Hybridisation processes in sympatric populations of pines Pinus sylvestris L., P. mugo Turra and P. uliginosa Neumann

Natural hybridisation was postulated between the closely related pine species Pinus sylvestris and the P. mugo complex, however no clear evidence on propagation of mature hybrids in nature has been documented so far. To test the hybridisation hypothesis we applied chloroplast DNA (cpDNA) markers and isozymes in the analyses of 300 individuals representing the variety of morphological forms in the sympatric populations of P. sylvestris, P. mugo and P. uliginosa at the peat bog complex in the Sudety Mts., Poland. Additionally, the haplotypes of paternally inherited cpDNA of 149 open pollinated progeny derived from seeds were compared to the haplotypes of parental trees to access the intensity and direction of contemporary hybridisation. The morphologically highly variable polycormic (multi-stemmed) hybrids between P. mugo and P. uliginosa were identified. The second group of hybrids was found among the monocormic (single-stemmed) P. sylvestris-like individuals carrying the cpDNA from P. mugo complex. Hybrids of P. sylvestris as a pollen donor and P. mugo or P. uliginosa as a mother were not found, either in the group of examined trees, or among the open pollinated progeny. The results indicate that numerous hybrids can exist in the sympatric population of the species studied and that gene flow can successfully proceed from P. mugo complex to P. sylvestris. Hybridisation and ecological selection seems to play a significant role in diversification and evolution of the investigated species.     

Cross-species amplification and characterization of microsatellite loci in Pinus mugo Turra

Pinus mugo (dwarf mountain pine) is an important component of European mountain ecosystems. However, little is known about the present genetic structure and population differentiation of this species at the DNA level, possibly due to a lack of nuclear microsatellite markers (SSR) developed for Pinus mugo. Therefore in this study we transferred microsatellite markers originally developed for Pinus sylvestris and Pinus taeda to Pinus mugo. This cross-species amplification approach is much faster and less expensive than isolation and characterization of new microsatellite markers. The transfer rates from the source species to Pinus mugo were moderately low (26%). There were no differences in microsatellite repeat motifs between the source species and Pinus mugo. Nuclear microsatellite markers successfully transferred to Pinus mugo can be applied to various genetic studies on this species, due to the high level of their polymorphism and high value of polymorphic information content.     

Genetic comparison between Pinus sylvestris and P. mugo using isozymes and chloroplast DNA

Pinus sylvestris and P. mugo populations from Poland and Czechoslovakia were compared using genetic variability at isozyme markers, chloroplast DNA variation, and mating system measurements. Two isozyme loci were found to differ between the species. P. mugo was as variable at isozyme loci as P. sylvestris. Diagnostic cpDNA fragments were found using the restriction enzyme Bcl-I. Populations that were morphologically classified as hybrids were found to be pure species, based both on isozyme and cpDNA results.     

Reciprocal controlled crosses between Pinus sylvestris and P. mugo verified by a species-specific cpDNA marker

A species-specific marker of cpDNA (paternally inherited in pines) was used to verify the hybrid origin of seedlings from controlled reciprocal crosses between Pinus sylvestris and P. mugo. A very low degree of compatibility between those two species has been revealed. In the three consecutive years of experiments, no filled seeds were obtained in the combination with P. mugo as the seed parent. From P. sylvestris as the seed parent and P. mugo as the pollen donor, we succeeded to obtain four filled seeds (about 1 %), but only in one year. The seedling obtained from the seeds had cpDNA haplotypes specific to P. mugo, which proves their hybrid origin. This method enables verification of the result of controlled crosses. The importance of the results has been discussed in the aspect of postulated natural hybridisation in sympatric populations of the two species.     

Secondary Metabolites from Pinus mugo Turra subsp. mugo Growing in the Majella National Park (Central Apennines,Italy)

In this study, we examined the composition regarding secondary metabolites of P. mugo Turra ssp. mugo growing in the protected area of Majella National Park, which is the southernmost station of the habitat of this species. Both the nonpolar and polar fractions were considered. In particular, the essential‐oil composition showed a high variety of compounds, and 109 compounds were detected, and 101 were identified, among which abietane‐type compounds have a taxonomic relevance. Abietanes were also isolated from the polar fraction, together with an acylated flavonol and a remarkably high amount of shikimic acid.     

