The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora.

Population genetic structure and genetic diversity levels are important issues to understand population dynamics and to guide forest management plans. The Brazil nut tree (Bertholletia excelsa Bonpl.) is an endemic species, widely distributed through Amazonian upland forests and also an important species for the local extractive economy. Our aim was to analyze the genetic structure of Brazil nut trees at both fine and large scales throughout the Amazon Basin, contributing to the knowledge base on this species and to generate information to support plans for its conservation. We genotyped individuals from nine sites distributed in five regions of the Brazilian Amazon using 11 microsatellite loci. We found an excess of heterozygotes in most populations, with significant negative inbreeding coefficients (f) for five of them and the finescale structure, when present, was very small. These results, as a consequence of self-incompatibility, indicate that conservation plans for B. excelsa must include the maintenance of genetic diversity within populations to ensure viable amounts of seeds for both economic purposes and for the local persistence of the species.

The effects of water deficit on photochemical parameters and expression of several candidate genes were investigated in drought-tolerant clone 73 of Coffea canephora submitted to slowly imposed water limitation. Under irrigation, this clone showed low values of stomatal conductance (g s) and of CO 2 assimilation rates (A) suggesting that it had a great efficiency in controlling stomatal closure and transpiration. After water withdrawal, this clone reached a −3.0 MPa after 15 days without irrigation and showed a slow decrease in the pre-dawn leaf water potential. Under drought, the suppression of A was accompanied by maintenance of photochemical quenching (q P) and internal to ambient CO 2 concentration (Ci/Ca) ratios as well as by a decrease of non-photochemical quenching (q N). This is confirmed by the transport rate/CO 2 assimilation (ETR/A) rates that suggested the participation of an alternative electron sink protecting the photosynthetic apparatus against photoinhibition. At the transcriptomic level, high up-regulation of genes encoding for a dehydrin (CcDH3), an ascorbate peroxidase (CcAPX1), a prephenate-dehydrogenase like protein (CcPDH1) and a non-symbiotic haemoglobin (CcNSH1) was also observed upon drought suggesting a strong induction of antioxidant and osmoprotection systems in this clone. High expression levels of gene-encoding ABA receptors (CcPYL3 and CcPYL7) under water limitation were also observed suggesting the involvement of the ABA signaling pathway in response to drought. All these results where compared to those previously obtained for drought-tolerant clones 14 and 120. Our results demonstrated the existence of different mechanisms amongst the drought-tolerant coffee clones regarding water deficit. Keywords Candidate gene. Coffea canephora. Drought. Gene expression. Real-time quantitative PCR. Water potential Abbreviations CG Candidate gene qPCR Quantitative polymerase chain reaction

To support the long-term ecological viability of restoration projects, it is necessary to reach adequate levels of genetic diversity in spontaneously recolonizing and reintroduced populations. The importance of genetic diversity in the long-term viability of populations is acknowledged, but still poorly monitored in restoration projects. This study aimed to estimate the genetic diversity and inbreeding levels of populations of a tree species widely used in restoration projects in the Atlantic Forest of Brazil, Centrolobium tomentosum, exploring the potential of active restoration in successfully reestablishing populations with higher chances of long-term perpetuation in agricultural landscapes. We used both nuclear and chloroplast microsatellite markers to assess genetic parameters in juveniles and adult individuals in two high-diversity restoration plantations (28 and 60 years old), one disturbed fragment, and one large and well conserved protected area. We observed similar levels of genetic diversity and inbreeding, for juveniles and adults, in both restored and natural populations. Surprisingly, haplotype diversity was higher in restoration sites. We also found private alleles in juveniles in both restoration areas, and this is evidence of gene flow between restored and neighboring natural populations. However, we observed negative effects of inbreeding on the effective population size of populations from the disturbed natural remnant and restoration areas. These results provide evidence of the capacity of restoration plantations for recovering high levels of genetic diversity and the importance of maintaining large and wellconserved forest remnants to be used as seed sources for restoration efforts.

Multiple ecological and life-history traits shape the fine-scale spatial genetic structure (FSGS) of a given population. The occurrence in core versus peripheral populations, levels of outcrossing, pollen and seed dispersal, and hybridization are important biological properties that influence the kinship of individuals within populations. We examined spatial genetic structure within 15 populations of Epidendrum fulgens and E. puniceoluteum distributed along a linear gradient of Brazilian coastal vegetation, including both allopatric and sympatric populations where the two orchid species hybridize. We analyzed 581 mapped specimens using nine simple sequence repeat loci, aiming to investigate how geographic distribution and hybridization shape within-population FSGS. A significant increase in FSGS was found towards peripheral populations, compared to core populations. Analysis of short-distance and long-distance components of FSGS identified biparental inbreeding and higher levels of FSGS at peripheral populations, when compared to core populations. In contrast, the relatively high density of reproductive adults in core populations potentially leads to highly overlapping seed and pollen movement, decreasing FSGS. Hybridization was an important factor shaping within-population spatial genetic structure at sympatric sites, decreasing the FSGS observed in parental species. Our results indicate that different ecological forces act in concert to create a gradient of FSGS along species distribution ranges, shaped by extensive levels of intraspecific and interspecific gene exchange.

ABSTRACT. Seven polymorphic microsatellite markers were developed and validated for Bertholletia excelsa (Brazil nut tree) population genetic studies. This species is a widespread monotypic Amazonian tree with high non-timber economic value. Unfortunately, Brazil nut production is currently less than 25% of historical production levels, because of extensive deforestation. All pairs of primers produced clearly interpretable and polymorphic bands. No linkage disequilibrium was observed in an analysis of 46 individuals from one population, three to seven alleles per locus were observed; the expected heterozygosity ranged from 0.378 to 0.978, with significant heterozygote excess for four loci. An analysis of individuals from two populations showed private alleles at all loci. These primer pairs will be useful for population studies, especially for comparing samples from different parts of the Amazon forest.

Long‐term ecological success of large‐scale restoration programs planned for the next decades will rely on genetic diversity (GD) of reintroduced or colonizing species, a limiting factor in highly fragmented landscapes. In small and isolated natural remnants or restoration areas, substantial reduction in population's size or connectivity may lead to local extinctions due to the accumulation of deleterious recessive alleles and ongoing reduction of fecundity, plant vigor, recruitment success, and adaptive potential. Despite the paramount role of GD for species persistence, its levels in restoration programs are poorly known. We assessed the GD of four model tree species (different succession stages, dispersal, and pollination syndromes) from the Brazilian Atlantic Forest, comparing two high‐diversity restoration plantations, one forest fragment and one conserved remnant. Contrary to the expectation that the plantation strategies adopted in the restoration programs could result in genetic composition homogenization, we found that restoration areas established heterogeneous genetic groups with similar levels of neutral GD and inbreeding to those observed in natural forest remnants. This pattern was consistent across the four functionally different tree species, despite some species idiosyncrasies. For instance, we observed lower allelic richness in early successional species in restoration sites, suggesting that some species may be more prone to reintroduction with lower GD. Thus, we advocate the use of high GD levels in restoration to support biodiversity conservation in human‐modified landscapes, thus reinforcing the role of ecological restoration for recovering the diversity of genes—the basic constituent of biodiversity.

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