Buzz-Pollination and Patterns in Sexual Traits in North European Pyrolaceae Author(s): Jette T. Knudsen and Jens Mogens Olesen Reviewed work(s): Source: American Journal of Botany, Vol. 80, No. 8 (Aug., 1993), pp. 900-913 Published by: Botanical Society of America Stable URL: http://www.jstor.org/stable/2445510 . Accessed: 08/08/2012 10:49

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http://www.jstor.org American Journalof Botany 80(8): 900-913. 1993.

BUZZ-POLLINATION AND PATTERNS IN SEXUAL TRAITS IN NORTH EUROPEAN PYROLACEAE1

JETTE T. KNUDSEN2 AND JENS MOGENS OLESEN Departmentof ChemicalEcology, University of G6teborg, Reutersgatan2C, S-413 20 G6teborg,Sweden; and Departmentof Ecology and Genetics,University of Aarhus, Ny Munkegade, Building550, DK-8000 Aarhus,Denmark

Flowerbiology and pollinationof Moneses uniflora, secunda, minor, P. rotundifolia,P. chlorantha, and Chimaphilaumbellata are describedand discussedin relationto patternsin sexualtraits and possibleevolution of buzz- pollinationwithin the group. The largenumber of pollengrains are packedinto units of monadsin Orthilia,tetrads in Monesesand Pyrola,or polyadsin . is thesole rewardto visitinginsects except in thenectar-producing 0. secundaand C. umbellata.Correlations are presentbetween several sexual traits. Pollen: ovule ratiosare verylow, indicatingefficient pollen transfer. Use ofpolyads may reduce the number of siring males. This trend may be counteracted by largerstigmas as in C. umbellata.Increasing pollen unit size mayenhance relatedness of seeds,reduce abortion, and improveseed set.This mayexplain the evolution, within the Pyrolaceae and otherfamilies, of higher ovule numbers with largerpollen units. The plantsare mainlypollinated by bumblebees. is visitedby nectar-collectors; Monesesuniflora, , P. rotundifolia,and P. chloranthaare visitedexclusively by pollen-buzzers; and Orthilia secundais visitedby both nectar-collectors and pollen-buzzers.Nectar secretion is suggestedto be an ancientattractant in thegroup with C. umbellatahaving the least specialized pollination system, and buzz-pollinationin M. unifloraand in the studiedPyrola regarded as an advancedsystem.

