Zorica S. Mitić

Geographically Related Variation in Epicuticular Wax Traits of Pinus nigra Populations from Southern Carpathians and Central Balkans – Taxonomic Considerations

The chemical composition of epicuticular waxes of nine populations from three Pinus nigra J. F. Arnold subspecies (namely subsp. nigra, subsp. banatica (Borbás) Novák, and subsp. pallasiana (Lamb.) Holmboe) from Southern Carpathians and central Balkan Peninsula were analyzed using GC/MS and GC/FID chromatography, and multivariate statistical techniques with respect to biogeography and taxonomy. In the needle waxes, four primary alcohols and 14 n‐alkanes ranging from C21 to C33 were identified, and the most abundant compounds were the four odd‐numbered n‐alkanes C27, C25, C23, and C29. Multivariate statistical analyses (CDA and CA) have shown existence of three P. nigra groups and suggested clinal differentiation as a mechanism of genetic variation across a geographic area: the first group consisted of the southernmost populations of subsp. pallasiana from Macedonia, the second consisted of the northernmost subsp. banatica populations from Romania, while all populations in Serbia described as three different subspecies (nigra, banatica, and pallasiana) formed the third group together with subsp. nigra population from Bosnia and Herzegovina. According to simple linear regression, geographic latitude and four bioclimatic parameters were moderately correlated with the contents of epicuticular wax compounds that are important in population discrimination, while stepwise multiple regression showed that latitude participated in most of the regression models for predicting the composition of the epicuticular waxes. These results agree with CDA and CA analysis, and confirmed the possibility of recognition of fine geographic differentiation of the analyzed P. nigra populations.

Needle Terpenes as Chemotaxonomic Markers in Pinus: Subsections Pinus and Pinaster

Chemical compositions of needle essential oils of 27 taxa from the section Pinus, including 20 and 7 taxa of the subsections Pinus and Pinaster, respectively, were compared in order to determine chemotaxonomic significance of terpenes at infrageneric level. According to analysis of variance, six out of 31 studied terpene characters were characterized by a high level of significance, indicating statistically significant difference between the examined subsections. Agglomerative hierarchical cluster analysis has shown separation of eight groups, where representatives of subsect. Pinaster were distributed within the first seven groups on the dendrogram together with P. nigra subsp. laricio and P. merkusii from the subsect. Pinus. On the other hand, the eighth group included the majority of the members of subsect. Pinus. Our findings, based on terpene characters, complement those obtained from morphological, biochemical, and molecular parameters studied over the past two decades. In addition, results presented in this article confirmed that terpenes are good markers at infrageneric level.

Terpenes are useful markers in differentiation of natural populations of relict pines Pinus heldreichii, P. nigra and P. peuce

Comparative analysis of terpene diversity and differentiation of relict pines Pinus heldreichii, P. nigra, and P. peuce from the central Balkans was performed at the population level. Multivariate statistical analyses showed that the composition of needle terpenes reflects clear divergence among the pine species from different subgenera: P. peuce (subgenus Strobus) vs. P. nigra and P. heldreichii (subgenus Pinus). In addition, despite the described morphological similarities and the fact that P. nigra and P. heldreichii may spontaneously hybridize, our results indicated differentiation of their populations naturally growing in the same area. In accordance with recently proposed concept of ‘flavonic evolution’ in the genus Pinus, we assumed that the terpene profile of soft pine P. peuce, defined by high amounts of six monoterpenes, is more basal than those of hard pines P. nigra and P. heldreichii, which were characterized by high content levels of mainly sesquiterpenes. In order to establish precise positions of P. heldreichii, P. nigra and P. peuce within the taxonomic and phylogenetic tree, as well as develop suitable conservation strategies and future breeding efforts, it is necessary to perform additional morphological, biochemical, and genetic studies.

