Philodendron adamantinum (Araceae) lures its single cyclocephaline scarab pollinator with specific dominant floral scent volatiles

Philodendron adamantinum (Araceae) lures its singlecyclocephaline scarab pollinator with specific dominantfloral scent volatiles

JULIANA PEREIRA1, CLEMENS SCHLINDWEIN2, YASMINE ANTONINI3,ARTUR CAMPOS DÁLIA MAIA4, STEFAN DÖTTERL5, CRISTIANE MARTINS1,DANIELA MARIA DO AMARAL FERRAZ NAVARRO4 and REISLA OLIVEIRA*3

1Programa de Pós Graduação em Ecologia de Biomas Tropicais, Universidade Federal de Ouro Preto– UFOP, Ouro Preto, MG, Brazil2Departamento de Botânica, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG, Brazil3Departamento de Biodiversidade, Evolução e Meio Ambiente, Universidade Federal de Ouro Preto –UFOP, Ouro Preto, MG 35400 000, Brazil4Departamento de Química Fundamental, Universidade Federal de Pernambuco – UFPE, Recife, PE,Brazil5AG Ökologie, Biodiversität und Evolution der Pflanzen, Universität Salzburg, Salzburg, Austria

Received 10 September 2013; revised 31 October 2013; accepted for publication 01 November 2013

Cyclocephline scarabs and their host plants are documented as highly specialized plant–pollinator associations,with various fine-tuned adaptations. We studied the association between Philodendron adamantinum, a speciesendemic to the Espinhaço Range in Minas Gerais, South-East Brazil, and its exclusive pollinators. We focusedon the pollination mechanism and reproductive success of P. adamantinum, analysed its floral scent composition,and performed field bioassays to verify the scent-mediated attraction of pollinators. The reproductive success ofP. adamantinum depends on the presence of Erioscelis emarginata (Scarabaeidae, Cyclocephalini), its sole polli-nator. At dusk, the inflorescences heat up to 18 °C above the surrounding ambient air temperature and give off astrong sweet odour, from which 32 volatile compounds were isolated. Dihydro-β-ionone, the major constituent inthe floral scent bouquet, lures individuals of E. emarginata when applied to scented artificial decoys, either aloneor blended with methyl jasmonate. We attribute the low fruit set of P. adamantinum at our study sites to pollinatorlimitation of small and isolated populations and geitonogamic pollen flow of vegetatively generated clonal plantgroups. The interaction between P. adamantinum and E. emarginata shows common traits typical of the knownplant–pollinator associations involving cyclocephaline scarabs: the asymmetrical dependence of plants on theirpollinators, and the scent-mediated interaction between flowers and beetles. In addition to updating the currentcatalogue of active compounds of cantharophilous pollination systems, further experimental studies shouldelucidate the role of the specific chemical compounds that attract pollinators along different time and biogeographicscales. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 679–691.

ADDITIONAL KEYWORDS: Campo Rupestre – Erioscelis emarginata – olfactory signalization – pollenlimitation.

INTRODUCTION

Since the work of Darwin (1862), it has been repeat-edly evidenced that the diversity of floral traitsis related to adaptations that promote reproductive

efficiency (Stebbins, 1970; Faegri & van der Pijl,1979). Because most angiosperms depend on interac-tion with animals for reproduction, the evolution ofconvergences and divergences on floral traits shouldrelate to how animals intermediate the ‘sexual ren-dezvous’ among plants. To ensure cross-pollination,plants have to emit advertising signals to attractvisitors and offer rewards to stimulate them to*Corresponding author. E-mail: [email protected]

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Biological Journal of the Linnean Society, 2014, 111, 679–691. With 4 figures

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proceed with the pollen transport service. Floralattributes like colour, shape, fragrance, and resourceproduction should match sensorial perception, mor-phology, and behaviour of frequent and efficient pollenvectors at a given locality. It is to be expected thatplants have been selected to use the pre-existingsensorial equipment of anthophilous animals to bestguide them to flowers. Thus, more specialized rela-tionships should encompass a fine-tuning betweenfloral advertisement and its perception by specificpollinators (Schiestl & Johnson, 2013).

Among cantharophilous flowers (pollinated bybeetles), such highly specialized associations are foundbetween scarab beetles of the tribe Cyclocephalini(Scarabaeidae, Dynastinae) and Araceae, besidesa few others families in the Neotropics (Gottsberger,1990). These flowers converge in showing protogyny,nocturnal anthesis, thermogenesis, intense nocturnalodour emission, sterile nutritive flower tissues asfood resources, and floral chambers (Gottsbergerand Amaral, 1984; Young, 1986; Gottsberger, 1990;Schatz, 1990; Gottsberger & Silberbauer-Gottsberger,1991; Croat, 1997; Gibernau et al., 1999; Gibernau &Barabé, 2002; Maia et al., 2010).

