Extensive size homoplasy at a microsatellite locus in the Japanese bumblebee, Bombus diversus

An extensive size homoplasy was found at microsatellite locus B11 of the bumblebee, Bombus diversus, in northern to central Honshu, Japan. A total of 16 alleles of different nucleotide sequences in five length morphs was obtained at B11 for this species. Of these alleles, five were 141 base pairs (bp) in length, five were 137 bp and four were 133 bp. Allele diversity in each length morph was high compared with previous studies. It is noteworthy that this extensive size homoplasy was found in a relatively small geographic area, in contrast to results from previous studies. Reconstruction of a median‐joining network revealed the complicated evolutionary process of the locus, involving insertion/deletion and point mutations. Preliminary estimation of the mutation rate of the B11 locus in B. diversus gives a value comparable to those estimated from experimental Drosophila populations. Effects of the extensive size homoplasy in population genetic studies is discussed.     

Floral scent evolution in the section Pseudophrys: pollinator‐mediated selection or phylogenetic constraints?

• Sexually deceptive orchid species from the Mediterranean genus Ophrys usually interact with one or a few pollinator species by means of specific floral scents. In this study, we investigated the respective role of pollinator‐mediated selection and phylogenetic constraints in the evolution of floral scents in the section Pseudophrys.
• We built a phylogenetic tree of 19 Pseudophrys species based on three nuclear loci; we gathered a dataset on their pollination interactions from the literature and from our own field data; and we extracted and analysed their floral scents using solid phase microextraction and gas chromatography‐mass spectrometry. We then quantified the phylogenetic signal carried by floral scents and investigated the link between plant–pollinator interactions and floral scent composition using phylogenetic comparative methods.
• We confirmed the monophyly of the section Pseudophrys and demonstrated the existence of three main clades within this section. We found that floral scent composition is affected by both phylogenetic relationships among Ophrys species and pollination interactions, with some compounds (especially fatty acid esters) carrying a significant phylogenetic signal and some (especially alkenes and alkadienes) generating dissimilarities between closely related Pseudophrys pollinated by different insects.
• Our results show that in the section Pseudophrys, floral scents are shaped both by pollinator‐mediated selection and by phylogenetic constraints, but that the relative importance of these two evolutionary forces differ among compound classes, probably reflecting distinct selective pressures imposed upon behaviourally active and non‐active compounds.

     

Floral scent composition predicts bee pollination system in five butterfly bush (Buddleja,Scrophulariaceae) species

Traditionally, plant–pollinator interactions have been interpreted as pollination syndrome. However, the validity of pollination syndrome has been widely doubted in modern studies of pollination ecology. The pollination ecology of five Asian Buddleja species, B. asiatica, B. crispa, B. forrestii, B. macrostachya and B. myriantha, in the Sino‐Himalayan region in Asia, flowering in different local seasons, with scented inflorescences were investigated during 2011 and 2012. These five species exhibited diverse floral traits, with narrow and long corolla tubes and concealed nectar. According to their floral morphology, larger bees and Lepidoptera were expected to be the major pollinators. However, field observations showed that only larger bees (honeybee/bumblebee) were the primary pollinators, ranging from 77.95% to 97.90% of total visits. In this study, floral scents of each species were also analysed using coupled gas chromatography and mass spectrometry (GC‐MS). Although the five Buddleja species emitted differentiated floral scent compositions, our results showed that floral scents of the five species are dominated by substances that can serve as attractive signals to bees, including species‐specific scent compounds and principal compounds with larger relative amounts. This suggests that floral scent compositions are closely associated with the principal pollinator assemblages in these five species. Therefore, we conclude that floral scent compositions rather than floral morphology traits should be used to interpret plant–pollinator interactions in these Asian Buddleja species.     

