Discovery and characterization of new microsatellite loci in Dipteryx alata Vogel (Fabaceae) using next-generation sequencing data.

The use of next-generation sequencing (NGS) technologies provides a great volume of genome sequence data even for non-model species. The development of microsatellite markers using these data is a relatively quick and easy process. Dipteryx alata Vogel (Fabaceae) is an arboreal species from the Cerrado biome and is considered an important plant genetic resource. Here, we report the development of microsatellite markers for D. alata using NGS data. DNA samples from four individuals were sequenced using the Illumina MiSeq platform and high-quality reads were assembled into contigs of the D. alata genome sequence. Microsatellite regions were identified using the IMEX webserver and primer pairs were designed using the Primer3 software. The amplification settings for each locus were optimized. Fluorescent-labeled primers were developed and used to genotype individuals derived from three natural populations of D. alata. Fifty-four microsatellite regions were identified, from which 27 were elected to primer design. Among the amplified loci, 11 were polymorphic, with the number of alleles ranging from 2 to 10. The expected heterozygosity under Hardy-Weinberg Equilibrium (HWE) per locus varied from 0.191 to 0.807. Genotype and allele frequencies for all loci agreed with those expected under HWE and linkage disequilibrium was not significant for all pairs of loci. The probabilities of exclusion of paternity and of combined identity were equal to 0.993 and 5.65 x 10-8, respectively. The markers developed in this study are useful to several types of population genetic studies with D. alata and, eventually, for closely related species.

Multi-objective optimization in systematic conservation planning and the representation of genetic variability among populations.

Biodiversity crises have led scientists to develop strategies for achieving conservation goals. The underlying principle of these strategies lies in systematic conservation planning (SCP), in which there are at least 2 conflicting objectives, making it a good candidate for multi-objective optimization. Although SCP is typically applied at the species level (or hierarchically higher), it can be used at lower hierarchical levels, such as using alleles as basic units for analysis, for conservation genetics. Here, we propose a method of SCP using a multi-objective approach. We used non-dominated sorting genetic algorithm II in order to identify the smallest set of local populations of Dipteryx alata (baru) (a Brazilian Cerrado species) for conservation, representing the known genetic diversity and using allele frequency information associated with heterozygosity and Hardy-Weinberg equilibrium. We worked in 3 variations for the problem. First, we reproduced a previous experiment, but using a multi-objective approach. We found that the smallest set of populations needed to represent all alleles under study was 7, corroborating the results of the previous study, but with more distinct solutions. In the 2nd and 3rd variations, we performed simultaneous optimization of 4 and 5 objectives, respectively. We found similar but refined results for 7 populations, and a larger portfolio considering intra-specific diversity and persistence with populations ranging from 8-22. This is the first study to apply multi-objective algorithms to an SCP problem using alleles at the population level as basic units for analysis.

Demographic history and the low genetic diversity in Dipteryx alata (Fabaceae) from Brazilian Neotropical savannas.

Genetic effects of habitat fragmentation may be undetectable because they are generally a recent event in evolutionary time or because of confounding effects such as historical bottlenecks and historical changes in species' distribution. To assess the effects of demographic history on the genetic diversity and population structure in the Neotropical tree Dipteryx alata (Fabaceae), we used coalescence analyses coupled with ecological niche modeling to hindcast its distribution over the last 21 000 years. Twenty-five populations (644 individuals) were sampled and all individuals were genotyped using eight microsatellite loci. All populations presented low allelic richness and genetic diversity. The estimated effective population size was small in all populations and gene flow was negligible among most. We also found a significant signal of demographic reduction in most cases. Genetic differentiation among populations was significantly correlated with geographical distance. Allelic richness showed a spatial cline pattern in relation to the species' paleodistribution 21 kyr BP (thousand years before present), as expected under a range expansion model. Our results show strong evidences that genetic diversity in D. alata is the outcome of the historical changes in species distribution during the late Pleistocene. Because of this historically low effective population size and the low genetic diversity, recent fragmentation of the Cerrado biome may increase population differentiation, causing population decline and compromising long-term persistence.

Development and characterization of new microsatellites for Eugenia dysenterica DC (Myrtaceae).