Among these traits, floral scents are crucial forthe long-distance attraction of pollinating beetles,as already suggested by Vogel (1963), and demon-strated more than 20 years ago by Gottsberger &Silberbauer-Gottsberger, 1991); however, the firstfloral scent compounds that elicit behaviouralresponses of specific cyclocephaline pollinators haveonly recently been identified (Dötterl et al., 2012;Gottsberger et al., 2012, 2013; Maia et al., 2012,2013a). Gottsberger (1990) interpreted the floraltraits of cyclocephaline scarab-pollinated flowers asadaptations to pre-existent preferences of the beetles,which apparently have not evolved major adaptationsrelated to their role as specialized pollinators, whencompared with non-anthophilous scarabs.

With about 700 described species, the megadiverseneotropical genus Philodendron Schott accounts foralmost 20% of the richness of Araceae (Mayo et al.,1997, Cusimano et al., 2011). The seven species ofPhilodendron for which pollination/reproductiveecology has been investigated so far are all pollinatedby cyclocephaline scarabs of the genera Cyclocephalaand Erioscelis (Gottsberger & Amaral, 1984; Schatz,1990; Gottsberger & Silberbauer-Gottsberger, 1991;Gibernau et al., 1999; Gibernau, Barabé & Labat,2000; Gibernau & Barabé, 2002; Maia et al., 2010).Croat (1997) even speculated that cyclocephalinescarabs could well be the sole pollinators of the vastmajority of Philodendron.

At Parque Estadual do Rio Preto, a Brazilian statenature reserve, we studied the pollination biologyof Philodendron adamantinum Schott. The species,

which is endemic to Serra do Espinhaço (theEspinhaço Mountain Range), in Minas Gerais, south-eastern Brazil (Mayo, 1991; Gonçalves, 2004), belongsto the subgenus Meconostigma, the least speciose(20 species) of the three subgenera of Philodendron(Gonçalves & Salviani, 2002, WCSP, 2013). Itsdiversity centre is South-East Brazil (Mayo, 1988;Mayo, Bogner & Boyce, 1997), and unlike species ofthe subgenera Philodendron and Pteromischum,which mostly occur in shaded sites, the majority ofMeconostigma prefer open habitats exposed to intenselight (Mayo, 1991).

Philodendron adamantinum is a rupicolous plantof montane habitats, where individuals grow underharsh environmental conditions of high thermic ampli-tude and severe seasonal drought. We addressed thefollowing questions: (i) which are the pollinators ofP. adamantinum; (ii) what is the pollination success atthe study site; (iii) does this plant share floral odourcomponents with other cantharophilous species; and(iv) are the major components of the floral bouquetcapable of attracting pollinators?

MATERIALS AND METHODSSTUDY SITE

The study was conducted at Parque Estadual doRio Preto (Rio Preto State Park), municipality ofSão Gonçalo do Rio Preto (18°00′23″S, 43°23′42″W),Minas Gerais, Brazil. The reserve is located inthe Espinhaço Mountain Range, and encompasses10 755 ha of semi-deciduous forest, cerrado, andrupestrian fields (Campo Rupestre). The climate ischaracterized by a hot and rainy summer fromNovember to March and a well-defined cooler dryseason from June to August (Köppen’s Aw category).The mean annual temperature is 18.9 °C and themean rainfall ranges from 223 to 8 mm averagemonthly precipitation (Instituto Estadual deFlorestas (IEF), 2004).

PLANT SPECIES

Philodendron adamantinum Schott is an ascendingherb, up to 1.5 m tall, that grows exclusively onquartzite and sandstone outcrops. The species exhib-its a peculiar life form: individuals show erect, aerialstems, from which arise several strong adventitiousroots that firmly position the plant erect and parallelwith steeply sloping rocks. Different to most semi-epiphytic species of Philodendron, the stems ofP. adamantinum are never directly attached to thesubstrate, and on some steeper slopes the roots dropseveral metres until they reach the rock surface. Theleathery leaves are sagittate, pinatissect, with longpetioles. Each leaf, inflorescence, and the whole shoot

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© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 679–691

apex are wrapped by a prophyll, which falls off withthe developing leaf and inflorescence. Inflorescencesarise from the base of the petiole, with one per leaf.Thus, stems present characteristic leaf, prophyll, andinflorescence scars.

Voucher specimens are deposited in the Herbarium‘Professor José Badini’ (OUPR), Universidade Federalde Ouro Preto (UFOP) (OUPR 25950).

MORPHOLOGY AND PHENOLOGY

The flowering period and the production of new leaveswere recorded once a month from 20 marked indi-viduals from November 2011 to November 2012.Leaves developed in the same month were markedwith the same colour code. A linear regression analy-sis was conducted to evaluate the influence of rainfallon the number of new leaves throughout the studyperiod.

Floral morphology was described using 11 inflores-cences fixed in formalin/acetic acid/alcohol (FAA). Wemeasured the length of the spathe, spadix, and thezones of fertile staminate flowers, sterile staminateflowers, and pistillate flowers. We counted the totalnumber of pistillate flowers and estimated the numberof fertile and sterile staminate flowers from ten inflo-rescences by counting the number of staminate flowerscontained in a demarcated 0.5-cm broad ring in bothzones, and calculated the total number of flowers inboth zones according to the lengths of each zone.

To describe the morphology of the pollen grains,we prepared slides with fresh grains (Louveaux,Maurizio & Vorwohl, 1978; Wittmann & Schlindwein,1995). Using an optical microscope, 30 pollen grainswere measured and characterized by shape, aperture,and ornamentation of the exine.

