Complex population genetic structure in the endemic Canary Island pine revealed using chloroplast microsatellite markers

The Canary archipelago, located on the northwestern Atlantic coast of Africa, is comprised of seven islands aligned from east to west, plus seven minor islets. All the islands were formed by volcanic eruptions and their geological history is well documented providing a historical framework to study colonization events. The Canary Island pine (Pinus canariensis C. Sm.), nowadays restricted to the westernmost Canary Islands (Gran Canaria, Tenerife, La Gomera, La Palma and El Hierro), is considered an old (Lower Cretaceous) relic from an ancient Mediterranean evolutionary centre. Twenty seven chloroplast haplotypes were found in Canary Island pine but only one of them was common to all populations. The distribution of haplotypic variation in P. canariensis suggested the colonization of western Canary Islands from a single continental source located close to the Mediterranean Basin. Present-day populations of Canary Island pine retain levels of genetic diversity equivalent to those found in Mediterranean continental pine species, Pinus pinaster and Pinus halepensis. A hierarchical analysis of variance (AMOVA) showed high differentiation among populations within islands (approximately 19%) but no differentiation among islands. Simple differentiation models such as isolation by distance or stepping-stone colonization from older to younger islands were rejected based on product-moment correlations between pairwise genetic distances and both geographic distances and population-age divergences. However, the distribution of cpSSR diversity within the islands of Tenerife and Gran Canaria pointed towards the importance of the role played by regional Pliocene and Quaternary volcanic activity and long-distance gene flow in shaping the population genetic structure of the Canary Island pine. Therefore, conservation strategies at the population level are strongly recommended for this species.Communicated by D.B. NealeA. Gómez and S.C. González-Martínez as joint authors     

Mitochondrial haplotype distribution,seed dispersal and patterns of postglacial expansion of ponderosa pine

Maternally inherited mtDNA in a secondary contact zone of ponderosa pine revealed a cline less than 10 km wide - much narrower than previously described. A survey of 76 populations gave no evidence either of intermixing or of a mosaic contact zone. Such sharp contact zones are consistent with diffusive range expansion, rather than long distance colonization. However, evidence for long distance seed dispersal events was found in two populations where haplotypes were observed far from their main area of occurrence. The results suggest a small number of long distance colonists with diffusive dispersal from these centres.     

Cytoplasmic composition in Pinus densata and population establishment of the diploid hybrid pine

Sequence and restriction site analyses of the paternally inherited chloroplast rbcL gene and maternally inherited mitochondrial nad1 fragments from the same set of populations and individuals were used to investigate cytoplasmic composition and population establishment of Pinus densata, a diploid pine that originated through hybridization between P. tabuliformis and P. yunnanensis. Two variable sites and three chlorotypes (TT, TC and GC) were detected on the rbcL gene of the three pines. P. densata harboured the three chlorotypes, two of which (TT, GC) were characteristic of the parental species, respectively. The third chlorotype (TC) was distributed extensively in seven of the 10 P. densata populations analysed, and might represent a mutation type or have been derived from an extinct parent. The distribution of chlorotypes, together with that of mitotypes, indicated that significant founder effect and backcross happened during the population establishment of the hybrid pine. P. tabuliformis and P. yunnanensis had acted as both mother and father donors, i.e. bi-directional gene flow existed between the two parental species in the past. Population differentiation of P. densata is high, as detected from the cytoplasmic genomes: GST = 0.533 for cpDNA and GST = 0.905 for mtDNA. The differences in cytoplasmic composition among the hybrid populations suggest that the local populations have undergone different evolutionary histories.     

Promotion of Female Strobili Flowering and Seed Production in two Japanese Pine Species by 6-Benzylaminopurine (BAP) Paste Application in a Field Seed Orchard

Treatment with 6-benzylaminopurine (BAP) to promote the flowering of female strobili and to increase seed production in a seed orchard was examined at different times of conebud differentiation for Japanese red pine (Pinus densiflora) and Japanese black pine (P. thunbergii), from late August to mid-September 1998. BAP was mixed with lanolin and Vaseline to prepare a paste (BAP, 2000 mg/L), which was used for a terminal bud treatment and also applied in a girdling treatment. The BAP treatment in early September transformed lateral buds on lower branches that would have normally differentiated as male strobili into female strobili. The highest percentages of female flowering branches were 21.1% (red) and 40.5% (black) produced by the terminal bud treatment, while control branch percentages were 5.0% and 7.1%, respectively. Girdling combined with BAP treatment further promoted female flowering branches to 32.3% (red) and 64.3% (black), while those of controls were 8.3% and 9.9%, respectively. Moreover, the numbers of female strobili per female flowering branch were 6 to 11-fold greater than controls. Mature seed cones were harvested in October two years later from which full seeds with a high germination ratio were recovered. Use of BAP treatment is thus highly effective for enhancing seed production in seed orchards of these two pine species.     

Effect of Pine Callus Elicitation by the Fusarium Strains of Various Pathogenicity on the Content of Phenolic Compounds

Pine (Pinus sylvestris L.) callus culture was treated with the mycelium extracts from six Fusarium strains. Previously, pine seedlings were infected with a spore suspension in order to test the pathogenicity of the used strains. Callus culture infection resulted in a decrease in the free proanthocyanidin (PA) and an increase in bound PA content. After treating the calli with all strains except F. oxysporum var. orthoceras, the lignin content became lower than the control one. The most considerable changes involved the p-hydroxybenzoic acid (HBA) content, and its greatest change was observed after treating the calli with F. nivale, when the HBA concentration (1229 g/g dry wt) exceeded fourfold the control one. There was a positive correlation (R = 0.81) between the HBA content in the callus culture cells treated with a fungal extract and the virulence of Fusarium strains. At the same time, there was an inverse correlation (R = –0.80) between the lignin content in a callus culture and the fungal virulence; the latter did not affect the contents of both free and bound PA.     

