Characteristics of gene pools of three Pinus pumila (Pall.) Regel populations at the range margins

Genetic variation of Siberian dwarf pine Pinus pumila (Pall.) Regel was characterized in three marginal populations in southwestern, southern and eastern parts of the natural species range (Transbaikalia, Primorye, Kamchatka) using isozyme analysis. Analysis involving 16 isozyme loci encoding ten enzyme systems was conducted. Our results confirm that P. pumila is among the most polymorphic species in the family Pinus. Three marginal populations exhibited high genetic variation (P95 = 68.8%, Ho = 0.247, He = 0.291). Populations heterogeneity and significantly high level of divergence in coniferous (F(ST) = 0.050, D(N) = 0.044) reflect their genetic originality. In summary, it was shown that the level of genetic variation characteristic for P. pumila in other parts of the not only is reproduced in the populations examined but even is close to maximum there.     

Dynamics of allozyme heterozygosity in Siberian dwarf pine Pinus pumila (Pall.) Regel populations of the Russian Far East: comparison of embryos and maternal plants

Siberian dwarf pine, or Japanese stone pine, Pinus pumila (Pall.) Regel is widespread in eastern Siberia and the Russian Far East; the species is bird-dispersed and has a unique spreading or shrub living form. A mixed mating system (predominant outcrossing with self-pollination and matings of close relatives) leads to the formation of partly inbred progenies in P. pumila, as in the majority of other conifers. The question arises as to whether inbred individuals persist in the reproductive part of a population, which can have negative genetic consequences. The ADH, FDH, FEST, GDH, GOT, IDH, LAP, MNR, MDH, PEPCA, 6-PGD, PGI, PGM, SKDH, and SOD isozyme systems were analyzed to study the dynamics of heterozygosity in four P. pumila natural populations from the Pacific region, optimal for the species. Samples were collected in northern Koryakia, southern Kamchatka (two samples), and the Kunashir Island (Kurils). Wright's fixation index was used to estimate the level of inbreeding in embryos from dormant seeds resulting from free pollination and in maternal plants. A substantial level of inbreeding (F(IS) = 0.124-0.342) was observed in the embryo samples but not in three out of the four adult samples. The inbreeding level at the reproductive age was high only in the sample from Koryakia, which was explained by a relatively young age of plants in the population frequently affected by fires. A general increase in heterozygosity, characteristic of other conifers as well, was attributed to elimination of inbred progenies and by balancing selection for heterozygotes, which is a key factor maintaining allozyme polymorphism in populations.     

Dynamics of allozyme heterozygosity in Siberian dwarf pine Pinus pumila (Pall.) Regel populations of the Russian Far East: Comparison of embryos and maternal plants

Siberian dwarf pine, or Japanese stone pine, Pinus pumila (Pall.) Regel is widespread in eastern Siberia and the Russian Far East; the species is bird-dispersed and has a unique crawling or shrub living form. A mixed mating system (predominant outcrossing with self-pollination and matings of close relatives) leads to the formation of partly inbred progenies in P. pumila, as in the majority of other conifers. The question arises as to whether inbred individuals persist in the reproductive part of a population, which can have negative genetic consequences. The ADH, FDH, FEST, GDH, GOT, IDH, LAP, MNR, MDH, PEPCA, 6-PGD, PGI, PGM, SKDH, and SOD isozyme systems were analyzed to study the dynamics of heterozygosity in four P. pumila natural populations from the Pacific region, optimal for the species. Samples were collected in northern Koryakia, southern Kamchatka (two samples), and the Kunashir Island (Kurils). Wright’s fixation index was used to estimate the level of inbreeding in embryos from dormant seeds resulting from open pollination and in maternal plants. A substantial level of inbreeding (F _IS = 0.124?0.342) was observed in the embryo samples but not in three out of the four adult samples. The inbreeding level at the reproductive age was high only in the sample from Koryakia, which can be explained by a relatively young age of plants in the population frequently affected by fires. A general increase in heterozygosity in the course of ontogeny, characteristic of other conifers as well, was attributed to elimination of inbred progenies and by balancing selection in favor of heterozygotes, which is a key factor maintaining allozyme polymorphism in populations.     