About 20,000 species in 72 familiesof floweringplants Moneses (one sp.) of perennialevergreen herbs and sub- have antherswith poricidal dehiscence, i.e., opening by shrubs.The familyoccurs mostlyin coniferforests in the apical pores, valves, or flaps(Buchmann, 1983). Many of northtemperate zone (Andersen, 1943). these species are presumedto be buzz-pollinatedby bees Some highertaxa, e.g., the Pyrolaceae,possess a variety (van der Pijl, 1954; Michener, 1962; sensu Buchmann, of what could be called evolutionarystable reprotypes, 1974). Using rapid contractionsof their indirect flight i.e., differentsets of sexual characterseach carried by a muscles, bees sonicate anthersand harvestpollen. These restrictedgroup of species, while others are extremely buzz-pollinated flowersshare a number of floraltraits. uniformin their expression. Species of the Pyrolaceae They are oftenopen bowl-shaped or withreflexed petals, displaya strikingvariation in floralform and traitsrelated small to average-sized,and oftenlack floralnectar (Buch- to theirpollination ecology. The inflorescencevaries among mann, 1983). Vaccinium,however, a largegenus of buzz- generain its structureand numberof .The pollinated species, has urceolatecorolla and nectar. varies interspecificallyin its size, form,anther dehiscence, Anthersmay forma cone or brush,and pollen grainsare and stylecurvature (Dahlgren et al., 1980). In addition, of small to medium size with a nonoily,smooth surface thereis a largevariation in pollen formand development (Buchmann, 1983). However, the featureshared by most (Takahashi, 1986a, b, 1987). Several authors have pub- buzz-pollinatedflowers is antherdehiscence by small api- lished pollination ecological informationon the Pyrola- cal pores. Poricidal anthersand deep and narrowcorolla ceae (e.g., Muller, 1881; Ekstam, 1894; Warnstorf,1896; tubes concealing nectar are two parallel ways to reduce Knuth, 1905; Silen, 1906a, b; Andres, 1910; Lovell and therange ofpotential flower resource harvesters -to buzz- Lovell, 1936; Knaben, 1944; Hagerup, 1954; Vogel, 1978; ers and long-tonguedsuckers, respectively.One of the Buchmann, 1983; Standley, Kim, and Hjersted, 1988). predominantlybuzz-pollinated families is thePyrolaceae. These authors have observed Hymenoptera (Bombus, The Pyrolaceae includes four genera, Pyrola (20-30 Halictidae), Diptera (Muscidae, Syrphidae), and Cole- spp.), Chimaphila(seven spp.), Orthilia(one sp.), and optera (e.g., Meligethes)as flowervisitors. A strikingfea- ture common to all these observations is the very low abundance of visitorsto species of the Pyrolaceae. Thus, ' Receivedfor publication 30 October1991; revisionaccepted 25 pollination by wind or selfinghas also been suggestedto March1993. play an importantrole in the reproductiveecology of the The authorsthank the reviewers, S. L. Buchmannand P. G. Kevan, Pyrolaceae (e.g., Knuth, 1905; Hagerup, 1954). formany helpful comments; U. Molau,P. N0rgaard,R. Olson,B. Ox- The aims of this studywere to describe and compare elman,and B. StAhlfor comments on earlierdrafts of the manuscript; L. Tollsten,G. Stenhagen,and H. Albomfor help with the nectar anal- the flowerbiology in relationto the pollinationsystem in yses;R. Erikssonfor help with UV-photographing; P. Johnsen, B. Ov- six NorthernEuropean species of Pyrolaceae,to describe ergaardNielsen, V. Mahler,and B. G. Svenssonfor assistence with and discuss patternsin theexpression of sexual characters, identificationof the insectvisitors; and E. Larssonfor permission to especially pollen unit size and pollination mechanism, campon his property.The studywas supportedfinancially by Marcus and to hypothesizeabout the origin of buzz-pollination LorentzenFoundation (JTK), Nordic Council (JTK), Uppsala Univer- withinthe group. However, since differentplant species sityEcological Research Station, Oland, Sweden (JTK), Danish Natural ScienceResearch Council (#1 1-9025-1, JMO), and CarlsbergFounda- may vary in total amount of resources available for re- tion(#88-0386, JMO). productionand in theirshared ancestry (comparative ob- 2 Authorfor correspondence. servationsare structuredby theircommon evolution[Fel- 900 August 1993] KNUDSEN AND OLESEN-SEXUAL PATTERNS AND POLLINATION 901

