Landscape barriers to pollen and seed flow in the dioecious tropical tree Astronium fraxinifolium in Brazilian savannah.

Gene flow studies provide information on gene exchange between populations, which is essential for developing genetic conservation strategies. Such analyses enable a better understanding of the life history and seed and pollen dispersal mechanisms of plant species. In this study, we investigate pollen and seed flow in a regenerant population of the pioneer species Astronium fraxinifolium in an area degraded during the construction of a hydroelectric dam. We mapped, sampled, sexed, and genotyped 386 individuals in the regenerant population (RP), as well as 128 adult trees located along two highways adjacent to the degraded area; one in Mato Grosso do Sul State (MS) and other in São Paulo State (SP). Parentage analyses was carried out for 370 individuals of the RP population, using as putative parents 348 individuals from RP and all 128 individuals sampled in MS and SP. Based on parentage analysis and eight microsatellite loci, our analyses revealed that for individuals of the RP with an identified father (pollen donor), 1.1% of the pollen was dispersed up to 532 m, while for those with an identified mother (seed donor), 0.5% of seeds were dispersed up to 4,782 m. However, a large proportion of pollen (76.5%) and seeds (57%) immigrated from trees outside the sampled populations. Pollen and seeds were dispersed through a pattern of isolation by distance. Genetic diversity was significantly similar between adults of both highway populations and individuals from RP, with significant levels of inbreeding detected only in RP. Our results demonstrate that the nearest trees contributed pollen and seeds for the recovery of the degraded area, indicating reproductive spatial isolation among the sampled populations due to the damming of the river. Such results help to understand the process of regeneration for A. fraxinifolium in regenerant populations to inform strategies for conservation and environmental recovery with this species.

Isolation of 27 polymorphic nuclear microsatellite markers for Roupala montana var. brasiliensis (Proteaceae).

Microsatellite primers pairs were developed for the Neotropical tree Roupala montana var. brasiliensis for use in studies on genetic diversity, mating system, and gene flow. Forty-two primer pairs were developed, resulting in 27 polymorphic loci, with two to 27 alleles per locus. The primer pairs were validated against 34 R. montana var. brasiliensis adult trees from four populations. The observed (H o) and expected (H e)heterozygosities ranged among loci from 0.061 to 0.930 (mean of 0.544) and from 0.116 to 0.950 (mean of 0.700), respectively. Null alleles were observed for ten loci. No genotypic linkage disequilibrium was detected in any pair of loci. This set of loci is suitable for population genetic studies of the species.

Detection and application of novel SSR markers from transcriptome data for Astronium fraxinifolium Schott, a threatened Brazilian tree species.

Astronium fraxinifolium is an endangered tree species from Brazil. Due to its significance in environmental reforestation, as well as the continued exploitation of its wood, it is necessary to develop management programs that support the conservation of the species. Simple sequence repeats (SSR) or microsatellite markers are widely used in population genetic studies across a range of diverse organisms. In this study, we present the first SSR markers developed for A. fraxinifolium as well as their frequency and distribution based on transcriptome data. From transcriptome data, we identified more than 100 thousand sequences presenting microsatellites, with a predominant distribution of trinucleotide repeats. From the initial screening, we selected 20 microsatellite loci which were validated and evaluated for genetic indices in two natural populations. All loci were polymorphic, ranging from four to 11 alleles per locus. The observed and expected heterozygosities ranged from 0 to 1.0 and from 0.533 to 1.0, respectively, while the genetic differentiation (GST = 0.363) was greater within than between populations. The developed SSR loci from RNA-Seq data provides a foundation for future studies on genetic diversity and population structure, mating system, and gene flow for A. fraxinifolium populations and related species, aiming at conservation and management.

SNP Markers as a Successful Molecular Tool for Assessing Species Identity and Geographic Origin of Trees in the Economically Important South American Legume Genus Dipteryx.