Comparative sequence analysis of the LEA gene fragment in Pinus sibirica Du tour and Pinus pumila (Pallas) Regel

A comparative sequence analysis of the LEA (Late Embryogenesis Abundant (LEA)-like gene) intron fragment was performed in Pinus sibirica and P. pumila differing in geographic origin. It was demonstrated that in P. sibirica this fragment was represented by two types of PCR products, 224 and 202 bp in size. Similarly, in accessions of P. pumila, two PCR products of 224 and 159 bp in size were identified. Comparison of 224-bp fragments in P. sibirica and P. pumila showed that they differed in single nucleotide substitutions. Analysis of the intron fragment in a plant, which was characterized as an interspecific hybrid based on morphological characters, showed that it had fragments of 224 and 159 bp in size. The sequence of 224-bp fragment was similar to that of the corresponding fragment in P. sibirica. The structure of the short fragment was the same as the structure of the corresponding fragment in P. pumila. The data obtained are discussed in terms of the use of the sequences examined for species taxonomic classification and for an analysis of species hybridization.     

Effect of Grazing on Restoration of Endemic Dwarf Pine (Pinus culminicola Andresen et Beaman) Populations in Northeastern Mexico

A pilot experiment designed to test the effect of cattle, small mammals, and elevation on the success of reforestation of an endemic dwarf pine species in northeastern Mexico was implemented. Pinus culminicola (Andresen et Beaman) grows only in four high peaks in the Sierra Madre Oriental and is under pressure from grazing, wildfires, and human activities such as mining, road development for timber extraction, and telecommunication and aerial navigation devices. We planted and monitored 2‐year‐old seedlings at three elevations within the natural distribution range of this species at Cerro El Potosí in Nuevo León, Mexico. At each elevation three treatments were established: (1) seedlings protected from cattle plus small mammals, (2) seedlings protected from cattle, and (3) seedlings with free access to cattle and small mammals. Seedling survival was approximately 50% in (1) after 4 years, but there were no surviving seedlings with free access to cattle. Elevation in general did not account for variation in survival. Seedling growth was poor during the 4 years, which implies that seedlings remain susceptible to grazing and trampling by cattle and small mammals. The implications for a large‐scale restoration program are discussed.     

Chloroplast microsatellites and mitochondrial nad1 intron 2 sequences indicate congruent phylogenetic relationships among Swiss stone pine (Pinus cembra), Siberian stone pine (Pinus sibirica), and Siberian dwarf pine (Pinus pumila)

We studied the phylogenetic relationships among the three stone pine species, Pinus cembra, P. sibirica, and P. pumila, using chloroplast microsatellites and mitochondrial nad1 intron 2 sequences. The three chloroplast microsatellite loci combined into a total of 18 haplotypes. Fourteen haplotypes were detected in 15 populations of P. cembra and one population of P. sibirica, five of which were shared between the two species, and the two populations of P. pumila comprised four species-specific haplotypes. Mitochondrial intron sequences confirmed this grouping of species. Sequences of P. cembra and P. sibirica were identical, but P. pumila differed by several nucleotide substitutions and insertions/deletions. A repeat region found in the former two species showed no intraspecific variation. These results indicate a relatively recent evolutionary separation of P. cembra and P. sibirica, despite their currently disjunct distributions. The species-specific chloroplast and mitochondrial markers of P. sibirica and P. pumila should help to trace the hybridization in their overlapping distribution area and to identify fossil remains with respect to the still unresolved postglacial re-colonization history of these two species.     

Pine sawfly Gilpinia albiclavata sp. nov. (Hymenoptera: Diprionidae) infesting Pinus pumila in the Japanese Alps

Gilpinia albiclavata Hara, sp. nov. is described from Honshu, Japan. Larvae feed on needles of Pinus pumila (Pinaceae). Since 2001, severe infestations of the sawfly have been often observed in the Japanese Alps. Notes on the immature stages and the life history are presented.     

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     

Embryonal Development of Siberian Pine (Pinus sibirica Du Tour) with the Annual Cycle of Ovulate Cone Development in the Western Sayan Mountains

Morphological and cytoembryological studies of unique forms of Siberian pine (Pinus sibirica Du Tour) with the annual cycle of ovulate cone development were carried out. The morphogenesis of female shoots in these anomalous trees with the annual cycle of development was shown to provide for an accelerated development and very rapid growth of megastrobili and accelerated formation of reproductive structures. Cytoembryological studies of ovules in these trees showed significant shortening of the free-nuclear stage of gametophyte development (up to 3 weeks instead of one year) and ultra-early formation of archegonia and egg cells. However, no fertilization of egg cells and the development of embryos occur in the anomalous forms; the egg-cell nucleus divides in the haploid state. Developing seeds are formed without embryo. The author hypothesizes that the acceleration of embryo development in the anomalous pine trees is caused by enhanced hormonal, carbohydrate, and nitrogen metabolism in ovule tissues.     

