Metrics details There are several studies that announce the inhibitory behavior of this sort of substance to strengthen the shield of metals which is one of the positive benefits of green inhibitors Araucaria heterophylla studied as a green corrosion inhibitor to avert the mild steel during the acidic cleaning The examination of this plant’s ability to control corrosion at different concentrations in the acidic solution used certain expert measures The electrochemical results suggested that Araucaria heterophylla had a stronger inhibitory effect on mild steel protection from corrosion heterophylla extract produced corrosion inhibition efficacy (about 83.94%) the mild steel corrosion current density dramatically decreased with the addition of Araucaria heterophylla extract going from 1.08 μA/cm2 for the sample without inhibitor to 0.17 μA/cm2 for the sample having 1000 ppm inhibitor and according to electrochemical study the inhibition efficiency was found around 83% Flavonoids were found in the plant’s leaves according to the high-performance thin-layer chromatography profile The FTIR picks analysis like N–H stretching secondary amine (3337.07 cm−1) CO–O–C–O stretching anhydride (1022.64 cm−1) defined that functional groups and heteroatom were present the suggested inhibitor’s components were identified Scanning electron microscopy suggests that a thin layer or passive film form on the surface Quantum chemical calculation also supports the experimental results These substances typically become adsorbed on a metal’s surface and avert the active locations of where corrosion occurs including their high cost and an unfavorable impact on the environment The water-soluble phytochemical elements found in plant extracts include proteins Different plant components from Araucaria species have been shown to be effective in treating a variety of conventional illnesses There many heterocyclic compounds which can behave good corrosion inhibitor but on the other hand they may harm to soil as well as human beings However the green corrosion does not do so The objective of the current study is to extract the leaves of A heterophylla and examine as corrosion inhibitor for mild steel in 1 M HCl using weight loss measures and electrochemical analysis scanning electron microscopy has been used to examine the adsorption of the inhibitor molecules on the surface of mild steel (SEM) To better comprehend the adsorption phenomenon all experimental results were connected with theoretical information 60-F254 silica gel plates with 10.0 × 10.0 cm size (MERCK KGaA) were employed in the analysis The samples were applied with a 100 μL syringe 15 μl of standard and 30 μl of plant extract samples were applied in bands of 10 mm width The chromatograms were developed after a saturation period of 30 min The mobile phase used in the analysis was toluene plates were detected with a UV spectrophotometer at 254 nm and 365 nm Hydrochloric acid purchased from sigma aldrich used to make 1 M HCl For inhibitor we form a stock solution of 1000 ppm solution of plant extract in 1 M HCl The weight loss of four-sided metal samples of size 2.5 cm × 2.5 cm was weighted according to the subsequent procedures: the polished samples from above were weighed they were immersed in the 1 M hydrochloric acidic medium at room temperature here wi is the value of weight loss in presence of inhibitor w0 is the value of weight loss in absence of inhibitor Through Eq. 3 we can calculate the corrosion rate of mild steel in acidic medium: Corrosion inhibition efficiency is calculated as follows, on the basis of EIS and PDP experiments (4, 5): Rct and \({\text{R}}_{\text{ct}}^{\text{o}}\) are corrosion current density JEOL (JSM6510) instrument was used to examine the surface morphology of M-steel samples with and without inhibitors To find out the method of bonding of plant extract and the metal surface was FTIR spectrum was carried out the pure plant extract and of layer created on the metal surface after absorption in acidic medium A typical chromatogram of bioactive chemicals found in the chloroform fraction extract of A. heterophylla. A typical chromatogram of bioactive chemicals found in the methanolic fraction extract of A. heterophylla. 3D spectra of methanolic extract of A.heterophylla Plot between inhibitor concentration Vs inhibition efficiency and corrosion rate at different temperature initiation energy is represented by Ea (kJ mol−1) R is the molar gas constant (8.314 J K−1 mol−1) where N is Avogadro’s number and h is Plank’s constant This result reveals that these types of plants (A heterophylla) have an inhibitory effect on the corrosion process by increasing the intrinsic limitations of the corrosion reaction while focusing on the electrostatic properties of plant extract on the surface Plot between ratio of ln corrosion rate/ temperature and temperature Vs 1/T The endothermic character of M-steel corrosion was indicated by the positive Ha values which implies that it is difficult to dissolve M-steel in the presence of A The negative values of Sa for both the pure 1 M HCl solution and A heterophylla solutions indicated that the activated complex in the rate-determining phase reflected an association instead of dissociation which implied that the transition caused less disturbance in the solution where θ = the surface coverage, and Cinh, concentrations of A. heterophylla leaf extract and Kads, equilibrium constant of the adsorption phenomena, respectively. Relation of surface cover and the concentration A The ΔGads values were examined to better understand the adsorption mechanism between the investigated A heterophylla leaf extract and the M-steel surface Three forms of adsorption have been reported in several studies whereas the adsorption phenomenon is physicochemical if the ΔGads values are between − 40 and − 20 kJ/mol and if the ΔGads values exceed − 20 kJ/mol It mimics the process of physical adsorption Nyquist plots for Mild steel in 1 M HCl medium with various dosages of A We can say that when we increase the concentration the charge transfer resistance increases. On the basis of that we found that the inhibition efficiency increases. Through the Table 5 we found that the double layer capacitance decreases All the values are calculated by equivalent circuit fit Also includes impedance characteristics linked to equivalent fits such as Constant Phase Element (CPE) and n values The circuit design includes a solution resistance (Rs) The CPE element used to calculate the constitution of a double-layer at the (metal)/electrolyte interface heterophylla with different concentration in 1 M HCl Tafel plot of mild steel in 1 M HCl solution without and with different dosages of A FTIR spectra of the A. heterophylla extract. FTIR spectra of mild steel immersed in acid solution containing A SEM analysis has emerged as one of the key techniques for analyzing substrates with and without corrosion. Figure 13 shows the morphology of surface of metal in the absence and Fig. 14 presence of A. heterophylla extract. SEM image for MS after immersion in 1 M HCl of A. heterophylla at 298 K temperature for 24 h. SEM image for MS after immersion in 1000 ppm of 1 M HCl of A heterophylla at 298 K temperature for 24 h The theoretical conclusions are quite equal to the outcomes of the experiments accord quite well with empirical observations that the considerable attachment of an inhibitor molecule onto a steel surface occurs at low quantities Information about charge transfer mechanisms can be gleaned from the dipole moment which measures the distribution of charge inside a substance The efficiency of compounds’ ability to control corrosion is increased by the greater electrostatic interactions that bigger dipole moments produce The DFT data analysis presented in Table 8 demonstrates that Ethyl iso-allocholate has a greater capacity to inhibit corrosion due to its more negative optimization energy (− 1385.820) and thermal energies point to improved stability and reactivity while favorable free energy profiles suggest effective corrosion inhibition heterophylla functions essentially as a good mild steel corrosion inhibitor Experiments using potentiodynamic polarisation demonstrate that the inhibition was of a mixed nature blocking both the cathodic hydrogen evolution and the anodic metal disintegration According to EIS and potentiodynamic polarization studies heterophylla leaves extract to a solution of 1 M HCl which cause causes a considerable drop in corrosion rate and increase in inhibitory efficiency PDP also suggest that inhibitor is mixed type in nature because both type of reaction controlled by the molecules present in the A Adsorption study demonstrates that it complied with a modified Langmuir isotherm and confirms monolayer adsorption and show mixed type adsorption physical as well as chemo adsorption The impulsiveness of adsorption is shown by the negative value of ΔGo The results of SEM and Langmuir adsorption isotherm suggested that the only mechanism by which corrosion is inhibited is through mixed type adsorption SEM shows monolayer formation on the metal surface which is called passive layer Data supporting the findings of this work are provided in the paper All other raw data that support this paper and other finding of this study are available from the corresponding author upon reasonable request Data and materials will be shared with no restrictions on the availability of raw or processed data via a material transfer agreement Abdelaziz, S. et al. 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The Araucaria is a tree native to South America (photo: Mariana Mira Vasconcellos/IB-USP) The study used advanced DNA sequencing techniques machine learning and analysis of fossil pollen records to investigate the history of this forest formation postdoctoral student at the Institute of Biosciences of the University of São Paulo (IB-USP) and first author of the study The researcher explains: “The expansion of the Araucaria population in the Southern Core began during the last Glacial Period long before the presumed date of human arrival in South America which contradicts the idea of an anthropogenic expansion we interpret this initial expansion as a response to the climatic changes of the Upper Pleistocene The wetter and colder climate of this period would also have caused the expansion of other Araucaria species in Australia and New Caledonia Although this may have occurred in response to a brief climate cooling event in the Holocene and increased summer monsoon rainfall in South America between 4,000 and 3,300 years ago a possible impact of human settlements in southeastern Brazil cannot be ruled out.” Vasconcellos points out that this should not be taken as a proven fact since archaeological evidence of human settlements in the Mantiqueira is scarce compared to that of the Southern Plateau of Brazil genetic analyses indicate that while the initial spread of the Araucarias was not the result of human activity their subsequent evolution was significantly influenced by anthropogenic activity This influence resulted in lower genetic diversity greater endogamy and increased long-distance gene flow suggesting that humans facilitated seed dispersal and germination through the consumption of pine nuts the greater the genetic differentiation between them That’s what you’d expect from a natural process But that isn’t what we observed in the Araucaria population in southern Brazil Trees separated by long distances are genetically very similar This suggests that someone traveled these distances taking the seeds from one place to another It could be animals that feed on pine nuts it could be humans,” argues the researcher He continues: “There are now several studies that show evidence of human management in the Amazon rainforest There are no signs of management on the ground But the Araucaria is a plant that doesn’t need management The pine nut falls to the ground and all it takes is a little rain for it to germinate The simple fact that humans eat pine nuts and carry the seeds on their expeditions would’ve been enough to spread the species The storage and transportation of seeds are still common practices among the indigenous peoples of the region.” there are a large number of archaeological sites called “underground houses.” It is believed that these structures built by populations of the Proto-Jê language family like the houses in today’s indigenous villages The study found that the areas where the underground houses are located coincide with the current distribution of the Araucaria forest suggesting that humans may have chosen these locations because of the abundance of resources provided by the plant species “The Araucaria has great cultural importance in southern Brazil including cave paintings and analysis of food remains and tartar found in human skull dentitions indicate that this tree was an important food source and may have been dispersed by indigenous peoples,” Vasconcellos points out The Araucaria is a tree native to South America But there are only two species on the continent: Araucaria angustifolia the two populations of Araucaria angustifolia the one in the south and the one in the Mantiqueira diverged around the time of the penultimate Glacial Maximum when part of the globe was covered by an extensive layer of ice with a strong impact on the area that corresponds to today’s southeastern region of Brazil the population of the south began to expand the Araucaria forest reached its greatest extent which caused icebergs to break off in the North Atlantic resulting in peaks of humidity during cold periods in Brazil “The data collected in the study suggest that the remaining populations of Araucaria which responded well to the long period of climatic stability in the Holocene are now facing serious threats due to their reduced population size forest fragmentation caused by recent anthropogenic activities which is causing higher temperatures in southeastern Brazil They therefore require effective conservation policies,” concludes Vasconcellos The study was coordinated by Ana Carolina Queiroz Carnaval of the City University of New York (CUNY) and Fabián Michelangeli of the New York Botanical Garden (NYBG), both institutions based in New York, USA. It was supported by FAPESP through the Thematic Project “Dimensions US-BIOTA São Paulo: integrating disciplines to predict the biodiversity of the Atlantic Forest in Brazil” The article “Evaluating the impact of historical climate and early human groups in the Araucaria Forest of eastern South America” can be accessed at: nsojournals.onlinelibrary.wiley.com/doi/10.1111/ecog.06756 O endereço abaixo não existe na globo.com An official website of the United States government Learn about updates on NSF priorities and the agency's implementation of recent executive orders well-preserved conifer fossils from Patagonia show that an endangered and celebrated group of tropical West Pacific trees has roots in the ancient supercontinent that once comprised Australia according to an international team of researchers "The Araucaria genus, which includes the well-known Norfolk Island pine, is unique because it's so abundant in the fossil record, and is still living today," said scientist Gabriella Rossetto-Harris of Penn State University and lead author of the study the Norfolk Island pine is also a popular houseplant you might recognize in a dentist's office or a restaurant." Norfolk Island is a tiny Australian island in the South Pacific Ocean Araucaria grew around the world starting about 170 million years ago in the Jurassic period Around the time of the dinosaur extinction 66 million years ago the conifer became restricted to certain parts of the Southern Hemisphere The timing of when and where these living lineages evolved is still being debated and the other three are spread across New Caledonia Many are now endangered or vulnerable species is usually thought to have evolved near its modern range in the West Pacific well after the Gondwanan supercontinent split up about 50 million years ago The National Science Foundation-funded researchers from Penn State and the Museo Paleontológico Egidio Feruglio in Chubut found the fossils at two sites in Patagonia -- Río Pichileufú which has a geologic age of about 47.7 million years with a geologic age of about 52.2 million years They analyzed the fossils' characteristics and compared them to modern species to determine what living group the fossils belonged in. Then they developed a phylogenetic tree to show the relationships between the fossils and living species. The scientists reported their findings in the American Journal of Botany "These results run counter to examples where molecular dating methods suggest an older age of origin than that suggested by the fossil record," said Christopher Schneider a program director in NSF's Division of Environmental Biology the new fossils from South America suggest a much older time of origin for the Australasian group of Araucaria and that the group was widespread across the Gondwanan supercontinent prior to its breakup That changes the biogeographic picture for the Araucaria Sign up for email updates whose tufted branches also give them the name candelabra tree are being pushed toward extinction as government agencies continue to ignore or even abet in their logging The araucaria (Araucaria angustifolia) is a species native to the cooler and higher-elevation regions of southern Brazil It can grow to a height of nearly 50 meters (164 feet) and has a distinctive canopy that makes it look like a giant living candelabra Though it’s been around for some 200 million years it faces extinction at human hands within the next five decades as its habitat in the ever-dwindling Atlantic Forest continues to be destroyed The National Environment Council (Conama) banned the logging of endangered tree species araucaria forests had disappeared from 98% of their historic range; once covering 182,000 square kilometers (70,300 square miles) they’re now confined to scattered patches of forest totaling just 3,600 km2 (1,400 mi2) The ban hasn’t stopped the illegal logging Fines issued by environmental law enforcement agencies between 2018 and 2021 amounted to 102 million reais ($21.4 million) for the deforestation of 255 km2 (98 mi2) of Atlantic Forest Illegally logged araucaria is commonly found during seizures according to the Public Prosecutor’s Office of Paraná state which coordinates national efforts to protect the species director of knowledge at SOS Mata Atlântica an NGO that has been mapping the vanishing remains of the Atlantic Forest in Brazil since 1989 “Since states still do not report the deforestation that they authorize there is mounting evidence that the vast majority of this logging is illegal,” Guedes Pinto says States in which previously well-conserved araucaria forests have virtually disappeared have not carried out surveys of the species in 20 years and still allow permits for public works projects that run through araucaria groves the construction of a total of 1,500 km (930 mi) of power lines led to thousands of the trees — 4,000 in Paraná alone — being felled Civil society organizations have turned to the courts to challenge this kind of authorized clearing “Technical criteria are flawed and there are no alternative locations for the works,” says João de Deus Medeiros a professor at the Federal University of Santa Catarina (UFSC) “Environmental agencies are complicit and they license what is most comfortable for entrepreneurs Seeking better alternative paths would reduce social and environmental impacts.” the state government allowed more than 600 trees to be chopped down Cases like these abound throughout southern Brazil according to the Society for Wildlife Research and Environmental Education says it has become customary for courts to authorize works of “public utility” that clear araucaria forests He adds that mitigating this logging by providing resources for protected areas or planting seedlings doesn’t compensate for the destruction of these consolidated “Undiscerning licenses are often granted and validated by economic interests,” says Borges who holds a master’s degree in zoology from the Federal University of Paraná eliminating what remains of araucaria forests risks our future quality of life and survival Protecting them is also in the public interest.” permits the logging of araucaria and other trees facing extinction There are proposals in the Brazilian Congress, supported by the agribusiness caucus and the administration of President Jair Bolsonaro, to weaken the Atlantic Forest Law in order to expand agribusiness including into primary and araucaria forests The idea is to fit the legislation into the Forest Code of 2012 eliminating the only federal regulation to protect a specific biome “Pressure is coming mainly from Paraná and Santa Catarina the leading states in deforestation of Atlantic Forest,” Guedes Pinto says because the Atlantic Forest Law adds value to the Forest Code Cutting it down would be an enormous threat to the climate biodiversity and water sources for regions where most Brazilians live.” The araucaria is listed in both Brazil and globally by the IUCN as threatened with extinction. National parks and other conservation units contain just 2.5% of the habitat considered suitable for araucaria, according to a 2019 study we need to save the araucaria forests that are outside of protected areas guaranteeing genetic diversity for the repopulation of the species,” says Ricardo Brites “But these forests continue to be deforested or degraded by the selective removal of trees which inspections and satellites don’t identify.” The current model of commercial management of natural forests is economically unviable and would destroy forests in their surroundings Quality wood requires at least three decades of growth but investments are high and long-term,” Brites says “These are some major obstacles that continue to fuel the logic that there is more money in breaking the law selling lumber and continuing to move forward with unsustainable production in the country,” says Medeiros from the Federal University of Santa Catarina In emailed responses to questions from Mongabay the state environmental agencies in Paraná and Santa Catarina both said that illegal logging of araucaria is a violation punishable by fines and that they work to replant the species with consideration for genetic variety But they also said that removal of araucaria trees (planted not native) can be permitted due to risk of trees falling on private or public structures or for works that are in the public and social interest The environmental agency in Santa Catarina added that the licensing of projects “has always been participatory after discussions with educational institutions private companies and the general public.” It added that “environmental legislation allows for endangered species to be cut down whether isolated individuals or those present in forest fragments (with proof of a lack of alternative locations and proof that the removal will not aggravate the risk of extinction in situ) and institutes the need for mitigation measures.” Wilson, O. J., Walters, R. J., Mayle, F. E., Lingner, D. V., & Vibrans, A. C. (2019). Cold spot microrefugia hold the key to survival for Brazil’s critically endangered araucaria tree. Global Change Biology, 25(12), 4339-4351. doi:10.1111/gcb.14755 Saraiva, D. D., Esser, L. F., Grasel, D., & Jarenkow, J. A. (2021). Distribution shifts, potential refugia, and the performance of protected areas under climate change in the araucaria moist forests ecoregion. Applied Vegetation Science, 24(4). doi:10.1111/avsc.12628 Banner image of araucaria trees in Serra da Bocaina National Park in São Paulo state. Image by Heris Luiz Cordeiro Rocha via Wikimedia Commons (CC BY-SA 3.0) This article was reported by Mongabay’s Brazil team and first published here on our Brazil site on March 15, 2022. The “fortress conservation” model is under pressure in East Africa, as protected areas become battlegrounds over history, human rights, and global efforts to halt biodiversity loss. Mongabay’s Special Issue goes beyond the region’s world-renowned safaris to examine how rural communities and governments are reckoning with conservation’s colonial origins, and trying to forge a path forward […] Volume 6 - 2018 | https://doi.org/10.3389/fevo.2018.00011 This article is part of the Research TopicEcology and Evolution of Plants under Domestication in the Neotropics View all 21 articles Araucaria forest is a sub-tropical component of the Atlantic Forest Biome occurring between 18 and 30° S latitude and 500–1,800 m altitude in Southern and Southwestern Brazil and Northwestern Argentina this forest has undergone non-sustainable exploitation and reduction in area dedicated to agricultural and forestry use smallholders continue using several resources from this forest The main system is geared toward the production of yerba mate (Ilex paraguariensis) under araucaria trees (Araucaria angustifolia) and social relevance for thousands of farmers Seeking evidence of domestication and conservation of the resources managed in this system we compared different landscapes on the Northern Plateau of Santa Catarina in Southern Brazil araucaria and caraguatá (Bromelia antiacantha)—we characterized management practices (interviews and guided tours) demographic structure (permanent plots in farming zones and in a protected area) and genetic diversity in populations from the same places Demographic structure and genetic diversity from different landscapes were compared to evaluate the system potential for conservation The results indicated that the three species are intentionally promoted with practices of protection in different ways and with different functions (caraguatá hedges and collection of pinhões—seed like nuts—of araucaria) Landscapes are managed for yerba mate harvesting and cattle raising with a consequent reduction in the density of most other plant species In all cases the genetic diversity of the species was high for most of the sampled properties and the set of farmers' populations did not differ from the protected area The set of populations of each species operates as a metapopulation and local management practices contribute to conservation the farmers' management systems and practices maintain the landscape with productive forest fragments favoring the conservation-by-use of these species The system requires these management practices which bring about changes in various species and are motivated by cultural and economic factors in order to maintain the landscapes domesticated These advances encourage a new perspective on the dynamics of supposedly natural ecosystems and the ecology of species as well as possibilities for conservation and use The marks of domestication processes on numerous species and landscapes with models specific to different cultures in several environments The fact of landscape and species domestication in different environments can be present and currently active at the interface of agricultural and forest environments and produced several legal restrictions to its use According to the Brazilian Institute of Geography and Statistics (IBGE, 2014), in 2014 Brazil produced 333,017 tons of native yerba mate, and it was the main non-timber product in terms of production volume (IBGE, 2014). Production of native yerba mate is carried out in 5,150 establishments (predominately family farmers) in Santa Catarina State (EPAGRI/CEPA, 2016) In this context we investigated landscapes with NYMS characterizing the principal practices and motivation of family farmers seeking evidence of landscape domestication and evaluating whether the system conserves the principal resources managed Our study has focused on three species in the NYMS: yerba mate (I and caraguatá (Bromelia antiacantha) If on the one hand these species represent significant resources for the subsistence of family farmers on the other hand they are interesting case studies to exemplify how these farmers play an active role in the conservation of plant populations in order to evaluate