Short Communication: Elucidation of bacterial community structure on thin-spined porcupine (Chaetomys subspinosus) spines by denaturing.

Thin-spined porcupines (Chaetomys subspinosus) are threatened with extinction and are categorized as vulnerable. This is because of alteration to and loss of their habitat and possible hunting activities in their distribution area. Their spines constitute one of their defense mechanisms, which can be fomites for pathogens to humans. However, little is known about such pathogens. The present study aimed to detect bacteria on spines of C. subspinosus, from the Una Biological Reserve, South of Bahia, northeastern Brazil, by analyzing metagenomic DNA, isolating bacterial culture, using the denaturing gradient gel electrophoresis (DGGE) technique, and sequencing. Six anatomical points were selected for withdrawing spine samples from an individual C. subspinosus. At all sample points, bacteria were detected by bacteriological culture and/or DGGE and sequencing of excised bands. When all samples were combined, standard PCR-DGGE analysis of bacteria present in the spines identified 15 distinct bands, thereby revealing a distinct bacterial community. The main pathogens identified through sequencing were Bacillus cereus, B. thuringiensis, B. anthracis, and B. pumilus. The present study demonstrated the isolation and identification of non-pathogenic and pathogenic bacteria on the spines of C. subspinosus.

Effect of selective logging on genetic diversity and gene flow in Cariniana legalis sampled from a cacao agroforestry system.

The fragments of the Atlantic Forest of southern Bahia have a long history of intense logging and selective cutting. Some tree species, such as jequitibá rosa (Cariniana legalis), have experienced a reduction in their populations with respect to both area and density. To evaluate the possible effects of selective logging on genetic diversity, gene flow, and spatial genetic structure, 51 C. legalis individuals were sampled, representing the total remaining population from the cacao agroforestry system. A total of 120 alleles were observed from the 11 microsatellite loci analyzed. The average observed heterozygosity (0.486) was less than the expected heterozygosity (0.721), indicating a loss of genetic diversity in this population. A high fixation index (FIS = 0.325) was found, which is possibly due to a reduction in population size, resulting in increased mating among relatives. The maximum (1055 m) and minimum (0.095 m) distances traveled by pollen or seeds were inferred based on paternity tests. We found 36.84% of unique parents among all sampled seedlings. The progenitors of the remaining seedlings (63.16%) were most likely out of the sampled area. Positive and significant spatial genetic structure was identified in this population among classes 10 to 30 m away with an average coancestry coefficient between pairs of individuals of 0.12. These results suggest that the agroforestry system of cacao cultivation is contributing to maintaining levels of diversity and gene flow in the studied population, thus minimizing the effects of selective logging.

Reduced genetic diversity in endemic Brazilian Lymania spp (Bromeliaceae) populations and implications for conservation.

We analyzed the genetic diversity of populations of two sympatric species of Lymania (Bromeliaceae), both endemic to the Atlantic rainforest of southern Bahia (Brazil). Lymania azurea has a restricted occurrence, while Lymania smithii has a wider distribution. Our aim was to provide genetic data to contribute to the design of more efficient conservation strategies for these bromeliads, possibly justifying inclusion in the official Brazilian list of Endangered Species. Up to now, L. azurea has been classified by the Brazilian Ministry of the Environment as "data deficient". We sampled four populations of L. azurea throughout its distribution area in southern Bahia and two populations of L. smithii in the same region. Genotyping was performed with 48 random amplified polymorphic DNA markers. Based on the Jaccard genetic similarity index, L. smithii has greater diversity than L. azurea. An analysis of molecular variation showed greater genetic variance within than between populations for both species. L. azurea was found to have 20% inbreeding, probably due to population fragmentation, with L. smithii showing only 10%. When we analyzed pairs of populations of L. azurea within a conservation unit, we found low population structure (ФST = 0.098), apparently due to a large degree of gene flow between them. In disturbed areas, we found a higher ФST (0.372). We found low genetic variability for L. azurea, probably as a consequence of habitat fragmentation, supporting the need for its inclusion in the Brazilian list of endangered flora.

Molecular genetic analysis of the yellow-breasted capuchin monkey: recommendations for ex situ conservation.

The yellow-breasted capuchin monkey, Cebus xanthosternos, is one of the most endangered species of the Brazilian Atlantic Forest. In situ conservation for this species is problematic due to habitat destruction; therefore, captive conservation has been considered as an alternative strategy. A Studbook for C. xanthosternos has been kept for more than 20 years; however, no genetic data has been collected. Our aim was to provide a preliminary assessment of the genetic variability of C. xanthosternos in captivity in Brazil and compare it with data from the wild. Microsatellite and mtDNA sequencing were carried out in 40 samples from five Brazilian institutions registered in the international Studbook and compared with 8 samples collected in a wild population from REBIO-Una/BA. DNA for analysis was extracted from hair, feces and blood. Our results showed that two of the five captive groups assessed had a genetic variability comparable to wild animals. However, the other three groups apparently require urgent management to improve its genetic variability. Considering that inbreeding effects are more pronounced in captivity due to lack of gene flow, our data indicate a need to increase population size by introducing newly rescued individuals into these captive groups. Our results are the first attempt to provide genetic information for captive C. xanthosternos in Brazil.

Genetic assessment of the Atlantic Forest bristle porcupine, Chaetomys subspinosus (Rodentia: Erethizontidae), an endemic species threatened with extinction.

