Isolation and characterization of microsatellite loci for Hymenaea courbaril and transferability to Hymenaea stigonocarpa, two tropical timber species

Hymenaea courbaril is a tropical timber species, intensely exploited and found in the Amazon, Atlantic Forest and Brazilian Cerrado biome. Nine highly polymorphic microsatellite loci were developed from a genomic library enriched for AG/TC repeats. In a total of 41 individuals, from two natural populations, seven to 13 alleles per locus were detected and expected heterozygosity ranged from 0.75 to 0.90. Seven loci were effectively transferred to Hymenaea stigonocarpa. High levels of polymorphism make the present primers useful for population genetic studies and are a powerful tool to investigate mating system, gene flow and spatial genetic structure.     

Assessment of the diversity of Paenibacillus species in environmental samples by a novel rpoB-based PCR-DGGE method

A specific PCR system based on the gene encoding the RNA polymerase beta subunit, rpoB, was developed for amplification and denaturing gradient gel electrophoresis (DGGE) fingerprinting of Paenibacillus communities in environmental samples. This gene has been previously proven to be a powerful identification tool for the discrimination of species within the genus Paenibacillus and could avoid the limitations of 16S rRNA-based phylogenetic analysis. Initially, the PCR system based on universal rpoB primers were used to amplify DNAs of different Paenibacillus species. A new reverse primer (rpoBPAEN) was further designed based on an insertion of six nucleotides in the Paenibacillus sequences analyzed. This semi-nested PCR system was evaluated for specificity using DNAs isolated from 27 Paenibacillus species belonging to different 16S rRNA-based phylogenetic groups and seven non-Paenibacillus species. The non-Paenibacillus species were not amplified using this PCR approach and one group of Paenibacillus species consisting of strains without the six-base insert also were not amplified; these latter strains were found to be distinct based on 16S rRNA gene phylogeny. In addition, a clone library was generated from the rpoB fragments amplified from two Brazilian soil types (Cerrado and Forest) and all 62 clones sequenced were closely related to one of the 22 sequences from Paenibacillus previously obtained in this study. To assess the diversity of Paenibacillus species in Cerrado and Forest soils and in the rhizosphere of different cultivars of maize, a PCR-DGGE system was used. The Paenibacillus DGGE fingerprints showed a clear distinction between communities of Paenibacillus in Forest and Cerrado soils and rhizosphere samples clustered along Cerrado soil. Profiles of cultivars CMS22 and CMS36 clustered together, with only 53% of similarity to CMS11 and CMS04. The results presented here demonstrate the potential use of the rpoB-based Paenibacillus-specific PCR-DGGE method for studying the diversity of Paenibacillus populations in natural environments.     

Geographical ecology and conservation of Eugenia L. (Myrtaceae) in the Brazilian Cerrado: Past,present and future

Human actions have caused the fragmentation of natural vegetation, habitat loss and climate change. The Cerrado, considered one of the global hotspots of diversity, has suffered great habitat loss due to these factors, which has been aggravated by the agricultural expansion that took place during the last 60 years. In this context, we chose species of the genus Eugenia L. (Myrtaceae) occurring in the Brazilian Cerrado to describe richness patterns and range loss, and determine conservation priorities for the Cerrado. Ecological niche models (ENMs) were applied to calculate the geographical range of each species in the past (Last Glacial Maximum – LGM, 21 000 years ago), present (PIP, representing current climatic conditions – 1760 years ago) and future (near future – NF, 2080–2100). These results were combined to calculate the richness of the group and also to estimate the range loss of these species in the future. Moreover, we evaluated the irreplaceability of areas for species conservation, aiming to maximize the biotic stability of Eugenia species. Our results showed that the highest species richness in the past was found in the southwestern region of the Cerrado and, currently, the richest regions are found in the central and southeastern areas. However, in the future, we predict a shift of the greatest values of richness towards the southeastern region, an area currently occupied by the Atlantic forest. Although areas with high conservation priorities were found scattered across the biome, this shift is worrisome due to the high fragmentation rate and intensive human occupation thoughout the Atlantic region. Thus, conservation efforts should focus on areas found within these limits.     

