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Teucrium montanum L. and T. capitatum L. are two plant species with overlapping distribution in the Balkan Peninsula, especially in Croatia, where several occurrences of their putative hybrid species T. × rohlenae have been recorded. Next-generation sequencing of both species and de novo assembly was carried out resulting in 120 contigs for T. montanum and 1685 contigs for T. capitatum assembled. The Dig-up primers pipeline was used for SSR mining of both assemblies, applying different criteria that resulted in 112 SSR candidates for testing. A subset of 41 SSRs was selected and after two rounds of testing, twelve SSRs were developed and characterized. A total of 232 alleles were detected with 5 to 29 alleles per locus, based on the test sample. The genetic diversity analysis of three Teucrium taxa from a single location revealed a higher level of diversity in T. montanum than in T. capitatum with intermediate values for the hybrid species. The NeighborNet diagram and genetic structure analysis grouped the taxa into two separate clusters, the first of which consisted exclusively of T. montanum, while the other was composed of intermixed T. capitatum and T. × rohlenae individuals. The availability of SSR markers for two Teucrium taxa will allow in-depth analysis of genetic diversity and structure, as well as molecular identification of their putative hybrids in the future. [ABSTRACT FROM AUTHOR]

Simple sequence repeats (SSRs) have become one of the most popular molecular markers and are used in numerous fields, including conservation genetics, population genetic studies, and genetic mapping. Advances in next-generation sequencing technology and the growing amount of genomic data are driving the development of bioinformatics tools for SSR marker design. These tools work with different combinations of input data, which can be raw reads or assemblies, and with one or more input datasets. We present here a new strategy and implementation of a simple standalone pipeline that utilizes more than one assembly for the in silico design of PCR primers for microsatellite loci in more than one species. Primers are tested in silico to determine if they are polymorphic, eliminating the need to test time-consuming cross-species amplification in the laboratory. The end result is a set of markers that are in silico polymorphic in all analyzed species and have great potential for the identification of interspecies hybrids. The efficiency of the tool is demonstrated using two examples at different taxonomic levels and with different numbers of input assemblies to generate promising, high-quality SSR markers. [ABSTRACT FROM AUTHOR]

The development of next-generation sequencing technology and the increasing amount of sequencing data have brought the bioinformatic tools used in genome assembly into focus. The final step of the process is genome annotation, which works on assembled genome sequences to identify the location of genome features. In the case of organelle genomes, specialized annotation tools are used to identify organelle genes and structural features. Numerous annotation tools target chloroplast sequences. Most chloroplast DNA genomes have a quadripartite structure caused by two copies of a large inverted repeat. We investigated the strategies of six annotation tools (Chloë, Chloroplot, GeSeq, ORG.Annotate, PGA, Plann) for identifying inverted repeats and analyzed their success using publicly available complete chloroplast sequences of taxa belonging to the asterid and rosid clades. The annotation tools use two different approaches to identify inverted repeats, using existing general search tools or implementing stand-alone solutions. The chloroplast sequences studied show that there are different types of imperfections in the assembled data and that each tool performs better on some sequences than the others. [ABSTRACT FROM AUTHOR]

The high-biodiversity phenomenon of the Balkan Peninsula is a result of the past and present topographic, climatic and geological conditions, as well as human impact. The Dinaric Alps, as a part of the Balkan Peninsula, harbour a high number of endemic species, some of them comprising a small number of populations, which are often endangered. We investigated spatial distribution, community composition, site ecology, genetic diversity and conservation of Degenia velebitica, a stenoendemic species of the north-western Dinaric Alps. Our results showed that D. velebitica is nowadays restricted to three localities with the area of occupancy of 48,560 m2, harbouring approximately 37,000 individuals. Degenia velebitica stands are differentiated into three well-characterized, floristically homogenous syntaxa, very distinct from the surrounding vegetation, suggesting their azonal occurrence and restriction to spatially highly fragmented microsites exposed to stormy winds. Spatial distances of populations, differences in D. velebitica community composition and site ecology are mirrored in genetic variation patterns of the populations, such as high-frequency down-weighted marker values in the north-western populations and high gene diversity in the south-eastern group of populations. The fact that we could not find a single D. velebitica individual at the locus classicus indicates the existence of the ever-growing fragmentation. The analyses of genetic structure using AFLP data recognized two main genetic groups of populations as evolutionary significant units that should be considered when planning protection measures. According to our IUCN Red List reassessment, D. velebitica should be treated as a critically endangered species that requires immediate conservation actions. [ABSTRACT FROM AUTHOR]