Changes of morphogenic competence in mature Pinus sylvestris L. buds in vitro

The effects of season and cold storage on morphogenic competence in mature Pinus sylvestris buds were investigated. Peroxidase and polyphenol oxidase activity were measured as markers of oxidative metabolism. No growth in vitro was observed on explants detached from the end of January until the beginning of March. Brachioblasts, each with a couple of needles, formed on 11% of the buds without macrostrobili that were detached in early April and introduced immediately into culture. Of the explants detached in late July, 15% formed shoots with brachioblasts and needles. The lowest activity of peroxidase and polyphenol oxidase in pine buds was observed from the end of April until the beginning of June when morphogenic competence of tissues started to increase. Development of bud explants detached in January was achieved by cold storage for 5 months. Low polyphenol oxidase and peroxidase activity coincided with increased morphogenic potential. Results suggest that reduced or stable activity of peroxidase and polyphenol oxidase is associated with an increased ability of tissues to start growth in vitro.     

DNA from bird-dispersed seed and wind-disseminated pollen provides insights into postglacial colonization and population genetic structure of whitebark pine (Pinus albicaulis)

Uniparentally inherited mitochondrial (mt)DNA and chloroplast (cp)DNA microsatellites (cpSSRs) were used to examine population genetic structure and biogeographic patterns of bird-dispersed seed and wind-disseminated pollen of whitebark pine (Pinus albicaulis Engelm.). Sampling was conducted from 41 populations throughout the range of the species. Analyses provide evidence for an ancestral haplotype and two derived mtDNA haplotypes with distinct regional distributions. An abrupt contact zone between mtDNA haplotypes in the Cascade Range suggests postglacial biogeographic movements. Among three cpSSR loci, 42 haplotypes were detected within 28 cpSSR sample populations that were aggregated into six regions. Analysis of molecular variance (amova) was used to determine the hierarchical genetic structure of cpSSRs. amova and population pairwise comparisons (FST ) of cpSSR, and geographical distribution of mtDNA haplotypes provide insights into historical changes in biogeography. The genetic data suggest that whitebark pine has been intimately tied to climatic change and associated glaciation, which has led to range movements facilitated by seed dispersal by Clark's nutcracker (Nucifraga columbiana Wilson). The two hypotheses proposed to explain the genetic structure are: (i) a northward expansion into Canada and the northern Cascades in the early Holocene; and (ii) historical gene flow between Idaho and the Oregon Cascades when more continuous habitat existed in Central Oregon during the late Pleistocene. Genetic structure and insights gained from historical seed movements provide a basis on which to develop recovery plans for a species that is at risk from multiple threats.     

Plant and fungal responses to elevated atmospheric carbon dioxide in mycorrhizal seedlings of Pinus sylvestris

The effects of elevated CO₂ concentration upon the mycorrhizal relationships of Scots pine (Pinus sylvestris) seedlings were investigated. Plants were grown for 4 months with their shoots exposed to ambient (C_AMB=360 μl l⁻¹) or elevated (C_ELEV=700 μl l⁻¹) CO₂ environments while their root systems, either colonised by the mycorrhizal fungi Paxillus involutus or Suillus bovinus, or left in the non-mycorrhizal condition, were maintained in sealed dishes. In one series of these plants the effects of C_ELEV upon the extent of mycorrhizal development and upon their growth and nutrition were determined, while another series were transferred from the dishes after 1 month, to transparent observation chambers before being returned to the two CO₂ environments. In these chambers, the effects of C_ELEV upon development of the external mycelial systems of the two mycorrhizal fungi was determined by measuring the advance of the hyphal fronts of the mycorrhizal fungi across non-sterile peat from the colonised plants. The dry mass and number of mycorrhizal tips were significantly higher in C_ELEV than in the C_AMB condition in plants colonised by both fungi in the dishes. Yields of whole plants and of shoots were higher in the C_ELEV treatment whether or not they were grown in the mycorrhizal condition, but the greater yields were not associated in these sealed systems with enhanced nutrient gain. The dry mass of non-mycorrhizal plants was greater than that of those colonised by mycorrhizal fungi under elevated CO₂. This is thought to be attributable to the energetic cost of production of the larger mycorrhizal systems in this treatment. The extent of development of the mycorrhizal mycelial systems of both fungi was greatly increased in C_ELEV relative to that in C_AMB environments. It is hypothesised that increased allocation of carbon to mycorrhizal root systems and their associated mycelia would provide the potential for enhancement of nutrient acquisition in open systems of greater fertility.     

Litter production and accumulation in Pinus patula plantations of the Mpumalanga Province,South Africa

Litter accumulates on the forest floor in Pinus patula Schlechtd. et Cham. plantations in Mpumalanga, South Africa especially in high altitude sites, as a result of an imbalance between litter production and decomposition. To determine which factor caused this increase we measured litter production in an age series of P. patula growing at low and high altitude sites. Litter production was shown to increase with increasing stand. The average annual litter production figure ranged from 3.64 to 5.89 t ha-1 yr-1 measured over a two-year period, with maximum litterfall occurring from mid-winter to early spring. Litter accumulation, as measured by litter depth, ranged from 39 to 121 mm. No significant relationship existed between litter production and altitude; however, litter accumulation was shown to increase with increasing stand age and increasing altitude. Increased litter production was, therefore, not responsible for the accumulation of litter in high altitude sites.