Ecological release in lizard assemblages of neotropical savannas

We compare lizard assemblages of Cerrado and Amazonian savannas to test the ecological release hypothesis, which predicts that niche dimensions and abundance should be greater in species inhabiting isolated habitat patches with low species richness (Amazonian savannas and isolated Cerrado patches) when compared with nonisolated areas in central Cerrado with greater species richness. We calculated microhabitat and diet niche breadths with data from 14 isolated Cerrado patches and Amazon savanna areas and six central Cerrado populations. Morphological data were compared using average Euclidean distances, and lizard abundance was estimated using the number of lizards captured in pitfall traps over an extended time period. We found no evidence of ecological release with respect to microhabitat use, suggesting that historical factors are better microhabitat predictors than ecological factors. However, data from individual stomachs indicate that ecological release occurs in these areas for one species (Tropidurus) but not others (Ameiva ameiva, Anolis, Cnemidophorus, and Micrablepharus), suggesting that evolutionary lineages respond differently to environmental pressures, with tropidurids being more affected by ecological factors than polychrotids, teiids, and gymnophthalmids. We found no evidence that ecological release occurs in these areas using morphological data. Based on abundance data, our results indicate that the ecological release (density compensation) hypothesis is not supported: lizard species are not more abundant in isolated areas than in nonisolated areas. The ecology of species is highly conservative, varying little from assemblage to assemblage. Nevertheless, increases in niche breadth for some species indicate that ecological release occurs as well. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Precipitation gradients,plant biogeography,and the incidence of drip-tips in Cerrado plant species

Examining how both climate and species distribution patterns correlate with leaf morphology can give insights into the ecological and evolutionary patterns that drive adaptive selection of leaf form and function. Drip-tips are a common feature of leaves in rain forest tree species; they are thought to be an adaptation that aids leaf drying and maximizes photosynthesis in areas with high-rainfall climates. We tested whether this macroecological pattern holds true across the precipitation gradients in a non-rain forest region—the woodland savannas of Brazil known as the Cerrado—and compared our results with previous studies from Amazonia. Drip-tips were, as expected, less common overall in the drier Cerrado than in Amazonia. In addition, within the Cerrado, drip-tips were more prevalent in areas with higher rainfall as well as in Cerrado sites that were closer to Amazonia. Moreover, species that occurred across both the Cerrado and Amazonia had drip-tips more often than species that were found only in the Cerrado. These findings support the hypothesis that drip-tips are adaptive and that either the cost of retaining drip-tips is low or that in drier regions they have other benefits.     

Heterologous Amplification of Microsatellite Loci from Mouse and Rat in Oryzomyine and Thomasomyine South American Rodents

Eleven heterologous primers were investigated in 119 individuals of 11 species of rodents of the Oryzomyine and Thomasomyine groups. The animals were collected at four sites of the Cerrado, a dryland biome located on the Brazilian Plateau, all of them being karyotyped and taxonomically allocated according to the karyotype. Four of these primers, R47, R65, R75 (from Rattus), and ATP (from Mus) cross-amplified in at least one of these taxa, giving products of seven, nine, one, and three bands, respectively. These values are of the same order as others obtained when heterologous primers were amplified in other orders of mammals. Of the 20 products amplified in these two rodent groups by these four primers, only 7 of the bands were seen in a heteromorphic state (one individual presenting two bands), in two species (Rhipidomys aff. leucodactylus and Oryzomys megacephalus). The others occurred as monomorphic bands.     

Mites from Cerrado fragments and adjacent soybean crops: does the native vegetation help or harm the plantation?

Aiming to recognize the distribution patterns of the mite fauna in soybean crops and to determine the existence of similarity in the composition of species between Cerrado fragments and these plantations, samplings were carried out in 10 areas located in Brazilian Mid-Western and Southeastern regions. Each area was comprised of one fragment of Cerrado sensu stricto and one adjacent soybean crop plantation, with simultaneous samplings carried out on both of them. From the total sampled species, 111 were recorded in the Cerrado and 25 in soybean crops. About 68 % were common in both environments and only eight were exclusively found in soybean. All the species of Phytoseiidae recorded in soybean were also found in the Cerrado. The tetranychid mite Mononychellus planki (McGregor) was the most abundant species (96.3 % of the total individuals) and the only one recorded in every sampled crop. The main occurrence of M. planki was verified in soybean crops using the transgenic cultivar ANTA 82. In general terms, it has been observed that Cerrado fragments do not shelter pest species that may attack soybean and can contribute to the increase of possible predator species in this agroecosystem.     

