly on inaccessible vertical sea cliffs. It’s represented by two subspecies: typical C. ragusina subsp. ragusina with pinnatifid basal leaves which is distributed from north to south Dalmatia (Dugi Island to Konavle), while C. ragusina subsp. lungensis has mainly entire basal leaves, present only on the islands of Dugi and Kornati archipelago. We used amplified fragment length polymorphisms (AFLPs) to access the genetic diversity, differentiation and structure of populations across its entire distribution range (on 210 individuals from 22 populations). The aims were to test taxonomic validity of existence of two subspecies, to investigate genetic structure, to search for variations in the cpDNA sequences using trnL-trnF and rpL32-trnL markers and to quantify the genetic diversity within and among populations of C. ragusina. The results obtained from analyses of molecular variance (AMOVA) showed that 36.09% of genetic variation can be attributed to differences among populations. Mantel test revealed positive correlation between genetic and geographical distance between pairs of populations. Our study implies that most previous taxonomic infra- specific concepts of C. ragusina were artificial and according to results obtained by BAPS and STRUCTURE there is no clear genetic evidence that support existence of subsp. lungensis as a separate taxon from typical subsp. ragusina. On total of 60 samples, 16 different chloroplast haplotypes were detected. Haplotypes network indicate very fragmented distribution of C. ragusina that can be consequence of evolutionary history and colonization of vertical rocky cliffs during the Pleistocene sea level changes. Recommendations for conservation purposes of C. ragusina based on the patterns of genetic diversity were proposed.

One of the main challenges in molecular ecology and population genetics is the detection of genes, or genomic regions, that have been targeted by natural selection. The aim of this study was to quantify the relative contribution of natural selection in shaping the genetic variation observed among Dalmatian sage (Salvia officinalis L.) populations. Genetic diversity of 25 Dalmatian sage populations from Croatia and Bosnia and Herzegovina, each consisting of 20 to 25 plants, was assessed using AFLP markers. Two alternative methods for the identification of FST outlier loci have been used: the frequentist methods implemented in Mcheza and the Bayesian method implemented in BayeScan. Moreover, the spatial analysis method as implemented in Samada was used to compute multiple univariate logistic regressions to test the probability of presence of an allelic variant for a polymorphic marker given the environmental conditions of the sampling locations. The climate data for the sampling locations were obtained from the WorldClim database. The ecological characteristics were described using 19 bioclimatic variables representing the annual trends, seasonal variations and extremes in temperature and precipitation. The comparison of the outlier marker loci detected by the three approaches could help to identify the factors that are responsible for the observed spatial structuring of genetic diversity in Dalmatian sage populations.

Dalmatian sage (Salvia officinalis L.) and Greek sage (Salvia fruticosa Mill.) grow sympatrically on the island of Vis. In Croatia, S. fruticosa is found only on the island of Vis where it forms small isolated population in the vicinity of Komiža, whereas S. officinalis is distributed throughout eastern Adriatic coastal region. Detailed morphological analysis revealed the existence of morphologically intermediate individuals that indicated natural hybridization between the two species. Until now, spontaneous hybrids of the species were not observed, although their hybrid derived from artificial crossing has been described as Salvia x auriculata Mill. In order to confirm natural hybridization between S. officinalis and S. fruticosa on genetic level, SSR and AFLP markers have been utilized. The obtained results strongly supported the ongoing hybridization events. Our findings will not only contribute to conservation efforts but will also provide a solid foundation for future plant breeding programs and agricultural exploitation.

