Clara de Vega

Yeasts in floral nectar: a quantitative survey

BACKGROUND AND AIMS One peculiarity of floral nectar that remains relatively unexplored from an ecological perspective is its role as a natural habitat for micro-organisms. This study assesses the frequency of occurrence and abundance of yeast cells in floral nectar of insect-pollinated plants from three contrasting plant communities on two continents. Possible correlations between interspecific differences in yeast incidence and pollinator composition are also explored. METHODS The study was conducted at three widely separated areas, two in the Iberian Peninsula (Spain) and one in the Yucatán Peninsula (Mexico). Floral nectar samples from 130 species (37-63 species per region) in 44 families were examined microscopically for the presence of yeast cells. For one of the Spanish sites, the relationship across species between incidence of yeasts in nectar and the proportion of flowers visited by each of five major pollinator categories was also investigated. KEY RESULTS Yeasts occurred regularly in the floral nectar of many species, where they sometimes reached extraordinary densities (up to 4 x 10(5) cells mm(-3)). Depending on the region, between 32 and 44 % of all nectar samples contained yeasts. Yeast cell densities in the order of 10(4) cells mm(-3) were commonplace, and densities >10(5) cells mm(-3) were not rare. About one-fifth of species at each site had mean yeast cell densities >10(4) cells mm(-3). Across species, yeast frequency and abundance were directly correlated with the proportion of floral visits by bumble-bees, and inversely with the proportion of visits by solitary bees. CONCLUSIONS Incorporating nectar yeasts into the scenario of plant-pollinator interactions opens up a number of intriguing avenues for research. In addition, with yeasts being as ubiquitous and abundant in floral nectars as revealed by this study, and given their astounding metabolic versatility, studies focusing on nectar chemical features should carefully control for the presence of yeasts in nectar samples.

Zooming-in on floral nectar: a first exploration of nectar-associated bacteria in wild plant communities

Floral nectar of some animal-pollinated plants usually harbours highly adapted yeast communities which can profoundly alter nectar characteristics and, therefore, potentially have significant impacts on plant reproduction through their effects on insect foraging behaviour. Bacteria have also been occasionally observed in floral nectar, but their prevalence, phylogenetic diversity and ecological role within plant-pollinator-yeast systems remains unclear. Here we present the first reported survey of bacteria in floral nectar from a natural plant community. Culturable bacteria occurring in a total of 71 nectar samples collected from 27 South African plant species were isolated and identified by 16S rRNA gene sequencing. Rarefaction-based analyses were used to assess operational taxonomic units (OTUs) richness at the plant community level using nectar drops as sampling units. Our results showed that bacteria are common inhabitants of floral nectar of South African plants (53.5% of samples yielded growth), and their communities are characterized by low species richness (18 OTUs at a 16S rRNA gene sequence dissimilarity cut-off of 3%) and moderate phylogenetic diversity, with most isolates belonging to the Gammaproteobacteria. Furthermore, isolates showed osmotolerance, catalase activity and the ability to grow under microaerobiosis, three traits that might help bacteria to overcome important factors limiting their survival and/or growth in nectar.

Permanent Genetic Resources added to the Molecular Ecology Resources Database 1 February 2010-31 March 2010.

This article documents the addition of 142 microsatellite marker loci to the Molecular Ecology Resources database. Loci were developed for the following species: Agriophyllum squarrosum, Amazilia cyanocephala, Batillaria attramentaria, Fungal strain CTeY1 (Ascomycota), Gadopsis marmoratus, Juniperus phoenicea subsp. turbinata, Liriomyza sativae, Lupinus polyphyllus, Metschnikowia reukaufii, Puccinia striiformis and Xylocopa grisescens. These loci were cross‐tested on the following species: Amazilia beryllina, Amazilia candida, Amazilia rutila, Amazilia tzacatl, Amazilia violiceps, Amazilia yucatanensis, Campylopterus curvipennis, Cynanthus sordidus, Hylocharis leucotis, Juniperus brevifolia, Juniperus cedrus, Juniperus osteosperma, Juniperus oxycedrus, Juniperus thurifera, Liriomyza bryoniae, Liriomyza chinensis, Liriomyza huidobrensis and Liriomyza trifolii.

