Gene expression profiling in the stem of young maritime pine trees: detection of ammonium stress-responsive genes in the apex

The shoots of young conifer trees represent an interesting model to study the development and growth of conifers from meristematic cells in the shoot apex to differentiated tissues at the shoot base. In this work, microarray analysis was used to monitor contrasting patterns of gene expression between the apex and the base of maritime pine shoots. A group of differentially expressed genes were selected and validated by examining their relative expression levels in different sections along the stem, from the top to the bottom. After validation of the microarray data, additional gene expression analyses were also performed in the shoots of young maritime pine trees exposed to different levels of ammonium nutrition. Our results show that the apex of maritime pine trees is extremely sensitive to conditions of ammonium excess or deficiency, as revealed by the observed changes in the expression of stress-responsive genes. This new knowledge may be used to precocious detection of early symptoms of nitrogen nutritional stresses, thereby increasing survival and growth rates of young trees in managed forests.     

Common environmental factors explain both ectomycorrhizal species diversity and pine regeneration variability in a post-fire Mediterranean forest

Natural seedling regeneration and establishment after stand replacing wildfires is influenced by a series of environmental and biological constraints. In this study, we characterized the diversity and structure of the ectomycorrhizal (ECM) fungal community associated with post-fire naturally regenerated maritime pine saplings, and individuate the environmental factors responsible for fungal species distribution. We also identify the main environmental factors responsible for maritime pine regeneration variability and assessed the relation between saplings performance and ECM fungal diversity indices. Fungal species were identified by direct sequencing of internal transcribed spacer regions. Five years after the disturbance event, a total of 30 taxa colonized the pine saplings. The ECM fungal community was dominated by ruderal species of the genus Rhizopogon (present in almost half of the samples). Almost one third of the identified ECM fungal species belonged to the family Thelephoraceae. Typical k-selected species like Amanita pantherina, Boletus aestivalis, Lactarius chrysorrheus, and Russula densifolia were found on pine saplings collected in proximity of unburnt pine trees, in correspondence with low erosion extents. Pine regeneration varied throughout the study areas and was enhanced at higher elevations, in correspondence with moderate slopes, shallower soils, and a reduced cover of ericaceous shrubs and bare ground. These conditions were found in close proximity to patches of pine trees that survived the disturbance event and were previously characterized by a higher pre-fire pine biomass. Even though no correlations were found between saplings performance and ECM fungal diversity indices, common environmental factors (i.e., ericaceous shrub cover, extent of erosion, slope, and soil depth) were responsible for shaping the ECM fungal distribution and for describing most of the explained regeneration variability.     

Spatial genetic structure in continuous and fragmented populations of Pinus pinaster Aiton

Habitat fragmentation, i.e., the reduction of populations into small isolated remnants, is expected to increase spatial genetic structure (SGS) in plant populations through nonrandom mating, lower population densities and potential aggregation of reproductive individuals. We investigated the effects of population size reduction and genetic isolation on SGS in maritime pine ( Pinus pinaster Aiton) using a combined experimental and simulation approach. Maritime pine is a wind-pollinated conifer which has a scattered distribution in the Iberian Peninsula as a result of forest fires and habitat fragmentation. Five highly polymorphic nuclear microsatellites were genotyped in a total of 394 individuals from two population pairs from the Iberian Peninsula, formed by one continuous and one fragmented population each. In agreement with predictions, SGS was significant and stronger in fragments ( Sp  = 0.020 and Sp  = 0.026) than in continuous populations, where significant SGS was detected for one population only ( Sp  = 0.010). Simulations suggested that under fat-tailed dispersal, small population size is a stronger determinant of SGS than genetic isolation, while under normal dispersal, genetic isolation has a stronger effect. SGS was always stronger in real populations than in simulations, except if unrealistically narrow dispersal and/or high variance of reproductive success were modelled (even when accounting for potential overestimation of SGS in real populations as a result of short-distance sampling). This suggests that factors such as nonrandom mating or selection not considered in the simulations were additionally operating on SGS in Iberian maritime pine populations.     

Comparison of maritime pine oil and fish oil effects on plasma lipoproteins in apolipoprotein E-deficient mice

The goal of this study was to compare the lipid lowering properties of maritime pine and fish oils in apolipoprotein E-deficient (KOE) mice, an animal model of hyperlipidemia. KOE mice were supplemented with either lard, fish or maritime pine oil (10% w/w) for one month. Compared to lard, fish and maritime pine oils decreased cholesterol (-31% and -52% respectively) and phospholipid (-41 and -52%) levels and increased triglyceride (+182% and +123%) levels. These lipid changes resulted in an enrichment in triglycerides and a depletion in cholesterol of VLDL+IDL plasma fraction as compared to lard-fed mice. These findings suggest that VLDL-triglyceride lipolysis is impaired in KOE mice fed fish or maritime pine oil.     