senstein,1985]), cross-taxontrends and patternsmust be petrumnigrum. One hundred floweringshoots were viewed with caution. In spite of that we are certainthat monitoredin July 1986. a comparative study of a closely related set of species LocalityB, 1,000 floweringshoots at the-LakeHomsjon obviously possessing a greatvariation in theirsexuality locality,Sweden. Sixtyflowering shoots were monitored might tell us how evolution has favored differentsuc- in July 1988. cessful solutions, i.e., reprotypes,within the ecological and phylogeneticconstraints of the species set. Flowerform - Inflorescenceand grossflower characters forall species were described and measured. MATERIALS AND METHODS Ultraviolet reflectancepatterns were revealed with a UV-transmittingKodak Wratten18A filter(Kevan et al., Plant species,study sites, and populations-Six taxa were 1973; Kevan, 1983). Spectralreflectance in the 300-700- studied: Moneses uniflora(L.) A. Gray, Orthiliasecunda nm rangeof innerand outerpetal surfaces,and of anthers (L.) House, Pyrola minor L., P. rotundifoliaL. ssp. ro- was obtained using a Unicam SP 8,000 recordingspec- tundifolia,P. chloranthaSwartz, and Chimaphilaum- trophotometerfitted with a SP 890 diffusereflectance unit. bellata (L.) W. Barton. To reveal the presenceof nectaries,serial cross sections Moneses uniflora:Locality A, 3,000 floweringshoots of paraffin-embeddedflowers were made. The sections in an area of 1,000 m2 in a forestdominated by Pinus were stained in safraninand fastgreen (Mattsson, 1968). sylvestris,Hasle Lystskov,Bornholm, Denmark (55?10'N, In 0. secunda,nectar productionwas measured with 14?42'E). The forestfloor was sparselycovered by herbs, 1- or 2-Almicropipettes on inflorescencesenclosed for3, especiallyDeschampsia flexuosa, Festuca rubra,Lotus 6, 9, 12, and 24 hr afterthey were depleted fornectar by corniculatus,0. secunda,P. minor,Sarothamnus sco- bumblebees. A seriesof flowers was testedfor nectar pres- parius, and Thymus serpyllum.One hundred and fifty- ence afterbumblebee visits and nothingor only minute eightflowers were monitoredin June-July1987. amounts were present,since nothingwas extractable.In Locality B, severalthousand flowering shoots in an area C. umbellata,standing crop of nectarwas measured with alongthe Northeastern shoreside of Lake Hornsjon,Oland, 1- or 2-idlmicropipettes after enclosure of flowersfor 24 Sweden (57013'N, 16?58'E). Dominant herbs were An- hr, the firstflowers were enclosed from06 to 06 hr and thyllisvulneraria, Lotus corniculatus,and Melampyrum the last from21 to 21 hr. Danish and Swedish Summer pratense.All otherScandinavian lowland Pyrolaceae spp. Time is 2 hr ahead of Greenwich Mean Time. Concen- were also present. One hundred and fiftyflowers were trationof sucrose equivalents in nectarof 0. secundawas monitoredin June-July1988. measured with a Bellingham and Stanley pocket refrac- Orthilia secunda: Locality C, 300 floweringshoots in tometermodified for small volumes of nectar (>0.5 4l; an area of 1,000 m2 in an 80-yr-oldstand of Picea abies % of sucrose was correctedto 20 C; Corbet, 1978). Con- and Abies normanniana, Klim