miRNA profiling in leaf and cork tissues of Quercus suber reveals novel miRNAs and tissue-specific expression patterns

The differentiation of cork (phellem) cells from the phellogen (cork cambium) is a secondary growth process observed in the cork oak tree conferring a unique ability to produce a thick layer of cork. At present, the molecular regulators of phellem differentiation are unknown. The previously documented involvement of microRNAs (miRNAs) in the regulation of developmental processes, including secondary growth, motivated the search for these regulators in cork oak tissues. We performed deep sequencing of the small RNA fraction obtained from cork oak leaves and differentiating phellem. RNA sequences with lengths of 19–25 nt derived from the two libraries were analysed, leading to the identification of 41 families of conserved miRNAs, of which the most abundant were miR167, miR165/166, miR396 and miR159. Thirty novel miRNA candidates were also unveiled, 11 of which were unique to leaves and 13 to phellem. Northern blot detection of a set of conserved and novel miRNAs confirmed their differential expression profile. Prediction and analysis of putative miRNA target genes provided clues regarding processes taking place in leaf and phellem tissues, but further experimental work will be needed for functional characterization. In conclusion, we here provide a first characterization of the miRNA population in a Fagacea species, and the comparative analysis of miRNA expression in leaf and phellem libraries represents an important step to uncovering specific regulatory networks controlling phellem differentiation.     

Very low mitochondrial variability in a stingless bee endemic to cerrado

Partamona mulata is a stingless bee species endemic to cerrado, a severely threatened phytogeographical domain. Clearing for pasture without proper soil treatment in the cerrado facilitates the proliferation of termite ground nests, which are the nesting sites for P. mulata. The genetic consequences of these changes in the cerrado environment for bee populations are still understudied. In this work, we analyzed the genetic diversity of 48 colonies of P. mulata collected throughout the species’ distribution range by sequencing two mitochondrial genes, cytochrome oxidase I and cytochrome B. A very low polymorphism rate was observed when compared to another Partamona species from the Atlantic forest. Exclusive haplotypes were observed in two of the five areas sampled. The sharing of two haplotypes between collection sites separated by a distance greater than the flight range of queens indicates an ancient distribution for these haplotypes. The low haplotype and nucleotide diversity observed here suggests that P. mulata is either a young species or one that has been through population bottlenecks. Locally predominant and exclusive haplotypes (H2 and H4) may have been derived from local remnants through cerrado deforestation and the expansion of a few colonies with abundant nesting sites.     

Alien grasses in Brazilian savannas: a threat to the biodiversity

African grasses used as forage are spreading fast in cerrado (Brazilian savanna) patches, probably displacing native species. An analysis of the graminoid species abundance was performed in Cerrado Pé-de-Gigante Reserve (São Paulo State, Brazil), where their relative frequency, density, dominance and the value of importance were assessed in two cerrado forms: cerrado sensu stricto (denser) and campo cerrado (open). Thirty-six transects were determined, along which 3240.5 m × 0.5 m herbaceous samples were taken. Ordination by CCA analysis was performed to detect gradients in the graminoid species distribution, according to shading, distance from the reserve border and aspect. Interspecific associations among the species were tested. A total of 93 species were sampled, predominantly Poaceae and Myrtaceae families. Two alien grasses were found, Melinis minutiflora and Brachiaria decumbens, with very high values of importance. Light availability proved to be the most important analyzed environmental factor related to graminoid distribution, strongly correlated with the abundance of M. minutiflora. Both alien grasses were negatively associated with most native graminoids, suggesting they exert a strong competitive pressure on the native herbaceous community. Attention must be taken to the introduction of alien species in the country.     

