Does acclimation in distinct light conditions determine differences in the photosynthetic heat tolerance of coffee plants?

Worldwide coffee production is threatened by climate change, which highlights the importance of heat tolerance studies. Here we tested the hypothesis that photosynthetic heat tolerance in coffee varieties changes according to acclimation to distinct light conditions. Furthermore, we tested if heat tolerance is associated with the habitat of origin of the coffee species. We evaluated heat tolerance using chlorophyll fluorescence in varieties of Coffea arabica (Mundo Novo and Catuai Amarelo) and C. canephora (Conilon) grown in a common garden under two conditions: high (HS) and low (LS) sunlight. Leaf traits associated with leaf cooling were evaluated in plants grown in LS and HS and associations of heat tolerance with these traits were determined. The varieties tested had high photosynthetic heat tolerance, with temperatures above 54 °C leading to a 50% reduction in Fv /Fm (T50 ). The heat tolerance of each Coffea variety was unaffected by growth in distinct light conditions. Leaves of plants grown in LS were larger and had a lower fraction of the leaf area occupied by stomata (nast ). Heat tolerance was positively associated with leaf size and negatively with nast . C. canephora exhibited higher heat tolerance than C. arabica. The limited plasticity of heat tolerance in response to acclimation under distinct light conditions contradicts the prediction that plants acclimated to HS would have higher photosynthetic heat tolerance than those acclimated to LS. Our results on heat tolerance among Coffea species/varieties in HS and LS indicate the possibility of selection of varieties for better acclimation to ongoing climate changes.

Phylogeography of Eriotheca species complex: insights into the origin and range expansion of apomictic and polyploid trees in Neotropical Savannas.

Polyploidy and whole genome duplication are major evolutionary drivers in plants. Climate variations during the Pleistocene have influenced distribution and range expansion worldwide. Similar trends have been reported for Cerrado plants, but no attempt has been made to link phylogeography with ploidy and breeding changes. Thus, we aimed to (i) assess ploidy and genome size of Eriotheca estevesiae Carv.-Sobr., and compare it with E. pubescens (Mart.) Schott & Endl. (Both included into the Eriotheca Stellate Trichome Species Complex - ESTSC). (ii) Subsequently, we investigated their phylogeography to see whether genetic structure and range expansion trends were similar to those previously described for the Cerrado biome. Finally (iii), we discuss whether ESTSC phylogeographic patterns could be associated with geographic parthenogenesis processes. Common cytogenetic techniques and flow cytometry were used to confirm chromosome number and genome size of E. estevesiae. We used three cpDNA regions to analyse 14 ESTSC Cerrado populations, for which we also obtained ploidy level and breeding information. We investigated haplotype diversity, population structure and tested neutrality, aiming to reconstruct phylogeographic scenarios. We found three ploidy levels and eight cpDNA haplotypes in ESTSC, one shared by most populations. Haplotype and ploidy distribution corroborated that E. pubescens, the widely distributed polyploid and apomictic species, may have originated from northern diploid and probably sexual E. estevesiae. Matrilinear cpDNA links support the idea that apomixis and polyploidy in ESTSC may have allowed range expansion during the Pleistocene, in a process analogous to the geographic parthenogenesis described elsewhere.

Leaf trait variation during ontogeny in the endangered Brazilian rosewood tree.

Knowledge of plant responses to environmental heterogeneity during ontogeny is important to elucidate the changes that occur to promote resource capture in tropical forests. We tested the hypothesis that expression changes in leaf metamer traits of Brazilian rosewood (Dalbergia nigra), from seedlings to emergent canopy trees, occur as new microclimate environments are achieved. We also tested the hypothesis that increased light heterogeneity in the understorey leads to higher plasticity in leaf traits of seedlings and saplings than in sun-exposed metamers of emergent trees subject to stressful conditions. We compared leaf metamer traits of 53 individuals including seedlings, saplings and emergent trees. We also evaluated the light heterogeneity in vertical strata and the variations in leaf traits within individuals (among metamers of the same individual). These were associated with height of the individuals. Compared to understorey plants, emergent trees presented larger metamers, with lower specific leaf area (SLA), lower investment in leaf area per total dry mass of metamer (LARm ), lower specific petiole length (SPL) and lower specific internode length (SIL). Higher phenotypic variation within individuals was observed in seedlings, which decreased as the trees grew taller. The results suggest the integration of ontogenetic changes in leaf traits under new microclimate conditions as the plants reach different vertical strata in the forest. Additionally, our results support the hypothesis that increased light heterogeneity in the understorey shaped higher phenotypic variation within individuals in juveniles and that stressful conditions in sun-exposed leaf metamers of emergent trees led to increased phenotypic stability.