Genetic diversity and structure of natural populations of Plathymenia reticulata (Mimosoideae), a tropical tree from the Brazilian Cerrado

Plathymenia reticulata is a tropical tree native to the Brazilian Cerrado, one of the most important and endangered ecosystems in Brazil. This species presents high-quality wood and potential for recovery of degraded areas. Despite its importance, almost nothing is known about its genetic or ecological features. Random amplified polymorphic DNA (RAPD) markers were used to investigate the genetic diversity and structure of six natural populations of P. reticulata. DNAs from 117 adult individuals were amplified with 10 random primers and Shannon's index and amova were used to evaluate the levels of genetic diversity within and among populations. Through 72 markers, 70.8% of which were polymorphic, it was possible to obtain 117 unique RAPD phenotypes. The levels of genetic variability found in the six populations of P. reticulata were considerable and most of the genetic variation was found between individuals within populations, although pairwise PH(ST) values indicated significant divergence between populations. The among-population component accounted for, respectively, 12.3% and 16% of the genetic variation, according to amova and Shannon's index. These results were compared with other genetic studies on plant species and such a level of differentiation among populations corresponds to that which has usually been observed for outcrossing plants. The importance of maintenance of the P. reticulata populations and implications of the analysis of adult individuals, considering the longevity of this species and the relatively recent Cerrado fragmentation, are discussed.     

Woody vegetation structure of Brazilian Cerrado invaded by Pteridium arachnoideum (Kaulf.) Maxon (Dennstaedtiaceae)

Biological invasion is one of the major components of global change. In the Brazilian Cerrado, the fern Pteridium arachnoideum (neotropical bracken) is a potentially invasive species which is expected to change the vegetation structure of savannas. We assessed effects of the dominance of P. arachnoideum on the diversity of woody Cerrado species. We compared three sites dominated by neotropical bracken with three sites without neotropical bracken. We used multivariate analyses of variance and rarefaction curves with confidence limits to test whether the groups of species found in invaded areas were different from those found in non-invaded ones. We also analyzed the density of individuals and species and the mean area at breast height per plot. We found significant floristic differences between the areas. The diversity and the densities in the invaded areas were lower than in non-invaded ones. However, we did not observe differences in the mean area at breast height of woody plants. Thus, the P. arachnoideum invasion is a potential threat to Cerrado biodiversity. Management efforts should be made to control the bracken, since its dominance in the plant community may lead to impoverishment and simplification of the savanna vegetation.     

Molecular genetic diversity and differentiation of populations of ‘somnus’ passion fruit trees (Passiflora setacea DC): Implications for conservation and pre-breeding

The ‘somnus’ passion fruit tree (Passiflora setacea) is native to the ‘Cerrado’ and ‘Caatinga’ biomes in Brazil and possesses agrobiological and commercial traits of interest. Studies examining the nature of genetic variability in natural populations are important for the utilization of these traits in conservation and breeding programs. In this study, we analyzed 12 populations of P. setacea from different locations distributed in three agro-ecological zones within the Bahia state of Brazil. Eleven ISSR primers and four pairs of RGA primers were used to assay 109 and 49 unique and reproducible loci, of which 108 (99%) and 49 (100%) were polymorphic. Although the level of genetic diversity in ‘somnus’ passion fruit trees was greater than that observed for other species of the same genus, preferential collection of certain populations, such as those located in the cities of Vitória da Conquista and Licinio de Almeida, is important when considering the fact that ‘somnus’ passion fruit trees occur in areas that are highly disturbed. This disturbance is primarily due to the deployment of pasture, predatory extraction and accelerated urban expansion. An analysis of molecular variance revealed a balance between the estimated genetic variation within and among populations. These data may be useful for developing strategies for preservation of this species in the Cerrado.     

