Apomictic Mountain Whitebeam (Sorbus austriaca, Rosaceae) Comprises Several Genetically and Morphologically Divergent Lineages.

The interplay of polyploidisation, hybridization, and apomixis contributed to the exceptional diversity of Sorbus (Rosaceae), giving rise to a mosaic of genetic and morphological entities. The Sorbus austriaca species complex from the mountains of Central and South-eastern Europe represents an allopolyploid apomictic system of populations that originated following hybridisation between S. aria and S. aucuparia. However, the mode and frequency of such allopolyploidisations and the relationships among different, morphologically more or less similar populations that have often been described as different taxa remain largely unexplored. We used amplified fragment length polymorphism (AFLP) fingerprinting, plastid DNA sequencing, and analyses of nuclear microsatellites, along with multivariate morphometrics and ploidy data, to disentangle the relationships among populations within this intricate complex. Our results revealed a mosaic of genetic lineages-many of which have not been taxonomically recognised-that originated via multiple allopolyploidisations. The clonal structure within and among populations was then maintained via apomixis. Our results thus support previous findings that hybridisation, polyploidization, and apomixis are the main drivers of Sorbus diversification in Europe.

Phytochemicals and Their Correlation with Molecular Data in Micromeria and Clinopodium (Lamiaceae) Taxa.

A study of the phytochemical and molecular characteristics of ten Micromeria and six Clinopodium taxa (family Lamiaceae) distributed in the Balkan Peninsula was carried out. The phytochemicals detected in essential oils by gas chromatography, mass spectrometry, and molecular data amplified fragment length polymorphism were used to study the taxonomic relationships among the taxa and the correlations between phytochemical and molecular data. STRUCTURE analysis revealed three genetic groups, while Bayesian Analysis of Population Structure grouped the studied taxa into 11 clusters nested in the groups obtained by STRUCTURE. Principal components analysis performed with the 21 most represented compounds in the essential oils yielded results that were partly consistent with those obtained by STRUCTURE and neighbour-joining analyses. However, their geographic distributions did not support the genetic grouping of the studied taxa and populations. The Mantel test showed a significant correlation between the phytochemical and genetic data (r = 0.421, p < 0.001). Genetic distance explained 17.8% of the phytochemical distance between populations. The current taxonomic position of several of the studied taxa is yet to be satisfactorily resolved, and further studies are needed. Such future research should include nuclear and plastid DNA sequences from a larger sample of populations and individuals.

Genome Size, Cytotype Diversity and Reproductive Mode Variation of Cotoneaster integerrimus (Rosaceae) from the Balkans.

Cotoneaster integerrimus represents a multiploid and facultative apomictic system of widely distributed mountain populations. We used flow cytometry to determine genome size, ploidy level, and reproduction mode variation of the Balkan populations, supplemented by analysis of nuclear microsatellites in order to address: (i) geographic distribution and variation of cytotypes among the populations; (ii) variation of reproduction mode and the frequency of sexuality; (iii) pathways of endosperm formation among the sampled polyploids and their endosperm balance requirements; (iv) genotypic diversity and geographic distribution of clonal lineages of polyploids. The prevalence of apomictic tetraploid cytotype followed by sexual diploids and extremely rare triploids was demonstrated. This prevalence of tetraploids affected the populations' structure composed from clonal genotypes with varying proportions. The co-occurrence of diploids and tetraploids generated higher cytotype, reproductive mode, and genotypic diversity, but mixed-ploidy sites were extremely rare. The endosperm imbalance facilitates the development and the occurrence of intermediate triploids in mixed-ploidy populations, but also different tetraploid lineages elsewhere with unbalanced endosperm. All these results showed that the South European populations of C. integerrimus have higher levels of cytotype and reproductive diversity compared to the Central European ones. Therefore, the South European populations can be considered as a potential reservoir of regional and global diversity for this species.

Micromorphological Traits of Balcanic Micromeria and Closely Related Clinopodium Species (Lamiaceae).

A study of the trichomes types and distribution and pollen morphology was carried out in nine Micromeria taxa (M. cristata ssp. cristata, M. cristata ssp. kosaninii, M. croatica, M. graeca ssp. graeca, M. graeca ssp. fruticulosa, M. juliana, M. kerneri, M. longipedunculata and M. microphylla) and five closely related Clinopodium species (C. dalmaticum, C. frivaldszkyanum, C. pulegium, C. serpyllifolium and C. thymifolium) from the Lamiaceae family of the Balkan Peninsula. By scanning electron microscope, non-glandular trichomes, peltate and capitate trichomes were observed on the calyx, leaves and stem of the studied species. Two subtypes of capitate trichomes were observed in Micromeria species: subtype 1 (consisting of a basal epidermal cell and an elliptically shaped head cell) and subtype 2 (consisting of a basal epidermal cell, two to three stalk cells and a round head cell). In Clinopodium species, three types of capitate trichomes were observed: subtype 1, subtype 3 (consisting of a basal epidermal cell, a short peduncle cell, and a single round head cell), and subtype 4 (consisting of a basal epidermal cell, a stalk cell, and an elongated head cell). These results support the recent transfer of Micromeria species from the section Pseudomelissa to the genus Clinopodium.

