Comprehensive conservation assessments reveal high extinction risks across Atlantic Forest trees.

Biodiversity is declining globally, yet many biodiversity hotspots still lack comprehensive species conservation assessments. Using multiple International Union for Conservation of Nature (IUCN) Red List criteria to evaluate extinction risks and millions of herbarium and forest inventory records, we present automated conservation assessments for all tree species of the Atlantic Forest biodiversity hotspot, including ~1100 heretofore unassessed species. About 65% of all species and 82% of endemic species are classified as threatened. We rediscovered five species classified as Extinct on the IUCN Red List and identified 13 endemics as possibly extinct. Uncertainties in species information had little influence on the assessments, but using fewer Red List criteria severely underestimated threat levels. We suggest that the conservation status of tropical forests worldwide is worse than previously reported.

Deep vicariance and frequent transoceanic dispersal shape the evolutionary history of a globally distributed fern family.

PREMISE: The historical biogeography of ferns is typically expected to be dominated by long-distance dispersal due to their minuscule spores. However, few studies have inferred the historical biogeography of a large and widely distributed group of ferns to test this hypothesis. Our aims were to determine the extent to which long-distance dispersal vs. vicariance have shaped the history of the fern family Blechnaceae, to explore ecological correlates of dispersal and diversification, and to determine whether these patterns differ between the northern and southern hemispheres. METHODS: We used sequence data for three chloroplast loci to infer a time-calibrated phylogeny for 154 of 265 species of Blechnaceae, including representatives of all genera in the family. This tree was used to conduct ancestral range reconstruction and stochastic character mapping, estimate diversification rates, and identify ecological correlates of diversification. RESULTS: Blechnaceae originated in Eurasia and began diversifying in the late Cretaceous. A lineage comprising most extant diversity diversified principally in the austral Pacific region around the Paleocene-Eocene Thermal Maximum. Land connections that existed near the poles during periods of warm climates likely facilitated migration of several lineages, with subsequent climate-mediated vicariance shaping current distributions. Long-distance dispersal is frequent and asymmetrical, with New Zealand/Pacific Islands, Australia, and tropical America being major source areas. CONCLUSIONS: Ancient vicariance and extensive long-distance dispersal have shaped the history of Blechnaceae in both the northern and southern hemispheres. The exceptional diversity in austral regions appears to reflect rapid speciation in these areas; mechanisms underlying this evolutionary success remain uncertain.

Human impacts as the main driver of tropical forest carbon.

Understanding the mechanisms controlling forest carbon storage is crucial to support "nature-based" solutions for climate change mitigation. We used a dataset of 892 Atlantic Forest inventories to assess the direct and indirect effects of environmental conditions, human impacts, tree community proprieties, and sampling methods on tree above-ground carbon stocks. We showed that the widely accepted drivers of carbon stocks, such as climate, soil, topography, and forest fragmentation, have a much smaller role than the forest disturbance history and functional proprieties of the Atlantic Forest. Specifically, within-forest disturbance level was the most important driver, with effect at least 30% higher than any of the environmental conditions individually. Thus, our findings suggest that the conservation of tropical carbon stocks may be dependable on, principally, avoiding forest degradation and that conservation policies focusing only on carbon may fail to protect tropical biodiversity.

Azepine-Indole Alkaloids From Psychotria nemorosa Modulate 5-HT2A Receptors and Prevent in vivo Protein Toxicity in Transgenic Caenorhabditis elegans.