Evidence of pollen transfer efficiency as the natural selection factor favoring a large corolla of Campanula punctata pollinated by Bombus diversus

Flowers exhibiting a large corolla encompassing a wide inner space, characteristic of bee-pollinated plant species, are postulated to be maintained by a natural selection mechanism related to their pollinators. To confirm the existence and to elucidate the mechanism of such selection, we investigated the effect of floral traits on male reproductive success (RS) and its components in experimental populations of Campanula punctata and C. microdonta (Campanulaceae) under pollination by Bombus diversus (Apidae). Larger corolla width significantly increased male RS per fruit, though there was no effect on pollen removal, showing, for the first time, that a wide corolla serves a reproductive function of enhancing pollen transfer efficiency (ratio of male RS to pollen removal). Selection by pollen transfer efficiency through an indirect effect of initial pollen number or postpollination success was experimentally ruled out. Several mechanisms are proposed to explain how this selection factor is exerted on C. punctata corollas. Received: 15 February 1997 / Accepted: 23 April 1997     

Pollination of Campomanesia phaea (Myrtaceae) by night‐active bees: a new nocturnal pollination system mediated by floral scent

• Bees are the most important diurnal pollinators of angiosperms. In several groups of bees a nocturnal/crepuscular habit developed, yet little is known about their role in pollination and whether some plants are adapted specifically to these bees. We used a multidisciplinary approach to investigate the reproductive biology and to understand the role of nocturnal/crepuscular bees in pollination of Campomanesia phaea (Myrtaceae), popularly named cambuci.
• We studied the floral biology and breeding system of C. phaea. We collected the floral visitors and tested the pollinators' effectiveness. We also determined the floral scents released at night and during daytime, and studied behavioural responses of crepuscular/nocturnal bees towards these scents.
• The flowers of cambuci were self‐incompatible and had pollen as the only resource for flower visitors. Anthesis lasted around 14 h, beginning at 04:30 h at night. The flowers released 14 volatile compounds, mainly aliphatic and aromatic compounds. We collected 52 species of floral visitors, mainly bees. Nocturnal and crepuscular bees (four species) were among the most frequent species and the only effective pollinators. In field bioassays performed at night, nocturnal/crepuscular bees were attracted by a synthetic scent blend consisting of the six most abundant compounds.
• This study describes the first scent‐mediated pollination system between a plant and its nocturnal bee pollinators. Further, C. phaea has several floral traits that do not allow classification into other nocturnal pollination syndromes (e.g. pollinator attraction already before sunrise, with pollen as the only reward), instead it is a plant specifically adapted to nocturnal bees.

     

The influence of relative plant density and floral morphological complexity on the behaviour of bumblebees

We assessed the combined effects of varying the relative density and the relative floral morphological complexity of plant species on the behaviour of their bumblebee pollinators. Three species of bumblebee (Bombus pascuorum, B. terrestris and B. hortorum) were observed foraging on experimental arrays consisting of pair-wise combinations of four plant species: Borago officinalis, Phacelia tanacetifolia (both with simple flowers), Antirrhinum majus and Linaria vulgaris (both with complex flowers). Plant arrangements consisted of either two simple-flower species, a simple with a complex species or two complex species. The number of plants in each array was constant, while the frequency of each species was manipulated so that it was either rare, equal or common compared with its competitor. Contrary to predictions, rare plants were actually at an advantage in terms of the number of bees attracted per plant. However, rare plants were at a disadvantage in terms of pollen wastage because foragers more often went to a flower of another species after visiting a rare plant. The behaviour of bees on each plant species was further affected by plant floral complexity and the identity of the other species in the array. The three bumblebee species were markedly different in their foraging behaviour and in their responses to varying floral density and complexity. Each species preferred particular flower species. The results are discussed with reference to resource partitioning among bumblebee species. Received: 29 July 1998 / Accepted: 5 October 1998     

Bumblebee-pollination and temporal change of the calyx tube length in Clematis stans(Ranunculaceae)