whether the NYMS can conserve genetic diversity of the studied species we analyzed genetic diversity in the yerba mate araucaria and caraguatá populations from NYMS and from a protected area Study area – Northern Plateau of Santa Catarina State including localization of Três Barras National Forest (Protected Area) This study integrates different field methodologies as well as demographic and genetic studies of the above-mentioned species in a Protected Area and on farmers' properties In all cases the plant populations sampled were the same as in the demographic and ethnobotanical studies The results are presented in a descriptive interpretative form and expressed as percentage of occurrence or average values with standard deviation sampling was carried out in two different situations on the farmers' properties according to the local management mentioned in the interviews paraguariensis gathering and few interventions (8 plots on 4 properties) and cattle grazing (19 plots on 5 properties) paraguariensis gathering and no other interventions (12 plots) 30 on farmers' properties and 30 in the PA was evaluated annually (2010–2012) in terms of number of pinhas (reproductive structures) per plant permanent plots of 20 × 40 m were used in forests and 20 linear meters to study the caraguatá hedges classified as vegetative or reproductive (considering the presence of inflorescences or infructescences) and leaf length was measured Bromelia antiacantha populations were studied in the following areas: harvesting of yerba and mowing (3 plots in Farmer 1 and 2 plots in another area in farmer 2) Unmanaged: areas that have been characterized for not having undergone any of the activities previously mentioned for at least 10 years previously in the case of farmer properties and 50 years in the PA (3 plots in farmer 3 and 12 plots in PA) Hedges: characterized as “strips”/linear bands of B established and administered by local farmers (26 plots in farmers 1 and 2) Considered the areas with most human interference The results are expressed as average frequencies estimated per hectare Three buffers were utilized in the electrophoresis process: Tris Citrate pH 7.5 (Tris 27 g.L−1 and citric acid 16.52 g.L−1) Morpholine Citrate pH 6.1 (7.68 g.L−1 citric acid) and Histidine pH 8.0 (105.82 g.L−1 sodium citrate tribasic) Enzymatic systems used for genetic characterization of Araucaria angustifolia Leaf tissues were collected from 50 individuals in each population paraguariensis sampling respected a minimum distance of 50 m between individuals adults/mature rosettes (leaves more than 2 m in length) were sampled respecting at least 15 m distance between them and avoiding the collection of ramets from the same plant In order to evaluate whether farmers conserve species genetic diversity for groups of populations inside the PA and on farmer's properties in order to evaluate possible differences between landscape units as described in the Demographic studies item all genetic descriptors were also estimated for groups of populations in each landscape unit Possible genetic differences between farmers' properties and PA, and between landscape units were tested through confidence intervals (95%) for Ân, jackknifing the values across loci in R language (R Development Core Team, 2015) and for ĤE and ĤO through 1,000 bootstraps of individuals within population/group, also in R language, but using the “PopGenKit” package (Paquette, 2012) The forest structure of the landscapes with NYMS in the region is presented in Table 2, which also includes information on the total number of species, density of the araucaria and the total number of individuals of Myrtaceae and yerba mate, considered as structuring species of these landscapes (Assis et al., 2010; Mello and Peroni, 2015) Structural characteristics of landscapes with Ilex paraguariensis the presence of cattle in many areas is a determining factor of this system due to the value added by the sale of livestock potreiro and invernada are areas with some degree of Araucaria forest cover and yerba mate management; in the first there is no livestock production Most of the NYMS areas (ervais) are located in places (soil topography) of excellent to good agricultural aptitude with high pressure for conversion into crops Yerba mate is cited as one of the main reasons for non-conversion in 80% of cases On the Northern Plateau (SC), yerba mate is managed in several situations; for instance, Marques (2014) identified 13 typologies (including situations with planted yerba mate) the main landscape is considered to be forest All farmers considered yerba mate important mainly because it is a low-risk activity associated with landscapes which have different uses the NYMS constitutes a stabilizing element on their property capable of generating resources for forest areas and linked to strong cultural aspects as well as being a pleasurable activity for the great majority of farmers These aspects reinforce the role of these family farmers as maintainers of Araucaria forest fragments nowadays An additional relevant aspect is the preference shown by Brazilian consumers for a product (ground tea-like leaves for chimarrão) with a milder flavor (de Oliveira Suertegaray, 2002) from herbs grown in shade (native yerba mate), implying higher prices paid by the industry for the raw material originating from this system (EPAGRI/CEPA, 2017) rather than generating significant income in relation to total gross income yerba mate constitutes an important reserve for 72% of the families with the characteristics of a savings account since the farmers tend to prune all the plants without worrying about leaving seed trees evaluated by a sample of 30 trees from farms was 11.1 pinhas/tree between 2010 and 2012 Average productivity over the years was 71.1 kg/ha 54.9 kg/ha in 2011 and 135.3 kg/ha in 2012 In the PA sample observations were made in the years 2011 and 2012 and the average production for these was 17.5 pinhas/tree with averages of 11.8 in 2011 and 23.2 pinhas/tree in 2012 The average productivity of 2011 and 2012 was 333.0 kg/ha varying from 224.3 kg/ha in 2011 to 441.7 kg/ha in 2012 The number of female plants producing pinhas varied between years; considering the entire period evaluated the average number of female plants in production was 13.9 on farms An important aspect is the great variation in pinhão productivity in different years of production, as already mentioned by Mattos (1994), Mantovani et al. (2004), and Vieira-da-Silva and Reis (2009), and even between relatively close sites (Zechini et al., 2012) This variation reflects the unpredictability of production that is also used by farmers as an argument for the lower value of pinhão as a trade product in the region Considering the management practices carried out by farmers the population structure of yerba mate and structure of the forest fragments (considering the set of structuring species—araucaria Landscapes without cattle: areas used with few anthropic interventions (no livestock presence and no mowing) such as pruning with shears every 3 years or less drastic such as “hand” pruning every 4 years Landscapes with cattle: areas used with more intense interventions Includes the same management characteristics for herbaceous plants as the previous group In both cases there are practices for protection of yerba mate plants including possible transplanting and enrichment with plants from the same forest fragment or seedlings produced by farmers Caraguatá has been used for a long time in the region, for various purposes (Filippon, 2014). The fruits of this plant are used for medicinal treatment of, for example, pulmonary diseases (bronchitis/asthma) and influenza (Filippon, 2009; Filippon et al., 2012a). Uses of the species for making liquor and jelly were also cited in Filippon (2014) but the most frequently cited use (60%) was for hedges Caraguatá hedges were very common in the region from 1900 to 1960 this kind of fencing was used especially for pig farming; animals can't pass through the hedge due to the high density of rosettes and the thorns in their leaves Caraguatá hedges are also used in other kinds of animal breeding like sheep Nowadays this type of pig breeding is no longer common; however Some farmers still plant new caraguatá hedges and taking advantage of the efficiency of this type of fencing to hold animals in and establish property boundaries The density of B. antiacantha rosettes varies between studied areas. The unmanaged area presented the greatest density of rosettes and reproductive rosettes per hectare when compared to the managed areas (Table 3). Hedges possess, on average, the highest population density of all the sampled areas (316 times greater than that of the population in the unmanaged area), and also the highest number of the reproductive rosettes (Table 3) Rosette density (per hectare) of Bromelia antiacantha in unmanaged made with rosettes harvested from the Araucaria forest fragments paraguariensis is extracted and where cattle graze Sometimes the ramets used in hedges are donations from neighbors who want to “clean” the area to improve pasture for cattle or want better conditions for working with yerba mate since the caraguatá thorns make the farmers' work more difficult On these occasions the number of rosettes removed is generally high and the farmers who receive them have an abundance of ramets to plant so they can generally choose the strongest lushest and youngest rosettes (0.8–1 m leaf length) This intention of making a hedge and the selection of rosettes allows us to highlight a domestication process These management practices (choosing rosettes and hedge maintenance) employed by local farmers generate a change in landscape tending toward greater productivity and convenience In these areas caraguatá is not the main focus but a means of adaptation of the landscape to facilitate the development of activities that generate income for the property such as the harvesting of yerba mate and cattle grazing management (mowing) of caraguatá can be seen as a consequence of this domesticated landscape for the production of yerba mate and/or cattle grazing Genetic descriptors for groups of populations in PA or on farmers' properties (the same for the three species) are presented in Table 4 the data indicate that genetic diversity of the three species is being better maintained on the farmers' properties This result can be interpreted as arising from the common effect of past overexploitation; however it is also an indication that the medium-term possibilities of maintaining diversity are similar in both situations reinforcing the possibility of conservation by use Genetic descriptors estimated for Araucaria angustifolia (A.a) and Bromelia antiacantha (B.a) populations grouped per occurrence in Protected Area (PA) or on Farmers' Properties The estimated values were lower than the mean values obtained in a large study in the SC (Reis et al., 2012) angustifolia populations (13 allozymic loci) The values found were lower than the SC average for ĤE (0.124) and total number of alleles (51) but similar in terms of regional (Northern Plateau) ĤE mean (0.104) and total number of alleles detected (30) there were no differences between the diversity maintained in PA and on farmers' properties the practices associated with the species are similar involving maintenance of the adult individuals and cone collection Unique alleles were found in two situations 4 in non-managed populations and 3 in populations with yerba mate management and cattle grazing Genetic descriptors estimated for Ilex paraguariensis in different landscape units Caraguatá populations presented some differences in heterozygosities (ĤO and ĤE) between landscapes. Mean values of ĤO and ĤE for hedges were significantly higher than those estimated for unmanaged landscape (Table 6) Hedges also presented higher numbers of alleles (k^ = 28) unique alleles (Âun = 3) and allelic richness (Ân = 2.15) than all other landscape units Fixation indexes were significantly different from zero in the unmanaged landscape the reduced number of rosettes when compared to the hedges may contribute to more crossing between relatives Genetic descriptors estimated for Bromelia antiacantha in different landscape units Âun and Â48 values and the non-significant f^ estimated for hedges could be related to the origin of the plants (from the same or different locations) used for hedge construction and to the selection of plants for hedges on many occasions neighbors donated the seedlings for hedges so any single hedge could have been made with seedlings from different populations possibly increasing their number of alleles and heterozygotes In this context, observing the genetic diversity indexes obtained for B. antiacantha, it can be inferred that the hedges presented greater genetic diversity than populations that had not been managed for over 50 years, in accordance with results from other studies (Otero-Arnaiz et al., 2005; Zizumbo-Villarreal et al., 2013) This aspect is possibly due to the way the hedges are constructed with rosettes from sites of natural occurrence of the species (forests) Demographic studies showed that either by greater exposure to light in the hedge or other factors such as soil and temperature, the hedge had more reproductive rosettes (Table 3) The rosettes may come from various populations/farms with different genetic characteristics which may contribute to greater diversity in hedges caraguatá fences can be seen as a metapopulation of the species where each population of a landscape unit is a subpopulation it is possible to argue that the decrease in genetic diversity as one of the indicators of domestication generally suggested in the literature must be taken into account in relation to management practices the fact that intensively managed populations present greater genetic diversity than unmanaged ones does not imply that they are considered less domesticated than the others in the sense of human intervention in the population and in the landscape the farmers' way of life related to the gathering of rosettes in different populations is a result of management of the ervais (NYMS) In this sense there is an opportunity to “collect seedlings” from an activity directed by other managements which generate income for the property This fact differentiates the caraguatá from other plants cultivated and used in hedges: the use of these seedlings is desired and in turn they are collected from different populations the fact that the yerba mate areas are managed together with the existence of farmers interested in making caraguatá hedges increases species diversity this domestication process has a tendency to increase genetic diversity These distinct fragments of a wider landscape form a unique situation that allows a high level of gene flow between the various units favoring maintenance of the genetic diversity of each species and of the whole Each landscape unit studied has structural peculiarities that influence ecological and genetic patterns The units also have an interconnection with allele movement (pollen seeds and individuals) mediated by fauna and man allowing the maintenance of diversity in the metapopulation such metapopulation is created and maintained for cultural and economic motives The system requires these management practices in the landscape with changes in various species motivated by cultural and economic factors in order to maintain domestication of these landscapes domesticated landscapes for the production of yerba mate did not lead to an important reduction in genetic variability since there is no conscious selection action in the species studied the perspective of conservation-by-use of the multispecies system described here depends heavily on valorization of cultural aspects of the region as well as on the economic valuation of yerba mate from this artisan system Mechanisms of certification of origin could be an alternative of great regional importance on-farm conservation of several autochthonous species from these Araucaria forests codirected AdCM thesis and wrote the manuscript analyzed genetic data and wrote the manuscript AGM: Conducted field work (including interviews) analyzed part of data and wrote the manuscript SF: Conducted field work (including interviews) analyzed part of data and provided comments on the manuscript AZ: Conducted field and laboratory work and analyzed part of data was responsible for resource acquisition and provided comments on the manuscript This research is the product of AZ master's thesis and AGM This study was supported by the Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC – process no 4448/2010-2 and 11939/2009) and Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA – Macroprojetos 2/2009) Conselho Nacional de Desenvolvimento Científico e Tecnológico provided a Productivity Scholarship for MdR (CNPq – 309128/2014-5) and NP Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) provided doctoral scholarships for TM and SF and a master's scholarship for AZ Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) supported AL The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest We are profoundly grateful to all farmers for their kindness in sharing their knowledge with us We would also like to thank Núcleo de Pesquisas em Florestas Tropicais (NPFT) Laboratório de Fisiologia do Desenvolvimento e Genética Vegetal (LFDGV) and Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) for logistical support The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fevo.2018.00011/full#supplementary-material use and management of ethnovarieties of Araucaria angustifolia (Bert.) 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Maurício S. Reis, bXNlZHJlekBnbWFpbC5jb20= Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish BRASIL — A serene orange hue cuts across the horizon with the first rays of sunlight as dawn breaks in the mountainous countryside of Brazil’s second-most southerly state the frost melts and water vapor rises from the ground The smoky landscape of a morning frost is a typical occurrence during winter in this highland region In the kitchen of Sara Aparecida and Silvino de Liz Rosa’s house a wood fire crackles as it warms the room while the couple serve themselves chimarrão the traditional hot drink of people in southern Brazil The day starts early on the Santo Antônio do Caveiras farm Just a few steps from the slightly ajar front door one can see the trunk of a 20-meter (65-foot) tall araucaria tree The tree is a female of the species and is nearly 45 years old it guards over the land below and is something of a family treasure since it produces more than 200 pine cones a year,” said the couple’s son As the owner of a neighboring farm just a few kilometers away in Morro do Bacheiro the family farmer and his father have been araucaria pine nut harvesters for as long as they can remember The practice is generations-old in the region which is home to the largest population of araucaria trees in the state In the area that forms the Planalto Serrano Catarinense the Araucaria angustifolia — also known as the Brazilian or Paraná pine — is the predominant species It is the main feature of the moist araucaria forests or mixed ombrophilous forests as they are officially known which form part of Brazil’s Atlantic Forest It is also the only naturally occurring forest of its kind in the country is a symbol of the region’s culture and cuisine This type of pine nut is also the driving part of a productive chain involving more than a dozen municipalities in Santa Catarina’s southwest São José do Cerrito and Bom Jardim da Serra account for roughly 75% of the araucaria pine nut market in Santa Catarina are working together to promote actions that aim to combine empirical and academic science while valuing traditional knowledge and know-how to strengthen sustainable production chains that preserve and restore native vegetation ensuring the subsistence of local communities generates income for hundreds of families in this highland region and provides opportunities to promote inclusive agriculture collective consciousness and environmental preservation Investment in real food combined with active community associations point the way forward for the people and forests of the region coordinator of the Vianei Center for Popular Education — an association that has been providing advice on family agriculture agroecology and food security in the region — said that a large part of current initiatives had been discussed for a long time “Everything is part of a process of sustainable development for the region as we’re talking about a chain that is still in a position of marginality in a market that is only interested in profit,” he said a value-added tax on sales and services that was a burden on the final price of their products and represented another obstacle to the formalization of the chain of production and retail “We’re talking about a production chain that is mostly informal The legal part of the industry makes up somewhere between 10% and 30%,” Magnanti said Hundreds of araucaria pine nut harvesters sell their products in both their natural and processed form A large part of the harvest is sold clandestinely with current regulations being one of the main obstacles in the production chain an auditor for the economic activity of a local municipal association the data available for analysis are rather unclear “Based on the invoices that passed through local councils in each municipality 234 producers were recorded in the year 2022 who sold 1,500 [metric tons] for an average of 3.37 reais [$0.70] per kilogram corresponding to a total of 6 million reais [$1.2 million],” the auditor said the financial turnover of the production chain came in at 20 million reais [$4.1 million] last year A survey carried out by the Agricultural Research and Rural Extension Company of Santa Catarina showed that 1,700 metric tons of araucaria pine nuts were sold in the municipality of Painel alone the latest census carried out by the Brazilian Institute of Geography and Statistics showed that 3,386 farming establishments said they have araucaria pine nuts on their property “That shows a projection of the overall productive capacity which can then be divided between the part that is registered for sale the part that is sold without being recorded as well as the part that is not sold because there is insufficient labor to carry out the harvest,” Branco explained with the harvest period falling between April and July the income that comes from extracting and processing araucaria pine nuts is hardly enough to support a family through the year who idealized the prototype for a machine that removes the shell from the nut The extraction of the kernel required additional manpower due to the hard bark of the nutshell Increasing capacity was an important step since processing the araucaria pine nuts once they have been shelled allows for them to be sold outside the harvest season Niehues lives in the community of São Pedro and is part of the Renascer Agroecological Association The project to facilitate and increase the productive capacity of the araucaria pine nut farmers was developed between 2014 and 2017 at the Federal University of Santa Catarina in partnership with the Ecoserra Ecological Cooperative from the city of Lages Urubici is also where the first processing plant for the araucaria pine nut was established we managed to get funding from the Ministry of Agrarian Development and Family Agriculture to set up a small facility on public land which belonged to the local council,” Magnanti said The Renascer Association and the Ecoserra Ecological Cooperative worked together to produce a rotation scheme for families in the community to carry out work on the facility The following step saw the distribution of approximately 15 kits among the farmers which included machines to shell and grind the nuts as well as machines that could be used to seal the plastic bags used to store the roasted nuts The aim of the project was to widen the scale of processing for the product the storage of unprocessed nuts can lead to farmers losing out both in terms of the quantity and quality of their product as they face the risk of their crop becoming infested by pests the number of legal plants that process the araucaria pine nut for sale are still extremely low in number According to data from the Intermunicipal Consortium of the Santa Catarina Mountains a total of 13 have been registered in municipalities such as São Joaquim processes nearly 30 metric tons of araucaria pine nut each harvest which is the edible part of the araucaria pine nut has been eaten for centuries by the inhabitants of the southern highlands of Brazil The most traditional way of preparing the nut is the sapecada which are set alight and burn quickly and intensely This ancient method was previously used by the Indigenous inhabitants of the region and later by the mule drivers who used to travel through the countryside transporting supplies for local traders The sapecada is still a favorite of those who work during the harvest On a day spent harvesting the nut in the forest a sapecada is the go-to dish to keep them well-fed while they work Machines are seldom used for the extraction transportation and processing of the araucaria pine nut on these farms The work to harvest the nut is carried out entirely manually and can be dangerous the properties belonging to Jaison and Silvino de Liz Rosa produced a crop weighing 10 metric tons from their 47 hectares (116 acres) of land and then come and pick it up from us here,” Silvino explained They sold their product at 5 reais ($1) per kilogram they take in around 600 pine cones per day or even make a [pine nut] flour that we can sell the whole year round That way it’ll be easier and we’ll run fewer risks,” said Jaison who has been involved in the harvests for 30 years The harvesters fearlessly climb the towering pine trees It takes a lot of skill to be able to climb 15-30 of these trees a day with each movement at every step being carefully measured After climbing 20-30 meters (about 65-100 feet) up the tree trunk the harvesters reach the crown of the tree and must then take care to make sure that the branch on which their feet are resting is not going to break which could lead them to fall and suffer serious injury the harvesters must also navigate themselves through the tree’s branches the harvesters are only able to get less than half of what the araucaria produces Wildlife feed off the scraps and husks that fall to the forest floor and are left behind after the harvest The azure jay (Cyanocorax caeruleus) is the species that most contributes to seed dispersal It will often bury the araucaria pine nuts but will not always remember to return to the spot where it previously hid the nut “There’s also the red-spectacled amazon [Amazona pretrei] that comes here in flocks from Rio Grande do Sul and only stays during the pine nut season A rich lexicon exists for the araucaria pine nut with every community giving the tree’s seed a different name according to the seed’s characteristics such as its appearance or stage of ripeness pinhão do tarde and pinhão de 25 de março are a sample of the various names the nuts are given The araucaria pine nut harvest season is officially opened by law from April 1 however the harvesters — who have an intimate relationship with and understanding of their environment — say that part of the harvest is lost because of this as some of the tree’s fruit has already ripened by March The fruit here refers to the bulbous pine cones that weigh 2 kilograms (4.4 pounds) and hold 100-150 pine nuts Pollination rates cause the size of the pine cones to vary greatly from tree to tree and from year to year with climatic conditions influencing whether the cones end up larger or smaller than average Among the rich variety of the araucaria pine nut the cajuvá is held to be the cream of the crop Beautifully shaped and at the ideal point of ripeness An infinite number of dishes are prepared with the araucaria pine nut with the entrevero — beef and pork with araucaria