FLOWER BIOLOGY, ANTHESIS, AND THERMOGENESIS

To determine the duration and the developmentalsequence of the inflorescences, we observed 15 inflo-rescences of different plant individuals from theopening of the spathe until its closure. The beginningand end of stigmatic receptivity was determined bythe addition of droplets of a 3% solution of H2O2

(Kearns & Inouye, 1993). The beginning of anthesisof staminate flowers, the functional male phase ofanthesis, was defined by anther dehiscence (pollenrelease).

THERMOGENESIS

The temperature of the spadix and the surroundingair was recorded in situ from four inflorescencesthroughout the entire flowering period at 5-minuteintervals. We used a Minipa MT-600 portable ther-

mometer equipped with data-logger and two probes(Minipa, Brazil): one was inserted about 0.5 cm intothe sterile staminate zone and the other was main-tained about 50 cm distant from the plant.

BREEDING SYSTEM

Controlled self- and cross-pollination experimentswere carried out to assess the breeding system ofP. adamantinum. To perform hand cross-pollination,20 inflorescences were bagged in pre-anthesis andpollinated during the functional female phase withpollen from conspecific plants from at least 600 maway, and remained bagged to prevent any access toflower visitors. Hand pollination was conductedbetween 18:30 and 19:00 h during anthesis of thepistillate flowers on the first day of open inflores-cences. Another 24 inflorescences were bagged inpre-anthesis and maintained in bags to test for spon-taneous self-pollination. For natural pollination (con-trols), 46 inflorescences accessible to flower visitorswere marked. All marked inflorescences were moni-tored, and the number of infructescences counted at30 days after flowering.

Moreover, we determined the pollen to ovule ratio(P/O) by counting the number of ovules of 30 pistillateflowers from ten inflorescences (N = 300 flowers), andmultiplying the average number of ovules per flowerwith the average total number of flowers per inflores-cence. The number of pollen grains of fertile stami-nate flowers was estimated from four flowers of sixinflorescences (N = 24 flowers). To remove the pollengrains from the anthers, flowers were immersed for5 days in 500 μl of 95% sulphuric acid at 24 °C(Chouteau, Barabé & Gibernau, 2006). After homog-enizing the mixture for 1 minute using a Vortex agi-tator (Vortex Q220; Quimis LTDA, Brazil), an aliquotof 50 μl was placed on a microscope slide and thenumber of pollen grains was counted under a micro-scope (100× magnification). In order to estimate thenumber of pollen grains per flower, the averagenumber of pollen grains in 24 flowers was extrapo-lated to 500 μl. The number of pollen grains perinflorescence was obtained by multiplying the numberof grains per flower by the number of fertile stami-nate flowers.

FLOWER VISITORS

Flower visitors of P. adamantinum were recorded for∼200 h, between November and December in 2011and 2012. The observations were conducted in variouspatches of the population, predominantly from 18:00to 21:00 h. Flowers were inspected to determine theoccupation frequency of flower visitors and the meannumber of visitors per inflorescence. The beetles were

POLLINATION OF PHILODENDRON ADAMANTINUM 681


captured manually in the inflorescences, individuallyconditioned in glass vials, and stored in a freezer at–20 °C. To analyse the pollen attached to the cuticlesof the beetles, each individual was washed in 70%ethanol and the liquid was then transferred to aPetri dish where it remained covered until completeevaporation. From each Petri dish, we removed fouraliquots to prepare microscope slides of pollen aspreviously described. We identified the grains usingthe pollen slide reference collection of the Laboratoryof Biodiversity (Universidade Federal de Ouro Preto)and Laboratory Plebeia (Bee and Pollination Ecology),at the Universidade Federal de Minas Gerais.

To survey the local fauna of nocturnal Cycloce-phalini at the study site, we installed three black-light traps at the proximities of patches withP. adamantinum during the flowering season for twonights in November and December 2011, and inJanuary 2012. The collected beetles were storedin 70% ethanol and identified in cooperation withspecialists. The beetles were deposited at the entomo-logical collection Pe. Jesus Santiago Moure atUniversidade Federal do Paraná.

VOLATILE COLLECTION AND CHEMICAL ANALYSES

Floral scent samples of five inflorescences were col-lected in situ using standard dynamic headspaceextraction methods (Raguso and Pellmyr, 1998),during the flowering seasons of 2011 and 2012. Odouranalyses were conducted by combined gas chromatog-raphy and mass spectrometry (GC-MS; for details onsampling and odour analyses, see Supporting Infor-mation, Appendix S1).

FIELD BIOASSAYS

To test whether the floral scent compounds wereattractive to flower visitors, we conducted bioassaysin situ using cones of filter paper impregnated withscent (100 μl synthetic compounds purchased fromSigma-Aldrich, of the highest available purity, mixedwith 100 μl of odourless mineral oil) and cones withjust solvent (100 μl hexane P.A. plus 100 μl mineraloil). The paper cones were scented with methyljasmonate, di-hydro-β-ionone, or the 1 : 1 mixtureof both. The cones were positioned upon the stemapex of different non-flowering individuals ofP. adamantinum. The biotests were performed from18:00 to 20:00 h on four occasions in December,during the beginning of the 2012 flowering season. Weconducted two series of bioassays: series 1, a four-choice test (N = 5), in which a control was testedagainst the two single compounds and a blend of thetwo compounds; and series 2, a three-choice test(N = 6), in which a control was tested against the twosingle compounds. The cones were installed at foursites, with the baits positioned at least 10 m distantfrom one another. We counted contact with the conesand collected the beetles when possible.

