Pollen transfer by hummingbirds and bumblebees, and the divergence of pollination modes in Penstemon

We compared pollen removal and deposition by hummingbirds and bumblebees visiting bird-syndrome Penstemon barbatus and bee-syndrome P. strictus flowers. One model for evolutionary shifts from bee pollination to bird pollination has assumed that, mostly due to grooming, pollen on bee bodies quickly becomes unavailable for transfer to stigmas, whereas pollen on hummingbirds has greater carryover. Comparing bumblebees and hummingbirds seeking nectar in P. strictus, we confirmed that bees had a steeper pollen carryover curve than birds but, surprisingly, bees and birds removed similar amounts of pollen and had similar per-visit pollen transfer efficiencies. Comparing P. barbatus and P. strictus visited by hummingbirds, the bird-syndrome flowers had more pollen removed, more pollen deposited, and a higher transfer efficiency than the bee-syndrome flowers. In addition, P. barbatus flowers have evolved such that their anthers and stigmas would not easily come into contact with bumblebees if they were to forage on them. We discuss the role that differences in pollination efficiency between bees and hummingbirds may have played in the repeated evolution of hummingbird pollination in Penstemon.     

SELECTION ON POLEMONIUM BRANDEGEEI (POLEMONIACEAE) FLOWERS UNDER HUMMINGBIRD POLLINATION: IN OPPOSITION,PARALLEL, OR INDEPENDENT OF SELECTION BY HAWKMOTHS?

Particular floral phenotypes are often associated with specific groups of pollinators. However, flowering plants are often visited, and may be effectively pollinated by more than one type of animal. Therefore, a major outstanding question in floral biology asks: what is the nature of selection on floral traits when pollinators are diverse? This study examined how hummingbirds selected on the floral traits of Polemonium brandegeei, a species pollinated by both hummingbirds and hawkmoths. In array populations of P. brandegeei, we measured pollen movement, and female (seeds set) and male (seeds sired) fitness under hummingbird pollination. We then compared the patterns of selection by hummingbirds with our previous study examining selection by hawkmoths. We documented contrasting selection on sex organ positioning through female function, with hummingbirds selecting for stigmas exserted beyond the anthers and hawkmoths selecting for stigmas recessed below the anthers. Furthermore, hummingbirds selected for longer and wider corolla tubes, and hawkmoths selected for narrower corolla tubes. Therefore, contrasting selection by hawkmoths and hummingbirds may account for variation in sex organ arrangements and corolla dimensions in P. brandegeei. We documented how floral traits under selection by multiple pollinators can result in either an intermediate “compromise” between selective pressures (sex organs) or apparent specialization (corolla tube length) to one pollinator.     

Risk‐averse inflorescence departure in hummingbirds and bumble bees: could plants benefit from variable nectar volumes?

Most hermaphroditic, many-flowered plants should suffer reduced fitness from within-plant selfing (geitonogamy). Large inflorescences are most attractive to pollinators, but also promote many flower visits during a single plant visit, which may increase selfing and decrease pollen export. A plant might avoid the negative consequences of attractiveness through modification of the floral display to promote fewer flower visits, while retaining attractiveness. This report shows that increasing only the variance in nectar volume per flower results in fewer flower visits per inflorescence by wild hummingbirds ( Selasphorus rufus ) and captive bumble bees ( Bombus flavifrons ) foraging on artificial inflorescences. Inflorescences were either constant (all flowers contained the same nectar volume) or variable (half the flowers were empty, the other half contained twice as much nectar as in the constant flowers). Both types of inflorescence were simultaneously available to foragers. Risk-averse foraging behaviour was expressed as a patch departure preference: birds and bees visited fewer flowers on variable inflorescences, and this preference was expressed when resource variability could be determined only by concurrent sampling. When variance treatments were clearly labelled with colour and offered to hummingbirds, the departure effect was maintained; however, when preference was measured by inflorescence choice, birds did not consistently prefer to visit constant inflorescences. The reduced visitation lengths on variable inflorescences by both birds and bees documented in this study imply that variance in nectar production rates within inflorescences may represent an adaptive trait to avoid the costs of geitonogamy.     