Assessment of genetic and chemical variability in Thymus caramanicus

Thymus caramanicus is an endemic species grown in Iran with interesting pharmacological and biological properties. In the present work, essential oil compositions and inter-simple sequences repeat (ISSR) markers were used to estimate the relationships among and within seven populations of T. caramanicus, belonging to three provinces in Iran. The studied individuals were distinguished on the basis of ISSR markers and constituents of essential oil. A total of 127 band positions were produced by 12 ISSR primers, of which 105 were found polymorphic with 82.68 % polymorphism. Genetic similarity values among individuals ranged between 0.15 and 0.82 which was indicative of a high level of genetic variation. On the basis of their genetic similarities, ISSR analysis allowed to group the samples into two main clusters. One of these included populations originated from Kerman and Isfahan provinces, and the other cluster consists of populations from Semnan province. Chemical compounds of essential oils were found variable in the various individuals and all samples were principally composed of phenolic constituents (carvacrol and/or thymol). As a consequence, the plants were classified into two major chemotypes including carvacrol and thymol/carvacrol. A relationship between genetic and chemical variability and geographic distribution has been observed in studied populations of T. caramanicus.     

Individual identification and distribution assessment of otters (Lutra lutra) through non-invasive genetic sampling: Recovery of an endangered species in the Basque Country (Northern Spain)

Non-invasive genetic techniques have proven to be cost-effective for monitoring and studying otter populations, largely due to the linearity of otter territories and the marking behavior of the species. After a severe decline, in the 60, the Eurasian otter is recovering in the Iberian Peninsula. However, the recovery pattern is not homogeneous and the species is still considered “Threatened” in many regions. During 2007–2010 a systematic non-invasive genetic sampling effort was carried out to determine the spatial distribution and to estimate the population size of an endangered otter population in northern Iberian Peninsula (Basque Country). Samples were identified to species level by sequencing a 226 bp mtDNA fragment prior to genotyping. Among the 132 obtained samples, 127 (98.4%) belonged to the study species, one sample was genetically identified as European mink (Mustela lutreola) and one as American mink (Neovison vison) while genetic species confirmation was not possible in the three remaining samples. These results provided novel and accurate data on species distribution, highlighting an overall increase of 25% in 10 × 10 UTM grids occupied by otter and a clear pattern of re-colonization upstream of the main rivers. All samples corresponding to otter were subsequently individually genotyped using a novel multiplex panel of 11 microsatellite markers and sexed by typing the sex-chromosome-related gene ZFX/ZFY. We obtained a complete individual genetic profile for 55 samples (genotyping success 43%), corresponding to 20 different individuals (11 females, 6 males, and 3 individuals of unknown gender). The mean otter density in occupied areas estimated to be 0.09 (0.06–0.12) individuals per river kilometer. The present study enabled us to obtain updated and relevant information about this elusive species’ distribution and population size, essential to define population status and to design successful and effective management and conservation programs.     

Vegetation structure: Fine scale relationships with soil in a cerrado site

There is a lack of understanding on factors influencing the occurrence of high species heterogeneity at fine scale in the Brazilian cerrado. Soil is a major determinant of vegetation in the Brazilian cerrado and an important candidate to influence species distribution at fine scales, since soil features vary at very small distances, whereas many environmental variables are relatively homogeneous at such scale. We tested plant-soil relationships at fine scales in a cerrado site. We placed 100 contiguous 25 m² plots, where we identified all woody individuals and measured several soil features. We did partial redundancy analysis, controlling for spatial autocorrelation, to test for relationships between soil features and floristic composition. We also did multiple regressions or spatial autoregressive models to test for relationships between soil features and: (1) the abundance of the five most common species, (2) total abundance, (3) richness, (4) evenness, and (5) diversity. We found weak relationships between soil and floristic composition, richness, and total abundance, which, coupled with also weak relationship found in another study with plant available water, indicate there is no major environmental variable influencing vegetation at fine scales, but several of them interacting. Organic matter was positively related with the abundance of Myrsine umbellata and was negatively related to evenness. Although a causal relationship cannot be inferred with certainty, the dominance of Myrsine umbellata seems to be related to a positive feedback with soil.     