Limited effects of fire disturbances on the species diversity and structure of ant-plant interaction networks in Brazilian Cerrado

Fire is one of the main natural disturbances in Tropical Savannas, changing the diversity of species, altering the structure of species interactions, and driving the evolution of adaptations. Here, we investigated the effects of fire disturbance on interactions between ants and plants with extrafloral nectaries in Cerrado (Brazilian Savanna). We carried out the study in two different ecosystems of Brazilian Cerrado 700 km apart; Woody Cerrado and Rupestrian Grasslands. We conducted a Before-After-Control-Impact (BACI) experiment, in which the impact was the disturbance caused by fire. In Woody Cerrado, we found no evidence of fire affecting the diversity and composition of plants or its interactions. Fire also did not affect ant diversity but changed the interaction pattern of its interactions by reorganizing the paired interactions between species (i.e., rewiring). However, this effect did not result in changes on the overall structure of the network. In Rupestrian Grasslands, fire also did not affect the diversity and composition of plant species or its interactions, but it did increase the number of ant species and change its composition, leading to a reorganization of the its paired interactions. However, these fire disturbances in the ant level did not affect the overall structure of the network. Our findings indicate that the structure of ant-plant interaction networks is robust to fire disturbances, more in Woody Cerrado than Rupestrian Grasslands, confirming our hypothesis that ant-plant interactions in Cerrado are adapted to fire disturbances. In sum, our study enhances the understanding of the effects of environmental disturbances and the stability of the ant-plant interactions in fire-adapted ecosystems such as Brazilian Cerrado.     

Isoenzyme Variation in Melipona rufiventris (Hymenoptera: Apidae, Meliponina) in Minas Gerais State, Brazil

The stingless bee Melipona rufiventris is an important pollinator in several Brazilian ecosystems. Originally widely distributed in Minas Gerais (MG) state, this species is becoming very rare. Therefore this species was included in the endangered species list of MG. We used isoenzyme data for a better understanding of the genetic structure of several M. rufiventris colonies. Samples of 35 colonies were collected from 12 localities and evaluated by nine enzymatic systems, which yielded 17 loci. M. rufiventris genetic variation was found to be low, typical of an endangered species. The proportion of polymorphic loci was 5.88% in both ecosystems. Only Est-4 was polymorphic in colonies from the Forest and Mdh-1 in colonies from the Cerrado. The expected heterozygosity ranged from 0.0068 in the Cerrado to 0.0078 in the Forest. Despite this, enzyme electrophoretic analyses provided a good idea of the diversity between samples from Cerrado and Forest which reinforce the existence of two different forms of M. rufiventris in MG, one present in the Cerrado and the other in Forest. This information is of great importance for the conservation of, M. rufiventris in MG.     

Isolation and characterization of microsatellite loci in the plant-ant Pseudomyrmex ferrugineus (Formicidae: Pseudomyrmecinae) and cross-testing for two congeneric species

To investigate the population structure of the obligate plant-ant Pseudomyrmex ferrugineus, we developed primers for 12 microsatellite loci. We tested the variability of the markers on 11 individuals from each of two populations (totalling 22 individuals) and found two to 12 alleles per locus and population. No deviations from Hardy-Weinberg equilibrium were detected. Observed and expected heterozygosities at each locus ranged from 0.00 to 0.50 and from 0.08 to 0.46, respectively. We also investigated suitability of these primers in two congeneric species.     

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.     

Compliance to Brazil's Forest Code will not protect biodiversity and ecosystem services

In striking contrast to heartening events in the adjacent Amazon, Brazil's Cerrado biome has seen continued deforestation over the past decade. Though approved in 2012, no study evaluated the impacts of new Brazilian Forest Code (FC) revision on biodiversity and ecosystem services. Here, we report the first assessment of the likely loss and gain in biodiversity and ecosystem services expected if the FC is properly enforced across 200 million hectares of the Cerrado. We also discuss the challenges associated to compliance with the law and present opportunities for conservation. Establishing restoration programmes in private properties with currently less native vegetation than required by the FC could create habitat for 25% more threatened species than now found in these places and could also increase water security and carbon stock in 56.6 MtC. More important, trading environmental reserve quotas coupled with the strategic expansion of protected areas on private and public land could definitely rescue the Cerrado from the brink.     

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2%) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3% of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson''s correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.     