Clematis stans is dioecious semi-arboreal, with pale purple–blue, nodding, tubulous flowers in a paniculate inflorescence. Both male and female flowers produce nectar from the base of the calyx tube during a flowering period of 3 or 4 days, and are pollinated by two bumblebee species, Bombus diversus and B. honshuensis, with different proboscis lengths. When the flowers open, four sepals constructing a calyx tube separate at the top and their respective tips gradually curl up, so that a tubular part shortens. Observations at two field sites showed that B. diversus (with a longer proboscis) most often visits the flowers with a longer calyx tube, and B. honshuensis (with a shorter proboscis) the flowers with a shorter calyx tube, i.e., later in the flowering period. By changing the calyx tube length, the flowers of C. stans accept the two bumblebee species with different proboscis length as pollinators and thus increase the chance of pollination for each flower. It was also found that the two bumblebee species prefer the male flowers to the female flowers, although the female flowers secrete more nectar as a reward than male flowers. This is likely because they visit the male flowers to collect pollen grains in addition to nectar. Electronic Publication     

Abundance,body size,and morphology of bumblebees in an area where an exotic species, <Emphasis Type="Italic">Bombus terrestris</Emphasis>, has colonized in Japan

An exotic bumblebee species, Bombus terrestris, has colonized in Japan and becomes dominant in some local communities. We examined the effects of land use and bumblebee abundance on the number and body size of bumblebees collected using window traps in a lowland area in the southern Ishikari district, Hokkaido. In 2004, we collected 922 bumblebees of six species using 70 traps at 17 sites. A statistical model fitted to the data demonstrated that dispersion from commercial B. terrestris colonies used in greenhouses positively affected the number of B. terrestris caught by each trap. This exotic species was abundant in sites where paddy fields were prevalent, but three native species, B. hypocrita, B. ardens, and B. diversus, were abundant in sites where farms and woodlands were widespread. The local abundance of B. terrestris was not associated negatively with the number and body size of native bumblebees. Thus, we did not find any competitive interactions between exotic and native bumblebees although habitat conditions seem to be common determinants of the bumblebee populations. A morphological analysis showed that B. terrestris had intermediate tongue length between B. hypocrita and B. ardens.     

Nitrate reduction by Citrobacter diversus under aerobic environment

A new aerobic denitrifier, Citrobacter diversus, was isolated from both nitrification and denitrification sludge. To monitor the variation in the concentration of nitrogen oxides, aerobic denitrification by C. diversus was carried out in a batch reactor. When the nitrate concentration was greater than 180 mg N l⁻¹, the nitrate reduction rate became stable. The effect of the C/N ratio on the denitrification activity was also investigated. The results showed that the optimum denitrification activity was obtained when the C/N ratio was 4–5. The range of the C/N ratio was higher than that for traditional anoxic denitrification. The effect of the dissolved oxygen concentration was further studied; and it was found that the range of dissolved oxygen concentrations, both for specific growth rates and for specific denitrification rates, was 2–6 mg⁻¹. From these results, it can be concluded that both the concentration of dissolved oxygen and the C/N ratio are key factors in the aerobic denitrification by C. diversus. Received: 23 November 1999 / Received revision: 4 February 2000 / Accepted: 13 February 2000     

Unusual structural and chemical characteristics of the Dufour gland in the ant Meranoplus diversus

Abstract The Dufour gland of both queens and workers of Meranoplus diversus is very large. The reservoir is lined with flat epithelial cells, which usually contain smooth endoplasmic reticulum but also show the unusual accumulation of electron‐dense inclusions. Chemically, the gland contains an unusual mixture of long‐chain fatty acids and their derivatives, including hydrocarbons, ketones and amines with long‐chain substituents, hydroquinone, and sugars. Some of the substances identified are already known as insect defensive compounds against other arthropods.     

Morphological variation in relation to flower use in bumblebees

To understand resource partitioning in a bumblebee community, we analyzed various morphological characters. A total of 1269 individuals of six bumblebee species, Bombus ardens, B. hypocrita, B. diversus, B. ignitus, B. honshuensis and B. beaticola, were examined and principal component analysis showed that the bumblebee species were clearly differentiated. Glossa, prementa and head lengths were positively correlated with the second component, and a longer proboscis was associated with a narrower body, which may help bees to intrude into and access deep‐lying nectar sources. Bombus diversus, with a long proboscis and narrow body, preferred flowers with a long corolla tube, whereas B. hypocrita and B. ignitus, which have short proboscises and wide bodies, visited flowers with short corollas or dish‐shaped flowers. Two pairs of consubgeneric species that have similar morphological characteristics, B. ardens and B. beaticola, and B. hypocrita and B. ignitus, divided flower resources by habitat selection and seasonal partitioning. For resource partitioning among bumblebee species, not only morphology but also other factors, such as habitat and seasonal preference, flower use, foraging behavior, and interspecific interactions, are responsible.     