Summary: Epigenetic modifications may play an important role in invasion and adaptation of clonal and invasive populations to different environments. The aim of this study was to analyse epigenetic diversity and structure within and among populations of invasive pathogenic fungus Cryphonectria parasitica from south‐eastern Europe, where one haplotype S12 dominates. The highest level of epigenetic diversity was found in haplotype S1, followed by S2, while the lowest level of epigenetic diversity was found in haplotype S12. Similar pattern of epigenetic diversity was detected in the control, genetically diverse Croatian population where S1 haplotype dominates. In four south‐eastern European populations, the highest level of epigenetic diversity was observed in the Italian population, the oldest population in the studied area, while the lowest diversity was found in most recently established Bulgarian population. This relationship between epigenetic diversity and population age implies the important role of epigenetic modifications on the process of invasion. Our data suggest that epigenetic differences might affect the success of expansion of certain haplotype into new regions. Understanding the role of epigenetic processes in expansion and (pre)adaptation of fungal plant pathogens, besides fundamental knowledge, can contribute to development of strategies for control of fungal spread and pathogenesis. [ABSTRACT FROM AUTHOR]

Olive growing in Croatia has a long tradition and is of great economic and social impact. The present study includes a set of 108 tree samples (88 samples corresponding to 60 presumed cultivars and 20 trees of unnamed ones) collected from 27 groves in the entire olive growing area, and is the most comprehensive survey to be conducted in Croatia. The genetic diversity, relationships, and structures of olive plants were studied using eight microsatellite loci. All loci were polymorphic and revealed a total of 90 alleles. A total of 74 different genotypes were detected that were subjected to further diversity and genetic relationship studies. The Fitch–Margoliash tree and Bayesian analysis of population structure revealed a complex relationship between the identified olive genotypes, which were clustered into three gene pools, indicating different origins of Croatian olive germplasms. Excluding the redundant germplasms, 44 different genotypes among the sampled trees of well-known cultivars and 16 new local germplasms were identified. In addition, we provide the etymology of 46 vernacular names, which confirms that the vast majority of traditional Croatian cultivars have common and widespread names. The results presented herein underline the importance of safeguarding local cultivars and conducting continuous surveys. [ABSTRACT FROM AUTHOR]

Altitudinal gradients in mountain environments impose selection pressures that could result in marked heterogeneity in genetic characteristics, population features and reproductive success. Most Cactaceae occur in arid and semi-arid areas of the American continent, from sea level to 4500 m a.s.l. and some species have wide altitudinal ranges. The aim of this work was to analyse how cytogenetic features (mitotic chromosomes analysed using molecular cytogenetic techniques, DNA content estimated by flow cytometry), amplified fragment length polymorphism genetic diversity, population features (population density and individual size) and reproductive success (seeds per plant and fruit production) are related to an altitudinal gradient in five sites of Gymnocalycium monvillei located between 878 and 2230 m a.s.l. Our results showed that the sites at three intermediate altitude have optimal conditions as greatest genetic diversity, highest population density and highest seed production, whereas extreme sites are characterized by greatest reproductive isolation and highest DNA content and seed mass. The results indicate that the species has various mechanisms to survive along altitudinal gradients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Amplified fragment length polymorphism (AFLP) markers were used to assess genetic relations of 32 Thymus populations belonging to seven species of genus Thymus, section Serpyllum, from Serbia. The collected species belong to three subsections: (1) Alternantes Klokov (Th. pulegioides L.), (2) Isolepides (Borbás) Halácsy (Th. glabrescens Willd., Th. marschallianus Willd., Th. pannonicus All.), and (3) Pseudomarginati (Braun ex Borbás) Jalas (Th. balcanus Borbás, Th. moesiacus Velen., Th. praecox Opiz). Analysis of molecular variance (AMOVA) revealed that most of the genetic diversity was attributable to differences among individuals within populations, while the remaining variability was almost equally distributed among species and among populations within species. The pairwise AMOVA analyses further showed that species differentiation was significant in all cases, except between Th. marschallianus and Th. pannonicus. All the individuals belonging to Th. pulegioides (subsection Alternantes) formed a well-supported clade, the most divergent from all other species in this study. This genetic differentiation of Th. pulegioides is supported by its morphological features because it is the only analyzed taxon having hairs only on the stem edges, and can easily be recognized by this character. According to our results, the subsection Isolepides is polyphyletic. Th. glabrescens is clearly separated from the other two species of this subsection (Th. marschallianus, Th. pannonicus). Although morphologically distinguishable by leaf indumentum, individuals belonging to Th. marschallianus and Th. pannonicus could not be differentiated at the molecular level. The species belonging to subsection Pseudomarginati formed a monophyletic clade, although not supported by high bootstrap value. The clade is further divided into three well-supported clades representing distinct species (Th. balcanus, Th. praecox and Th. moesiacus). In our study, the AFLP markers demonstrated that they could be suitable for studying complex genetic relationships, including frequent interspecies hybridization events, although strict reticulate evolutionary history could not be determined. All the population genetic parameters in the section Serpyllum suggest that the reproductive incompatibility between the species is very weak, which indicates a strong reticulating system, especially in the case when two or more species occur sympatrically. [Copyright &y& Elsevier]