The evolution of signal–reward correlations in bee- and hummingbird-pollinated species of Salvia

Within-individual variation in floral advertising and reward traits is a feature experienced by pollinators that visit different flowers of the same plant. Pollinators can use advertising traits to gather information about the quality and amount of rewards, leading to the evolution of signal–reward correlations. As long as plants differ in the reliability of their signals and pollinators base their foraging decisions on this information, natural selection should act on within-individual correlations between signals and rewards. Because birds and bees differ in their cognitive capabilities, and use different floral traits as signals, we tested the occurrence of adaptive divergence of the within-individual signal–reward correlations among Salvia species that are pollinated either by bees or by hummingbirds. They are expected to use different floral advertising traits: frontal traits in the case of bees and side traits in the case of hummingbirds. We confirmed this expectation as bee- and hummingbird-pollinated species differed in which specific traits are predominantly associated with nectar reward at the within-individual level. Our findings highlight the adaptive value of within-individual variation and covariation patterns, commonly disregarded as ‘environmental noise’, and are consistent with the hypothesis that pollinator-mediated selection affects the correlation pattern among floral traits.     

Intrafloral patterns of color and scent in Capparis spinosa L. and the ghosts of its selection past

Premise

Capparis spinosa is a widespread charismatic plant, in which the nocturnal floral habit contrasts with the high visitation by diurnal bees and the pronounced scarcity of hawkmoths. To resolve this discrepancy and elucidate floral evolution of C. spinosa, we analyzed the intrafloral patterns of visual and olfactory cues in relation to the known sensory biases of the different visitor guilds (bees, butterflies, and hawkmoths).

Methods

We measured the intrafloral variation of scent, reflectance spectra, and colorimetric properties according to three guilds of known visitors of C. spinosa. Additionally, we sampled visitation rates using a motion-activated camera.

Results

Carpenter bees visited the flowers eight times more frequently than nocturnal hawkmoths, at dusk and in the following morning. Yet, the floral headspace of C. spinosa contained a typical sphingophilous scent with high emission rates of certain monoterpenes and amino-acid derived compounds. Visual cues included a special case of multisensory nectar guide and color patterns conspicuous to the visual systems of both hawkmoths and bees.

Conclusions

The intrafloral patterns of sensory stimuli suggest that hawkmoths have exerted strong historical selection on C. spinosa. Our study revealed two interesting paradoxes: (a) the flowers phenotypically biased towards the more inconsistent pollinator; and (b) floral display demands an abundance of resources that seems maladaptive in the habitats of C. spinosa. The transition to a binary pollination system accommodating large bees has not required phenotypic changes, owing to specific eco-physiological adaptations, unrelated to pollination, which make this plant an unusual case in pollination ecology.     

Plant–pollinator interactions in the understory of a lowland mixed dipterocarp forest in Sarawak

Pollination biology of 41 plants species of 21 families blooming in the forest understory was investigated in a lowland mixed diplerocarp forest in Lambir Hills National Park, Sarawak. Among these species, 29 species (71%) were pollinated by bees, four (10%) by nectariniid birds, three by small dipterans, and others by moths, butterflies, syrphid flies, wasps, and beetles. The 29 bee-pollinated species consisted of five distinct pollination guilds: ten species pollinated by medium traplining bees (two Amegilla species), nine by small traplining bees (three halictid and a xylocopine species), two by stingless bees and beetles, seven by stingless bees, and one by megachilid bees. The bees constituting the first two guilds were shade-loving, swiftly flying, long-tongued trapliners. Proboscis lengths of these pollinators correlated with flower depth of the host plant. Pollination systems in the forest understory were distinguished from that in the canopy by the prevalence of specific interactions, the number of traplining solitary bees, and lack of pollination systems by mass-recruiting eusocial bees, large Xylocopa bees, thrips, bats, and wind. These characteristics are largely similar between the Palaeotropics and the Neotropics through convergence of nectarivorous birds (spiderhunters vs. hummingbirds) and traplining bees (Amegilla vs. euglossine bees).     