Bjerg, NorthernJutland, centrationof sucrose equivalents in nectarof C. umbellata Denmark (57?6'N, 9011 'E). Dominant herbswere Mone- and sugar composition of nectar fromboth 0. secunda ses uniflora,Thalictrum minus, and Violatricolor. Thirty and C. umbellatawere analyzed in frozensamples using floweringshoots were monitoredin June-July1986. High PerformanceLiquid Chromatography(column: Nu- Locality B with several thousand floweringshoots at cleosil NH2, 5 mm, 20 cm x 4.6 mm I.D., mobile phase: the Lake Hornsjon locality, Sweden. Thirty flowering 75% acetonitrilein waterat 1.0 ml per min,detector LDC shoots were monitoredin June-July1988. RI refractomonitor). Pyrola minor:Locality C, 2,000 floweringshoots in an Pollen samples fromall species (except P. chlorantha) area of 1,000 m2 at the Klim locality, Denmark. Fifty were acetolyzed and mounted for scanningelectron mi- floweringshoots were monitoredin June-July1986. croscopy formorphology (description of exine sculptur- Locality B with several thousand floweringshoots at ing,cf. Walker and Doyle, 1975). Size measurementswere the Lake Hornsjon locality, Sweden. Thirty flowering performedby lightmicroscopy (Oldfield, 1959). Dryness shoots were monitoredin June-July1988. or wettabilityof pollen was tested by squeezing pollen :Locality D, 150 floweringshoots into waterto observe ifpollen grainsseparated from each in an area of 100 m2 in a meadow with some Pinus syl- other. Viability of pollen was estimated as percentage vestris,Tastum, Northwestern Jutland, Denmark (56031 'N, pollen grains with stainable callose contentusing lacto- 901'E). Dominant herbs were Cirsiumpalustre, Filipen- phenol-anilineblue. Fresh pollen grainswere taken up in dula ulmaria,Lotus corniculatus, L. uliginosus,and Pedic- standardJKJ- and Sudan IV-testsolutions and examined ularispalustris. Seventy-four flowering shoots were mon- for starchand lipid reactions (Baker and Baker, 1979a, itored in June-July1986. 1983). Locality B with several thousand floweringshoots at Flowers of 0. secunda,P. minor,P. chlorantha,and C. the Lake Hornsjon locality, Sweden. Thirty flowering umbellatawere stained in neutralred solution for 5-24 shoots were monitoredin July1988. hr to detectany scent-emittingparts (Vogel, 1963; Stem, :Locality B, 500 floweringshoots at Curry,and Whitten,1986). Similardata fromM. uniflora, the Lake Hornsj6n locality,Sweden. Forty-sixflowering P. rotundifolia,P. norvegica,and P. media have been shoots were monitoredin June-July1988. published in Knudsen and Tollsten (1991). In addition, Chimaphila umbellata: Locality E, 1,500 flowering organolepsywas performedon flowersof all species. shoots in an area of 12 m2 in an old dune plantation Flower longevityof pollinated and unpollinatedemas- dominated by P. sylvestris,Blykobbe, Bornholm, Den- culated flowerswas measured and compared in C. um- mark(5 5?8'N, 14042'E). The forestfloor was sparselycov- bellataand M. uniflora.Half of each sample of emas- eredby Calluna vulgaris, Deschampsia flexuosa, and Em- culated flowersof each species was cross pollinated. 902 AMERICAN JOURNAL OF BOTANY [Vol. 80

