Genetic structure of Araucaria angustifolia (Araucariaceae) populations in Brazil: implications for the in situ conservation of genetic resources

The distribution of the genetic variation within and among natural populations of A. ANGUSTIFOLIA growing in different regions in Brazil was assessed at microsatellite and AFLP markers. Both markers revealed high gene diversity ( H = 0.65; AR = 9.1 for microsatellites and H = 0.27; P = 77.8 % for AFLPs), moderate overall differentiation ( RST = 0.13 for microsatellites and FST = 0.10 for AFLPs), but high divergence of the northernmost, geographically isolated population. In a Bayesian analysis, microsatellite data suggested population structure at two levels: at K = 2 and at K = 3 in agreement to the geographical distribution of populations. This result was confirmed by the UPGMA dendrogram based on microsatellite data (bootstrap support > 95 %). Non-hierarchical AMOVA revealed high variation among populations from different A POSTERIORI defined geographical groups. The genetic distance between sample locations increased with geographical distance for microsatellites ( R = 0.62; P = 0.003) and AFLPs ( R = 0.32; P = 0.09). This pattern of population differentiation may be correlated with population history such as geographical isolation and postglacial colonization of highlands. Implications of the population genetic structure for the conservation of genetic resources are discussed.     

Cell-to-cell trafficking patterns in cell lines of Araucaria angustifolia (Brazilian pine) with contrasting embryogenic potential

Plant Cell, Tissue and Organ Culture (PCTOC) - The acquisition of embryogenic competence involves aspects at a cellular level related to cell communication and signaling. For Araucaria...     

Initial growth of Brazilian pine (Araucaria angustifolia) under equal soil volumes but contrasting rooting depths

Araucaria angustifolia is a critically endangered tall tree species of valuable wood, and field observations led to the suggestion that limitations imposed to the vertical growth of its tap root system greatly restrict the height of mature individuals. However, experimental studies dealing with the effects of soil depth on the species growth are mostly lacking. This study evaluated and compared the growth responses of young plants of A. angustifolia to distinct rooting depths but same soil volumes. Seeds were planted in pots of different heights and diameters, all containing 3 liters of soil mixture. Plants were submitted to four available rooting depths: 65 (T1), 35 (T2), 20 (T3), and 10 (T4) cm. There were eight experimental units in each treatment, arranged in a randomized complete block design, each block containing two units per treatment. Contrary to what was expected, the T3 and T4 plants had accumulated more mass and attained the same height as the other two groups, after a 10-month growth period in a green house. Those plants also had thicker stems, longer shoot branches, and thicker and longer lateral roots, which were interpreted as compensatory responses to increase plant anchorage and stability. The inverse relationship between rooting depth and plant mass was attributed to a down-regulation of shoot growth because or restricted lateral space and/or poor soil aeration of the longer and narrower pots. This experiment allowed us to demonstrate that is not the possibility of the tap root to grow deep into the soil that ensures a better growth to plants of A. angustifolia: provided that the offer of soil volume and resources are the same, the vertical extension of the tap root does not result in greater growth of the plants. In fact, much greater growth impairment is expected from lateral than from vertical restriction to root growth.     

Reproductive biology of the “Brazilian pine” (Araucaria angustifolia – Araucariaceae): Development of microspores and microgametophytes

Araucaria angustifolia Bert. (O. Ktze), also known as the “Brazilian pine”, is native to the South of Brazil. This species has a long reproductive cycle, taking about 29–34 months. Its cones begin to develop in early January and remain dormant from March to July. In August, they become active again and microsporogenesis occurs, which proceeds until September. From September to October, microgametogenesis is established and pollination occurs from October to November. Meiosis is asynchronous, with simultaneous cytokinesis, and the tetrads are of the tetrahedral and isobilateral type. During gametogenesis, microgametophytes gradually develop an axial row of cells that are isolated by internal callose and undergo four mitotic cycles until pollen dispersal. In mature pollen grains, the vegetative cells do not possess a cell wall, but maintain strong internal polarization. The pollen of A. angustifolia is suboblate, without apertures or air sacs. Histochemical analysis of the sporoderm was also performed and when compared to other conifer families, showed the most simplified intine structure among the group. Embryological characteristics analyzed during the phenological phases of this species showed certain peculiarities, knowledge about which may be helpful contributing to the management and conservation of A. angustifolia.     