POLLINATION AND SEED PRODUCTION IN IPOMOPSIS AGGREGATA: DIFFERENCES AMONG AND WITHIN FLOWER COLOR MORPHS

The pollination of red, pink, and white color morphs of Ipomopsis aggregata was evaluated to assess whether ethological isolation based on pollinator color discrimination may occur. We observed animal visitors, assessed pollen delivery, seed set per fruit, percentage of flowers setting fruit, nectar production, and timing of flower opening for different color morphs in the Front Range of Colorado. Based on traditional zoophilous flower classifications, we expected hummingbirds to pollinate red-flowered I. aggregata subsp. collina and hawkmoths to pollinate white-flowered I. aggregata subsp. Candida. However, ethological isolation does not appear to occur among color morphs of I. aggregata in the Front Range. Hummingbirds visited red-flowered plants in excess overall, and, to a lesser extent, so did hawkmoths. Both hummingbirds and hawkmoths visited all color morphs and probably transferred pollen among them. Pollen delivery data and a day-night bagging experiment also suggest that pollinators do not necessarily behave as predicted by flower classifications. In addition, there is little evidence for major differences between red, white, and pink flowers in any aspects of reproductive biology. Indeed, most variation occurs within a given color morph.     

Pollination ecotypes of Satyrium hallackii (Orchidaceae) in South Africa

Intraspecific variation in floral traits may reflect adaptive shifts in the pollination system of a plant. This idea was tested by examining the pollination biology of Satyrium hallackii H. Bolus., an orchid which has spurs varying from 8 to 36 mm among populations in southern Africa. Field observations showed that the short-spurred form ( S. hallackii subsp. hallackii ) in coastal fynbos habitats is pollinated by bees, while the long-spurred form ( S. hallackii subsp. ocellatum ) in grassland habitats is pollinated primarily by hawkmoths and, secondarily, by long-tongued flies. The shift between hawkmoths and bees as pollinators may have been promoted by an ecological gradient in South Africa: carpenter bees are common in coastal fynbos habitats, but rare in grassland habitats where there are few available nesting sites. On the other hand, hawkmoths are common in grassland habitats, but rare in fynbos where there are few palatable host-plants. The formation of pollination ecotypes across pollinator gradients has probably been a major factor in the diversification of South African plants with specialized pollination systems.     

Variation in pollinator preference between two Ipomopsis contact sites that differ in hybridization rate

Pollinator-mediated reproductive isolation is often a principal factor in determining the rate of hybridization between plant species. Pollinator preference and constancy can reduce interspecific pollen transfer between otherwise interfertile, coflowering species. The importance of this ethological isolation can be assessed by comparing the strength of preference and constancy of pollinators in contact sites that differ in the frequency of hybrid individuals. We observed visitation by hummingbirds and hawkmoths in natural single-species patches and artificial mixed-species arrays in two Ipomopsis aggregata/I. tenuituba contact sites-one with few hybrids, and one in which hybrids are abundant. Pollinator preference and constancy were stronger at the low-frequency hybrid site, especially for hawkmoths (Hyles lineata). Hawkmoths at the low-frequency hybrid site showed significant preference and constancy for I. tenuituba, while at the high-frequency site hawkmoths visited both species equally. One hypothesis that might explain these differences in hawkmoth foraging is that warmer nights at the low-frequency hybrid site allow for nocturnal foraging where the light-colored corollas of I. tenuituba have a visibility advantage. These differences in hawkmoth behavior might in turn affect hummingbirds differently at the two sites, through changes in nectar resources, leading to greater pollinator-mediated isolation at the low-frequency hybrid site. Our results suggest that differences in pollinator behaviors between sites can have both direct and indirect effects on hybridization rates between plant species.     

The influence of distinct pollinators on female and male reproductive success in the Rocky Mountain columbine

Although there are many reasons to expect distinct pollinator types to differentially affect a plant's reproductive success, few studies have directly examined this question. Here, we contrast the impact of two kinds of pollinators on reproductive success via male and female functions in the Rocky Mountain columbine, Aquilegia coerulea . We set up pollinator exclusion treatments in each of three patches where Aquilegia plants were visited by either day pollinators (majority bumble bees), by evening pollinators (hawkmoths), or by both (control). Day pollinators collected pollen and groomed, whereas evening pollinators collected nectar but did not groom. Maternal parents, potential fathers and progeny arrays were genotyped at five microsatellite loci. We estimated female outcrossing rate and counted seeds to measure female reproductive success and used paternity analysis to determine male reproductive success. Our results document that bumble bees frequently moved pollen among patches of plants and that, unlike hawkmoths, pollen moved by bumble bees sired more outcrossed seeds when it remained within a patch as opposed to moving between patches. Pollinator type differentially affected the outcrossing rate but not seed set, the number of outcrossed seeds or overall male reproductive success. Multiple visits to a plant and more frequent visits by bumble bees could help to explain the lack of impact of pollinator type on overall reproductive success. The increase in selfing rate with hawkmoths likely resulted from the abundant pollen available in experimental flowers. Our findings highlighted a new type of pollinator interactions that can benefit a plant species.     

