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.     

A morphometric analysis of spontaneous and artificial hybrids of Pinus mugo ×sylvestris (Pinaceae)

Hybridization among Pinus mugo and P.sylvestris is visualized in Wells' distance diagrams using either equally or differently weighted characters. Parents and hybrids are better resolved in diagrams using differently weighted characters. Putative hybrids from mixed, planted or naturalized stands of P. mugo and P. sylvestris in SW and NW Jutland, Denmark, are compared with artificial F₁ and spontaneous material of P. mugo × sylvestris (P. × rhaetica ) from the Alps and the Pyrenees. The natural hybrids are primary hybrids, rather than introgressants.     

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.     

Seed quality in hybrid swarm populations of Pinus mugo Turra and P. sylvestris L.

Crop characteristics involving cone size, average number of seeds per cone, and seed germinability have been studied for three hybrid swarm populations of Pinus mugo and P. sylvestris, two pure P. mugo populations and one pure P. sylvestris population, in northern Slovakia. Generally, reproduction capability characteristics were significantly reduced in hybrid swarms in relation to the control populations. Although there were no consistent differences between hybrid swarms and control populations in the average number of seeds per cone or cone size, seed germination rates and full seed production in hybrid swarms were lower than for the control population of P. sylvestris and one of the pure populations of P. mugo. Based on these data a conclusion has been drawn postulating partial rather than full fertility of the hybrid swarm populations of P. mugo and P. sylvestris.     

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.     

A new species-specific nuclear DNA marker for identification of hybrids between Atlantic salmon and brown trout

The banding pattern of Alu I-digested prepro-gonadotropin releasing hormone gene is species-specific in Atlantic salmon and brown trout and can be applied for identification of their hybrids.     

Genetic variation of F1 hybrids from controlled crosses between Pinus montana var. rostrata and Pinus sylvestris in morphological needle traits

One hundred and seventeen hybrids, representing ten families from controlled crosses between Pinus montana var. rostrata and Pinus sylvestris, were examined in respect to six morphological traits of two-year-old needles. The biometric data obtained from the measurements provided a basis for multivariate statistical analyses, including discriminant analysis and Mahalanobis distances as principal methods. The analysed families formed two complexes, which were significantly different. The main traits responsible for the distinction were numbers of stomatal rows on both sides of the needles.     

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.     

Initiation of somatic embryogenesis in Pinus banksiana, P. strobus, P. pinaster, and P. sylvestris at three laboratories in Canada and France

During 2002–2004, three laboratories in Canada and France collaborated to improve initiation of somatic embryogenesis (SE) in jack pine (Pinus banksiana Lamb.), eastern white pine (P. strobus L.), maritime pine (P. pinaster Ait.), and Scots pine (P.␣sylvestris L.), giving particular attention to the effects of (1) N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) versus various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA), (2) differences in basal nutrient media, i.e., macro- and microelements, and (3) gelling agent concentration. The work was carried out separately at␣each laboratory, but the details of media compositions were shared and tested on their respective species. Results indicate that the developmental stage of the zygotic embryo (ZE) and genotype effects had a large influence on SE initiation, and that genetic effects were consistent over time. Different species responded differently to PGR types and concentration, basal nutrient media, trace elements, and their combinations. Currently, our best initiation rates based on a selected group of genotypes, optimal development stage of ZE, and medium are 3.9% for jack pine, 54.6% for eastern white pine, 76.2% for maritime pine, and 19.7% for Scots pine.     

Cuticular Analysis – a method to distinguish the leaves of Pinus sylvestris L. (Scots Pine) from those of Pinus mugo s. str. (Dwarf Mountain-pine)

Fragments of leaf needles from the early Holocene were found in peat sediments of the Adršpašskoteplické skály nature reserve in northeast Bohemia. These remains were identified from macroscopic characters as belonging to Pinus sp. (pine), and they were identified to species level by cuticular analysis. This identification method uses the number and shape of subsidiary stomatal cells and has been verified by comparision of both fossil and modern material. Taxonomic characters have been discovered which distinguish the leaves of Pinus sylvestris (Scots Pine) from those of P. mugo s. str. (Dwarf Mountain-pine) on the basis of stomatal density, crypt morphology and length of the crypt. This method is helpful in palaeobotany and archaeobotany for identifying most such leaf remains. Received June 28, 2001 / Accepted June 18, 2002     

Lophodermium pini-mugonis sp. nov. on needles of Pinus mugo from the Alps based on morphological and molecular data

Lophodermium pini-mugonis, collected on needles of Pinus mugo from German Alps, is described as a species new to science. It is characterized by subcuticular ascomata with a wrinkled surface and a somewhat untidy outline, a complex structure of lip cells, and ellipsoidal conidia. An analysis of the internal transcribed spaces of rDNA showed that Lophodermium pini-mugonis is, sister to Lophodermium autumnale and distantly related to other Lophodermium species on pines. The hypothesis of cospeciation of Lophodermium species with members of the Pinaceae is discussed.     