DNA fingerprinting (AFLP) and chemical analyses of essential oils were utilized to define the extent of variation existing in the genus Ocimum. Research was carried out on 22 Ocimum accessions representing seven species. Concerning the essential oil composition of all investigated accessions, 115 compounds were identified. UPGMA cluster analysis, based on Euclidian distances of essential oil constituents between all pairs of accessions, showed four well-supported clusters ( O. tenuiflorum, O. basilicum/O. africanum, O. basilicum, and O. americanum/O. africanum). Relating to the essential oil composition of all of the investigated accessions, 17 compounds were identified as the main ones, and according to them 13 chemotypes were determined. AFLP relationships were determined by neighbor-joining (NJ) cluster analysis based on Dice's distance matrix and by maximum parsimony (MP) analysis. O. basilicum, O. americanum/O. africanum, O. tenuiflorum, and O. gratissimum represented four clusters supported with high bootstrap values. A neighbor-net diagram allowed the visualization of apparently conflicting data by revealing relationships between genotypes and chemotypes. Concerning the O. africanum species, two distinct chemotypes, geranial/neral (accession 11) and estragol (accession 10), have been established, while all the studied O. americanum accessions belong to the geranial/neral chemotype. This could be additional evidence that O. americanum is one of the parents of O. africanum. Furthermore, the fact that the O. africanum accession (10) as well as O. basilicum 'Purpurascens' and O. basilicum 'Erevanskii' accessions belong to the estragol chemotype supports the theory that O. africanum is one of the parents of these two O. basilicum accessions. [ABSTRACT FROM AUTHOR]

Twenty-eight basil accessions including six Ocimum species and six botanical varieties or cultivars of O. basilicum were studied using molecular markers, nuclear DNA content, and chromosome counting. This is the first study reporting the nuclear DNA content in the genus Ocimum. The results supported the existence of more infrageneric groups within the genus. The section Ocimum was further divided into two separate clades. The first clade contained the accessions belonging to different botanical varieties and cultivars of O. basilicum as well as O. minimum, indicating that the separate species rank of O. minimum was not justified. The second clade, comprising O. americanum, O. africanum, and two O. basilicum var. purpurascens accessions, could represent a set of allopolyploid species sharing some common parental genomes. O. tenuiflorum was the most divergent species according to genetic distance; it had the smallest genome size, organized in small chromosomes, and the lowest chromosome number. Chromosome data obtained in our research could indicate that the basic chromosome number for species belonging to section Ocimum is x = 12. This suggestion implies that species belonging to O. basilicum clade are tetraploids, while species belonging to O. americanum clade are hexaploids. It seems that the basic chromosome number for O. gratissimum could be x = 10 and for O. tenuiflorum x = 9. The differences in genome size and chromosome number among Ocimum species indicate that evolution of their genomes was accompanied by both sequence deletion/amplification and chromosome rearrangements and polyploidization. [ABSTRACT FROM AUTHOR]

The relationships among western Balkan and some amphi-Adriatic endemic campanulas were studied using RFLP analysis of the 1.8-kb trnT - trnF chloroplast DNA region. The results were compared to those of a comparative floral morphometry study performed earlier on a similar sample. Maximum parsimony and distance methods produced very similar results to those of the floral morphometry, both of which point to a clear separation of the taxa into three main clades. The first isophyllous, or Garganicae series, clade comprised Campanula poscharskyana, C. portenschlagiana, C. garganica, C. reatina and two C. fenestrellata-subspecies. The second "isophylloid" clade consisted of C. pyramidalis agg., C. waldsteiniana agg. and C. carpatica. The third, heterophyllous clade was very uniform, comprising undistinguishable taxa of the C. rotundifolia complex and alpine endemic C. cochleariifolia. Interestingly the Ligurian stenoendemic C. isophylla was clearly separated from the members of the ser. Garganicae, C. reatina was the most distant taxon in the Garganicae clade, the controversial species Asyneuma pichleri was nested inside the Campanula core, while Campanula carpatica was closer to the "isophylloids" than to the sect. Rapunculus Boiss. In order to resolve the taxonomic uncertainty among the closely related heterophyllous taxa more refined molecular methods, e.g. AFLP or microsatellites, should be applied. [ABSTRACT FROM AUTHOR]

