Study of grapevine solute transporters involved in berry quality. A biochemical and molecular approach

Tese de Doutoramento em Biologia de PlantasDue to its sessile nature, plants must take up mineral nutrients and water from the soil, and fix carbon in leaves from atmospheric CO2. The resulting photoassimilates must be precisely delivered to each plant organ, including leaves, roots and fruits. The present dissertation explored key biochemical mechanisms involved in the transport and compartmentation of sugars and water in grapevine. The fixation of carbon in chloroplasts, the synthesis of sugars in leaves and their transport to sink tissues are the most important biochemical steps in plant productivity, and have been investigated for several decades. Nonetheless, this is still a very relevant research topic because our knowledge is fragmented and the ongoing climate changes became an additional threat that may potentially affect plant productivity, in particular in grapevine. Plastids, which are characteristic of the plant kingdom, have pivotal roles in several important physiological processes. In grapevine, they are responsible for the accumulation of starch in woody tissues, roots, flowers and berries, and they are involved in the synthesis of secondary compounds. The first part of the present work was dedicated to the characterization of plastidial metabolism in grapevine, and focused on two plastidial glucose-6-phosphate/phosphate translocators (GPT), VvGPT1 and VvGPT2. It was found that three different splicing variants identified for VvGPT2 (VvGPT2α, VvGPT2β and VvGPT2Ω) were more expressed in the leaves. Contrarily, VvGPT1 was more expressed in mature berries, canes and flowers. Confocal microscopy revealed that VvGPT1 and VvGPT2Ω are localized in the plastidial envelope, and the transformation of pgi1-1 Arabidopsis mutant showed that these grapevine transporters mediate the uptake of glucose-6-phosphate into the plastid. In grape cell suspensions, ABA, light and galactinol, together with sucrose and fructose, increased the transcript abundance of VvGPT1. Furthermore, elicitation with MeJA dramatically increased the expression of VvGPT1 and VvPAL1, suggesting a role for GPTs in the production of secondary compounds in grapevine. Given its importance, this research line deserves further attention, particularly regarding the biochemical mechanisms underlying starch accumulation in grapevine amyloplasts during the winter, and its remobilization during spring to allow a fast vegetative growth. Besides sugar, water content is also an important parameter for berry quality, which is particularly intermingled with sugar status during berry development. Their content in the fruit is tightly regulated by the activity of sugar and water transporters (aquaporins – AQPs) in response to the environment. Berry and wine quality are directly affected by the berry water content at harvest. The second part of the present dissertation was devoted to the characterization of two AQPs in grapevine, VvSIP1 (Small Basic Intrinsic Protein 1) and VvXIP1 (Uncharacterized –X- Intrinsic Protein 1). Results showed that VvSIP1 was expressed in leaves and berries from field-grown vines, and in leaves and stems from in vitro plantlets, but not in roots. When expressed in tobacco mesophyll cells and in the yeast Saccharomyces cerevisiae, fluorescent-tagged VvSIP1 was localized at the ER. Stoppedflow spectroscopy showed that VvSIP1-enriched ER membrane vesicles from yeast exhibited higher water permeability and lower Ea for water transport than control vesicles, indicating the involvement of protein mediated water diffusion. This aquaporin was able to transport water but not glycerol, urea, sorbitol, glucose and inositol. VvSIP1-His-tag was solubilized and purified to homogeneity from yeast ER membranes and the reconstitution of the purified protein in phosphatidylethanolamine liposomes confirmed its water channel activity. XIPs are a new group of MIP