COMPETITION BETWEEN HERMIT HUMMINGBIRDS PHAETHORNINAE AND INSECTS FOR NECTAR IN A COSTA RICAN RAIN FOREST

Hummingbirds in the tropical rainforests of southwestern Costa Rica face intense competition from stingless bees Trigona that steal nectar from hummingbird flowers. Here we document both interference and exploitative competition between bees and hummingbirds at scarlet, hummingbird pollinated flowers of Passiflora vitifolia. Aggressive stingless bees prevented Long-tailed Hermit Hummingbirds Phaethornis superciliosus from feeding at nearly one-third of the passion flowers approached. In exclusion experiments, bees and hummingbirds each removed most of the nectar from treated flowers. Experimental exclusion of bees also increased hummingbird use of both natural and artificial flowers.     

Bimodal pollination system of the bromeliad Aechmea nudicaulis involving hummingbirds and bees

In order to compare the effectiveness of birds and insects as pollinators, we studied the floral biology of the bromeliad Aechmea nudicaulis (L.) Grisebach in the biome of the Atlantic rain forest, southern Brazil. On Santa Catarina Island, flowering extends from mid-September to the end of December, with diurnal anthesis. The reproductive system is obligatory xenogamy, thus pollinator-dependent. Flowers secrete 31.84 μl of nectar per day, with a mean sugar concentration of 23.2%. Highest nectar volume and sugar concentration occur at the beginning of anthesis. Most floral traits are characteristic for ornithophily, and nectar production appears to be adapted to the energy demand of hummingbirds. Continued secretion of the sucrose-dominated nectar attracts and binds visitors to inflorescences, strengthening trapline foraging behaviour. Experiments assessing seed set after single flower visits were performed with the most frequent visitors, revealing the hummingbird Thalurania glaucopis as the most effective pollen vector. In addition, bees are also functional pollinators, as substantiated by their high visitation frequency. We conclude that this pollination system is bimodal. Thus, there is redundancy in the pollination service provided by birds and bees, granting a high probability of successful reproduction in Ae. nudicaulis.     

Mobility of Impatiens capensis flowers: effect on pollen deposition and hummingbird foraging

Flexible pedicels are characteristic of birdpollinated plants, yet have received little attention in studies of hummingbird-flower interactions. A major implication of flexible pedicels is that flowers may move during pollination. We examined whether such motion affected interactions between ruby-throated hummingbirds (Archilochus colubris) and jewelweed (Impatiens capensis) by increasing pollen deposition and by altering the effectiveness of nectar removal. For I. capensis, flower mobility enhanced pollen deposition: birds had significantly longer contact with anthers and more pollen deposited on their bills and crowns when foraging at mobile flowers than at flowers that had been experimentally immobilized. In contrast, flower mobility imposed a cost on hummingbirds by significantly increasing their handling times and reducing their extraction rates relative to their interactions with immobile flowers. Field observations indicated that the motion observed during hummingbird visits did not occur when bees (Bombus spp., Apis mellifera) visited I. capensis flowers, which suggests that the mobility of I. capensis flowers is an adaptation for hummingbird pollination.     

Why do some nectar foragers perch and others hover while probing flowers?

Diurnal hawkmoths, Hemaris fuciformis, and bumblebees, Bombus pasquorum, were observed foraging for nectar in flowers of Viscaria vulgaris. The hawkmoths hovered in front of the flowers, while the bees perched on them. The hawkmoths had a faster probing rate than the bees, and consequently also had higher gross and net rates of energy gain. A model is presented that shows that hovering only yields a higher net rate of energy gain (NREG) than perching when nectar volumes are high due to low competition for the resource. The difference in NREG of perchers and hoverers decreases with an increase of competition, and eventually perching yields the highest NREG. This is an effect of the higher cost of hovering. The results suggest that hovering can only evolve as a pure evolutionarily stable strategy (ESS) if competition is reduced, for example by co-evolutionary specializations with plants. The possibility that it has evolved as a mixed ESS (i.e. individuals can both hover and perch depending on the resource level) is discussed. The evolution of optimal foraging strategies is discussed, and it is pointed out that the rate of gain of an animal is independent of the strategy used when all competing foragers use the same strategy, but competitively superior strategies will nevertheless evolve because they are ESSs. Competition between strategies with different energy costs are special, because resource availability determines which strategy is competitively superior. A high-cost strategy can only evolve as a pure ESS at high resource levels, or as a mixed ESS at intermediate levels.     