RESULTSPHENOLOGY

Philodendron adamantinum showed a well-definedflowering period from late November to the end ofJanuary, within the rainy season. The production ofnew leaves was positively linked to precipitation(r2 = 0.59; P < 0.001), and peaked in the wettestmonths (November and December; Fig. 1).

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Nov Dec Jan Fev Mar Abr May Jun Jul Ago Sep Out Nov

new leaves

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Figure 1. Phenology of Philodendron adamantinum at Parque Estadual do Rio Preto (Brazil), from November 2011 toNovember 2012.



INFLORESCENCE MORPHOLOGY

The spadix of P. adamantinum was about 13 cm inlength and contained, on average, 5.060 ± 1.068 uni-sexual flowers. About 70% were fertile staminateflowers on the upper portion, 26% were sterile stami-nate flowers, and 4% were pistillate flowers at thebasal portion. The staminate flowers occupy ∼41% ofthe total length of the inflorescence, the staminodesoccupy ∼42%, and the pistillate flowers occupy ∼17%(see Table S1). Philodendron adamantinum haspsilate, inaperturate, spheroidal pollen grains, with adiameter of 86 ± 7.4 μm (n = 30).

FLOWER BIOLOGY

‘Anthesis’ of inflorescences lasted 36 h over two con-secutive days, and began between 07:00 and 09:00 hwith the aperture of the spathe (Fig. 2A). The inflo-rescences were protogynous, and stigmas of pistillateflowers were receptive around 16:30 h (at the begin-ning of anthesis of pistillate flowers). During theopening process of the spathe, the spadix and spathecontinuously parted from each other until theyformed an angle of 80° at about 18:30 h (Fig. 2B),when the spadix began to warm up. At this time, theinflorescence emanated an intense odour (Fig. 2C).On the following day, at about 11:00 h, drops of yellowresin were exudated from the inner surface of thespathe (Fig. 2D). At around 15:30 h, the directionalliberation of copious strings of pollen from the basalto the apical flowers in the fertile staminate zone tookplace (Fig. 2E). The spathe closed gradually, and atdusk the floral chamber underneath the constriction

containing pistillate and sterile staminate flowerswas already inaccessible (Fig. 2F).

THERMOGENESIS

The inflorescences of P. adamantinum showed twoperiods of heat production. The first occurred duringthe functional female phase, between 10:00 and22:00 h, with a peak between 18:00 and 19:00 h(42.4 ± 1.14 °C, which is 18.8 ± 0.72 °C above the airtemperature; N = 4; see Fig. S1). The second periodof heat production was during the male phase,between 03:00 and 18:00 h, when the temperatureof the spathe was 10 °C above the environmentaltemperature. Thermogenesis was active throughoutthe anthesis, and although there were distinct peaksassociated with the female and male phases, thetemperatures of the spadix dropped to equal thoseof the ambient air only from 18:00 h onward (SeeFig. S1).

FLORAL ODOURS

During the functional female phase, the inflores-cences emitted an intense sweet odour with citricovertones. Headspace analyses revealed the presenceof 39 volatile compounds from seven main classes,based on molecular structure and biosynthesis(Knudsen et al., 2006). Monoterpenes (16 compounds)and irregular terpenes (nine compounds) were therichest compounds classes (Table 1). Dihydro-β-iononewas the major volatile compound, representingalmost 90% (89.67 ± 3.38%) of the total relativecomposition of the floral odour bouquet of P.

Figure 2. Sequence of floral events in the course of inflorescence development, from the opening to the closing of thespathe, in Philodendron adamantinum. The inflorescence is a functional unit, comparable with a single flower. Theopening and closing of the spathe are equivalent to the beginning and the end of anthesis of a single flower.



Table 1. Chemical compounds of the floral odour bouquet of Philodendron adamantinum (Araceae) and relativecontributions (N = 5)

Kovats retention index % (± SD)

AliphaticsEsters

Methyl-(E)-2-octenoate 1171 0.01*AromaticsEsters

methyl salicylate 1194 0.02 ± 0.01†methyl ρ-anisate 1375 0.02 ± 0.01†Miscellaneous cyclic compounds

Carbocyclics(Z)-jasmone 1400 0.01*methyl jasmonate 1651 1.69 ± 0.46methyl epijasmonate 1681 0.12 ± 0.06†