pine nuts fried in lard — and paçoca — a araucaria pine nut-based farofa or toasted and seasoned flour mixture that is used as an accompaniment to many Brazilian dishes — being the most popular Araucaria pine nuts are also used to make croquetas the Santa Catarina araucaria pine nut was added to the Brazilian edition of the Ark of Taste an Italian body that aims to promote local and sustainable growing and harvesting of food safeguarding products that are part of a diverse human food chain “Slow Food was a chance to make an international connection the araucaria pine nut market was just starting to grow Slow Food put an ingredient that was new to many in the shop window the pine nut producing region became a Slow Food Presidium Food heritage and food security are linked to forest management by traditional populations The Santa Catarina highlands were once a borderless land inhabited by Indigenous peoples from the Xokleng The eldest members of these groups speak of how during the period in which the rural areas of the state were occupied known as bugreiros or “Indian hunters,” to wipe out the Indigenous population of the region The mark of this dark episode in the region’s history has been left on the land referring to the community whose name means “bloodshed” or “slaughter” in Portuguese local farmers have come to see the importance of keeping the forest standing as well as of integrating different tree and plant species which they already intuitively did on their properties without knowing that it was an agroforestry system Managing the forests has allowed the landscape to be protected Facing this is the expansion of the agricultural frontier as it encroaches ever farther into the forest generates a number of obstacles to the evolution of the productive chain The cultural landscape of the Santa Catarina highlands is faced by a number of major threats such as the conversion of forests into pasture the leasing of land for pine plantations to be created monocultural sowing of grains such as soybeans in rural areas and the construction of small-scale hydroelectric plants “The araucaria can be found above an altitude of 500 meters [1,640 feet] [above the town of] Rancho Queimado This whole part of the state had a different history of occupation from other parts of Santa Catarina It was an area that was part of the province of São Paulo It only became part of the state much later A large part of the fragments of forested land that are home to the araucaria are found on private estates The vegetation that once covered almost 200,000 square kilometers (77,220 square miles) of high-altitude land has been decimated the predatory exploitation of natural resources that powered southern Brazil’s timber industry saw the area of araucaria forests fall by 98% It was only in 2001 that the National Environment Council (CONAMA) outlawed the felling of the araucaria — a tree species that has been around since the time of the dinosaurs There are fossilized remnants of the araucaria that have been dated to 250 million years ago only 1% of the 3% of remaining areas belong to the original forest cover The species is critically endangered and was included in the official list of endangered Brazilian species and in the Red List of the International Union for Conservation of Nature (IUCN) It is forecast to go extinct within the next 50 years if no action to preserve and recuperate the species is taken According to Marciano Coelho Correa, a director at the Ecoserra Ecological Cooperative, clandestine felling of the araucaria still goes on I mean that they need to be backed up with the science and the law so that they can promote the management of the trees on their properties.” The araucaria pine nut harvesters who manage the resources of the forests play a vital role in the construction of the landscapes of the region plant management integrated with small-scale agro-ecological crops characterizes the conservation of an agroforestry system feijoa trees and Mimosa scabrella grow among the forest “The connection between araucaria forests and agroecology changes the lives of those who depend on the land as it gives people the chance to make use of things that are important to them,” Correa explained developed by the postgraduate program in Agricultural and Natural Ecosystems at the Federal University of Santa Catarina seeks to formulate conservation strategies through use for the maintenance of the species in the landscape and culture economic and social aspects aims to contribute to the development of reference units in agroforestry management with araucaria The ongoing project has received funding from FAPESC the Research Support Foundation of the State of Santa Catarina and has partnerships with the Vianei Education Center the Environmental Institute of Santa Catarina and the Santa Catarina State University campus in the city of Lages On the majority of the properties that are part of the focus area of the study at least 50% of the area is covered by araucaria trees Silvino’s farm has 24,000 trees — coming in at 600 per hectare and three times more than what would be expected in an araucaria forest that had not undergone any type of intervention “They have been carrying out activities that interfere with the landscape for a very long time What they would need to add to what they have traditionally done is thinning out [the forest] in areas where there is a high density of trees,” Siminski said What the study argues is that the conditions that farmers in that region have are different “The harvesters aren’t going to turn their land into soybean plantations or cattle-ranching pastures What they want is to manage the araucaria numbers in a way that allows them to carrying on producing pine nuts in the long term And legislation needs to be modernized to attend to this situation Social and economic well-being and the defense of life are the basic concepts of agroecology the farm of Joelce da Rosa Damas and Maria Elizabete Oliveira Damas is part of the Acolhida na Colônia ecological agrotourism route The locals open their doors to the visitors holds the valorization of the countryside way of life as its guiding principle The agroextractivist couple collected nearly 80 bags weighing 50 kilograms (110 pounds) each in 2022 “Some of it I got from our farm [9 hectares in size] and the rest I got working as a sharecropper on my neighbor’s farm,” Damas said He built the community’s sheds that are used for storing and processing the araucaria pine nuts and has also made a machine that can separate the pine kernel from the husk based on the teachings he received from an old farmworker the harvesters would make a ladder in the trunk of the araucaria tree itself with a machete This method has since been dropped by the harvesters of today for it is even more risky than the current way of scaling the trees Joelce changed from his traditional methods to an agroecological approach after being hospitalized three times with a serious infection because of the use of chemical products They were really traumatic experiences for me and my family No money can replace the health and quality of life of the plants the animals that feed on them and those who make their living from the land,” he said The return to an integrative and systemic understanding changed how the farmer saw and related to nature “Most of our income comes from selling apples Faced with the necessity of creating a structure that transcended the local economy the rural producers came together to create the Ecoserra Ecological Cooperative Organic agriculture in the region has been strengthened by the territorial brand for twenty years “There was little demand for a large volume of products for sale We needed to have an intermediary that would go beyond direct sales especially in terms of the institutional market through which we have access to public policies such as the Food Acquisition Program and the National School Meals Program,” explained Marciano Coelho Correa which is responsible for the annual distribution of 40-80 metric tons of every harvest and off-season product including araucaria pine nuts processed in 500-gram (1.1-lb) and 1 kg (2.2-lb) bags the farmers sell their products in the local market grocery stores and they supply federal government programs “It’s important to remember that the vast majority of the araucaria pine nut harvest circulates [through the economy] via middlemen,” Natal Magnanti said São Joaquim and Capão Alto sell their goods to the CEASA or to other regions in Santa Catarina or to states such as Paraná Another vital form of distribution is the direct sale of produce at roadside stalls located on kilometer 182 of the BR-282 highway is literally flowing with araucaria pine nuts the owner Antônio Milton Amarante has been selling the araucaria pine nut to a whole range of consumers “At my stall I sell all kinds of farm products but the araucaria pine nut accounts for 90% of my income From the first day of the harvest season onward does not close his stall for even a single day This article was first reported by Mongabay’s Brazil team and published here on our Brazil site on Apr The “fortress conservation” model is under pressure in East Africa as protected areas become battlegrounds over history and global efforts to halt biodiversity loss Mongabay’s Special Issue goes beyond the region’s world-renowned safaris to examine how rural communities and governments are reckoning with conservation’s colonial origins and trying to forge a path forward […] Metrics details Wildfire regimes are being altered in ecosystems worldwide The density of reptiles responds to fires and changes to habitat structure Some of the most vulnerable ecosystems to human-increased fire frequency are old-growth Araucaria araucana forests of the southern Andes We investigated the effects of wildfires on the density and richness of a lizard community in these ecosystems considering fire frequency and elapsed time since last fire During the 2018/2019 southern summer season we conducted 71 distance sampling transects to detect lizards in Araucaria forests of Chile in four fire “treatments”: (1) unburned control We found that the density and richness of lizards are impacted by wildfire frequency and time of recovery mediated by the modification of habitat structure The lizard community varied from a dominant arboreal species (L pictus) in unburned and long-recovered stands to a combination of ground-dwelling species (L araucaniensis) in areas affected by two fires Araucaria forests provided key habitat features to forest reptiles after fires but the persistence of these old-growth forests and associated biodiversity may be threatened given the increase in fire frequency An assessment of the effects of forest fires on a lizard community should include this habitat component which is modified by fires and can simultaneously act as an important resource for several coexisting species We expected that fires would modify lizard richness and density in Araucaria forests determined by functional traits associated with natural history (e.g. we expected: (1) a negative impact on arboreal and CWD-dependent species in areas affected by more than one fire; (2) higher density of arboreal reptiles with longer time elapsed since last fire; and (3) a negative effect on overall lizard density and richness in areas affected by more than one fire Information on the response of animal communities to altered wildfires in old-growth forests with low fire frequency is scarce This study aims contributing to our understanding and predictions on global consequences of the widespread alterations of fire regimes Modeling results for lizard density in relation to meaningful habitat covariates and “treatments'' for each lizard species Leftmost column shows graphic representations of covariates and fire treatments (from top to bottom: upper canopy cover Graphics are ordered by columns according to each modeled species: (A) slender lizard (L pictus); and (C) araucana and ribboned lizards (“ground lizards” Grey area shows estimated 95% confidence intervals N/A (not applicable) indicate uninformative covariates according to AIC results for each species’ models Total density (a) and richness (b) in each treatment for the lizard community in Araucaria forests from the Araucarias Biosphere Reserve (ABR) in the Andes of La Araucanía Region Density estimates are based on predictions from best HDS models for each species For richness results we used data from the 7 species detected Understanding the consequences of altered wildfire regimes on terrestrial biodiversity is critical, as these disturbances become more frequent at a global scale45 Here we reported the consequences of an altered fire regime on the understudied lizard community from the endangered Araucaria araucana forests in southern Chile Our results suggest that both the density and richness of the studied lizard community are impacted by wildfire frequency and time of recovery the lizard community varied from a dominant arboreal species in unburned and long-recovered stands to a combination of ground-dwelling species in areas affected by two successive fires allowing greater light and solar radiation inside the forest and increasing the growth and reproduction of L This process would also explain the higher density of the arboreal L tenuis in burned forests with short recovery times we detected both species in all burned treatments their populations declined considerably in the burned twice condition the simultaneous positive response to fire by arboreal and ground lizards resulted in the overall increase in density of lizards among fire treatments but the composition of species varied considerably among them we observed a clear differentiation in species composition between the burned twice treatment (where L lemniscatus dominated) and the long and short-term recovery treatments (where L future research may test alternative methodologies (i.e may allow to control possible confounding variables rising from geographic differences and increasing sampling units and treatments prescribed fires are illegal in native forests of Chile and due to the relatively novelty of fire regime changes in temperate forests of the southern Andes it is not yet possible to survey Araucaria forests burned three or more consecutive times landscape-scale natural experiments are the only current option to investigate the consequences of fires on this forest ecosystem we sampled all available area for the long-recovered treatment Future research should focus on surveying lizards in our treatments in a multi-season study to better understand the dynamics of reptile assemblages after fires and to survey reptiles in Araucaria stands burned more than twice This is the first study showing how wildfires modify the lizard community of Araucaria forests in southern Chile considering both fire frequency and time elapsed since last fire The structure of the resulting community depended on habitat structure and species’ functional traits Our results provide evidence of the resilience of the detected species after fire disturbances Two fires could even be beneficial to the threatened lizard L informed inferences about conservation outcomes should consider different taxa as they may respond differently to these disturbances Araucaria forests granted key habitat features to forest reptiles after fire disturbances but the persistence of these old-growth forests and associated biodiversity may be threatened by the current upward trend in fire frequency and ranging from approximately 400 to 3000 m.a.s.l All treatments were located between 1014 and 1469 m.a.s.l Protected areas are delimited by dashed black lines Forest conditions of the four “treatments”: (a) unburned control [old growth forest with 5,000 year old araucarias]; (b) “long-term recovery” (16 years) [abundant CWD evident understory and canopy regrowth]; (c) “short-term recovery” (3 years) [abundant CWD understory and canopy regrowth is still scarce]; (d) “burned twice” (3 years of recovery) [scarce CWD understory regrowth is scarce and canopy almost nonexistent] unclear distributional delimitations/co-occurrence and ecological and morphological similarities we will consider them the same for the analyses (hereafter which overlapped with reptile sampling units Each plot was divided in four quadrants with the aid of two tape measures and we visually estimated: understory cover (% to avoid recording same individuals in different sampling units which assumes the measurement of distance in discrete intervals and that sample unit i has local latent abundance Ni This model also assumes a multinomial distribution for the detection frequencies in each of the J distance classes which is conditional to the latent abundance Ni Transect-specific covariates are used to model λ and σ parameters using the log link function Support and permissions to investigate in public protected areas (Tolhuaca National Park Malleco National Reserve and China Muerta National Reserve) were given by National Forest Service of Chile (CONAF) and in private areas were given by the Pewenche Quinquén Community The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request Fire regime: History and definition of a key concept in disturbance ecology Interactions between successive forest fires across a temperate mountain region Tamm Review: Shifting global fire regimes: Lessons from reburns and research needs Cambio climático y su impacto potencial en la ocurrencia de incendios forestales en la zona centro-sur de Chile (33°–42° S) Efectos del fuego sobre la avifauna del cerro Cayumanque First-order fire effects on animals: Review and recommendations Ecosystem responses to fire: Identifying cross-taxa contrasts and complementarities to inform management strategies A 55-year-old 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in banksia woodlands: How does the fire-free period influence reptile communities? Reptiles in monterey pine plantations of the coastal range of central Chile Contrasts in short- and long-term responses of Mediterranean reptile species to fire and habitat structure Responses of reptiles to fire in transition zones are mediated by bioregion affinity of species Changes in the structure of assemblages of three liolaemus lizards (Iguania liolaemidae) in a protected area of south-central Chile affected by a mixed-severity wildfire Lizard assemblages in a fragmented landscape of central Chile How many common reptile species are fire specialists A replicated natural experiment highlights the predictive weakness of a fire succession model The ecological roles of logs in Australian forests and the potential impacts of harvesting intensification on log-using biota A long-term experiment reveals strategies for the ecological restoration of reptiles in scattered tree landscapes Coarse woody debris is affected by the frequency and intensity of historical harvesting and fire in an open eucalypt forest Biases in the current knowledge of threat status in lizards Addressing knowledge gaps in reptile conservation Habitat structure and proximity to forest edge affect the abundance and distribution of forest-dependent birds in tropical coastal forests of southeastern Madagascar small mammal and bird communities in the arid spiny forest Evaluating the role of fire disturbance in structuring small reptile communities in temperate forests Effect of altitude on thermal responses of Liolaemus pictus argentinus in Argentina Geographic variation and acclimation effects on thermoregulation behavior in the widespread lizard Liolaemus pictus Forest-fire regimes affect thermoregulatory opportunities for terrestrial ectotherms Response of a reptile guild to forest harvesting Realized niche modelling uncovers contrasting responses to fire according to species-specific biogeographical affinities of amphibian and reptile species A case study using reptiles from semi-arid Australia Distribución histórica y actual de la lagartija Liolaemus pictus (Dumeril & Bibron 1837) (Liolaemidae) y nuevo límite continental sur de distribución Thermal niche evolution across replicated Anolis lizard adaptive radiations Paradise burnt: How colonizing humans transform landscapes with fire thermal environment and wind intensity on the thermal ecology of Brazilian sand lizards in a restinga remnant Why do some species have geographically varying responses to fire history? Megafires: An emerging threat to old-forest species Socioeconomic factors drive fire-regime variability in the mediterranean basin Estructura y Dinámica (Editorial Universitaria S.A. Short-term response of soil microorganisms nutrients and plant recovery in fire-affected Araucaria araucana forests Diferencias en la composición florística en bosques de Araucaria-Nothofagus afectados por distintas severidades de fuego Incendios catastróficos en bosques andinos de Araucaria-Nothofagus: Efecto de la severidad y respuesta de la vegetación Sinopsis bioclimática y vegetacional de Chile (Editorial Universitaria S.A. Análisis de la afectación y severidad de los incendios forestales (2017) Rodent assemblage composition as indicator of fire severity in a protected area of south-central Chile Lizards on ice: Evidence for multiple refugia in Liolaemus pictus (Liolaemidae) during the last glacial maximum in the southern Andean beech forests Biotic homogenization: Loss of avian functional richness and habitat specialists in disturbed Andean temperate forests Beyond species richness: An empirical test of top predators as surrogates for functional diversity and endemism Modeling abundance effects in distance sampling Improving estimates of bird density using multiple-covariate distance sampling Unmarked: An R package for fitting hierarchical models of wildlife occurrence and abundance R: A Language and Environment for Statistical Computing (2019) Hierarchical distance sampling to estimate population sizes of common lizards across a desert ecoregion A Practical Information-Theoretical Approach (Springer Package ‘nlme’: Linear and Nonlinear Mixed Effects Models (2020) Package ‘AICcmodavg’: Model Selection and Multimodel Inference Based on (Q)AIC(c) (2020) Simultaneous inference in general parametric models Download references We acknowledge the National Forest Service of Chile (CONAF) for logistic support and the Pewenche Quinquén Community for allowing us to work in their land Marín provided valuable support and collaboration throughout the project The authors would also like to thank Constanza Arévalo for her work in reviewing the English of the manuscript This work was supported by Columbia University President’s Global Innovation Fund the ANID/Apoyo a la Formación de Redes Internacionales entre Centros de Investigación (REDES150047) Internationalization Grant Agreement (PUC1566-MINEDUC) and ANID/FONDECYT de Iniciación (11160932) JTI acknowledges the support from ANID PIA/BASAL FB0002 and the ANID—Millennium Science Initiative—CESIEP Code NCS13_004 JI acknowledges the support from ANID BECAS/DOCTORADO NACIONAL 21212206 Programa de Doctorado en Ecosistemas Forestales y Recursos Naturales & Instituto de Conservación Facultad de Ciencias Forestales y Recursos Naturales ECOS (Ecosystem-Complexity-Society) Co-Laboratory Center for Local Development (CEDEL) & Center for Intercultural and Indigenous Research (CIIR) Millennium Nucleus Center for the Socioeconomic Impact of Environmental Policies (CESIEP) & Center of Applied Ecology and Sustainability (CAPES) Department of Earth and Environmental Sciences designed field surveys and planned analyses; J.I. wrote the manuscript and prepared figures and illustrations contributed critically to drafts and have given their approval for publication Download citation DOI: https://doi.org/10.1038/s41598-021-02169-3 This story is part of the Landscape News series Forgotten Forests The Scottish-American environmental hero John Muir is famous for his activism to preserve the sequoias of the American West he traveled to the other end of the Americas in search of a different forest giant he had only seen in photographs: the araucaria In the misty highlands of Southern Brazil, he walked among the distinctive flat-topped conifers for a week, writing in his diary: “Rainy morning Wondrous sight.” He had recently visited the Amazon but the araucarias were the trees he’d wanted to see before he died the most interesting forest he had ever seen Brazil’s araucarias belong to an ancient family, the Araucariaceae, which were found worldwide during the Jurassic and Cretaceous periods (200–65 million years ago). Some research suggests their energy-rich slow-fermenting leaves were a key food source for sauropod dinosaurs the family’s three surviving genera are largely confined to the Southern Hemisphere And like the dinosaurs that once roamed among them many of these odd-looking trees are now facing extinction there are 20 species in the Araucaria genus the hoop pine of Australia and Papua New Guinea Another 22 species belong to the Agathis genus found in the South Pacific and Southeast Asia the most famous of which is New Zealand’s colossal kauri tree And Australia’s Wollemia genus contains just one species a spectacular ‘living fossil’ only discovered in 1994 with its last stronghold a hidden canyon in a top-secret location near Sydney A 2018 study ranked the world’s gymnosperm species (non-flowering plants) according to their evolutionary history and extinction risk Of the top four identified as the highest conservation priority three were Araucariaceae — the Wollemi pine Despite the ancient family’s geographical spread almost half of its living species – 19 out of 45 – are only found on one small Pacific archipelago about 1,000 kilometers from Australia: New Caledonia These mountainous tropical islands are home to five Agathis species and 14 Araucaria. “These plants are amazing, they don’t look like anything else,” says Robert Nasi, director general of the Center for International Forestry Research For research while obtaining his master’s degree Nasi mapped the distribution of the different Araucariaceae species across the islands Some grow only at the very top of New Caledonia’s spine of wet misty mountains others on rocky upraised reefs near the shore “Then you have all the weird ones that look very much like there is a dinosaur waiting behind,” Nasi says “You’re looking to see what sort of antediluvian monster is going to pop out the other side.” But with each adapted to a specific environment, New Caledonia’s Araucariaceae are vulnerable. One species, Araucaria scopulorum, is known only from a handful of locations designated for nickel mining one of the country’s most important industries And climate change poses risks for many of the species “I don’t think they will disappear within our lifetime but I don’t think they will last very long,” says Nasi “A species that is restricted to a 200-meter elevation band on top of a mountain on one island in the Pacific probably doesn’t have a bright future under climate change.” More effort should be put into propagating New Caledonia’s botanical treasures and growing them in living collections Nasi says – and the last stands of the most endangered species should be fully protected from mining and other disturbances Kauri trees can live for more than 2,000 years and grow to be at least 50 meters.  