若干种子处理方法对偃松种子生活力的影响

为了提高偃松种子生活力,采用水浸、NaOH溶液浸泡、PEG6000（PEG,聚乙二醇）溶液浸泡、低温冷冻、吸湿/回干5种方法对偃松种子进行处理,研究不同处理中各因素（如溶液浓度、处理时间与温度）对偃松种子生活力的影响,并采用靛蓝染色法测定偃松种子的生活力。结果表明：10%PEG6000溶液、初始温度60℃、浸泡36 h时,偃松种子生活力最强;水浸、低温处理和吸湿/回干处理方法也能提高偃松种子生活力;然而4%~12%NaOH溶液浸泡处理会导致偃松种子生活力下降。因此可将PEG6000溶液处理种子的方法应用于偃松种子萌发及人工种苗培育的实践中,使偃松资源得到更好的可持续开发与利用。     

Indole-3-acetic acid producing root-associated bacteria on growth of Brazil Pine (Araucaria angustifolia) and Slash Pine (Pinus elliottii)

Araucaria forests in Brazil today correspond to only 0.7 % of the original 200 km² of natural forest that covered a great part of the southern and southeastern area of the Atlantic Forest and, although Araucaria angustifolia is an endangered species, illegal exploitation is still going on. As an alternative to the use of hardwoods, Pinus elliottii presents rapid growth and high tolerance to climatic stress and low soil fertility or degraded areas. Thus, the objective of this study was to evaluate the effect of IAA-producing bacteria on the development of A. angustifolia and P. elliottii. We used five bacterial strains previously isolated from the rhizosphere of A. angustifolia, which produce quantities of IAA ranging from 3 to 126 μg mL^?1. Microbiolized seeds were sown in a new gnotobiotic system developed for this work, that allowed the quantification of the plant hormone IAA produced by bacteria, and the evaluation of its effect on seedling development. Also, it was shown that P. elliottii roots were almost as satisfactory as hosts for these IAA producers as A. angustifolia, while different magnitudes of mass increases were found for each species. Thus, we suggest that these microbial groups can be helpful for the development and reestablishment of already degraded forests and that PGPR isolated from Araucaria rhizosphere have the potential to be beneficial in seedling production of P. elliottii. Another finding is that our newly developed gnotobiotic system is highly satisfactory for the evaluation of this effect.     

Rangewide Genetic Diversity in Natural Populations of Chinese Pine (Pinus tabulaeformis)

Thirteen natural populations from throughout the range of the Chinese pine (Pinus tabulaeformis Carr.) were examined using inter-simple sequence repeat markers to characterize the genetic structure at the species level and to compare the extent and distribution of genetic variation among central, intermediate, and marginal populations. Although the total genetic variation in the Chinese pine was mainly maintained within populations, the genetic differentiation among populations was significant (P < 0.001). The genetic divergence was significantly correlated with geographic distance (P < 0.05). Genetic diversity tended to decrease from the central to intermediate and marginal populations. The marginal populations had significantly lower intrapopulation genetic diversity than central populations (P < 0.05). Cluster analysis based on Nei’s unbiased genetic distances confirmed the difference among four central populations and the rest. Both historical and contemporary factors may have played key roles in shaping the spatial genetic structure of this species.     

Preformed mRNA and Light-induced Germination of Pine (Pinus thunbergii) Seed

The mRNAs were isolated from dry, dark-imbibed and light-imbibedpine (Pinus thunbergii) seeds, in which germination is inducedupon exposure to light, and their translational activities ina wheat embryo cell-free system were examined. A portion ofthe mRNA extracted from each type of seed appeared to be poly(A)⁺RNA.A significant translational activity was already present inthe RNA fraction isolated from the dry pine seeds. The preformedmRNA seems not to be translated in vivo during the dark-imbibition,since most of the in vivo protein synthesis did not occur untilthe seeds were exposed to light. The SDS polyacrylamide gelelectrophoregrams of the polypeptides synthesized in vitro inresponse to either the preformed mRNA or the mRNAs from thedark-imbibed or light-imbibed seeds were qualitatively identical;thus it seems that the preformed mRNA is conserved during darkimbibition, and that its translation is initiated after theexposure of the imbibed seeds to light. (Received August 10, 1981; Accepted May 15, 1982)     

Nursery Culture Impacts Cold Hardiness in Longleaf Pine (Pinus palustris) Seedlings

Success in restoring longleaf pine ecosystems depends on outplanting high‐quality longleaf pine seedlings. One important and relatively understudied attribute of seedling quality is cold hardiness. A suite of trials was conducted to investigate the influence of common nursery cultural practices on longleaf pine cold hardiness. Cold hardiness was increased with higher rates of nitrogen, unaffected by copper coating containers, greater for foliage than for root‐collar tissue, and tended to increase with increases in container size.     