Dipteryx timber has been heavily exploited in South America since 2000s due to the increasing international demand for hardwood. Developing tools for the genetic identification of Dipteryx species and their geographical origin can help to promote legal trading of timber. A collection of 800 individual trees, belonging to 6 different Dipteryx species, was genotyped based on 171 molecular markers. After the exclusion of markers out of Hardy-Weinberg equilibrium or with no polymorphism or low amplification, 83 nuclear, 29 chloroplast, 13 mitochondrial single nucleotide polymorphisms (SNPs), and 2 chloroplast and 5 mitochondrial INDELS remained. Six genetic groups were identified using Bayesian Structure analyses of the nuclear SNPs, which corresponded to the different Dipteryx species collected in the field. Seventeen highly informative markers were identified as suitable for species identification and obtained self-assignment success rates to species level of 78-96%. An additional set of 15 molecular markers was selected to determine the different genetic clusters found in Dipteryx odorata and Dipteryx ferrea, obtaining self-assignment success rates of 91-100%. The success to assign samples to the correct country of origin using all or only the informative markers improved when using the nearest neighbor approach (69-92%) compared to the Bayesian approach (33-80%). While nuclear and chloroplast SNPs were more suitable for differentiating the different Dipteryx species, mitochondrial SNPs were ideal for determining the genetic clusters of D. odorata and D. ferrea. These 32 selected SNPs will be invaluable genetic tools for the accurate identification of species and country of origin of Dipteryx timber.

Long-distance pollen and seed dispersal and inbreeding depression in Hymenaea stigonocarpa (Fabaceae: Caesalpinioideae) in the Brazilian savannah.

Hymenaea stigonocarpa is a neotropical tree that is economically important due to its high-quality wood; however, because it has been exploited extensively, it is currently considered threatened. Microsatellite loci were used to investigate the pollen and seed dispersal, mating patterns, spatial genetic structure (SGS), genetic diversity, and inbreeding depression in H. stigonocarpa adults, juveniles, and open-pollinated seeds, which were sampled from isolated trees in a pasture and trees within a forest fragment in the Brazilian savannah. We found that the species presented a mixed mating system, with population and individual variations in the outcrossing rate (0.53-1.0). The studied populations were not genetically isolated due to pollen and seed flow between the studied populations and between the populations and individuals located outside of the study area. Pollen and seed dispersal occurred over long distances (>8 km); however, the dispersal patterns were isolated by distance, with a high frequency of mating occurring between near-neighbor trees and seeds dispersed near the parent trees. The correlated mating for individual seed trees was higher within than among fruits, indicating that fruits present a high proportion of full-sibs. Genetic diversity and SGS were similar among the populations, but offspring showed evidence of inbreeding, mainly originating from mating among related trees, which suggests inbreeding depression between the seed and adult stages. Selfing resulted in a higher inbreeding depression than mating among relatives, as assessed through survival and height. As the populations are not genetically isolated, both are important targets for in situ conservation to maintain their genetic diversity; for ex situ conservation, seeds can be collected from at least 78 trees in both populations separated by at least 250 m.

Assessing the Ability of Chloroplast and Nuclear DNA Gene Markers to Verify the Geographic Origin of Jatoba (Hymenaea courbaril L.) Timber.

Deforestation-reinforced by illegal logging-is a serious problem in many tropical regions and causes pervasive environmental and economic damage. Existing laws that intend to reduce illegal logging need efficient, fraud resistant control methods. We developed a genetic reference database for Jatoba (Hymenaea courbaril), an important, high value timber species from the Neotropics. The data set can be used for controls on declarations of wood origin. Samples from 308 Hymenaea trees from 12 locations in Brazil, Bolivia, Peru, and French Guiana have been collected and genotyped on 10 nuclear microsatellites (nSSRs), 13 chloroplast SNPs (cpSNP), and 1 chloroplast indel marker. The chloroplast gene markers have been developed using Illumina DNA sequencing. Bayesian cluster analysis divided the individuals based on the nSSRs into 8 genetic groups. Using self-assignment tests, the power of the genetic reference database to judge on declarations on the location has been tested for 3 different assignment methods. We observed a strong genetic differentiation among locations leading to high and reliable self-assignment rates for the locations between 50% to 100% (average of 88%). Although all 3 assignment methods came up with similar mean self-assignment rates, there were differences for some locations linked to the level of genetic diversity, differentiation, and heterozygosity. Our results show that the nuclear and chloroplast gene markers are effective to be used for a genetic certification system and can provide national and international authorities with a robust tool to confirm legality of timber.