proteins recently identified, so their precise physiological role has remained elusive. In our study, VvXIP1-RFP protein co-localized with ZmTIP2;1-YFP in the tonoplast of transiently transformed Nicotiana bethamiana leaves. Sopped-flow spectrometry performed with microsomal vesicles from yeast expressing pVV214-VvXIP1 showed that VvXIP1 is unable to transport water but transports glycerol. Plate growth assays showed that this AQP is also able to transport copper, boron and H2O2. Transcriptional analysis showed a much higher steady-state expression of VvXIP1 in leaves than in berries, canes or flowers from field grown grapevine. Furthermore, VvXIP1 transcripts were downregulated in leaves from plants treated with the copper-based fungicide Bordeaux mixture, and in vines under severe water deficit. In agreement, VvXIP1 was downregulated by ABA and salt stress in in vitro cultured grape cells. Much work is still needed to fully elucidate the physiological role of SIPs and XIPs in grapevine in particular towards the confirmation of their involvement in intracellular compartmentation of specific solutes like heavy metals. Thus, this avenue of research is still wide open.Devido à sua natureza séssil, as plantas obtêm do solo os nutrientes minerais e a água e fixam o carbono nas folhas a partir do CO2 atmosférico. A água e os solutos orgânicos e inorgânicos são distribuídos com precisão pelos diferentes tecidos da planta, incluindo as folhas, as raízes e os frutos. O presente trabalho de doutoramento visou o estudo de mecanismos bioquímicos envolvidos no transporte e compartimentação de açúcares e de água nos tecidos/células de videira (Vitis vinifera L.) A fixação de carbono, a síntese de açúcares nas folhas e o seu transporte para os tecidos heterotróficos constituem os passos bioquímicos mais relevantes envolvidos na produtividade das plantas. Embora estes aspetos fisiológicos tenham sido alvo de investigação ao longo das últimas décadas o nosso conhecimento é ainda fragmentado, e as modificações climatéricas em curso representam uma limitação adicional à produtividade das plantas, em particular da videira. Os plastídios, que são organelos característicos do reino vegetal, desempenham papéis fundamentais em diversos mecanismos fisiológicos. Na videira, os plastídios são responsáveis pela acumulação de amido nos tecidos lenhosos, raízes, flores e frutos, e estão ainda envolvidos na síntese de compostos secundários. A primeira parte do presente trabalho foi dedicada à caracterização do metabolismo plastidial na videira, tendo-se focado em dois antiportadores plastidiais de glucose-6- fosfato com fosfato (GPT), VvGPT1 e VvGPT2. Os resultados demonstraram que três variantes de splicing alternativo do gene VvGPT2 (VvGPT2α, VvGPT2β e VvGPT2Ω), são mais expressos nas folhas do que noutros órgãos. Pelo contrário, o gene VvGPT1 demonstrou uma expressão mais elevada em bagos maduros, varas e flores do que nas folhas. Estudos de microscopia confocal demonstraram que as proteínas VvGPT1 e VvGPT2Ω localizam-se na membrana plastidial, e estudos de transformação estável do mutante de Arabidopsis pgi1-1 confirmaram o seu papel na incorporação de glucose-6- fosfato. Estudos desenvolvidos com células em suspensão sugeriram que alguns fatores ambientais, como a luz e sinais hormonais ou endógenos, como o ABA ou os níveis de açúcar (incluindo o galactinol, a sacarose e a frutose), conduzem a um aumento do número de transcritos do VvGPT1. Adicionalmente, a eliciação com MeJA conduziu a um aumento acentuado da expressão dos genes VvGPT1 e VvPAL1, sugerindo que os GPTs desempenham um papel importante na produção de compostos secundários na videira. Dada a sua importância, esta linha de investigação merece estudos mais aprofundados no futuro, em particular no que diz respeito aos mecanismos bioquímicos de remobilização do amido armazenado durante o inverno nos amiloplastos, permitindo o rápido crescimento dos gomos caulinares na primavera. A água constitui