Genetic Structure of Marchalina hellenica (Hemiptera: Margarodidae) Populations from Turkey: Preliminary mtDNA Sequencing Data

The scale insect Marchalina hellenica (Gennadius) (Hemiptera: Margarodidae) contributes to the production of pine honey in Turkey and Greece via the honeydew excreted when it feeds on pine trees. Although it is an insect of prime economic importance, there is no information on its genetic structure. Preliminary data were obtained based on sequencing analysis of 12s rDNA and COI mtDNA gene segments from samples from four areas of Turkey. Sequences of the 12s rDNA gene segment from Greek samples available in GenBank were also included. No variability was detected concerning the COI mtDNA gene segment analysis, although 13 haplotypes were revealed based on the 12s rDNA gene segment. The most distant population was from Mudanya-Bursa Province (Turkey). Further research is necessary on the genetic structure and variability of M. hellenica populations from the two neighboring countries.     

A comparative analysis of craniological variability of Eurasian (sable and pine marten) and North American (American marten) species of the <Emphasis Type="Italic">Martes</Emphasis> genus

This paper studies the craniological variability of closely related species—the pine marten Martes martes, the American marten M. americana, and the sable M. zibellina. The study included the data on 22 nonmetric characters of skulls in 2344 individuals. The studied Martes species were revealed to have an evident species-specific manifestation of the investigated craniological characters. Sables stand apart most of all in the interspecific comparison of populations. The degree of epigenetic variability is somewhat higher in the sable than in the pine marten. The American marten significantly differs from Eurasian species in the manifestation of the nonmetric skull characters, which is noted when analyzing even a small number of samples.     

High rate of adaptive evolution in two widespread European pines

Comparing related organisms with differing ecological requirements and evolutionary histories can shed light on the mechanisms and drivers underlying genetic adaptation. Here, by examining a common set of hundreds of loci, we compare patterns of nucleotide diversity and molecular adaptation of two European conifers (Scots pine and maritime pine) living in contrasted environments and characterized by distinct population genetic structure (low and clinal in Scots pine, high and ecotypic in maritime pine) and demographic histories. We found higher nucleotide diversity in Scots pine than in maritime pine, whereas rates of new adaptive substitutions (ω_a), as estimated from the distribution of fitness effects, were similar across species and among the highest found in plants. Sample size and population genetic structure did not appear to have resulted in significant bias in estimates of ω_a. Moreover, population contraction–expansion dynamics for each species did not affect differentially the rate of adaptive substitution in these two pines. Several methodological and biological factors may underlie the unusually high rate of adaptive evolution of Scots pine and maritime pine. By providing two new case studies with contrasting evolutionary histories, we contribute to disentangling the multiple factors potentially affecting adaptive evolution in natural plant populations.     

Rangewide variation of the maritime pine bast scale matsucoccus feytaudi duc. (Homoptera: matsucoccidae) in relation to the genetic structure of its host

The bast scale Matsucoccus feytaudi is a specific pest of maritime pine, but the damage inflicted by the insect on the host trees is variable, ranging from no apparent effect to severe decline of the maritime pine stands. Rangewide variation of mitochondrial DNA among M. feytaudi populations was analysed by polymerase chain reaction-restriction fragment length-single-strand conformation polymorphism (PCR-RFLP-SSCP) analysis and the results compared with the genetic information already available for its host. Three main nonoverlapping lineages can be distinguished in M. feytaudi. The phylogeography of the pest population is clearly related to the history of its host. Most local associations could result from common evolution while others must be interpreted as intraspecific host shifts. Because the distribution of cultivated tree species is greatly influenced by humans, much may be learned concerning their genetic structure from the indirect study of their specific pests.     

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.     

Somatic embryogenesis from different tissues of Spanish populations of maritime pine

The somatic embryogenesis (SE) capacity of megagametophytes belonging to Continental and Mediterranean Spanish provenances of maritime pine (Pinus pinaster Aiton) was studied, noting factors (megagametophyte developmental stage and culture medium) that enhanced the induction and establishment of SE lines. In both provenances, initiation and establishment of embryogenic calli was higher on megagametophytes in which the dominant zygotic embryo had begun to develop. In the Mediterranean provenance, however, SE lines were also established from megagametophytes enclosing zygotic embryos with well-developed cotyledons. A modified Litvay medium (mLV) containing 9.9???M 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4???M 6-benzyladenine (BA) was superior to DCR medium containing 13.6???M 2,4-D and 4.4???M BA for SE induction, but there were no differences between media in terms of the number of SE lines established after 4?months in culture (153 vs. 155 established SE lines, for mLV and DCR media respectively). Of the 26 embryogenic lines tested for maturation, 15 (58?%) produced cotyledonary somatic embryos and 75?% of these gave rise to plants on germination medium. SE-like cultures from adult maritime pine trees were also initiated, but embryogenic lines could not be established. This is the first report on the production of SE in maritime pine of Continental and Mediterranean origin. The micropropagation protocols presented here provide an important tool for the vegetative multiplication of selected families and breeding programs for maritime pines from Spain.     