Beyond the pollination syndrome: nectar ecology and the role of diurnal and nocturnal pollinators in the reproductive success of Inga sessilis (Fabaceae)

Inga species present brush‐type flower morphology allowing them to be visited by distinct groups of pollinators. Nectar features in relation to the main pollinators have seldom been studied in this genus. To test the hypothesis of floral adaptation to both diurnal and nocturnal pollinators, we studied the pollination ecology of Inga sessilis, with emphasis on the nectar secretion patterns, effects of sequential removals on nectar production, sugar composition and the role of diurnal and nocturnal pollinators in its reproductive success. Inga sessilis is self‐incompatible and pollinated by hummingbirds, hawkmoths and bats. Fruit set under natural conditions is very low despite the fact that most stigmas receive polyads with sufficient pollen to fertilise all ovules in a flower. Nectar secretion starts in the bud stage and flowers continually secreting nectar for a period of 8 h. Flowers actively reabsorbed the nectar a few hours before senescence. Sugar production increased after nectar removal, especially when flowers were drained during the night. Nectar sugar composition changed over flower life span, from sucrose‐dominant (just after flower opening, when hummingbirds were the main visitors) to hexose‐rich (throughout the night, when bats and hawkmoths were the main visitors). Diurnal pollinators contributed less than nocturnal ones to fruit production, but the former were more constant and reliable visitors through time. Our results indicate I. sessilis has floral adaptations, beyond the morphology, that encompass both diurnal and nocturnal pollinator requirements, suggesting a complementary and mixed pollination system.     

Ornithophilous canopy species in the Atlantic rain forest of southeastern Brazil

ABSTRACT.   In tropical ecosystems, birds play a relevant role in plant reproduction. Although hummingbirds are regarded as the most important vertebrate pollinators in the Neotropics, the possible role of perching birds as pollinators has been neglected. From 2003 to 2005, we observed 68 species of plants visited by birds in an Atlantic rainforest in southeastern Brazil, including three canopy species: Spirotheca rivieri (Malvaceae, Bombacoidea), Schwartzia brasiliensis (Marcgraviaceae), and Psittacanthus dichrous (Loranthaceae). Flowers of these three species were visited by 15 different species of perching birds and by hummingbirds. The flowers of these three plants are colorful, ranging from deep red or purple to orange. Spirotheca rivieri blooms during the austral winter and Schwartzia brasiliensis during the summer. The flowers of these two species produce copious amounts of dilute nectar in easily accessible structures and both species appear to depend primarily on perching birds as pollinators, with hummingbirds being secondary or minor pollen vectors. The tubular, narrow flowers of P. dichrous are produced during the austral summer and are visited primarily by hummingbirds. Perching birds also visit the flowers, but destroy them. Our results suggest that previous estimates of the number of perching birds that feed on nectar may be too low and that flowers pollinated by perching birds may be more common in the canopy of Neotropical forests than previously thought.     

Nectar, floral morphology and pollination syndrome in Loasaceae subfam. Loasoideae (Cornales)