Intensity and the ratios of compounds in the scent of snapdragon flowers affect scent discrimination by honeybees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Floral scent is used by pollinators during foraging to identify and discriminate among flowers. The ability to discriminate among scents may depend on both scent intensity and the ratios of the concentrations of the volatile compounds of a complex mixture rather than on the presence of a few compounds. We used four scent-emitting cultivars of snapdragon (Antirrhinum majus) to test this hypothesis by examining the ability of honeybees to differentiate among their scents. Each cultivar produced three monoterpenes (myrcene, E--ocimene, and linalool) and five phenylpropanoids (methylbenzoate, acetophenone, dimethoxytoluene, cis-methylcinnamate, and trans-methylcinnamate). Cultivars were reliably classified by their scents in a canonical discriminant analysis. Honeybees were unable to discriminate among the scents of flowers of the same cultivar in our assay. The ability of honeybees to discriminate among the scents of different cultivars was a function of the intensity of the floral scent. Discrimination was also correlated to the distance among the scents described by the discriminant analysis; the cultivars that had the greatest differences observed in the discriminant analysis were the easiest to discriminate. Our results show that honeybees are capable of using all of the floral volatiles to discriminate subtle differences in scent.     

Floral integration,phenotypic covariance structure and pollinator variation in bumblebee‐pollinated Helleborus foetidus

By analysing patterns of phenotypic integration and multivariate covariance structure of five metric floral traits in nine Iberian populations of bumblebee‐pollinated Helleborus foetidus (Ranunculaceae), this paper attempts to test the general hypothesis that pollinators enhance floral integration and selectively modify phenotypic correlations between functionally linked floral traits. The five floral traits examined exhibited significant phenotypic integration at all populations, and both the magnitude and the pattern of integration differed widely among populations. Variation in extent and pattern of integration was neither distance‐dependent nor significantly related to between‐population variation in taxonomical composition and morphological diversity of the pollinator assemblage. Patterns of floral integration were closer to expectations derived from consideration of developmental affinities between floral whorls than to expectations based on a pollinator‐mediated adaptive hypothesis. Taken together, results of this study suggest that between‐population differences in magnitude and pattern of floral integration in H. foetidus are probably best explained as a consequence of random genetic sampling in the characteristically small and ephemeral populations of this species, rather than reflecting the selective action of current pollinators.     

Flexibility of Pollinator-Flower Relationship in Isodon umbrosus and I. effusus (Lamiaceae) and its Relation to Fruit-set and Seed-set

Isodon umbrosus was pollinated by two bumblebee pollinators, Bombus honshuensis and B. diversus. B. diversus was a secondary pollinator when both pollinators were present, but in the absence of B. honshuensis, B. diversus pollinated I. umbrosus, and the resulting seed production was comparable to when visited by the two bumblebee species together. Not only the high visitation frequency of B. diversus but also the low ratio of seed to ovules (= average seed set × fruit set) of I. umbrosus compensated for the low pollination efficiency of B. diversus. Since, the low pollination efficiency of B. diversus, based on the finding that the low average seed set of I. umbrosus did not require a definite morphological match between flowers and pollinators, and did not require very frequent visitations to each flower. In other words, Isodon umbrosus could briefly maintain constant seed production under the condition of the low ratio of seeds to ovules (the low seed set and the low fruit set) and a large number of flowers without B. honshuensis as legitimate pollinator. On the other hand, I. effusus was exclusively pollinated by B. diversus and produced almost the same volume of seeds as I. umbrosus. Isodon effusus had fewer flowers than I. umbrosus, but this smaller number of flowers was compensated for by the high ratio of seeds to ovules in I. effusus, which was maintained by frequent visitations of the morphologically matched pollinator, B. diversus. Received 17 September 1999/ Accepted in revised form 10 February 2000     

