Antioxidant and Antiproliferative Activities of Kale (Brassica oleracea L. Var. acephala DC.) and Wild Cabbage (Brassica incana Ten.) Polyphenolic Extracts.

Brassicaceae are rich in healthy phytochemicals that have a positive impact on human health. The aim of this study was to analyze the phenolic compounds and antioxidant and anticancer potential of traditional Croatian kale (Brassica oleracea L. var. acephala DC.) and wild cabbage (Brassica incana Ten.) extracts. The phenolic groups and antioxidant activity were determined by spectrophotometry, selected phenolic compounds (ferulic acid, sinapic acid, salicylic acid, kaempferol, and quercetin) were analyzed by LC-MS/MS, and anticancer potential was evaluated in vitro using HeLa cells. The extracts of both plant species are rich in phenolic compounds and showed significant antioxidant activity at similar levels. LC-MS/MS detected sinapic acid as the most abundant phenolic acid, followed by ferulic acid, while salicylic acid was present at lower concentrations. A comparative analysis showed that wild cabbage contained significantly more sinapic acid, while kale contained more kaempferol and quercetin. Both Brassica extracts at a concentration of 50 µg mL-1 showed an antiproliferative effect on HeLa cells, while they did not affect the proliferation of normal human skin fibroblasts. Wild cabbage extract also showed an antiproliferative effect on HeLa cells at a lower applied concentration of 10 µg mL-1 of extracts. The clonogenic analysis also revealed the inhibitory effect of the extracts on HeLa colony growth.

Biological Potential of Fruit and Leaves of Strawberry Tree (Arbutus unedo L.) from Croatia.

The strawberry tree fruit and leaf are a rich source of minerals, easily digestible sugars, dietary fibers, vitamins (especially vitamin C) and many bioactive compounds of significant functional value. Due to their favorable chemical composition, fruits have recently become increasingly popular in consumption. The aim of this study was to determine the physical-chemical composition, content of bioactive compounds, and also the antioxidant capacity of the fruit and leaves of wild strawberry tree populations among the Adriatic coast in Croatia, as well as to investigate the influence of location on the content of specific bioactive compounds. According to the obtained results, both fruit and leaves are pronouncedly high in vitamin C content, the average value for fruits amounted to 224.21 mg/100 g FW, while that for leaves amounted to 138.08 mg/100 g FW. Additionally, significantly high values of total polyphenolic compounds were recorded both in fruits (average value of 637.94 mg GAE/100 g FW) and especially in leaves (average value of 2157.01 mg GAE/100 g FW). Several pigments from different categories were determined in the fruit depending on fruit maturity, including: total anthocyanins, ß-carotene and lycopene; while in leaves chlorophylls and carotenoids. Given the high content of different bioactive compounds high values of antioxidant capacity were determined (the average value for fruits was 2269.96 µmol TE/kg and for leaves, 2237.16 µmol TE/kg). Location strongly influenced the physical-chemical composition and also the content of specialized metabolites; populations collected from southern areas (central and south Dalmatia) of the Adriatic coast tended to have higher amounts of vitamin C, total phenols, total anthocyanins and ß-carotene.

Disentangling relationships among the diploid members of the intricate genus Knautia (Caprifoliaceae, Dipsacoideae).

The genus Knautia (Caprifoliaceae, Dipsacoideae) encompasses 40-60 species mainly distributed in western Eurasia, with highest species diversity in the Alps and the Balkan Peninsula. It is traditionally regarded as one of the taxonomically most challenging European genera due to the widespread occurrence of polyploidy, the high incidence of hybridisation and the maintenance of morphologically intermediate forms. A prerequisite for assessing the complex spatiotemporal diversification of a polyploid group is a comprehensive hypothesis of the phylogenetic relationships among its diploid members. To this end, DNA sequence data (nrDNA ITS and plastid petN(ycf6)-psbM) combined with AFLP fingerprinting were performed on 148 diploid populations belonging to 35 taxa. Phylogenies obtained by maximum parsimony and Bayesian analyses were used to test the monophyly of the genus and its three sections Trichera, Tricheroides and Knautia, to provide insights into its evolutionary history and to test previous hypotheses of inter- and intrasectional classification. Both nuclear and chloroplast datasets support the monophyly of Knautia and its three sections, with ambiguous placement of K. cf. degenii. The majority of species belong to the nearly exclusively perennial section Trichera (x=10). Within section Trichera all markers revealed largely unresolved phylogenetic relationships suggesting rapid radiation and recent range expansion. In addition, extensive sharing of plastid haplotypes across taxa and wide geographic ranges of plastid haplotypes and ribotype groups were observed. The molecular data are partly at odds with the traditional informal grouping of taxa within section Trichera. Whereas the traditional groups of K. dinarica, K. drymeia and K. montana can be maintained, the new, smaller and well supported Midzorensis and Pancicii Groups as well as the SW European Group are separated from the heterogeneous traditional K. longifolia group. The former groups of K. arvensis, K. dalmatica, K. fleischmannii and K. velutina are clearly polyphyletic. Their diploid members have to be rearranged into the Xerophytic Group, the Carinthiaca Group, and the Northern and Southern Arvensis Groups. The annual sections Tricheroides (x=10) and Knautia (x=8) with only a few taxa are resolved in the ITS and plastid trees on long branches as early diverging lineages within the genus.

Genome Size Variation in Dianthus sylvestris Wulfen sensu lato (Caryophyllaceae).