Gene flow between cultivated and wild gene pools is common in the contact zone between agricultural lands and natural habitats and can be used to study the development of adaptations and selection of novel varieties. This is likely the case in the northern Adriatic region, where centuries-old cultivated orchards of sweet chestnut (Castanea sativa Mill.) are planted within the natural distribution area of the species. Thus, we investigated the population structure of several orchards of sweet chestnuts. Furthermore, the genetic background of three toponymous clonal varieties was explored. Six genomic simple sequence repeat (gSSR) and nine EST-derived SSR (EST-SSR) loci were utilized in this research, and both grafted and non-grafted individuals were included in this study. Five closely related clones were identified, which represent a singular, polyclonal marron variety, found in all three cultivation areas. Furthermore, many hybrids, a result of breeding between cultivated and wild chestnuts, have been found. Analyzed semi-wild orchards defined by a diverse genetic structure, represent a hotspot for further selection and could result in creation of locally adapted, high-yielding varieties. [ABSTRACT FROM AUTHOR]

In the eastern Adriatic flora the genus Pistacia L. (Anacardiaceae) is represented with P. lentiscus L., P. terebinthus L., their hybrid P. × saportae Burnat and the cultivated P. vera L. In addition, an endemic putative taxon from Mt. Biokovo was described in 1985 as P. calcivora Radić. Our aim was to resolve relationships between the four putative indigenous taxa of this genus in Croatia (P. lentiscus, P. terebinthus, P. × saportae and P. calcivora) based on genetic (AFLP) and morphological data. Specifically, we aimed to determine the presence of the hybrid taxon P. × saportae and to validate the controversial taxonomic status of P. calcivora at the molecular and morphological levels. Our combined results indicate the presence of two well separated groups of populations. The first group included all individuals of P. terebinthus and the individuals initially assigned to the potential taxon P. calcivora based on leaf morphology, suggesting that there is no support for the described taxon P. calcivora and that it should be considered as a synonym for P. terebinthus. The second group corresponded to P. lentiscus and included the majority of the presumed hybrid individuals of P. × saportae. However, four hybrid individuals were confirmed at the molecular level and were placed between the two parental taxa in the phylogenetic tree, confirming the presence of P. × saportae in Croatia. Although confirmed P. × saportae individuals were genetically closer to P. lentiscus, they were morphologically more similar to P. terebinthus, hindering their correct identification in the field. [ABSTRACT FROM AUTHOR]

Campanula fenestrellata subsp. fenestrellata and C. fenestrellata subsp. istriaca belong to the morphologically and phylogenetically well established amphi-Adriatic group Campanula ser. Garganicae. The two morphologically differentiated endemic taxa are distributed on the north-eastern Adriatic islands and coast. In order to study the relationships among C. fenestrellata s.l. populations and to gain insight into genetic basis of taxonomic separation established on morphology, we examined amplified fragment length polymorphisms (AFLPs) as well as nuclear and plastid DNA sequences (ITS and trnL–trnF). With the sequence data, no structure and no distinction between the two subspecies were observed. The AFLPs separated populations geographically and inferred high number of most likely clusters. The northern and the southernmost populations were assigned to uniform separate clusters indicating the longer isolation of respective populations, while the admixed populations were evident in the central area where the two taxa geographically overlap, indicating the presence of gene flow. Distinct fragmentation patterns and the existence of several temporally continuous microrefugia during the Quaternary climatic oscillations likely promoted strong genetic differentiation within the species range, while secondary postglacial contacts are reflected in observed admixture. Although the two taxa exhibited differences in ecological preferences, niche conservatism was detected. Overall, phylogeographic reconstruction of C. fenestrellata s.l. populations highlights the importance of microrefugia in shaping the intraspecific genetic variation as well as of melting-pot areas in rearranging such variation as a result of a more recent admixture. [ABSTRACT FROM AUTHOR]

Salvia officinalis is a perennial species, native and endemic to the Western Balkans and the Apennine Peninsula. Due to its medicinal and aromatic properties, it is used in pharmaceutical, cosmetic, and food industries. The main objectives of the study were to infer the genetic structure of S. officinalis populations in the northern and central parts of the eastern Adriatic coast, to detect the phylogeographical barriers among the putative microrefugia and to assess the genetic diversity among the resulting ancestral clusters. Twenty-five populations were assessed using amplified fragment length polymorphism markers. High polymorphism and high diversity within populations were typical for this outcrossing long-lived species. The Fitch–Margoliash tree based on Nei’s genetic distance matrix showed that most of the populations tended to group in accordance with the geographical position of their collecting sites. Spatial analysis of the genetic diversity revealed a typical pattern of isolation by distance. Very low overall among-population differentiation and detection of only three private alleles indicate that there has been high gene flow among populations. By using Bayesian Analysis of Population Structure on population level, two distinct ancestral clusters were obtained. It is likely that these two ancestral clusters were separated for a longer period by Pleistocene glaciation, although the subsequent fast recolonization resulted in diminished genetic differences. High rarity of northern and southern populations of the investigated area indicates that S. officinalis presumably survived in both northern and southern microrefugia and expanded from there resulting in secondary contact zones, characterized by lower rarity and equal genetic diversity. [ABSTRACT FROM AUTHOR]