Early growth of Brazilian pine (Araucaria angustifolia[Bertol.] Kuntze) in response to soil compaction and drought

Soil compaction leads to changes in soil physical properties such as density, penetration resistance and porosity, and, by consequence, affects root and plant growth. The initial growth of Brazilian pine is considered as being more affected by soil physical than chemical conditions, and the presence of a well-developed tap root system has been associated with this fact. A greenhouse experiment was conducted in order to evaluate the impact of soil compaction on the growth of Brazilian pine seedlings and on their susceptibility to a simulated drought period. In the first phase of the experiment, the effects of three levels of soil compaction on root morphology and plant growth were examined. Soil cylinders were artificially compacted in PVC tubes. Pre-germinated seeds were planted, and 147 days later 10 plants from each treatment were harvested for analysis. Higher values of soil density were associated with a shorter and thicker tap root. Growth of lateral roots and shoots remained unaffected at this stage. In the second phase, half of the plants (12) in each compaction treatment were drought-stressed by withholding water for a period of 77 days. Increased soil compaction again resulted in reduced length and increased diameter of the main tap root. This time, the effects were also extended to the lateral roots. Shoot extension growth and overall plant mass, however, increased with soil compaction. This greater mass accumulation in plants growing under increased soil compaction may be attributed to a more intimate contact between roots and soil particles. Drought stress reduced both root and shoot growth, but root mass was more negatively affected by drought stress in plants growing under high levels of soil compaction. Future investigations on the effects of soil compaction on the initial growth of Brazilian pine should include a wider range of compaction levels to better establish the relationship between soil physical parameters and plant growth.     

Changes in IAA, tryptophan and activity of soluble peroxidase associated with zygotic embryogenesis in Araucaria angustifolia (Brazilian pine)

In A. angustifolia seeds the highest values of freeIAA occurred in the embryonic axis, at the initial phases of development. Thesevalues decreased sharply coincident with the increase of IAA with thedifferentiation of cotyledons and seed elongation. During seed development,tryptophan concentrations varied inversely with free IAA and directly withconjugated IAA. An increase in peroxidase activity was followed by a decreaseinfree IAA in the embryo axis, and in conjugated IAA in the megagametophyte.Megagametophyte tissues did not exhibit significant variation in free IAAduringseed development. Following the stage where cotyledons arise, tryptophandecreased in the megagametophyte and increased in the cotyledons and embryonicaxis.     

Root and Crown Rot of Brazilian Pine (Araucaria angustifolia) Caused by Phytophthora cinnamomi

In an area reforested with Brazilian pine (Araucaria angustifolia) located in Paraná State, southern Brazil, 20‐ to 40‐year‐old trees representing 0.2% of the surveyed area had symptoms of root and crown rot, yellowing and browning of leaves from the uppermost branches and death. Three Phytophthora isolates obtained from diseased plant tissue were tested against 1‐year‐old Brazilian pine seedlings and found to display positive pathogenicity. Based on their morphological and physiological characteristics, the isolates were identified as Phytophthora cinnamomi. A GenBank BLAST search of partial sequences from the β‐tubulin and elongation factor‐1α genes, as well as the ITS regions and 5.8S gene of rDNA, confirmed the species identification. This is the first report of the involvement of this pathogen on the aetiology of Brazilian pine root and crown rot.     

Cold stress on Araucaria angustifolia embryogenic cells results in oxidative stress and induces adaptation: implications for conservation and propagation

