Pollination biology and breeding system of Zeyheria montana (Bignoniaceae)

The reproductive biology of Zeyheria montana was studied through field observations of flower visitors and floral events, controlled manual pollinations, and observations of pollen tube growth and ovule penetration by fluorescence microscopy. Analysis of secretory areas of the nectar chamber was made by flower dissections and histology of serial sections. The flower lasted 6–8 days, but pollen exposure and stigma receptivity occurred only up to the end of the first and second days, respectively. Pollination was effected by several species of hummingbirds, especially Colibri serrirostris. The flowers present a rudimentary, non-functional disc, and secretion of nectar is performed by corolla-borne glandular trichomes. Only hand cross-pollinated and natural-pollinated flowers set fruits. Artificially self-pollinated and non-pollinated flowers dried off after anthesis without presenting any swelling of the ovary. Almost all the ovules in selfed and crossed pistils were penetrated 96 h after pollination. However, a delay in ovule penetration in self-pollinated pistils was verified, which indicates the occurrence of late-acting self-incompatibility.We are grateful to Fundo de Apoio ao Ensino e à Pesquisa (UNICAMP) for financial support, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the fellowship granted to the first author (this work is part of a Ph.D. thesis presented to UNICAMP, Campinas, SP, Brazil).     

Micropropagation of Turbinicarpus laui Glass et Foster, an endemic and endangered species

Summary In a short period of time, a large number of adventitious shoots were regenerated from longitudinal sections of in vitro-germinated seedlings of the endangered Mexican cactus, Turbinicarpus laui. The induction medium consisted of Murashige and Skoog salts, supplemented with 6-benzylaminopurine (BA) and α-naphthaleneacetic acid (NAA) in a wide range of combinations. The most effective concentrations of growth regulators for shoots initiation were 8.8–13.32 μM BA with 0–2.68 μM NAA. After 3–4 mo., individualized shoots were rooted on half-strength Murashige and Skoog medium and then transferred to soil to acclimatize under greenhouse conditions. In vitro strategies play a key role in the conservation and propagation of this commercially important endangered species of cactus.     

Phylogeography and disjunct distribution in Lychnophora ericoides (Asteraceae), an endangered cerrado shrub species

Background and Aims

Lychnophora ericoides (Asteraceae) presents disjunct geographical distribution in cerrado rupestre in the south-east and central Brazil. The phylogeography of the species was investigated to understand the origin of the disjunct geographical distribution.

Methods

Populations in the south and centre of Serra do Espinhaço, south-east Brazil and on ten other localities in Federal District and Goiás in central Brazil were sampled. Analyses were based on the polymorphisms at chloroplast (trnL intron and psbA-trnH intergenic spacer) and nuclear (ITS nrDNA) genomes. From 12 populations, 192 individuals were sequenced. Network analysis, AMOVA and the Mantel test were performed to understand the relationships among haplotypes and population genetic structure. To understand better the origin of disjunct distribution, demographic parameters and time to most recent common ancestor (T_MRCA) were estimated using coalescent analyses.

Key Results

A remarkable differentiation between populations from the south-east and central Brazil was found and no haplotype was shared between these two regions. No significant effect of isolation by distance was detected. Coalescent analyses showed that some populations are shrinking and others are expanding and that gene flow between populations from the south-east and central Brazil was probably negligible.

Conclusions

The results strongly support that the disjunct distribution of L. ericoides may represent a climatic relict and that long-distance gene flow is unlikely. With an estimated time to most recent common ancestor (T_MRCA) dated from approx. 790 655 ± 36 551 years bp (chloroplast) and approx. 623 555 ± 55 769 years bp (ITS), it was hypothesized that the disjunct distribution may be a consequence of an expansion of the geographical distribution favoured by the drier and colder conditions that prevailed in much of Brazil during the Kansan glaciation, followed by the retraction of the distribution due to the extinction of populations in some areas as climate became warmer and moister.Key words: Disjunct geographical distribution, climatic relict, vicariance, coalescent analysis, phylogeography, endemism, Asteraceae, Lychnophora ericoides, Cerrado, cerrado rupestre, ITS, cpDNA     

Ovary peltate trichomes of Zeyheria montana (Bignoniaceae): developmental ultrastructure and secretion in relation to function

BACKGROUND AND AIMS: Nectar production in the Bignoniaceae species lacking a nectariferous functional disc is ascribed to trichomatic glands around the ovary base and/or on the inner corolla wall. Nevertheless, knowledge about the secretion and function of these glands is very incomplete. The purpose of this paper is to study, from a developmental viewpoint, the ultrastructure, histochemistry and secretory process of the peltate trichomes on the ovary of Zeyheria montana, a species in the Bignoniaceae which has a rudimentary disc. METHODS: Samples of the gynoecium at various developmental stages were fixed and processed for light and electron microscopy. Histochemistry and cytochemistry tests were performed to examine the chemical composition of exudates. Thin layer chromatography was used to determine the presence of alkaloids and terpenes in gynoecium and fruit extracts, and in fresh nectar stored in the nectar chamber. KEY RESULTS: Peltate trichomes at different developmental stages appear side by side from floral budding up to pre-dispersal fruit. Large plastids with an extensive internal membrane system consisting of tubules filled with lipophilic material, abundant smooth endoplasmic reticulum, few Golgi bodies, lipophilic deposits in the smooth endoplasmic reticulum and mitochondria, and scattered cytoplasmic oil droplets are the main characteristics of mature head cells. The secretion which accumulates in the subcuticular space stains positively for hydrophilic and lipophilic substances, with lipids prevailing for fully peltate trichomes. Histochemistry and thin layer chromatography detected terpenes and alkaloids. Fehling's test to detect of sugars in the secretion was negative. CONCLUSIONS: The continuous presence and activity of peltate trichomes on the ovary of Z. montana from early budding through to flowering and fruiting set, and its main chemical components, alkaloids and terpenes, suggest that they serve a protective function and are not related to the floral nectar source or to improving nectar quality.     

Micropropagation of endangered endemic Brazilian bromeliad Cryptanthus sinuosus (L.B. Smith) for in vitro preservation

An efficient liquid culture system for plant regeneration from leaflessstem–root axes of Cryptanthus sinuosus L. B. Smith(Bromeliaceae) was established. High regeneration rates (93%) were achieved inMurashige and Skoog's medium without growth regulators. Whole plants wereobtained in a single-step procedure, resulting in the production of 25.3± 3.6 plants/explant after 6 months of culture. Incubationof plant material at 35 ± 3 °C resulted in an increaseof 60% in the regeneration efficiency compared with tissues incubated at 28± 2 °C. Moreover, after 5–6 sub-cultures in thesame medium, the axes originated bud clusters that could be continuouslymultiplied and gave rise to 19.4 ± 3.2 whole plants per gram of freshmatter. It was estimated that the liquid culture system described is potentiallyable to produce about 4500 plants/explant/year. Rates of 98% acclimatizationwere achieved. The use of plants produced following this method for populationreinforcement and for in vitro preservation programs ofendangered populations is suggested.     

New Stenella and Parastenella species from the Brazilian cerrado

Five new Stenella species were found on native cerrado plants. Stenella erythroxyli-campestris, S. erythroxyli-suberosi and S. erythroxylicola were associated with plant species belonging in the family Erythroxylaceae; S. cyrtopodii was found infecting the rare Cyrtopodium eugenii (Orchidaceae), and S. ocoteae occurred on Ocotea sp. (Lauraceae). Finally Parastenella callisthenis-fasciculatae was collected on a Vochysiaceae (viz. Callisthene fasciculate) endemic to the cerrado.     

Isolation and characterization of 13 microsatellite loci from Incarvillea mairei (Bignoniaceae), an endemic species to the Himalaya-Hengduan mountains region

Incarvillea mairei (H. Léveillé) Grierson (Bignoniaceae) is an endemic species to the Himalaya-Hengduan Mountains region. Here, we developed 13 microsatellite markers from I. mairei using a modified biotin-streptavidin capture method. Number of alleles per locus (NA) ranged from 3 to 7 with an average of 4.615. The observed (H _O) and expected (H _E) heterozygosities were from 0.050 to 0.800 and from 0.249 to 0.815, respectively. Additionally, among the 13 identified microsatellite markers, 12 of them were successfully amplified in other three congeneric species, and most of them showed polymorphic. Obtained evidences suggest that these markers provide a useful tool for further study of the population genetic structure and the breeding system in this species or/and infra-generic species.     

New hyphomycete genera on Qualea species from the Brazilian cerrado

As part of studies on cerrado fungi three new hyphomycetes are described in association with trichomes on leaves of native species of Qualea (Vochysiaceae). These are: Trichomatomyces gen. nov. (type species: T. byrsonimae comb. nov.), Trichosporodochium gen. nov. (type species: T. cerradensis sp. nov.), and Phaeoidiomyces gen. nov. (type species: P. qualeae).     

Micropropagation of an endangered plant species: Coronopus navasii (Brassicaceae)

Plantlets of Coronopus navasii, an endangered species from SE Spain, were successfully regenerated from shoot and root segments excised from young seedlings. Initiation of multiple buds and development of leaves were obtained in MS modified medium plus l mg/l BAP and 0.1 mg/l NAA. Rooting was achieved by transfer of the isolated shoots to fresh MS medium without plant growth regulators. Plant survival of 47% was obtained six weeks after removal from in vitro culture conditions.Abbreviations BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - MS medium see Murashige and Skoog 1962     

Population genetic structure of Monimopetalum chinense (Celastraceae), an endangered endemic species of eastern China

BACKGROUND AND AIMS: Monimopetalum chinense (Celastraceae) standing for the monotypic genus is endemic to eastern China. Its conservation status is vulnerable as most populations are small and isolated. Monimopetalum chinense is capable of reproducing both sexually and asexually. The aim of this study was to understand the genetic structure of M. chinense and to suggest conservation strategies. METHODS: One hundred and ninety individuals from ten populations sampled from the entire distribution area of M. chinense were investigated by using inter-simple sequence repeats (ISSR). KEY RESULTS: A total of 110 different ISSR bands were generated using ten primers. Low levels of genetic variation were revealed both at the species level (Isp=0.183) and at the population level (Ipop=0.083). High clonal diversity (D = 0.997) was found, and strong genetic differentiation among populations was detected (49.06 %). CONCLUSIONS: Small population size, possible inbreeding, limited gene flow due to short distances of seed dispersal, fragmentation of the once continuous range and subsequent genetic drift, may have contributed to shaping the population genetic structure of the species.     

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.     

Pollination biology of two species ofKielmeyera (Guttiferae) from Brazilian cerrado vegetation

The pollination biology and breeding systems ofKielmeyera coriacea andK. speciosa, two sympatric woody species common in the cerrado vegetation of C. Brazil, were studied. Both species have similar nectarless, polystemonous Papaver-type flowers which are visited by a similar spectrum of insects, though they bloom in different seasons and are thus phenologically isolated. Large carpenter bees seem to be the most important pollinators and these and other bees effect buzz pollen retrieval despite the fact that anthers are not poricidal. Both species ofKielmeyera possess strong xenogamous breeding systems. The presence of staminate flowers and andromonoecy inK. coriacea, as well as the longevity ofK. speciosa flowers are discussed as alternative strategies to improve pollination success and reproductive efficacy.     

Physiological characteristics underpinning successful cryopreservation of endemic and endangered species of Bromeliaceae from the Brazilian Atlantic Forest

The germination requirements and the basis of the optimal water content before and after cryopreservation were studied for ten endangered Brazilian species of Bromeliaceae. Constant and alternating temperature regimes were used to determine the best conditions for seed germination. The relationship between seed water content and relative humidity was evaluated using water sorption isotherms at 15 °C. Seeds were cryostored at four water contents (3, 5, 7 and 9%) and three storage periods (0, 180 and 365 days), and loss in viability and vigour were estimated. Fresh seeds of all species showed maximum germination in < 30 days at temperatures between 20 and 30 °C, indicating the absence of a physical/morphological dormancy. A sigmoidal relationship between seed water content and relative humidity was observed with no apparent differences in sorption characteristics among the species. The optimum water content for cryopreservation of most of these species was c. 7%. Ultra‐drying (3% seed water content) had a detrimental effect on seed viability and vigour. Our experiments suggested orthodox storage behaviour for all species of Bromeliaceae examined as they are able to survive desiccation and freezing. This study has shown the feasibility of ex situ conservation in seed cryobanks of endangered bromeliads from the Brazilian Atlantic Forest to support future reintroduction of these species in nature. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 567–576.     

Micropropagation of Citrus halimii – an endangered species of South-east Asia

A successful system of direct organogenesis is described for the wild citrus tree, Citrus halimii Stone which used in vitro seedling explants cultured on Murashige and Skoog medium supplemented with 0.4–11.1 μM 6-benzyladenine. Hypocotyl was the best explant for multiple shoots regeneration. Maximum number of shoots was obtained on medium with 2.2–11.1 μM 6-benzyladenine. Rooting of regenerated shoots was best on Murashige and Skoog medium supplemented with 2.7 μM α-naphthalenacetic acid. In vitro and ex vitro rooted plantlets survived (on average 83.3%) after being transferred to the soil mixture consisting of soil, sand and organic material (1: 1: 1) and kept in the glasshouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Limonoids from the endemic Brazilian species Raulinoa echinata

Phytochemical survey of stems and leaves of the South Brazilian endemic Raulinoa echinata Cowan, Rutaceae led to the isolation of five limonoid derivatives: the widespread limonin, limonexic acid, kihadalactone B, a methoxylated limonexic acid derivative and a degraded limonoid structurally related to fraxinellone. The two latter compounds have been isolated for the first time. These compounds displayed weak inhibitory activity when assayed in vitro against trypomastigote forms of Trypanosoma cruzi. In this paper, the isolation, structure elucidation and bioactivity of these compounds are reported.     

Genetic variability of an endangered Bromeliaceae species (Pitcairnia albiflos) from the Brazilian Atlantic rainforest

Pitcairnia albiflos is a Bromeliaceae species endemic to Brazil that has been included as data-deficient in the extinction risk list of Brazilian flora. We analyzed genetic variability in P. albiflos populations using RAPD markers to investigate population structure and reproductive mechanisms and also to evaluate the actual extinction risk level of this species. Leaves of 56 individuals of P. albiflos from three populations were collected: Urca Hill (UH, 20 individuals), Chacrinha State Park (CSP, 24 individuals) and Tijuca National Park (TNP, 12 individuals). The RAPD technique was effective in characterizing the genetic diversity in the P. albiflos populations since it was possible to differentiate the populations and to identify exclusive bands for at least two of them. Even if there is low genetic diversity among them (CSP-UH = 0.463; CSP-TNP = 0.440; UH-TNP = 0.524), the populations seem to be isolated according to the low genetic diversity observed within them (H(pop) CSP = 0.060; H(pop) UH = 0.042; H(pop) TNP = 0.130). This fact might be the result of clonal and self-reproduction predominance and also from environmental degradation around the collection areas. Consequently, it would be important to protect all populations both in situ and ex situ to prevent the decrease of genetic variability. The low genetic variability among individuals of the same population confirms the inclusion of this species as critically endangered in the risk list for Brazilian flora.     

Genetic structure among morphotypes of the endangered Brazilian palm Euterpe edulis Mart (Arecaceae)

Euterpe edulis (Arecaceae) Mart has high ecological and economic importance providing food resources for more than 58 species of birds and 20 species of mammals, including humans. E. edulis is the second most exploited nontimber product from Brazilian Atlantic Forest. Due to overexploitation and destruction of habitats, E. edulis is threatened by extinction. Euterpe edulis populations have large morphological variations, with individuals having green, red, or yellow leaf sheath. However, no study has related phenotypic distinctions between populations and their levels of genetic structure. Thus, this study aimed to evaluate the diversity and genetic structure of different E. edulis morphotypes. We sampled 250 adult individuals in eight populations with the different morphotypes. Using 14 microsatellite markers, we access genetic diversity through population genetic parameters calculated in the GenAlex program and the diveRsity package in R. We used the Wilcoxon test to verify population bottlenecks and the genetic distance of Nei and Bayesian analysis for genetic clusters. The eight populations showed low allele richness, low observed heterozygosity, and high inbreeding values (f). In addition, six of the eight populations experienced genetic bottlenecks, which would partly explain the low genetic diversity in populations. Cluster analysis identified two clusters (K = 2), with green morphotype genetically distinguishing from yellow and red morphotypes. Thus, we show, for the first time, a strong genetic structure among E. edulis morphotypes even for geographically close populations.     

Antifungal activity of essential oils of Croton species from the Brazilian Caatinga biome

Aims: To find new antifungal agents among essential oils from Brazilian Croton species. Methods and Results: Plant leaves were steam distilled and the obtained essential oils were analyzed by gas chromatography/mass spectroscopy. The main constituents were estragole and anethole for Croton zehntneri, methyl-eugenol and bicyclogermacrene for Croton nepetaefolius and spathulenol and bicyclogermacrene for Croton argyrophylloides. The antifungal activity of essential oils was evaluated against Candida albicans, Candida tropicalis and Microsporum canis by the agar-well diffusion method and the minimum inhibitory concentration (MIC) by the broth microdilution method. Essential oils of Croton species demonstrated better activity against M. canis. Among the three plants C. argyrophylloides showed the best results, with MIC ranging from 9 to 19 μg ml⁻¹. The acute administration of the essential oil up to 3 g kg⁻¹ by the oral route to mice was devoid of overt toxicity. Conclusions: The studied essential oils are active in vitro against the dermatophyte M. canis and present relative lack of acute toxicity in vivo. Significance and Impact of the Study: Because of its antifungal activity and low toxicity, the essential oils of studied Croton species are promising sources for new phytotherapeutic agents to treat dermatophytosis.