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TABLE 2. Numberof pollen grains, pollen units (PU), and ovulesperflower, pollen: ovule ratio (P:O), andpollen unit: ovule ratio (PU:O)

No. of pollen grains x 103 No. of pollen units x 103 No. of ovules Species Mean Range Mean Range Mean Range P/O PU/O M. uniflora 681 437-994 170 109-248 9,750 6,225-14,525 70 17 0. secunda 179 123-228 179 123-228 2,150 1,475-2,575 83 83 P. minor 98 60-120 25 15-30 4,575 3,275-6,000 21 5 P. rotundifolia 490 327-751 123 82-188 5,975 3,450-9,900 82 21 P. chlorantha 324 185-524 81 46-131 6,025 4,250-9,250 54 13 C. umbellata 343 175-571 7 44-143 10,700 9,265-12,725 32 <<1 N = 10 in each case.

Ramets weretagged and censusedevery or everysecond 1988), P. chlorantha(9 m2 plot with 45 floweringshoots day during flowering,and number of open flowersand in 1988), and C. umbellata(1.6 m2plot with206 flowering floweranthesis monitoredin 1988. shoots in 1986, and 2.3 m2 plot with 54 floweringshoots in 1988). Buzz sounds wererecorded with a tape recorder, Breeding system-Type of breeding system was re- and replayed at slow speed in order to measure buzzing vealed by insectexclosure and emasculation tests.Nylon time. Flower handlingtimes were measured witha stop- bags mounted on a frame of wire were used as watch fromtape-recorded dictations of landingand take- exclosures.Buds were emasculatedprior to opening.Five offof individual bees on C. umbellata. treatmentswere performedon flowersof each species: 1) Specimens of all visitor species were caught for iden- untreatedbagged flowers(autodeposition); 2) artificially tification,and pollen loads of specimenscollected in 1986 self-pollinated,bagged flowers(compatibility); 3) emas- and 1987 wereinspected. Additionally, pollen loads from culated,artificially cross-pollinated, bagged flowers(com- bumblebeesvisiting other flowering bee plantsat thestudy patibility);4) emasculated,bagged flowers (apomixis); and sites in 1986 and 1987 were sampled in orderto estimate 5) open-pollinated,unbagged flowers (control). Pollen for species constancy.Each corbicularpollen load was boiled artificialcross pollination was taken fromplants at least in 10% NaOH, dehydrated,and stained for2-5 hr in 1% 100 m fromthe experimental . Artificial pollination safraninein 50% alcohol. Loads were then rinsed three was performedby applying the pollen content of two times in 70% alcohol, twice in acetone, and finallytwice anthersto the stigma.Pyrola minor and 0. secundaonly in ethylacetate. This rinsingprocedure was necessaryin received treatments1 and 5. Fruitswere harvested6 wk orderto removewaxy materials and separatepollen grains. aftertreatment, and pollinationsuccess was estimatedby Vouchers of are in the firstauthor's collection in scoringseeds as eithermatured or aborted. In treatments the Departmentof Chemical Ecology,University of Go- 2 and 3 on M. uniflora,data on pollen tube growthwere teborg. used instead. Pollinated stigmas were fixedin Camoy's solution (1:3 glacial-aceticacid: 99% ethanol; Johansen, Statistics-Average values are presentedwith standard 1940), softenedin 8 M NaOH, stained in leuco-aniline deviation (?SD). Nonparametrictests were applied when blue solution,dissected, mounted in 10% glycerol-leuco- data were assured not to be normallydistributed. Product aniline blue, and finallyinspected for pollen tube growth moment correlationcoefficients (r) and Spearman rank in styleby fluorescencemicroscopy. correlationcoefficients (p) were calculated, with 0.4 < r Pollen: ovule ratio(P:O) was used as a relativemeasure or p < 0.7 indicatingmodest, 0.7 < r or p -< 0.9 strong, ofoutcrossing (Cruden, 1977). Ten buds fromeach species and r or p > 0.9 very strongcorrelations (Fowler and were collected and preservedin 70% ethanol. Two sta- Cohen, 1990). All analyseswere performed with the Mac- mens per bud were dissected in a 4-ml aqueous solution intosh StatView (Feldman, 1987) and SYSTAT (Wilkin- with detergent,stirred, and number of pollen grains in son, 1989) packages. ten subsamples of 20,ul counted. These counts had a low coefficientof variance forall species (except C. umbellata RESULTS because its pollen is in polyads). The same countingpro- cedurewas, however,applied to all species. Placentas were Flowerform and phenology-Quantitative and quali- dissected and halved, and ovules were spread on a slide, tativeinformation on a seriesof floral sexual traitsis listed mounted in glycerol,and counted. These countingswere in Tables 1, 2. Flower formis shown in Fig. 1 (excluding used to calculate the P:O ratios. P. chlorantha,which is similar to P. rotundifolia).The generavaried in inflorescencetype and flowerproduction. Pollinators -At least 5 d (from5 to 22 hrat mostcensus Flower abortion (no. of flowersaborted x 100/no. of days) were spenton each species recordingflower visitors. flowers)was low (< 10%) in all species exceptC. umbellata Flower visitors and theirbehavior were recorded in M. where it was 29%. The most importantagents causing uniflora(2.5 m2 plot with 158 floweringshoots in 1987, abortion were dipteranlarvae. and observational walks along a predeterminedpath in Four species have dish-shapedflowers, but 0. secunda 1988), 0. secunda(observational walks in 1986, and 1 and P. minorhave more funnel-shapedones. Symmetry m2 plot with 30 floweringshoots in 1988), P. minor(10 ofthe flowers varied. Pyrolaminor and C. umbellatahave m2 plot with 600 floweringshoots in 1986, and obser- actinomorphicflowers but the resthave more or less zy- vational walks in 1988), P. rotundifolia(4 m2 plot with gomorphicflowers. The two nectar-producers,0. secunda 22 floweringshoots in 1986, and observationalwalks in and C. umbellata,have a flowerdepth of 4.6 ? 0.4 mm 904 AMERICAN JOURNAL OF BOTANY [Vol. 80

Pma12 mm 2Ikm Os Pr

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Cu

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2 mm

Fig. 1. Flowersand antherinversion during opening of flowers of the Pyrolaceae. Mu, Moneses uniflora, Os, Orthiliasecunda, Pm, Pyrolaminor, Pr, P. rotundifolia,and Cu, Chimaphilaumbellata. Drawn from liquid material.