Amplification of repetitive DNA from Nicotiana plumbaginifolia in asymmetric somatic hybrids between Nicotiana sylvestris and Nicotiana plumbaginifolia

Summary Asymmetric somatic hybrids were obtained between a chlorophyll-deficient mutant of Nicotiana sylvestris (V42) and a nitrate-reductase (NR)-deficient line of N. plumbaginifolia (cnx20 or Nia26), using each of the parents alternately as the irradiated donor. Irradiation doses applied ranged from 10 to 1,000 Gy of gamma-rays. Hybrid selection was based on complementation of NR deficiency with wild-type NR genes. To aid in the analysis of somatic hybrids, species-specific repetitive DNA sequences from N. plumbaginifolia (NPR9 and NPR18) were cloned. NPR18 is a dispersed repetitive sequence occupying about 0.4% of the N. plumbaginifolia genome. In turn, NPR9, which is part of a highly repetitive DNA sequence, occupies approximately 3% of the genome. The species-specific plant DNA repeats, together with cytological analysis data, were used to assess the relative amount of the N. plumbaginifolia genome in the somatic hybrids. In fusion experiments using irradiated N. plumbaginifolia, an increase in irradiation dose prior to fusion led to a decrease in N. plumbaginifolia nuclear DNA content per hybrid genome. For some hybrid lines, an increase in the quantity of repetitive sequences was detected. Thus, hybrid lines 1NV/21, 100NV/7, 100NV/ 9, and 100NV/10 (where N. plumbaginifolia was the irradiated donor) were characterized by amplification of NPR9. In the reverse combination (where N. sylvestris was the irradiated donor), an increase in the copy number of NPR18 was determined for hybrid clones 1VC/2, 1VC/3, 100VC/2 and oct100/7. Possible reasons for the amplification of the repeated sequences are discussed.     

The spatial relationship between post‐crop remaining trees and the establishment of saplings in Pinus sylvestris stands in Spain

Question: What is the spatial relationship between remaining trees and the establishment and development of recruited saplings? Location: The Pinus sylvestris forest Pinar de Valsaín, in the Sistema mountain range (central Spain). Methods: Three 0.5 ha plots have been analysed. The saplings were located in a 2 m x 2 m grid, characterizing their spatial pattern through a nested ANO VA. The spatial pattern of stems was analysed using the L(d) function. To analyse the spatial relationship between stems belonging to different cohorts, the intertype L_rs(d) function was used. Finally a new function K_rx(d) is presented as a method to analyse the relationship between the spatial distribution of stems and the sapling density (a sampled continuous variable). Results: The mother trees show cluster pattern at scales of ca. 12 m ‐ 22 m, leading to a spatial pattern at 14 m–16 m for the saplings during the regeneration period. At the beginning of the shelter phase, saplings less than 1.30 m in height show spatial repulsion from the old crop at distances above 10 m, whereas taller saplings show repulsion at shorter distances, due to the suppression of sapling development near the mother trees. At the end of the regeneration period, saplings < 1.30 m appear under the last remaining mother tree canopies. Conclusions: In the stands analysed, located at the southern limit of Pinus sylvestris distribution, this species behaves as half‐shade tolerant. This study shows that the K_rx(d) function might be widely applied to analyse the relationship between patterns that occur at different scales or between a point pattern and a continuous variable, being a useful tool for analysing some forest processes.     

Molecular identification and relative abundance of cryptic Lophodermium species in natural populations of Scots pine,Pinus sylvestris L.

The multi-locus phylogenetic species recognition approach and population genetic analysis of Amplified Fragment Length Polymorphism (AFLP) markers were used to delineate Lophodermium taxa inhabiting needles of Scots pine (Pinus sylvestris) in native pinewoods within Scotland. These analyses revealed three major lineages corresponding to the morphological species Lophodermium seditiosum and Lophodermium conigenum, fruiting on broken branches, and Lophodermium pinastri, fruiting on naturally fallen needles. Within L. pinastri three well supported sister clades were found representing cryptic taxa designated L. pinastri I, L. pinastri II, and L. pinastri III. Significant differences in mean growth rate in culture were found among the cryptic taxa. Taxon-specific primers based on ITS sequences were designed and used to classify over 500 Lophodermium isolates, derived from fallen needles of P. sylvestris in three Scottish and one French pinewood site, into the three L. pinastri cryptic taxa. Highly significant differences in the relative abundance of the three taxa were found among the Scottish pinewood sites, and between the French and all of the Scottish sites.     

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.     

Species-specific DNA sequences for identification of somatic hybrids between Lycopersicon esculentum and Solanum acaule

Summary Species-specific highly repeated DNA sequences can be used to screen the progeny of protoplast fusions combining different species. Such probes are easy to clone and can be detected by fast methods, e.g., hybridization to total genomic DNA. Furthermore, due to their high copy number, hybridization signals are strong and represent more than one locus, unlike isozymes or resistance markers. After cloning and screening for species-specific DNA sequences we characterized the highly repeated DNA sequences of the solanaceous species Solanum acaule and Lycopersicon esculentum var. gilva. DNA sequencing and hy ridization revealed a prominent, tandemly arranged satellite DNA repeat of 162 bp in Lycopersicon esculentum and a different satellite repeat of 183 bp, also tandemly organized, in Solanum acaule. Each repeat is absent in the respective other species. Therefore, we have used these DNA repeats as markers to distinguish regenerated interspecific somatic hybrids from the respective fusion partners. These hybrids were clearly identified by Southern hybridization and dot-blot assays to the respective ³²P-labelled satellite DNA.