DNA microsatellite markers for Swartzia glazioviana (Fabaceae), a threatened species from the Brazilian Atlantic Forest.

PREMISE OF THE STUDY: Development and characterization of a set of DNA microsatellite markers for Swartzia glazioviana (Fabaceae), a naturally rare and threatened tree species, were carried out to investigate its conservation genetics. METHODS AND RESULTS: Through an enriched genomic library procedure, 10 DNA microsatellite loci were isolated and characterized for the species. The mean expected heterozygosity was 0.776 (0.424-0.894). Cross-species amplifications of these loci were successfully tested for six congener taxa (S. apetala var. apetala, S. flaemingii, S. langsdorffii, S. macrostachya, S. myrtifolia var. elegans, and S. simplex var. continentalis). CONCLUSIONS: The 10 polymorphic microsatellite markers developed are quite informative and will provide a valuable resource to study the population and conservation genetics of S. glazioviana and other Swartzia species.

Conservation implications of the mating system of the Pampa Hermosa landrace of peach palm analyzed with microsatellite markers.

Peach palm (Bactris gasipaes) is cultivated by many indigenous and traditional communities from Amazonia to Central America for its edible fruits, and is currently important for its heart-of-palm. The objective of this study was to investigate the mating system of peach palm, as this is important for conservation and breeding. Eight microsatellite loci were used to genotype 24 open-pollinated progenies from three populations of the Pampa Hermosa landrace maintained in a progeny trial for genetic improvement. Both the multi-locus outcrossing rates (0.95 to 0.99) and the progeny level multi-locus outcrossing rates (0.9 to 1.0) were high, indicating that peach palm is predominantly allogamous. The outcrossing rates among relatives were significantly different from zero (0.101 to 0.202), providing evidence for considerable biparental inbreeding within populations, probably due to farmers planting seeds of a small number of open-pollinated progenies in the same plot. The correlations of paternity estimates were low (0.051 to 0.112), suggesting a large number of pollen sources (9 to 20) participating in pollination of individual fruit bunches. Effective population size estimates suggest that current germplasm collections are insufficient for long-term ex situ conservation. As with most underutilized crops, on farm conservation is the most important component of an integrated conservation strategy.

Paternity analysis reveals significant isolation and near neighbor pollen dispersal in small Cariniana legalis Mart. Kuntze populations in the Brazilian Atlantic Forest.

Throughout the world, large trees are increasingly rare. Cariniana legalis is the tallest tree species of the Brazilian Atlantic Forest, reaching up to 60 m in height. Due to extensive deforestation of the Atlantic Forest, remnant C. legalis populations are small and spatially isolated, requiring the development of strategies for their conservation. For in situ and ex situ genetic conservation to be effective, it is important to understand the levels and patterns of spatial genetic structure (SGS), and gene flow. We investigated SGS and pollen flow in three small, physically isolated C. legalis stands using microsatellite loci. We measured, mapped, and sampled all C. legalis trees in the three stands: 65 trees from Ibicatu population, 22 trees from MGI, and 4 trees from MGII. We also collected and genotyped 600 seeds from Ibicatu, 250 seeds from MGI, and 200 seeds from MGII. Significant SGS was detected in Ibicatu up to 150 m, but substantial levels of external pollen flow were also detected in Ibicatu (8%), although not in MGI (0.4%) or MGII (0%). Selfing was highest in MGII (18%), the smallest group of trees, compared to MGI (6.4%) and Ibicatu (6%). In MGI and MGII, there was a strong pattern of mating among near-neighbors. Seed collection strategies for breeding, in situ and ex situ conservation and ecological restoration, must ensure collection from seed trees located at distances greater than 350 m and from several forest fragments.

Development and characterization of 32 microsatellite loci in Genipa americana (Rubiaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for the tree species Genipa americana (Rubiaceae) for further population genetic studies. • METHODS AND RESULTS: We identified 144 clones containing 65 repeat motifs from a genomic library enriched for (CT)8 and (GT)8 motifs. Primer pairs were developed for 32 microsatellite loci and validated in 40 individuals of two natural G. americana populations. Seventeen loci were polymorphic, revealing from three to seven alleles per locus. The observed and expected heterozygosities ranged from 0.24 to 1.00 and from 0.22 to 0.78, respectively. • CONCLUSIONS: The 17 primers identified as polymorphic loci are suitable to study the genetic diversity and structure, mating system, and gene flow in G. americana.