Genetic diversity of isoenzymes in mountain pine (Pinus mugo Turra) in natural populations in the Ukrainian Carpathian mountains

Electrophoretic spectra of GOT, GDH, DIA, MDH, SOD, FDH, ADH, ACP, IDH enzymes in the megagametophytes of seeds of 69 mountain pine (Pinus mugo Turra) trees from natural populations of the Ukrainian Carpathian mountains have been described. 19 loci products had efficient electrophoretic separation. The analysis of alleles segregation of the heterozygous trees on the whole confirms monogenic inheritance of the discovered variants.     

Genetic variation and differentiation of peat-bog and dry-meadow populations of the dwarf mountain pine Pinus mugo Turra in the highlands of the Ukrainian Carpathians

Based on electrophoretic analysis of 21 isozyme loci controlling 10 enzyme systems, the intra- and inter-population variation was studied in two peat-bog and three dry-meadow populations of the dwarf mountain pine Pinus mugo Turra from the highlands of the Ukrainian Carpathians. In the studied samples (a total of 164 trees), on average 62% of the studied genes were polymorphic; the mean heterozygosity was 21.3%. The dry-meadow populations differed from the peat-bog populations by allele and genotype diversity and by heterozygosity although the indices characterizing population heterogeneity (Fst and Gst) were small (0.027 and 0.032, respectively). Nei's genetic distances between the populations ranged of 0.011 to 0.032 with the mean of 0.018.     

Essential‐Oil Variability in Natural Populations of Pinus mugo Turra from the Julian Alps

The composition and variability of the terpenes and their derivatives isolated from the needles of a representative pool of 114 adult trees originating from four natural populations of dwarf mountain pine (Pinus mugo Turra ) from the Julian Alps were investigated by GC‐FID and GC/MS analyses. In total, 54 of the 57 detected essential‐oil components were identified. Among the different compound classes present in the essential oils, the chief constituents belonged to the monoterpenes, comprising an average content of 79.67% of the total oil composition (74.80% of monoterpene hydrocarbons and 4.87% of oxygenated monoterpenes). Sesquiterpenes were present in smaller amounts (average content of 19.02%), out of which 16.39% were sesquiterpene hydrocarbons and 2.62% oxygenated sesquiterpenes. The most abundant components in the needle essential oils were the monoterpenes δ‐car‐3‐ene, β‐phellandrene, α‐pinene, β‐myrcene, and β‐pinene and the sesquiterpene β‐caryophyllene. From the total data set of 57 detected compounds, 40 were selected for principal‐component analysis (PCA), discriminant analysis (DA), and cluster analysis (CA). The overlap tendency of the four populations suggested by PCA, was as well observed by DA. CA also demonstrated similarity among the populations, which was the highest between Populations I and II.     

DNA marker based identification of spontaneous hybrids between Pinus mugo and P. sylvestris at the Lithuanian sea‐side

The objective was to identify spontaneous hybrids between P. mugo and P. sylvestris using organelle DNA markers in sympatric zones at the sea‐side spit of Kursiu Nerija in western Lithuania. A field inventory was carried out over the entire Lithuanian part of the spit and 203 individuals morphologically intermediate between P. sylvestris and P. mugo were tested for their male parent with chloroplast DNA PCR‐RFLP markers and for their female parent with mitochondrial DNA PCR markers. Unfortunately, the mitochondrial DNA Nad7 marker failed to identify the female parent species in our study. However, the chloroplast DNA PCR‐RFLP marker revealed that out of 203 tested individuals only 23 had a paternity different than that indicated by morphology. Of these, 13 individuals were morphologically identified as P. sylvestris but possessed cpDNA of P. mugo (putative hybrids with P. sylvestris (female) ×P. mugo (male parent), and 10 individuals morphologically identified as P. mugo possessed cpDNA of P. sylvestris and may be hybrids with P. mugo (female) ×P. sylvestris (male parent). The remainder of the 177 individuals identified in the field inventory were were considered as pure species. In conclusion, our study indicates ongoing spontaneous hybridization between P. mugo and P. syvestris in Kursiu Nerija. Human impact via seed transfer on altered hybridization rates as well as evolutionary consequences are discussed.     