Pyrans2,2,6-trimethy-6-vinyldihydro-2H-pyran-3(4H)-one 1107 0.03 ± 0.01†

TerpenoidsMonoterpenes

α-pinene 932 0.05 ± 0.02sabinene 973 0.09 ± 0.06β-pinene 975 0.04 ± 0.01β-myrcene 992 0.03†limonene 1028 0.04eucalyptol 1030 2.13 ± 0.42γ-terpinene 1059 0.02linalool oxide <cis-> (furanoid) 1073 0.03 ± 0.02linalool oxide <trans-> (furanoid) 1088 0.07 ± 0.04linalool 1100 0.17 ± 0.15linalool oxide <trans-> (pyranoid) 1175 0.01*terpinen-4-ol 1178 0.02 ± 0.01†α-terpineol 1191 0.01*methyl citronellate 1262 0.02 ± trmethyl geranate 1325 0.33 ± 0.11geranyl acetone 1455 0.05 ± 0.01†

Irregular terpenes6-methyl-5-hepten-2-one 988 0.01*(E)-4,8-dimethyl-1,3,7-nonatriene 1117 0.02†theaspirane isomer I 1300 0.06 ± 0.02theaspirane isomer II 1317 0.07 ± 0.01cyclo-β-ionone 1334 0.08 ± 0.01†dihydro-β-ionone 1445 89.67 ± 3.38dihydro-β-ionol 1448 0.45 ± 0.07(E)-8(9)-dehydro-4(5)-dihydrotheaspirone 1458 4.15 ± 3.22β-ionone 1488 0.04 ± 0.03

Unidentified compoundsm/z 124,40,69,50,68 1341 0.02 ± trm/z 40,44,43,123,149 1405 0.01†m/z 121,136,95,43,93 1418 0.07 ± 0.02m/z 119,121,43,120,91 1422 0.03 ± 0.03†m/z 123,109,208,110,40 1530 0.03 ± 0.01m/z 109,123,208,43,82 1676 0.30*m/z 109,123,208,43,82 1679 0.41 ± 0.37

Total number of compounds 39

*Compounds present in only one sample.†Compounds present in two samples.



adamantinum. Three other compounds togetheraccounted for 8% of the total relative scent discharge:(E)-8,9-dehydro-4,5-diydrotheaspirone (4.15% ± 3.22),eucalyptol (2.13% ± 0.42), and methyl jasmonate(1.69% ± 0.46).

BREEDING SYSTEM

Philodendron adamantinum depends on pollinatorsto set fruit, because no fruits were produced by any ofthe bagged unmanipulated flowers (n = 24 inflores-cences). In cross-pollination, more infructescencesdeveloped fruits than by natural pollination (85% of20 inflorescences and 11% of 45 inflorescences, respec-tively; λ2 = 34.471; P < 0.001). The P/O ratio perflower and inflorescence were high, averaging about251.7 and 4710, respectively (See Table S2).

FLOWER VISITORS

During the female phase, the inflorescences ofP. adamantinum were exclusively visited by beetlesof Erioscelis emarginata (Mannerheim, 1829)(Scarabaeidae, Dynastinae). Up to four beetles perinflorescence (1.70 ± 1.92 SD) were recorded in nine of20 marked inflorescences. Besides pollen grains ofP. adamantinum, 44% of the beetles also carriedpollen of sympatric Philodendron uliginosum (subge-nus Meconostigma), which grows along riverbankhabitats with the direct influence of water-saturatedsoils.

The beetles arrived between 18:30 and 19:00 h atthe inflorescences of P. adamantinum, and immedi-ately entered the floral chambers below the constric-tion of the spathe, where they consumed the sterilestaminate flowers. Fertile staminate flowers and pis-tillate flowers were not damaged by the feeding activ-ity. The beetles remained inside the floral chambersduring the whole night and the whole daylight periodof the following day. At about 18:00–18:30 h of thesecond day of anthesis, the beetles left the floralchambers and moved upwards because of the gradualclosing of the spathe from base to apex. During thisprocess, the fertile staminate flowers already hadpresented their huge quantity of pollen grains on thesurface of the spadix and plenty of sticky oily dropswere secreted from the inner surface of the spathe.Thus, the beetles, which contacted the sticky drops,got powdered with pollen grains before they left theclosing inflorescences in search of newly opened inflo-rescences in the female phase.

During the pollen-presenting phase, stinglesshoney worker bees of Trigona spinipes (Fabricius,1793) and Trigona hyalinata (Lepeletier, 1836)(Apidae) visited the inflorescences to collect pollenand resin. The bees neither visited inflorescences

during the functional female phase nor entered thefloral chamber. The survey of night-active insectsconducted at the study site using ultraviolet lighttraps did not yield the capture of any beetle ofE. emarginata nor individuals of any other species ofcyclocephaline scarabs.

BIOTESTS WITH FLORAL ODOUR COMPOUNDS

The biotests were carried out with the major com-pound of the flower bouquet, the irregular terpenedihydro-β-ionone, and with the carbocyclic compoundmethyl jasmonate. The tests showed that bothcompounds, pure or mixed, attracted beetles ofE. emarginata, the pollinators of P. adamantinum.Contacts with baits of a mixture of both compoundswere more frequent, however (Fig. 3). We did notrecord any individual of E. emarginata approachingthe controls, and no other insect species was attractedto the scented baits. The period of beetle attractionwas restricted to dim light conditions at dusk, from18:30 to 19:00 h.