Campanula hawkinsiana and C. ramosissima are among the poorly investigated Campanula species distributed in the Balkan Peninsula. According to recent phylogenetic studies these species are closely related, and they belong to the section Decumbentes along with the two species from the Iberian Peninsula. However, none of these published studies included a larger number of populations of C. hawkinsiana and C. ramosissima, nor were they studied in detail at the morphological level. During our floristic investigations in the Balkan Peninsula, we observed a great morphological variability within the populations of C. ramosissima. The most morphologically specific and diverse are C. ramosissima populations from Montenegro. Considering all this, in this study, both molecular (AFLP fingerprinting and DNA sequencing) and classical morphometric analyses were applied with the aim to get insight into the genetic diversity and morphological variability of the populations from Montenegro and geographically close populations of both species from the section Decumbentes in the western part of the Balkan Peninsula. Significant morphological differences and a distinct position based on molecular phylogenetic evidence indicate that the plants from Montenegro should be recognized as a new subspecies, described here as Campanula ramosissima subsp. megapetala subsp. nov. [ABSTRACT FROM AUTHOR]

Biotic stress caused by virus infections induces epigenetic changes in infected plants and animals, but this is the first report on methylation pattern changes in a fungus after mycovirus infection. As a model pathosystem for mycovirus-host interactions, we used Cryphonectria hypovirus 1 (CHV1) and its host fungus Cryphonectria parasitica, in which deregulation of methylation cycle enzymes upon virus infection was observed previously. Six CHV1 strains of different subtypes were transferred into three different C. parasitica isolates in order to assess the effect of different CHV1 strains and/or subtypes on global cytosine methylation level in infected fungus, using methylation-sensitive amplification polymorphism (MSAP). Infection with CHV1 affected the methylation pattern of the C. parasitica genome; it increased the number and diversity of methylated, hemi-methylated, and total MSAP markers found in infected fungal isolates compared to virus-free controls. The increase in methylation levels correlated well with the CHV1-induced reduction of fungal growth in vitro, indicating that C. parasitica genome methylation upon CHV1 infection, rather than being the defensive mechanism of the fungus, is more likely to be the virulence determinant of the virus. Furthermore, the severity of CHV1 effect on methylation levels of infected C. parasitica isolates depended mostly on individual CHV1 strains and on the combination of host and virus genomes, rather than on the virus subtype. These novel findings broaden our knowledge about CHV1 strains which could potentially be used in human-aided biocontrol of chestnut blight, a disease caused by C. parasitica in chestnut forest ecosystems and orchards. [ABSTRACT FROM AUTHOR]

We reconstructed the relationships among populations of Campanula secundiflora s.l. and closely related and geographically close populations of C. austroadriatica and C. versicolor. Based on analyses of microsatellite DNA data, the investigated populations have high overall genetic diversity and abundant allelic variation over seven investigated loci. Bayesian model-based clustering identified four clearly differentiated genetic groups of populations. The genetic differentiation was reflected by morphological differentiation, allowing us to propose a new taxonomic treatment of the constituents of C. secundiflora s.l. The populations distributed in south-western Serbia and north-eastern Montenegro were included in C. secundiflora. A new species, Campanula montenegrina sp. nov., distributed in the continental part of Montenegro and the northern part of Albania is described. [ABSTRACT FROM AUTHOR]

Studying the population-genetic and phylogeographic structures of a representative species of a particular geographical region can not only provide us with information regarding its evolutionary history, but also improve our understanding of the evolutionary processes underlying the patterns of species diversity in that area. By analysing eight highly polymorphic microsatellite loci and two chloroplast DNA regions, we have investigated the influence of Pleistocene climate fluctuations on the evolutionary history of Salvia officinalis L. (common sage). The populations with the highest genetic diversity were located in the central parts of the Balkan distribution range. A large group of closely related haplotypes was distributed throughout the Balkans and the central Apennines, while the private lineage occupied the southern Apennines. In addition, two highly differentiated lineages were scattered only over the Balkans. The results suggest that a single refugium of the studied species from the last glacial period was located in the central part of the range in the Balkans. Numerous microrefugia, probably spanning several glaciation cycles, were scattered across the Balkans, while colonisation of the Apennines from the Balkans occurred at least on two occasions. [ABSTRACT FROM AUTHOR]