Isolation of high quality and polysaccharide-free DNA from leaves of Dimorphandra mollis (Leguminosae), a tree from the Brazilian Cerrado.

Dimorphandra mollis (Leguminosae), known as faveiro and fava d'anta, is a tree that is widely distributed throughout the Brazilian Cerrado (a savanna-like biome). This species is economically valuable and has been extensively exploited because its fruits contain the flavonoid rutin, which is used to produce medications for human circulatory diseases. Knowledge about its genetic diversity is needed to guide decisions about the conservation and rational use of this species in order to maintain its diversity. DNA extraction is an essential step for obtaining good results in a molecular analysis. However, DNA isolation from plants is usually compromised by excessive contamination by secondary metabolites. DNA extraction of D. mollis, mainly from mature leaves, results in a highly viscous mass that is difficult to handle and use in techniques that require pure DNA. We tested four protocols for plant DNA extraction that can be used to minimize problems such as contamination by polysaccharides, which is more pronounced in material from mature leaves. The protocol that produced the best DNA quality initially utilizes a sorbitol buffer to remove mucilaginous polysaccharides. The macerated leaf material is washed with this buffer until there is no visible mucilage in the sample. This protocol is adequate for DNA extraction both from young and mature leaves, and could be useful not only for D. mollis but also for other species that have high levels of polysaccharide contamination during the extraction process.

Phenotypic plasticity to light of two congeneric trees from contrasting habitats: Brazilian Atlantic Forest versus cerrado (savanna).

The Brazilian Atlantic Forest is a typically multi-layer tropical forest, while cerrado (savanna) is a patchy habitat with different physiognomy. Despite these differences, both habitats have high light heterogeneity. Functional traits of Dalbergia nigra and D. miscolobium from the Atlantic Forest and cerrado, respectively, were evaluated under shade (25% of full sunlight) and full sunlight in a nursery experiment. We hypothesised that both species should benefit from high phenotypic plasticity in relation to light. Plasticity was estimated using the relative distance phenotypic index (RDPI). D. miscolobium had lower shoot growth under both light conditions, suggesting it has low competitive capacity in the forest environment, which could explain its limited ability to expand over areas of Atlantic Forest. The studied species exhibited photoprotection strategies under high light and improved light capture under low light. Stomatal conductance, ETR(max) (maximum electron transport rate), PPFD(sat) (saturating photosynthetically active photon flux density), chlorophyll and carotenoid content had higher RDPI than stem morphological traits. Although both species showed considerable phenotypic plasticity, D. miscolobium had higher RDPI for eight of 11 evaluated traits. This high plasticity could be one of the factors that explain the occurrence of this species in a wide range of environmental conditions, from open grassland to dense woodlands, and it could also reflect its adaptation to high light. D. nigra also had considerable plasticity and good growth performance in both shade and full sunlight, but its absence in areas of cerrado suggests that factors other than light limit its occurrence in these habitats.

Phylogeography of the endangered rosewood Dalbergia nigra (Fabaceae): insights into the evolutionary history and conservation of the Brazilian Atlantic Forest.