Abortive embryogenesis in hybrid swarm populations of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L. and <Emphasis Type="Italic">Pinus muga</Emphasis> Turra

Comparative study on fertilization process in Pinus sylvestris, Pinus mugo and in their putative hybrid swarm individuals was done involving pre-zygotic and post-zygotic stages. The amount of surviving ovules from open pollination reflecting the mode of interaction between pollen grains and nucellar tissue of an ovule averaged at 8.1 of sound ovules per conelet in Pinus sylvestris, 7.3 ovules in the hybrid swarm population and at 4.9 ovules in Pinus mugo. A strong correlation was observed between the number of surviving ovules and the proportion of germinating seeds in the compared species and hybrids. Normal course of embryogenesis in Pinus sylvestris and Pinus mugo contrasted with increased frequency of disturbances observed in the hybrid swarm individuals. The differential survival rates of the ovules and deviations from typical pattern of embryogenesis are discussed from the standpoint of cross-ability relationship between Pinus sylvestris and Pinus mugo.     

Dry-matter Production by Pinus sylvestris L.

This paper contains the data for the gross weight and relativeproportions of canopy, bole, and roots in a series of twelveeven-aged forest stands. From these it is possible to describe the growth curves of treesof Pinus sylvestris in Britain under the observed conditionsand the production of dry matter per unit area of such standsof trees. The following general results are given: regressionsrelating weight to bole, height, and diameter; the relativegrowth of leaves, roots, and bole; annual increments of drymatter; relative growth-rates per unit leaf area or per unitdry weight per annum. Some comparative data are given for Pinus nigra (one plantation)which is more productive than Pinus sylvestris.     

Cryptic hybrids between Pinus uncinata and P. sylvestris

We tested the performance of molecular markers and biometric traits in the identification of hybrids between closely related mountain pine (Pinus uncinata) and Scots pine (Pinus sylvestris). A plastid DNA marker and a set of morphological and anatomical needle traits were applied in analyses of individuals from several sympatric stands of the species and a single‐species' population from southern Europe, used as a reference. A polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) marker from the plastid trnL–trnF region and morphological and anatomical traits clearly discriminated between the pure species. Significant differences were found between P. uncinata and P. sylvestris, mostly in the shape of epidermal cells and the number of stomata. Four putative hybrids with P. sylvestris morphology, but with P. uncinata plastid DNA haplotypes, were found in a population from Sierra de Gúdar near Valdelinares, the southernmost locality of the latter species in eastern Spain. Discrimination analyses between and within populations placed these individuals on the edge of an agglomeration of P. sylvestris individuals. The results suggest that hybridization between the species is rare, but can result in cryptic hybrids morphologically similar to the maternal species. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163 , 473–485.     

Plastids and glycolipids in the stemwood of Pinus sylvestris L.

Summary The ultrastructure of plastids in xylem ray parenchyma cells of Pinus sylvestris L. was studied and compared with the glycolipid composition of the stemwood. Seasonal changes of the ultrastructure were studied by taking samples regularly throughout the year. The plastids resemble amyloplasts. They usually have one large starch grain, and considerable variation in structure and starch content was observed, especially in the innermost sapwood and in the sapwood-heartwood transition zone. Electron-dense deposits were observed attached to the plastid membranes and envelopes, especially in the transition zone, from April to November. The plastids were aggregated near the nucleus and the starch disappeared during the winter (January–March). The glycolipids, monogalactosyldiacylglycerol (MGDG) and di-galactosyldiacylglycerol (DGDG), were present only in the sapwood, in trace amounts. The glycolipid content was slightly greater in the outer sapwood than in the sapwood-heartwood transition zone. DGDG was the dominant lipid of the two.     

Mineral Content of Plantations of Pinus sylvestris L.

Data are given of the percentage composition and total contentsof Na, K, Ca, Mg, P, and N in trees forming an age series ofplantations of Pinus sylvestris. From these data, calculationsare made of the total nutrient uptake by the trees, both annuallyand for the whole rotation, the removal of nutrients in thecrop, the immobilization of nutrients within the trees, andthe return of nutrients to the soil by litter fall and unharvestedmaterial from selective thinning and cropping. Comparisons aremade of the nutrient uptake by the trees and typical agriculturalcrops, and the results are considered in terms of the maintenanceof soil fertility.     

Glucomannan-synthase activity in differentiating cells of Pinus sylvestris L.