Lavandin (Lavandula × intermedia Emeric ex Loisel.) was brought to the Island of Hvar (Croatia) in the 1920s, coinciding with the beginning of large-scale cultivation of lavandin in France. Although the cultivation of lavandin and the production of essential oils are of great importance worldwide, the genetic diversity of lavandin has been little studied. We performed an AFLP-based genetic analysis that included the landraces 'Bila' and 'Budrovka' and two lavandin cultivars from France 'Grosso' and 'Abrialis', as well as the parental species of the hybrid (L. angustifolia and L. latifolia). Distance-based cluster analysis revealed the existence of the third landrace, named 'Budrovka Sveti Nikola'. This result was confirmed by the model-based cluster analyses implemented in STRUCTURE and BAPS, where the optimal number of clusters was three. 'Budrovka' clearly separated from all other samples, while 'Bila' and 'Budrovka Sveti Nikola' showed some degree of admixture, indicating ancestral polyclonality. The landrace 'Bila' showed higher polymorphism than 'Budrovka' and 'Budrovka Sveti Nikola'. Analysis of molecular variance (AMOVA) showed that genetic diversity (56.63%) was higher within landraces than among (43.37%). This research will provide a basis for conservation of the Island landraces and will help in the establishment of a high-quality regional brand. [ABSTRACT FROM AUTHOR]

The article presents the phylogeny and biogeography of isophyllous species of Campanula in the Mediterranean area. Phylogenetic relationships of Campanula groups were examined using the sequence data from the nuclear internal transcribed spacer (ITS). It was found that the Rapunculus clade, members of Isophylla group, did not form a monophyletic group. Age estimates of the garganica clade was provided by molecular dating analysis while its dispersal was provided by dispersal-vicariance analysis.

Immortelle (Helichrysum italicum (Roth) G. Don; Asteraceae) is a perennial plant species native to the Mediterranean region, known for many properties with wide application mainly in perfume and cosmetic industry. A total of 18 wild H. italicum populations systematically sampled along the eastern Adriatic environmental gradient were studied using AFLP markers to determine genetic diversity and structure and to identify loci potentially responsible for adaptive divergence. Results showed higher levels of intrapopulation diversity than interpopulation diversity. Genetic differentiation among populations was significant but low, indicating extensive gene flow between populations. Bayesian analysis of population structure revealed the existence of two genetic clusters. Combining the results of FST - outlier analysis (Mcheza and BayeScan) and genome-environment association analysis (Samβada, LFMM) four AFLP loci strongly associated with the bioclimatic variables Bio03 Isothermality, Bio08 Mean temperature of the wettest quarter, Bio15 Precipitation seasonality, and Bio17 Precipitation of driest quarter were found to be the main variables driving potential adaptive genetic variation in H. italicum along the eastern Adriatic environmental gradient. Redundancy analysis revealed that the partitioning of genetic variation was mainly associated with the adaptation to temperature oscillations. The results of the research may contribute to a clearer understanding of the importance of local adaptations for the genetic differentiation of Mediterranean plants and allow the planning of appropriate conservation strategies. However, considering that the identified outlier loci may be linked to genes under selection rather than being the target of natural selection, future studies must aim at their additional analysis. [ABSTRACT FROM AUTHOR]

Sideritis scardica Giseb. is a subalpine/alpine plant species endemic to the central part of the Balkan Peninsula. In this study, we combined Amplified Fragment Length Polymorphism (AFLP) and environmental data to examine the adaptive genetic variations in S. scardica natural populations sampled in contrasting environments. A total of 226 AFLP loci were genotyped in 166 individuals from nine populations. The results demonstrated low gene diversity, ranging from 0.095 to 0.133 and significant genetic differentiation ranging from 0.115 to 0.408. Seven genetic clusters were revealed by Bayesian clustering methods as well as by Discriminant Analysis of Principal Components and each population formed its respective cluster. The exception were populations P02 Mt. Shara and P07 Mt. Vermio, that were admixed between two clusters. Both landscape genetic methods Mcheza and BayeScan identified a total of seven (3.10%) markers exhibiting higher levels of genetic differentiation among populations. The spatial analysis method Samβada detected 50 individual markers (22.12%) associated with bioclimatic variables, among them seven were identified by both Mcheza and BayeScan as being under directional selection. Four bioclimatic variables associated with five out of seven outliers were related to precipitation, suggesting that this variable is the key factor affecting the adaptive variation of S. scardica. [ABSTRACT FROM AUTHOR]