Short-toothed sage (Salvia brachyodon Vandas) is Illyrian-Adriatic stenoendemic and one of the rarest plant species of the Dinaric karst. S. brachyodon grows in Croatia and Bosnia and Herzegovina/Montenegro and only three localities have been confirmed at the present time: (1) Mt. Zmijino brdo (Sv. Ilija) near Orebić on the peninsula of Pelješac, Croatia, (2) Mt. Konavoska brda near Velji do, north of Cavtat in Konavle region, Croatia, and (3) Mt. Orjen, near Vrbanje, at the border of Bosnia and Herzegovina and Montenegro. It has been categorized as near thretened (NT) species in Croatia and as endangered (EN) in Montenegro. The habitats of S. brachyodon greatly differ among localities and the distinctions at the morphological and molecular levels may represent the adaptation of the plant to the specific habitat conditions. Heritable epigenetic variation could influence the course of evolution in plants, as it can affect the processes of adaptation and divergence through selection of stable epigenetic variants without involvement of genetic variation. In order to understand the true importance of epigenetic processes in a stenoendemic plant of a very narrow range size existing epigenetic variation has been assessed and compared to genetic variation. The aim of this research was to determine genetic and epigenetic diversity and differentiation of the three known short- toothed sage populations by using molecular markers. Microsatellite markers (SSRs) and amplified fragment-length polymorphysims (AFLPs) were used to investigate the genetic diversity and structure within and among sampled natural plant populations while the methylation-sensitive amplified polymorphisms (MSAPs) were used to assess the epigenetic diversity.

Lavandin (Lavandula x intermedia Emeric ex Loisel.) is a spontaneous hybrid of L. angustifolia Mill. and L. latifolia Medik. widely cultivated in Mediterranean areas for the production of essential oil. The island of Hvar is well-known as a centre of commercial production and oil distillation in Croatia. Two landraces of lavandin are grown on the island Hvar, 'Budrovka' and 'Levanda'. Despite the economic importance and historical value, information on lavandin genetic diversity is not available. The AFLP based genetic analysis included landraces 'Levanda' and 'Budrovka' from Hvar, 'Levanda' from Vis, two most famous lavandin cultivars from France ('Grosso' and 'Abrialis') and putative parents of the hybrid (L. angustifolia and L. latifolia). The results of the genetic analysis revealed that both 'Budrovka' and 'Levanda' are polyclonal landraces. It seems that groups of closely related genotypes originated from different hybridization events while the intra-group variability could be attributed to the accumulation of somatic mutations. Polyclonality is maintained by vegetative propagation of different genotypes opening up future possibilities for the selection of potential superior clones.

Sideritis scardica Griseb. is an endangered plant species, endemic to the Balkan peninsula. Aerial parts of Sideritis scardica Griseb. are used in traditional medicine as a herbal tea for treatment of lung and gastrointestinal disorders, antimicrobial infections and rheumatic disorders. The plants are extensively gathered from natural resources and due to often overexploitation many wild populations are seriously threatened. Therefore, the design of conservation strategies is crucial, thus requiring detailed analysis of the level and distribution of genetic diversity. For this purpose 12 natural S. scardica populations from Greece and FYR of Macedonia were analyzed using Amplified fragment length polymorphism (AFLP) markers. Analysis of molecular variance (AMOVA) indicated that the most genetic variation was found within populations (65.02%). Gene diversity ranged from 0.088 (Mt. Kozuf ; MKD) to 0.123 (Mt. Vermio ; GR). Populations tended to group together according to their geographical origin as revealed by distance and model based methods. Isolation by distance analysis indicated that 52.2% of population differentiation could be explained with isolation by distance. The obtained results provide basic information for the efficient conservation strategy development for S. scardica and introduction into agricultural cultivation.

Section Incani DC. (= Sect. Proselius Bunge ; Sect. Holophyllum Boriss.) of the genus Astragalus L. (Fabaceae) comprises 135-150 species and represents species richest group of taxa among medifixed hairy Astragalus species (subgenus Cercidothrix Bunge), with the Armeno-Iranian floristic province of the Irano-Turanian phytogeographical region in the Middle and Greater Middle East being diversification centre. The species of this section are distributed in Asia, Middle East, Caucasus, Nort Africa and Europe. Based on molecular evidence, taxa of the section Incani represent a monophyletic clade among modifixed hairy Astragalus taxa. Most recent monographic treatment of the European taxa listed only three species (A. incanus L., A. monspessulanus L. and A. spruneri Boiss.) and several other taxa ranked on subspecies level or bellow, distributed mostly in Circum-Mediterranean area. Despite being only a poor reflection of species diversity within the Astragalus section Incani, complex and ambiguous systematics and nomenclature of European species reflect recognition of different number of taxa, most probably based on diagnostic features of dubious taxonomic relevance. Our research aims at more accurate taxa circumscriptions and their distribution ranges as well as examination of taxonomic relevance of characters used in current systematic treatments. Here, the first results of morphometric and AFLP fingerprinting data based on comprehensive sampling across the whole distribution range of the Astragalus section Incani in Europe are presented.