Application of a novel Paenibacillus-specific PCR-DGGE method and sequence analysis to assess the diversity of Paenibacillus spp. in the maize rhizosphere

In this study, a Paenibacillus-specific PCR system, based on the specific primer PAEN515F in combination with bacterial primer R1401, was tested and used to amplify specific fragments of the 16S rRNA gene from rhizosphere DNA. The amplicons were used in a second (semi-nested) PCR for DGGE, in which bacterial primers F968GC and R1401 were used. The resulting products were separated into community fingerprints by DGGE. To assess the reliability of the method, the diversity of Paenibacillus species was evaluated on the basis of DNA extracted directly from the rhizospheres of four different cultivars of maize (Zea mays), i.e. CMS04, CMS11, CMS22 and CMS36, sown in two Brazilian field soils (Cerrado and Várzea). In addition, a clone library was generated from the PCR-generated 16S rDNA fragments, and selected clones were sequenced.The results of the bacterial community analyses showed, at the level of clone libraries, that considerable diversity among Paenibacillus spp. was present. The most dominantly found sequences clustered into 12 groups, each one potentially representing a species complex. Sequences closely affiliated with the P. macerans and P. azotofixans complexes were found in all samples, whereas other sequences were scarcer. Clones affiliated with the latter species complex were most abundant, representing 19% of all clones analysed.The Paenibacillus fingerprints generated via semi-nested PCR followed by DGGE showed a clear distinction between the maize plants grown in Cerrado versus Várzea soils. Thus, soil type, instead of maize cultivar type, was the overriding determinative factor that influenced the community structures of the Paenibacillus communities in the rhizospheres investigated. At a lower level (subcluster), there was a trend for maize cultivars CMS11 and CMS22 on the one hand, and CMS36 and CMS04 on the other hand, to cluster together, indicating that these respective pair of cultivars were similar in their Paenibacillus species composition. This trend was tentatively linked to the growth characteristics of these maize cultivars. These results clearly demonstrated the efficacy of the Paenibacillus-specific PCR-DGGE method in describing Paenibacillus species diversity in rhizosphere soils.     

Unexpectedly high genetic diversity and divergence among populations of the apomictic Neotropical tree Miconia albicans

• Since tropical trees often have long generation times and relatively small reproductive populations, breeding systems and genetic variation are important for population viability and have consequences for conservation. Miconia albicans is an obligate, diplosporous, apomictic species widespread in the Brazilian Cerrado, the savanna areas in central Brazil and elsewhere in the Neotropics. The genetic variability would be, theoretically, low within these male‐sterile and possibly clonal populations, although some variation would be expected due to recombination during restitutional meiosis.
• We used ISSR markers to assess genetic diversity of M. albicans and to compare with other tropical trees, including invasive species of Melastomataceae. A total of 120 individuals from six populations were analysed using ten ISSR primers, which produced 153 fully reproducible fragments.
• The populations of M. albicans presented mean Shannon's information index (I) of 0.244 and expected heterozygosity (H_e) of 0.168. Only two pairs of apparently clonal trees were identified, and genetic diversity was relatively high. A hierarchical amova for all ISSR datasets showed that 74% of the variance was found among populations, while only 26% of the variance was found within populations of this species. Multivariate and Bayesian analyses indicated marked separation between the studied populations.
• The genetic diversity generated by restitutional meiosis, polyploidy and possibly other genome changes may explain the morpho‐physiological plasticity and the ability of these plants to differentiate and occupy such a wide territory and different environmental conditions. Producing enormous amounts of bird‐dispersed fruits, M. albicans possess weedy potential that may rival other Melastomataceae alien invaders.

     

East-west divergence in central Brazilian Cerrado revealed by cpDNA sequences of a bird-dispersed tree species

Population genetic studies of plant species can contribute to understanding the evolutionary history of the Brazilian Cerrado, a highly threatened hotspot and the richest tropical savanna in the world. Therefore, this study aimed at investigating the diversity and genetic structure of Byrsonima coccolobifolia (Malpighiaceae), one of the most common tree species in the Brazilian Cerrado, focusing mainly on the central area of the biome. This is a bird-dispersed species, and studies targeting species with this seed dispersal syndrome are scarce. First, we performed a careful screening of cpDNA markers to identify regions suitable for intraspecific genetic studies, and then we investigated the diversity and genetic structure in ten populations of B. coccolobifolia. The cpDNA regions selected (trnS-trnG and trnH-trnK) revealed considerable divergence among populations and a striking geographical structure, separating populations in two groups, east and west. Although seed dispersal by birds is expected to reach long distances, our results strongly corroborate studies targeting trees with limited seed dispersal. This suggests that historical fragmentation of Cerrado (possibly during cold and dry periods of the Quaternary) might have limited long distance dispersal events after climate amelioration even in bird-dispersed species, especially while Cerrado was expanding but still fragmented.