(N= 10,range 3.9-5.1) and 3.0 ? 0.3 mm(N= 10,range Formof pollen grains is shownin Figs. 3-8. Pollenunits 2.7-3.5), respectively.Corolla colors are whiteto green. (PU) are monadsin 0. secunda,tetrads in Monesesand Chimaphilaumbellata, however, has pinkflowers. UV- Pyrola,and polyadsin C. umbellata.Most polyadscom- reflectionfrom the flowers of all specieswas weak. Sta- prise more than 50 pollen grains.Exine sculpturingis mens,filaments, pore areas, and petalbases reflectedUV verrucatein all species,but C. umbellatain whichit is to varyingdegrees (Table 1; Fig. 2). In all species,anthers psilate-foveolate.Single grains in polyadsare intercon- reflectedmore UV thanpetals. The insideand outsideof nectedby exine bridges and tectumcoalescences (Fig. 8). petalsreflected light to the same degree.Thus colorsof A fewdyads were observed in 0. secunda(Fig. 4). Pollen bothsides were probably equally important in attracting grainsize rangesfrom 19 to 30 ,umand PU size ranges flowervisitors. The colorsof anthers are yellow and pink from19 to 42 ,im,excluding the polyads of C. umbellata. and theyellow ones contrasted especially vividly with the Viabilityof pollen,indicated by stainabilityof callose corolla. tissue,was 100% in all species,except in C. umbellata The poreopenings of the anthers are positioned directly whereit was 96.5% (N = 743 pollengrains). Pollen grains on thethecae in 0. secundaand P. minor,whereas they ofall speciescontained both starch and lipids.The starch are born on the end of prominenttubes in the other reactionwas, however,weak in P. rotundifoliaand C. species.The anthertubes of P. rotundifoliaand C. um- umbellata. bellataare distinctly shorter than those of M. unifloraand P:O ratiosranged from 21 to 83 and PU:O ratiosfrom P. chlorantha(0.1-0.5 vs. 0.6-0.8 mm;Table 1). < 1 to 83 (Table 2). Spearmanrank correlation coefficients August 1993] KNUDSEN AND OLESEN-SEXUAL PATTERNS AND POLLINATION 905

100 _

80-

Myoneseuniflora Op op

100

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Fig. 2. Spectralreflectance curves of ip, innerpetal surface; op, outerpetal surface; and a, anthersof species of the Pyrolaceae. betweennine different floral traits are givenin Table 3. base of theovary. In 0. secunda,the average amount of Size of pollen,seed, and flowerwere strongly positively nectarper flower and itsconcentration in sucroseequiv- correlated.Number of pollengrains per flower was pos- alentsirrespective of theproduction period was 0.45 ? itivelycorrelated with flower and pollen size. Thus no 0.437,uland 43 ? 19.0%(N= 32). Nectarconcentration negativetrade-off between pollen size and numberwas wasnegatively correlated with volume (N= 32,r= -0.55, found.Number of ovules per flowerwas stronglyposi- P = 0.011; Fig. 9a). No significantcorrelation of volume tivelycorrelated with size of flower,seed, pollen,and withtime of day or ofconcentration with time of day was pollen unit,and negativelycorrelated with number of found (N = 32, r[volume] = 0.31, P = 0.085; flowersper inflorescence. Style length and P:O ratiowere r[concentration]= -0.11, P = 0.55). In C. umbellata,the notcorrelated to any of the floral traits. Number of flowers averageamount of nectar and itsconcentration was 2.60 was stronglynegatively correlated to flowersize. Seed size ? 1.892 ,l and 34 ? 21.2% (N= 39). Significantcorre- was stronglynegatively correlated to numberof flowers lationswere present of nectarvolume on timeof day (N and stronglypositively correlated to pollenunit size. = 39, r = -0.48, P = 0.0019), of nectar concentration Nectarieswere only present in 0. secundaand C. um- on time of day (N = 39, r = 0.34, P = 0.037), and of bellata,in whicha nectar-secretingdisc was foundat the nectarconcentration on volume(N= 38, r -0.37, P 906 AMERICAN JOURNAL OF BOTANY [Vol. 80

IV~~~~~~~~

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Figs.3-8. Scanningelectron micrographs of pollen grains and pollenunits. 3. A monadof Orthiliasecunda. 4. A dyadof Orthiliasecunda. 5. A tetradof Pyrola rotundifolia. 6. A tetradof Moneses uniflora. 7. A tetradof P. minor.8. A polyadof Chimaphilaumbellata. Bar = 10 ,um.