Campanula tommasiniana is a typical chasmophyte occupying calcareous rock crevices and cracks along the wide range of various ecological gradients demonstrating high degree of ecological plasticity and stress tolerance with regards to abiotic factors. Generally, three ecologically and floristically distinct groups of stands were recognized and typified according to sigmatistic approach: (a) Seslerio juncifoliae-Campanuletum tommasinianae ass. nov., with stands occupying higher elevated sites fully exposed to sun and strong winds ; (b) Seslerio automnalis-Campanuletum tommasiniana ass. nov., representing stands predominantly developed within the thermophytic beech stands, semi- to fully-shaded by the nearby tree canopy ; (c) Cystopteri fragilis-Campanuletum tommasinianae, sciophytic, moist and cold adapted stands with high frequency and coverage of bryophytes. Results of DCA analyses using unimodal model suggest that Campanula tommasiniana is primarily a plant of open and exposed sites of higher elevation despite being most frequently found in rock crevices within the thermophytic and altimontane beech forest. With regard to distinct groups of stands, genetic (AFLP) and epigenetic (MSAP) diversities of the species were assessed, compared and discussed.

Adriatic basin is relatively isolated phylogeographical region within Mediterranean area with numerous endemic taxa. Molecular analysis based on AFLP markers was used to resolve relationships between endemic taxa belonging to the Adriatic Limonium cancellatum complex. The AFLP data indicate that L. cancellatum, L. dictyophorum, L. liburnicum, L. apulum, L. diomedeum, L. vestitum, L. brusnicense and L. subanfractum belong to the L. cancellatum complex. While Ionian taxa from Greek islands together with south Italian L. japygicum does not belong to the L. cancellatum complex. They form independent Mediterranean group of L. bocconei. Current taxonomical status and division of endemic Limonium taxa is not congruent with AFLP data. Our results revealed the presence of three genetically and morphologically differentiated groups within L. cancellatum complex along the eastern Adriatic coast where L. cancellatum and L. liburnicum forms one group (Istria, Kvarner, north Dalmatia), L. apulum and L. dicyophorum forms the second group (southern Adriatic) and the third group is represented by L. vestitum, L. brusnicense and L. subanfractum (central Dalmatian islands). The third group could be the result of secondary contacts after post-glacial range expansion. Hybridogen origin of some populations of L. subanfractum, L. dictyophorum and L. cancellatum on remote islands was detected. Highly significant correlation between genetic and geographical distance was established.

Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip.) is a perennial plant species endemic to the east coast of the Adriatic Sea. Secondary metabolite of Dalmatian pyrethrum is a natural insecticide pyrethrin. Pyrethrin is effective on a broad range of insect pest and low in toxicity to humans and worm blooded animals. Amplified fragment length polymorphism (AFLP) markers were used to investigate genetic diversity within and among 25 wild populations of Dalmatian pyrethrum. Six primer combinations produced 985 bands of which 935 (94.9%) were polymorphic. Analysis of the molecular variance (AMOVA) revealed that the most of the AFLP diversity was distributed among individuals within populations (85.89%). Bayesian analysis of population structure enabled identification of two dominant genetic units. Spatial analysis of genetic diversity indicated that 3.72% of genetic differentiation resulted from isolation by distance, while 12.55% of genetic differentiation among populations followed the pattern of isolation-by-adaptation. The results have shown that wild Dalmatian pyrethrum populations are valuable source of genetic diversity that could be exploited in future plant breeding programs. Moreover, the results will have great contribution in the development of adequate conservation strategies.

