Isolation and characterization of 12 microsatellite loci for Rhamnus alaternus (Rhamnaceae)

Twelve polymorphic microsatellite loci were developed for Rhamnus alaternus using an enriched-library approach. We detected 69 alleles in 49 individuals genotyped (mean number of alleles per locus was 4.79) in two different populations. The values of observed and expected heterozygosities ranged from 0.045 to 0.963 and 0.089 to 0.873 respectively. Levels of polymorphism and the exclusionary power of the developed markers render them readily applicable for studies of contemporary pollen and seed gene flow through parentage analyses.     

Sex ratio, reproductive investment and flowering phenology in dioecious Rhamnus alaternus (Rhamnaceae)

Guitián, J. 1995. Sex ratio, reproductive investment and flowering phenology in dioecious Rhamnus alaternus (Rhamnaceae). - Nord. J. Bot. 15: 139–143. Copenhagen. ISSN 0107–055X.
During 1992 and 1993 I investigated the reproductive biology of the dioecious Mediterranean shrub Rhamnus alaternus in a population in the northwest Iberian Peninsula. Reproductive investment was estimated as the mean total dry weight of reproductive organs per branch tip. I also estimated number of flowers produced per plant and population sex ratio, and investigated the spatial distribution of the sexes and flowering phenology. The sex ratio was 1:1, and the spatial distribution of the sexes was random. Male plants produced 2.6 times more flowers than female plants, but the overall reproductive investment by females was 5- to 9-fold higher. In both years of study, male plants commenced flowering first. The male and female flowering peaks coincided closely (in late March) in both years. The results of this study suggest that male and female R. alaternus differ most notably in the amount of resources allocated to reproduction.     

Anti-genotoxic and free-radical scavenging activities of extracts from (Tunisian) Myrtus communis

The effect of extracts from leaves of Myrtus communis on the SOS reponse induced by Aflatoxin B1 (AFB1) and Nifuroxazide was investigated in a bacterial assay system, i.e. the SOS chromotest with Escherichia coli PQ37. Aqueous extract, the total flavonoids oligomer fraction (TOF), hexane, chloroform, ethyl acetate and methanol extracts and essential oil obtained from M. communis significantly decreased the SOS response induced by AFB1 (10 microg/assay) and Nifuroxazide (20 microg/assay). Ethyl acetate and methanol extracts showed the strongest inhibition of the induction of the SOS response by the indirectly genotoxic AFB1. The methanol and aqueous extracts exhibited the highest level of protection towards the SOS-induced response by the directly genotoxic Nifuroxazide. In addition to anti-genotoxic activity, the aqueous extract, the TOF, and the ethyl acetate and methanol extracts showed an important free-radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. These results suggest the future utilization of these extracts as additives in chemoprevention studies.     

Reproductive lability in pre-dispersal biology ofRhamnus alaternus L. (Rhamnaceae)

Summary Rhamnus alaternus L. is a dioecious, fleshy-fruited shrub, typical of Mediterranean vegetation, which has been recorded from the region since the tropical Tertiary. Reproductive biology of this species has been studied in Southern Italy over a period of 4 years with both field and laboratory experiments. In all the studied populations male plants predominate over the females, have a higher flower density and flower regularly every year while females are usually alternate-bearing. A strong negative association between fruit formation and vegetative growth has been found indicating that the presence of fruits on a branch reduces the growth of new shoots. This phenomenon, together with alternate-bearing, is evidence of a strong competition between vegetative growth and reproductive activity and indicates resource limitation. Flower morphology and quantity of pollen produced are typical of insect pollinated species, and flies of the Empididae and Muscidae were trapped bearing pollen at female plants but pollen is also dispersed by wind. Field experiments on pollination success showed that the exclusion of insects as pollen vectors produces a significant decrease in fruit development. Excess pollen provided by hand-pollination increases fruit set over open-pollination and reproductive success in the field is therefore pollen limited. The variability of sex expression, male fitness, fruit production, regrowth and pollination presented here suggests that these predispersal characteristics ofR. alaternus have been subject to evolutionary change since the tropical Tertiary climate was replaced by a Mediterranean one. This points to the possibility that reproductive, as well as morphological and physiological characteristics, may be equally selected for in extreme environments such as the Mediterranean.     

Flavonoids from Rhamnus alaternus L. (Rhamnaceae): Kaempferol 3-O-β-isorhamninoside and rhamnocitrin 3-O-β-isorhamninoside protect against DNA damage in human lymphoblastoid cell and enhance antioxidant activity

Kaempferol 3-O-β-isorhamninoside (K3O-ir) and rhamnocitrin 3-O-β-isorhamninoside (R3O-ir), were isolated from Rhamnus alaternus L leaves. The genotoxic and antigenotoxic properties of these compounds were investigated by assessing the induction and inhibition of the genotoxicity induced by the direct-acting mutagen, hydrogen peroxide (H₂O₂), using the “comet assay.” K3O-ir and R3O-ir exhibited a preventive effect against H₂O₂ induced DNA damages in human lymphoblastoid TK6 cells and its derivative the p53 deficient cell line NH32.These two flavonoids, also investigated for their antioxidant capacities, using different antioxidant tests, such as: Cuprac, Frap and reducing power assays, revealed significant activity through their capacities to transfer electrons.     

Critical stages in the recruitment process of Rhamnus alaternus L

BACKGROUND AND AIMS: Rhamnus alaternus is a Mediterranean shrub commonly used in reforestation programs. Although several aspects of its reproductive biology have been studied, little is known about the importance of the different recruitment stages in the overall regeneration process of this species, which limits its proper use in Mediterranean forests and shrubland management. The aim of the present work was to quantify the importance of the different recruitment stages in the regeneration process of R. alaternus. METHODS: Two populations of Rhamnus alaternus on the island of Mallorca that differ in climatic conditions, type of habitat and sex ratio were studied. The importance of seed production, seed dispersal and predation, seedling emergence and seedling survival for the regeneration of this species were quantified. KEY RESULTS: In both populations, fruit set and fruit removal by animals were not critical stages, since almost half of the flowers became mature fruits and 90% of those were dispersed. Most seeds were deposited under female conspecifics (86 and 47%, at Lloret and Esporles, respectively), and very few were found in open inter-spaces (1 and 5%). Post-dispersal seed predation (mostly by ants and rodents) was very high in both populations. Seedling emergence took place during autumn and early winter and it ranged from 31 to 68% depending upon year and microhabitat. The majority of emerged seedlings died during the first year, mainly due to desiccation; such mortality was influenced by rainfall and differed among microhabitats (varying from 67 to 100%). The general spatial distribution of seed rain was concordant with the seedling emergence and survival pattern in both populations. CONCLUSIONS: The recruitment of Rhamnus alaternus appeared to be mainly limited by seed and seedling survival, regardless of the type of habitat in which the species is found.     

Leaf domatia in the section Alaternus (Miller) DC. of the genus Rhamnus (Rhamnaceae)

MARTINEZ-SOLIS, I., IRANZO, J., ESTRELLES, E. & IBARS, A. M., 1993. Leaf domatia in the section Alaternus (Miller) D C, of the genus Rhamnus (Rhamnaceae). Anatomical and morphological study of leaves from three species of Rhamnus {R. alaternus, R. myrtifolius and R. ludovici-salvatoris) which comprise the section Alaternus (Miller) DC. from the Iberian Peninsula and Balearic Islands, have revealed the presence of domatia, which are macroscopic infundibuliform structures. Although not all the leaves of one single plant showed domatia, all the individuals of each species examined presented these structures.     

Genetic variability of the narrow endemic Rhamnus persicifolia Moris (Rhamnaceae) and its implications for conservation

Rhamnus persicifolia Moris is an endemic small tree belonging to the Rhamnus cathartica group, growing along mountainous streams of Central-Eastern Sardinia (Italy). ISSR markers were used to detect the genetic diversity within and among six populations representative of the species distribution range. In spite of the limited distribution of this endemic taxon, fairly high levels of genetic diversity were detected. Percentage of polymorphic bands (PPB), gene diversity (H_S and H_T) and Shannon information measure (Sh) were calculated both at population (PPB = 30.70%, H_S = 0.1105, Sh = 0.1646) and at species level (PPB = 68.42%, H_T = 0.2066, Sh = 0.3139).The existence of a spatial distribution of genetic diversity in R. persicifolia was revealed by a low gene flow, a fairly high level of genetic differentiation (G_ST = 0.4583) among populations and a positive correlation between genetic and geographic distances (Mantel test, r = 0.71, p = 0.016). The spatial genetic structure was also confirmed with BAPS analysis. Our results show that a certain level of isolation by distance and sex-ratio bias may explain the distribution of genetic diversity among populations.Conservation measures are suggested on the basis of the genetic diversity detected, by implementing an integrated in situ and ex situ conservation program for each population, in order to ensure effective protection for this endemic species.     

Microsatellite primers in Rhamnus cathartica (Rhamnaceae) and applicability in related taxa to assess hybridization events

? Premise of the study: Microsatellite primers were developed in the introduced shrub Rhamnus cathartica L., to investigate potential hybridization events with related taxa as a stimulus of invasive spread. ? Methods and Results: Using a non-radioactive protocol, 15 primer sets were identified in North American and European populations of R. cathartica. The primers amplified di-, tri- and pentanucelotide repeats with 1-14 alleles per locus. Most primers also amplified in R. lanceolata, R. smithii, and/or Frangula alnus (previously R. frangula). ? Conclusions: These results indicate the utility of primers in R. cathartica for future genetic studies as well as their potential applicability in other related taxa.     

Exclusive frugivory and seed dispersal of Rhamnus alaternus in the bird breeding season

We studied avian frugivory and seed dispersal in a dioecious shrub, Rhamnus alaternus, focusing on the quantitative and qualitative components of effectiveness. The study took place at three locations in the northeast of the Iberian Peninsula, and examined bird behaviour, intensity of feeding, and the consequences for seedling emergence. The coincidence between the bird breeding season and fruit ripening of R. alaternus in the absence of other ripe fruit, generates a monospecific interaction. The extant frugivorous species were mainly legitimate seed dispersers and their abundance was low. Sylvia melanocephala and S. undata were the most important at one site whereas S. atricapilla, Erithacus rubecula and Turdus merula predominated at the other two sites. Fruit handling took place directly on the branches. Bird species used microhabitats differently as first post-feeding perch, which usually was a short distance away. The low density of frugivorous birds in all localities, among others factors, resulted in satiation of the disperser community and many mature fruits unconsumed. Both adults and juveniles feed upon the plants and their foraging patterns are similar. Adults of S. melanocephala were observed to feed fruit to nestlings and consequently a second phase of dispersal potentially arises from the transport of fecal sacs. Pulp removal and passage through the digestive tract increased the probability of seedling emergence. This plant-dispersal interaction has important consequences, both positive and negative for the plant. Positively, the fruiting of R. alaternus at a time when other ripe fruits are not available avoids interspecific competition for seed dispersers. In addition, a low density of seed rain may reduce intraspecific competition. Negatively, the low density and small size of the breeding frugivorous bird community limit fruit handling and removal away from the parent plants, while the territorial behaviour of birds at that time of the year reduces the potential distances of seed dispersal.     

Myrmecochory and short-term seed fate in Rhamnus alaternus: Ant species and seed characteristics

Benefits conferred on plants in ant-mediated seed dispersal mutualisms (myrmecochory) depend on the fate of transported seeds. We studied the effects of elaiosome presence, seed size and seed treatment (with and without passage through a bird's digestive tract) on short-term seed fate in Rhamnus alaternus. In our study, we define short-term seed, or initial, seed fate, as the location where ants release the seeds after ant contact with it. The elaiosomes had the most influence on short-term fate, i.e. whether or not seeds were transported to the nest. The workers usually transported big seeds more often than small ones, but small ants did not transport large seeds. Effect of seed size on transport depended on the ant species and on the treatment of the seed (manual extraction simulating a direct fall from the parent plant vs. bird deposition corresponding to preliminary primary dispersal). Probability of removal of elaiosome-bearing seeds to the nest by Aphaenogaster senilis increased with increasing seed weight.     

Acaricidal and cytotoxic activities of extracts from selected genera of Australian Lamiaceae

Crude foliar extracts of 67 species from six subfamilies of Australian Lamiaceae were screened by whole organism contact toxicity on the polyphagous mite Tetranychus urticae Koch (Acari: Tetranychidae) by using a Potter precision spray tower. Cytotoxicity assessments against insect cell lines from Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) and Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) also were made. The Spodoptera cell line was more susceptible to extracts than the Drosophila cellline. No direct correlation was observed between the two screening methods, but several interesting relationships were identified. Extracts from subfamilies Ajugoideae, Scutellarioideae, Chloanthoideae, Viticoideae and Nepetoideae showed acaricidal activity, whereas only those from Ajugoideae and Nepetoideae displayed potent cytotoxic effects. A range of activities was observed for the 25 species of Plectranthus, 14 of which showed moderate-to-high contact toxicity against T. urticae. Overall, the lowest toxicity was observed for extracts from the plant subfamily Prostantheroideae, which showed little contact toxicity or cytotoxicity for the 18 extracts studied.     