The intensive exchange and diffusion of plant material over the whole Mediterranean Basin including the East Adriatic region, has contributed to the confusion in denomination of olive varieties, and formation of a large number of synonyms and homonyms. In the present study, we report on clonal diversity and genetic relationships among a total of 190 olive trees sampled along the East Adriatic coast in Slovenia (SVN), Croatia (HRV) and Montenegro (MNE). Samples belonged to the six East Adriatic olive varieties with homonymic denomination: ‘Istrska belica’ (SVN), ‘Bjelica’ (HRV), ‘Žutica’ (MNE), ‘Črnica’ (SVN), ‘Crnica’ (HRV), and ‘Crnica’ (MNE). Results showed polymorphism in all twelve microsatellite markers (DCA3, DCA9, DCA11, DCA14, DCA16, DCA18, EMO03, EMO90, GAPU101, GAPU103A, GAPU71B and UDO99-019), revealing a total of 83 alleles. A total of 36 different genotypes in the panel of 190 samples were found. Genetic profiling of six homonymic East Adriatic olive varieties revealed four different varieties: 1) ‘Istrska belica’ (SVN), 2) ‘Bjelica’ (HRV)/‘Žutica’ (MNE), 3) ‘Crnica’ (HRV)/‘Crnica’ (MNE) and 4) ‘Črnica’ (SVN). Detailed clonal and genetic relationship analyses demonstrated genetic uniformity of ‘Istrska belica’ (SVN), negligible low diversity of ‘Črnica’ (SVN), and moderate diversity of ‘Crnica’ (HRV)/‘Crnica’ (MNE). Polyclonal property was assigned to only ‘Bjelica’ (HRV)/‘Žutica’ (MNE) variety. High genetic similarity between ‘Bjelica’ (HRV)/‘Žutica’ (MNE), and ‘Crnica’ (HRV)/‘Crnica’ (MNE), with different distribution of clones within them, reflects the past selection and propagation processes in Croatia and Montenegro. For the first time, international synonymy of ‘Žutica’ (MNE) and ‘Bjelica’ (HRV), and homonymy of ‘Istrska belica’ (SVN) and ‘Bjelica’ (HRV)/‘Žutica’ (MNE), as well as ‘Črnica’ (SVN) and ‘Crnica’ (MNE, HRV) were reliably confirmed with DNA markers. [ABSTRACT FROM AUTHOR]

The long tradition of green bean (Phaseolus vulgaris L.) cultivation in Croatia has resulted in the development of a landraces adapted to specific areas and chosen to suit different dietary habits. The hypothesis of our study was that nutritional quality (content of proteins, sugars, fibres, calcium, potassium and phosphorus in fresh pods) of 14 widely grown Croatian green bean accessions is affected by origin and morphotype. The analysed accessions were grown in non-replicated field plots where morphotypes were classified. The gene pool of adscription (Andean vs. Mesoamerican) of each accession was determined with the phaseolin marker, whereas nutritional quality traits were analysed according to standard analytical methods. The relationships among nutritional quality traits of fresh pods were assessed by Pearson's correlation coefficient. The univariate ANOVA and a post hoc Tukey test (p≤0.05) were carried out for each trait to compare the values among accessions. Accessions were grouped according to phaseolin type (I, II or III) and morphological traits (growth habit, pod colour and width). Four accessions had phaseolin type I ("S") predominant in the Mesoamerican while the rest of accessions belong to the Andean gene pool characterized by the phaseolin type II ("H" or "C") or III ("T"). Significant differences among accessions grouped by phaseolin type were found in all nutritional quality traits. The accessions of the Mesoamerican gene pool had higher dry matter content and crude protein content of fresh pods, while those of the Andean gene pool had higher total sugars content. Accessions of indeterminate growth habit had higher dry matter and crude protein content as well as phosphorus and potassium content than determinate ones, but display lower total sugar and calcium content. Outstanding accessions: S538, S541, S506 and S545 could be used as valuable sources of bioactive nutrients in future breeding programs. [ABSTRACT FROM AUTHOR]

Ocimum basilicum L. exhibits a great variety of cultivars grown for various purposes. The aim of this research was to analyse the chemical composition of 85 O. basilicum accessions cultivated throughout the world using gas chromatography (GC/FID and GC/MS). All accessions were previously categorized into six morphotypes based on 24 morphological traits: True basil, Small-leaf, Lettuce-leaf, Purple basil A, Purple basil B and Purple basil C morphotype. A total of 77 volatiles were observed, with seven of them being in concentrations higher than 10% in at least one accession (1,8-cineole, linalool, linalool acetate, methyl chavicol, eugenol, trans -methyl cinnamate, trans -α-bergamotene). Based on the essential oil composition of 85 O. basilicum accessions, we propose the intraspecific characterization of O. basilicum into five chemotypes: (A) High-linalool, (B) Linalool/ trans -α-bergamotene, (C) Linalool/methyl chavicol, (D) Linalool/ trans -methyl cinnamate and (E) High-methyl chavicol chemotype. Groups based on morphological and biochemical traits do not necessarily coincide with one another. Due to great morphological and biochemical diversity, both morphological and chemical characterizations are crucial for the effective management of germplasm collections to facilitate the use of the accessions in future breeding programs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article discusses a study that described new Campanula species endemic to Central Albania and was named C. skanderbegii of the Campanula ser. Garganicae. Topics covered include phylogentical and morphological differntiation of C. skanderbegii from other species and similarity of corolla shape with C. portenschlagiana thriving in Central Dalmatia, Croatia. Results indicated rarity of C. skanderbegii and its inclusion in the list of critically endangered species.