They are considered a taonga A vast kauri forest once carpeted the north of the country, but demand for timber and agricultural land after Europeans arrived led to the destruction of 99.5 percent of it by the early 20th century Pockets remain in protected areas and on private land. But kauri are now facing a new enemy – a soil-borne fungus-like pathogen named Phytophthora agathidicida There is no cure, but scientists and Māori are working together, trying to combine microbiology with traditional knowledge in an effort to save the trees and it might have disappeared from the wild altogether at the end of 2019 if it weren’t for a daring Wollemi-like trees were only known from the ancient pollen record a forestry scientist at the Australian National University in Canberra “We knew there was something like that tree that had existed They were thought to have become extinct millions of years ago abseiled into a canyon in the Wollemi National Park in the Blue Mountains northwest of Sydney and came across a towering tree he didn’t recognize It had fern-like leaves and bark covered in chocolate-colored bubbles “It was really like finding a living dinosaur,” says Brack around 100 Wollemi pines have been found in four groves in the same canyon system their location kept secret to protect them from vandalism or the accidental introduction of phytophthora Genetic analysis has revealed the population has extremely low genetic diversity In an effort to preserve the Wollemi for future generations, horticulturists at the Australian Botanic Garden at Mount Annan devised a way to clone the trees. Since 2006 they have been grown by home gardeners and botanical gardens around Australia and the world The Royal Botanic Garden Sydney has just launched an international citizen science project, I Spy a Wollemi Pine to try to track the different environments the species can grow in It eventually engulfed more than 444,000 hectares the largest forest fire to start from a single ignition point in Australian history.  Brack followed its progress and worried it would be the death knell for the Wollemi pine in the wild there’s no way they could have survived that fire.” But in January, the New South Wales government announced they had saved the trees in a secret rescue mission Aircraft dropped water bombs and fire retardant in a ring around the grove Firefighters were winched into the gorge by helicopter to set up and operate an irrigation system to keep the ground moist but the fire passed on and the rest of the population was unharmed the Wollemi pine is no longer in danger of disappearing But many mysteries remain about the species’ genetics and biology and how this one small stand survived through the millennia Those answers might only come from the wild population and the interactions the trees have with the soil More is known about the Araucaria species that Muir so admired They are the dominant species in a forest type that once stretched 200,000 square kilometers across Brazil’s three southern states – Rio Grande do Sul Santa Catarina and Parana – as well as part of Argentina’s Misiones province the araucaria sheltered a hyperdiverse range of endemic palms the leaves of which are used across southern South America to make the ubiquitous hot drink known as mate (Spanish) or chimarrão (Portuguese) today’s Indigenous Kaingang and Xokleng-Laklãnõ people Recent archeological evidence indicates that around 1,000 years ago, the Southern Jê helped the araucaria forests expand from a smaller natural distribution to cover almost the entire plateau humans began to have the opposite effect on the forest clearing it for timber and making way for agriculture just 3 to 5 percent of the forest’s original extent remains Logging araucaria is now illegal. But research in 2019 found that climate change is likely to drive the species even closer to extinction Oliver Wilson from the University of Reading and colleagues used modeling to combine climate data with high-resolution topography and vegetation maps just 3.5 percent of remnant forest will be suitable for araucaria moist areas where the trees might be able to hold on – but found that more than a third of these areas have already been deforested.  Araucaria have survived significant climate changes before (They’ve been around for 100 million years after all.) But while in the past the forests were able to shift up and down the South American continent as the climate fluctuated “They live at the southernmost extent of Brazil’s southern highlands They’re trapped to the west by deforestation to the south by low elevations and to the north by changing climates It’s like climate and human actions together are closing in on them targeted interventions could ensure its survival in the wild That could include increasing protection in the most climatically suitable spots and adjusting regulations to encourage landowners to retain and restore araucaria in cattle-grazing landscapes We might also be able to discover more about how the Southern Jê managed the forest they increased these forests in the face of a climate that wasn’t helping them do so maybe we can learn lessons to help the forest persist.” Our mission is to make them freely accessible to everyone We believe that lasting and impactful change starts with changing the way people think That’s why we amplify the diverse voices the world needs to hear – from local restoration leaders to Indigenous communities and women who lead the way not only are you supporting the world’s largest knowledge-led platform devoted to sustainable and inclusive landscapes but you’re also becoming a vital part of a global movement that’s working tirelessly to create a healthier world for us all Every donation counts – no matter the amount Filmmaker Guille Isa speaks to poet Donatella Gasparro on how documentaries are crucial for cultural conservation during the climate crisis local forest farmers are taking on land degradation and bureaucracy – and winning GLF Live with Analì Bustos and Marlon Webb ThinkLandscape is a multimedia platform bringing you original knowledge-backed news and feature stories about climate and landscape solutions from around the world Metrics details The essential oil isolated by hydrodistillation of the oleogum resin of Araucaria heterophylla has been analyzed by GC–MS Twenty-four components accounting to 99.89% of the total detected constituents of this essential oil were identified The major ones were: caryophyllene oxide (14.8%) trans-verbenol (5.88%) and α-pinene oxide (5.18%) The in vitro inhibitory activities of this oil against aldose reductase COX-2 and SARS-CoV-2 Mpro enzymes were evaluated This revealed promising inhibitory activity of the essential oil against both aldose reductase and BuCHE enzymes The molecular docking study of the major components of the Araucaria heterophylla essential oil was carried out to correlate their binding modes and affinities for aldose reductase and BuCHE enzymes with the in vitro results the in vitro inhibitory activity of the essential oil attributed to the synergistic effect between its components and the in silico study suggested that compounds containing epoxide and hydroxyl groups may be responsible for this activity This study is preliminary screening for the oil to be used as antidiabetic cataract and Alzheimer’s disease therapeutics and further investigations may be required the use of plant-derived natural products with therapeutic significance attracts great attention due to their availability This motivates further search into traditional medicine to explore highly effective and safer natural inhibitors for some important enzymes involved in certain disorders and diseases In the framework of continuing research on Araucaria heterophylla tree this study is concerned with analyzing the essential oil isolated from the oleogum resin exuded from its trunk discovering potential anti-diabetic cataract anti-inflammatory and anti-COVID-19 leads based on the evaluation of the ability of the major components of this essential oil to inhibit certain enzymes as aldose reductase COX-2 and SARS-CoV-2 Mpro using in vitro and in silico studies this study is the second one to study the composition of EO of the oleogum resin of A heterophylla collected from Egyptian culture it is the first one to report the inhibitory activity of this essential oil against aldose reductase and BUCHE enzymes GC–MS chromatogram of essential oil of Araucaria heterophylla oleogum resin differences in some components and in some percentage of others was found The EO of the previous study showed that the most abundant component was α-pinene (44.88%) followed by germacrene-D (10.25%) it showed low percentage of oxygenated components (5.66%) compared to the EO of our study (40.66%) In addition to caryophyllene oxide that exhibited very low percentage (0.33%) it was the most abundant component in our study (14.82%) heterophylla oleogum resin showed the highest inhibitory activity against aldose reductase and BUCHE enzymes compared to the other two enzymes under investigation thus molecular docking study was carried out to demonstrate the major 8 components of the EO binding modes and their affinities for these two enzymes thus exploring the components of the EO responsible for the inhibitory activities against these enzymes and their mode of action Docking results of the major compounds of the essential oil & the standard epalrestat in the active site of aldose reductase enzyme (3RX2) 2D interactions of (a) Standard epalrestat Other essential oil major components showed weaker binding interactions with aldose reductase active site This may be due to their non-polar structure They only bound to target by hydrophobic bonds it could be predicted that the activity of Araucaria heterophylla essential oil against aldose reductase enzyme (IC50 = 0.133 µg/mL) that was more potent than the reference epalrestat itself (IC50 = 0.165 µg/mL) may be attributed to trans-verbenol and alpha-pinene oxide components and in a less extent to caryophyllene oxide Docking results of the major compounds of the essential oil & the standard rivastigmine in the active site of BuChE enzyme (4BDS) 2D interactions of (a) Standard rivastigmine Other essential oil major components showed poor interactions with BuChE active site This may be due to their lipophilic character and absence of any hydrogen bonding functional groups in their structures They only bound to target by hydrophobic interactions it could be predicted that the BuChE inhibition of essential oil (IC50 = 0.154 µg/mL) nearly similar to rivastigmine (IC50 = 0.078 µg/mL) trans-verbenol and alpha-pinene oxide components our findings shed light toward the potential use of the EO from A heterophylla as a green source of aldose reductase and BUCHE inhibitor This study is the first one that reports the in vitro aldose reductase COX-2 and SARS-CoV-2 Mpro inhibitory activities of Araucaria heterophylla essential oil Our findings attracted the attention to the possibilities of using the EO of A heterophylla as a new medicinal natural inhibitor against aldose reductase and BUCHE enzymes as it exhibited promising significant inhibitory activity compared to the standards this EO may be suggested for treatment of diabetic cataract as well as Alzheimer’s disease The oleogum resin of cultivated Araucaria heterophylla (Salisb.) were collected from the Gardens of Al Montazah The plant identity was confirmed by Associate Prof Department of Vegetables & Floriculture Voucher specimens were coded as Ah-1–2021 and kept in Pharmacognosy Department This extraction was repeated three times to afford 5 ml of essential oil The oil was dried over anhydrous sodium sulfate then stored at + 4 °C in the dark until tested All procedures were conducted in accordance to the relevant institutional and international guidelines and legislation GC/MS analysis was carried out using an Agilent 19091S-433 system with a mass selective detector film thickness: 0.25 μm); injection mode: splitless; split-flow: 10 ml/min; splitless time: 0.80 min; injector and detector temperature: 250 °C; oven temperature was programmed as follows: 60 °C for 2 min and then 5 °C/min programmed at 240 °C; the carrier gas was helium with a constant flow of 1 mL/min; injection volume: 1 µL of diluted essential oil (1% w/v in CH2Cl2) For measuring the aldose reductase activity: (Catalog # K369-100) colorimetric kit was used epalrestat was included as a positive control (155 S The principle of the assay depends on the ability of aldose reductase enzyme to catalyze the oxidation of NADPH to NADP The absorption of NADPH at 340 nm was measured in a mixture containing NADPH the enzyme and its substrate in addition to test samples For measuring butyryl choline esterase activity: (Catalog Number EIABCHEF (192 tests) fluorescent kit was used rivastigmine was included as a positive control (Life Technologies Corporation | Carlsbad CA 92,008 USA | Toll Free in USA 1 800 955 6288) The substrate butyryl thiocholine chloride is hydrolyzed by BuChE to give butyrate and thiocholine thiocholine reacts with 5,5-Dithiobis [2-nitrobenzoic acid] (DTNB) to give yellow-colored 2-nitro-5-thiobenzoate which can be detected spectrophotometrically at 412 nm For measuring COX-2 activity: (Catalog # K547-100) fluorometric kit was used celecoxib was included as a positive control (155 S The assay is based on the fluorometric detection of prostaglandin G2 the intermediate product generated from arachidonic acid by the action of COX enzyme at 535/587 nm For measuring main protease (SARS-CoV-2): (Catalog #79,955–1) fluorometric kit was used tipranavir was included as a positive control (6042 Cornerstone Court West The principle of the assay depends on the C-terminal of the peptide substrate being linked to a fluorophor (Edans) and the N-terminal has a fluorescence quencher (Dabcyl) that quenches the fluorescence signal of Edans the peptide substrate exhibits low fluorescence because the fluorescence intensity of Edans in the C-terminal is quenched by the Dabcyl in the N-terminal of the substrate Statistical analysis of the data was performed using GraphPad Instat version 8 software package ((GraphPad Software Inc Statistical tests used were one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparisons test for statistical comparison between parametric data The results were expressed as percentage inhibition Inhibitory activity (%) = (1 − A test/A control) × 100 A test is the reading* in the presence of test substance and A control is the reading* of control Molecular docking was used as a tool that predicting the binding mode of the essential oil major components with the targeted enzymes The molecular docking studies in both two- & three-dimensional visualizations were used to predict the binding affinity in Kcal/mol between the essential oil major components and the enzymes active sites then comparing their results with a reference molecular docking studies helped to pick up the essential oil components that are responsible for the activity These studies were done only on two enzymes aldose reductase and BUCHE because of their promising in vitro results The datasets used during the current study available from the corresponding author upon request Purification and biological effects of Araucaria angustifolia (Araucariaceae) seed lectin Phytochemical and ethno-pharmacological review of the genus Araucaria—Review Chemical constituents and biological activities of Araucaria angustifolia (Bertol.) 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hallmarks of cancer and adaptation to the tumor microenvironment In vitro and in silico studies of SARS-CoV-2 main protease Mpro inhibitors isolated from Helichrysum bracteatum A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography Constituents of the essential oil of Mentha microphylla C Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry Vol 456 (Allured Publishing Corporation Carol Stream IL Yield and chemical composition of Citrus essential oils as affected by drying pretreatment of peels Lemongrass essential oil components with antimicrobial and anticancer activities Metabolomic profiling of three Araucaria species and their possible potential role against COVID-19 A caryophyllene oxide and other potential anticholinesterase and anticancer agent in Salvia verticillata subsp The molecular basis for inhibition of sulindac and its metabolites towards human aldose reductase Crystal structures of human cholinesterases in complex with huprine W and tacrine: Elements of specificity for anti-Alzheimer’s drugs targeting acetyl-and butyryl-cholinesterase Soxhlet extraction versus hydrodistillation using the Clevenger apparatus: A comparative study on the extraction of a volatile compound from Tamarindus indica seeds Effects per se of organic solvents in the cerebral acetylcholinesterase of rats and docking studies of some novel chalcones as selective COX-2 inhibitors Learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-nCoV Inhibition of aldose reductase by ginsenoside derivatives via a specific structure activity relationship with kinetics mechanism and molecular docking study biological evaluation and molecular modeling simulations of new heterocyclic hybrids as multi-targeted anti-Alzheimer’s agents Download references Esam Rashwan (Head of the confirmatory diagnostic unit VACSERA-EGYPT) is acknowledged for carrying out the biological assays Open access funding provided by The Science Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB) operated the hydrodistillation of plant materials revised of the manuscript and took over the publishing process G.A.: Participated in the identification of the oil components writing the manuscript and took over the publishing process M.A.S.: did the molecular docking study of the major components of the Araucaria heterophylla essential oil to explain their binding modes and affinities for aldose reductase and BuCHE enzymes Download citation DOI: https://doi.org/10.1038/s41598-023-38143-4 Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology was better known to Guardian crossword solvers as Araucaria on the strength of his success in winning the Observer's crossword setting competition two years running he was taken on by the then Manchester Guardian as one of its small band of crossword setters The first clue in his first puzzle for the Manchester Guardian set the standard for what was to follow: "Establishment cut to the bone setters were anonymous but were then given noms de plume Araucaria is the botanical name for the monkey puzzle tree ("Monkey" was a term of endearment in the Graham family and echoed Desmond Morris's The Naked Ape.) later principal of Cuddesdon Theological College and then bishop of Brechin Graham would share the Times crossword puzzle with his parents and together they would engage in cryptic word play and in setting simple puzzles in 1939 with a scholarship and passed part one of the classical tripos he was sent for aircrew training to Rhodesia and failed his pilot's course He flew as a navigator/bomb aimer in some 30 sorties in Baltimores and Bostons in Italy being mentioned in dispatches after baling out behind enemy lines and successfully hiding with an Italian family until rescued by the Americans He returned to King's in 1946 to read theology went on to Ely Theological College and was ordained in the Church of England in 1948 After serving in East Dulwich as a curate and St Chad's College in 1952 he married Ermesta and moved to Aldershot as senior curate and then His final living was as rector of Houghton and Wyton in Cambridgeshire Soon he was producing eight cryptic puzzles a month for the Guardian (including three bank holiday "specials" a year) and later also contributing six a month to the Financial Times where he was Cinephile (an anagram of Chile pine the monthly subscription magazine that he founded in 1984 For many years he also set the Guardian's daily Quick crosswords In the last 20 years of his life he embraced new technology eventually setting up his own website and finding himself to his astonishment overwhelmed by private requests for "bespoke" puzzles to mark some event or anniversary the speed at which he worked and his clarity of mind scarcely diminished and he remained a strikingly handsome man until the end such an output might have led to plodding repetition but the joy of Araucaria's crosswords was that they were always new-coined Even into his 90s custom could not stale his infinite variety This was largely due to his refusal to accept the strict Ximenean straitjacket conceived by AF Ritchie (Afrit in the Listener) and codified by DS Macnutt (Ximenes of the Observer) assisted by Alec Robins (Custos of the Guardian) Graham followed his own instincts and solvers largely accepted them because they led to such enjoyable results Afrit's central injunction for cluing was: "I need not mean what I say but I must say what I mean." Araucaria had no quarrel with the rule but insisted that it had to be applied flexibly convey its meaning so that the solver had a fair chance of solving it and of knowing that the answer arrived at was correct it had to stick narrowly to the laws of grammar and syntax there had to be some sort of understanding between setter and solver about what was "fair" and Araucaria once articulated his difference with extreme Ximeneans thus To clue the word CAIN (who killed Abel) his device might be to insert an "I" into "CAN" which has the slang meaning of "prison" His clue might be: "Having committed a murder I am in prison." A Ximenean would object that the "definition" in the clue for Cain is unfair because "Cain" is a noun and "Having committed a murder" is not; and because here "I" is a letter of the alphabet and not a personal pronoun it should be followed grammatically by "is" not "am" So a strict Ximenean would require some clue like: "Being guilty of murder I must be put in prison." In Araucaria's view Guardian solvers would find that his clue was fair – and better Araucaria did not invent "themed" cryptic puzzles the 250th anniversary of the death of Bach he produced a grid full of more references to the composer than one would have thought humanly possible But an Araucaria-themed puzzle might equally concentrate on the heroes of the struggle against apartheid in South Africa or the sea areas of the British shipping forecast where the clues are presented in alphabetical order of their solutions and the solver has to fit these into the grid "jigsaw-wise First: "O hark the herald angels sing the boy's descent producing WHILE SHEPHERDS WATCHED THEIR FLOCKS BY NIGHT ALL SEATED ON THE GROUND; second: "Poetical scene with surprisingly chaste Lord Archer vegetating" for THE OLD VICARAGE For all his repute as the most admired and renowned crossword setter in the English language Graham was an essentially shy and humble man He derived huge happiness from his second marriage but after Margaret died of a heart attack in 1993 had the strength to continue to pursue a full and active life living alone but being very much part of the social and church activity of the village of Somersham in Cambridgeshire stealthily giving away much of his not very large income political views were definitely of the left ("the Guardian is my paper though I find it a bit conservative") As an adult his paper was the old liberal News Chronicle before it was the Manchester Guardian On principle he would never have set puzzles for a Murdoch paper He expected little or nothing from Tony Blair's New Labour and even less from the present coalition Typically, he decided to announce the fact that he was suffering from non-operable cancer in a puzzle set for the December 2012 issue of 1 Across, which was reprinted in the Guardian on 11 January 2013 read: "Araucaria has cancer of the oesophagus which is being treated with palliative care." The solutions to other clues included Macmillan nurse somehow," he responded when asked why be had gone public in this way He was quite overcome by the volume and warmth of the tributes that flowed in as a result of this singular puzzle He gave great entertainment to a great many readers for more than half a century and inspired a younger generation of crossword setters Graham was made MBE in 2005 for his services to crosswords He is survived by his brothers Stephen and Martin his sister Mary and stepdaughters Jane and Judith born 16 February 1921; died 26 November 2013 This article was amended on 28 November 2013 The earlier version gave the wording of a clue as "Poetic scene with chaste Lord Archer Metrics details Despite the high diversity of the Southern South American environments the patterns and processes driving both their species diversity and demographic history are still poorly known and are a challenging task we evaluate plastid DNA sequences of the conifer species Araucaria angustifolia aiming to (i) assess the species genetic structure within its main range of occurrence (ii) infer its population demographic history looking for evidence of southward expansion (iii) search for evidence of glacial refugia within the species distribution area and (iv) discuss some conservation and management strategies for this species revealing the presence of three distinct genetic groups across the geographic range of the species and structuring the populations into Northern Our results suggest the occurrence of post-glacial expansion of A as well as the existence of at least three refugia within the species occurrence area Testing the occurrence of historical demographic expansion we suggest that genetic groups experienced fluctuations in effective size associated to a structured distribution of populations The identification of three genetic groups in this study corroborates the proposition of using the geographic distribution of A angustifolia for selecting in situ conservation areas for planning seed collection for ex situ conservation as well as for the delineation of seed zones This species presents a latitudinal discontinuous distribution with a large gap dividing the occurrence area of the species in two main geographical zones in Brazil: (i) a northernmost zone that is formed by scattered medium to large forest formations at high altitudes in the Southeastern region of the country and (ii) a southernmost zone which is composed by a much wider area originally formed by large continuous forest formations in the Southern region of Brazil This zone includes the Argentinian and Paraguayan patches of forest with araucaria The gap area between the northern and southern occurrence areas of A angustifolia lacks register of recent population presence and only ecological simulations suggest the possibility of occurrence of the species in this area Since these studies are based only on data from southernmost populations without inclusion of samples representing the northern genetic group and weakly representing the most central populations in the Paraná State Aiming to evaluate distribution patterns of genetic variation among natural populations of A angustifolia across most of the species range we conducted a large-scale genetic analysis based on the sequence variation of three intergenic regions of plastid DNA (ptDNA) this study aimed to test the above presented hypotheses about the current distribution of A angustifolia genetic groups by exploring these ptDNA sequences Signatures of recent demographic expansion (e.g genetic divergence among populations associated to geographic distances and haplotype network presenting a star-like form (i.e several haplotypes originated from a single ancestor haplotype) would suggest that the current distribution of this species can be accepted as the result of southward expansion a strong genetic structure among genetic groups and a haplotype network containing more than one putative ancestor haplotype would imply that the current distribution of A angustifolia genetic groups is a consequence of the expansion of populations from different glacial refugia in the northern and in the southern zones of the species occurrence Following this logic and considering the results of previous studies that suggest geographic patterns of genetic differentiation in populations of A angustifolia influenced by Quaternary shifts on the distribution of the genetic variants this study intended to (i) evaluate the genetic structure of A angustifolia within its range of occurrence; (ii) infer the population demographic history of this species looking for evidence of southward expansion; (iii) search for evidence of glacial refugia within the species distribution area of A angustifolia; and (iv) discuss some conservation and management strategies for this species based on its current genetic structure and inferred evolutionary history The alignment of the three intergenic plastid regions generated in the present study encompasses 2,382 bp, with 16 variable sites and two indels, revealing low variation (Table 1) Twelve of these sites exhibit