The bristle-spined porcupine, Chaetomys subspinosus, an endemic rodent from Atlantic Forest, was considered to be abundant in the recent past, but population reductions due to habitat loss and expansion of human activities caused this species to be included in the "vulnerable" category of the World Conservation Union Red List. We performed the first genetic assessment in natural populations of this focal species along its geographical distribution. Thirty-five non-invasive samples (hair) were collected from three natural populations in the Brazilian States of Sergipe, Bahia and Espírito Santo. Genetic similarity obtained by Jaccard's index, based on dominant RAPD and ISSR markers, varied between 25 and 100%. Four clusters, mainly coincident with the geographical distribution of the populations, were observed. Analysis of molecular variance based on 47 polymorphic loci showed that there was 15.99% genetic variability among populations and 84.01% within populations. The estimated genetic structure among populations (Φ(ST)) was 0.16. The populations may have formed a continuum along the past distribution of the Atlantic rainforest but historical events of human occupation resulted in recent divergence among sampled populations.

Single nucleotide polymorphisms from Theobroma cacao expressed sequence tags associated with witches' broom disease in cacao.

In order to increase the efficiency of cacao tree resistance to witches' broom disease, which is caused by Moniliophthora perniciosa (Tricholomataceae), we looked for molecular markers that could help in the selection of resistant cacao genotypes. Among the different markers useful for developing marker-assisted selection, single nucleotide polymorphisms (SNPs) constitute the most common type of sequence difference between alleles and can be easily detected by in silico analysis from expressed sequence tag libraries. We report the first detection and analysis of SNPs from cacao-M. perniciosa interaction expressed sequence tags, using bioinformatics. Selection based on analysis of these SNPs should be useful for developing cacao varieties resistant to this devastating disease.

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.

DNA extraction from bristles and quills of Chaetomys subspinosus (Rodentia: Erethizontidae) using a novel protocol.

DNA extraction protocols are as varied as DNA sources. When it comes to endangered species, it is especially important to pay attention to all details that ensure the completion of the study goals and effectiveness in attaining useful data for conservation. Chaetomys subspinosus (Rodentia: Erethizontidae) is a secretive arboreal porcupine endemic to certain ecosystems of the Brazilian Atlantic Forest. A multidisciplinary study (including genetic data) was performed to create a management plan for the conservation of this species. Individuals from natural populations of the states of Bahia, Espírito Santo and Sergipe were sampled. To obtain a reliable and abundant amount of starting material, non-destructive methods were tested, extracting DNA from the bristles and quills that comprise most of this animal's hide. This method has also been innovative in adapting a DNA extraction protocol traditionally used for plants. Digestion using proteinase K was followed by protein precipitation with CTAB, a chloroform-isoamyl alcohol cleaning and DNA precipitation with isopropyl alcohol. This protocol supplies good-quality DNA for genetic analysis with molecular markers based on PCR.

DNA extraction from bristles and quills of Chaetomys subspinosus (Rodentia: Erethizontidae) using a novel protocol

DNA extraction protocols are as varied as DNA sources. When it comes to endangered species, it is especially important to pay attention to all details that ensure the completion of the study goals and effectiveness in attaining useful data for conservation. Chaetomys subspinosus (Rodentia: Erethizontidae) is a secretive arboreal porcupine endemic to certain ecosystems of the Brazilian Atlantic Forest. A multidisciplinary study (including genetic data) was performed to create a management plan for the conservation of this species. Individuals from natural populations of the states of Bahia, Espírito Santo and Sergipe were sampled. To obtain a reliable and abundant amount of starting material, non-destructive methods were tested, extracting DNA from the bristles and quills that comprise most of this animal’s hide. This method has also been innovative in adapting a DNA extraction protocol traditionally used for plants. Digestion using proteinase K was followed by protein precipitation with CTAB, a chloroform-isoamyl alcohol cleaning and DNA precipitation with isopropyl alcohol. This protocol supplies good-quality DNA for genetic analysis with molecular markers based on PCR.

Genetic structure, mating system, and long-distance gene flow in heart of palm (Euterpe edulis Mart.).

We report a detailed analysis of the population genetic structure, mating system, and gene flow of heart of palm (Euterpe edulis Mart.-Arecaceae) in central Brazil. This palm is considered a keystone species because it supplies fruits for birds and rodents all year and is intensively harvested for culinary purposes. Two populations of this palm tree were examined, using 18 microsatellite loci. The species displays a predominantly outcrossed mating system (tm = 0.94), with a probability of full sibship greater than 70% within open-pollinated families. The following estimates of interpopulation genetic variation were calculated and found significant: FIT = 0.17, FIS = 0.12, FST = 0.06, and RST = 0.07. This low but significant level of interpopulation genetic variation indicates high levels of gene flow. Two adult trees were identified as likely seed parents (P > 99.9%) of juveniles located at a distance of 22 km. Gene flow over such distances has not been reported before for tropical tree species. The establishment and management of in situ genetic reserves or ex situ conservation and breeding populations for E. edulis should contemplate the collection of several hundreds open-pollinated maternal families from relatively few distant populations to maximize the genetic sampling of a larger number of pollen parents.

Tricuspid dura mater bioprostheses: more than 20-year follow-up of 3 patients.

Replacement of the tricuspid valve is sometimes necessary. We report 3 consecutive patients with tricuspid insufficiency who underwent valve replacement with glycerol-preserved, homologous dura mater cardiac bioprostheses between 1971 and 1973. The first 2 patients are well 28 and 27 years later; the last patient was lost to follow-up after 20 years. We conclude that preservation of homologous dura mater bioprostheses in glycerol may reduce rates of thromboembolism, thrombosis, and structural dysfunction during the late postoperative period.