Nemorosine A (1) and fargesine (2), the main azepine-indole alkaloids of Psychotria nemorosa, were explored for their pharmacological profile on neurodegenerative disorders (NDs) applying a combined in silico-in vitro-in vivo approach. By using 1 and 2 as queries for similarity-based searches of the ChEMBL database, structurally related compounds were identified to modulate the 5-HT2A receptor; in vitro experiments confirmed an agonistic effect for 1 and 2 (24 and 36% at 10 µM, respectively), which might be linked to cognition-enhancing properties. This and the previously reported target profile of 1 and 2, which also includes BuChE and MAO-A inhibition, prompted the evaluation of these compounds in several Caenorhabditis elegans models linked to 5-HT modulation and proteotoxicity. On C. elegans transgenic strain CL4659, which expresses amyloid beta (Aß) in muscle cells leading to a phenotypic paralysis, 1 and 2 reduced Aß proteotoxicity by reducing the percentage of paralyzed worms to 51%. Treatment of the NL5901 strain, in which α-synuclein is yellow fluorescent protein (YFP)-tagged, with 1 and 2 (10 µM) significantly reduced the α-synuclein expression. Both alkaloids were further able to significantly extend the time of metallothionein induction, which is associated with reduced neurodegeneration of aged brain tissue. These results add to the multitarget profiles of 1 and 2 and corroborate their potential in the treatment of NDs.

Quality Control of Herbal Medicines: From Traditional Techniques to State-of-the-art Approaches.

Herbal medicines are important options for the treatment of several illnesses. Although their therapeutic applicability has been demonstrated throughout history, several concerns about their safety and efficacy are raised regularly. Quality control of articles of botanical origin, including plant materials, plant extracts, and herbal medicines, remains a challenge. Traditionally, qualitative (e.g., identification and chromatographic profile) and quantitative (e.g., content analyses) markers are applied for this purpose. The compound-oriented approach may stand alone in some cases (e.g., atropine in Atropa belladonna). However, for most plant materials, plant extracts, and herbal medicines, it is not possible to assure quality based only on the content or presence/absence of one (sometimes randomly selected) compound. In this sense, pattern-oriented approaches have been extensively studied, introducing the use of multivariate data analysis on chromatographic/spectroscopic fingerprints. The use of genetic methods for plant material/plant extract authentication has also been proposed. In this study, traditional approaches are reviewed, although the focus is on the applicability of fingerprints for quality control, highlighting the most used approaches, as well as demonstrating their usefulness. The literature review shows that a pattern-oriented approach may be successfully applied to the quality assessment of articles of botanical origin, while also providing directions for a compound-oriented approach and a rational marker selection. These observations indicate that it may be worth considering to include fingerprints and their data analysis in the regulatory framework for herbal medicines concerning quality control since this is the foundation of the holistic view that these complex products demand.

The erosion of biodiversity and biomass in the Atlantic Forest biodiversity hotspot.

Tropical forests are being deforested worldwide, and the remaining fragments are suffering from biomass and biodiversity erosion. Quantifying this erosion is challenging because ground data on tropical biodiversity and biomass are often sparse. Here, we use an unprecedented dataset of 1819 field surveys covering the entire Atlantic Forest biodiversity hotspot. We show that 83-85% of the surveys presented losses in forest biomass and tree species richness, functional traits, and conservation value. On average, forest fragments have 25-32% less biomass, 23-31% fewer species, and 33, 36, and 42% fewer individuals of late-successional, large-seeded, and endemic species, respectively. Biodiversity and biomass erosion are lower inside strictly protected conservation units, particularly in large ones. We estimate that biomass erosion across the Atlantic Forest remnants is equivalent to the loss of 55-70 thousand km2 of forests or US$2.3-2.6 billion in carbon credits. These figures have direct implications on mechanisms of climate change mitigation.

Bioactive Azepine-Indole Alkaloids from Psychotria nemorosa.

Phytochemical investigation of the alkaloid extract of the aerial parts of Psychotria nemorosa led to the isolation and characterization of 10 azepine-indole alkaloids, i.e., cimitrypazepine (1), fargesine (2), nemorosines A (3), and B (12), nemorosinosides A-F (4-9), as well as two ß-carboline derivatives, 10-hydroxyisodolichantoside (10) and 10-hydroxydolichantoside (11), an isoxazole alkaloid, nemorosinoside G (13), serotonin (14), bufotenine (15), and (S)-gentianol (16). Compounds 3-13 have not yet been described. These compounds were isolated by semipreparative HPLC, and their structures were determined by means of HRMS, NMR, and ECD measurements. In addition, the monoamine oxidase-A (MAO-A), MAO-B, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) inhibitory activities were evaluated. Alkaloids 1-3 inhibited the MAO-A activity with IC50 values of 1.4, 1.4, and 0.9 µM, respectively.