Highlights: [•] Tribe Alysseae is monophyletic. Four major evolutionary lineages are revealed with ITS, ndhF and trnL-F data. [•] Genera Alyssum, Fibigia, Clypeola and Alyssoides are not monophyletic. Monotypic genera Leptoplax and Physocardamum are embedded in Bornmuellera. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The European Mediterranean region is one of the world's major centres of biodiversity. Research on plant species diversity in this region has neglected the area along the eastern part of the Adriatic basin in comparison to the other Mediterranean areas. The main aim of this study was to focus on this neglected area, by supplying data which is at present lacking in order to discuss the species-area relationship (SAR), floristic richness and endemism of the Adriatic islands and coast. Floristic data for 106 Croatian islands collected by different authors were integrated, systematised and presented in a form usable by other researchers worldwide. The Power (Arrhenius) function was used for modelling (by non-linear regression) the SAR. Residuals around the regression curve (as indicators of floristic diversity without the influence of island area) were calculated for each island. The proportion of endemics in the total island flora varies from 0% up to 28.6%, and 17.5% for narrow endemics. Floristic richness of the broader region was estimated (on the basis of SAR extrapolation) on 1807 species for all Croatian islands, and on 2797 species for the entire Mediterranean area in Croatia. [ABSTRACT FROM AUTHOR]

• 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. [ABSTRACT FROM AUTHOR]

• Genetic variability of wild red raspberry populations. • Relationship with the most common cultivated cultivars. • Amplified fragment length polymorphism (AFLP) markers. • AFLP markers show that the cultivars are clearly distinct from wild populations. • The most of the genetic diversity is attributable to differences between individuals. The red raspberry (Rubus idaeus L.) is one of the most important fruit species in Serbia; there are currently approximately 11000 ha under cultivation. Wild red raspberry populations can be an important source of genes for breeding new raspberry varieties. This study was carried out to determine the genetic variability of wild populations, as well as their relationship with the most common cultivated cultivars using amplified fragment length polymorphism (AFLP) markers. In this study were included 128 individuals from seven wild populations and seven cultivar specimens from a nursery ('Mekeer', 'Willamette', 'Polka', 'Polana', 'Loganberry', 'Tayberry', 'Black Jewel'). Four AFLP combinations yielded a total of 247 polymorphic bands in 135 R. idaeus specimens. Analysis of AFLP markers showed that the cultivars are clearly distinct from wild populations. Population P4 (Mt. Ozren) had the highest gene diversity, the highest number of private markers and the highest rarity index. Analysis of molecular variance among and within wild populations showed that the most of the genetic diversity was attributable to differences between individuals within populations. A Mantel test showed that differences in altitude play the crucial role in structuring of the genetic diversity of red raspberry populations. Altitude contributed more to the population differentiation than spatial distance. Populations of wild raspberry from the territory of Serbia are characterized by high diversity, and should be protected and used as a resource in selection and breeding processes for the production of new varieties, with improved properties in terms of better yield and stronger resistance to external factors. [ABSTRACT FROM AUTHOR]

The inheritance of phenotypic, genetic and epigenetic traits in hybridization events is difficult to predict, as numerous evolutionary, ecological, and genetic factors can play a crucial role in the process of hybridization. In the middle Adriatic island of Vis, we investigated hybridization between Salvia officinalis and S. fruticosa at morphological, genetic and epigenetic levels. SSR results revealed that hybrid individuals were characterized by diploid set of chromosomes suggesting homoploid hybridization. A well-defined group that mostly comprised of F1 generation individuals was detected. For the majority of analysed morphological characteristics, hybrids were placed in-between parental taxa, while at the same time, values of different genetic parameters were mostly higher in hybrids than in parental species. The results revealed a high contrast in the levels of phenotypic variability and epigenetic excitation between parental taxa. Environmental niche modelling confirmed that in the studied location S. officinalis experiences optimal climatological conditions, while S. fruticosa struggles with unsuitable conditions. Very low levels of gene flow between the parental species were detected. In addition, contrasting levels of epigenetic excitation in the studied groups clearly demonstrated the importance of an epigenetic response to an altered environment and confirmed the trans-generational nature of the epigenetic changes. [ABSTRACT FROM AUTHOR]