Maristerra R. Lemes

Population genetic structure of mahogany (Swietenia macrophylla King, Meliaceae) across the Brazilian Amazon, based on variation at microsatellite loci: implications for conservation

Mahogany (Swietenia macrophylla, Meliaceae) is the most valuable and intensively exploited Neotropical tree. No information is available regarding the genetic structure of mahogany in South America, yet the region harbours most of the unlogged populations of this prized hardwood. Here we report on the genetic diversity within and the differentiation among seven natural populations separated by up to 2100 km along the southern arc of the Brazilian Amazon basin. We analysed the variation at eight microsatellite loci for 194 adult individuals. All loci were highly variable, with the number of alleles per locus ranging from 13 to 27 (mean = 18.4). High levels of genetic diversity were found for all populations at the eight loci (mean HE = 0.781, range 0.754–0.812). We found moderate but statistically significant genetic differentiation among populations considering both estimators of FST and RST, θ = 0.097 and ρ = 0.147, respectively. Estimates of θ and ρ were significantly greater than zero for all pairwise population comparisons. Pairwise ρ‐values were positively and significantly correlated with geographical distance under the isolation‐by‐distance model. Furthermore, four of the populations exhibited a significant inbreeding coefficient. The finding of local differentiation among Amazonian mahogany populations underscores the need for in situ conservation of multiple populations of S. macrophylla across its distribution in the Brazilian Amazon. In addition, the occurrence of microgeographical genetic differentiation at a local scale indicates the importance of maintaining populations in their diverse habitats, especially in areas with mosaics of topography and soil.

Genetic structure of Mesoamerican populations of Big‐leaf mahogany (Swietenia macrophylla) inferred from microsatellite analysis

While microsatellites have been used to examine genetic structure in local populations of Neotropical trees, genetic studies based on such high‐resolution markers have not been carried out for Mesoamerica as a whole. Here we assess the genetic structure of the Mesoamerican mahogany Swietenia macrophylla King (big‐leaf mahogany), a Neotropical tree species recently listed as endangered in CITES which is commercially extinct through much of its native range. We used seven variable microsatellite loci to assess genetic diversity and population structure in eight naturally established mahogany populations from six Mesoamerican countries. Measures of genetic differentiation (FST and RST) indicated significant differences between most populations. Unrooted dendrograms based on genetic distances between populations provide evidence of strong phylogeographic structure in Mesoamerican mahogany. The two populations on the Pacific coasts of Costa Rica and Panama were genetically distant from all the others, and from one another. The remaining populations formed two clusters, one comprised of the northern populations of Mexico, Belize and Guatemala and the other containing the southern Atlantic populations of Nicaragua and Costa Rica. Significant correlation was found between geographical distance and all pairwise measures of genetic divergence, suggesting the importance of regional biogeography and isolation by distance in Mesoamerican mahogany. The results of this study demonstrate greater phylogeographic structure than has been found across Amazon basin S. macrophylla. Our findings suggest a relatively complex Mesoamerican biogeographic history and lead to the prediction that other Central American trees will show similar patterns of regional differentiation.

Extreme long‐distance dispersal of the lowland tropical rainforest tree Ceiba pentandra L. (Malvaceae) in Africa and the Neotropics

Many tropical tree species occupy continental expanses of rainforest and flank dispersal barriers such as oceans and mountains. The role of long‐distance dispersal in establishing the range of such species is poorly understood. In this study, we test vicariance hypotheses for range disjunctions in the rainforest tree Ceiba pentandra, which is naturally widespread across equatorial Africa and the Neotropics. Approximate molecular clocks were applied to nuclear ribosomal [ITS (internal transcribed spacer)] and chloroplast (psbB‐psbF) spacer DNA sampled from 12 Neotropical and five West African populations. The ITS (N = 5) and psbB‐psbF (N = 2) haplotypes exhibited few nucleotide differences, and ITS and psbB‐psbF haplotypes were shared by populations on both continents. The low levels of nucleotide divergence falsify vicariance explanations for transatlantic and cross‐Andean range disjunctions. The study shows how extreme long‐distance dispersal, via wind or marine currents, creates taxonomic similarities in the plant communities of Africa and the Neotropics.

Optimal sampling strategy for estimation of spatial genetic structure in tree populations

Fine-scale spatial genetic structure (SGS) in natural tree populations is largely a result of restricted pollen and seed dispersal. Understanding the link between limitations to dispersal in gene vectors and SGS is of key interest to biologists and the availability of highly variable molecular markers has facilitated fine-scale analysis of populations. However, estimation of SGS may depend strongly on the type of genetic marker and sampling strategy (of both loci and individuals). To explore sampling limits, we created a model population with simulated distributions of dominant and codominant alleles, resulting from natural regeneration with restricted gene flow. SGS estimates from subsamples (simulating collection and analysis with amplified fragment length polymorphism (AFLP) and microsatellite markers) were correlated with the ‘real’ estimate (from the full model population). For both marker types, sampling ranges were evident, with lower limits below which estimation was poorly correlated and upper limits above which sampling became inefficient. Lower limits (correlation of 0.9) were 100 individuals, 10 loci for microsatellites and 150 individuals, 100 loci for AFLPs. Upper limits were 200 individuals, five loci for microsatellites and 200 individuals, 100 loci for AFLPs. The limits indicated by simulation were compared with data sets from real species. Instances where sampling effort had been either insufficient or inefficient were identified. The model results should form practical boundaries for studies aiming to detect SGS. However, greater sample sizes will be required in cases where SGS is weaker than for our simulated population, for example, in species with effective pollen/seed dispersal mechanisms.