DISCUSSIONFLORAL VOLATILES AS KEY COMPONENTS IN

CANTHAROPHILOUS POLLINATION SYSTEMS INVOLVING

CYCLOCEPHLINE SCARABS

Our results show that the floral bouquet ofP. adamantinum is dominated by dihydro-β-ionone, avolatile irregular terpene that alone or in combinationwith methyl jasmonate is responsible for the attrac-tion of beetles of E. emarginata, the sole pollinators ofthe aroid in the investigated populations. This con-firms recent results about the outstanding role ofspecific floral scent volatiles in highly specialized pol-lination systems involving cyclocephaline scarabs(Dötterl et al., 2012; Gottsberger et al., 2012, 2013;Maia et al., 2012, 2013b). At a short distance, themilk-white coloured adaxial surface of the spathemight have a visual role in guiding the beetles tothe floral chamber. Without olfactory trails, however,it is unlikely that the beetles would ever findtheir host flowers from afar under crepuscular/nocturnal dim light conditions (Gottsberger &Silberbauer-Gottsberger, 1991).

After recent findings of cyclocephaline scarab-attractive floral compounds isolated from Magnoliaovata (Magnoliaceae; methyl-2-methylbutanoate;Gottsberger et al., 2012), Caladium bicolor (Araceae),several species of Annona (Annonaceae; 4-methyl-5-vinylthiazole; Maia et al., 2012), Philodendron aff.bipinnatifidum (named ‘P. form. selloum’ in theP. bipinnatifidum complex, see Gottsberger et al.,2013; Araceae; 4-metoxystyrene; Dötterl et al.,2012), and Philodendron acutatum and Taccarum ulei



[Araceae; (S)-2-hydroxy-5-methyl-3-hexanone; Maiaet al., 2013b], we present here dihydro-β-ionone andmethyl jasmonate as two more volatile compoundswith proven biological activity in the attraction ofthese anthophilous beetles. Dihydro-β-ionone, whichaccounts for about 90% of the total scent emission offemale-phase inflorescences of P. adamantinum, isalso the dominating compound in headspace floralscent extracts of P. acutatum Schott in the AtlanticForest of Pernambuco, Brazil (Maia et al., 2010);moreover, it is the second most common compoundof Taccarum ulei (Araceae) at the same locality(Maia et al., 2013a). In these studies, field biotestswith dihydro-β-ionone traps did not lure anycyclocephaline scarabs, however, nor did they lureany other insect. This might indicate that a particularfloral scent compound can exhibit different biologi-cal attractiveness towards different cyclocephalinespecies, even if this compound is predominant orco-dominant in the floral bouquet of the host plants.Scented traps containing (S)-2-hydroxy-5-methyl-3-hexanone, in contrast, successfully lured beetles ofCyclocephala cearae Höhne, 1923, which are pollina-tors of T. ulei (Maia et al., 2013a), and Cyclocephala

celata Dechambre, 1980, which are pollinators ofCaladium bicolor, P. acutatum, and T. ulei (Maia &Schlindwein, 2006, Maia et al., 2010, 2013a): resultsapparently demonstrating that the same compound iscapable of attracting more than one species. Ourstudy plant is overall most similar to P. acutatum(Fig. 4), but does not share a pollinator with thisspecies. It would be interesting to check whetherE. emarginata would also be attracted to dihydro-β-ionone at the P. aff. bipinnatifidum site.

Before our study, Erioscelis emarginata, the uniquepollinator of P. adamantinum, was recorded exclu-sively as the sole pollinator in inflorescences ofP. aff. bipinnatifidum in populations occurring about750 km south-west of our study site, which had ledto the assumption that the relationship of thesecyclocephaline scarabs and that aroid was speciesspecific (Gottsberger & Amaral, 1984, Gottsbergeret al., 2013). The dominant floral volatile of thisspecies is the aromatic compound 4-methoxystyrene,which alone or in combination with co-dominant3,4-dimethoxystyrene and (Z)-jasmone is capable ofluring beetles of E. emarginata (Dötterl et al., 2012).The floral bouquet of P. aff. bipinnatifidum does not

Figure 3. Number of contacts of beetles of Erioscelis emarginata (Scarabaeidae, Cyclocephalini) with scent baitscontaining dihydro-β-ionone, methyl jasmonate, a mixture of both compounds, and controls (scentless baits) during fournights in December 2012.



contain the dominant compound of P. adamantinum(dihydro-β-ionone), and in turn 4-methoxystyrene isabsent from the floral scent of P. adamantinum(Dötterl et al., 2012). Methyl jasmonate, a carbocycliccompound that occurs in the floral scents of severalangiosperm families (Knudsen et al., 2006), was alsorecorded in the floral bouquets of P. aff. bipinna-tifidum, P. bipinnatifidum (1–3%; Dötterl et al., 2012;Gottsberger et al., 2013), and in trace quantities inCaladium bicolor and Annona coriacea (Maia et al.,2012), which are also cyclocephaline scarab-pollinatedplants. This reveals that different substances canbe used by different host-plant species to attractthe same (and sole) pollinator cyclocephaline scarabspecies.