Floral volatile alleles can contribute to pollinator‐mediated reproductive isolation in monkeyflowers (Mimulus)

Pollinator‐mediated reproductive isolation is a major factor in driving the diversification of flowering plants. Studies of floral traits involved in reproductive isolation have focused nearly exclusively on visual signals, such as flower color. The role of less obvious signals, such as floral scent, has been studied only recently. In particular, the genetics of floral volatiles involved in mediating differential pollinator visitation remains unknown. The bumblebee‐pollinated Mimulus lewisii and hummingbird‐pollinated Mimulus cardinalis are a model system for studying reproductive isolation via pollinator preference. We have shown that these two species differ in three floral terpenoid volatiles – d ‐limonene, β‐myrcene, and E‐β‐ocimene – that are attractive to bumblebee pollinators. By genetic mapping and in vitro analysis of enzyme activity we demonstrate that these interspecific differences are consistent with allelic variation at two loci, LIMONENE‐MYRCENE SYNTHASE (LMS) and OCIMENE SYNTHASE (OS). Mimulus lewisii LMS (MlLMS) and OS (MlOS) are expressed most strongly in floral tissue in the last stages of floral development. Mimulus cardinalis LMS (McLMS) is weakly expressed and has a nonsense mutation in exon 3. Mimulus cardinalis OS (McOS) is expressed similarly to MlOS, but the encoded McOS enzyme produces no E‐β‐ocimene. Recapitulating the M. cardinalis phenotype by reducing the expression of MlLMS by RNA interference in transgenic M. lewisii produces no behavioral difference in pollinating bumblebees; however, reducing MlOS expression produces a 6% decrease in visitation. Allelic variation at the OCIMENE SYNTHASE locus is likely to contribute to differential pollinator visitation, and thus promote reproductive isolation between M. lewisii and M. cardinalis. OCIMENE SYNTHASE joins a growing list of ‘speciation genes’ (‘barrier genes’) in flowering plants.     

Parallel chemical switches underlying pollinator isolation in Asian Mitella

Floral scents are among the key signals used by pollinators to navigate to specific flowers. Thus, evolutionary changes in scents should have strong impacts on plant diversification, although scent‐mediated plant speciation through pollinator shifts has rarely been demonstrated, despite being likely. To examine whether and how scent‐mediated plant speciation may have occurred, we investigated the Asimitellaria plant lineage using multidisciplinary approaches including pollinator observations, chemical analyses of the floral scents, electroantennographic analyses and behavioural bioassays with the pollinators. We also performed phylogenetically independent contrast analyses of the pollinator/floral scent associations. First, we confirmed that the pairs of the sympatric, cross‐fertile Asimitellaria species in three study sites consistently attract different pollinators, namely long‐tongued and short‐tongued fungus gnats. We also found that a stereoisomeric set of floral volatiles, the lilac aldehydes, could be responsible for the pollinator specificity. This is because the compounds consistently elicited responses in the antennae of the long‐tongued fungus gnats and had contrasting effects on the two pollinators, that is triggering the nectaring behaviour of long‐tongued fungus gnats while repelling short‐tongued fungus gnats in a laboratory experiment. Moreover, we discovered that volatile composition repeatedly switched in Asimitellaria between species adapted to long‐tongued and short‐tongued fungus gnats. Collectively, our results support the idea that recurrent scent‐mediated speciation has taken place in the Asimitellaria–fungus gnat system.     