two variants two sites show three variants and two sites have singleton polymorphism Red circles are haplotypes exclusive to the North group; green circles are haplotypes exclusive to the Central group; blue circles are haplotypes exclusive to the Southern group; haplotypes shared by the Central and Southern groups represent their frequency in each genetic group with the green and blue colors representing the Central and Southern groups The sizes of the circles correspond to the frequency of each haplotype over all populations and the length of the lines is proportional to the number of mutations All clades presented high posterior probability support (PP ≥ 0.89) we adopt hereafter a geographical-genetic partitioning of populations in three groups: a Northern group (formed by populations from the northernmost occurrence area) a Southern group (composed by populations from the southernmost occurrence area) and a Central group geographically intermediate between northernmost and southernmost populations but genetically distinct from both Northern and Southern genetic groups The estimations of genetic diversity were θπ = 1.947 and θS = 2.306 for the pooled dataset, θπ = 1.644 and θS = 1.542 for the Northern group, θπ = 1.539 and θS = 1.026 for the Central group and θπ = 0.848 and θS = 1.283 for the Southern group (Table 1) The hierarchical AMOVA analysis (Table 2) revealed clear structuring with high differentiation among groups (ΦCT = 0.52 The differences between the Northern and Central/Southern groups Northern and Central groups and Northern and Southern groups were also high and statistically significant (ΦCT = 0.45 while the differences between the Central and Southern groups were moderate (ΦCT = 0.48 Mismatch distribution for the Northern Estimations of the raggedness (rg) and of the sum of squared deviations (SSD) index are presented with their respective significance where ***p < 0.001 and ns = not significant Tajima’s D and Fu´s FS tests (Table 3) presented non-significant (p > 0.13) negative values for the Northern and Southern groups (D(N) = −0.04 FS(S) = −3.40) and positive values for the Central group (D(C) = 1.02 and FS(C) = 1.75) Bayesian Skyline plots derived from ptDNA sequences of Araucaria angustifolia The analysis was performed for each of the determined genetic group The thick solid line represents the mean effective population size Ne while the 95% HPD (highest posterior density) limits are shown by the blue area angustifolia based on the climatic conditions of 20,000 years before present The approximate location of the putative Rio de Janeiro/Espírito Santo (RJ/ES) and South refuges are indicated by red arrows The blue arrow indicates the putative north-to-south corridor connecting the Northern and the Central regions (B) Predicted occurrence based on current climatic conditions suggesting the correspondence of the current marker phylogeographic signature to the actual species phylogeography defined by the configurations of haplotype distribution can be explained by both the occurrence of southward expansion and the existence of further unidentified glacial refugia suggesting rapid and recent expansion of such populations from a single southern refuge enabling gene flow that would explain the haplotypes shared between these two groups originating some populations of the Central genetic group These Central populations might further have mixed with Southern populations originating the existing genetic structure Populations undergoing demographic expansion usually present a unimodal mismatch distribution while a multimodal distribution is expected for populations at demographic equilibrium reflecting the stochasticity of gene trees A unimodal mismatch distribution was observed for all genetic groups but significant estimations of Harpending’s raggedness and SSD indexes were observed only for the Central and Southern groups angustifolia have to be interpreted in the proper context of its population structure allied to a structured distribution of populations The present study confirms high differentiation regarding the Northern group compared to the Central and Southern ones and used a much larger and wider sampling strategy than previously reported This differentiation is of great significance for the conservation of A angustifolia genetic resources since it is possible that it reflects not only in the intergenic ptDNA regions investigated but also in genes of adaptive significance PCR products were purified by precipitation using a 20% Polyethylene Glycol 8000 + 2.5 mM NaCl solution followed by washing in 80% ethanol and checking under UV in a 1% agarose gel stained with GelRed® (BiotiumTM) Sequencing reactions consisted of 1.0 μL of PCR product (100–200 ng μL−1) 0.35 μL of primer (10.0 μM) and 4.0 μL of DYEnamicTM ET dye terminator® mix (GE Healthcare some sequencing reactions were performed using 1.0 μL of PCR product (100–200 ng μL−1) 3.5 μL of 5X Sequencing Buffer and 1.0 μL of BigDye TerminatorTM v3.1 (Applied Biosystems Each purified PCR product was subjected to DNA sequencing by capillary electrophoresis (CE) from both the forward and reverse directions using a MegaBACE 1000 DNA Sequencing System (GE Healthcare and an ABI3500XL Genetic Analyzer (Applied Biosystems) Sequences were extracted using the programs Sequence Analyzer v 4.0 (GE Healthcare) and Sequencing Analysis v5.4 (Applied Biosystems) Alignments and final concatenation were performed using the program CLC Genomics Workbench v.8 (Qiagen Bioinformatics DNA sequences were deposited in the NCBI database under ID numbers MH223675 – MH225411 This program finds the best number of geographic groups (K-value) by maximizing FCT value between K groups of geographically adjacent populations The number of geographic groups K was set from 2 to 15 and estimations were performed using the Tamura 2-parameters model A run length of 2.5 × 106 generations was used under the HKY nucleotide substitution model The geographic data from the 39 sampled populations and additional points obtained from the literature were employed which totaled 49 points of species occurrence distributed from 21°13′S to 30°30′S latitude and 43°46′W to 54°00′W longitude covering the main distribution area of the species A Jackknife test was employed to determine the prediction power of each variable by setting the variable aside from the test and generating its percentage of contribution to the model All employed parameters were setting as follow: convergence threshold = 10−5; maximum iterations = 500; regularization multiplier = 1; application of a random seed duplicate presence records removal and logistic probabilities used for the output The model training was performed using 80% of species records and 20% was used to test the model Data files were downloaded at their original 2.8125° resolution and resampled to 30” resolution via bilinear interpolation Species paleodistribution modeling was conducted using the same parameters used for modeling current species distribution Biodiversity hotspots for conservation priorities Biodiversity hotspots: A shortcut for a more complicated Concept underlie the origin of diversity in toads endemic to the Brazilian Atlantic Forest Post-glacial recolonization of European biota Biological Journal of the Linnean Society 68 Genetic consequences of climatic oscillations in the Quaternary Philosophical Transactions of the Royal Society of London B: Biological Sciences 359 Phylogeographic Structure of White Oaks Throughout the European Continent Historical climate modeling predicts patterns of current biodiversity in the Brazilian Atlantic forest Environmental drivers of diversity in Subtropical Highland Grasslands Inheritance and linkage relationships of isoenzymes variants of Araucaria angustifolia (Bert.) O Genetic structure of Araucaria angustifolia (Araucariaceae) in Brazil: implications for the in situ conservation of genetic resources The role of gene flow in shaping genetic structures of the subtropical conifer species Araucaria angustifolia Phylogeography of Brazilian pine (Araucaria angustifolia): integrative evidence for pre-Columbian anthropogenic dispersal Phylogeographical patterns shed light on evolutionary process in South America Multiple ice-age refugia in a southern beech of South America as evidenced by chloroplast DNA markers Araucariaceae: flora ilustrada catarinense State of the Araucaria angustifolia forests: native species in critical danger South and Southeast Brazilian grasslands during Late Quaternary times: a synthesis charcoal and multivariate analysis of the Cambará do Sul core in southern Brazil Evidences of delayed size recovery in Araucaria angustifolia populations after post-glacial colonization of highlands in Southeastern Brazil Uncoupling human and climate drivers of late Holocene vegetation change in southern Brazil A simulated annealing approach to define the genetic structure of populations Forest Refuges: evidences form woody angiosperms in Biological diversification in the Tropics (ed T.) 137–158 (Columbia University Press 1982) Phylogeographic inferences concerning evolution of Brazilian Passiflora actinia and P elegans (Passifloraceae) based on ITS (nrDNA) variation Range-wide patterns of nuclear and chloroplast DNA diversity in Vriesea gigantea (Bromeliaceae) Large-scale phylogeography of the disjunct Neotropical tree species Schizolobium parahyba (Fabaceae-Caesalpinioideae) The impact of Quaternary climate oscillations on divergence times and historical population sizes in Thylamys opossums from the Andes Biological Journal oj the Linnean Society 58 Integrating approaches towards the conservation of forest genetic resources: a case study of Araucaria angustifolia Stability Predicts Genetic Diversity in the Brazilian Atlantic Forest Hotspot Selecting the best-fit model of nucleotide substitution Genetic evidence for Near-Eastern origins of European cattle Hitchhiking under positive Darwinian selection The confounding effect of population structure on Bayesian skyline plot inferences of demographic history The impact of sampling schemes on the site frequency spectrum in nonequilibrium subdivided populations The population demography of Betula maximowicziana in relation to the last glacial period: its admixture-like genetic structure is the result of simple population splitting not admixing The genetics and conservation of Araucaria angustifolia: I Genetic structure and diversity of natural populations by means of non-adaptive variation in the state of Santa Catarina The genetics and conservation of Araucaria angustifolia: III DNA extraction protocol and informative capacity of RAPD markers for analysis of genetic diversity in natural populations PCR-RFLP analysis of non-coding regions of cpDNA in Araucaria angustifolia (Bert.) O A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G + C content biases MEGA6: Molecular Evolutionary Genetics Analysis version 6.0 Median-joining networks for inferring intraspecific phylogenies Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space Statistical Method for Testing the Neutral Mutation Hypothesis by DNA Polymorphism On the number of segregating sites in genetic models without recombination Statistical Properties of New Neutrality Tests Against Population Growth DnaSPv5: a software for comprehensive analysis of DNA polymorphism data Population Growth Makes Waves in the Distribution of Pairwise Genetic Differences Signature of ancient population growth in a low-resolution mitochondrial DNA mismatch distribution New Statistical Tests of Neutrality for DNA Samples from a Population BEAST 2: A Software Platform for Bayesian Evolutionary Analysis Maximum entropy modeling of species geographic distributions Novel methods improve prediction of species’ distributions from occurrence data Very High Resolution Interpolated Climate Surfaces For Global Land Areas Rethinking receiver operating characteristic analysis applications in ecological niche modeling Deutsches Klimarechenzentrum Modellbetreuungsgruppe The ECHAM3 atmospheric general circulation model Download references This work was supported by the Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina (FAPESC 14848/2011-2) as well as by CNPq through the postdoctoral fellowship awarded to VMS (Process 152143/2007-6) Authors thank Nathan Smith for performing the language review of the manuscript Nucleus of Molecular Ecology and Plant Micropropagation Valdir Marcos Stefenon & Rafael Plá Matielo Lemos Graduate Program on Plant Genetic Resources Gustavo Klabunde & Rubens Onofre Nodari and elaborated the final version of the manuscript after the co-authors review analyzed data and reviewed drafts of the manuscript performed the MaxEnt analyses and reviewed drafts of the manuscript collected samples and reviewed drafts of the manuscript designed the experiments and provided reagents/materials/analysis tools and reviewed drafts of the manuscript Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-019-39308-w Genetic Resources and Crop Evolution (2022) surprisingly well-preserved conifer fossils from Patagonia which includes the well-known Norfolk Island pine is unique because it’s so abundant in the fossil record and still living today,” said Gabriella Rossetto-Harris a doctoral student in geosciences at Penn State and lead author of the study the Norfolk Island pine is also a popular houseplant that you might recognize in a dentist’s office or a restaurant.” Araucaria grew all around the world starting about 170 million years ago in the Jurassic period. Around the time of the dinosaur extinction 66 million years ago, the conifer became restricted to certain parts of the Southern Hemisphere, said co-author Peter Wilf, professor of geosciences and associate in the Earth and Environmental Systems Institute (EESI) and the timing of when and where these living lineages evolved is still debated is usually thought to have evolved near its modern range in the West Pacific well after the Gondwanan supercontinent split up starting about 50 million years ago Researchers from Penn State and the Museo Paleontológico Egidio Feruglio found the fossils at two sites in Patagonia – Río Pichileufú They analyzed the fossil characteristics and compared them to modern species to determine to which living group the fossils belonged Then they developed a phylogenetic tree to show the relationships between the fossil and living species They reported their findings in a recent issue of the American Journal of Botany Unlike the monkey puzzle trees of the living South American group of Araucaria needle-like leaves and cone remains that closely resemble the Australasian Norfolk Island pine group They also found a fossil of a pollen cone attached to the end of a branch “The new discovery of a fossil pollen cone still attached to a branch is rare and spectacular,” said Rossetto-Harris, who is also an EESI Environmental Scholar “It allows us to create a more complete picture of what the ancestors of these trees were like.” The researchers used 56 new fossils from Río Pichileufú to expand the taxonomic description of Araucaria pichileufensis a species first described in 1938 using only a handful of specimens scientists have lumped together the Araucaria fossils found at Río Pichileufú and Laguna del Hunco as the same species,” Rossetto-Harris said that although both species belong to the Norfolk pine group of Araucaria there is a difference in conifer species between the two sites.” The researchers named the new species from Laguna del Hunco Araucaria huncoensis The fossils are about 30 million years older than many estimates for when the Australasian lineage evolved The findings suggest that 52 million years ago before South America completely separated from Antarctica and during the first few million years after separation was underway relatives of Norfolk Island pines were part of a rainforest that stretched across Australasia and Antarctica and up into Patagonia The change in the Araucaria species from the older Laguna del Hunco site to the younger Río Pichileufú site may be a response to the climatic cooling and drying that occurred after South America first became isolated “We’re seeing the last bits of these forests before the Drake Passage between Patagonia and Antarctica began to really widen and deepen and set forth a lot of big climatic changes that would eventually cause this version of Araucaria to go extinct in South America but survive in the Australian rainforest and later spread and thrive in New Caledonia,” Rossetto-Harris said The study shows how tiny details can provide the definition needed to reveal big important stories about the history of life and Penn State provided funding for this project The reaction online is extraordinarily heartfelt and moving Some admitted to completing their crosswords through tears; others devoted their comments to trying to explain why Araucaria has meant so much to them over the years It is a "brain-joy" doing his puzzles "No other setter comes near to attaining his fusion of knowledge humour and consistency," said another it's an intimate exercise," said brighton2k wit and imagination being teased and excited by the setter I've never seen crosswords as a challenge: they're a unique way of engaging with another person's mind I imagine the person being just the same." The Reverend John Galbraith Graham – aka Araucaria the Latin name for the monkey puzzle tree – has for the past 18 years lived in a tiny cottage in the village of Somersham frost-rimed fields stretch for miles around calm and quiet except for two clocks ticking a hurried who set his first puzzle for the Guardian 55 years ago Partly this is because his default expression is a smile that lights up his face because they have spent much of the last half-century puzzling out your character I suppose I have a vague picture in my mind of a sort of idealised solver who is a combination of everybody I've loved When I meet or correspond with any of them because they don't always react the way I think they're going to." The cryptic crossword turns 100 this year, and like every established religion or party, has its own schisms: in the stand-off between Ximenian (named after a setter called Ximenes, and requiring an absolute adherence to set rules) and Libertarian crosswords which depends more on the solver's trust in the setter; trust that the answers will be in the reasonable purview of the average educated reader with a decent amount of life experience Crossword buffs such as the historian and former Guardian columnist David McKie point to the layers of interest that make up the peculiar joy of an Araucaria puzzle "The first thing is if you look at the clues they're entertaining to read just on their own without starting to solve them." He's also having invented his own forms of the crossword And then there's his range: a recent crossword contained references to A Winter's Tale ("exit while he reads the papers with half a mind to keeping up to date he did once devote a crossword to the Spice Girls says he sees crossword clues in everything in a way – you can be reading a beautiful poem and then something occurs to you in the middle that this could be a big part of a crossword clue And of course the enjoyment of the poem is spoilt completely." He actively enjoys setting crosswords I love the way the word invention both means discovering something and producing something new Clues are not something you've invented in the sense that they're completely new – they're something you discover The art of the crossword is getting all this stuff into a form that makes sense to people and brings the connection to them." But he resists the quote attributed to the famous setter Barbara Hall who also devised codes for the navy during the second world war that crosswords can be a refuge from the world you know." If he wanted a pleasure to disappear into it turns out; especially the Shakespeare of A Midsummer Night's Dream – an answer which of joy in words and in the worlds each word can hold Graham is the oldest of six children; his father when she was not managing her large household but she also wrote a companion to children's worship about four brilliant uncles born in the 1880s once said in an interview that: "They were a vicarage family and vicarages were the intellectual powerhouses of 19th-century England." Although his family did not hit the same heights The six children had to find their own entertainment and they often found it in wordplay: charades and puzzles One of the online comments on his announcement of cancer is from a nephew: "The young John used to grab the Times as soon as it was delivered to his father's home put it on the upright piano in the drawing room and stand to excitedly solve the whole thing before breakfast – he was around eight or nine at the time." Anyone who did that was mentioned in dispatches." He found refuge with an Italian family full of rats" and then in a hide in a field He took Italian lessons from a schoolteacher billeted with the family; he taught her English and Latin and couldn't understand the maths lectures "I was saying me prayers," he told Young trying to distance himself from the sincerity by slipping into uncharacteristic demotic It's the only time something [like that] happened to me but it did seem as though I was being spoken to." it was to work in a parish in south London this was a Church of England parish that demanded celibacy from its priests We got married at the end of those three years and I went as a curate to a parish." They were married for 26 years though many of the latter years were increasingly unhappy and eventually he left to be with the woman who would eventually become his second wife The church intervened again: divorced priests could not then serve in the ministry and so he had to leave his job and his home really – it's always been the church's teaching that marriage is indissoluble sex has always been a problem for the church It still is." He must have resented it I had to choose between keeping to the rulebook but there was never much doubt about what I was going to do." felt it would not look good if a priest was seen to be moonlighting; suddenly it became his only source of income "I was living with Margaret for seven years When Nesta died [in the mid-1980s] I was no longer living in sin So they recognised the second marriage" and finally allowed him to return to the ministry You remember the earlier part of your life better than the middle part He had had his suspicions about what was wrong with him and his official diagnosis of oesophageal cancer He seems to have met it with a striking measure of calm Although he was exhausted for a couple of months I haven't been able to do anything." But a few days ago he was given iron tablets I hope I'm going to feel very fit and happy Until – until whatever happens next." This article was amended on 21 January 2013 Barbara Hall worked predominantly for the Sunday Times She did not work at Bletchley Park during the second world war EDITOR’S NOTE: The opinions expressed in this piece are solely those of the author and do not reflect the views of Latino USA By Oliver Wilson, University of Reading For hundreds of thousands of years, the distinctive candelabra shapes of Araucaria trees (Araucaria angustifolia) have defined landscapes at the southern edge of Brazil’s Atlantic Forest. Humans have never known a world without these majestic evergreens. But my new research conducted with colleagues in Brazil and Reading suggests that their extinction could be just a generation or two away At a glance you might mistake Brazil’s Araucaria for its sister species, the monkey puzzle tree found in Chile and Argentina. But the two have inhabited South America as separate species for aeons, after diverging some 28m years ago If you compressed those 28m years into 24 hours North and South America wouldn’t become one land mass until 9.30pm Araucaria trees have been revered for as long as humans have lived in southern Brazil’s highlands. Their starchy, nutrient-rich nuts (known as pinhão) underpinned the diets of indigenous groups before European arrival The trees also hold great cultural importance. For example, the Kaingang people’s Kikikoi funerary ritual requires Araucaria knots to keep bonfires burning and a trough made of an Araucaria trunk to hold Kiki The Xokleng people even used to define the boundaries of a year by the coming and going of pinhão Sadly, the short-sighted culture of consumption that drove Araucaria’s dramatic decline hasn’t gone away: human-caused climate breakdown is now threatening to tip the species into extinction. Araucaria trees are adapted to relatively cool, constantly moist conditions—conditions that are disappearing as the planet heats and normal rainfall patterns become disrupted Using data on current and predicted temperatures and rainfall, as well as high-resolution maps that include small-scale terrain features, we modeled the likely fate of Araucaria in the coming decades. We found that projected climatic changes are likely to significantly loosen Araucaria’s grip on its current strongholds in southern Brazil Our most optimistic scenario predicts an 85 percent loss of the tree’s most suitable habitat by 2070 and several scenarios predicted that this habitat would vanish altogether we were able to identify some potential “microrefugia” for the Araucaria—areas where the trees have at least a three-in-four chance of enduring long into the future These are mainly found in colder spots in the landscape – places like sheltered slopes or river valleys where cool even as the wider region becomes more inhospitable While none of our findings are good news for Brazil’s Araucaria There are steps that we can take to ensure its continued survival in Brazil’s southern highlands And for truly long-term conservation planning which looks beyond the next decades, or even the coming centuries, we could learn a lesson or two from the past. Evidence suggests that indigenous people helped Araucaria forests expand beyond their natural boundaries around 1,000 years ago although how and to what extent isn’t yet clear Investigating how millennia of climate change and centuries of human actions combined to shape the present-day Araucaria forests may reveal ways of helping them survive the grave challenges of the coming decades We are living through the most turbulent point in the long history of Brazil’s Araucaria Our actions in the next split-second of its 24-hour life will determine whether or not future generations have the chance to treasure this ancient icon This article is republished from The Conversation under a Creative Commons license. Read the original article Concern Over Amazon Rainforest Fires Reaches Globe Misreading the Story of Climate Change and the Maya How Climate Change Is Driving Emigration From Central America and website in this browser for the next time I comment Δdocument.getElementById( "ak_js_1" ).setAttribute( "value" This site uses Akismet to reduce spam. Learn how your comment data is processed. Get Your Copy! Order Your Almanac Today! If you are short on space (or energy) for a large Christmas tree why not try a Norfolk Island Pine (Araucaria heterophylla) These potted pines used at Christmas can remain as wonderful houseplants throughout the year Norfolk pines are easy to find at your local garden center or big box store in pots making them a perfect Christmas tree for hanging ornaments.  Even small ones look great decorated with tiny LED lights and lightweight ornaments or they’re the perfect size for a tabletop We have a Norfolk pine that we now decorate every Christmas with all the “tasteful” holiday jewelry I have amassed over the years If I need a pair of Santa earrings as I head out the door Caring for your Norfolk Island pine couldn’t be easier Buy one for the holidays and enjoy it all year long Your plant can spend the summer outside in a shady spot where it will thrive in the humidity and rainfall.  