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.

Endemic Veronica saturejoides Vis. ssp. saturejoides-Chemical Composition and Antioxidant Activity of Free Volatile Compounds.

Chemical profile and antioxidant activity of the species Veronica saturejoides Vis. ssp. saturejoides (Plantaginaceae)-which is endemic to Croatia, Bosnia and Herzegovina and Montenegro -were investigated. Volatile compounds produced by glandular trichomes (composed of one stalk cell and two elliptically formed head cells according to scanning electron microscope investigation) were isolated from the plants collected in two locations. Additionally, as a part of specialized metabolites, total polyphenols, total tannins, total flavonoids and total phenolic acids were determined spectrophotometrically. In the lipophilic volatile fractions-essential oils, the most abundant compounds identified were hexahydrofarnesyl acetone, caryophyllene oxide and hexadecanoic acid. In total, the class of oxygenated sesquiterpenes and the group of fatty aldehydes, acids and alcoholic compounds dominated in the essential oils. In the hydrophilic volatile fractions-hydrosols, the most abundant compounds identified were trans-p-mentha-1(7),8-dien-2-ol, allo-aromadendrene and (E)-caryophyllene. A group of oxygenated monoterpenes and the sesquiterpene hydrocarbons dominated in the hydrosols. Antioxidant activity of essential oils and hydrosols was tested with two methods: 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC). Essential oils showed higher antioxidant activity than hydrosols and showed similar antioxidant activity to Rosmarinus officinalis essential oil. Obtained results demonstrate that this genus is a potential source of volatiles with antioxidant activity.

Epigenetic Differentiation of Natural Populations of Lilium bosniacum Associated with Contrasting Habitat Conditions.

Epigenetic variation in natural populations with contrasting habitats might be an important element, in addition to the genetic variation, in plant adaptation to environmental stress. Here, we assessed genetic, epigenetic, and cytogenetic structure of the three Lilium bosniacum populations growing on distinct habitats. One population was growing under habitual ecological conditions for this species and the other two were growing under stress associated with high altitude and serpentine soil. Amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses revealed that the three populations did not differentiate genetically, but were clearly separated in three distinct clusters according to DNA methylation profiles. Principal coordinate analysis showed that overall epigenetic variation was closely related to habitat conditions. A new methylation-sensitive amplification polymorphism scoring approach allowed identification of mainly unmethylated (φST = 0.190) and fully CpG methylated (φST = 0.118) subepiloci playing a role in overall population differentiation, in comparison with hemimethylated sites (φST = 0.073). In addition, unusual rDNA repatterning and the presence of B chromosomes bearing 5S rDNA loci were recorded in the population growing on serpentine soil, suggesting dynamic chromosome rearrangements probably linked to global genome demethylation, which might have reactivated some mobile elements. We discuss our results considering our earlier data on morphology and leaf anatomy of several L. bosniacum populations, and suggest a possible role of epigenetics as a key element in population differentiation associated with environmental stress in these particular lily populations.

When sexual meets apomict: genome size, ploidy level and reproductive mode variation of Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina.

BACKGROUND AND AIMS: Allopolyploidy and intraspecific heteroploid crosses are associated, in certain groups, with changes in the mating system. The genus Sorbus represents an appropriate model to study the relationships between ploidy and reproductive mode variations. Diploid S. aria and tetraploid apomictic S. austriaca were screened for ploidy and mating system variations within pure and sympatric populations in order to gain insights into their putative causalities. METHODS: Flow cytometry was used to assess genome size and ploidy level among 380 S. aria s.l. and S. austriaca individuals from Bosnia and Herzegovina, with 303 single-seed flow cytometric seed screenings being performed to identify their mating system. Pollen viability and seed set were also determined. KEY RESULTS: Flow cytometry confirmed the presence of di-, tri- and tetraploid cytotype mixtures in mixed-ploidy populations of S. aria and S. austriaca. No ploidy variation was detected in single-species populations. Diploid S. aria mother plants always produced sexually originated seeds, whereas tetraploid S. austriaca as well as triploid S. aria were obligate apomicts. Tetraploid S. aria preserved sexuality in a low portion of plants. A tendency towards a balanced 2m : 1p parental genome contribution to the endosperm was shared by diploids and tetraploids, regardless of their sexual or asexual origin. In contrast, most triploids apparently tolerated endosperm imbalance. CONCLUSIONS: Coexistence of apomictic tetraploids and sexual diploids drives the production of novel polyploid cytotypes with predominantly apomictic reproductive modes. The data suggest that processes governing cytotype diversity and mating system variation in Sorbus from Bosnia and Herzegovina are probably parallel to those in other diversity hotspots of this genus. The results represent a solid contribution to knowledge of the reproduction of Sorbus and will inform future investigations of the molecular and genetic mechanisms involved in triggering and regulating cytotype diversity and alteration of reproductive modes.