Genetic Diversity in Cymbopogon Species using PCR-Based Functional Markers

Genetic diversity among 25 accessions (involving 8 species, 2 interspecific hybrids and one hybrid mutant) of medicinally important genus Cymbopogon was assessed using 17 PCR-based functional markers, that were designed from members of three different multigene families. We developed 16 primer pairs from two multigene families, 8 primer pairs each from cytochrome P450 and UDP-glucosyltransferase (UGT); one primer pair was derived from 5S rRNA gene family. A total of 119 fragments were visualized, of which 108 (91%) were polymorphic. The level of diversity among different taxa/accessions observed during the present study was, however, low relative to the diversity level obtained due to RAPD markers in two earlier studies. The pattern of genetic diversity neither matched with the known taxonomic classification, nor did it always match with the distribution of chemical constituents of the essential oils available in these accessions. Thus, present investigation though revealed poor correlation between the molecular and chemical diversity, these gene-based markers may prove useful in the development of perfect markers for association mapping of genes involved in controlling agronomically important traits.     

Soil chemical factors and grassland species density in Emas National Park (central Brazil)

Studies of grasslands on specific soil types suggest that different nutrients can limit biomass production and, hence, species composition and number. The Brazilian cerrado is the major savanna region in America and once covered about 2 million km(2), mainly in the Brazilian Central Plateau, under seasonal climate, with wet summer and dry winter. In view of the importance of soil chemical factors in the distribution of the vegetation forms within the Cerrado domain and which may influence the number of species, we analyzed some soil characteristics in three herbaceous vegetation forms -- hyperseasonal cerrado, seasonal cerrado, and wet grassland -- in Emas National Park, a core cerrado site, to investigate the relationship between number of species and soil characteristics. We collected vegetation and soil samples in these three vegetation forms and submitted the obtained data to multiple linear regression. We found out that aluminum and pH were the best predictors of species density, the former positively related to species density and the latter negatively related. Since the predictable variation in species density is important in determining areas of conservation, we can postulate that these two soil factors are indicators of high species density areas in tropical grasslands, which could be used in selecting priority sites for conservation.     

How does a Psittacanthus robustus Mart. population structure relate to a Vochysia thyrsoidea Pohl. host population?

Distribution of parasitic plants is directly linked with the distribution of host species and behavioral patterns of seed dispersers. Psittacanthus robustus (Loranthaceae) is a neotropical hemiparasite that mainly colonizes species of the family Vochysiaceae. Vochysia thyrsoidea is the main host of P. robustus and is commonly found in areas of cerrado rupestre (rocky savanna), an abundant vegetation in our study site. We conducted the study in the ecological park Parque Ecológico Quedas do Rio Bonito (PEQRB), over an area of 2.82 ha of cerrado rupestre. The objective of this work was to investigate population structure, parasitic behavior (mistletoe grip height and circumference of host branch), and spatial distribution of Psittacanthus robustus on a population of Vochysia thyrsoidea. We sampled 267 V. thyrsoidea individuals and found that the population had a random distribution pattern. Seventy-nine individuals (29.6% of the sample population) hosted the hemiparasite, to a total of 193 P. robustus individuals. The number of mistletoe individuals per host plant ranged between 1 and 12. The V. thyrsoidea individuals most infested with mistletoes were those reaching greater heights. The correlation between height of host plant and preferred grip height was highly significant, with the preferred grip height being the uppermost portions of host plants. The crown size of P. robustus individuals ranged between 10 and 230 cm. The main disperser of P. robustus fruit was found to be swallow-tanager Tersina viridis viridis. Its activities led to a clumped pattern of spatial distribution of the hemiparasite along with higher infestation in larger trees.     