Genetic diversity of 25 Dalmatian sage populations from Croatia and Bosnia and Herzegovina, each consisting of 20 to 25 plants, was assessed using AFLP markers. Four AFLP primer combinations yielded 559 polymorphic markers. Allelic frequencies at AFLP marker loci in each population were calculated from the observed fragment frequencies assuming Hardy- Weinberg equilibrium. Gene diversity ranged from 0.162 to 0.199 with an average value of 0.178. The overall genetic differentiation among populations was very low, FST = 0.042, ranging from 0.006 to 0.081. The analysis of molecular variance indicated that most of the genetic diversity was attributable to differences among individuals within populations (92%). The Fitch-Margoliash tree based on Nei's genetic distance matrix showed that most of the populations tended to group together in accordance to geographical position of their collecting sites. Spatial analysis of genetic diversity revealed a typical pattern of isolation-by-distance.

AFLP markers were used for genetic diversity analysis of 40 basil accessions representing eight species (O. americanum L., O. basilicum L., O. champechianum Mill., O. x citriodorum Vis., O. gratissimum L., O. kilimandscharicum Baker ex Gürke O. minimum L., O. tenuiflorum L.). Genetic relationships were determined by Neighbor Joining cluster analysis based on Dice's distance matrix between accessions. High bootstrap support values for the branches separating species were observed as well as grouping of morphologically similar accessions within the O. basilicum cluster. The position of O. minimum accession within the O. basilicum cluster indicate that at genomic level O. minimum is closely related to other taxa of O. basilicum. O. minimum should be considered as a variety of O. basilicum, although it possesses a limited number of dwarfing genes giving it clearly distinguishable morphological features. The accessions of O. americanum and O. x citriodorum grouped together with two accessions of O. basilicum var. purpurascens. The explanation could be the hybrid origin of O. x citriodorum (= O. basilicum x O. americanum) as well as of these two accessions of var. purpurascens (= O. basilicum var. purpurascens s. stricto x O. x citriodorum). AFLP markers can contribute in resolving existing problems concerning basil taxonomy at both inter- and intra-specific levels.

Basil (O. basilicum L.) is one of the most popular aromatic and medicinal plants grown in the world. High levels of both morphological and chemical variability exist within the species. Long-term traditional uses and wide distribution throughout the world, as well as traditional selection and breeding efforts, have contributed to that variability within the species. Morphological descriptors and AFLP markers were utilized to define the extent of variation existing in the species analyzing 24 accessions from the Collection of medicinal and aromatic plants of the Department of Seed Science and Technology, Faculty of Agriculture, University of Zagreb, Croatia. Phenotypic dissimilarities between pairs of accessions were calculated and the UPGMA dendrogram was constructed. A number of clearly defined clusters have been detected giving a good representation of traditional taxonomic relationships. Genetic relationships were determined by Neighbour-Joining cluster analysis based on Dice's distance matrix between accessions. In general, morphologically similar accessions grouped together and a high congruence between trees was observed. Both AFLP markers and morphological descriptors can contribute in resolving existing problems concerning intraspecific classification in O. basilicum. In addition, morphological descriptors provide an inexpensive and reliable method for routine screening of accessions in order to monitor and manage basil germplasm collections.