Targeted Isolation of Monoterpene Indole Alkaloids from Palicourea sessilis.

Phytochemical investigation of the alkaloid extract of Palicourea sessilis by LC-HRMS/MS using molecular networking and an in silico MS/MS fragmentation approach suggested the presence of several new monoterpene indole alkaloids. These compounds were isolated by semipreparative HPLC, and their structures confirmed by means of HRMS, NMR, and ECD measurements as 4-N-methyllyaloside (3), 4-N-methyl-3,4-dehydrostrictosidine (4), 4ß-hydroxyisodolichantoside (6), and 4α-hydroxyisodolichantoside (7), as well as the known alkaloids alline (1), N-methyltryptamine (2), isodolichantoside (5), and 5-oxodolichantoside (8). In addition, the acetylcholinesterase inhibitory activity of the compounds was evaluated up to 50 µM.

Toxicological profile and acetylcholinesterase inhibitory potential of Palicourea deflexa, a source of ß-carboline alkaloids.

Palicourea genus is chemically and taxonomically close to Psychotria genus, a well-known source of neuroactive alkaloids. It has been previously reported the pharmacological potential of these alkaloids in some targets related to the neurodegenerative process. In this context, this study was carried out in order to evaluate the toxic effects and acetylcholinesterase (AChE) inhibitory potential of Palicourea deflexa fraction of total alkaloids (FTA). P. deflexa FTA was analyzed by means of HPLC-DAD and HRMS-ESI. We performed toxicological screening through Fish Embryo Toxicity (FET) test using zebrafish embryo and abnormal developmental phenotypes were recorded daily. For AChE inhibition, zebrafish brains were used as enzymatic source and formation of thiolate dianion of 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) was used to monitor acetylthiocholine hydrolysis. Lineaweaver-Burk double reciprocal plots were used to indicate mode of inhibition. Chemical analysis of the P. deflexa FTA allowed the identification of the main compound as harman-3-carboxylic acid. This fraction was evaluated in vivo for its toxicological effect. The zebrafish embryo test indicated that the FTA has a lethal concentration of 50% (LC50)=72.18µg/mL. Further, the FTA was evaluated for its AChE inhibitory profile, demonstrating an inhibitory concentration of 50% (IC50) of 50.65µg/mL. Lineaweaver-Burk double reciprocal plots indicated a mixed mode of inhibition. It is reported for the first time the toxicological and pharmacological profile of the alkaloid fraction of Palicourea deflexa in zebrafish models.

Molecular phylogeny of the fern family Blechnaceae (Polypodiales) with a revised genus-level treatment.

Blechnaceae, a leptosporangiate fern family nested within eupolypods II, comprises 200-250 species, typically divided among seven to nine genera. Despite recent molecular studies of the family, it still lacks a modern taxonomic update based on broad sampling from the two centres of diversity-the Neotropics and Australasia/Oceania. To test generic circumscriptions, we have assembled the broadest dataset thus far, from three plastid regions (rbcL, rps4-trnS, trnL-trnF) and with taxonomic sampling focused on both major diversity centres. Our sampling includes 156 taxa and 178 newly generated sequences. We recognize three subfamilies, each corresponding to a highly supported clade across all analyses (maximum parsimony, Bayesian inference and maximum likelihood). The genera Salpichlaena, Stenochlaena and Telmatoblechnum are monophyletic, while Blechnum is polyphyletic, because Brainea, Doodia and Sadleria all nest within it. We outline and explain a plan to resolve the polyphyly of Blechnum by recognizing additional, monophyletic, segregate genera.

The use of chemometrics to study multifunctional indole alkaloids from Psychotria nemorosa (Palicourea comb. nov.). Part II: Indication of peaks related to the inhibition of butyrylcholinesterase and monoamine oxidase-A.

