Pollination Syndromes in Mediterranean Orchids—Implications for Speciation,Taxonomy and Conservation

The Mediterranean flora is spectacularly rich in orchid species that have evolved remarkable adaptations to their environment. Orchids have complex and delicate interactions with their pollinators, which makes them particularly prone to local extinction. Conservation actions should be encouraged for a range of endangered Mediterranean orchid species, but the current taxonomic confusion in several genera and the apparent disagreement among orchid taxonomists make the situation particularly confusing from a conservation perspective. In this review, we document how the different pollination syndromes of Mediterranean orchids (nectar reward, shelter offering, food deception and sexual deception) can have a profound impact on the type of reproductive barriers among species, on floral phenotypic variation as we perceive it, on potentially related processes of species sorting and extinction and, consequently, should have a strong influence on the related conservation management programs. We also highlight that the majority of Mediterranean orchids are pollinated by specialised bees often occupying otherwise narrow ecological niches (e.g. pollen specialisation, brood cell parasites, specific nesting site). This condition makes the orchid-pollinator interactions very fragile and several orchid species prone to local extinction. We illustrate this phenomenon by a selection of case studies that show how the adequate integration of the ecological requirements/traits of the orchids and their associated pollinators into conservation actions could help protect endangered species and ensure the sustainability of the often complex local pollination web.     

A framework for comparing pollinator performance: effectiveness and efficiency

Measuring pollinator performance has become increasingly important with emerging needs for risk assessment in conservation and sustainable agriculture that require multi‐year and multi‐site comparisons across studies. However, comparing pollinator performance across studies is difficult because of the diversity of concepts and disparate methods in use. Our review of the literature shows many unresolved ambiguities. Two different assessment concepts predominate: the first estimates stigmatic pollen deposition and the underlying pollinator behaviour parameters, while the second estimates the pollinator's contribution to plant reproductive success, for example in terms of seed set. Both concepts include a number of parameters combined in diverse ways and named under a diversity of synonyms and homonyms. However, these concepts are overlapping because pollen deposition success is the most frequently used proxy for assessing the pollinator's contribution to plant reproductive success. We analyse the diverse concepts and methods in the context of a new proposed conceptual framework with a modular approach based on pollen deposition, visit frequency, and contribution to seed set relative to the plant's maximum female reproductive potential. A system of equations is proposed to optimize the balance between idealised theoretical concepts and practical operational methods. Our framework permits comparisons over a range of floral phenotypes, and spatial and temporal scales, because scaling up is based on the same fundamental unit of analysis, the single visit.     

The flower biology ofCephalanthera longifolia (Orchidaceae)—pollen imitation and facultative floral mimicry

Solitary bees (Halictus sp.) were found to be the effective pollinators ofCephalanthera longifolia. In the same foraging flight the bees also visit flowers ofCistus salviifolius which has a similar colour pattern.Cephalanthera offers no reward to its pollinators, but orange papillae on its labellum successfully imitate pollen ofCistus. AsCephalanthera also attracts pollinators in the absence ofCistus, this is regarded as facultative floral mimicry.     

Buzz-pollination in three nectariferousBoraginaceae and possible evolution of buzz-pollinated flowers

Buzz-pollination was observed in three nectariferousBoraginaceae spp.:Onosma gigantea Lam.,Trichodesma africana (L.)R. Br. andT. boissieri Post. An evolutionary pathway from usual nectariferous flowers to typical buzz-pollinated flowers is suggested.     

Volatiles of two chemotypes of Majorana syriaca L. (Labiatae) as olfactory cues for the honeybee

Summary A study of olfactory discrimination by honeybee has been set up in order to understand the role of volatiles of the aromatic plant Majorana syriaca in attracting pollinating insects. The honeybee's response to volatiles from leaves and inflorescences of two M. syriaca chemotypes, which differ in the thymol carvacrol ratio of their volatiles, was tested using a bioessay method based on associative conditioning and recruitement techniques. Behavioural data show that a honeybee identifies and reacts selectively to olfactory signals from leaves and from inflorescences of the two chemotypes. Such data suggest that the volatiles from all parts of the aromatic plant M. syriaca may have a role in attracting pollinators. The vegetative parts produce a volatile emission which attracts the pollinators from a distance towards the whole plant. At close range these are directed by the flower signal which is amplified by the volatiles of bracts and leaves in the inflorescence.     