= 0.021; Fig.9b-d). The proportionsof fructose, glucose, and 41 samples,respectively). Nectar of bothspecies is and sucrosein nectarof 0. secundawere 18%:24%:58% thussucrose-dominated (sensu Baker and Baker,1979b, (ratioby weight:sucrose: hexose = 1.4) and in C. um- 1990). bellata 22%:12%:66% (sucrose: hexose = 1.9) (N = 10 Monesesuniflora and P. rotundifoliaemitted a readily August 1993] KNUDSEN AND OLESEN-SEXUAL PATTERNS AND POLLINATION 907

TABLE 3. Correlationmatrix of sexual traits in thePyrolaceaea

#Flowers/ inflorescence Flower size Pollen size PU-size Style length Seed size # Pollen #Ovules Flowersize (p) -0.83 (P) 0.06 Pollensize (p) -0.64 0.81 (P) 0.09 0.07 PU-size (p) -0.23 0.69 0.73 (P) 0.23 0.13 0.13 Stylelength (p) 0.31 -0.09 -0.22 -0.43 (P) 0.49 0.84 0.62 0.34 Seed size (p) -0.81 0.83 0.90 0.90 -0.49 (P) 0.07 0.06 0.05 0.05 0.27 #Pollen (p) -0.54 0.78 0.84 0.43 0.29 0.60 (P) 0.15 0.08 0.06 0.45 0.52 0.20 #Ovules (p) -0.77 0.93 0.84 0.86 -0.26 0.94 0.66 (P) 0.05 0.04 0.06 0.06 0.56 0.04 0.14 P/O ratio (p) 0.21 -0.03 -0.20 -0.68 0.12 -0.52 0.31 -0.37 (P) 0.63 0.94 0.65 0.13 0.70 0.25 0.48 0.41 a The tablegives the Spearman rank correlation coefficients (p). HO P(p) > 0.05; HA # HOis tested.P (probability-value)ofthe correlation. perceivablefloral scent, but P. minorand C. umbellata spectively).The generallylonger flowering time of both emittedonly a faintscent, and 0. secundaand P. chlor- emasculatedunpollinated and cross-pollinatedflowers anthahad no detectable(to thehuman nose) scentat all. comparedto open-pollinatedflowers (Table 1) probably In all speciesthe area around pore openings, anther tubes, indicatesa treatmenteffect on longevityof theflowers. and stigmastained deeply with neutral red. The coloration Floweringphenology and flowerlongevity are givenin ofC. umbellatamasked any reaction of neutral red. How- Fig. 10 and Table 1. The floweringseason overlapped for ever,neutral red is not an absoluteindicator of osmo- O. secunda,P. minor,and P. rotundifolia.Moneses uni- phoricactivity (Stem, Curry, and Whitten, 1986). Staining floraand P. chloranthawere slightly earlier, and C. um- of stigmasmight result from their layer of exudates.In bellatalater. From the plethora of nullhypotheses (e.g., addition,anthers of P. minorand upperparts of the petals Stronget al., 1984),we chosethe one formulatedby Poole in M. uniflora,P. minor,and P. chloranthawere com- and Rathcke(1979) and used theirtest. Peak flowering pletelystained. ofeach of the six species was independently and randomly Anthersin youngbuds were centered around the ovary positionedalong the season definedfrom the beginning withtheir pore openings pointing toward the base ofthe of theflowering of thefirst species to theend of thelast flower.In latebud stageor duringopening of the flower, species.The samplestatistic P (sensuPoole and Rathcke, the anthersinverted and thepore openingspointed out 1979) herecalled O(p) is thesample variance of the dis- fromthe flower (Fig. 1).The area around the pore openings tancesbetween peak floweringdates betweenadjacent ofthe anthers changed color from yellow to dark-yellow- species.Its expectedvalue was (E(p)) underthe null hy- orangewhen pores opened in M. uniflora,P. minor,and pothesis(see Poole andRathcke, 1979). The ratioof O(p)! P. rotundifolia.Only a slightcolor change was observed E(p) wasthus a measureof aggregation of flowering times. in 0. secundaand P. chlorantha. The ratiois approaching0.0 if the floweringtimes are Flowerlongevity is givenin Table 1. In P. rotundifolia regularlydistributed, 1.0 ifthey are random,and above and C. umbellata,time of flower opening was monitored. 