Canopy Herbivory and Insect Herbivore Diversity in a Dry Forest–Savanna Transition in Brazil

This study aimed to compare canopy herbivore diversity and resultant insect damage to vegetation in two distinct and adjacent ecosystems, specifically a dry forest ecosystem and a cerrado (savanna) ecosystem that occur together in an abrupt transition zone in southeastern Brazil. In the dry forest, the canopy was reached using a single rope climbing technique, whereas the shorter canopy of the cerrado was assessed using a 7 m ladder. Insect specimens were collected by beating the foliage, and 20 representative leaves were collected to calculate the specific leaf mass (SLM) and leaf area loss through herbivory. Also, we collected ten soil samples from each habitat to determine soil nutrient content. We sampled 118 herbivorous insects from ten families, mostly in dry forest trees (96 individuals belonging to 31 species). A higher abundance of chewing and sap-sucking insects were observed in dry forest trees than in cerrado trees. The same pattern was observed for the richness of chewers, with a higher degree of diversity of chewers found in dry forest trees than in cerrado trees. Herbivorous insects were not affected by SLM regardless of guild and habitat. However, we observed a negative correlation between the herbivory rate and the specific leaf mass (SLM). The cerrado trees showed a higher SLM and lower herbivory rates than trees occurring in the dry forest. These results suggest that herbivory rates in the transition dry forest–cerrado may be driven by soil nutrient content, which is thought to influence leaf sclerophylly.
Abstract in Portuguese is available at http://www.blackwell-synergy.com/loi/btp .     

Changes in cerrado vegetation after disturbance by frost (São Paulo State,Brazil)

We report changes after frost in cerrado species populations and community structure in Assis, São Paulo State, Brazil and consider the possible impacts of frost on cerrado types and their distribution. Four permanent plots (10 m × 10 m) were established and 736 individuals were marked with enumerated plastic labels and measured one week after the frost. Frost damage for each individual was assessed: 0 - not affected; 1 - slightly affected; 2 - substantially affected; and 3 - strongly affected. The frost impact on vegetation structure was high, reducing tree canopy cover from 88% to 19% in the upper stratum and that of the lower stratum from 48% to 8.5%. Floristic richness did not change, even though a small number (3%) of individuals of some species died. Conversely, the relative dominance of species changed dramatically in the short-term because of varying susceptibility to frost of different species. The largest reductions in crown cover were observed in Xylopia aromatica and Vochysia tucanorum. Of the 57 species recorded, 15% were unaffected, 19% had only their leaves damaged, 25% had some of their leaves and branches damaged, and 41% had all their aerial parts killed. The majority of individuals in the community belonged to frost tolerant species. The regeneration of the stand structure was remarkably rapid; height and canopy cover of the lower stratum recovered completely after five months, and those of the arboreal stratum showed a recovery of about 80% after 11 months. We consider that the frequency and intensity of frosts can maintain more open forms of cerrado vegetation even in sites where both water and nutrient availability could support denser vegetation. It is also probable that some frost-resistant heliophyte species are confined to areas where frosts prevent the development of denser cerrado vegetation. Much of the characteristic floristic composition of the southern region of the cerrado biome may be related to the selection of frost-resistant species. Our study shows, however, that a full complement of cerrado plants is capable of occupying and colonizing frost susceptible sites.     

Effects of genetic variability and habitat of Qualea parviflora (Vochysiaceae) on herbivory by free-feeding and gall-forming insects