Microorganisms transported by ants induce changes in floral nectar composition of an ant-pollinated plant

PREMISE OF THE STUDY Interactions between plants and ants abound in nature and have significant consequences for ecosystem functioning. Recently, it has been suggested that nectar-foraging ants transport microorganisms to flowers; more specifically, they transport yeasts, which can potentially consume sugars and alter nectar composition. Therefore, ants could indirectly change nectar sugar profile, an important floral feature involved in the plant-pollinator mutualism. But this novel role for ants has never been tested. We here investigate the effects of nectarivorous ants and their associated yeasts on the floral nectar sugar composition of an ant-pollinated plant. METHODS Differences in the nectar sugar composition of ant-excluded and ant-visited flowers were examined in 278 samples by using high-performance liquid-chromatography. The importance of the genetic identity and density of ant-transported basidiomycetous and ascomycetous yeasts on the variation of nectar traits was also evaluated. KEY RESULTS Ant visitation had significant effects on nectar sugar composition. The nectar of ant-visited flowers contained significantly more fructose, more glucose, and less sucrose than the nectar of ant-excluded flowers, but these effects were context dependent. Nectar changes were correlated with the density of yeast cells in nectar. The magnitude of the effects of ant-transported ascomycetes was much higher than that of basiodiomycetes. CONCLUSIONS Ants and their associated yeasts induce changes in nectar sugar traits, reducing the chemical control of the plant over this important floral trait. The potential relevance of this new role for ants as indirect nectar modifiers is a rich topic for future research into the ecology of ant-flower interactions.

A multi-year study of factors affecting fruit production in Aristolochia paucinervis (Aristolochiaceae)

Pollen limitation, resource limitation, fruit abortion, and predation have all been proposed as factors explaining low fruit set in hermaphroditic plants. We conducted a 5-year study combining field observations and pollination experiments to determine the causes of the low fruit set in Aristolochia paucinervis, a Mediterranean species with a specialized pollination system in two populations in SW Spain. Fruit initiation was markedly low, and between 28.6 and 75.0% of the flowering stems did not initiate any fruit. In most flowers, the number of germinated pollen grains was less than the number of ovules, and supplemental pollination significantly increased fruiting, indicating deficient pollination. In A. paucinervis, autonomous self-pollination seems to be a decisive factor in fruit production because the number of germinated pollen and the fruit set from flowers bagged before anthesis were similar to those in free-pollinated flowers. Only in 2005 did flowers that were successfully pollinated outnumber ripened fruits, suggesting that other factors limit fruiting. We found a significant positive correlation between tuber mass and fruit set. Deficient pollination and lack of resources could explain the low fruit set, but the relative consequences seem to vary spatially and temporarily.

Endozoochory by beetles: a novel seed dispersal mechanism

BACKGROUND AND AIMS Due in part to biophysical sized-related constraints, insects unlike vertebrates are seldom expected to act as primary seed dispersers via ingestion of fruits and seeds (endozoochory). The Mediterranean parasitic plant Cytinus hypocistis, however, possesses some characteristics that may facilitate endozoochory by beetles. By combining a long-term field study with experimental manipulation, we tested whether C. hypocistis seeds are endozoochorously dispersed by beetles. METHODS Field studies were carried out over 4 years on six populations in southern Spain. We recorded the rate of natural fruit consumption by beetles, the extent of beetle movement, beetle behaviour and the relative importance of C. hypocistis fruits in beetle diet. KEY RESULTS The tenebrionid beetle Pimelia costata was an important disperser of C. hypocistis seeds, consuming up to 17·5 % of fruits per population. Forty-six per cent of beetles captured in the field consumed C. hypocistis fruits, with up to 31 seeds found in individual beetle frass. An assessment of seeds following passage through the gut of beetles indicated that seeds remained intact and viable and that the proportion of viable seeds from beetle frass was not significantly different from that of seeds collected directly from fruits. CONCLUSIONS A novel plant-animal interaction is revealed; endozoochory by beetles may facilitate the dispersal of viable seeds after passage through the gut away from the parent plant to potentially favourable underground sites offering a high probability of germination and establishment success. Such an ecological role has until now been attributed only to vertebrates. Future studies should consider more widely the putative role of fruit and seed ingestion by invertebrates as a dispersal mechanism, particularly for those plant species that possess small seeds.