Microsatellite markers were developed for population genetic analyses of the Neotropical tree Eugenia dysenterica DC (Myrtaceae), after construction of a shotgun genomic library for microsatellite discovery. Nine primers were designed, of which 5 yielded amplified product. These primers were polymorphic for 97 individuals collected in 3 distinct localities. The number of alleles per locus (primer) ranged from 3 to 11 and expected heterozygosities varied from 0.309 to 0.884. The probability of locus identity was ~1.88 x 10(-4) and the probability of paternity exclusion was ~0.9367. The 5 microsatellite primer pairs may be suitable for population genetic studies such as parentage and fine-scale genetic analyses of this species.

Transferability and characterization of microssatellite loci in Anacardium humile A. St. Hil. (Anacardiaceae).

Microsatellite markers were transferred from the cashew, Anarcadium occidentale, to Anacardium humile (Anacardiaceae), a Neotropical shrub from the Brazilian savanna, that produces an edible nut and pseudo-fruit. We tested 14 microsatellite primers from A. occidentale on A. humile. Polymorphism of each microsatellite locus was analyzed based on 58 individuals from three populations. Twelve loci amplified successfully and presented 2 to 9 alleles; expected heterozygosity ranged from 0.056 to 0.869. These 12 microsatellite loci provide a new tool for the generation of fundamental population genetic data for devising conservation strategies for A. humile.

Geographic shifts in climatically suitable areas and loss of genetic variability in Dipteryx alata ("Baru" Tree; Fabaceae).

Many species are expected to suffer strong shifts in their geographic ranges due to climate changes in the next 50 years, with severe consequences for biodiversity patterns and population structure. We used here an ensemble forecast approach for obtaining species' range in which multiple species distribution models and climatic models were combined to model loss of genetic variability in Baru, Dipteryx alata (Fabaceae), an economically important Neotropical tree native to the Cerrado of Brazil. We estimated a series of genetic parameters (number of alleles per locus, expected heterozygosity under Hardy-Weinberg equilibrium and mutation-drift equilibrium) for this species based on eight microsatellite loci. We then recalculated these parameters assuming that local populations in areas of low future environmental suitability will go extinct. All genetic parameters remained approximately constant up to a 50% threshold of climatic suitability in the future; after this critical threshold there is an abrupt reduction in all parameters, although the magnitude of shift is only about 10% of current values, on average. Thus, despite the shifts in geographic range and climatically suitable areas towards southeastern Brazil, our analyses do not predict a strong loss of genetic diversity in D. alata because of the broad tolerance of this species, which ensures large future ranges, contrasting with other Cerrado species that have been analyzed in a similar manner.

Combining multiple models to predict the geographical distribution of the Baru tree (Dipteryx alata Vogel) in the Brazilian Cerrado.

The Brazilian Cerrado is a biome of great biodiversity, but detailed information about the diversity and distribution of species in this region is still insufficient for both testing ecological hypotheses and for conservation purposes. Among native plants in the Cerrado, Dipteryx alata Vogel (commonly known as the "Baru" tree), has a high potential for exploitation. The aims of this paper were to predict the potential spatial distribution of D. alata in the Brazilian Cerrado utilising five different niche modelling techniques. These techniques usually provide distinct results, so it may be difficult to choose amongst them. To adjust for this uncertainty, we employ an ensemble forecasting approach to predict the spatial distribution of the Baru tree. We accumulated a total of 448 occurrence points and modelled the subsequent predicted occurrences using seven climatic variables. Five different presence-only ecological niche modelling techniques (GARP, Maxent, BIOCLIM, Mahalanobis Distance and Euclidean Distance) were used and the performance of these models was compared using Receiver Operating Characteristics (ROC) and the Area Under the Curve (AUC). All models presented AUC values higher than 0.68, and GARP presented the highest AUC value, whereas Euclidean Distance presented the lowest. The ensemble forecasting approach suggested a high suitability for the occurrence of the Baru tree in the Central-Western region of the Brazilian Cerrado. Our study demonstrated that modelling species distribution using ensemble forecasting can be an important computational tool for better establishing sampling strategies and for improving our biodiversity knowledge to better identify priority areas for conservation. For the Baru tree, we recommend priority actions for conservation in the central region of the Cerrado Biome.

Genetic characterization of natural populations of the medicinal plant Palicourea coriacea (Rubiaceae) with molecular markers.