BACKGROUND AND AIMS: Loasaceae subfam. Loasoideae are mostly distributed in South America (sea level to over 4500 m) with a wide range of animals documented as pollinators. The aim was to investigate correlations between nectar parameters, flower morphology, pollination syndrome and phylogeny. METHODS: Nectar was collected from 29 species from seven genera in the subfamily. Concentration and volumes were measured and the amount of sugar calculated. Correlations of nectar data were plotted on a ternary graph and nectar characteristics compared with flower visitors, floral morphology and phylogenetic data. KEY RESULTS: Sugar concentrations are generally higher than reported for most plant families in the literature. The species investigated can be roughly grouped as follows. Group I: plants with approx. 1.5(-3.5) microL nectar with (40-)60-80% sugar and 0.19-2 mg sugar flower-1; with small, white, star-shaped corollas, pollinated by short-tongued bees. Groups II, III and IV: plants with mostly orange, balloon-, saucer-, bowl- or bell-shaped corollas. Group II: plants with approx. 9-14 microL nectar with 40-60% sugar and 4-10 mg sugar flower-1; mostly visited by long-tongued bees and/or hummingbirds. Group III: plants with 40-100 microL nectar with 30-40% sugar and 14-36 mg sugar flower-1, mostly visited by hummingbirds. Group IV: geoflorous plants with 80-90 microL with 10-15% sugar and 8.5-12 mg sugar flower-1, presumably visited by small mammals. Groups II and III include species visited by bees and/or hummingbirds. CONCLUSIONS: Pollinator switches from short-tongued bees via long-tongued bees to hummingbirds appear to have taken place repeatedly in the genera Nasa, Loasa and Caiophora. Changes in nectar amount and concentration appear to evolve rapidly with little phylogenetic constraint.     

Pollinator identity and spatial isolation influence multiple paternity in an annual plant

The occurrence and extent of multiple paternity is an important component of variation in plant mating dynamics. However, links between pollinator activity and multiple paternity are generally lacking, especially for plant species that attract functionally diverse floral visitors. In this study, we separated the influence of two functionally distinct floral visitors (hawkmoths and solitary bees) and characterized their impacts on multiple paternity in a self‐incompatible, annual forb, Oenothera harringtonii (Onagraceae). We also situated pollinator‐mediated effects in a spatial context by linking variation in multiple paternity to variation in plant spatial isolation. We documented pronounced differences in the number of paternal sires as function of pollinator identity: on average, the primary pollinator (hawkmoths) facilitated mating with nearly twice as many pollen donors relative to the secondary pollinator (solitary bees). This effect was consistent for both isolated and nonisolated individuals, but spatial isolation imposed pronounced reductions on multiple paternity regardless of pollinator identity. Considering that pollinator abundance and pollen dispersal distance did not vary significantly with pollinator identity, we attribute variation in realized mating dynamics primarily to differences in pollinator morphology and behaviour as opposed to pollinator abundance or mating incompatibility arising from underlying spatial genetic structure. Our findings demonstrate that functionally distinct pollinators can have strongly divergent effects on polyandry in plants and further suggest that both pollinator identity and spatial heterogeneity have important roles in plant mating dynamics.     

Fitness consequences of a clock pollinator filter in Nicotiana attenuata flowers in nature

Nicotiana attenuata flowers, diurnally open,emit scents and move vertically to interact with nocturnal hawkmoth and day-active hummingbird pollinators. To examine the fitness consequences of these floral rhythms, we conducted pollination trials in the plant's native habitat with phase-shifted flowers of plants silenced in circadian clock genes. The results revealed that some pollination benefits observed under glasshouse conditions were not reproduced under natural field conditions. Floral arrhythmicity increased pollination success by hummingbirds, while reducing those by hawkmoths in the field. Thus, floral circadian rhythms may influence a plant's fitness by filtering pollinators leading to altered seed set from outcrossed pollen.     

Consequences of floral visits by ants and invasive honeybees to the hummingbird‐pollinated,Caribbean cactus Melocactus intortus

The pink, tubular, nectariferous flowers of Melocactus intortus (Cactaceae) in Puerto Rico are visited by native hummingbirds (Anthracothorax dominicus), but also by invasive honeybees (Apis mellifera) and ants (Solenopsis sp.). We sought to determine if the bees and ants significantly alter the pollination of M. intortus by measuring pollinator effectiveness. Using traditional estimates of effectiveness (visitation rate and seed set), our results show that hummingbirds were the most effective pollinators as expected. Bees and ants were less effective, and their contributions were one‐fourth to one‐tenth of that observed for hummingbirds. We then modified this measure of effectiveness by adding two components, fitness of progeny and temporal availability of visitors, both of which refine estimates of flower visitor effectiveness. With these new estimations, we found that the effectiveness values of all three animal visitors decreased; however, the role of hummingbirds as the principal pollinator was maintained, whereas the effectiveness values of bees and ants approached zero. By these new measures of overall pollinator effectiveness, the invasive honeybees and ants have little effect on the reproductive success of M. intortus.