Genetic races associated with the genera and sections of host species in the holoparasitic plant Cytinus (Cytinaceae) in the Western Mediterranean basin

* Speciation via race formation is an important evolutionary process in parasites, producing changes that favour their development on particular host species. Here, the holoparasitic plant Cytinus, which has diverse host species in the family Cistaceae, has been used to study the occurrence of such races. * Amplified fragment length polymorphism (AFLP) analyses were performed on 174 individuals of 22 populations parasitizing 10 Cistaceae species in the Western Mediterranean basin. * Neighbour-joining, multivariate ordination analyses, and individual-based Bayesian analyses, clustered Cytinus populations into five well-characterized genetic races that, overall, agreed with the taxonomic sections of their hosts. In the AMOVA, among-races differences accounted for almost 50% of the genetic variation. The isolation-by-distance model was not supported by a Mantel test among Cytinus populations (r = 0.012; P = 0.456). All races showed low within-population genetic diversity, probably as a result of restricted pollen flow aggravated by flowering asynchrony, restricted seed dispersion, or stochastic processes. * The genetic differentiation among the five races of Cytinus is congruent with the view that these races are well-characterized lineages that have evolved independently as a result of selective pressures imposed by their hosts. This pattern, with genetically distinctive groups associated with the infrageneric sections of the host species, has not been reported previously for parasitic angiosperms.

Presence of yeasts in floral nectar is consistent with the hypothesis of microbial-mediated signaling in plant-pollinator interactions

Olfactory floral signals are significant factors in plant-pollinator mutualisms. Recently, unusual fermentation odours have been described in the nectar and flowers of some species. Since yeasts are common inhabitants of many angiosperms nectars, this raises the possibility that nectar yeasts may act as causal agents of fermentation odours in flowers and, therefore, as possible intermediate agents in plant signaling to pollinators. A recent field study has reported that nectar yeasts were quite frequent in floral nectar across three different regions in Europe and America, where they reached high densities (up to 105 cells/mm3). Yeast incidence in floral nectar differed widely across plant host species in all sampling sites. A detailed study currently in progress on one of the species surveyed in that study (Helleborus foetidus, Ranunculaceae) has detected that, in addition to interespecific differences in yeast incidence, there is also a strong component of variance in yeast abundance that takes place at the subindividual level (among flowers of the same plant, among nectaries of the same flower). If yeast metabolism is eventually proved to contribute significantly to floral scent, then multilevel patchiness in the distribution of nectar yeasts (among species, among individuals within species, and among flowers and nectaries of the same individual) might contribute to concomitant multilevel variation in plant signaling and, eventually, also in pollination success, pollen flow and plant fitness.

Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi sp. nov. and Rosenbergiella epipactidis sp. nov., three novel bacterial species isolated from floral nectar

The taxonomic status of nine strains of the family Enterobacteriaceae isolated from floral nectar of wild Belgian, French, South African and Spanish insect-pollinated plants was investigated following a polyphasic approach. Confirmation that these strains belonged to the genus Rosenbergiella was obtained from comparative analysis of partial sequences of the 16S rRNA gene and other core housekeeping genes (atpD [ATP synthase β-chain], gyrB [DNA gyrase subunit B] and rpoB [RNA polymerase β-subunit]), DNA-DNA reassociation data, determination of the DNA G+C content and phenotypic profiling. Two strains belonged to the recently described species Rosenbergiella nectarea, while the other seven strains represented three novel species within the genus Rosenbergiella. The names Rosenbergiella australoborealis sp. nov. (with strain CdVSA 20.1(T) [LMG 27954(T)=CECT 8500(T)] as the type strain), Rosenbergiella collisarenosi sp. nov. (with strain 8.8A(T) [LMG 27955(T)=CECT 8501(T)] as the type strain) and Rosenbergiella epipactidis sp. nov. (with strain 2.1A(T) [LMG 27956(T)=CECT 8502(T)] as the type strain) are proposed. Additionally, the description of the genus Rosenbergiella is updated on the basis of new phenotypic and molecular data.

Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria.