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.

Validation of EST-derived microsatellite markers for two Cerrado-endemic Campomanesia (Myrtaceae) species.

We assessed the transferability of 120 EST-derived Eucalyptus microsatellite primers to Campomanesia adamantium and C. pubescens. Both species are berry trees native to the Brazilian Cerrado, and population genetic information is poor. Twelve markers were used to analyze the genetic variability of four sampled populations. Regarding DNA extraction, we sampled leaf tissues from two populations of each species (80 individuals). Of the 120 primers evaluated, 87 did not amplify any PCR products, and 21 rendered nonspecific amplification. Twelve primers were successfully transferred, providing a low combined probability of genetic identity for both species (5.718 x 10(-10) for C. adamantium; 1.182 x 10(-11) for C. pubescens) and a high probability of paternity exclusion (0.99939 for C. adamantium; 0.99982 for C. pubescens). The average number of alleles in the polymorphic loci was 6.8 for C. adamantium and 7.8 for C. pubescens, ranging from 2 to 16 alleles per locus. The observed heterozygosity values for C. adamantium and C. pubescens were 0.504 and 0.503, respectively, and the expected heterozygosity values for C. adamantium and C. pubescens were 0.517 and 0.579, respectively. The populations exhibited structured genetic variability with qP values of 0.105 for C. adamantium and 0.249 for C. pubescens. Thus, we concluded that these 12 microsatellite markers, transferred from Eucalyptus, were efficient for population genetic studies of C. adamantium and C. pubescens.

Exhaustive search for conservation networks of populations representing genetic diversity.

Conservation strategies routinely use optimization methods to identify the smallest number of units required to represent a set of features that need to be conserved, including biomes, species, and populations. In this study, we provide R scripts to facilitate exhaustive search for solutions that represent all of the alleles in networks with the smallest possible number of populations. The script also allows other variables to be added to describe the populations, thereby providing the basis for multi-objective optimization and the construction of Pareto curves by averaging the values in the solutions. We applied this algorithm to an empirical dataset that comprised 23 populations of Eugenia dysenterica, which is a tree species with a widespread distribution in the Cerrado biome. We observed that 15 populations would be necessary to represent all 249 alleles based on 11 microsatellite loci, and that the likelihood of representing all of the alleles with random networks is less than 0.0001. We selected the solution (from two with the smallest number of populations) obtained for the populations with a higher level of climatic stability as the best strategy for in situ conservation of genetic diversity of E. dysenterica. The scripts provided in this study are a simple and efficient alternative to more complex optimization methods, especially when the number of populations is relatively small (i.e., <25 populations).

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.

Development of microsatellite markers for Hancornia speciosa Gomes (Apocynaceae).

Herein, we describe 34 microsatellite loci developed using an enrichment genomic library for the tree species Hancornia speciosa Gomes (Apocynaceae). Thirty-five individuals were genotyped using 34 primers to analyze the polymorphisms at each locus. The number of alleles per locus ranged from 4 to 20. The average number of alleles was 8.11, and the expected heterozygosity ranged from 0.62 to 0.94. These microsatellite primers will be useful in population genetics studies for this species.

Isolation and characterization of microsatellite loci from the tick Amblyomma aureolatum (Acari: Ixodidae).

Amblyomma aureolatum (Pallas) is the main vector of the bacterium Rickettsia rickettsii, the etiological agent of Brazilian spotted fever. This disease is the most lethal human spotted fever rickettsiosis in the world. Microsatellite loci were isolated from a dinucleotide-enriched library produced from A. aureolatum sampled in Southeastern Brazil. Eight polymorphic microsatellites were further characterized among 38 individuals sampled from São Paulo metropolitan region. The number of observed alleles ranged from 2 to 9, observed heterozygosity was 0.184-0.647, and expected heterozygosity was 0.251-0.747. Cross-species amplifications suggested that these loci will be useful for other Amblyomma species.

Geographical genetics of Pseudoplatystoma punctifer (Castelnau, 1855) (Siluriformes, Pimelodidae) in the Amazon Basin.

Geographical genetics allows the evaluation of evolutionary processes underlying genetic variation within and among local populations and forms the basis for establishing more effective strategies for biodiversity conservation at the population level. In this study, we used explicit spatial analyses to investigate molecular genetic variation (estimated using 7 microsatellite markers) of Pseudoplatystoma punctifer, by using samples obtained from 15 localities along the Madeira River and Solimões, Amazon Basin. A high genetic diversity was observed associated with a relatively low FST (0.057; P < 0.001), but pairwise FST values ranged from zero up to 0.21 when some pairs of populations were compared. These FST values have a relatively low correlation with geographic distances (r = 0.343; P = 0.074 by Mantel test), but a Mantel correlogram revealed that close populations (up to 80 km) tended to be more similar than expected by chance (r = 0.360; P = 0.015). The correlogram also showed a exponential-like decrease of genetic similarity with distance, with a patch-size of around 200 km, compatible with isolation-by-distance and analogous processes related to local constraints of dispersal and spatially structured levels of gene flow. The pattern revealed herein has important implications for establishing strategies to maintain genetic diversity in the species, especially considering the threats due to human impacts caused by building large dams in this river system.

Kin structure and parallel dispersal in the black-and-gold howler monkey Alouatta caraya (Platyrrhini, Atelidae).

We here investigated the kin structure and pattern of dispersal in the black-and-gold howler monkey (Alouatta caraya, Platyrrhini, Atelidae) based on genotype differences at nine microsatellite loci of 48 individuals from eight social groups along the riparian forest of the Tocantins River, Brazil. The genetic diversity (HE = 0.647) was similar to or higher than previously reported values in other Alouatta species. Given that no spatial kinship structure was detected, we found no evidence that dispersal was constrained by distance within the spatial scale analyzed (<25 km). Although no evidence was found for sex-biased dispersal, our results strongly suggest that extra-group copulations are common in A. caraya, and that both males and females disperse from their natal group.

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.

Isolation and characterization of 10 microsatellite loci for Pallas' long-tongued bat Glossophaga soricina (Phyllostomidae).

Glossophaga soricina is a widespread Neotropical nectarivorous bat. We characterized 10 microsatellite loci isolated from a shotgun genomic library. We analyzed tissues from wing membrane of 67 individuals collected from two populations of Central Brazil (Brasília and Alto Paraíso). The number of alleles per locus ranged from 2 to 20, and the observed and expected heterozygosities ranged from 0.015 to 0.666 and from 0.016 to 0.915, respectively. The high combined probability of genetic identity (4.369 x 10(-8)) and probability of paternity exclusion (0.996) showed that these microsatellite loci would be useful for population genetic structure and parentage studies in natural populations of G. soricina.

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.

Heterologous amplification and characterization of microsatellite markers in the Neotropical fish Leporinus friderici.

Leporinus friderici, native to the Amazon Basin and popularly known as "piau-três-pintas", has great ecological and economic importance; it is widely fished and consumed throughout much of tropical South America. Knowledge of the genetic diversity of this native species is important to support management and conservation programs. We evaluated microsatellite loci amplification, using heterologous primers, in 31 individuals of L. friderici. These samples were collected from natural populations of the Araguaia River basin, in central Brazil, and the DNA was extracted from samples of muscle tissue. Eight loci were successfully analyzed. Six of them were polymorphic, and the number of alleles ranged from three to 10. Values of expected heterozygosities for these polymorphic loci ranged from 0.488 to 0.795. Exclusion probability (0.983), the identity probability (0.000073), and the mean genetic diversity values were high, showing that these microsatellite markers are suitable for assessing the genetic variability of L. friderici populations. There is a growing interest in studies that evaluate the genetic variability of natural populations for various purposes, such as conservation. Here, we showed that a viable alternative to the costly development of specific primers for fish populations is simply testing for heterologous amplification of microsatellite markers available from research on other species.

Development of microsatellite markers for the endangered Neotropical tree species Tibouchina papyrus (Melastomataceae).

We isolated and characterized 12 microsatellite loci for Tibouchina papyrus (Melastomataceae), an endangered species with narrow and disjunct range, endemics to a few localities in "cerrado rupestre" from Central Brazil. These microsatellites were obtained by sequencing of a genomic shotgun library for primer design. Leaves from 96 individuals collected in the three known local populations were genotyped using the 12 primers designed to analyze the polymorphisms at each locus. The number of alleles per locus ranged from one to six; two loci were monomorphic. Among the polymorphic loci, expected heterozygosities ranged from 0.161 to 0.714. Combined paternity exclusion probability was 0.957 and combined genetic identity (0.051) was high for studies on parentage. Tibouchina papyrus is a rare and endemic tree species of outcrop quartzite and sandstone soils, with highly isolated populations, which may have lead to the low degree of polymorphism that we detected. Also, motifs of most loci are larger than dinucleotide, which typically display lower levels of polymorphism.

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.

Isolation and characterization of microsatellite markers in the armored catfish Hypostomus gymnorhynchus (Loricariidae).

We isolated and characterized 10 microsatellite loci in the armored catfish (Hypostomus gymnorhynchus, Loricariidae), using a genomic shotgun library to obtain the repetitive sequences. Twenty-four primers were designed and 14 individuals of H. gymnorhynchus from the Caiapó River, in central Brazil, were genotyped using these primers to analyze the polymorphism at each locus. All loci showed low polymorphism, with a low number of alleles per locus (1 or 2), except locus Hg_E19, which had 11 alleles. Expected heterozygosities for polymorphic loci ranged from 0.182 to 0.901. Combined paternity exclusion probability (0.857) was low and combined genetic identity (0.0026) was high, when we examined parentage. The low degree of polymorphism that we detected may be due to the small sample size and the small microsatellite size, despite the large motif size.

Development of microsatellite markers for Hoplias malabaricus (Erythrinidae).

We identified 14 microsatellite loci for the wolf fish, Hoplias malabaricus (Erythrinidae), from a genomic shotgun library. Twenty-five primers were designed, and 48 individuals of H. malabaricus from four localities of northwest Goiás, in central Brazil, were genotyped to characterize the polymorphism at each locus. Fourteen primers amplified clearly interpretable products using a single PCR protocol; six loci were polymorphic, but with a low number of alleles per locus (2 or 3). Expected heterozygosities for polymorphic loci ranged from 0.136 to 0.505. Combined paternity exclusion probability (0.638) was low and combined genetic identity (0.056) was high in studies of parentage. The low polymorphism may be due to the small microsatellite size and the large size of the motifs.

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.

Genetic diversity of natural populations of Anemopaegma arvense (Bignoniaceae) in the Cerrado of São Paulo State, Brazil.

Catuaba (Anemopaegma arvense), a Bignoniaceae species endemic to Cerrado regions, shows anticancer properties and is widely used as a stimulant in traditional medicine. We evaluated the genetic diversity of seven populations found in the State of São Paulo, using random amplified polymorphic DNA markers. After optimization of the amplification reaction, 10 selected primers produced 70 reproducible bands, with 72.8% polymorphism. The greatest genetic diversity was observed within populations (71.72%). Variation estimates, theta(B) (0.2421) and Phi(ST) (0.283), obtained by inter- and intra-populational analysis of genetic variability of catuaba, indicated considerable population structure. However, the r value 0.346 (P = 0.099), calculated by the Mantel test, indicates that the genetic diversity among populations is not strongly structured in geographical space, although there appears to be a tendency towards structuring.