Psychotria nemorosa is chemically characterized by indole alkaloids and displays significant inhibitory activity on butyrylcholinesterase (BChE) and monoamine oxidase-A (MAO-A), both enzymes related to neurodegenerative disorders. In the present study, 43 samples of P. nemorosa leaves were extracted and fractionated in accordance to previously optimized methods (see Part I). These fractions were analyzed by means of UPLC-DAD and assayed for their BChE and MAO-A inhibitory potencies. The chromatographic fingerprint data was first aligned using correlation optimized warping and Principal Component Analysis to explore the data structure was performed. Multivariate calibration techniques, namely Partial Least Squares (PLS1), PLS2 and Orthogonal Projections to Latent Structure (O-PLS1), were evaluated for modelling the activities as a function of the fingerprints. Since the best results were obtained with O-PLS1 model (RMSECV=9.3 and 3.3 for BChE and MAO-A, respectively), the regression coefficients of the model were analyzed and plotted relative to the original fingerprints. Four peaks were indicated as multifunctional compounds, with the capacity to impair both BChE and MAO-A activities. In order to confirm these results, a semi-prep HPLC technique was used and a fraction containing the four peaks was purified and evaluated in vitro. It was observed that the fraction exhibited an IC50 of 2.12µgmL(-1) for BChE and 1.07µgmL(-1) for MAO-A. These results reinforce the prediction obtained by O-PLS1 modelling.

The use of chemometrics to study multifunctional indole alkaloids from Psychotria nemorosa (Palicourea comb. nov.). Part I: Extraction and fractionation optimization based on metabolic profiling.

Extraction methods evaluation to access plants metabolome is usually performed visually, lacking a truthful method of data handling. In the present study the major aim was developing reliable time- and solvent-saving extraction and fractionation methods to access alkaloid profiling of Psychotria nemorosa leaves. Ultrasound assisted extraction was selected as extraction method. Determined from a Fractional Factorial Design (FFD) approach, yield, sum of peak areas, and peak numbers were rather meaningless responses. However, Euclidean distance calculations between the UPLC-DAD metabolic profiles and the blank injection evidenced the extracts are highly diverse. Coupled with the calculation and plotting of effects per time point, it was possible to indicate thermolabile peaks. After screening, time and temperature were selected for optimization, while plant:solvent ratio was set at 1:50 (m/v), number of extractions at one and particle size at ≤180µm. From Central Composite Design (CCD) results modeling heights of important peaks, previously indicated by the FFD metabolic profile analysis, time was set at 65min and temperature at 45°C, thus avoiding degradation. For the fractionation step, a solid phase extraction method was optimized by a Box-Behnken Design (BBD) approach using the sum of peak areas as response. Sample concentration was consequently set at 150mg/mL, % acetonitrile in dichloromethane at 40% as eluting solvent, and eluting volume at 30mL. Summarized, the Euclidean distance and the metabolite profiles provided significant responses for accessing P. nemorosa alkaloids, allowing developing reliable extraction and fractionation methods, avoiding degradation and decreasing the required time and solvent volume.

Structure of mixed ombrophyllous forests with Araucaria angustifolia (Araucariaceae) under external stress in Southern Brazil.

This study is part of the Floristic and Forest Inventory of Santa Catarina, conceived to evaluate forest resources, species composition and structure of forest remnants, providing information to update forest conservation and land use policy in Southern Brazilian State of Santa Catarina (95 000 km2). In accordance to the Brazilian National Forest Inventory (IFN-BR), the inventory applies systematic sampling, with 440 clusters containing four crosswise 1 000m2 plots (20 x 50m) each, located on a 10 x 10km grid overlaid to land use map based on classification of SPOT-4 images from 2005. Within the sample units, all woody individuals of the main stratum (DBH > or = 10cm) are measured and collected (fertile and sterile), if not undoubtedly identified in field. Regeneration stratum (height > 1.50m; DBH < 10cm) is registered in 100m2 in each sample unit. Floristic sampling includes collection of all fertile trees, shrubs and herbs within the sample unit and in its surroundings. This study performs analysis based on 92 clusters measured in 2008 within an area of 32320km2 of mixed ombrophyllous forests with Araucaria angustifolia located at the state's high plateau (500m to 1 560m above sea level at 26 degrees 00'-28 degrees 30' S and 49 degrees 13'-51 degrees 23' W). Mean density (DBH > or = 10cm) is 578 individuals/ha (ranging from 85/ha to 1 310/ha), mean species richness in measured remnants is 35 (8 to 62), Shannon and Wiener diversity index (H') varies between 1.05 and 3.48. Despite high total species diversity (364 Magnoliophyta, five Coniferophyta and one tree fern) and relatively high mean basal area (25.75m2/ha, varying from 3.87 to 68.85m2/ha), the overwhelming majority of forest fragments are considered highly impacted and impoverished, mostly by logging, burning and extensive cattle farming, turning necessary more efficient protection measures. Basal area was considered an appropriate indicator for stand quality and conservation status.

