Analysis of Mendelian inheritance and genetic linkage in microsatellite loci of Eucalyptus urophylla S.T. Blake.

Eucalyptus urophylla is an important species in the Brazilian forest sector due to its rapid growth rates and resistance to disease. The aim of this study was to verify Mendelian inheritance, genetic linkage, and genotypic disequilibrium for 15 microsatellite loci, with the goal of producing a robust set of genetic markers. Mendelian inheritance and genetic linkage analyses were carried out using genotypes from maternal trees, and their open-pollinated seeds and genotypic disequilibrium were assessed using adult trees. By comparing heterozygous maternal genotypes and their seeds, we found no significant deviations from the expected 1:1 Mendelian segregation and the expected 1:1:1:1 segregation hypothesis for pairwise loci. For adult trees, we did not find strong evidence of genotypic imbalance for pairwise loci. Our results indicated that the analyzed set of microsatellite loci could be used to carry out analyses of genetic diversity, mating system, and parentage in E. urophylla.

Mendelian inheritance, genetic linkage, and genotypic disequilibrium for nine microsatellite loci in Cariniana estrellensis (Raddi) Kuntze (Lecythidaceae).

Cariniana estrellensis is one of the largest trees found in Brazilian tropical forests. The species is typical of advanced stages of succession, characteristic of climax forests, and essential in genetic conservation and environmental restoration plans. In this study, we assessed Mendelian inheritance, genetic linkage, and genotypic disequilibrium in nine microsatellite loci for a C. estrellensis population. We sampled and genotyped 285 adult trees and collected seeds from 20 trees in a fragmented forest landscape in Brazil. Based on maternal genotypes and their seeds, we found no deviation from the expected 1:1 Mendelian segregation and no genetic linkage between pairwise loci. However, for adults, genotypic disequilibrium was detected for four pairs of loci, suggesting that this result was not caused by genetic linkage. Based on these results, we analyzed microsatellite loci that are suitable for use in population genetic studies assessing genetic diversity, mating system, and gene flow in C. estrellensis populations.

Contemporary pollen and seed dispersal in natural populations of Bertholletia excelsa (Bonpl.).

Due to the nutritional content and commercial value of its seeds, Bertholletia excelsa is one of the most important species exploited in the Amazon region. The species is hermaphroditic, insect pollinated, and its seeds are dispersed by barochory and animals. Because the fruit set is dependent on natural pollinator activity, gene flow plays a key role in fruit production. However, to date, there have been no studies on pollen and seed flow in natural populations of B. excelsa. Herein, we used microsatellite loci and parentage analysis to investigate the spatial genetic structure (SGS), realized pollen and seed dispersal, and effective pollen dispersal for two B. excelsa populations in the Brazilian Amazon forest. Two plots were established in natural forests from which adults, juveniles, and seeds were sampled. Realized and effective pollen flow was greater than realized seed flow. The distance of realized pollen dispersal ranged from 36 to 2060 m, and the distance of realized seed dispersal ranged from 30 to 1742 m. Both pollen and seeds showed a dispersal pattern of isolation by distance, indicating a high frequency of mating among near-neighbor trees and seed dispersal near to mother trees. Both populations present SGS up to 175 m, which can be explained by isolation by distance pollen and seed dispersal patterns. Our results suggested that fragmentation of these forest populations may result in a significant decrease in gene flow, due to the isolation by distance pollen and seed dispersal patterns.

Short Communication Mendelian inheritance, linkage, and genotypic disequilibrium in microsatellite loci of Hymenaea stigonocarpa Mart. ex Hayne (Fabaceae-Caesalpinioideae).

Hymenaea stigonocarpa is a deciduous and monoecious Neotropical tree species pollinated by bats. Due to overexploitation and habitat destruction, the population size has drastically diminished in nature. No previous study has investigated Mendelian inheritance, linkage, and genotypic disequilibrium in the available microsatellite markers in this species. So, our aim was to estimate these parameters using six microsatellite loci in a sample of 470 adults and 219 juveniles from two populations of H. stigonocarpa. In addition, 30 seeds per tree from 35 seed-trees were collected. Each seed was kept record of the seed-trees and fruit origin. Based on the six microsatellite loci, we found that only 10.6% of the cases showed significant deviations from Mendelian segregation and 15.3% showed linkage. We detected no evidence of genotypic disequilibrium between the loci in the adult trees or juveniles. Thus, our results suggest that these loci can be used with great accuracy in future genetic analyses of H. stigonocarpa populations.

Long-term impacts of selective logging on two Amazonian tree species with contrasting ecological and reproductive characteristics: inferences from Eco-gene model simulations.

The impact of logging and subsequent recovery after logging is predicted to vary depending on specific life history traits of the logged species. The Eco-gene simulation model was used to evaluate the long-term impacts of selective logging over 300 years on two contrasting Brazilian Amazon tree species, Dipteryx odorata and Jacaranda copaia. D. odorata (Leguminosae), a slow growing climax tree, occurs at very low densities, whereas J. copaia (Bignoniaceae) is a fast growing pioneer tree that occurs at high densities. Microsatellite multilocus genotypes of the pre-logging populations were used as data inputs for the Eco-gene model and post-logging genetic data was used to verify the output from the simulations. Overall, under current Brazilian forest management regulations, there were neither short nor long-term impacts on J. copaia. By contrast, D. odorata cannot be sustainably logged under current regulations, a sustainable scenario was achieved by increasing the minimum cutting diameter at breast height from 50 to 100 cm over 30-year logging cycles. Genetic parameters were only slightly affected by selective logging, with reductions in the numbers of alleles and single genotypes. In the short term, the loss of alleles seen in J. copaia simulations was the same as in real data, whereas fewer alleles were lost in D. odorata simulations than in the field. The different impacts and periods of recovery for each species support the idea that ecological and genetic information are essential at species, ecological guild or reproductive group levels to help derive sustainable management scenarios for tropical forests.

Mendelian inheritance, genetic linkage, and genotypic disequilibrium at microsatellite loci in Genipa americana L. (Rubiaceae).

Genipa americana is a tropical tree species that is widely distributed in the humid tropical and subtropical regions of Central and South America. This study investigated Mendelian inheritance, genetic linkage, and genotypic disequilibrium at six microsatellite loci developed for G. americana. Adult trees (188) and regenerants (163) were sampled and genotyped in a fragmented population of the species. We also genotyped open-pollinated seeds from 12 seed-trees during reproductive events in 2010 and 2011. Significant deviations from the expected 1:1 Mendelian segregation were detected in 29.5% of the tests. Significant genetic linkage between pairwise loci was detected in 54.4% of the tests, but no genotypic disequilibrium was detected between pairwise loci for adult trees and regenerants. Overall, the results indicate that the six loci analyzed may be used in studies of G. americana's genetic diversity and structure, its mating system, and in parentage analyses.

Edge effects enhance selfing and seed harvesting efforts in the insect-pollinated Neotropical tree Copaifera langsdorffii (Fabaceae).

Edge effects may affect the mating system of tropical tree species and reduce the genetic diversity and variance effective size of collected seeds at the boundaries of forest fragments because of a reduction in the density of reproductive trees, neighbour size and changes in the behaviour of pollinators. Here, edge effects on the genetic diversity, mating system and pollen pool of the insect-pollinated Neotropical tree Copaifera langsdorffii were investigated using eight microsatellite loci. Open-pollinated seeds were collected from 17 seed trees within continuous savannah woodland (SW) and were compared with seeds from 11 seed trees at the edge of the savannah remnant. Seeds collected from the SW had significantly higher heterozygosity levels (Ho=0.780; He=0.831) than seeds from the edge (Ho=0.702; He=0.800). The multilocus outcrossing rate was significantly higher in the SW (tm=0.859) than in the edge (tm=0.759). Pollen pool differentiation was significant, however, it did not differ between the SW (=0.105) and the edge (=0.135). The variance effective size within the progenies was significantly higher in the SW (Ne=2.65) than at the edge (Ne=2.30). The number of seed trees to retain the reference variance effective size of 500 was 189 at the SW and 217 at the edge. Therefore, it is preferable that seed harvesting for conservation and environmental restoration strategies be conducted in the SW, where genetic diversity and variance effective size within progenies are higher.

Mendelian inheritance, genetic linkage, and genotypic disequilibrium at nine microsatellite loci of Cariniana legalis (Mart.) O. Kuntze.

Cariniana legalis is one of the largest tropical trees with a wide distribution in the Brazilian Atlantic rainforest. We investigated the Mendelian inheritance, genetic linkage, and genotypic disequilibrium at seven microsatellite loci specifically isolated for C. legalis, and at two previously developed heterologous microsatellite loci. Forty to 100 open-pollinated seeds were collected from 22 seed-trees in two populations. Using the Bonferroni correction, no remarkable deviations from the expected Mendelian segregation, linkage, or genotypic disequilibrium were detected in the nine loci studied. Only 3.7% of the tests were significant for investigations of the Mendelian proportions. On the other hand, only 2.8% of tests for linkage detection showed significance. In addition, among all pairwise tests used for investigating linkage disequilibrium, significance was found in 9.7% of the locus pairs. Our results show clear evidence that the nine simple sequence repeat loci can be used without restriction in genetic diversity, mating system, and parentage analyses.

Genetic diversity and mating system of Copaifera langsdorffii (Leguminosae/Caesalpinioideae).

Copaifera langsdorffii, locally known as copaíba, is a valuable tropical tree with medicinal properties of its oil. We studied the genetic variation, genetic structure, and the mating system of trees in stands of C. langsdorffii (Leguminosae/Caesalpinioideae) located in an extensive area between the Pardo and Mogi-Guaçu basins in São Paulo State, Brazil, and their offspring, conserved in an ex situ germplasm bank at the University of São Paulo in Ribeirão Preto, SP, Brazil, using six microsatellite loci. Leaves were collected from 80 seed trees and from 259 offspring and their DNA extracted. A total of 140 and 175 alleles were found in the seed trees and their offspring, respectively. Low genetic differentiation was observed between stands, indicating intense gene flow due to efficient pollen dispersion vectors. An estimation of the outcrossing rate showed that these stands are outcrossed (tm = 0.98, P > 0.05). The mean variance of the effective population size of each family in two of the stands was 3.69 and 3.43, while the total effective population size retained in the germplasm bank was between 81 and 96. The paternity correlation was low, ranging from 0.052 to 0.148, demonstrating that the families implanted in this germplasm bank are composed predominantly of half-sibs.

Genetic structure in fragmented populations of Solanum lycocarpum A. St.-Hil. with distinct anthropogenic histories in a Cerrado region of Brazil.

Solanum lycocarpum is a woody tree widely distributed in the Cerrado that reaches high population densities in disturbed environments. We examined the genetic diversity and population differentiation of six S. lycocarpum populations with different degrees of human disturbance in order to determine if they are negatively affected by anthropogenic activity. Three populations located in southern and three located in southeastern regions of Goiás State, Central Brazil, were genotyped with five microsatellite markers. The population located in a protected area had higher number of alleles (26) than the remaining populations (19 to 21 alleles). It indicates that extensive and continuous areas of preserved native vegetation contribute positively to the conservation of genetic diversity, even with S. lycocarpum that easily adapts to disturbed environments. The three southeastern populations, although fragmented, had preserved native vegetation and were not significantly different from each other (θp = 0.002). All other population pairs compared were significantly divergent (θp varied from 0.03 to 0.11 between pairs, P < 0.05). We found three distinct sets of allele frequencies. The three southeastern populations shared similar gene pools, as well as the two disturbed southern populations, which are secondary vegetation. The southern population located in protected area had the most dissimilar gene pool. In conclusion, populations showing a higher degree of human disturbance tends to show a larger population differentiation than expected from the isolation by distance model, which in the current scenario of the Cerrado destruction points out to a threat to the long-term conservation of the species.

Mendelian inheritance, linkage and genotypic disequilibrium in microsatellite loci isolated from Hymenaea courbaril (Leguminosae).

The Neotropical tree Hymenaea courbaril, locally known as Jatobá, is a valuable source of lumber and also produces comestible and medicinal fruit. We characterized Mendelian inheritance, linkage and genotypic disequilibrium at nine microsatellite loci isolated from H. courbaril, in order to determine if they would provide accurate estimates of population genetic parameters of this important Amazon species. The study was made on 250 open-pollinated offspring originated from 14 seed trees. Only one of nine loci presented significant deviation from the expected Mendelian segregation (1:1). Genotypic disequilibrium between pairwise loci was investigated based on samples from 55 adult and 56 juvenile trees. No genetic linkage between any paired loci was observed. After Bonferroni's corrections for multiple tests, we found no evidence of genotypic disequilibrium between pairs of loci. We conclude that this set of loci can be used for genetic diversity/ structure, mating system, gene flow, and parentage analyses in H. courbaril populations.

Understanding the genetic diversity, spatial genetic structure and mating system at the hierarchical levels of fruits and individuals of a continuous Theobroma cacao population from the Brazilian Amazon.

Understanding the mating patterns of populations of tree species is a key component of ex situ genetic conservation. In this study, we analysed the genetic diversity, spatial genetic structure (SGS) and mating system at the hierarchical levels of fruits and individuals as well as pollen dispersal patterns in a continuous population of Theobroma cacao in Pará State, Brazil. A total of 156 individuals in a 0.56 ha plot were mapped and genotyped for nine microsatellite loci. For the mating system analyses, 50 seeds were collected from nine seed trees by sampling five fruits per tree (10 seeds per fruit). Among the 156 individuals, 127 had unique multilocus genotypes, and the remaining were clones. The population was spatially aggregated; it demonstrated a significant SGS up to 15 m that could be attributed primarily to the presence of clones. However, the short seed dispersal distance also contributed to this pattern. Population matings occurred mainly via outcrossing, but selfing was observed in some seed trees, which indicated the presence of individual variation for self-incompatibility. The matings were also correlated, especially within (Ρ(p(m))=0.607) rather than among the fruits (Ρ(p(m))=0.099), which suggested that a small number of pollen donors fertilised each fruit. The paternity analysis suggested a high proportion of pollen migration (61.3%), although within the plot, most of the pollen dispersal encompassed short distances (28 m). The determination of these novel parameters provides the fundamental information required to establish long-term ex situ conservation strategies for this important tropical species.

Low levels of realized seed and pollen gene flow and strong spatial genetic structure in a small, isolated and fragmented population of the tropical tree Copaifera langsdorffii Desf.

Over the past century, the Brazilian Atlantic forest has been reduced to small, isolated fragments of forest. Reproductive isolation theories predict a loss of genetic diversity and increases in inbreeding and spatial genetic structure (SGS) in such populations. We analysed eight microsatellite loci to investigate the pollen and seed dispersal patterns, genetic diversity, inbreeding and SGS of the tropical tree Copaifera langsdorffii in a small (4.8 ha), isolated population. All 112 adult trees and 128 seedlings found in the stand were sampled, mapped and genotyped. Seedlings had significantly lower levels of genetic diversity (A=16.5±0.45, mean±95% s.e.; H(e)=0.838±0.006) than did adult trees (A=23.2±0.81; H(e)=0.893±0.030). Parentage analysis did not indicate any seed immigration (m(seeds)=0) and the pollen immigration rate was very low (m(pollen)=0.047). The average distance of realized pollen dispersal within the stand was 94 m, with 81% of the pollen travelling <150 m. A significant negative correlation was found between the frequency and distance of pollen dispersal (r=-0.79, P<0.01), indicating that short-distance pollinations were more frequent. A significant SGS for both adults (∼50 m) and seedlings (∼20 m) was also found, indicating that most of the seeds were dispersed over short distances. The results suggested that the spatial isolation of populations by habitat fragmentation can restrict seed and pollen gene flow, increase SGS and affect the genetic diversity of future generations.

Verifying the geographic origin of mahogany (Swietenia macrophylla King) with DNA-fingerprints.

Illegal logging is one of the main causes of ongoing worldwide deforestation and needs to be eradicated. The trade in illegal timber and wood products creates market disadvantages for products from sustainable forestry. Although various measures have been established to counter illegal logging and the subsequent trade, there is a lack of practical mechanisms for identifying the origin of timber and wood products. In this study, six nuclear microsatellites were used to generate DNA fingerprints for a genetic reference database characterising the populations of origin of a large set of mahogany (Swietenia macrophylla King, Meliaceae) samples. For the database, leaves and/or cambium from 1971 mahogany trees sampled in 31 stands from Mexico to Bolivia were genotyped. A total of 145 different alleles were found, showing strong genetic differentiation (δ(Gregorious)=0.52, F(ST)=0.18, G(ST(Hedrick))=0.65) and clear correlation between genetic and spatial distances among stands (r=0.82, P<0.05). We used the genetic reference database and Bayesian assignment testing to determine the geographic origins of two sets of mahogany wood samples, based on their multilocus genotypes. In both cases the wood samples were assigned to the correct country of origin. We discuss the overall applicability of this methodology to tropical timber trading.

Patterns of pollen and seed dispersal in a small, fragmented population of the wind-pollinated tree Araucaria angustifolia in southern Brazil.

Paternity analysis based on eight microsatellite loci was used to investigate pollen and seed dispersal patterns of the dioecious wind-pollinated tree, Araucaria angustifolia. The study sites were a 5.4 ha isolated forest fragment and a small tree group situated 1.7 km away, located in Paranalpha State, Brazil. In the forest fragment, 121 males, 99 females, 66 seedlings and 92 juveniles were mapped and genotyped, together with 210 seeds. In the tree group, nine male and two female adults were mapped and genotyped, together with 20 seeds. Paternity analysis within the forest fragment indicated that at least 4% of the seeds, 3% of the seedlings and 7% of the juveniles were fertilized by pollen from trees in the adjacent group, and 6% of the seeds were fertilized by pollen from trees outside these stands. The average pollination distance within the forest fragment was 83 m; when the tree group was included the pollination distance was 2006 m. The average number of effective pollen donors was estimated as 12.6. Mother-trees within the fragment could be assigned to all seedlings and juveniles, suggesting an absence of seed immigration. The distance of seedlings and juveniles from their assigned mother-trees ranged from 0.35 to 291 m (with an average of 83 m). Significant spatial genetic structure among adult trees, seedlings, and juveniles was detected up to 50 m, indicating seed dispersal over a short distance. The effective pollination neighborhood ranged from 0.4 to 3.3 ha. The results suggest that seed dispersal is restricted but that there is long-distance pollen dispersal between the forest fragment and the tree group; thus, the two stands of trees are not isolated.