A new procedure to asses pollen viability

We tested pollen viability of eight species using four vital dyes, a new peroxidase test together with three other established methods (MTT, Baker’s and X-Gal), to determine their potential to differentiate fresh pollen from pollen heated for 2 h and 24 h at 80°C (killed pollen) and compared the results with in vitro germination. We found that two of three dyes previously employed to determine viability also stained killed pollen, while the new peroxidase test and MTT did not. We suggest that the latter two are the best methods to test pollen viability, since they do not normally stain either killed or aborted pollen. Received: 7 May 1999 / Revision accepted: 25 October 1999     

Pollen viability and longevity: Practical, ecological and evolutionary implications

The present article reviews the various definitions and terminology of pollen viability and longevity as well as the various tests of its assessment. We compare the advantages and the disadvantages of each method and suggest some practical implications as revealed by the extensive data. We recognize eight main hypotheses concerning the ecology and the evolution of pollen longevity and critically evaluated them according to the literature. The hypotheses are grouped as follows: (1) Desiccation risk-carbohydrate content; (2) Pollen packaging; (3) Pollen competitive ability; (4) Pollinator activity-stigma receptivity duration; (5) Self-pollination chance; (6) Pollen exposure schedule; (7) Pollen travel distance, and (8) Pollen removal chance.     

High yield of biologically active recombinant human amelogenin using the baculovirus expression system

The amelogenins are secreted by the ameloblast cells of developing teeth; they constitute about 90% of the enamel matrix proteins and play an important role in enamel biomineralization. Recent evidence suggests that amelogenin may also be involved in the regeneration of the periodontal tissues and that different isoforms may have cell-signalling effects. During enamel development and mineralization, the amelogenins are lost from the tissue due to sequential degradation by specific proteases, making isolation of substantial purified quantities of full-length amelogenin challenging. The aim of the present study was to express and characterize a recombinant human amelogenin protein in the eukaryotic baculovirus system in quantities sufficient for structural and functional studies. Human cDNA coding for a 175 amino acid amelogenin protein was subcloned into the pFastBac HTb vector (Invitrogen), this system adds a hexa-histidine tag and an rTEV protease cleavage site to the amino terminus of the expressed protein, enabling effective one-step purification by Ni2+-NTA affinity chromatography. The recombinant protein was expressed in Spodoptera frugiperda (Sf9) insect cells and the yield of purified his-tagged human amelogenin (rHAM+) was up to 10 mg/L culture. Recombinant human amelogenin (rHAM+) was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, peptide mapping, and MS/MS sequencing. Production of significant amounts of pure, full-length amelogenin opened up the possibility to investigate novel functions of amelogenin. Our recent in vivo regeneration studies reveal that the rHAM+ alone could bring about regeneration of the periodontal tissues; cementum, periodontal ligament, and bone.     

Monitoring histone deacetylase inhibition in vivo: noninvasive magnetic resonance spectroscopy method

Histone deacetylase inhibitors (HDACis) are emerging as promising and selective antitumor agents. However, HDACis can lead to tumor stasis rather than shrinkage, in which case, traditional imaging methods are not adequate to monitor response. Consequently, novel approaches are needed. We have shown in cells that (19)F magnetic resonance spectroscopy (MRS)-detectable levels of the HDAC substrate Boc-Lys-TFA-OH (BLT) are inversely correlated with HDAC activity. We extended our investigations to a tumor xenograft model. Following intraperitoneal injection of BLT, its accumulation within the tumor was monitored by in vivo (19)F MRS. In animals treated with the HDACi suberoylanilide hydroxamic acid (SAHA), tumoral BLT levels were higher by 77% and 132% on days 2 and 7 of treatment compared with pretreatment levels (n = 6; p < .05). In contrast, tumoral BLT levels remained unchanged in control animals and in normal tissue. Thus, (19)F MRS of BLT detected the effect of HDACi treatment as early as day 2 of treatment. Importantly, tumor size confirmed that SAHA treatment leads to inhibition of tumor growth. However, difference in tumor size reached significance only on day 6 of treatment. Thus, this work identifies BLT as a potential molecular imaging agent for the early noninvasive MRS detection of HDAC inhibition in vivo.     

Delayed Selfing in an Alpine Biennial Gentianopsis paludosa (Gentianaceae) in the Qinghai-Tibetan Plateau

Delayed selfing could provide ovules with an opportunity to be fertilized as a means of "pollination assurance" before the flowers wilt. It could, thus, be regarded as an adaptation to unpredictable pollinator environments. Within the alpine biennial Gentianopsis paludosa, the showy flowers and herkogamy at the early stage of a flower's life cycle may favor outcrossing. As the flower ages, anthers contact the central stigma due to the elongation of all filaments, resulting in autonomous selfing. Flower visitors are extremely rare in a high altitude population; and examination of the mating system indicates that G. paludosa is self-pollinated under natural conditions in this population. While at the lower altitude, the bumblebee visitation rate is relatively high but possibly unreliable. Stigma receptivity is the highest on the third day of anthesis, and decreases thereafter. Pollen viability is the highest when flowers open, and gradually decreases later. Self-pollination of G. paludosa occurs at the late stage of a flower's lifecycle when stigma receptivity and pollen viability have both decreased, suggesting delayed selfing and assurance of seed production. This delayed selfing could assure seed production under the constraints of pollinator scarcity, but ensure outcrossing when pollinators were available. Such a flexible pollination mechanism is highly adaptive in the alpine environment of the Qinghai-Tibetan Plateau.