Effects of the Argentine ant Linepithema humile on seed dispersal and seedling emergence of Rhamnus alaternus

We studied influence of the Argentine ant's Linepithema humile occurrence on seed dispersal of Rhamnus alaternus (Rhamnaceae). Rhamnus alaternus is a fleshy fruit plant dispersed primarily by birds; the seeds have an elaiosome attractive to ants. The observations were made in two study plots of Mediterranean cork-oak forests (one invaded and the other not invaded by L. humile ) over two years. For R. alaternus , presence of L. humile was associated with of the following: reduction of seed transport to ant nests (80.2% of seed offered were removed in non-invaded vs 7.1% in invaded area), a shorter seed dispersal distance (mean=90.2 cm in non-invaded vs 1.1 cm in invaded area), increase in seed retention time on the soil surface (median time in non-invaded was 50 vs 209.2 min in invaded area) and increased vulnerability to predation. In addition there was lower probability of seedling emergence, due to little elaiosome removal from the seeds (82.0% of emergence for seeds without elaiosome and 57.3% for seeds with elaiosome). These results, similar to findings obtained in South African fynbos, confirm that the Argentine ant invasion can strongly affect ecosystem processes in the Mediterranean biome.     

Leaf expansion in Rhamnus alaternus L. by leaf morphological, anatomical and physiological analysis

Morphological, anatomical and physiological traits of Rhamnus alaternus during leaf expansion were analysed. Bud break occurred when mean air temperature was 14.1 ± 1.2°C, and it was immediately followed by the increase of leaf area and leaf dry mass. The highest leaf expansion rates happened during the first 22 days of the process. Leaf area and leaf dry mass reached the steady-state value 46 and 62 days after bud break, respectively. Net photosynthesis increased from bud break to full leaf expansion, and total chlorophyll content had the same trend, confirmed by the correlation between the two variables. Leaf dark respiration peaked during the first 11 days of leaf expansion, then decreased and reached a steady-state value 34 days after bud break. R. alaternus completed cell division and cell enlargement of the epidermal tissue 28 days after bud break, and the ones of the mesophyll tissue at full leaf expansion. The results underline that morphological, anatomical and physiological leaf traits in R. alaternus are indicative of a less sclerophyllous species (i.e. higher specific leaf area) compared with other Mediterranean evergreen species. Moreover, the higher fraction of mesophyll volume occupied by the intercellular air spaces, and the ability to end the leaf expansion process before air temperature might be a limiting factor, makes R. alaternus closer to the mesophyte species.     

Antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian medicinal plant Eremophila neglecta

Aims: To determine the antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian plant Eremophila neglecta. Methods and Results: Antimicrobial activities of serrulatane compounds 8,19‐dihydroxyserrulat‐14‐ene ( 1 ) and 8‐hydroxyserrulat‐14‐en‐19‐oic acid ( 2 ) were tested against Gram‐negative and Gram‐positive bacteria including human and veterinary pathogens and some multidrug‐resistant isolates. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the compounds were determined by broth microdilution assay. Both compounds exhibited antibacterial activity against all Gram‐positive test strains. They showed antimycobacterial activity against isolates of Mycobacterium fortuitum and Mycobacterium chelonae. Of the five Gram‐negative bacteria tested, only Moraxella catarrhalis showed susceptibility to the compounds. Cytotoxic activities were tested in the Vero cell line. Compound 1 showed more activity than 2 in both antibacterial and cytotoxicity assays with cytotoxicity at concentrations similar to the MBC. Conclusions: Serrulatane compounds showed significant activity against medically important bacteria, with 1 exhibiting stronger antibacterial activity. However, they also displayed toxicity to mammalian cells. Significance and Impact of the Study: Serrulatanes are of interest as novel antibacterial compounds for use in biomedical applications; this study reports data obtained with a range of bacterial strains and mammalian cells, essential for assessing the capabilities and limitations of potential applicability of these compounds.