Genome size (GS) is an important characteristic that may be helpful in delimitation of taxa, and multiple studies have shown correlations between intraspecific GS variation and morphological or environmental factors, as well as its geographical segregation. We estimated a relative GS (RGS) of 707 individuals from 162 populations of Dianthus sylvestris with a geographic focus on the Balkan Peninsula, but also including several populations from the European Alps. Dianthus sylvestris is morphologically variable species thriving in various habitats and six subspecies have been recognized from the Balkan Peninsula. Our RGS data backed-up with chromosome counts revealed that the majority of populations were diploid (2n = 30), but ten tetraploid populations have been recorded in D. sylvestris subsp. sylvestris from Istria (Croatia, Italy). Their monoploid RGS is significantly lower than that of the diploids, indicating genome downsizing. In addition, the tetraploids significantly differ from their diploid counterparts in an array of morphological and environmental characteristics. Within the diploid populations, the RGS is geographically and only partly taxonomically correlated, with the highest RGS inferred in the southern Balkan Peninsula and the Alps. We demonstrate greater RGS variation among the Balkan populations compared to the Alps, which is likely a result of more pronounced evolutionary differentiation within the Balkan Peninsula. In addition, a deep RGS divergence within the Alps likely points to persistence of the alpine populations in different Pleistocene refugia.

Stability in the South, Turbulence Toward the North: Evolutionary History of Aurinia saxatilis (Brassicaceae) Revealed by Phylogenomic and Climatic Modelling Data.

The Balkan Peninsula played an important role in the evolution of many Mediterranean plants and served as a major source for post-Pleistocene colonisation of central and northern Europe. Its complex geo-climatic history and environmental heterogeneity significantly influenced spatiotemporal diversification and resulted in intricate phylogeographic patterns. To explore the evolutionary dynamics and phylogeographic patterns within the widespread eastern Mediterranean and central European species Aurinia saxatilis, we used a combination of phylogenomic (restriction-site associated DNA sequencing, RADseq) and phylogenetic (sequences of the plastid marker ndhF) data as well as species distribution models generated for the present and the Last Glacial Maximum (LGM). The inferred phylogenies retrieved three main geographically distinct lineages. The southern lineage is restricted to the eastern Mediterranean, where it is distributed throughout the Aegean area, the southern Balkan Peninsula, and the southern Apennine Peninsula, and corresponds to the species main distribution area during the LGM. The eastern lineage extends from the eastern Balkan Peninsula over the Carpathians to central Europe, while the central lineage occupies the central Balkan Peninsula. Molecular dating places the divergence among all the three lineages to the early to middle Pleistocene, indicating their long-term independent evolutionary trajectories. Our data revealed an early divergence and stable in situ persistence of the southernmost, eastern Mediterranean lineage, whereas the mainland, south-east European lineages experienced more complex and turbulent evolutionary dynamics triggered by Pleistocene climatic oscillations. Our data also support the existence of multiple glacial refugia in southeast Europe and highlight the central Balkan Peninsula not only as a cradle of lineage diversifications but also as a source of lineage dispersal. Finally, the extant genetic variation within A. saxatilis is congruent with the taxonomic separation of peripatric A. saxatilis subsp. saxatilis and A. saxatilis subsp. orientalis, whereas the taxonomic status of A. saxatilis subsp. megalocarpa remains doubtful.

Centaurealovricii, a new species of C. sect. Centaurea (Asteraceae) from Croatia.

A new species, Centaurealovricii, is described and illustrated from the island of Vis (Dalmatia, Croatia). It occurs on northwest-facing calcareous cliffs near the sea, where it grows with several other rare endemic species. Centaurealovricii is morphologically similar to C.glaberrima and C.divergens of C.sect.Centaurea, from which it differs in having more succulent leaves with larger and less incised leaflets, bigger capitula, larger phyllaries with more developed appendages and denser and undulate fimbriae, larger florets, bigger achenes, and longer pappus. Its morphological features, distribution, ecology, conservation status and taxonomic affinities are examined. In addition, a new iconography and lectotypification for C.glaberrima and C.divergens is provided.

Loncomelos koprulense (Asparagaceae), a new species from southern Turkey.

A new species, Loncomelos koprulense (Asparagaceae), is described and illustrated from southern Turkey. It is a very rare endemic species growing on small semi-rocky escarpments within the Köprülü Kanyon in the province of Antalya. Morphologically for its hairy leaves, L. koprulense shows some relationships with L. malatyanum and L. tardum, species localized in Anatolia too. The chromosome number of the new species is 2n = 2x = 22. Geographical distribution map for L. koprulense, L. malatyanum and L. tardum is provided.

On the identity of Thymushumifususvar.aureopunctatus (Lamiaceae) and taxonomic notes on the Th.richardii complex.

The name Thymushumifususvar.aureopunctatus, described from Bosnia and Herzegovina, is lectotypified, and its taxonomic value is discussed. Thymusrichardiisubsp.richardii is currently considered an endemic subspecies common to Mallorca (Balearic Islands) and Bosnia and Herzegovina from the Balkan Peninsula. Specimens identified as Th.richardii from both Balearic Islands and Bosnia and Herzegovina were studied to determine if they are indeed the same taxonomic entity. Detailed micromorphological observations and morphometric analysis, suggest that the Balkan plants (Th.humifususvar.aureopunctatus) and the Majorcan populations (Th.richardiisubsp.richardii) are clearly separate entities. For the former name, based on morphological, phytochemical, biogeographical and present results, we propose the subspecific rank, as Th.richardiisubsp.aureopunctatuscomb. & stat. nov. Full descriptions of all five subspecies currently accepted within Th.richardii are provided.

Morphological, genetic and epigenetic aspects of homoploid hybridization between Salvia officinalis L. and Salvia fruticosa Mill.

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.