Norfolk Island pine is native to tiny Norfolk Island located in the South Pacific east of Australia It is a valuable export and treasured as a symbol of the island with the tree on its flag it grows to be a 200-foot tall tree with a trunk 10 feet in diameter But fear not! As a houseplant, the Norfolk Pine is slow growing—only 2 to 3 inches a year The plant rarely gets over 5 feet tall when grown in a pot.  gardeners in Zones 9 to 11 are discouraged from planting them outside because the trees will eventually outgrow their welcome and will need to be removed. A subtropical evergreen it is not really a pine but a prehistoric conifer that once fed the dinosaurs The plants we see for sale are grown from seed collected from cones high in mature trees The seeds germinate fast—in 9 to 13 days—and then are grown on for several years before the plants make it to the store.  you can root a cutting from the top of a plant but be aware that cutting the top will make that plant bush out and lose its pyramidal shape.  A cutting taken from the tip of a side branch will always grow sideways instead of vertically Learn more about how Christmas trees grow My tree is dyeing on one side Howland I save it how much water do I give it When we lived in Hawaii we always had a Norfolk pine for Christmas freshly cut at the farm of a guy my husband worked with It stayed green as lone as we had it and no needles dropped It is a memory I'll never forget and I have a pot with four small trees that I'll repot after Christmas Do you know if the Norfolk pine is poisonous or harmful to cats I'd love to get one but concerned since my kitty likes to chew on things In reply to Hi! Do you know if the… by Diane Anderson (not verified) In reply to Apparently not. I have had… by Cheri (not verified) I beg to differ as to the toxicity of the Norfolk Island Pine's toxicity to pets The first site I just looked at said it was fine for cats but when I specifically typed in "toxic to pets" rather than just cats I came up with numerous posts that said it was bad for both dogs and cats Please investigate thoroughly if you care about your fur babies ©2025 Yankee Publishing Inc., An Employee-Owned Company1121 Main Street | P.O Journal of King Saud University - ScienceCitation Excerpt :Administration of E.O showed a significant (p < 0.05) increase in amplitude and frequency of contraction at 50 and 150 mcg/ml (Table 2) the spontaneous contraction in rabbit jejunum is mediated by intracellular calcium ions (Khan et al. The responses showed by higher doses of A.L and E.O suggests that the herbal extracts might have reduced the contraction of calcium ions intracellularly in intestinal smooth muscles to reduce the amplitude and frequency of spontaneous contractions Food ChemistryCitation Excerpt :The high caffeic acid content observed in this study might have been converted from p-coumaric acid after dynamic digestion homogentisic acid was reported to possess bound form (Schmeda-Hirschmann et al. After the mixing and grinding during dynamic digestion the homogentisic acid in bound form was suggested to be converted into free form Food ChemistryCitation Excerpt :It was tentatively characterized as caffeoyl-(epi)catechin hexoside which showed the characteristic neutral loss of the hexosyl moiety were also found (Schmeda-Hirschmann et al. Food Research InternationalCitation Excerpt :C-2 (7.56 ± 0.2 μg/mL) and MeOH extracts (8.5 ± 0.2 μg/mL) showed moderate inhibitory activity Previous studies reported that polyphenolic compounds had good inhibitory activity against α-glucosidase (Apostolidis et al. and similar research was reported by Schmeda-Hirschmann et al LC–MS analysis revealed that the extracts were rich in flavonoid and phenolic acid compounds which is a potent α-glucosidase inhibitor (Yuca Please upgrade your browser to improve your experience We're always trying to bring you more amazing hotels But not every hotel can immediately connect their reservation system with ours we provide real-time rates and availability via a partnership with Booking.com You'll still enjoy the full fabulous MICHELIN Guide experience when you book including our legendary service and delightful style This hotel offers rates & availability on request only Submit a request below and a travel specialist will assist We’re not currently taking reservations for this hotel Please explore other hotels in our selection Non-members can add the privileges at checkout through our 30 day free trial By continuing I accept the Terms & Condition and Privacy Policy. I would like to receive Newsletter from MICHELIN Guide Save lists of your favorite restaurants & hotels You can find them in many places around the world - tall lean conifers that can't seem to grow straight And now scientists have figured out that the direction these Cook pines (Araucaria columnaris) lean is always towards the equator Scientists have measured these trees across five continents and documented a species with a leaning pattern that appears to be hemisphere-dependent Cook pines originally come from New Caledonia, a tropical archipelago in the southwest Pacific Ocean. The trees were first classified during Captain James Cook's second mission to circumnavigate the globe These stately pines are a popular choice for parks and gardens in many parts of the world They can grow up to 60 metres tall (197 feet) they have a characteristic narrow appearance But even more characteristic is a propensity for a drunken-looking slant "When grown outside of its native range, this species has a pronounced lean so ubiquitous that it is often used as the identifying characteristic for the species," the researchers write in their paper Leaning pines on the campus of the University of California It started out as an anecdotal observation - one of the researchers botanist Matt Ritter from California Polytechnic State University But A. columnaris are also commonly grown in Australia, where one of them has even become an infamous leaning Christmas tree in the town of Lismore colleagues told him that the tilt in the southern hemisphere is directed towards the north Ritter and his team gathered measurements from 256 trees across 18 regions on five continents including the species' native range in New Caledonia The researchers excluded any trees whose growth could be impacted by another object as well as the compass direction and extent of the lean Cook pines turned out to be more systematic in their leanings than anyone could have expected "We uncovered a surprisingly consistent pattern of hemisphere-dependent directional leaning in A. columnaris," the team reports and the opposite direction in the southern hemisphere Less than 9 percent of the trees measured didn't conform to this pattern too - the further away the trees grew from the equator it could have something to do with sunlight Many plants, including conifers like these pines, are known for their propensity to lean towards a light source when it's not directly above the shoot - a characteristic known as phototropism But there's a different plant characteristic that helps trees stay upright - their ability to detect gravity at a molecular level and therefore direct roots and shoots in the correct directions (towards and away from the ground Even if a baby tree develops a tilt towards the sun as the plant matures it tends to correct this asymmetry and grow upwards unless there's an environmental force preventing this such as really strong prevailing winds in one direction And they even appear to be unique in this regard because other Araucaria species from New Caledonia can stand up straight no matter where in the world you plant them It's possible that Cook pines have a genetic quirk that allows them to lean seeking out more sunlight in latitudes other than their native range But scientists think that gravity and even Earth's magnetic field could be playing a role "The mechanisms underlying directional lean of A. columnaris may be related to an adaptive tropic response to the incidence angles of annual sunlight, gravity, magnetism, or any combination of these," they write the researchers are hoping to investigate further They think that further studies of the species could even lead to discovery of little-known mechanisms that plants use to respond to environmental cues The study was published in Ecology Guardian solvers would open the Saturday paper to find no numbers in the grid and know they were in for a different kind of challenge: one of Araucaria's "alphabetical jigsaws": a list of clues for which the answers begin with A which must be fitted into the grid "jigsaw-wise Here then are 26 of my favourite John Graham clues: 26 answers from A to Z They're mainly from the Guardian but pick up on Graham's other outlets One category of clue that's missing is those that have probably brought the greatest pleasure of all: those in the personalised puzzles commissioned by fans for the birthdays of friends and relatives We start, appropriately, with one from 1 Across the the monthly crossword magazine co-founded by Araucaria in 1984 And here's one from the Financial Times which reflects the political undercurrent which meshes with the erudition and wordplay in Graham's work: 14d/24d Consequence of gripe led into Nye's basis for the welfare state (9,4) ...as clued in a special puzzle for Newsnight "2" refers to the answer at two down 29ac Film lover's FT version of the 2′s 10 (9) the tree otherwise known as … Araucaria araucana Now, here's a clue from a puzzle by Paul 24ac A lesser figure in Jude the Obscure – have you read this somewhere before Except it was also the exact same clue as had appeared the day before, also at 24 across, in an Araucaria puzzle For many readers, Araucaria was their Steve Bell: the reason they bought the Guardian; a sense of humour which helped form the identity of the paper. And his clues which made reference to the Guardian were always fondly amusing: Time for a colloquial answer Accommodation urgently needed with an easy lot of pieces (4,2,1,7) And a miniature masterclass in concision: Sandy Balfour's book Pretty Girl in Crimson Rose (8) is a love letter to the crossword, as well as a kind of autobiography. Araucaria is one of its heroes, and it ends with a Guardian puzzle by Araucaria that retells the book in cryptic form 19d It isn't so funny to be given the elbow (7) We're already at I and we haven't yet had one of those distinctive incredibly long answers. Let's put that right: 6d Funny thing afoot – shy vernal youths go contemplating girls: Thus (in catalectic trochees) poem by 19 unfurls (2,3,6,1,5,4,5,7,5,2,8,2,4) And let's also see one from the many occasions – April Fool's Day anniversaries – marked with themed clues and puzzles: 9ac/7d Dickensian whisky makes Christmas music (6,5) Of course, the imagery in an Araucaria puzzle is not always so wholesome: 12ac Prevent passing of legislation to reduce police numbers Or, indeed: It's impossible not to lose some time pleasurably pondering Bayreuth, when the feast in question here … 8d Traditional feast of Wagner's work gets us up into its sequel (9,6) Now, I don't believe we've yet had an excruciating pun: 23ac Bike burnt by Eliot makes line for the Irish (6) Today would be a good time to listen to Araucaria's Desert Island Discs although Kirsty Young appeared to find this superb early clue … When the solver imagined Araucaria's working environment, the imagery tended to be calm: the retired churchman assembling anagrams with Scrabble tiles and checking the Oxford Book of English Verse. It's instructive, then, to learn … 7ac For first option there's no end — keep going spent more time than can ever be justified waiting for the correct department in a call centre It's the Guardian – so, the reader wonders, is this a typo Even with a cryptic device as established as the spoonerism, Araucaria played fast and loose, swapping the middles of words or whatever seemed most likely to raise a smile. Here's one which works more conventionally while cunningly exploiting two different senses of "fast": 24ac Embarrassed Spooner broke fast and went fast (3-5) Sometimes the answer would yield easily enough from the definition … 16ac People like Lolita — it's a difficult thing to do (3,7) … but it would take a while for the wordplay to become apparent and raise a smile Brace yourself antiphlogistine will be needed (3,4,2,4,2,5,4,4,10) For T, of course, there is also that famous other long one The GNU is, like the EMU and the ORCA a word much beloved of crossword setters. Here Araucaria uses it as a frame for some odd zoological imagery: 14d Not red and not quite blue bone in antelope's back (10) Araucaria's themed puzzles are underrepresented here because the intricacies of the related words are designed to work in a puzzle as a whole. Here's a flavour, though, of how one about Alfred Hitchcock 1ac I turn green in front of 25's work (7) Having omitted that one, we must include this one: O hark the herald angels sing the Boy's descent which lifted up the world And we'll close with three from those alphabetical jigsaws. First: Penultimately: Y Farmers' circle in Middle Eastern country (6) And There will always be more - many more - in the Araucaria archive A: ASPICB: BEVERIDGE PLANC: CINEPHILED: DEJA VUE: ENAMOUREDF: FLAT AS A PANCAKEG: GRADIENTH: HUMERUSI: IN THE SPRING A YOUNG MAN'S FANCY LIGHTLY TURNS TO THOUGHTS OF LOVEJ: JINGLE BELLSK: KILL THE BILLL: LONG GRASSM: MOTHERING SUNDAYN: NORTONO: ORTHODONTICP: PRESS ONQ: QUEENR: RED-FACEDS: SEX KITTENST: THE ROAD TO HELL IS PAVED WITH GOOD INTENTIONSU: UNBLUSHINGV: VERTIGOW: WHILE SHEPHERDS WATCHED THEIR FLOCKS BY NIGHT ALL SEATED ON THE GROUNDX: XANADUY: YEOMENZ: ZEUGMA Metrics details Loss of genetic diversity reduces the ability of species to evolve and respond to environmental change Araucaria araucana is an emblematic conifer species from southern South America with important ethnic value for the Mapuche people (Pehuenche); the Chilean Government has catalogued its conservation status as vulnerable Climatic fluctuations were potentially a major impact in the genetic variation within many tree species the restricted geographic distribution of A araucana in Chile appears to be a consequence of the Last Glacial Maximum (LGM) strong human intervention has also affected the geographical distribution and population sizes of A Reduction of population size may cause loss of genetic diversity which could affect frequency of adaptive loci The aims of this study were to know the existence of potential loci under selection and populations with genetic demographic disequilibrium in the Chilean distribution of A we have investigated potential loci under selection and genetic demographic disequilibrium within seven Chilean populations of Araucaria araucana Correlation of 41 outlier loci with the environmental variables of precipitation and temperature reveals signatures of selection whereas 227 neutral loci provide estimates of demographic equilibrium and genetic population structure Three populations are recommended as priorities for conservation it is possible not only to identify potential loci under selection but also to correlate these loci with environmental parameters which vary greatly within the geographical distribution (Andean and Coastal) of Araucaria araucana Source Software licensed under the GPL inkscape v. 1 (https://inkscape.org/) Right-lower box shows the Chilean geographical context of studied sites Below-barplot indicates the individual genetic composition of K = 3 genetic group including outlier loci with adaptative significance amplified fragment length polymorphism (AFLP) markers were employed for the following objectives: (1) to detect potential loci under selection (2) to correlate loci under selection with climatic parameters and (3) to assess if existing populations of A araucana are in genetic demographic disequilibrium The results from the present work show the existence of loci under selection that are correlated with climatic variables of temperature and precipitation some localities are revealed to be in genetic demographic disequilibrium These two aspects have high relevance for making decisions on the establishment of protected areas for this species temperatures range from −5 °C to −10 °C in the winter to 30 °C in the summer the range of temperature variation is lower than that in the Andes Mountains from −1 °C in the winter to 9 °C in the summer [43; pg and the distribution of neutral genetic variation within and among Coastal and Andean populations (Group 2) strengthen the need for conservation strategies within the species Araucaria Village is in a non-protected area and hence completely exposed to human intervention which could lead to serious negative factors for reproductive dynamics of this species resulting in loss of loci with adaptive value considering that outlier loci (probably under selection) were documented for this locality in the present research we suggest the following localities should be included as priority protected areas: Villa Las Araucarias and Cañicú-Ralco It would also be highly desirable to expand geographic sampling in future studies especially on the western slope areas that have been recolonized from glacial refugia in the Andes range and which have shown different frequencies of outlier loci in response to temperature and precipitation pulverized leaves obtained by grinding the tissue with liquid nitrogen were suspended in HEPES buffer and centrifuged at 10,000 rpm for 5 min Extracted DNA was treated with RNase at 37 °C for 30 min The quality of the extracted DNA was assessed by running an aliquot on a 1% agarose gel DNA concentration was quantified by UV spectrophotometry (UV 160 Shimadzu) and samples were stored at −20 °C until needed Each AFLP fragment was coded as either present = 1 or absent = 0 in all samples All AFLP experiments were done in the Department of Botany and Biodiversity Research Outlier loci with higher or lower FST values than under neutral expectations and which fell outside 95% confidence levels were assumed to be under directional or balancing selection Bayesian credible intervals (95%) for Fst values were calculated which is sensitive to signals of population expansion Shi, M. 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K for permission to collect plant material in National Parks Mecesup Program (Programa de Mejoramiento de la Calidad y Equidad de la Educación Superior) UCO 9906; Molecular Systematic Laboratory of University of Vienna Austria (supporting laboratory facilities) and CONAF (Corporación Nacional Forestal): Biobío and Araucanía Regions Facultad de Ciencias Naturales y Oceanográficas Patricio López-Sepúlveda & Eduardo Ruiz-Ponce Department of Botany and Biodiversity Research G.F.: Obtaining partial AFLPs markers in Concepción, participation in redacting manuscript and making AFLP analyses, made lab protocol, prepared Fig. 1 T.F.S.: reviewed manuscript and discussed data Download citation DOI: https://doi.org/10.1038/s41598-021-98662-w Bioorganic ChemistryCitation Excerpt :In the context of a circular economy working with biological waste (biowaste) is crucial because using such bioactive waste provides an efficient economical and environmentally friendly source for the production of useful compounds high antioxidant content has been described in several species of the genus Araucaria [13–18] only a few works deal with its residue extracts and biowaste which are usually released into the environment [19–22] scientists at City University of Hong Kong (CityU) discovered that the spreading direction of different liquids deposited on the same surface can be steered solving a challenge that has remained for over two centuries This breakthrough could ignite a new wave of using 3D surface structures for intelligent liquid manipulation with profound implications for various scientific and industrial applications such as fluidics design and heat transfer enhancement Chair Professor in the Department of Mechanical Engineering (MNE) of CityU the research team found that the unexpected liquid transport behaviour of the Araucaria leaf provides an exciting prototype for liquid directional steering Their findings were published in the prestigious scientific journal Science under the title "Three-dimensional capillary ratchet-induced liquid directional steering" Araucaria is a species of tree popular in garden design Its leaf consists of periodically arranged ratchets tilting towards the leaf tip with both transverse and longitudinal curvature on its upper surface and a relatively flat visited a theme park in Hong Kong with Araucaria trees the special surface structure of the leaf caught his attention Special leaf structure enables liquid to spread in different directions "The conventional understanding is that a liquid deposited on a surface tends to move in directions that reduce surface energy Its transport direction is determined mainly by the surface structure and has nothing to do with the liquid's properties such as surface tension," said Professor Wang But the research team found that liquids with different surface tensions exhibit opposite directions of spreading on the Araucaria leaf in stark contrast to conventional understanding the team designed an Araucaria leaf-inspired surface (ALIS) with 3D ratchets of millimetre size that enable liquids to be wicked (i.e moved by capillary action) both in and out of the surface plane They replicated the leaf's physical properties with 3D printing of polymers They found that the structures and size of the ratchets especially the re-entrant structure at the tip of the ratchets are crucial to liquid directional steering the research team discovered that one frontier of liquid is "pinned" at the tip of the 3D ratchet Since the ratchet's tip-to-tip spacing is comparable to the capillary length (millimetre) of the liquid the liquid can go backward against the ratchet-tilting direction the surface tension acts as a driving force and enables the liquid to move forward along the ratchet-tilting direction First observation of liquid "selecting" directional flow we demonstrated directional transport of different liquids on the same surface successfully addressing a problem in the field of surface and interface science that has existed since 1804," said Professor Wang "The rational design of the novel capillary ratches enables the liquid to 'decide' its spreading direction based on the interplay between its surface tension and surface structure It was like a miracle observing the different directional flows of various liquids This was the first recorded observation in the scientific world." their experiments showed that a mixture of water and ethanol can flow in different directions on the ALIS A mixture with less than 10% ethanol propagated backwards against the ratchet-tilting direction while a mixture with more than 40% ethanol propagated towards the ratchet-tilting direction Mixtures of 10% to 40% ethanol moved bidirectionally at the same time "By adjusting the proportion of water and ethanol in the mixture we can change the mixture's surface tension allowing us to manipulate the liquid flow direction," said Dr Zhu Pingan Controlling spreading direction by adjusting surface tension The team also found out that the 3D capillary ratchets can either promote or inhibit liquid transport depending on the tilting direction of the ratchets When the ALIS with ratchets tilting upwards was inserted into a dish with ethanol the capillary rise of ethanol was higher and faster than that of a surface with symmetric ratchets (ratchets perpendicular to the surface) When inserting the ALIS with ratchets tilting downwards Their findings provide an effective strategy for the intelligent guidance of liquid transport to the target destination opening a new avenue for structure-induced liquid transport and emerging applications heat transfer enhancement and smart liquid sorting "Our novel liquid directional steering has many advantages long-distance transport with self-propulsion And the ALIS can be easily fabricated without complicated micro/nanostructures," concluded Professor Wang Professor Wang is the corresponding author of the paper Associate Professor at Dalian University of Technology (DUT) who was also a former postdoc under Professor Wang’s supervision Associate Professor in CityU's Department of Biomedical Sciences Dr Zheng Huanxi and Dr Li Jiaqian (now a postdoc in HKU) and Professor Wang Liqiu from The University of Hong Kong The research received funding support from the Research Grants Council and the Innovation and Technology Fund in Hong Kong the Shenzhen Science and Technology Innovation Council the National Natural Science Foundation of China Fundamental Research Funds for the Central Universities and the Star Ocean Outstanding Talents Program https://www.cityu.edu.hk/research/stories/2021/09/17/worlds-first-discovery-liquid-directional-steering-bio-inspired-surface 10.1126/science.abg7552 Three-dimensional capillary ratchet-induced liquid directional steering are not responsible for the accuracy of news releases posted to EurekAlert by contributing institutions or for the use of any information through the EurekAlert system Copyright © 2025 by the American Association for the Advancement of Science (AAAS) Metrics details Skin infections are among the bacterial infections that present significant therapeutic challenges due to antibiotic resistance herbal products clutched a significant attention as safe replacements for other medications but their low aqueous solubility and poor bioavailability are considered major challenges which could be circumvented via formulation Araucaria Heterophylla possesses pharmacological activities such as antioxidant and antibacterial actions and this study aimed to standardize the extract of the plant against 4ʹʹʹmethoxyamentoflavone (as a main component of the extract) through a validated UPLC-MS/MS method and evaluate its antibacterial activity which was followed by loading the standardized extract into a nanoemulsion to form a phytonanoemulsion (PNE) where the design analysis and optimization were performed through a simplex lattice design The optimized PNE (PNE 3) was then loaded into HPMC/Pluronic F-127 gel (in ratio 1:4) to sustain the release of the active constituent The heightened penetrability of PNE 3 gel was visualized via confocal laser scanning microscopy and its prolonged effect was proved thru an in vivo study conducted on male Wistar rats A histopathological study revealed the safety of the formulation when applied topically PNE gel could be a potentially broad-spectrum antibacterial drug delivery system NEs are intended to be incorporated into gel bases to increase their retention at the site of application the objective of this work was to conduct a comprehensive study including the extraction and the standardization of the methanolic extract of A Heterophylla via a validated UPLC-MS/MS method an approach to develop a suitable topical phytonanoemulsion (PNE) gel system to enhance the biological activity of the standardized extract of A Heterophylla (SAH) was attempted through a simplex lattice design using Design expert® software to optimize the PNE systems To test the enhanced penetration potentiality of the developed formulation the penetration of the optimized PNE gel was investigated via a confocal laser scanning microscopy study the deposition of SAH into the rat’s skin; following the topical application of SAH a histopathological study was conducted on rats to validate the biosafety of the optimized PNE gel Samples of non-flowering aerial parts of cultivated A Heterophylla were collected from Prince Mohamed Ali's palace garden (Cairo where the collection procedure was in compliance with the national and international guidelines and legislation The identification of the plant material was confirmed by Dr Therese Labib (senior head of specialists in plant identification) heterophylla) was deposited in the herbarium of the Pharmacognosy Department Germany) for Column chromatography and Sephadex LH-20 (GE Healthcare Bio-Sciences AB Uppsala Analytical precoated TLC silica gel 60 GF 254 and Preparative TLC silica gel G (E-Merck and deionized water were used all over the work 1H and 13C NMR were recorded using a Varian Mercury instrument (1H- and Tetramethylsilane (TMS) was included as an internal