Palicourea coriacea (Rubiaceae) is a herbaceous, perennial species typical of the Cerrado; it is popularly known as "douradinha", because of its yellow flowers. It is utilized in popular medicine, mainly for the treatment of renal diseases. We used RAPD markers to evaluate the genetic structure of nine natural populations of P. coriacea, totaling 168 individuals, collected in the States of Goiás and Bahia. This species showed a high level of genetic diversity, with He values varying between 0.259 and 0.338, with an overall mean of 0.296. Analysis by AMOVA revealed that 23% of the total variability was between populations and 77% was within populations. The estimate of apparent gene flow (Nm) was 0.83. Analyses of the fixation index (f ) using a Bayesian approach yielded a mean value of 0.98, suggesting that this is an autogamous species. Analyses of genetic divergence and spatial pattern of the populations utilizing theta(B) and Phi(ST) matrices, pair to pair, revealed no correlation between geographic distance and genetic distance; the nine populations grouped randomly, without relation to their geographic origin. The hypothesis that geographically close populations should be genetically close was discarded based on the Mantel test; the correlation was 0.155 (P = 0.23). The degree of interpopulational differentiation was relatively high, which allows us to recommend a strategy of sampling for the ex situ conservation of genetic variability, utilizing a larger number of populations. For in situ conservation, we suggest preservation of a larger number of areas in the Cerrado, where this species naturally occurs.

Optimizing reproducibility evaluation for random amplified polymorphic DNA markers.

The random amplified polymorphic DNA (RAPD) technique is often criticized because it usually shows low levels of repeatability; thus it can generate spurious bands. These problems can be partially overcome by rigid laboratory protocols and by performing repeatability tests. However, because it is expensive and time-consuming to obtain genetic data twice for all individuals, a few randomly chosen individuals are usually selected for a priori repeatability analysis, introducing a potential bias in genetic parameter estimates. We developed a procedure to optimize repeatability analysis based on RAPD data, which was applied to evaluate genetic variability in three local populations of Tibochina papyrus, an endemic Cerrado plant found in elevated rocky fields in Brazil. We used a simulated annealing procedure to select the smallest number of individuals that contain all bands and repeated the analyses only for those bands that were reproduced in these individuals. We compared genetic parameter estimates using HICKORY and POPGENE softwares on an unreduced data set and on data sets in which we eliminated bands based on repeatability of individuals selected by simulated annealing and based on three randomly selected individuals. Genetic parameter estimates were very similar when we used the optimization procedure to reduce the number of bands analyzed, but as expected, selecting only three individuals to evaluate the repeatability of bands produced very different estimates. We conclude that the problems of repeatability attributed to RAPD markers could be due to bias in the selection of loci and primers and not necessarily to the RAPD technique per se.

Transferability of short tandem repeat markers for two wild Canid species inhabiting the Brazilian Cerrado.

The maned wolf (Chrysocyon brachyurus) and the crab-eating fox (Cerdocyon thous) are two wild-canid species found in the Brazilian Cerrado. We tested cross-amplification and transferability of 29 short tandem repeat primers originally developed for cattle and domestic dogs and cats on 38 individuals of each of these two species, collected in the Emas National Park, which is the largest national park in the Cerrado region. Six of these primers were successfully transferred (CSSM-038, PEZ-05, PEZ-12, LOCO-13, LOCO-15, and PEZ-20); five of which were found to be polymorphic. Genetic parameter values (number of alleles per locus, observed and expected heterozygosities, and fixation indices) were within the expected range reported for canid populations worldwide.

Transferability of short tandem repeat markers for two wild Canid species inhabiting the Brazilian Cerrado

The maned wolf (Chrysocyon brachyurus) and the crab-eating fox (Cerdocyon thous) are two wild-canid species found in the Brazilian Cerrado. We tested cross-amplification and transferability of 29 short tandem repeat primers originally developed for cattle and domestic dogs and cats on 38 individuals of each of these two species, collected in the Emas National Park, which is the largest national park in the Cerrado region. Six of these primers were successfully transferred (CSSM-038, PEZ-05, PEZ-12, LOCO-13, LOCO-15, and PEZ-20); five of which were found to be polymorphic. Genetic parameter values (number of alleles per locus, observed and expected heterozygosities, and fixation indices) were within the expected range reported for canid populations worldwide.