Particulate membrane preparations have been isolated from cambial cells, and from differentiating and differentiated xylem cells of the main stem of pine trees. These preparations synthesise a 14 glucomannan from guanosine 5-diphosphate-mannose. The polysaccharide and the synthase have been characterized and the K_m and V_max for the synthase determined as 85 M and 52.9 M·min^-1, respectively. The enzymic activity was inhibited by the addition of guanosine 5-diphosphate-D-glucose so that the presence of an epimerase on the particulate fraction in conjunction with the synthase probably allowed the heteropolymer to be formed with the optimal ratio of the concentrations of the nucleoside-diphosphate sugar donors. No evidence for a polyprenyl-phosphate derivative as an intermediate during the polymer synthesis was obtained. Part of the control mechanism for the deposition of the large amounts of the glucomannan during the secondary thickening of the tracheids of the vascular system is by an increase in the amount of synthase activity at the endomembrane system of the cells. This probably occurs by an increase in the amount of enzyme which is modulated by gene regulation during differentiation.Abbreviations GDP guanosine 5-diphosphate - GLC gasliquid chromatography     

Soluble carbohydrates of Pinus sylvestris L. sapwood and heartwood

Summary The amounts of glucose, fructose, sucrose, arabinose/galactose, raffinose/stachyose and starch were investigated in the outer sapwood, innermost sapwood, transition zone and heartwood of four stems of Pinus sylvestris L. The samples were taken in October and the determination of the compounds was done enzymatically. It was not possible to distinguish arabinose from galactose and raffinose from stachyose. The amounts of glucose, fructose and sucrose were greatest in the outer sapwood and decreased gradually towards the innermost sapwood and the heartwood. In the outermost heartwood glucose, fructose and sucrose were only present in trace amounts. Raffinose/stachyose showed highest concentrations in the outer sapwood and decreased towards the heartwood. In contrast, the concentrations of arabinose/galactose increased towards the heartwood and the greatest amount was found in the inner heartwood. When identified by thin-layer chromatography (TLC), arabinose was found to be present in greater amounts than galactose. The amount of starch decreased markedly towards heartwood. However, the amounts of sugars in all the studied stems was very variable. The changes in the amounts of carbohydrates in the different zones of the stems and the possible relationships of these phenomena with heartwood formation are discussed.     

The refilling of embolized xylem in Pinus sylvestris L.

Abstract. The hypothesis that ray parenchyma cells are actively involved in the refilling of embolized xylem of Pinus sylvestris L. was tested by killing the ray parenchyma and comparing rehydration of killed stems with that of control material. Killing of ray parenchyma was achieved using hot water or sodium azide. In most experiments, the available water for refilling was at negative water potential. Experiments were done on three kinds of plant material: small branch segments, potted seedlings and small potted trees. In all experiments, there was no indication that the azide-killed xylem was slower to refill than the control material and it was concluded that the parenchyma has no role in the refilling process, which therefore must be purely physical or physicochemical. Stems treated with hot water did not refill; we suggest that this may be caused by high temperatures decreasing the water permeability of the tracheid wall. The refilling of small branch segments may be explained by surface tension forces (capillary action), which inside the tracheid lumen may lower the water potential down to -9.7kPa; this may be enough to draw in water from the available water that in experiment one was at -2kPa. In the case of seedlings or saplings, capillary action cannot explain refilling, because the xylem water potentials were always lower than those estimated from tracheid radii. Condensation of water during diurnal cycles of warming and cooling is also unlikely to contribute to refilling significantly. To account for refilling in these cases, it is supposed that the tracheid wall may be chemically active and able to lower the water potential below the value expected by capillarity.     

Genetic structure, variability and differentiation of Pinus sylvestris L. populations in the Ukrainian Carpathian Mountains and Rastoch'e

On the basis of electrophoretic analysis of 9 enzymous systems encoded by 20 gene loci the level of intra- and inter-population variation of two relict populations of Pinus sylvestris L. in the Ukrainian Carpathians and two ones in Rastochiye was studied. The less allele representation and the lower level of heterozygosity are typical for the Carpathian populations. Fst and Gst, parameters of populations subdivision, were not high--0.020 and 0.022 correspondingly and the coefficient DN was 0.008 in average. The results of the cluster analysis showed that only the populations of Rastochiye were united in one group indicating their genetic affinity.