standard UPLC-MS/MS method was adopted for the separation using a Shimadzu LC-2040C 3D PLUS Nexera-i series LC system coupled with a triple quadrupole 8040 mass spectrometer The separation was achieved on Shim-pack GIST-HP C18 (150 mmL.x3.0 mmI.D. and data acquisition was performed using LabSolutions software (Shimadzu Two pathogenic bacterial strains; a Gram-positive Staphylococcus aureus (ATCC 6358) and a Gram-negative Escherichia coli (ATCC 25923) in addition to the fungus Candida albicans (ATCC 10231) were used as test organisms Bacterial cultures were sub-cultured on Mueller Hinton agar (MERCK; Germany) and the fungus was sub-cultured on potato dextrose agar (MERCK; Germany) Maisine and Capryol 90 were acquired from Gattefossé (St-Priest Cremophor EL and Pluronic F127 were procured from BASF Co Transcutol and hydroxypropyl methylcellulose (HPMC,15000 cp) were bought from Sigma Aldrich Chemical Co Phosphate salts (potassium and sodium) were purchased from Merck (Darmstadt All methods were carried out in accordance with relevant guidelines and regulations Heterophylla (1 kg) were finely powdered and extracted with methanol and then left at 25 ± 2 \(^\circ\)C with frequent agitation The procedure was performed two times and the combined extract was evaporated under vacuum at 40 °C to form a green residue (135 g) A part of the residue (80 g) was analyzed via silica gel column chromatography The elution was conducted using dichloromethane (CH2Cl2) and then with dichloromethane-methanol (CH2Cl2-MeOH) in the direction of increasing polarity up to 20% MeOH in CH2Cl2 The process resulted in the separation of 50 portions (100 mL each) The collected portions were scrutinized by TLC using the solvent system CH2Cl2-MeOH (7:1) then followed by spraying with either ferric chloride (FeCl3) or 20% sulphuric acid in ethanol All portions eluted with (92:8) CH2Cl2-MeOH were alike and upon TLC analysis they were found to comprise a compound (A) The combined portions were repetitively chromatographed on silica gel TLC plates and eluted with CH2Cl2-MeOH (7:1) Repeated purification on Sephadex LH-20 column eluted with MeOH The physicochemical properties of 4ʹʹʹmethoxyamentoflavone were determined using the Swiss ADME tool A primary stock solution of standard 4ʹʹʹMethoxyamentoflavone of concentration 5.0 mg/mL was prepared by dissolving 50 mg of the standard powder in an ethanol/deionized water mixture in a 10 mL volumetric flask which was then filtered via a millipore filter (0.2 µm) The primary stock solution was diluted with deionized water to prepare standard working solutions (3 mg/mL) Ten milligrams of the dried extract were reconstituted in 10 mL ethanol (70% v/v) and sonicated till completely dissolved It was made up to the final volume (100 mL) with ethanol (70% v/v) then filtered via a millipore filter (0.2 µm) where this solution was employed for the standardization of the extract The extract and the standard compound were analyzed by UPLC connected to triple quadruple 8040 MS The used UPLC column was Shim-pack GIST-HP C18 (150 mmL Gradient elution was performed where the employed mobile phases were: phase A: 0.1% formic acid in acetonitrile; phase B: 0.1% formic acid The mobile phase gradient was set as follows: 90% B from 0 to 1 min at a flow rate of 0.5 mL/min; then linearly decreased to 60% B from 1 to 2.5 min; this was followed by decreasing to 30% B while the flow rate was increased to 1 mL/min from 2.5 to 3.5 min maintained at 30% B and flow rate of 1 mL/min from 3.5 to 4.5 min then increased to 90% B at a flow rate of 0.5 mL/min from 4.5 to 5.5 min which was kept maintained from 5.5 to 7 min The analysis was operated at the selected ion monitoring (SIM) mode The detection was accomplished on the triple quadrupole mass spectrometer equipped with an electrospray ionization mass (ESI) interface at – 3.5 kV in a negative ionization mode Desolvation line (DL) temperature and heat block temperatures of 250 \(^\circ\)C and 400 \(^\circ\)C were applied The flow rates of nebulizing gas and drying gas were set at 3 L/min and 15 L/min The analytical run time was 7 min and the full scan covered the mass range from 100 to 1000 m/z the precision of the method was checked by applying the proposed method to determine three concentrations (15 and 200 µg/mL) of the analyte thrice intra-daily and then calculating the relative standard deviations (RSD) the process was repeated inter-daily (intermediate precision) on three different days for the three selected concentrations thrice Determination of the limit of quantitation (LOQ) and limit of detection (LOD) can be accomplished through several approaches as per ICH recommendations the determination of the signal-to-noise ratio is achieved by linking measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected A signal-to-noise ratio between 3:1 is mostly considered acceptable for estimating the detection limit whereas a ratio of 10:1 is used to estimate the quantitation limit A concentration of 20 mg/mL of SAH was prepared and 200 μL was added to the first well then a 100 μL of SAH was subjected to two-fold serial dilution in each well to obtain concentrations of 10 The preparation of the inoculum was performed by fine-tuning the turbidity from an overnight microbial culture to a value equivalent to McFarland 0.5 which was then diluted to a final concentration of 106 CFU/mL 100 μL of the inoculum was mixed with comparable volumes of the two-fold serially diluted SAH in a microplate and DMSO in amounts corresponding to the highest quantity presented were set as controls The incubation was performed for 24 h at 37 °C and the MIC was established as the lowest concentration showed no visible growth The mean value of three replicates was calculated The agar well diffusion method was employed to examine the antifungal activity of SAH35 Albicans ATCC 10,231 was accustomed spectrophotometrically at 530 nm so that a final concentration matching the 0.5 McFarland standard was attained The produced inhibition zones were determined after incubation of the plates at 28 °C for 24 h Standard antibiotic amphotericin and diluted DMSO were used as positive and negative controls To determine the minimum fungicidal concentration which is defined as the lowest concentration showing no growth on potato dextrose agar after 48 h of incubation serial two-fold dilutions of SAH were prepared with potato dextrose agar to obtain final concentrations ranging from 30 to 1.6 mg/mL Aliquots were allocated in 96-well microplates Albicans was prepared to a final concentration of 2 × 103 CFU/mL in potato dextrose agar medium and then 100 μL of the suspension was added to each well a 100 μL was transferred on potato dextrose agar plates and incubated for 48 h The corresponding pseudo-ternary phase diagram was plotted via Tri-plot software (Ver and the transparent NEs region was contoured The optimization of the systems was achieved through a simplex lattice design employing Design expert® software (Version 7 and the Smix (C)) were defined as the independent variables Based on the region which corresponds to the clear systems in the pseudo-ternary phase diagram the higher and lesser percentages of formulating constituents were set as follows: and Percentage Transmittance (%T) measurements The average DS and PDI of the prepared PNEs were assessed via the dynamic light scattering technique (Zetasizer Nano ZS %T of the freshly prepared PNEs was measured at 550 nm via a UV–visible spectrophotometer (Shimadzu All measurements were carried out in triplicates and recorded as average ± standard deviation The test was executed via the dialysis membrane method in an incubator shaker (Unimax Samples (0.5 g) of SAH and PNEs (containing SAH equivalent to 100 mg) were packed in cellulose dialysis membranes which were sealed tightly from both sides and then immersed in bottles filled with 50 mL phosphate buffer (pH 6) containing 10% ethanol to maintain the sink condition The incubator shaker was operated at 60 strokes per minute and the temperature was maintained at 32 ± 2 \(^\circ\)C Aliquots (2 mL) were withdrawn from each bottle at specified time intervals and replenished immediately with a fresh medium The content of SAH in the samples was analyzed via UPLC-MS/MS method as stated previously under “UPLC-MS/MS analysis” section the viscosity of NE systems is practically low and to increase the formulation retention at the affected parts the viscosity of the systems has to be elevated through its incorporation in a gel base the plain gel base was prepared by mixing hydroxypropyl methylcellulose (HPMC) and pluronic F127 at a ratio of 1:4 (w/w) on a magnetic stirrer for 30 min at room temperature then the optimized PNE (1 g) was added to the gel base (1 g) and mixed on a magnetic stirrer for 10 min at room temperature where 1 g of the preparation was loaded in between two glass slides and a 200 g weight was situated on the upper glass slide for 1 min to uniform the thickness the area up to which the formulation was able to spread (spreadability) was measured and the spreadability was calculated via the equation: A = area of formulation spread on the slide and T = time (60 s) Aliquots of the supernatants were withdrawn and assayed via the UPLC-MS/MS method mentioned earlier under “UPLC-MS/MS analysis” section The area under the curve (AUC0-24) in the plot of the amount of SAH deposited (µg/cm2) versus time (h) was calculated via the PK solver integrated into Microsoft Excel which gives an idea about the degree of SAH dermal deposition upon the administration of the tested preparations The differences in the values of AUC0-24 were statistically checked by one-way ANOVA tailed by Tukey’s HSD (Honest Significant Difference) test where the difference was established to be significant at a P value less than 0.05 Rotatory microtome was used to slice sections of tissue (4 µm) which were then loaded on glass slides and finally tainted by Hematoxylin and Eosin The tissue samples were then visualized with a light microscope and photographed simultaneously Chemistry: 1H and 13C NMR data of compounds A is shown in Table 2 The elucidation and identification of the compound structure were performed via different spectroscopic analyses and the results were compared to the published literature The compound was identified as 4'" methoxy amentoflavone (I) Compound A 1H NMR and 13 C NMR data Selected Ion Chromatogram and retention time of 4ʹʹʹmethoxyamentoflavone standard (middle) and Selected Ion Chromatogram and retention time of SAH (bottom) and (b) Blank samples appeared free from any interfering substances at the retention time and m/z of the studied compound both poor aqueous solubility and large molecular weight of the compound result in low absorption due to the difficulty to cross lipid membrane it was found that 4ʹʹʹmethoxyamentoflavone was the major component where one gram of SAH contains 20 mg 4ʹʹʹmethoxyamentoflavone SAH antibacterial and antifungal activity were evaluated (I) Growth inhibition zones of Staphylococcus aureus ATCC (6358) and E compared to the positive control (Doxycycline) and the negative control (DMSO) (II) Pseudo-ternary phase diagram of oils (Maisine and Capryol 90 Surfactant/CoSurfactant mixtures (Smix) (Cremophore EL/Transcutol The defined percentages of the oils (5–25%) and the Smix (50–70%) are represented by the black triangle and (III) 3D surface plots demonstrating the effect of the independent variables; the oil (X1) and the Smix (X3) on (a) Droplet size in nm (DS) and (d) The cumulative percent of SAH released in 8 h (Q8) The choice of oils and Smix was based on the solubility of the herbal extract in different oils, surfactants, and cosurfactants (data not shown). A pseudo-ternary phase diagram was plotted after the preparation of different systems by changing the concentration of the oils, Smix, and water, Fig. 2II The contoured region in the pseudo-ternary phase diagram indicates the nanoemulsion region which helps to choose the optimum concentration of oil Systems containing more than 30% of the oil phase did not lie within the nano-emulsification region and Smix concentration lower than 50% resulted in turbid systems where higher amounts of Smix were required to emulsify the incorporated oils and the Smix (50–70%) were selected (represented by the black triangle) which were further included as independent variables in a simplex lattice design A simplex lattice experimental design produced 14 runs (including 4 replicates) generated by Design-Expert® software, the composition of PNEs (loaded with 20% SAH) and the corresponding responses are represented in Table 1 According to the analysis of the obtained data by the software and as per the highest R2 and the lowest predicted residual error sum of squares (PRESS) the droplet size (DS)(Y1) and polydispersity index (PDI)(Y2) followed a quadratic model while percentage transmittance (%T) (Y3) and the cumulative percent of drug released after 8 h (Q8) (Y4) followed a linear model Analysis of Variance (ANOVA) was carried out by the software to generate the polynomial equation of the responses The final equations (in terms of actual components) for the measured responses were as follows; where the sign and the value of the variable coefficient reflect the effect of the studied variables The positive sign of the variable coefficient designates synergistic effects and the negative sign designates an antagonistic effect of the factors it should be noted that the formulation of NE requires an optimum combination of all components and varying the proportion of any of them would alter the overall equilibrium of the system and thus its measured characteristics (I) (A) Release profiles of SAH and PNEs simulating skin surface pH) at 32 ± 0.5 \(^\circ\)C and (II) TEM micrograph of the optimized PNE 3 (Left) and its size distribution graph (Right) TEM micrograph of PNE 3 is displayed in Fig. 3II (left side), and it revealed a spherical droplet shape with narrow size distribution comparable to that obtained by the Zetasizer, Fig. 3II (right side) where no signs of aggregation or coalescence were observed The in vitro drug release profile of PNE 3 gel is illustrated in Fig. 3IB It could be observed that incorporation of PNE 3 into a gel base resulted in sustaining the release of SAH for 24 h which could be discussed in light of the increased viscosity that retarded the release of the drug Regarding the results of the stability study PNE 3 gel showed no signs of precipitation or change in color there was a nonsignificant difference (P > 0.05) in the values of pH and the viscosity between the stored samples at 40 °C ± 2 °C/75% RH ± 5% RH for 3 months and the freshly prepared ones (I) Confocal laser scanning micrographs (CLSMs) obtained after 8 h following the application of Rhodamine B dye aqueous solution (A) (II) The plot of the amount of SAH deposited per unit area in rat skin after the application of SAH and (III) Histopathological photomicrographs of skin tolerance study; showing histopathological sections (hematoxylin- and eosin-stained) of (a) Untreated rat’s skin (group A); showing a normal histological structure of the epidermis and the skin appendages and (b) Rat’s skin treated with SAH-loaded PNE 3 gel (group B) showing an apparent normal architecture of the epidermis and the dermis at 50 µm The capability of a delivery system to shuttle a drug through the skin layers can be evaluated via the in vivo deposition study. Figure 4II displays the plot of the amount of SAH deposited per unit area in rat skin after the application of SAH It can be observed that PNE 3 gel was capable of depositing elevated amounts of SAH in the dermal tissue related to PNE 3 and SAH The calculated AUC0-24 of PNE 3 gel (4398.98 ± 126.55 µg.h/cm2) was found to be pointedly higher (P < 0.05) than that of PNE 3 (2696.23 ± 201.74 µg.h/cm2) and that of SAH (1384.75 ± 54.53 µg.h/cm2) where PNE 3 gel was able to increase the AUC by 2.63 folds and 3.18 folds compared to PNE 3 and SAH it could be said that PNE 3 gel enhanced the skin deposition of SAH upon topical application via higher viscosity and retention potentialities of the gel base and improved penetration of the stratum corneum barrier because of the oil and Smix content of PNE The histological micrographs of the rat skin of the control group (Group A) and the group treated with PNE 3 gel (Group B) are displayed in Fig. 4III Group (A) showed normal skin histological structure of epidermis The skin of group (B) treated with PNE 3 gel did not display any significant changes in the microscopic structure of the skin where the surface epithelium lining and the dermis structure of the skin were intact The ultra-structure of skin morphology was not altered and the epithelial cells looked generally unaffected the histopathological results confirmed the safety of the topical application of PNE 3 gel on the skin Heterophylla was standardized against 4ʹʹʹmethoxyamentoflavone after being isolated and identified using 1H and 13C NMR A validated UPLC-MS/MS method was developed for both standardization and further quantitative analysis of SAH it was found that 4ʹʹʹmethoxyamentoflavone is a major constituent where one gram of SAH contained 20 mg of 4ʹʹʹmethoxyamentoflavone The antibacterial and antifungal activity results revealed that the MIC of SAH was 0.156 mg/mL in the case of standard S SAH was not effective against the standard strain of C.albicans A simplex lattice design was adopted for the optimization of PNEs The optimized formula (PNE 3) showed minimal DS and PDI with maximum %T and extended-release profile for 8 h PNE 3 was then loaded in a gel base (HPMC: Pluronic F-127 in ratio 1:4) which showed shear-thinning rheological behavior PNE 3 gel exhibited enhanced skin penetration compared to PNE 3 and SAH via the CLSM study The in vivo study confirmed the improved deposition of SAH after the application of PNE 3 gel compared to PNE 3 and SAH the safety profile of PNE 3 gel was proved by the normal skin structure observed after histopathological examination it could be concluded that PNE 3 gel (loaded with 20% SAH) is a promising topical broad-spectrum antibacterial formulation for the treatment of skin infections All data generated or analyzed during this study are included in this published article Exposure and health effects of bacteria in healthcare units: An overview Shortened courses of antibiotics for bacterial infections: a systematic review of randomized controlled trials Traditional herbal medicine candidates as complementary treatments for COVID-19: A review of their mechanisms Ethnopharmacology and integrative medicine—Let the history tell the future and biological activities of the Araucariaceae family—A review Patterns of pollen and seed dispersal in a small fragmented population of the wind-pollinated tree Araucaria angustifolia in southern Brazil characterization and utilization of polysaccharide of Araucaria heterophylla gum for the synthesis of curcumin loaded nanocarrier Inhibitory activity of methanolic extract of araucaria heterophylla leaves against gram negative bacteria anticancer and antimicrobial properties of Araucaria heterophylla grown in Egypt Could Araucaria heterophylla resin extract be used as a new treatment for toxoplasmosis? 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Volume 7 - 2019 | https://doi.org/10.3389/fevo.2019.00082 This article is part of the Research TopicAnimal-Mediated Dispersal in Understudied SystemsView all 15 articles While Psittaciformes (parrots and allies) are well-recognized as highly-mobile seed predators their role as seed dispersers has been overlooked until very recently It remains to be determined whether this role is anecdotic or is a key mutualism for some plant species We recently found that the large nut-like seeds of the two South American Araucaria tree species (Araucaria araucana in Andean forests and Araucaria angustifolia in Atlantic forests both observational and experimental work demonstrated that dispersed seeds can germinate faster after partial predation by parrots even larger-seeded (17.5 g) congeneric Australian species (A cunninghamii (a sympatric species with small winged seeds We found that sulfur-crested cockatoos (Cacatua galerita) consumed large amounts of seeds from all of the A 30% of the seeds they removed from the mother tree carrying the seeds to distant perches for handling or dropped them while flying Dispersal distances ranged between 10 and 153 m (mean = 61 m) Most seeds handled for consumption (93%) were fully eaten but others were dropped intact (3%) or only partially eaten (4%) and germination was confirmed for both intact and partially-eaten dispersed seeds seeds dropped by cockatoos facilitated secondary seed dispersal by conspecifics and another three bird species We found no evidence of other primary dispersal species for A winged seeds of Araucaria cunninghamii were only dispersed through barochory and anemochory The seed weight of the three Araucaria species dispersed by zoochory is strongly related to the body mass of their main seed-disperser parrot species These results support a role for parrots as key dispersers of the three large-seeded Araucaria species around the world and suggest that large seeds may have evolved–at least partially–as an adaptation that allows trees to attract parrots and benefit from their long-distance seed dispersal services Surveys replicated our previous work on seed dispersal of Araucaria species in South America see (Tella et al., 2016a,b) we drove a car at low speed through unpaved and secondary roads to increase the chances of finding highly mobile flocks of foraging parrots When we located parrots feeding on Araucaria trees we stopped and observed them with telescopes from a distance to avoid disturbance we recorded dispersal rates by counting the number of seeds consumed in the mother tree and the number of seeds transported in flight to distant perches we could only see parrots flying with seeds in their beaks without evaluating the actual dispersal rates Dispersal distances were measured with a laser rangefinder (Leica Geovid 10x42x range: 10–1,300 m) as the distance from the mother tree to the perching site (exact distance) or up to where the seed-carrying flying parrot went out of sight (minimum distance) we looked underneath them for additional dispersed seeds and measured the distance to the nearest seeding tree (i.e. we conservatively recorded a minimum dispersal distance) These perching sites were repeatedly used by parrots (cockatoos) so we can satisfactorily assume that seeds found there were moved by them but not by secondary disperser species We also recorded whether the dispersed seeds were fully consumed Intact and partially-eaten seeds were apparently viable (i.e. bidwillii were consumed by unidentified mammals so we restricted our sampling to 50–100 seeds per tree the midpoint was used when a range of weights was provided for a given species During field work in the Bunya Mountains, we only observed sulfur-crested cockatoos (Cacatua galerita) in a highly mobile flock of ca. 70 individuals, preying upon A. bidwillii seeds (Figure 1A). Cockatoos perched on the large female cones to extract the seeds (Figure 1B), and opened the pericarp in a characteristic way to gain access to the seed content and consume it (Figure 1C) This allowed us to clearly identify the seeds preyed upon by this species Individuals from another four parrots species (12 galahs Eolophus roseicapilla 10 Australian king parrots Alisterus scapularis and 3 red-winged parrots Aprosmictus erythropterus) were also observed We observed Torresian crows (Corvus orru) obtaining the remainders of seeds discarded by sulfur-crested cockatoos in the same tree canopy and Australian ravens Corvus coronoides and Australian brush-turkeys (Alectura lathami) taking seeds dropped by cockatoos under the trees The latter two species repeatedly pecked the seeds but seemed unable to open the pericarp for obtaining seed fragments to ingest them so they acted as secondary seed dispersers when moving the seeds to distant sites A sulfur-crested cockatoo handling a Bunya pine (A bidwillii) seed (A) after extracting it from the large and strong female cone (B) Note the size of the cone related to the size of the seeds Cockatoos open the pericarp to consume the endosperm in a characteristic way (C) Entire (D) and partially-eaten (E) seeds germinating after dispersal by cockatoos (Pictures: GB and JT) bidwillii seeds transported by sulfur-crested cockatoos (A) Distribution of the exact and minimum dispersal distances recorded (n = 241) (B) Kaplan-Meyer estimate of the dispersal function About half (52 out of 109) of the well-spaced A. bidwillii trees inspected produced seeds during the study season. All of these seeding trees were previously visited by cockatoos, as indicated by the characteristically predated seeds (Figure 1C) we found below their canopies We only found the remainders of a single case of barochory when the entire mature female cone falls to the ground and the contained seeds disaggregate due to strong impact almost all the seeds produced by the 52 trees were preyed upon dropped (intact or partially eaten) in situ or dispersed by cockatoos before naturally falling to the ground The proportion of seeds preyed upon (97.8%) or partially eaten by cockatoos (0.3%) found under the canopies (n = 1,283 seeds) differed from those regarding dispersed seeds (see above due to a lower proportion of undamaged and partially-eaten seeds This was probably due to the predation of the seeds dropped by cockatoos by terrestrial mammals attracted by the residual material they left we found clear evidence of seed predation by wild boars (Sus scrofa) rodents and unidentified mammals under 15.5% (5.8 bidwillii population in the Bunya Mountains we could only inspect 14 adult trees in the northern relict population and none of them produced seeds successfully cunninghamii seeds by any of the species of parrots recorded in the Bunya Mountains (see above) or in the Lamington-Canungra area where we also recorded a number of foraging granivorous parrots (140 sulfur-crested cockatoos 3 pale-headed rosellas Platycercus adscitus and 2 yellow-tailed black cockatoos Calyptorhynchus funereus) We observed sulfur-crested cockatoos feeding on fresh and dry branches and on gum of several A We inspected a sample of 2,550 seeds from 42 seeding trees (13 in Lamington–Canungra area and 29 in the Bunya Mountains) No seeds showed signs of predation by parrots and only 0.8% of them had been preyed upon by rodents These trees were isolated or separated >40 m from the closest seeding tree thus avoiding the possibility that seeds found under a particular tree came from another tree Most seeds were dispersed by barochory (i.e. they were found just below the mother canopy tree) while in a few cases the wind seemed responsible of dispersing seeds in a radius of up to 30 m (anemochory) Amazons exclusively act as primary seed dispersers with moderate dispersal rates and the largest dispersal distances recorded for Araucaria species galerita seems to be the only primary seed-disperser parrot of A showing moderate dispersal rates and dispersal distances compared to the rest of parrot species Comparison of seed dispersal traits by parrots of the three Araucaria species dispersed by zoochory Relationship between the seed weight of three Araucaria species (Aar bidwillii) and the body mass of parrot species that disperse them (red circles) Blue circles represent parrot species that have a small (for Aan) or null seed dispersal role (for Ab) The diagonal line represents the expected perfect correlation between seed size and parrot size cunninghamii (Ac) is depicted (green dot) as a reference since no parrots disperse its seeds (they are only dispersed by barochory and anemochory) feeding on seeds and suggested that they could also act as dispersal vectors are known to be shaped by seed predators/dispersers at short temporal and small spatial scales in conifers the comparison within areas of the genetic arrangement of seedlings and juveniles with that of centenary adults would further deepen the