Specific Features of Development of Male Generative Organs in Clones of the Siberian Stone Pine (Pinus sibirica De Tour) of Different Origins in the Krasnoyarsk Forest-Steppe

The development of male generative organs of the Siberian stone pine clones of different origin was analyzed in the Krasnoyarsk forest-steppe for many years. Geographical and individual variability was described as concerns the number of microstrobils and pollen variability. The beginning of flowering, abundance of microstrobils, and their regular formation were shown to markedly vary and, all other conditions being equal, be determined by the presence of clones, their hereditary features, and environmental conditions, as well by the influence of stock.     

Factors Affecting the Emission of Monoterpenes from Red Pine (Pinus densiflora)

The mechanism of monoterpene emission from Pinus densiflora was studied using an environmentally controlled gas cabinet. It was found that monoterpene emission rate increases exponentially with temperature and is also influenced by light. These observations were explained reasonably by a mechanism whereby monoterpene emission rate depends on the monoterpene amount in the leaf oil and its saturated vapor pressure.     

Allozyme evidence for polyzygotic polyembryony in Siberian stone pine (Pinus sibirica Du Tour)

Approximately 4000 mature seeds from 350 trees in nine populations (12–75 trees per population) of Siberian stone pine were investigated for multiple embryos (polyembryony). Haploid megagametophytes and embryos were genotyped for eight allozyme loci. Eight-yone seeds (2.11%) had more than 1 embryo. Of these, 71 seeds had 2 embryos (1.85%), 6 seeds had 3 embryos (0.16%), 3 seeds had 4 embryos (0.08%) and 1 seed had 6 embryos (0.026%). Allozyme comparison of megagametophytes and embryos could distinquish two types of polyembryony in 56 of the 81 seeds. In 28 seeds (50%) the polyembryony was polyzygotic (independent fertilizations of more than one egg cell in the ovule); 25 seeds (45%) had most likely monozygotic polyembryony (genetically identical embryos resulting from the cleavage of a single proembryo) and 3 seeds had both genetically different and genetically identical embryos. To the best of our knowledge, this is the first genetic evidence for the form of polyembryony in conifer seeds.     

Studies on Sugars and Starch in Light- and Dark-Germinated Seeds of Scots Pine (Pinus silvestris)

Stachyose, raffinose and sucrose have been tentatively identified in ungerminated Scots pine seeds with paper chromatography and TLC. After the onset of the germination process even glucose and fructose were detected. No free galactose has been found. The occurrence and distribution of starch has been investigated with histochemical technique. The same substances have been quantitatively studied during the first 24 hours of the germination in light and darkness. This was done with an enzymatic technique alone or in combination with TLC. The evidence of a light influence on the transformations of starch, glucose and fructose has been discussed in relation to the phytochrome mechanism.     

Plasticity in Dendroclimatic Response across the Distribution Range of Aleppo Pine (Pinus halepensis)

We investigated the variability of the climate-growth relationship of Aleppo pine across its distribution range in the Mediterranean Basin. We constructed a network of tree-ring index chronologies from 63 sites across the region. Correlation function analysis identified the relationships of tree-ring index to climate factors for each site. We also estimated the dominant climatic gradients of the region using principal component analysis of monthly, seasonal, and annual mean temperature and total precipitation from 1,068 climatic gridpoints. Variation in ring width index was primarily related to precipitation and secondarily to temperature. However, we found that the dendroclimatic relationship depended on the position of the site along the climatic gradient. In the southern part of the distribution range, where temperature was generally higher and precipitation lower than the regional average, reduced growth was also associated with warm and dry conditions. In the northern part, where the average temperature was lower and the precipitation more abundant than the regional average, reduced growth was associated with cool conditions. Thus, our study highlights the substantial plasticity of Aleppo pine in response to different climatic conditions. These results do not resolve the source of response variability as being due to either genetic variation in provenance, to phenotypic plasticity, or a combination of factors. However, as current growth responses to inter-annual climate variability vary spatially across existing climate gradients, future climate-growth relationships will also likely be determined by differential adaptation and/or acclimation responses to spatial climatic variation. The contribution of local adaptation and/or phenotypic plasticity across populations to the persistence of species under global warming could be decisive for prediction of climate change impacts across populations. In this sense, a more complex forest dynamics modeling approach that includes the contribution of genetic variation and phenotypic plasticity can improve the reliability of the ecological inferences derived from the climate-growth relationships.