Structure of mixed ombrophyllous forests with Araucaria angustifolia (Araucariaceae) under external stress in Southern Brazil

This study is part of the Floristic and Forest inventory of Santa Catarina, conceived to evaluate forest resources, species composition and structure of forest remnants, providing information to update forest conservation and land use policy in Southern Brazilian State of Santa Catarina (95 000km²). In accordance to the Brazilian National Forest inventory (IFN-BR), the inventory applies systematic sampling, with 440 clusters containing four crosswise 1 000m² plots (20x50m) each, located on a 10x10km grid overlaid to land use map based on classification of SPOT-4 images from 2005. Within the sample units, all woody individuals of the main stratum (DBH≥10cm) are measured and collected (fertile and sterile), if not undoubtedly identified in field. Regeneration stratum (height>1.50m; DBH<10cm) is registered in 100m² in each sample unit. Floristic sampling includes collection of all fertile trees, shrubs and herbs within the sample unit and in its surroundings. This study performs analysis based on 92 clusters measured in 2008 within an area of 32 320km² of mixed ombrophyllous forests with Araucaria angustifolia located at the state’s high plateau (500m to 1 560m above sea level at 26º00’-28º30’ S and 49º13’-51º23’ W). Mean density (DBH≥10cm) is 578 individuals/ha (ranging from 85/ha to 1 310/ha), mean species richness in measured remnants is 35 (8 to 62), Shannon and Wiener diversity index (H’) varies between 1.05 and 3.48. Despite high total species diversity (364 Magnoliophyta, five Coniferophyta and one tree fern) and relatively high mean basal area (25.75m²/ha, varying from 3.87 to 68.85m²/ ha), the overwhelming majority of forest fragments are considered highly impacted and impoverished, mostly by logging, burning and extensive cattle farming, turning necessary more efficient protection measures. Basal area was considered an appropriate indicator for stand quality and conservation status. Rev. Biol. Trop. 59 (3): 1371-1387. Epub 2011 September 01.

Este estudio es parte del inventario Florístico Forestal de Santa Catarina, realizado para evaluar los recursos forestales, la composición de especies y la estructura de remanentes de bosque, y proporciona información para actualizar la conservación de los bosques y políticas de uso de la tierra en el estado brasileño de Santa Catarina (95 000km²). El inventario se aplica al muestreo sistemático, de 440 conglomerados en cuatro parcelas de 1 000m² cada una, situados en una red de 10x10km. Dentro de las parcelas, todos los individuos leñosos (DAP≥10cm) fueron medidos. El estrato de regeneración (altura>1.50m, DAP<10cm) se registra en 100m² en cada conglomerado. Este estudio realiza un análisis de 92 conglomerados medidos en 2008 dentro de un área de 32 320km² de bosques ombrófilos mixtos con Araucaria angustifolia ubicados en el altiplano del estado. La densidad media (DAP≥10cm) es de 578 individuos/ha (desde 85/ha hasta 1 310/ha), la media de la riqueza de especies en los remanentes es de 35 (8-62), la diversidad (H’) de Shannon y Wiener varía entre 1.05 y 3.48. A pesar de la alta diversidad total de especies (364 Magnoliophyta, cinco Coniferophyta y un helecho arborescente) y el alto promedio del área basal (25.75m²/ha, variando de 3.87 a 68.85m²/ha), la mayoría de los fragmentos de bosque se consideran altamente impactados por la tala, quema y ganadería extensiva, por lo tanto es necesario más medidas eficaces de protección.