The use of a single pollinator species by severalsympatric and/or allopatric plant species with differ-ent floral scent bouquets implies that the beetlesare sensitive to different fragrant compounds,and that the pollinator must be provided with innatepreferences to different scents of different plantspecies. As the four E. emarginata-luring compoundsfrom P. adamantinum and P. aff. bipinnatifidum be-long to three different chemical classes (terpenoids,miscellaneous cyclic compounds, and methoxylatedaromatics), according to Knudsen et al. (2006),the use of these bioactive mediation compoundsdoes not result from a step-by-step adaptation toslightly altered substances of the same biochemicalpathway.

O

OH

2

T. ulei

O

O

O

O

O

S

N

OO

O

M. arborescens

C. bicolor

P. aff.

P. acutatum

C. colasi

C. celata

C. cearae

E. emarginata

C. variolosa

1

7

6

5

3

4

Araceae scents2D stress: 0.04

Figure 4. Nonmetric multidimensional scaling (NMDS) plots of the relative floral scent profile of five species of Araceaepollinated by cyclocephaline scarabs. The plants are presented with their pollinators. Key: C., Caladium; M.,Montrichardia; P., Philodendron; T., Tacarum. Chemical structures refer to components that contributed most to theseparation of species: 1, dihydro-β-ionone (Maia et al., 2010, this study); 2, (S)-2-hydroxy-5-methyl-3-hexanone (Maiaet al., 2013b); 3, 4-methyl-5-vinylthiazole (Maia et al., 2012); 4, 1,3,5-trimethoxybenzene (Gibernau et al., 2003);5, (Z)-jasmone; 6, (Z)-2-pentenyl acetate (Gottsberger et al., 2013); 7, 4-methoxystyrene (Dötterl et al., 2012).



ASYMMETRIC POLLINATION ASSOCIATIONS OF

CYCLOCEPHALINE SCARABS AND THEIR HOST PLANTS

The pollination association of cyclocephaline scarabsand their host plants to reproduce and set fruit isasymmetric. Plants of P. adamantinum in the popula-tion studied are completely dependent on the presenceof its single pollinator species, E. emarginata.An identical situation with this pollinator specieswas also found for P. aff. bipinnatifidum (Gottsbergeret al., 2013), and for P. uliginosum growing sympa-trically with P. adamantinum and in a population inParque Nacional da Serra do Cipó, a nature reserve∼150 km south of Parque Estadual do Rio Preto (CSchlindwein, ACD Maia, AT Carvalho, R Oliveira,pers. observ.). This ‘dangerous’ local dependence onone or sometimes two highly effective, specializedpollinator species seems to be the rule for allcyclocephaline scarab-pollinated Araceae (Gottsberger,1999; Gibernau et al., 1999, 2000, 2003; Maia &Schlindwein, 2006; Maia et al., 2010). In contrast, theanimal counterpart of the association appears to beless specific and not entirely dependent on a single hostplant. Gottsberger (1989) convincingly showed that sixsympatric Annona species in south-west Minas Geraiswere pollinated by only two species of Cyclocephala,but that their staggered flowering periods favouredintraspecific pollen flow and reproductive isolation.At a different study site, one of these two species,Cyclocephala atricapilla, is the single pollinator ofPhilodendron mello-barretoanum (Gottsberger &Silberbauer-Gottsberger, 2006, Gottsberger et al.,2013), underlining the asymmetric character of theserather uniformly shaped inter-relationships.

An asymmetric pattern of pollinator and specifichost plants is also found in the scent-driven associa-tions of male euglossine bees and their perfumeflowers, totalling almost 200 bee species that interactwith and pollinate more than four times as many plantspecies with perfume flowers (mostly orchids; Roubik& Hanson, 2004; Nemésio & Rasmussen, 2011), whichall depend on these male bees (Dressler, 1982; Roubik& Hanson, 2004). In general, males of one species areresponsible for the pollination of numerous orchidspecies, which occur within the usually wide distribu-tion range of a species of Euglossini. Generally, only afew euglossine bee species locally occur as the mainpollinators of a given orchid species (Dodson &Frymire, 1961; Dressler, 1982; Ackerman, 1983;Carvalho & Machado, 2002; Martini, Schlindwein &Montenegro, 2003). Asymmetry might simply be theconsequence of pollinators being able to move, whereastheir associated plants are sessile.

An advantage of the multiple use of a single polli-nator species by various plant species could be theassurance and maintenance of robust pollen vector

populations. At the study area, P. adamantinumoccurs in highly specific hostile habitats, occupied byonly a few drought-resistant plant species, leavingmost of the open rock outcrops unoccupied. Philoden-dron adamantinum populations are in generalrestricted to isolated spots, and number only a fewdozens of individual plants. The same is also true forthe aquatic P. uliginosum, which occurs in low densi-ties along water streams in the study area.

Growing exposed on rocks with almost no soil,plants of P. adamantinum showed a flowering perioddefined by the strongly seasonal rainfall regime,which is typical for the Cerrado (Lenza & Klink,2006). During the dry season, the formation of newleaves ceases and the plants gradually reduce theirtranspiratory surface, a phenomenon that reaches itspeak at the end of the dry season, when only one ortwo leaves remain per plant. With the spring rains,the plants become stronger, form new leaves, andproduce inflorescences, one from each leaf, with adelay of 2 weeks after the first rains. The well-definedflowering period of P. adamantinum might coincidewith the highly seasonal adult activity of E. emar-ginata (Gottsberger & Silberbauer-Gottsberger, 2006).Its life cycle is unknown, but it is likely to be stronglyaffected by the extreme climatic seasonality.

POLLINATOR LIMITATION

The reproductive success of P. adamantinum wasextraordinarily low: 80% lower than that of handcross-pollinated inflorescences. At first sight, this isprobably because of the low population density ofE. emarginata and the occupation of only 45% ofinflorescences, with, on average, only two beetles vis-iting each inflorescence. The aroid Montrichardiaarborescens that grows in swamps, also a constrainedhabitat for terrestrial beetles, had similar reproduc-tive characteristics, with an inflorescences occupationrate of 57% by, on average, two beetles (Gibernauet al., 2003). In other species of Araceae pollinated byCyclocephalini, records of dozens of beetles per inflo-rescence are reported (Gottsberger, 1990; Gibernauet al., 2000; Gibernau & Barabé, 2002; Maia et al.,2013a). This reinforces the aforementioned possibleshortcoming of the maintenance of a viable pollinatorpopulation based on just a single host-plant species,especially if this species occurs in low populationdensity. Moreover, interspecific pollen flow might alsobe responsible for the low fruit set, considering thatalmost half of the flower-visiting beetles also carriedpollen grains of P. uliginosum. Another reason forthe naturally low fruit set at the study site mightbe a high degree of pollen flow among neighbour-ing plant individuals. The rupiculous habitat ofP. adamantinum contributes to the naturally isolated



distribution. Moreover, settlement and developmentof new plants is rare at the study sites. Because ofthe growth plasticity of P. adamantinum, vegetativereproduction is common, which facilitates the forma-tion of clonal groups. Normally, the main stem rami-fies and the original older stem and root system getslost, and disintegrates during the dry season, whichleads to independent clonal plants forming, eachwith a single stem. Pollen flow from neighbouringindividuals is often geitonogamic, therefore, and maycause lower fruit set, especially under low pollinatordensity. Therefore, it would be interesting to checkwhether low-distance pollen flow and isolation ofsmall spots of P. adamantinum diminish fruit set.Furthermore, we found several inflorescences infestedwith non-identified Curculionidae, which feed on thecentre of the spadix, causing the premature death ofthe whole inflorescences. The impact of these insects,however, was not quantified.

We conclude that the highly specific asymmetricassociation between P. adamantinum and E. emar-ginata results in the complete dependence of theplant on the highly specialized pollination servicesprovided by the insect mutualist, and that the lowpopulation density of the plant at the study site is themain reason for its low fruit set. To shed light on thepuzzle of the interactions between cyclocephalinebeetles and their host plants, the key characteristicsof the chemical signalling between flowers and polli-nators, and the evolutionary patterns of the hostplant selection involved, it is necessary to: (1) surveythe relationships of cyclocephaline scarabs with theirhost plants; (2) analyse the floral scent composition ofthe plants involved; (3) determine the electrophy-siological antennal responses of the beetles to thefloral bouquets; (4) conduct biotests with the maincompounds of the floral scents to determine the inter-action channels between plants and pollinatorbeetles; and (5) determine the innate and acquiredcapacity of scent perception in the beetles. Possibly,various other specific bioactive compounds will befound with the results of a broader scent screening ofangiosperms pollinated by cyclocephaline scarabs.

ACKNOWLEDGEMENTS

We thank Eduardo Gomes Gonçalves (UniversidadeCatólica de Brasília) for kindly identifying the plantspecies, Luciana Iannuzzi (Universidade Federal dePernambuco) and Paschoal Grossi (UniversidadeFederal do Paraná) for identifying the beetles,Eduardo Gonçalves, Elder Paiva, Rubem Avila, andespecially Marc Gibernou, for constructive suggestionson the article, Instituto Estadual de Florestas (IEF) forthe permission to work in the nature reserve, andespecially Antônio Almeida Tonhão and the staff of

Parque Estadual do Rio Preto for logistic support. Thestudy was supported by Coordenação de Aperfeiçoa-mento de Pessoal de Nível Superior (CAPES). J.A.P.received a grant from UFOP, R.O. received a grantfrom CAPES, and A.C.D.M., C.S., and Y.A. receivedgrants from Conselho Nacional de Pesquisa (CNPq).

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SUPPORTING INFORMATION

Additional supporting information may be found in the online version of this article at the publisher’s web-site:

Figure S1. Temperature of spadix (black line) and surrounding air (grey line) during the 2 days of floweringof four inflorescences (A–D) of Philodendron adamantinum.Table S1. Measurements of the inflorescence architecture and numbers of fertile and sterile staminate andpistillate flowers per inflorescence of Philodendron adamantinum.Table S2. Number of ovules and pollen grains per flower, pollen/ovule ratio of single flowers, and pollen/ovuleratio per inflorescence of Philodendron adamantinum.



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Philodendron adamantinum (Araceae) lures its single cyclocephaline scarab pollinator with specific dominant floral scent volatiles