The Brazilian rosewood (Dalbergia nigra) is an endangered tree endemic to the central Brazilian Atlantic Forest, one of the world's most threatened biomes. The population diversity, phylogeographic structure and demographic history of this species were investigated using the variation in the chloroplast DNA (cpDNA) sequences of 185 individuals from 19 populations along the geographical range of the species. Fifteen haplotypes were detected in the analysis of 1297 bp from two non-coding sequences, trnV-trnM and trnL. We identified a strong genetic structure (F(ST)=0.62, P<0.0001), with a latitudinal separation into three phylogeographic groups. The two northernmost groups showed evidence of having maintained historically larger populations than the southernmost group. Estimates of divergence times between these groups pointed to vicariance events in the Middle Pleistocene (ca. 350,000-780,000 years ago). The recurrence of past climatic changes in the central part of the Atlantic forest, with cycles of forest expansion and contraction, may have led to repeated vicariance events, resulting in the genetic differentiation of these groups. Based on comparisons among the populations of large reserves and small, disturbed fragments of the same phylogeographic group, we also found evidence of recent anthropogenic effects on genetic diversity. The results were also analysed with the aim of contributing to the conservation of D. nigra. We suggest that the three phylogeographic groups could be considered as three distinct management units. Based on the genetic diversity and uniqueness of the populations, we also indicate priority areas for conservation.

An efficient protocol for tissue sampling and DNA isolation from the stem bark of Leguminosae trees.

Traditionally, molecular studies of plant species have used leaves as the source of DNA. However, sampling leaves from tall tree species can be quite difficult and expensive. We developed a sequence of procedures for using stem bark as a source of DNA from Leguminosae trees of the Atlantic Forest and the Cerrado. Leguminosae is an important species-rich family in these two highly diverse and endangered biomes. A modified CTAB protocol for DNA isolation is described, and details of the procedures for sampling and storage of the bark are given. The procedures were initially developed for three species, and then their applicability for 15 other species was evaluated. DNA of satisfactory quality was obtained from the bark of all species. The amounts of DNA obtained from leaves were slightly higher than from bark samples, while its purity was the same. Storing the bark frozen or by drying in silica gel yielded similar results. Polymerase chain reaction amplification worked for both plastid and nuclear genomes. This alternative for isolating DNA from bark samples of trees facilitates field work with these tree species.

Contrasting genetic diversity and differentiation of populations of two successional stages in a Neotropical pioneer tree (Eremanthus erythropappus, Asteraceae).

Eremanthus erythropappus, commonly known as "candeia", is an abundant pioneer tree species, forming dense populations known as "candeial", but it is also found in forests at middle stages of succession. Trees from forests are bigger and occur in lower density than in the "candeial". The objectives of the present study were to investigate if the decrease in population density during successional process is accompanied by 1) changes in within-population genetic diversity, and 2) differentiation of populations. Eight populations, four of early successional stage ("candeial") and four of middle successional stages (forest), were analyzed with RAPD markers. The genetic diversity found was high compared to other tree species analyzed with RAPD markers. AMOVA revealed that most of the genetic variations of E. erythropappus were found within populations (85.7%), suggesting that this species is predominantly outcrossing. The relatively low differentiation among the populations can be attributed to small distances among the populations analyzed (0.2 to 10.8 km). No indication that populations from middle successional habitats show lower genetic variation than populations from early successional stages was found. The percentage of polymorphic fragments (82.8 and 84.8%) and the Shannon indexes (0.442 and 0.455) were similar in "candeial" and forest, respectively. These results suggest that if an increase in selection intensity occurred during succession, it did not result in a decrease in genetic diversity or that the selection effect was balanced by other factors, such as gene flow. Higher significant differentiation among E. erythropappus populations from "candeial" in relation to that among populations from forest was also not detected.

Isolation and characterization of microsatellite loci for Hymenaea courbaril and transferability to Hymenaea stigonocarpa, two tropical timber species.

Hymenaea courbaril is a tropical timber species, intensely exploited and found in the Amazon, Atlantic Forest and Brazilian Cerrado biome. Nine highly polymorphic microsatellite loci were developed from a genomic library enriched for AG/TC repeats. In a total of 41 individuals, from two natural populations, seven to 13 alleles per locus were detected and expected heterozygosity ranged from 0.75 to 0.90. Seven loci were effectively transferred to Hymenaea stigonocarpa. High levels of polymorphism make the present primers useful for population genetic studies and are a powerful tool to investigate mating system, gene flow and spatial genetic structure.