Monitoring genetic diversity in tropical trees with multilocus dominant markers

Since no universal codominant markers are currently available, dominant genetic markers, such as amplified fragment length polymorphism (AFLP), are valuable tools for assessing genetic diversity in tropical trees. However, the measurement of genetic diversity (H) with dominant markers depends on the frequency of null homozygotes (Q) and the fixation index (F) of populations. While Q can be estimated for AFLP loci, F is less accessible. Through a modelling approach, we show that the monolocus estimation of genetic diversity is strongly dependent on the value of F, but that the multilocus diversity estimate is surprisingly robust to variations in F. The robustness of the estimate is due to a mechanistic effect of compensation between negative and positive biases of H by different AFLP loci exhibiting contrasting frequency profiles of Q. The robustness was tested across contrasting theoretical frequency profiles of Q and verified for 10 neotropical species. Practical recommendations for the implementation of this analytical method are given for genetic surveys in tropical trees, where such markers are widely applied.

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.

Chloroplast DNA microsatellites reveal contrasting phylogeographic structure in mahogany (Swietenia macrophylla King, Meliaceae) from Amazonia and Central America

Big-leaf mahogany (Swietenia macrophylla King) is one of the most valuable and overharvested timber trees of tropical America. In order to better characterize geographic patterns of genetic variation, we performed a phylogeographic analysis of S. macrophylla based on six polymorphic chloroplast genome simple sequence repeat loci (cpSSRs) analyzed in 16 populations (N = 245 individuals) distributed across Central America and the Brazilian Amazon. Of the 31 total cpDNA haplotypes identified, 16 occurred in Central America and 15 in Amazonia with no single haplotype shared between the two regions. Populations from Central America showed moderate differentiation (FST = 0.36) while within population genetic diversity was generally high (mean Nei’s HE = 0.639). In contrast, the Amazonian populations were strongly differentiated (FST = 0.91) and contained relatively low genetic diversity (mean HE = 0.176), except for one highly diverse population (HE = 0.925) from eastern Amazonia. Spatial analysis of molecular variance (SAMOVA) identified a single Central American phylogroup and four Amazonian phylogroups, indicating stronger phylogeographic structure within Amazonia. The results demonstrate distinctive regional patterns of S. macrophylla differentiation, and the first evidence of a strong phylogeographic break between Central American and South American mahogany populations. We suggest that the frequent occurrence of hurricanes in Central America, the differences in the glacial histories and in the duration and intensity of anthropogenic disturbance during the late Holocene may have played important roles in the geographic structuring of cpDNA lineages in the two regions. The high private haplotype diversity in Brazilian populations suggests that cpSSRs can be used as DNA barcodes for regional timber certification.

Low plant density enhances gene dispersal in the Amazonian understory herb Heliconia acuminata.

In theory, conservation genetics predicts that forest fragmentation will reduce gene dispersal, but in practice, genetic and ecological processes are also dependent on other population characteristics. We used Bayesian genetic analyses to characterize parentage and propagule dispersal in Heliconia acuminata L. C. Richard (Heliconiaceae), a common Amazonian understory plant that is pollinated and dispersed by birds. We studied these processes in two continuous forest sites and three 1‐ha fragments in Brazils Biological Dynamics of Forest Fragments Project. These sites showed variation in the density of H. acuminata. Ten microsatellite markers were used to genotype flowering adults and seedling recruits and to quantify realized pollen and seed dispersal distances, immigration of propagules from outside populations, and reproductive dominance among parents. We tested whether gene dispersal is more dependent on fragmentation or density of reproductive plants. Low plant densities were associated with elevated immigration rates and greater propagule dispersal distances. Reproductive dominance among inside‐plot parents was higher for low‐density than for high‐density populations. Elevated local flower and fruit availability is probably leading to spatially more proximal bird foraging and propagule dispersal in areas with high density of reproductive plants. Nevertheless, genetic diversity, inbreeding coefficients and fine‐scale spatial genetic structure were similar across populations, despite differences in gene dispersal. This result may indicate that the opposing processes of longer dispersal events in low‐density populations vs. higher diversity of contributing parents in high‐density populations balance the resulting genetic outcomes and prevent genetic erosion in small populations and fragments.

Development and characterization of microsatellite markers for the Brazil nut tree Bertholletia excelsa Humb. & Bonpl. (Lecythidaceae).

Twelve polymorphic microsatellite markers were developed for the Brazil nut (Bertholletia excelsa), one of the most valuable non‐timber forest products from the Amazon, based on enrichment protocol. Six to 18 (mean 10.4) alleles per locus were identified and the expected heterozygosity ranged from 0.663 to 0.923 based on a screen of 40 individuals from one population of B. excelsa. The combined probabilities of genetic identity (8.39 × 10−17) and paternity exclusion (0.999999) indicated that multilocus genotypes are likely to be unique allowing precise analyses of genetic structure, gene flow, and mating system of this economically important species.

Cross-amplification and characterization of microsatellite loci for three species of Theobroma (Sterculiaceae) from the Brazilian Amazon

This study reports on the cross-species amplification of 23 microsatellite markers previously developed for Theobromacacao L. (Sterculiaceae), source of chocolate in three economically important Amazonian species of Theobroma (T. grandiflorum, T. subincanum, T. sylvestre). Thirteen of the 23 microsatellite loci tested were polymorphic across the three species at 2–13 alleles per locus. The observed heterozygosity per locus varied from 0.18 to 0.84 and expected heterozygosity ranged from 0.28 to 0.87. The high level of transferability and genetic information content of these microsatellite loci indicate their usefulness for population genetic, mating system and breeding studies of these economically important Amazonian fruit trees.