Genetic structure of Pinus pinaster Ait. populations in Morocco revealed by nuclear microsatellites

Pinus pinaster is one of the most popular conifers used for reforestation in Morocco and represents an economically and ecologically important species for the region. In this study, nuclear microsatellites (ncSSRs) are used to compare genetic structure and diversity estimates of natural populations of Moroccan maritime pine. Samples were collected among 10 natural populations distributed in three biogeographically different regions, the Rif Mountain, the Middle and the High Atlas. Forty-five nuclear alleles at seven variable loci were found with a mean of 6.4 alleles per locus. A number of private alleles (17.1%) were shown in populations from Rif and Middle Atlas. Moreover, in Morocco, P. pinaster showed a lower genetic diversity than in other parts of its geographic range. Significant departures from Hardy–Weinberg equilibrium with excess homozygosity are observed indicating a high level of mating inside populations. Genetic diversity was structured with high variability among populations (Fst = 12%). Results show a correlation between genetic and geographic distances with an R-squared of 0.436. Two clusters were found using STRUCTURE, whereas three main clusters can be distinguished based on genetic distances of phylogenetic tree. Genetic relationships among maritime pine populations in Morocco appear to be related to historical, ecological as well as anthropogenic factors, suggesting the need for conservation strategies at the population level.     

Spatial pattern and variability in soil N and P availability under the influence of two dominant species in a pine forest

The presence of a legume in a nitrogen (N)-limited forest ecosystem may not only create ??islands of N fertility?? but also affect the phosphorus (P) availability. The main objective of this study was to compare the effect of a pine (Pinus canariensis) and a leguminous (Adenocarpus viscosus) species on the spatial pattern and variability of different labile organic-N (microbial biomass-N [MB-N] and dissolved organic-N [DON]), as well as inorganic-N (IN) and ?CP fractions (NH₄-N, NO₃-N, and PO₄-P), in a forest soil of the Canary Islands (Spain). Assuming some litter quantity and quality differences between these two species, we expected to find higher soil labile organic-N concentrations under isolated individuals of P. canariensis than under isolated individuals of A. viscosus. We also expected to find higher concentrations and spatial dependence (percentage of total variance explained by spatial autocorrelation) of NO₃-N beneath A. viscosus than beneath P. canariensis canopies, and higher spatial scaling of soil variables under the influence of P. canariensis canopies than under the presence of A. viscosus individuals. Moreover, we tested whether the soil variables measured under isolated individuals of both species showed a different spatial variability than the same soil variables measured under overlapping pine canopies inside a pine forest. To test these hypotheses, soil samples under isolated mature individuals of each species were collected in the winter and summer, whereas under a pine forest canopy, the sampling was performed only in the winter. The winter MB-N and DON concentrations were significantly higher beneath the pine individual, whereas the winter NO₃-N, NO₃-N-to-IN ratio, and PO₄-P were significantly higher under the leguminous individual; these differences were not observed in the summer samples. We found higher spatial ranges under the pine than under the legume canopy in the winter sampling, and the spatial dependence of NO₃-N was twice as high beneath the legumes as under the pines at both sampling dates. The soil spatial variability was higher (up to 17 times higher) under isolated individuals than inside the pine forest. The results of this study suggest that both the morphological and physiological characteristics of P. canariensis and A. viscosus, as well as the spatial pattern of P. canariensis, may influence the spatial pattern and variability of soil resources.     

Genetic polymorphism in maritime pine (Pinus pinaster Ait.) assessed by two-dimensional gel electrophoresis of needle,bud, and pollen proteins

Using two-dimensional polyacrylamide gel electrophoresis, the genetic variation of proteins was examined in three organs (needle, bud, and pollen) from 18 trees of maritime pine. Three types of variation were noted: presence/absence, staining intensity, and position variation of the spots. Of the 902 polypeptides scored in the three organs, 245 (27.2%) were polymorphic. Moreover, among these variable spots, 117 were found in a single organ, demonstrating an increased polymorphism of the organ-specific polypeptides (56.0% vs 18.4% for the organ-unspecific polypeptides). Finally, a positive correlation was found between variability level and subunit molecular weight for spots showing position variation but not for spots showing presence/absence or staining intensity variations. Possible explanations for this observation are discussed.Correspondence to: N. Bahrman     

Selection of haploid cell lines from megagametophyte cultures of maritime pine as a DNA source for massive sequencing of the species

The potential of haploid tissues for genetic studies in conifers is hampered by the lack of abundant and homogeneous plant material suitable for DNA isolation. In this work we have determined factors promoting haploid callus induction and proliferation from megagametophytes of Oria 6, a genotype of Pinus pinaster Aiton (maritime pine) from the natural population Sierra de Oria (Almería, Spain), selected based on its response to extreme drought conditions. The generation of haploid callus was restricted to megagametophytes isolated from light-brown cones with no dehydrated seeds collected in September. Culture medium composition did not significantly affect callus induction, but a modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine favored further multiplication. The ploidy status of the callus lines was determined by flow cytometry and seven polymorphic microsatellites. A total of sixteen haploid callus lines were established and one of these is being used as a source of DNA for massive sequencing of maritime pine genome.