• Background and Aims Differences in the chemical and physical traits of plants caused by both genetic and habitat characteristics may influence attack by herbivores. Leaves of Qualea parviflora (Vochysiaceae), a common tree of different habitats in the Brazilian Neotropical savannas (cerrado), are susceptible to severe attack by herbivorous free-living and gall-forming insects. Attack by free-living and gall-forming insects within and between populations of Q. parviflora were examined and it was determined to what extent genetic variability (detected by RAPD markers), phenotypic characteristics of the plants and habit traits influence the number of free-living and gall-forming insect species and individuals attacking the plants, and the intensity of attack.• Methods On four occasions in 2000, leaves were sampled from ten individual trees in each of three types of vegetation in the cerrado: campo sujo, cerrado sensu stricto and cerradão at the Ecological Station of Pirapitinga (ESP), in Três Marias, north-western Minas Gerais, Brazil. Genetic variability was detected by RAPD markers, and concentrations of nutrients, phenols and tannins, sclerophylly and pre-dawn water potential of leaves were measured. Water and nutrient contents in the soil below each tree characterized the habitat. The free-living and gall-forming herbivorous insects were determined.• Key Results Of 69 RAPD markers analysed, 41 were polymorphic and were used for analyses of genetic variability of Q. parviflora. Most of the variability occurred within habitats, accounting for 97·65 % of the genetic variability. Plants in the cerrado sensu stricto and campo sujo were the most similar. There were no significant associations between genetic similarity and the chemical and physical traits of Q. parviflora, or with habitat, nor was there significant correlation between phenotypic and habitat traits. Increasing concentrations of tannins and sulphur, C : N ratio and sclerophylly correlated with increasing percentage of leaf area damaged by herbivores. Decreased sclerophylly, concentration of tannins and C : N ratio, and increased concentration of nutrients in leaves correlated with increased severity of attack by gall-forming insects.• Conclusions Nutrient concentration in the soil had more influence, indirectly, on free-feeding insects than did composition of Q. parviflora leaves. However, gall-forming insects are affected more by leaf quality, attacking fewer sclerophyllous leaves, with larger nutrient but smaller tannin concentrations.Key words: Cerrado, genetic variability, gall attack, herbivory, insect galls, plant quality, Qualea parviflora, RAPD, Vochysiaceae     

Volatile and molecular characterization of two Portuguese endemic species: Angelica lignescens and Melanoselinum decipiens

Chemical and genetic differences of 11 individuals of Angelica lignescens and 3 individuals of Melanoselinum decipiens were studied to determine whether volatile components could be used as taxonomical markers and to examine the correlation between molecular and chemical markers. The volatiles were isolated from each individual by distillation–extraction and analyzed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS). The same plants were analyzed by Inter Simple Sequence Repeat (ISSR) using 14 arbitrary primers. The main component of volatile fraction isolated from vegetative aerial parts of A. lignescens was limonene (57–86%) while in M. decipiens the main components detected were β-pinene (38–47%) and sabinene (1–33%). Cluster analyses based both on the chemical composition of volatile fraction and on molecular markers grouped the 14 accessions in two main groups, corresponding to each of the two species under study. Considering the species together, a moderate Pearson's correlation of r = 0.61 was obtained between the two analyses.     

Suberins of Malus pumila stem and root corks

The suberin contents of the isolated superficial cork layers of Malus pumila stems and root ranged from 15 to 35% of the dry weight. The qualitative composition of the aliphatic monomers obtained after alkaline depolymerization of the extractive-free corks was similar but some quantitative differences were found according to cultivar and age of the cork layer. 1-Alkanols (mainly 22:0, 24:0 and 26:0), alkanoic acids (mainly 22:0 and 24:0), α, ω-alkanedioic acids (mainly 16:0, 18:1 (9) and 18:0) and ω-hydroxyalkanoic acids (mainly 18:1 (9) and 22:0) were major constituents of all the samples examined and together they comprised 40–50% of the total monomeric mixture. The remainder was composed mainly of 9,10-epoxy-18-hydroxy-and 9,10,18-trihydroxyoctadecanoic acids. The corresponding dibasic acids, 9,10-epoxy- and 9,10-dihydroxyoctadecane-1,18-dioic, were minor components as were C₁₆ and C₁₈ dihydroxyalkanoic acids (mainly 10,16-dihydroxyhexadecanoic and 10,18-dihydroxyoctadecanoic acids, respectively). The root suberin dittered from that of the stem in containing larger amounts of 9,10-epoxy- 18-hydroxyoctadecanoic and 18-hydroxyoctadec-9-enoic acids.     