Contrasting genetic diversity and differentiation of populations of two successional stages in a neotropical pioneer tree (Eremanthus erythropappus, Asteraceae)

Eremanthus erythropappus, commonly known as “candeia”, is an abundant pioneer tree species, forming dense populations known as “candeial”, but it is also found in forests at middle stages of succession. Trees from forests are bigger and occur in lower density than in the “candeial”. The objectives of the present study were to investigate if the decrease in population density during successional process is accompanied by 1) changes in within-population genetic diversity, and 2) differentiation of populations. Eight populations, four of early successional stage (“candeial”) and four of middle successional stages (forest), were analyzed with RAPD markers. The genetic diversity found was high compared to other tree species analyzed with RAPD markers. AMOVA revealed that most of the genetic variations of E. erythropappus were found within populations (85.7%), suggesting that this species is predominantly outcrossing. The relatively low differentiation among the populations can be attributed to small distances among the populations analyzed (0.2 to 10.8 km). No indication that populations from middle successional habitats show lower genetic variation than populations from early successional stages was found. The percentage of polymorphic fragments (82.8 and 84.8%) and the Shannon indexes (0.442 and 0.455) were similar in “candeial” and forest, respectively. These results suggest that if an increase in selection intensity occurred during succession, it did not result in a decrease in genetic diversity or that the selection effect was balanced by other factors, such as gene flow. Higher significant differentiation among E. erythropappus populations from “candeial” in relation to that among populations from forest was also not detected.

Comparative analysis of different DNA extraction protocols in fresh and herbarium specimens of the genus Dalbergia.

Five published DNA extraction protocols were compared for their ability to produce good quality DNA from fresh and herbarium leaves of several species of the genus Dalbergia. The leaves of these species contain high amounts of secondary metabolites, which make it difficult to perform a clean DNA extraction and thereby interfering with subsequent PCR amplification. The protocol that produced the best DNA quality in most of the Dalbergia species analyzed, utilizes polyvinylpyrrolidone to bind the phenolic compounds, a high molar concentration of NaCl to inhibit co-precipitation of polysaccharides and DNA, and LiCl for removing RNA by selective precipitation. The DNA quality of herbarium specimens was worse than that for fresh leaves, due to collecting conditions and preservation of samples. We analyzed 54 herbarium specimens, but the recovered DNA allowed successful PCR amplification in only eight. For the genus Dalbergia, the herbarium is an important source of material for phylogenetic and evolutionary studies; due to the occurrence of the different species in various geographical regions in Brazil, it is difficult to obtain fresh material in nature. Our results demonstrated that for Dalbergia species the methods used for the collection and preservation of herbarium specimens have a mayor influence on DNA quality and in the success of phylogenetic studies of the species.

Comparative analysis of different DNA extraction protocols in fresh and herbarium specimens of the genus Dalbergia

Five published DNA extraction protocols were compared for their ability to produce good quality DNA from fresh and herbarium leaves of several species of the genus Dalbergia. The leaves of these species contain high amounts of secondary metabolites, which make it difficult to perform a clean DNA extraction and thereby interfering with subsequent PCR amplification. The protocol that produced the best DNA quality in most of the Dalbergia species analyzed, utilizes polyvinylpyrrolidone to bind the phenolic compounds, a high molar concentration of NaCl to inhibit co-precipitation of polysaccharides and DNA, and LiCl for removing RNA by selective precipitation. The DNA quality of herbarium specimens was worse than that for fresh leaves, due to collecting conditions and preservation of samples. We analyzed 54 herbarium specimens, but the recovered DNA allowed successful PCR amplification in only eight. For the genus Dalbergia, the herbarium is an important source of material for phylogenetic and evolutionary studies; due to the occurrence of the different species in various geographical regions in Brazil, it is difficult to obtain fresh material in nature. Our results demonstrated that for Dalbergia species the methods used for the collection and preservation of herbarium specimens have a mayor influence on DNA quality and in the success of phylogenetic studies of the species

Variability in fruit and seed morphology among and within populations of Plathymenia (Leguminosae-Mimosoideae) in areas of the Cerrado, the Atlantic forest, and transitional sites.