Long-distance dispersal in a fire- and livestock-protected savanna.

Savannas are highly diverse and dynamic environments that can shift to forest formations due to protection policies. Long-distance dispersal may shape the genetic structure of these new closed forest formations. We analyzed eight microsatellite loci using a single-time approach to understand contemporary pollen and effective seed dispersal of the tropical tree, Copaifera langsdorffii Desf. (Fabaceae), occurring in a Brazilian fire- and livestock-protected savanna. We sampled all adult trees found within a 10.24 ha permanent plot, young trees within a subplot of 1.44 ha and open-pollinated seeds. We detected a very high level of genetic diversity among the three generations in the studied plot. Parentage analysis revealed high pollen immigration rate (0.64) and a mean contemporary pollen dispersal distance of 74 m. In addition, half-sib production was 1.8 times higher than full-sibs in significant higher distances, indicating foraging activity preference for different trees at long distances. There was a significant and negative correlation between diameter at breast height (DBH) of the pollen donor with the number of seeds (r = -0.640, P-value = 0.032), suggesting that pollen donor trees with a higher DBH produce less seeds. The mean distance of realized seed dispersal (recruitment kernel) was 135 m due to the large home range dispersers (birds and mammals) in the area. The small magnitude of spatial genetic structure found in young trees may be a consequence of overlapping seed shadows and increased tree density. Our results show the positive side of closed canopy expansion, where animal activities regarding pollination and seed dispersal are extremely high.

Development of microsatellite primers for Jatropha curcas (Euphorbiaceae) and transferability to congeners.

PREMISE OF THE STUDY: Microsatellite primers were developed for Jatropha curcas (Euphorbiaceae), a tree species with large potential for biofuel production, to investigate its natural genetic diversity and mating system to facilitate the establishment of tree improvement and conservation programs. METHODS AND RESULTS: Using a protocol for genomic library enrichment, 104 clones containing 195 repeat motifs were identified. Primer pairs were developed for 40 microsatellite loci and validated in 41 accessions of J. curcas from six provenances. Nine loci were polymorphic revealing from two to eight alleles per locus, and six primers were able to amplify alleles in the congeners J. podagrica, J. pohliana, and J. gossypifolia, but not in other Euphorbiaceae species, such as Hevea brasiliensis, Manihot esculenta, or Ricinus communis. CONCLUSIONS: The primers developed here revealed polymorphic loci that are suitable for genetic diversity and structure, mating system, and gene flow studies in J. curcas, and some congeners.

Mating system of a population of Myracrodruon urundeuva F.F. & M.F. Allemão using the fAFLP molecular marker

The mating system and genetic diversity were studied in a natural population of Myracrodruon urundeuva originating from 30 open-pollinated trees at the Paulo de Faria Ecological Station, SP, Brazil. The progenies were planted on the Teaching and Research Farm of the Ilha Solteira Engineering School, UNESP. Using the fAFLP molecular marker, eleven loci were selected to study the mating system. The mating system was analyzed using the multilocus mixed-mating model. The estimates of genetic divergence between pollen and ovule allele frequencies were significant for eight loci, suggesting nonrandom outcrossing. The estimates of the multilocus outcrossing rate revealed that M. urundeuva possesses a mating system with a predominance of outcrossing events (theta = 0.940 ± 0.086). The estimates of coancestry among plants within progenies (theta = 0.185) was higher than that expected for half-sib progenies (0.125) and the indirect estimate of the correlation of outcrossed paternity within progeny arrays (r p) was 0.403, suggesting that progenies have a high proportion of full-sibs. Result analysis suggests the need for the application of biometric models that take into account deviations from random outcrossing in the estimations of genetic parameters for quantitative traits and the need for retaining large sample sizes in order to preserve genetic variability.

Mating system in a natural population of Theobroma grandiflorum (Willd. ex Spreng.) Schum., by microsatellite markers

The aim of this research was to study the mating system of a natural population of Theobroma grandiflorum (cupuassu) from Nova Ipixuna, Pará state, using microsatellite markers. ...