Use of Volatile Organic Components in Scat to Identify Canid Species

Abstract: Identification of wildlife species from indirect evidence can be an important part of wildlife management, and conventional methods can be expensive or have high error rates. We used chemical characterization of the volatile organic constituents (VOCs) in scat as a method to identify 5 species of North American canids from multiple individuals. We sampled vapors of scats in the headspace over a sample using solid-phase microextraction and determined VOC content using gas chromatography with a flame ionization detector. We used linear discriminant analysis to develop models for differentiating species with bootstrapping to estimate accuracy. Our method correctly classified 82.4% (bootstrapped 95% CI = 68.8–93.8%) of scat samples. Red fox (Vulpes vulpes) scat was most frequently misclassified (25.0% of scats misclassified); red fox was also the most common destination for misclassified samples. Our findings are the first reported identification of animal species using VOCs in vapor emissions from scat and suggest that identification of wildlife species may be plausible through chemical characterization of vapor emissions of scat. (JOURNAL OF WILDLIFE MANAGEMENT 72(3):792–797; 2008)     

Expression of DNA methyltransferases is involved in Quercus suber cork quality

Cork oak (Quercus suber) is an important Portuguese species, mainly due to the economic value of the cork it produces. Cork results from phellogen, a meristematic tissue, which can locally produce lenticels or have discontinuities, originating “defects”: pores and nail inclusions that are detrimental to cork industrial use. Epigenetic processes control plant development and its deregulation can lead to altered phenotypes; therefore, the study of epigenetic players in the phellogen is important to understand the emergence of cork's defects. DNA methyltransferases (DNMTs) and one protein associated to MET1 (DMAP1) were characterized in Q. suber, and their gene expression was analyzed in phellogen and contiguous differentiating cell layers of trees producing high and low quality cork, after the evaluation of their defects by physical and image analysis methods. All classes of DNMTs (MET, DRM, and CMT) with the respective canonical motifs were identified in Q. suber. The expression analyses of these genes showed that QsDRM2 was the most active methyltransferases in the cells analyzed, and that all the genes were differentially expressed in trees with distinct cork quality, with a tendency for higher expression levels in low quality producers. Interestingly, the global methylation level was higher in cells with low expression of DNA methyltransferases. A positive and significant correlation was obtained between QsDMAP1 gene expression and the percentage of cork defects. This work provides the first evidence that cork quality in Q. suber is likely influenced by epigenetic mechanisms.     

Development of microsatellite markers for Qualea grandiflora (Vochysiaceae), a typical species of the Brazilian cerrado

? Premise of the study: Microsatellite primers were developed to investigate genetic diversity and population structure of Qualea grandiflora, a typical species of the Brazilian cerrado. ? Methods and Results: Eight microsatellite loci were isolated using an enrichment cloning protocol. These loci were tested on a population of 110 individuals of Q. grandiflora collected from a cerrado fragment in S?o Paulo State, Brazil. The loci polymorphism ranges from seven to 19 alleles and the average heterozygosity value is 0.568, while the average polymorphic information content is 0.799. ? Conclusions: The developed markers were found to be highly polymorphic, indicating their applicability to studies of population genetic diversity in Q. grandiflora.     

Analysis of the novel VNTR polymorphisms ofMUC8 gene

Analysis of mucin genes has identified the presence of several features that may represent important functional domains in mucin glycoproteins. In the central region of each mucin, there are a variable number of tandem repeats (VNTR; minisatellites). However, their genomic levels are unclear because of complex genomic properties. We report here the distribution of VNTR and polymorphic analysis ofMUC8. We searched for VNTR ofMUC8 using the Tandem Repeat Finder program and found nine VNTR motif. Six (MUC8 MS1～MS6) among the nine VNTRs were evaluated in this study. Each VNTR inMUC8 region was analyzed in genomic DNA obtained from 200 unrelated individuals and multi-generational families. All VNTRs (MUC8 MS1, -MS2, -MS3, -MS4,-MS5 and -MS6) were genotyped as polymorphic. The degree of polymorphism within theMUC8-MS5 showed the highest heterozygosity (h = 0.786) in theMUC8 region. In order to perform a segregation analysis of the VNTRs inMUC8, we analyzed genomic DNA obtained from two generations of five families and from three generations of two families. Six of the polymorphic VNTRs were transmitted through meiosis following a Mendelian inheritance, which suggests that polymorphic VNTRs could be useful markers for paternity mapping and DNA fingerprinting.