Morphological, genetic and phytochemical variation of the endemic Teucrium arduini L. (Lamiaceae).

Analysis of the morphological traits of leaves, genetic variability (analyzed by AFLP) and chemical composition of essential oils (analyzed by GC-MS) was conducted on eleven populations of the endemic Illyric-Balcanic species Teucrium arduini L. in Croatia, Bosnia and Herzegovina, and Montenegro. Average blade length and width ranged from 20.00 to 31.47mm and from 11.58 to 15.66mm, respectively. Multivariate analysis (PCA, UPGMA) of morphological traits distinguished two continental Bosnian populations from the remaining populations. AFLP analysis separated the investigated populations into two groups based primarily on geographical distance. Essential oil analysis showed a total of 52 compounds, with two chemotypes distinguished based on the essential oil profile. The first was a sesquiterpene chemotype, with ß-caryophyllene, germacrene D or caryophyllene oxide as the major compounds, while the second was an oxygenated monoterpene chemotype, with pulegone and piperitone oxide as the main components. The Mantel test showed a stronger correlation between the morphological traits and AFLP than between the essential oil profile and AFLP. The test also showed a stronger association between the essential oil profile and geographical position than between the morphological traits and geographical position.

Micromorphological traits and essential oil contents of Micromeria kerneri Murb. and M. juliana (L.) Benth. (Lamiaceae).

The chemical composition of the essential oil (analysed by GC and GC-MS), the types and distribution of trichomes and pollen morphology (analysed by scanning electron microscopy) were investigated in two closely related species, Micromeria kerneri Murb. and Micromeria juliana (L.) Benth. (Lamiaceae) from Southeast Europe as a contribution to their taxonomy. The essential oil of M. kerneri was characterized by a high concentration of oxygenated sesquiterpenes, with caryophyllene-oxide as the major compound. Caryophyllene-oxide was also the major component of the essential oil of M. juliana from all localities, except from Mt Krivosije (Montenegro), where piperitone oxide was the major constituent. Non-glandular trichomes, peltate trichomes, and two types of capitate trichomes (type 1 composed of one basal epidermal cell, and one head cell with subcuticular space; type 2 composed of one basal epidermal cell, two stalk cells, and one head cell with subcuticular space) were observed on leaves, the calyx and on the stem. Pollen of both species had six apertures (hexacolpate) set in the equatorial pollen belt (zonocolpate) and showed medium reticulate ornamentation. Multivariate analysis (PCA and UPGMA) of essential oil components clearly separated the investigated M. kerneri and M. juliana populations, and confirmed the opinion that they are different taxa. On the other hand, micromorphological traits between these species were the same. Nevertheless, definitive conclusions about the taxonomic relationships among these species will require genetic analysis.

Micromorphological and chemotaxonomical traits of Micromeria croatica (Pers.) Schott.

A study on the types and distribution of trichomes, pollen morphology, chemical composition of essential oil (analyzed by GC-FID and GC/MS), and the content of macroelements (Na, K, Ca, and Mg) and trace elements (B, Fe, Cu, Mn, Zn, Al, Pb, Cr, Cd, Ni, Hg, and As) analyzed by ICP-AES (=inductively coupled plasma atomic emission spectroscopy) was conducted on Micromeria croatica (Pers.) Schott. Non-glandular trichomes, peltate trichomes (typical hairs of Lamiaceae), small capitate trichomes (composed of one basal epidermal cell and one head cell), and larger capitate trichomes (composed of one basal epidermal cell, two to three stalk cells, and one head cell with subcuticular space) were observed on leaves, stems, the calyx, and corolla. Pollen of M. croatica had six apertures (hexacolpate) which were set in the equatorial pollen belt (zonocolpate) and showed medium reticulate ornamentation (hetrobrochate type). The essential oil contained from 27 to 39 constituents, the most representative of which were caryophyllene oxide and E-caryophyllene. Among the investigated macroelements, the content of K was highest (8730-10080 mg/kg). The content of trace elements ranged from 0.12 mg/kg (Cr) to 78.00 mg/kg (Fe), while the content of Cd, Ni, Hg, and As were lower than the limit of quantification.