Dalmatian sage (Salvia officinalis L.) is a perennial subshrub belonging to Lamiaceae family. It is a native of the northern shore region of the Mediterranean, and grows wild in large masses in the calcereous, karstic mountains of Dalmatia, Herzegovina, Montenegro and Albania. It is used as an herb with beneficial healing properties and its dried leaf (Salviae folium) is an authorized drug in most pharmacopoeias. Genetic diversity and structure of ten Dalmatian sage populations in Croatia has been analysed using AFLP markers. Five populations were collected in Istria and Kvarner (Šušnjevica, Kamenjak, Stara Baška, Cres, Lošinj) and five in Dalmatia (Hvar, Vis, Pelješac, Mljet, Konavle). Each population consisted of 25 plants. The four AFLP primer combinations yielded 1389 polymorphic fragments. Shannon's information index as a measure of genetic diversity within populations ranged from 0.327 (Vis) to 0.370 (Lošinj). The analysis of molecular variance was used to partition the total genetic variance into among and within populations. The most of the genetic diversity was attributable to differences among individuals within populations (93%) in concordance with the patterns expected for an outcrossing and moderately long-lived species. Allelic frequencies at AFLP marker loci in each population were calculated from the observed frequencies of fragments assuming Hardy-Weinberg equilibrium, using the Bayesian approach. A low level of genetic differentiation among populations (FST = 0.039) was observed. Neighbor-joining tree based on Nei's genetic distance matrix among populations revealed two clades supported by a high bootstrap value in accordance with geographical origin of the populations (Istra and Kvarner vs. Dalmatia). Regression analysis of geographic distances and pairwise FST values revealed a typical pattern of isolation-by-distance.

U planinskim se područjima povećanjem nadmorske visine mijenjaju klimatski i ekološki uvjeti, a što se zrcali u promjenama u sastavu vegetacije. Budući da se može pretpostaviti kako će vrste sa širokom rasprostranjenošću biti pod manjim utjecajem klimatskih promjena, nego što je to slučaj s usko rasprostranjenim vrstama, proučavanje vrsta tolerantnih na promjenu nadmorske visine osobito je zanimljivo u svjetlu današnjih klimatskih promjena. Cilj je ovog diplomskog rada provesti preliminarna populacijsko-genetička istraživanja triju prirodnih populacija vrste G. monvillei (Cactaceae) s različitih nadmorskih visina upotrebom AFLP-biljega (eng. Amplified Fragment Lenght Polymorphism markers). Nakon usporedbe dobivenih rezultata s rezultatima već provedenih morfoloških i fizioloških istraživanja, definirat će se opsežnija AFLP istraživanja na većem broju prirodnih populacija. Uočena genetska raznolikost, vrijednosti DW-indeksa (eng. frequency down-weighted marker), rezultati analize PCoA (eng. Principal Coordinate Analysis) i STRUCTURE analize ukazuju na dugu vremensku izoliranost populacije s najviše nadmorske visine i slobodan protok gena kod dviju populacija na nižim nadmorskim visinama. Jedinke populacija s nadmorske visine od 1250 m, zbog utvrđene najveće genetičke raznolikosti, globularne građe klijanaca, otpornosti na povišenu temperaturu i sušu, velikih plodova i velikog broja sjemenki, najizglednije su u ekološkom optimumu vrste te bi se vjerojatno najlakše prilagodile budućim klimatskim promjenama. U skladu s tim ova populacija predstavlja evolucijski signifikantnu jedinicu koju treba uzeti u obzir u budućim programima zaštite. In mountain regions, with increasing altitude, climate and ecological conditions change, which reflects in changes of vegetation composition. Since it can be assumed that widespread species will be less affected by climate change, than it is the case with narrowly distributed species, the study of species tolerant to altitude changes is particularly interesting in the light of today's climate change. The aim of this graduation thesis was to conduct a preliminary population genetic study of three natural populations of G. monvillei (Cactaceae) from different altitudes using AFLP (Amplified Fragment Lenght Polymorphism) markers. After comparing obtained results with the already existing results of morphological and physiological studies, a more extensive AFLP studies are going to be defined on a larger number of natural populations. The observed genetic diversity, DW index values (frequency down-weighted marker), PCoA (Principal Coordinate Analysis) and STRUCTURE analyses indicated long-time isolation of the population from the highest altitude and free gene flow in the two populations from lower altitudes. Due to the highest genetic diversity, globular seedlings, resistance to high temperatures and drought, large fruits and large numbers of seeds, the population at 1250 m a.s.l. probably represents an ecological optimum of the species and it would most likely be the easiest population to adapt to future climate changes. Therefore, this population can be recognized as an evolutionarily significant unit in future conservation programs.