Araucaria angustifolia (Bert.) O. Kuntze is a species critically endangered of extinction and its development and propagation is strongly affected by abiotic stress. We have previously shown the activation of uncoupling protein in A. angustifolia embryogenic stem cells subjected to cold stress. Now, we have furthered those studies by exposing these cells to cold stress (4?±?1?°C for either 24 or 48?h) and evaluating parameters associated with oxidative stress and alterations in the cellular and mitochondrial responses. Cold stress affect the H₂O₂ levels and lipid peroxidation increased after both stress condition, an effect associated with the decrease in the activities of peroxidases, catalase and ascorbate/dehydroascorbate ratio. On the other hand, the activities of ascorbate peroxidase, monodehydroascorbate and dehydroascorbate reductases increased as an indication of adaptation. Another important impact of cold stress conditions was the decrease of external alternative NAD(P)H dehydrogenases activity and the increase of mitochondrial mass. These results show that cold stress induces oxidative stress in A. angustifolia embryogenic cells, which results in activation of the glutathione-ascorbate cycle as a compensation for the decrease in the activities of catalase, peroxidases, and external NAD(P)H dehydrogenases. Our results contribute to the understanding of the pathways that gymnosperms employ to overcome oxidative stress, which must be explored in order to improve the methods of conservation and propagation of A. angustifolia.     

Development of mucilage cells of Araucaria angustifolia (Araucariaceae)

The roles of mucilage cells were investigated through morphological and cytological analysis during leaf development in young Araucaria angustifolia plants. Differentiation began in leaf primordia in the shoot apex, when the young cells underwent a greater increase in volume in comparison with other mesophyll cells. The mucilage polysaccharides were synthesized by dictyosomes, from where they were taken by large vesicles and released into a cavity formed by detachment of the tonoplast, which was separated from the cytoplasm. At the end of differentiation, the cell was completely filled with mucilage, a gel consisting of a denser reticular structure surrounding less dense regions. The nucleus and cytoplasm were degenerated in mature cells. The A. angustifolia mucilage cells presented some cytological resemblances to the mucilage cells of members of some dicotyledonous families; however, differences in the dictyosomes and the secretion route were observed. Translocation and water storage of solutes was suggested by the use of the hydroxy pyrenetrisulfonic acid tri-sodium salt apoplastic tracer. The tonoplast detachment, dechromatinization, nuclear condensation, and general degeneration of the membrane systems observed during maturity indicated a programmed cell death process, one not yet described for angiosperm mucilage cells.     

Cloning and expression of embryogenesis-regulating genes in Araucaria angustifolia (Bert.) O. Kuntze (Brazilian Pine)

Angiosperm and gymnosperm plants evolved from a common ancestor about 300 million years ago. Apart from morphological and structural differences in embryogenesis and seed origin, a set of embryogenesis-regulating genes and the molecular mechanisms involved in embryo development seem to have been conserved alike in both taxa. Few studies have covered molecular aspects of embryogenesis in the Brazilian pine, the only economically important native conifer in Brazil. Thus eight embryogenesis-regulating genes, viz., ARGONAUTE 1, CUP-SHAPED COTYLEDON 1, WUSCHEL-related WOX, S-LOCUS LECTIN PROTEIN KINASE, SCARECROW-like, VICILIN 7S, LEAFY COTYLEDON 1, and REVERSIBLE GLYCOSYLATED POLYPEPTIDE 1, were analyzed through semi-quantitative RT-PCR during embryo development and germination. All the eight were found to be differentially expressed in the various developmental stages of zygotic embryos, seeds and seedling tissues. To our knowledge, this is the first report on embryogenesis-regulating gene expression in members of the Araucariaceae family, as well as in plants with recalcitrant seeds.     

Realized pollen and seed dispersal within a continuous population of the dioecious coniferous Brazilian pine [Araucaria angustifolia (Bertol.) Kuntze]

Extensive realized pollen and seed flow across populations reduces inbreeding and spatial genetic structure (SGS) and increases the genetic diversity and effective size within populations. Inbreeding, SGS and realized patterns of pollen and seed dispersal of the dioecious, wind pollinated Araucaria angustifolia were investigated based on microsatellite loci. The study was conducted in a 7.2 ha plot established within a continuous Araucaria Forest in Southern Brazil. In the plot, all 290 adult trees were mapped, measured (diameter at breast height, dbh), sexed, sampled and genotyped. We also sampled, measured (total height), mapped and genotyped 223 juveniles. A total of 86 alleles were detected in the sample (n = 513). Adults and juveniles showed a positive and significant fixation index (minimum of 0.096), suggesting inbreeding or Wahlund effect. Juveniles presented a significant aggregated distribution which was associated with a genetic aggregation (significant SGS up to 20 m), indicating that near neighbor trees may be related due the limited seed dispersal. However, the intensity of SGS was not significantly higher among juveniles (Sp = 0.0041) than adults (Sp = 0.0026). Realized pollen and seed immigration into the plot was low (pollen = 6 %; seeds = 5 %) and the patterns of pollen and seed dispersal were similar. Pollen was dispersed over long distances (343 m), but 50 % was dispersed up to 124 m. Seeds also reached long distances (318 m), with 50 % being dispersed up to 133 m. Our results are discussed in terms of auto-ecology and the genetic conservation of A. angustifolia populations.     

Isolation and characterization of microsatellite markers for Araucaria angustifolia (Araucariaceae)

Araucaria angustifolia is a dioecious tree species that occurs in the southern part of Brazil. Because of the intense exploitation of the species due to its valuable wood, only 2% of the original population still remains. Twenty‐nine species‐specific and highly polymorphic microsatellite loci were developed from a genomic library enriched for AG/TC repeats. Levels of polymorphism were evaluated using a total of 16 adult trees from a natural population. An average of 8.1 alleles per locus was detected, and expected heterozygosity ranged from 0.63 to 0.72.     

Native tree regeneration in native tree plantations: understanding the contribution of Araucaria angustifolia to biodiversity conservation in the threatened Atlantic Forest in Argentina

Deforestation is a global process that has strongly affected the Atlantic Forest in South America, which has been recognised as a threatened biodiversity hotspot. An important proportion of deforested areas were converted to forest plantations. Araucaria angustifolia is a native tree to the Atlantic Forest, which has been largely exploited for wood production and is currently cultivated in commercial plantations. An important question is to what extent such native tree plantations can be managed to reduce biodiversity loss in a highly diverse and vulnerable forest region . We evaluated the effect of stand age, stand basal area, as a measure of stand density, and time since last logging on the density and richness of native tree regeneration in planted araucaria stands that were successively logged over 60 years, as well as the differences between successional groups in the response of plant density to stand variables. We also compared native tree species richness in planted araucaria stands to neighbouring native forest. Species richness was 71 in the planted stands (27 ha sampled) and 82 in native forest (18 ha sampled) which approximate the range of variation in species richness found in the native forests of the study area. The total abundance and species richness of native trees increased with stand age and time since last logging, but ecological groups differed in their response to such variables. Early secondary trees increased in abundance with stand age 3–8 times faster than climax or late secondary trees. Thus, the change in species composition is expected to continue for a long term. The difference in species richness between native forest and planted stands might be mainly explained by the difference in plant density. Therefore, species richness in plantations can contribute to local native tree diversity if practices that increase native tree density are implemented.     

Integrating approaches towards the conservation of forest genetic resources: a case study of Araucaria angustifolia

In the past years the conservation of forest genetic resources has been widely recognized because of the risk of adverse effects associated with global changes in the environment. Since little effort has been made towards conservation of these resources, the assessment and the integration of information and knowledge so far generated are valuable tools in order to promote their conservation. In the present work the Brazilian conifer Araucaria angustifolia was used as a case study, integrating information, published between 1980 and 2007, about genetics, biotechnology, palaeobotany, and archeology. These studies were interpreted towards the conservation needs and strategies of the species germplasm. The main conclusions for the conservation of A. angustifolia genetic resources are: (1) despite the reduction of genetic diversity by unsustainable forest exploitation, the genetic system of A. angustifolia seems to be efficient to protect this species against rapid losses of its genetic diversity; (2) conservation measures starting now may be efficient, since the genetic structures of existing populations is more largely influenced by the past evolutionary process than by human disturbance occurred in the last century; (3) there are opportunities to save the genetic resources of the species, if simple measures are taken to safeguard the continued existence of the few remaining araucaria forests. Emerged as a general conclusion that the use of sound scientific arguments supported by different areas of the science could be a very powerful instrument in persuading decision-makers, also by governments, taking into account forest genetic resources as a primary issue for the future.     

Purification and biological effects of Araucaria angustifolia (Araucariaceae) seed lectin

This paper describes the purification and characterization of a new N-acetyl-d-glucosamine-specific lectin from Araucaria angustifolia (AaL) seeds (Araucariaceae) and its anti-inflammatory and antibacterial activities. AaL was purified using a combination of affinity chromatography on a chitin column and ion exchange chromatography on Sephacel-DEAE. The pure protein has 8.0kDa (SDS-PAGE) and specifically agglutinates rabbit erythrocytes, effect that was independent of the presence of divalent cations and was inhibited after incubation with glucose and N-acetyl-d-glucosamine. AaL showed antibacterial activity against Gram-negative and Gram-positive strains, shown by scanning electron microscopy. AaL, intravenously injected into rats, showed anti-inflammatory effect, via carbohydrate site interaction, in the models of paw edema and peritonitis. This lectin can be used as a tool for studying bacterial infections and inflammatory processes.     

Updating embryonic ontogenesis in Araucaria angustifolia : from Burlingame (1915) to the present

This study addresses gaps in our understanding of pre-fertilization and archegonia development and reinterprets embryonic ontogenesis from Burlingame (Bot Gaz 59:1–39, 1915) to the present based on timescale and structural features allowing us to determine functionally and developmentally accurate terminology for all these stages in A. angustifolia. Different from previous reports, only after pollination, pre-fertilization tissue development occurs (0–13 months after pollination (MAP)) and gives rise to a mature megagametophyte. During all this period, pollen is in a dormant state at the microphyla, and pollen tube germination in nucellus tissue is only observed at the stage of archegonia formation (13 MAP) and not at the free nuclei stage as reported before. For the first time, 14 months after pollination, a fertilization window was indicated, and at 15 MAP, the polyzygotic polyembryony from different archegonia was also seen. After that, subordinated proembryo regression occurs and at least three embryonic developmental stages of dominant embryo were characterized: proembryogenic, early embryogenic, and late embryogenic (15–23 MAP). Along these stages, histochemical and ultrastructural analyses suggest the occurrence of cell death in suspensor and in cap cells of dominant embryo that was not previously reported. The differentiation of meristems, procambium, pith, and cortex tissues in late embryogenic stage was detailed. The morphohistological characterization of pre-fertilization and embryonic stages, together with the timescale of megastrobili development, warranted a referential map of female reproductive structure in this species.     

Genetic diversity of Chinese water deer (Hydropotes inermis inermis): implications for conservation

The Chinese water deer (Hydropotes inermis inermis) is endemic to China. Historically, the species was widely distributed, but now, habitat loss and poaching have reduced its range and number drastically. In order to provide useful information for its conservation, we have investigated the genetic diversity and population structure of the Chinese water deer by analyzing the 403 bp fragment of the mitochondrial DNA (mtDNA) control region (D-loop). Eighteen different haplotypes were detected in 40 samples. Overall, Chinese water deer have a relatively high-genetic diversity compared to other rare cervid species, with a haplotype diversity of 0.923+/-0.025 and nucleotide diversity of 1.318 +/- 0.146%. No obvious phylogenetic structure among haplotypes was found for samples of different origin. An analysis of molecular variance (AMOVA) showed significant differentiation between the Zhoushan and the mainland population (F(ST)= 0.088, P < 0.001; Phi( ST ) = 0.075, P = 0.043), which suggests that exchanges of individuals between Zhoushan and the mainland should be avoided. We also recommend that a breeding center be set up for the mainland population.     

Genetic diversity of the endangered Brazilian endemic herb Cunila menthoides Benth. (Lamiaceae) and its implications for conservation

Cunila menthoides is classified as a xylopodiferous subshrub found in fragmented environments threatened by local agriculture and pasture activities. The species presents strong and pleasant smell, and is commonly used in south Brazilian folk medicine. Based on the species’ medicinal and aromatic potential, the aim of this work was to examine the genetic variation within and among C. menthoides populations, focusing on the conservation of this species. In this context, six C. menthoides populations collected in Rio Grande do Sul, Brazil, were analyzed by ISSR. The populations were sorted as four clusters according to geographic distribution. The six C. menthoides populations analyzed presented low genetic variability, indicating that each population derives from a limited number of plants in a low gene flow scenario. In situ and ex situ conservation approaches may be useful in this species. Conservation strategies for the species should be based on in situ and ex situ approaches.