Breeding system and bumblebee drone pollination of an explosively pollen‐releasing plant,Meliosma tenuis (Sabiaceae)

• Explosive pollen release is a mechanism used by some angiosperms that serves to attach pollen to a pollinator's body. It is usually adopted by species with zygomorphic tubular flowers and pollinated by birds and bees. The tree genus Meliosma (Sabiaceae, Proteales) has unique disc‐like flowers that are externally actinomorphic, but internally zygomorphic, and release pollen explosively.
• To elucidate the adaptive significance of explosive pollen release, we observed flowering behaviour, the breeding system and pollinator visits to flowers of the Japanese species Meliosma tenuis in a temperate forest. Flowers bloomed in June and were nectariferous and protandrous. Explosive pollen release was triggered by slight tactile stimuli to anther filaments or staminodes in male‐stage flowers.
• Because pollen cannot come into contact with the pistils enclosed by staminodes, M. tenuis is functionally protandrous. Artificial pollination treatments revealed that M. tenuis is allogamous. The dominant flower visitors were nectar‐seeking drones of the bumblebee species Bombus ardens (Apidae). The drones’ behaviour, pollen attachment on their bodies and fruit set of visit‐restricted flowers suggest that they are the only agent triggering the explosive pollen release mechanism, and are the main pollinator of M. tenuis.
• The finding that bumblebee workers rarely visit these flowers suggests that the explosive pollen release has another function, namely to discourage pollen‐harvesting bumblebee workers.

     

Floral trait differentiation in Anacamptis coriophora: Phenotypic selection on scents,but not on colour

Current divergent selection may promote floral trait differentiation among conspecific populations in flowering plants. However, whether this applies to complex traits such as colour or scents has been little studied, even though these traits often vary within species. In this study, we compared floral colour and odour as well as selective pressures imposed upon these traits among seven populations belonging to three subspecies of the widespread, generalist orchid Anacamptis coriophora. Colour was characterized using calibrated photographs, and scents were sampled using dynamic headspace extraction and analysed using gas chromatography–mass spectrometry. We then quantified phenotypic selection exerted on these traits by regressing fruit set values on floral trait values. We showed that the three studied subspecies were characterized by different floral colour and odour, with one of the two predominant floral volatiles emitted by each subspecies being taxon‐specific. Plant size was positively correlated with fruit set in most populations, whereas we found no apparent link between floral colour and female reproductive success. We detected positive selection on several taxon‐specific compounds in A. coriophora subsp. fragrans, whereas no selection was found on floral volatiles of A. coriophora subsp. coriophora and A. coriophora subsp. martrinii. This study is one of the first to document variation in phenotypic selection exerted on floral scents among conspecific populations. Our results suggest that selection could contribute to ongoing chemical divergence among A. coriophora subspecies.     

Sexual and temporal variations in floral scent in the subdioecious shrub Eurya japonica Thunb

In many flowering plants, floral scents are a significant trait for visitors, playing an important role in attracting pollinators and/or detracting herbivores. The evolution of flowering plants from hermaphroditism to dioecy is often accompanied by sexual dimorphism in floral scent. In this study, floral scents emitted by different sexual morphs of the subdioecious shrub Eurya japonica Thunb. were collected using a dynamic headspace method, and sexual and temporal variations were evaluated by gas chromatography–mass spectrometry (GC–MS). Two volatiles, α‐pinene and linalool, were identified as the major components of floral scents in females, hermaphrodites, and males. The males emit higher amounts of floral scents, particularly α‐pinene, compared to females or hermaphrodites. Floral scents emitted by males generally decrease as flowers enter senescence, whereas those from females or hermaphrodites do not significantly differ. Intraspecific variations in floral scents of subdioecious species provided by this study would contribute to better understanding of sexual dimorphism in floral scent.     

Floral scents of hawkmoth-pollinated flowers in Japan

Similarity among the floral scents of hawkmoth-pollinated plants was investigated with headspace samplings. Six of seven plant species belonging to different families were found to be rich in isoprenoids, among which linalool was the most common compound. Linalool showed rhythmicity with a nocturnal increase inLonicera japonica. These findings suggest that linalool is a common attractant for nocturnal hawkmoths. However, the composition of other isoprenoids, benzenoids and fatty acid derivatives varied markedly among the plant species examined. There was a significant correlation between species composition of flower-visiting hawkmoths and specific floral scents, suggesting that attractiveness to each hawkmoth species is dependent upon floral scent.