um parâmetro fundamental para a qualidade do bago de uva e os seus níveis encontram-se particularmente relacionados com a concentração dos açúcares durante o desenvolvimento e amadurecimento do fruto. Tem sido demonstrado que a atividade de transportadores membranares de açúcares e de água (aquaporinas – AQPs) regula com precisão a concentração destes compostos em resposta às condições ambientais. Em bagos maduros, o conteúdo em água afeta diretamente a qualidade do fruto e do vinho. A segunda parte do presente trabalho de doutoramento foi dedicada à caracterização de duas AQPs de videira, VvSIP1 (Small Basic Intrinsic Protein 1) e VvXIP1 (Uncharacterized – X - Intrinsic Protein 1). Os resultados demonstraram que o gene VvSIP1 foi expresso em folhas e bagos de videiras cultivadas em condições de campo, bem como em folhas e caules, mas não em raízes, de plântulas crescidas in vitro. Estudos de expressão transiente em células do mesófilo de tabaco e em Saccharomyces cerevisiae indicaram que a proteína de fusão VvSIP1-GFP localiza-se no retículo endoplasmático (RE). Ensaios de espectrometria de stopped-flow demonstraram que vesículas de membrana de RE purificadas de leveduras transformadas com a construção VvSIP1-GFP exibem uma maior taxa de permeabilidade à água e uma menor energia de ativação do que vesículas obtidas de leveduras controlo. Estes resultados sugeriram o envolvimento de um canal proteico no transporte de água. Ensaios de especificidade mostraram que a proteína VvSIP1 não transporta glicerol, ureia, sorbitol, inositol e glucose. A proteína quimérica VvSIP1-6his foi purificada a partir do RE de leveduras transformadas e a sua atividade transportadora reconstituída em lipossomas de fosfatidiletanolamina. Estes resultados permitiram demonstrar cabalmente que a proteína VvSIP1 é um canal transportador de água. As aquaporinas XIP foram identificadas recentemente, pelo que o seu verdadeiro papel fisiológico é ainda pouco claro. Estudos de expressão transiente em folhas de tabaco mediada por Agrobacterium, demonstraram que a proteína de fusão VvXIP1-RFP colocaliza com a proteína ZmTIP2;1-YFP, sugerindo tratar-se de uma proteína da membrana vacuolar. Estudos de espectrometria de stopped-flow com vesículas de membrana de leveduras transformadas com o vector pVV214-VvXIP1 demonstraram que a VvXIP1 é impermeável à água mas transporta glicerol. Experiências de crescimento em meio sólido mostraram ainda que esta AQP medeia o transporte de cobre, boro e H2O2. Em videiras cultivadas em condições de campo, estudos de PCR quantitativo em tempo real demonstraram que os níveis de transcritos do VvXIP1 são mais abundantes em folhas do que nos frutos, varas e flores. Em folhas de videiras tratadas com o fungicida à base de cobre calda bordalesa, bem como de videiras submetidas a stresse hídrico severo, a expressão do VvXIP1 é inibida. Em linha com estes resultados, a expressão do VvXIP1 foi reprimida pelo ABA e por stresse salino em culturas celulares. A elucidação completa dos papéis fisiológicos das SIPs e das XIPs na videira requer ainda trabalho de investigação intenso, em particular com vista à confirmação do seu envolvimento na compartimentação intracelular de solutos específicos, como metais pesados, pelo que esta linha de investigação se mantém aberta.Fundação para a Ciência e Tecnologia, Bolsa de Doutoramento SFRH/BD/75257/2010; Fundação para a Ciência e Tecnologia - projeto PTDC/AGR-ALI/100636/2008 - “GrapeBerryFactory - Sugars, acids, phenolics and water on grape development and ripening”; Fundação para a Ciência e Tecnologia - projeto PTDC/AGR-AAM/099154/2008 - “AQUAVITIS - Compreender o transporte de água em Vitis vinifera: caracterização bioquímica de Aquaporinas através da sua expressão heteróloga em leveduras”; FEDER/COMPETE - Operational Competitiveness Programme – projeto Europeu "INNOVINE - Combining innovation in vineyard management and genetic diversity for a sustainable European viticulture" (FP7-KBBE-2012-6-311775

The effect of high-temperature on sugar transport in grape cells

Dissertação de mestrado em Fisiologia Molecular de PlantasVitis vinifera is a major crop worldwide and in Portugal. Berry content in sugars, organic acids, phenolics and aroma compounds are fundamental for fruit and wine quality. These compounds are accumulated/metabolized during the development of the berry. In particular, berry sugar content is directly related to the final alcoholic content of wine, and regulates the development of its aromatic and organoleptic properties. Massive sugar accumulation in berry mesocarp occurs after véraison due to a combined action of monosaccharide (MSTs) and disaccharide transporters (DSTs). High-temperatures affect berry set and development and alter the normal sugar content of the fruit. Also, peaks of high temperature, nowadays more and more frequent, may stop the ripening progress. We have been exploring the mechanisms involved in sugar import and compartmentation into the berry. VvHT1 (Vitis vinifera Hexose Transporter 1) is a high affinity plasma membrane H+-dependent symporter with broad specificity for monosaccharides abundant at early stages of berry development. The expression of this transporter is tightly regulated by sugars at transcriptional and post-translational levels. In the present study we aimed at the elucidation of the effect of high temperature and temperature fluctuations on sugar transport in grape cells. Results showed that a temperature treatment of 38ºC for 12 h decreased by 40% the Vmax of 14C-glucose transport in CSB (Cabernet Sauvignon Berry) cells. Contrarily, abscisic (ABA) and salicylic acid (SA) stimulated sugar uptake by 28.7% and 62.5%, respectively. ABA and SA also stimulated 14C-glucose accumulation in intact grape berries by 88.7% and 67.8%, respectively. The downregulation of glucose uptake mediated by high temperature corroborated the observed decrease of the VvHT1 levels in the plasma membrane. Moreover, after hightemperature treatment the intracellular ROS levels and lipid peroxidation increased by 97% and 29%, respectively. Proteomic analysis of the plasma membrane of CSB cells, allowed the identification of several proteins up-regulated in response to high temperature. It is hypothesised that intracellular ROS levels can mediate this cellular response to high-temperature. To study the recycling and turnover of VvHT1 in response to high-temperature a VvHT1-GFP expression clone was produced and a protocol for transient protoplast transfection is currently being optimized.A videira (Vitis vinifera) é uma espécie agrícola de elevada importância ao nível mundial e em Portugal. O conteúdo do bago em açúcares, ácidos orgânicos, compostos fenólicos e aromáticos determina a qualidade final do fruto e do vinho. Estes compostos são acumulados/metabolizados durante o desenvolvimento do fruto. O conteúdo do bago em açúcares condiciona o teor alcoólico do vinho, além de regular o desenvolvimento das suas propriedades aromáticas e organolépticas. A acumulação massiva de açúcares que ocorre no mesocarpo após a fase de pintor (véraison) resulta da acção combinada de transportadores membranares de mono (MST) e de dissacarídeos (DST). Temperaturas elevadas podem afectar a frutificação e o desenvolvimento do bago, bem como alterar o seu conteúdo normal em açúcares. Adicionalmente, picos de temperatura, muito frequentes no contexto das modificações climáticas em curso, podem comprometer o processo de amadurecimento. O nosso grupo de investigação tem dedicado atenção particular ao estudo dos mecanismos envolvidos no transporte e compartimentação de açúcares no bago. O transportador da membrana plasmática VvHT1 (Vitis vinifera Hexose Transporter 1), expresso nas fases iniciais do desenvolvimento do bago, medeia a incorporação da glucose e de outros monossacarídeos por um mecanismo de simporte com H+. A expressão deste transportador é finamente regulada pelo teor em açúcares ao nível transcricional e pós-transcricional. No presente trabalho foi estudado o efeito de temperaturas elevadas, e de flutuações de temperatura, no transporte de açúcares em células de videira. Os resultados demonstram que um pico de temperatura de 38ºC aplicado durante 12 h reduz em 40% a Vmax de transporte da 14C-glucose em culturas celulares (CSB, Cabernet Sauvignon Berry). Contrariamente, o ácido abcísico (ABA) e o ácido salicílico (SA) estimularam o transporte de açúcar em 28,7% e 62,5%, respectivamente. O ABA e o SA também estimularam a incorporação de 14C-glucose em bagos intactos em 88,7% e 67,8%, respectivamente. A repressão do transporte da glucose causada por temperaturas elevadas correlacionou-se com a detecção de níveis diminuídos do VvHT1 na membrana plasmática. Adicionalmente, a exposição a elevadas temperaturas aumentou os níveis intracelulares de ROS e de peroxidação lipídica em 97% e 29%, respectivamente. Uma análise de proteómica efectuada em membranas plasmáticas de células CSB, permitiu a identificação de diversas proteínas especificamente expressas em resposta a temperaturas elevadas. É discutido que o aumento observado dos níveis intracelulares de ROS podem mediar esta resposta celular a elevadas temperaturas. No sentido de estudar a reciclagem e turnover do transportador VvHT1 em resposta a temperaturas elevadas, foi construído um clone de expressão VvHT1-GFP e optimizado um protocolo de transformação de protoplastos.Fundação para a Ciência e a Tecnologia (FCT) - PTDC/AGR-ALI/100636/200

The first insight into the metabolite profiling of grapes from three vitis vinifera L. cultivars of two controlled appellation (DOC) regions

The characterization of the metabolites accumulated in the grapes of specific cultivars grown in different climates is of particular importance for viticulturists and enologists. In the present study, the metabolite profiling of grapes from the cultivars, Alvarinho, Arinto and Padeiro de Basto, of two Portuguese Controlled Denomination of Origin (DOC) regions (Vinho Verde and Lisboa) was investigated by gas chromatography-coupled time-of-flight mass spectrometry (GC-TOF-MS) and an amino acid analyzer. Primary metabolites, including sugars, organic acids and amino acids, and some secondary metabolites were identified. Tartaric and malic acids and free amino acids accumulated more in grapes from vines of the DOC region of Vinho Verde than DOC Lisboa, but a principal component analysis (PCA) plot showed that besides the DOC region, the grape cultivar also accounted for the variance in the relative abundance of metabolites. Grapes from the cultivar, Alvarinho, were particularly rich in malic acid and tartaric acids in both DOC regions, but sucrose accumulated more in the DOC region of Vinho Verde.This work was supported by European Union Funds (FEDER/COMPETE-Operational Competitiveness Programme) and by the Portuguese national funds (FCT—Portuguese Foundation for Science and Technology) under the project FCOMP-01-0124-FEDER-022692, the research project FCOMP-010124-FEDER-008760 (ref. FCT PTDC/AGR-ALI/100636/2008), and the PhD grants, no. SFRH/BD/ 64587/2009 to VM and SFRH/BD/74257/2010 to HN This work also benefited from the networking activities within the European project, INNOVINE (ref. 311775), and the European COST action (FA1106 “QualityFruit” and FA1003 “East-West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding”).info:eu-repo/semantics/publishedVersio

Solute transport across plant cell membranes

CanaBQ is a Journal of the Portuguese Biochemical Society (http://canalbq.spb.pt/index.html)The transport of solutes across cell membranes, including organic nutrients, such as sugar, osmolytes, ions or metabolic waste products, is of extreme importance in all living systems. Up to 14% of the genome of all organisms represents information for transport proteins, which reflects the importance of such process. Transporters are also involved in the transduction of environmental and endogenous signals. Several transport systems have been identified and fully characterised at both molecular and biophysical levels in a wide variety of living organisms, from bacteria to humans, with the bacterial lactose permease (LacY) being a good example of such successful studies. The majority of transporter proteins are very well conserved throughout living systems, and some of them, such as sugar transporters, belong to a large family (SP, Sugar Porter). In higher plants, the photoassimilated carbon is transported from mature leaves throughout the phloem, mainly in the form of sucrose, as in the grapevine, or mannitol, as in the olive tree, to heterotrophic organs such as developing leaves, flowers, fruits and roots, which rely on its supply for their growth and development. Thus, the unlocking of the mechanisms of photoassimilate transport into plant sink tissues, as well as their regulation, has an important basic and applied relevance. Moreover, as most living organisms, plants also face a continuous battle against adverse environmental factors like increasing soil salinity, heat and drought. In this context, solute transport also has a relevant role in plant defence. For instance, the efficient exclusion of Na+ excess from the cytoplasm and vacuolar Na+ accumulation are the most important steps towards the maintenance of ion homeostasis under salt stress. The production, transport and accumulation of compatible solutes like mannitol are also important plant responses to salinity and drought. Like animals, where important diseases such as depression and hypertension are commonly treated with drugs targeted to specific transporters, plants have also benefited from the extensive and ongoing study of membrane transport. The present review provides an overview on the investigation that has been conducted in our laboratory under the scope of this fascinating topic.This work was supported by the Portuguese Foundation for Science and Technology (FCT) (research project ref. PTDC/AGR-ALI/100636/2008; to A. Conde, grant ref. SFRH/BD/47699/2008; to V. Martins, grant ref. SFRH/BD/64587/2009; to H. Noronha, grant ref. SFRH/BD/75257/2010, to N. Fontes, grant ref. SFRH/ BD/23169/2005, and to C. Conde, grant ref. SFRH/ BPD/34998/2007). We are also grateful to BabeliUM, the Language Centre of the University of Minho, namely Ana Teresa Correia, for revising the English version of the manuscript

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Geography and ecology shape the phylogenetic composition of Amazonian tree communities

AimAmazonia hosts more tree species from numerous evolutionary lineages, both young and ancient, than any other biogeographic region. Previous studies have shown that tree lineages colonized multiple edaphic environments and dispersed widely across Amazonia, leading to a hypothesis, which we test, that lineages should not be strongly associated with either geographic regions or edaphic forest types.LocationAmazonia.TaxonAngiosperms (Magnoliids; Monocots; Eudicots).MethodsData for the abundance of 5082 tree species in 1989 plots were combined with a mega-phylogeny. We applied evolutionary ordination to assess how phylogenetic composition varies across Amazonia. We used variation partitioning and Moran's eigenvector maps (MEM) to test and quantify the separate and joint contributions of spatial and environmental variables to explain the phylogenetic composition of plots. We tested the indicator value of lineages for geographic regions and edaphic forest types and mapped associations onto the phylogeny.ResultsIn the terra firme and várzea forest types, the phylogenetic composition varies by geographic region, but the igapó and white-sand forest types retain a unique evolutionary signature regardless of region. Overall, we find that soil chemistry, climate and topography explain 24% of the variation in phylogenetic composition, with 79% of that variation being spatially structured (R2 = 19% overall for combined spatial/environmental effects). The phylogenetic composition also shows substantial spatial patterns not related to the environmental variables we quantified (R2 = 28%). A greater number of lineages were significant indicators of geographic regions than forest types.Main ConclusionNumerous tree lineages, including some ancient ones (>66 Ma), show strong associations with geographic regions and edaphic forest types of Amazonia. This shows that specialization in specific edaphic environments has played a long-standing role in the evolutionary assembly of Amazonian forests. Furthermore, many lineages, even those that have dispersed across Amazonia, dominate within a specific region, likely because of phylogenetically conserved niches for environmental conditions that are prevalent within regions

Geography and ecology shape the phylogenetic composition of Amazonian tree communities

Aim: Amazonia hosts more tree species from numerous evolutionary lineages, both young and ancient, than any other biogeographic region. Previous studies have shown that tree lineages colonized multiple edaphic environments and dispersed widely across Amazonia, leading to a hypothesis, which we test, that lineages should not be strongly associated with either geographic regions or edaphic forest types. Location: Amazonia. Taxon: Angiosperms (Magnoliids; Monocots; Eudicots). Methods: Data for the abundance of 5082 tree species in 1989 plots were combined with a mega-phylogeny. We applied evolutionary ordination to assess how phylogenetic composition varies across Amazonia. We used variation partitioning and Moran\u27s eigenvector maps (MEM) to test and quantify the separate and joint contributions of spatial and environmental variables to explain the phylogenetic composition of plots. We tested the indicator value of lineages for geographic regions and edaphic forest types and mapped associations onto the phylogeny. Results: In the terra firme and várzea forest types, the phylogenetic composition varies by geographic region, but the igapó and white-sand forest types retain a unique evolutionary signature regardless of region. Overall, we find that soil chemistry, climate and topography explain 24% of the variation in phylogenetic composition, with 79% of that variation being spatially structured (R$^{2}$ = 19% overall for combined spatial/environmental effects). The phylogenetic composition also shows substantial spatial patterns not related to the environmental variables we quantified (R$^{2}$ = 28%). A greater number of lineages were significant indicators of geographic regions than forest types. Main Conclusion: Numerous tree lineages, including some ancient ones (>66 Ma), show strong associations with geographic regions and edaphic forest types of Amazonia. This shows that specialization in specific edaphic environments has played a long-standing role in the evolutionary assembly of Amazonian forests. Furthermore, many lineages, even those that have dispersed across Amazonia, dominate within a specific region, likely because of phylogenetically conserved niches for environmental conditions that are prevalent within regions

Geographic patterns of tree dispersal modes in Amazonia and their ecological correlates

Aim: To investigate the geographic patterns and ecological correlates in the geographic distribution of the most common tree dispersal modes in Amazonia (endozoochory, synzoochory, anemochory and hydrochory). We examined if the proportional abundance of these dispersal modes could be explained by the availability of dispersal agents (disperser-availability hypothesis) and/or the availability of resources for constructing zoochorous fruits (resource-availability hypothesis). Time period: Tree-inventory plots established between 1934 and 2019. Major taxa studied: Trees with a diameter at breast height (DBH) ≥ 9.55 cm. Location: Amazonia, here defined as the lowland rain forests of the Amazon River basin and the Guiana Shield. Methods: We assigned dispersal modes to a total of 5433 species and morphospecies within 1877 tree-inventory plots across terra-firme, seasonally flooded, and permanently flooded forests. We investigated geographic patterns in the proportional abundance of dispersal modes. We performed an abundance-weighted mean pairwise distance (MPD) test and fit generalized linear models (GLMs) to explain the geographic distribution of dispersal modes. Results: Anemochory was significantly, positively associated with mean annual wind speed, and hydrochory was significantly higher in flooded forests. Dispersal modes did not consistently show significant associations with the availability of resources for constructing zoochorous fruits. A lower dissimilarity in dispersal modes, resulting from a higher dominance of endozoochory, occurred in terra-firme forests (excluding podzols) compared to flooded forests. Main conclusions: The disperser-availability hypothesis was well supported for abiotic dispersal modes (anemochory and hydrochory). The availability of resources for constructing zoochorous fruits seems an unlikely explanation for the distribution of dispersal modes in Amazonia. The association between frugivores and the proportional abundance of zoochory requires further research, as tree recruitment not only depends on dispersal vectors but also on conditions that favour or limit seedling recruitment across forest types