Morphological variability of Tragopogon pterodes Pančić ex Petrović achenes: taxonomic evaluation of heterocarpy

Abstract This study presents the carpological heterogeneity of Tragopogon pterodes, an endemic plant species from Balkan Peninsula. The goal of this paper was to determine the variability of morphological characters of achenes and assess the degree of their morphological differentiation relative to the position they occupy in the receptacle. Analysis included four different populations of T. pterodes and examination of 18 (8 qualitative and 10 quantitative) characters of achenes in total. Data analysis employed basic statistic, univariate (ANOVA, Tukey HSD) and multivariate (CDA, CA) analyses to check the level of heterocarpy in this species. The results indicate possible morphological differentiation of three groups of achenes (outer, median and inner achenes), depending of their position on the receptacle. The members of all three types were described, illustrated and compared. The variability of investigated carpological characters in this species is providing important information prior to their further taxonomic treatment.

Chemotaxonomic Considerations of the n-Alkane Composition in Pinus heldreichii, P. nigra, and P. peuce

The n‐alkane composition in the leaf cuticular waxes of natural populations of Bosnian pine (Pinus heldreichii), Austrian pine (P. nigra), and Macedonian pine (P. peuce) was compared for the first time. The range of n‐alkanes was wider in P. nigra (C16 – C33) than in P. heldreichii and P. peuce (C18 – C33). Species also diverged in abundance and range of dominant n‐alkanes (P. heldreichii: C23, C27, and C25; P. nigra: C25, C27, C29, and C23; P. peuce: C29, C25, C27, and C23). Multivariate statistical analyses (PCA, DA, and CA) generally pointed out separation of populations of P. nigra from populations of P. heldreichii and P. peuce (which were, to a greater or lesser extent, separated too). However, position of these species on the basis of n‐alkane composition was in accordance neither with infrageneric classification nor with recent molecular and terpene investigations.

Epicuticular waxes provide insights into chemical differentiation of natural populations of Pinus mugo Turra sensu stricto

Phytochemical diversity and differentiation of nine native populations of Pinus mugo sensu stricto from Julian Alps, Southern Carpathians and Balkan Peninsula were analyzed in regard to their epicuticular wax compounds. GC/MS and GC‐FID analyses of the hexane extracts of needle samples, collected from 118 individuals of P. mugo, revealed the presence of six diterpenes, three primary alcohols and 10 n‐alkanes ranging from C19 to C29 (except n‐alkane C21). According to simple linear regression, the contents of epicuticular wax compounds showed generally weak correlations with tested bioclimatic, orographic and geographic parameters, leading to assumption about their genetic conditioning. The multivariate statistical analyses suggested the existence of two chemical entities: the Alpine and the South Carpathian, while the Balkan populations appeared heterogeneous as three of them belonged to the Alpine and one to the South Carpathian group. The obtained results are largely consistent to those previously published in reference to morpho‐anatomical and molecular characters of P. mugo, supporting the hypothesis of colonization of Balkan Peninsula from two different glacial refugia: from the Alps (across the Dinarides) and from the Southern Carpathians. Extending this study to the entire range of P. mugo complex should provide a clearer picture of its phytochemical diversity and differentiation.

Epidermal structures and composition of epicuticular waxes of Sedum album sensu lato (Crassulaceae) in Balkan Peninsula

Abstract Sedum album is a succulent plant, with phenotypic variability resulting in delimitation of a number of taxa with diverse taxonomic recognition. We analyzed the taxa from Sedum album in broad sense (S. album, S. micranthum, S. athoum and S. serpentini) from Balkan Peninsula, to check whether the variability of their epidermal structures and epicuticular wax composition corresponds to their taxonomic reliability. Epidermal structures and epicuticular waxes were analyzed using light and scanning electron microscopy, gas chromatography-mass spectrometry and gas chromatography-flame ionization detector chromatography and multivariate statistical techniques. Analyses of studied taxa have shown that significant systematic characters include adaxial epidermal and guard cell length, stomatal frequency and subsidiary cells area on abaxial epidermis. The content of n-alkanes C29, C30, C27, C32 and C33 was shown to be valuable criterion for their phytochemical delimitation. The comparison of epicuticular wax microstructures has shown no significant differences. Results indicate presence of two (micromorphology) or three (phytochemistry) well-defined groups of populations, with S. serpentini manifesting a considerable level of separation toward the others. Sedum athoum have shown weak to moderate degree of differentiation, while S. micranthum has shown general resemblance to S. album.