disruption of dispersal processes at long-term temporal scales This study relies on observational data obtained in areas unrestricted to people and thus did not require special permits JT wrote a first draft of the manuscript and all authors contributed to improve it Heinsohn for his initial advice on preparing the field work expedition Gleiser and two reviewers for their valuable comments to the manuscript Loro Parque Fundacion funded the publication of this paper The first millennium-age Araucaria Araucana Baños-Villalba Seed dispersal by macaws shapes the landscape of an Amazonian ecosystem Primary seed dispersal by three neotropical seed-predating primates (Cacajao melanocephalus ouakary Genetic variation in the vulnerable and endemic Monkey Puzzle tree Reciprocal selection causes a coevolutionary arms race between crossbills and lodgepole pine Internal seed dispersal by parrots: an overview of a neglected mutualism Parrots as key multilinkers in ecosystem structure and functioning CrossRef Full Text | Google Scholar Ecological functions of parrots: an integrative perspective from plant life cycle to ecosystem functioning CrossRef Full Text | Google Scholar Boehning-Gaese Importance of primary and secondary seed dispersal in the Malagasy tree Commiphora guillaumini CrossRef Full Text | Google Scholar The nutritional composition of Australian Aboriginal bushfoods Google Scholar Substantial resource reallocation during germination of Araucaria bidwillii (bunya pine) an Australian rainforest conifer with large seeds and cryptogeal germination 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Southern conifer family Araucariaceae: history and value for paleoenvironmental reconstruction doi: 10.1146/annurev.ecolsys.32.081501.114059 CrossRef Full Text | Google Scholar Survival Analysis: Techniques for Censored and Truncated Data Google Scholar “The evolutionary ecology of seed size,” in Seeds: the Ecology of Regeneration in Plant Communities Google Scholar Flotation preferentially selects saccate pollen during conifer pollination Predation and protection in the macroevolutionary history of conifer cones Variation in seed size is structured by dispersal syndrome and cone morphology in conifers and other nonflowering seed plants Cities may save some threatened species but not their ecological functions CrossRef Full Text | Google Scholar CrossRef Full Text | Google Scholar Montesinos-Navarro Network structure embracing mutualism-antagonism continuums increases community robustness 1984 Cityproductividad de semillas de Araucaria araucana (Mol.) C el Area de Lonquimay—IX Región Tesis para optar al TítulodeIngenieroForestal.FacultaddeCienciasAgrarias,Veterinarias y Forestales their determinants and consequences for recruitment Fast Growing Timber Trees of the Lowland Tropics: the Araucarias Google Scholar Population Structure and the Conservation Status of Araucaria Bidwillii Hook The IUCN Red List of Threatened Species 2013: e.T31355A2805113 Genetic Diversity and Divergence Within and Among Natural Populations of Araucaria in Eastern Australia differentiation and conservation in Araucaria bidwillii (Araucariaceae) CrossRef Full Text | Google Scholar Differential levels of genetic diversity and divergence among populations of an ancient Australian rainforest conifer CrossRef Full Text | Google Scholar R Core Team (2015). A Language and Environment for Statistical Computing. R Foundation for Statistical Computing. Vienna. Available online at: https://www.R-project.org/ CrossRef Full Text | Google Scholar Rodent handling of Araucaria araucana seeds CrossRef Full Text | Google Scholar CrossRef Full Text | Google Scholar “The role of native rodents in seed dispersal and seed predation of the Bunya Pine (Araucaria bidwillii),” in: 22nd Annual Conference of the Australian and New Zealand Society for Comparative Physiology and Biochemistry (ANZSCPB 2005) Google Scholar “Maintaining the ancient bunya tree (Araucaria bidwillii Hook.) - dispersal and mast years,” in Presentation Given at the 5th Southern Connection Conference (Adelaide Google Scholar Pollen dispersal and gene flow by pollen in Araucaria angustifolia CrossRef Full Text | Google Scholar Patterns of genetic diversity in southern and southeastern Araucaria angustifolia (Bert.) 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited *Correspondence: José L. Tella, dGVsbGFAZWJkLmNzaWMuZXM= By Richa Malhotra Never mind the Leaning Tower of Pisa – this is the leaning tower of pines Cook pines are towering trees that were once restricted to their native home of New Caledonia subtropical and temperate regions around the world The trees often have slightly tilting trunks Scientists have now noted a bizarre pattern in their tilt: they lean south in their northern range and north in their southern range Matt Ritter at California Polytechnic State University in San Luis Obispo was writing up a description of the Cook pine (Araucaria columnaris) for a book on the urban trees of California when he realised that the pines always leaned south So he rang up a colleague in Australia to see if that was the case there It turned out it was – but this time the pines leaned north “We got holy-smoked that there’s possibly a tree that’s leaning toward the equator wherever it grows,” says Ritter He and his colleagues studied 256 Cook pines scattered across five continents They collected tree data at 18 locations between latitudes of 7 and 35° north The team estimated that the trees tilt by 8.55 degrees on an average – about double the tilt of the Leaning Tower of Pisa The trees also slant more the further they are from the equator in both hemispheres “It’s a shockingly distinct pattern,” says Ritter One tree in South Australia slants at 40 degrees Trees normally correct for such asymmetry in their growth but for some unknown reason the Cook pine is unable to do so “We could be just dealing with an artefact of its genetics that we are seeing now when we have spread it all over the world,” he says it could be an adaptation to catch more sunlight at higher latitudes “The tilting phenomenon is not unusual,” says Steven Warren of the US Forest Service in Utah. In 2016, he reported that the inflorescence of the yucca palm found in the US always points south thus cutting the cost of transporting nutrients to its flowers “this is the first time I have heard of a tree doing this” Winslow Briggs at Stanford University finds the north-south leaning pattern “fascinating.” Some pines Journal reference: Ecology, DOI: 10.1002/ecy.1850 is a species of conifer native to New Caledonia that tends to tilt sideways when planted outside its natural habitat First classified by Johann Reinhold Forster a botanist accompanying Captain James Cook on his second voyage to circumnavigate the globe as far south as possible the araucaria columnaris soon became popular all around the world thanks to its distinctive narrowly conical shape and its height (up to 60 meters) these evergreen giants are planted as ornamental trees in various areas with warm and temperate climate on five continents and they generally don’t attract too much attention but in some cases they have one noticeable particularity – they lean heavily to one side and when there are more of them planted in the same area they all lean in the same direction… with one known exception being challenging environments where competition for light or nutrients and mechanical stress can cause trees to crow crookedly that didn’t seem to be the case with araucaria columnaris as their leaning tendency was noticed even very favorable environments Scientists were puzzled by the growth of araucaria columnaris trees for years American botanist Matt Ritter set out to find out the cause of the unusual phenomenon and started documenting the growth of Cook pines in North America He noticed that all the observed specimens leaned their tops towards the south and shared his findings with a friend and colleague from Australia all the New Caledonia pine trees seemed to lean north Ritter and a number of colleagues traveled the world looking for araucaria columnaris trees and documented their leaning tendencies the scientists concluded that the Cook pines’ lean is non-random: trees in the northern hemisphere lean south and those in the southern hemisphere lean north “Additionally, the magnitude of the lean is more pronounced at higher latitudes in both hemispheres. Our data and the pattern that we describe here elucidate the fact that plants are responding to their global environment in a way not yet fully understood,” the scientists noted in a paper published in 2017 while in the Northern they bent to the south and only around the equator did they grow almost vertically the angle of inclination of the araucaria columnaris was directly related to the distance from the equator The cause of directional lean in this species is unclear but Ritter and his colleagues hypothesize that it “may be related to an adaptive tropic response to the incidence angles of annual sunlight the pronounced lean in Araucaria columnaris trees is rare in other species including other Araucaria native to New Caledonia Please press and hold the button until it turns completely green If you believe this is an error, please contact our support team 147.45.197.102 : 9d7f2556-9311-4882-8cbc-7cf69495 Thank you!We have emailed you a PDF version of the article you requested You can also addnewsletters@iflscience.comto your safe senders list to ensure you never miss a message from us IFLScience HomeNo Matter Where They Are On Earth These Trees Lean Toward The EquatorComplete the form below to listen to the audio version of this article IFLScience needs the contact information you provide to us to contact you about our products and services You may unsubscribe from these communications at any time For information on how to unsubscribe, as well as our privacy practices and commitment to protecting your privacy, check out ourPrivacy Policy IFLScience HomeJoin for Exclusive FacebookemailJames is a published author with four pop-history and science books to his name. He specializes in history, strange science, and anything out of the ordinary. FacebookemailDOWNLOAD PDF VERSION Image credit: Dmitry Rukhlenko/shutterstock.com a group of researchers made a strange discovery: No matter where they are on Earth Cook pines (Araucaria columnaris) have a distinct lean to them according to the researchers that discovered the equatorial lean this species has a pronounced lean so ubiquitous that it is often used as the identifying characteristic for the species." Matt Ritter, now director at Cal Poly Plant Conservatory, was writing about the pines for a book when he noticed that the trees he had studied all seemed to lean to the south, New Scientist reported Ritter began ringing colleagues around the world to see which way their cook pines were leaning and soon found that they all seemed to be leaning toward the equator Ritter and his team measured 256 trees across five continents on a range of latitudes to examine the phenomenon further "We demonstrate that the Cook pines’ lean is non-random: trees in the Northern Hemisphere lean south and those in the Southern Hemisphere lean north," the team wrote in their study the magnitude of the lean is more pronounced at higher latitudes in both hemispheres." the trees lean 8.05 degrees toward the equator but the leans were more pronounced further away from the equator and only 9 percent of trees moved away from the predicted direction.  trees grow vertically in response to the opposing influences of gravity (gravitropism) and light (phototropism) "The leading hypothesis for the molecular mechanism of gravitropism is that sedimentation of amyloplasts on actin microfilaments activates a signal transduction pathway resulting in asymmetric transport of auxin in the stem causing it to straighten itself parallel with the gravity vector," the study authors write In more competitive or challenging environments While young plants may grow toward the Sun they tend to correct themselves as they grow due to the influence of gravity It's not clear why cook pines have their distinctive lean though the team suggests it could be an unhelpful non-adaptive quirk of their genetics or something else "The mechanisms underlying directional lean of Araucaria columnaris may be related to an adaptive tropic response to the incidence angles of annual sunlight or any combination of these," the team concludes "It is interesting that the pronounced lean in A including other Araucaria native to New Caledonia It is possible that the biophysical constraints of leaning for a tall tree ultimately prevail over any benefits of additional light interception gained by leaning." The team adds that the phenomenon needs more study as the "plants are responding to their global environment in a way not yet fully understood." The study is published in Ecology animalsChatty Dolphins Might Know More Language Than Just Each Other's Names, Reveals New Study1 hour agolink to article animalsWhere Have All The Penis Bones Gone? We’re Looking At You, Natural History Museums2 days ago36link to article T. Rex Leather, Glow-In-The-Dark Gas Clouds, And Musical Sea Lionslink to article The Five Most Common Headacheslink to article © 2025 IFLScience. All Rights Reserved. RSS Volume 9 - 2018 | https://doi.org/10.3389/fpls.2018.01275 This article is part of the Research TopicGenetic Regulatory Mechanisms Underlying Developmental Shifts in Plant EvolutionView all 19 articles The megagametophyte of mature seeds of Araucaria angustifolia consists of cells with thin walls we describe the process of mobilization of reserves and analyzed the dismantling of the tissue during germination using a range of well-established markers of programmed cell death (PCD) including: morphological changes in nuclei and amyloplasts TUNEL reaction and DNA electrophoresis demonstrate that DNA fragmentation in nuclei occurs at early stages of germination which correlates with induction of specific nucleases The results of the present study add knowledge on the dismantling of the megagametophyte of genus Araucaria a storage tissue that stores starch as the main reserve substance by revealing new insights into the role of nucleases and the expression patterns of putative nuclease genes during germination To describe the PCD of the megagametophyte during germination of A in the present study we analyzed the mobilization of reserves at different times following imbibition and investigated the characteristics that define the process of PCD and autophagy such as activation of Cys-EPs nuclear fragmentation and internucleosomal DNA cleavage we analyzed genes of S1 nuclease-like endonucleases and Staphylococcus nuclease-like (SN) endonucleases and a gene with a DNase-RNase domain not classified as S1 or as Tudor because it lacks these domains Araucaria angustifolia seeds were collected from trees grown in natural populations in the Botanical Garden “Arturo E Argentina (34°40′S 58°39′W) Seeds were surface-disinfected with 5% NaClO for 15 min and then allowed to germinate onto imbibed perlite in a growth chamber under controlled conditions of 16 h light/8 h dark cycles at 25°C and the megagametophytes were either used fresh or milled after freeze-drying and the flours stored at -80°C until use Experiments reported here were repeated with at least three independent biological replicates; the results were comparable across experiments Samples were collected at 0, 14, 28, and 42 DAG and prepared for microscopy according to López-Fernández and Maldonado (2013a) by fixation in 4% paraformaldehyde 0.1 M phosphate buffered saline (PBS) pH 7.2 for 24 h at 4°C the samples were dehydrated in an acetone series and then embedded in Technovit 8100 (Kulzer and Co. The sections were stained with 0.5% toluidine blue O (Sigma-Aldrich Megagametophytes at 0 and 42 DAG following germination were stained with 1% Evans Blue for 1 min and photographed under a dissecting microscope The megagametophytes from A. angustifolia seeds were homogenized in liquid nitrogen with pestle and mortar, and total RNA was extracted using the protocol described by Chang et al. (1993) The quantity and purity of the RNA samples were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific United States); samples with 260/280 nm and 260/230 nm ratios between 1.8–2.2 and 1.6–2.2 The integrity of the samples was confirmed by electrophoresis on a 1.5% (w/v) agarose gel Total RNA was treated with DNase I (New England Biolabs) first-strand cDNA was synthesized using M-MuLV Reverse Transcriptase (New England Biolabs) and d(t) 20 oligonucleotide following the manufacturer’s instructions Gene expression was evaluated through semiquantitative RT-PCR. Nuclease primers were designed using Primer3Plus Program (Untergasser et al., 2007). The endogenous normalization was performed using Ubiquitin 1 gene (Schlögl et al., 2012). The primer sequences are shown in Table 1 The PCR reactions were conducted in a total volume of 25 μL containing 5 μL of 1:10 diluted cDNA 0.1 μM for a specific sense and anti-sense primers 5 μL 10× PCR buffer (Invitrogen) and 0.5 mM MgCl2 The thermal cycle conditions used were: 94°C for 3 min 60°C for 30 s and 72°C for 1 min The numbers of cycles were specific for each pair of primers The PCR products had a length between 160 and 247 bp The RT-PCR products were resolved on 1.5% (w/v) agarose gel and stained with ethidium bromide (0.5 μg/mL) the gels were incubated overnight in 25 mM sodium acetate-acetic acid buffer [pH 5.5 0.2 mM DTT and 1% (v/v) Triton X-100] or 10 mM Tris–HCl neutral buffer [pH 8.0 0.2 mM DTT and 1% (v/v) Triton X-100] at 37°C In-gel assays in the presence of cations were performed as above in buffer containing 0.1 mM ZnSO4 or 10 mM CaCl2 the gels were washed for 5 min in cold stop buffer [10 mM Tris–HCl (pH 8.0) Nuclease activity was detected as a negatively stained band revealed by staining the gels with 0.01 mg/mL ethidium bromide and photographed using the Box GeneSnap software from Syngene The band intensity was analyzed using the Gel-Pro Analyzer Software (Media Cybernetics Inc.) All SDS-PAGE results were replicated a minimum of three times Genomic DNA was isolated by the cetyl-trimethyl-ammonium-bromide (CTAB) method (Doyle, 1991) 200 mg of three different megagametophytes were ground with liquid nitrogen into a fine powder and mixed with 400 μL CTAB solution [1.4 M NaCl; 2% (w/v) PVPPM40,000 The mix was incubated for 15 min at 70°C An equal volume of chloroform:isoamyl alcohol mixture (24:1) was added and the mixture was centrifuged for 10 min at 10,000 g The upper aqueous phase was removed and the total DNA was precipitated by addition of 700 μL 70% (v/v) ethanol DNA was recovered by centrifugation for 2 min at 10,000 g The yield and quality of the DNA obtained were assessed in a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific 20 μg of each sample was separated on a 2% (w/v) agarose gel and stained with ethidium bromide (0.5 μg/mL) A Thermo Fisher ScientificTM DNA GeneRulerTM 100 bp was used as a reference The protein concentrations were determined as described by Bradford (1976) using a Quick Start Bradford Protein Assay Kit 1 (500–0201; Bio-Rad United States Laboratories) and bovine serum albumin (BSA) as a standard (Bio-Rad Laboratories Nuclear DNA fragmentation was detected by TdT-mediated dUTP nick-end labeling (TUNEL) according to the protocol provided by the manufacturer (In situ cell detection kit TMR red tissue sections from proximal to embryo area were treated with 0.05% Tween 20 in PBS for 15 min at room temperature to facilitate penetration of the labeling reagents The slides were incubated in the TUNEL reaction mix at 37°C for 60 min sections were incubated without the Terminal deoxynucleotide Transferase (TdT) enzyme from the reaction mixture; to obtain positive controls sections were pretreated with DNase I (image not shown) were calculated from 100 nuclei randomly selected At least 5 semi thin sections of a different megagametophyte tissue were observed The membrane was then incubated with a secondary alkaline phosphatase-conjugated goat anti-rabbit antibody (Sigma A3587 Germany) diluted 1:5000 in TTBS for 1:30 h at room temperature The secondary antibody was detected with NBT/BCIP (Promega Immunolocalization was carried out according to the protocol described by Schmid et al. (1999) and López-Fernández and Maldonado (2013b) after blocking with 1% (w/v) BSA in PBS for 90 min the slides were incubated with anti-Cys-EP (dilution 1:100 in 0.1% BSA/PBS) overnight at 4°C After washing in PBS plus 0.05% (v/v) Tween 20 (PBST) three times for 10 min the slides were incubated with a fluorescent anti-rabbit ALEXA 488 IgG (Invitrogen United States) antibody applied 1:1000 in 0.1% BSA/PBS for 1 h at room temperature in the dark sections were examined by epifluorescence and light microscopy Images were obtained by epifluorescence and light microscopy with an Axioskop 2 microscope (Carl Zeiss All images were captured with an EOS 1000D camera (Canon analyzed using the AxioVision 4.8.2 software package (Carl Zeiss and compiled (Photoshop version CS6; Adobe Systems Rhodamine filters (excitation 520–560 nm emission 570–620 nm) and DAPI filters (excitation 340–390 nm emission 420–470 nm) were used to examine samples by TUNEL assay whereas Alexa filters (excitation 450–490 nm emission 515–565 nm) were used to examine samples for immunofluorescence assays During germination, the megagametophyte changed its color progressively from white and bright to brownish. Figure 1A shows the progressive cell death observed in the megagametophyte from the innermost layers to the outer ones using Evans Blue dye: living cells are able to exclude the dye (and thus cells remain unstained) whereas dead cells lose membrane integrity and stain blue only the remnants of the cell layers close to the embryo appear stained and no extensive cell death is observed until well after the mobilization of reserves has finalized cell death has been initiated in the entire tissue (A) Evans Blue staining at 0 and 42 DAG in the megagametophyte after extraction of the embryo In each case the figure is a representative result of observation of at least 6 seeds Scale bar = 0.5 cm (B) Changes occurring from the proximal to distal zones in the megagametophyte Adjacent to proximal sections; in c collapsed cell area the double white asterisk indicates crushed cells and the black arrow indicates amyloplast discharged into central vacuole Each image is a representative result of observation of at least 30 semi-thin sections of megagametophyte tissue at different DAG The nuclear dismantling was associated with cytoplasmic events during plant PCD. However, chromatin was partially adhered to the nuclear membrane until very advanced the processes of mobilization of reserves (Figures 1Cd–h) Analysis of DAPI-stained nuclei by fluorescence microscopy exhibited the progressive changes in the nuclear morphology Analyses were repeated at least three times on independent biological samples Bands are indicated by arrowheads (B) TUNEL assay (left column) and DAPI staining (central column) were performed on LRW tissue sections at 0 Merged images (right column) confirmed that DAPI co-labeled TUNEL-positive nuclei Each image is a representative result of observation of at least 5 semi-thin sections of megagametophyte tissue at different DAG The protein extracts from 0, 14, 28, and 42 DAG were separated by SDS–PAGE and electrophoretically transferred to a PVDF membrane. The western blot clearly showed the presence of the immature and mature forms (approximately 45 and 38 kDa, respectively) of Cys-EP (Figure 3A) which corresponds to the active form of CysEP neither of the two bands appeared revealed (A) Western blot analysis with anti-RcCys-EP as primary antibody and 42 DAG were separated on a 12% polyacrylamide gel and then transferred to a PVDF membrane (B) In situ immunolocalization of Cys-EP in megagametophytes of A Merge bright field and fluorescence imaging of longitudinal sections shows Cys-EP selectively localized to the proximal sector of the embryo (a) and adjacent to the proximal sector of the embryo (b) at 14 DAG; note labeling in the cytoplasm next to the cell wall (b) Each image is a representative result of observation of at least 5 semi-thin sections of a different megagametophyte tissue The in situ accumulation pattern of Cys-EP at 14 DAG was studied on longitudinal sections of the megagametophyte from the proximal and adjacent sectors of the embryo to the proximal cell layers. As mentioned above, the cell walls progressively lost rigidity (Figure 1B). Figure 3B indicates that Cys-EP immunolocalized in ricinosomes mixed with vacuolar content and highly vesiculated cytoplasm (a); in vacuolated cells (i.e. Cys-EP localized in the parietal cytoplasm (A) Determination of nuclease activity in A and 42 days after germination (DAG) by using the in-gel activity assay with ssDNA or dsDNA as substrate See section “Materials and Methods” for detailed reaction conditions The three parameters exhibited a marked decrease initiated before 14 DAG Data are represented as the mean ± standard error from three independent biological replicates Correlated with the increase in DNA-activity, there was a reduction in both DNA and RNA contents, especially at 14 DAG (Figure 4B). During germination, changes were detected in the RNA and soluble protein contents, which, at 42 DAG, reached values close to zero. In addition, the DNA content decreased drastically, reaching values close to 5 μg/g at 42 DAG (i.e., 2.6 times when compared to the control) (Figure 2A) All these sequences were identified from a leaf by transcriptomic analysis which indicates that they come from genes that are expressed at least in leaf tissue Ac114 also showed 67.6% identity to BBD2 (Uniprot Acc Q93VH2) and 63.7% identity to BBD1 (Uniprot Acc (A) In silico analysis of putative nuclease in Araucaria angustifolia Representation of the Ac520 protein showing the Staphylococcal nuclease domains at the N terminus and a Tudor domain toward the C terminus (ii) Representation of the Ac343 protein showing the S1-P1 Nuclease domain (iii) Representation of the Ac114 protein showing the DNase-Rnase domain (B) Expression levels of the putative nuclease genes during the germination process in A and Ac343 genes by semiquantitative RT-PCR a fragment of the Ubiquitin 1 gene was simultaneously amplified The results are representative of at least three independent biological replicates Expression of the Ac520 and Ac114 genes increased along the germination process with higher expression levels at 28 and 42 DAG whereas that of the Ac343 gene was only detected at 28 and 42 DAG although this issue needs further investigation the other bands that we detected correspond to a precursor protease a C-terminally truncated active form and to degradation products In the present study, two Zn2+-dependent nucleases of 35 and 43 kDa were induced at 28–42 DAG at acid pH. Nucleases of the S1/P1 family are thought to be similar to nucleases type I, showing maximal activity at acidic pH and Zn2+ dependency (Sugiyama et al., 2000) the Araucariaceae are well defined by the rbcL sequence and their monophyly is supported by a bootstrap value of 100% Ac520 also exhibited identity to TUDOR ribonucleases (Uniprot Acc The results of the present study add knowledge on the dismantling of the megagametophyte of mature starchy seeds in species of the genus Araucaria ML-F coordinated the field work and sampling SM and ML-F wrote the first draft of the paper All authors contributed to discussing the results and editing the paper This work was supported by Universidad de Buenos Aires (UBACYT 20020100100232 to SM) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET 7879/14 IA to ML-F and 810/13.P IP 0465 to SM) The Supplementary Material for this article can be found online at: 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Correa, Favre, Rodríguez, Maldonado and López-Fernández. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Sara Maldonado, c2FyYW1AYmcuZmNlbi51YmEuYXI= †These authors have contributed equally to this work