Plathymenia reticulata is a tree species that occurs in two different Brazilian biomes, the Cerrado (a savannah environment), and the Atlantic Forest. In the present study, we evaluated morphological variation within and among five populations located in these vegetation types and in transitional sites in order to test the hypothesis that habitat selective pressures, being different in the Cerrado and the Atlantic Forest, would cause adaptive differences in morphological traits in individuals occurring under these different circumstances. Thirteen morphological traits of fruits, seeds, and of the membranous endocarp were obtained from 30 fruits and 20 seeds from each of nine to 10 individuals per population. Significant variation was found for all traits while comparing individuals within populations, and most traits varied significantly among populations as well. Some traits differed significantly between forest and Cerrado populations, while transition sites showed intermediate patterns and higher within-population variation. Contrary to our hypothesis, variation in seed size and mass among populations from different habitats was not significant. However, as predicted, the membranous endocarp was shorter for the seeds from Cerrado populations than from the Atlantic Forest. These data suggest the existence of P. reticulata ecotypes from Cerrado and Atlantic Forest; the relevance to Plathymenia evolution and to its wide ecological distribution is discussed.

Applicability of RAPD markers on silver-stained polyacrylamide gels to ascertain genetic diversity in Peripatus acacioi (Peripatidae; Onychophora)

RAPD (random amplification of polymorphic DNA) molecular markers can be utilized for analyzing genetic variability in populations for which only a few or no molecular markers are available. They were used in a study of an endangered species, Peripatus acacioi, found in the Tripuí Ecological Station, in Ouro Preto, MG, Brazil. The ecological station was specifically created to protect this velvet worm species, the first of this group found in Brazil. For an initial evaluation of the genetic diversity of this species, DNA samples from the lobopods of four individuals, collected at random, were analyzed using RAPD. Each reaction was run with a different primer (Operon RAPD 10-mer Kits), totaling 13 primers (OPC2, OPC3, OPC4, OPC6, OPC8, OPC10, OPC11, OPL2, OPL7, OPL11, OPL13, OPL18, and OPL19). Due to the low amplification yield, RAPD fragments were separated in polyacrylamide gels and stained with silver nitrate. Numerous bands were observed. Fifty-five of the amplified bands proved to be reproducible, both in terms of presence and intensity. Among these, 27 were variable and 28 were constant. The average number of bands per gel was 4.2. Nine of the 13 primers tested allowed the identification of constant and variable bands among these four individuals. RAPD analysis of genetic variation using silver-stained polyacrylamide gel electrophoresis provided measures of band sharing among the individuals, and therefore could be used in population genetics studies of P. acacioi.

Genetic diversity and structure of natural populations of Plathymenia reticulata (Mimosoideae), a tropical tree from the Brazilian Cerrado.

Plathymenia reticulata is a tropical tree native to the Brazilian Cerrado, one of the most important and endangered ecosystems in Brazil. This species presents high-quality wood and potential for recovery of degraded areas. Despite its importance, almost nothing is known about its genetic or ecological features. Random amplified polymorphic DNA (RAPD) markers were used to investigate the genetic diversity and structure of six natural populations of P. reticulata. DNAs from 117 adult individuals were amplified with 10 random primers and Shannon's index and amova were used to evaluate the levels of genetic diversity within and among populations. Through 72 markers, 70.8% of which were polymorphic, it was possible to obtain 117 unique RAPD phenotypes. The levels of genetic variability found in the six populations of P. reticulata were considerable and most of the genetic variation was found between individuals within populations, although pairwise PH(ST) values indicated significant divergence between populations. The among-population component accounted for, respectively, 12.3% and 16% of the genetic variation, according to amova and Shannon's index. These results were compared with other genetic studies on plant species and such a level of differentiation among populations corresponds to that which has usually been observed for outcrossing plants. The importance of maintenance of the P. reticulata populations and implications of the analysis of adult individuals, considering the longevity of this species and the relatively recent Cerrado fragmentation, are discussed.