1.0 if theyare aggregated.We foundthat O(p) = 0.012 Flowersof P. rotundifoliaopened at differenttimes during and E(p) = 0.014 (X2 = 5.14, df= 6, 0.05 < P < 0.5). theday; themajority, however, opened during the after- The null hypothesiswas confirmed,and the flowering noon and evening(05-09 hr: 8%, 09-13 hr: 9%, 13-17 timeswere thus randomly distributed. However, the result hr:29%, 17-21 hr:29%, and 21-05 hr:25% of censused dependson thechoice of null hypothesis. If one extends flowers;N = 140).Most of its flowers withered during the the growingseason, the ratioincreases and at a certain night(05-09 hr: 12%, 09-13 hr: 3%, 13-17 hr:2%, 17- pointthe flowering times become significantly aggregated. 21 hr:29%, and 21-05 hr:54%; N= 87). In C. umbellata, The degreeof dichogamy within the species group was 85% and 90% of the flowers(N = 40 and N = 96) opened difficultto ascertain.Flowers of M. uniflora,P. rotundi- and witheredduring the night (19-09 hr),respectively. A folia, and P. chloranthawere suspected to be homoga- seasonalchange in flowerlongevity was monitoredin C. mous/protogynous,but the other three species were pro- umbellata;it changed significantly during the season. Those tandrous(Table 3). Orthiliasecunda and C. umbellata flowersthat opened at thebeginning and end ofthe flow- opened theiranthers a fewdays priorto anthesis.The eringseason became 9.3 ? 2.7 d old (N = 31), butthose stigmabecame receptive 1-2 d afteranthesis. open in themiddle of the season lasted 6.7 ? 1.8 d (N= 118, t-test, P < 0.0001). Unpollinated,emasculated flow- ersof M. unifloraand C. umbellatapersisted significantly Breedingsystem -The specieswere self-compatible to longer(26.6 ? 3.6 d, N= 11; 12.0 ? 2.0 d, N= 24, some degree(data missingon 0. secundaand P. minor), respectively)than those cross pollinated and emasculated butPyrola minor had a largeseed-set after autodeposition (13.6 ? 1.5 d, N= 10; 8.0 ? 1.8 d, N= 31, respectively; ofpollen,and is thusself-compatible (Table 4). Allspecies, Mann-WhitneyU-test, P < 0.0001 and P < 0.001, re- exceptC. umbellata,had a higherseed-set after open- 908 AMERICAN JOURNAL OF BOTANY [Vol. 80

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5 10 15 20 25 0.0 2.0 4.0 6.0 8.0 10.0 Productionperiod (h) Nectarvol. (ii) Fig. 9. a. Nectarconcentration as a functionof volumein Orthiliasecunda. b, c. Nectarvolume and concentrationas functionsof a 24-hr productionperiod in Chimaphilaumbellata. d. Nectarconcentration as a functionof volume in C. umbellata. pollination(treatment 5). None of the specieswas apo- atelystarted to sucknectar by insertingits proboscis be- mictic. tweenthe filaments. Of thebumblebees visiting C. um- bellata,28% weremales and theyused, both in 1986and Pollinators-Four ordersof insectsvisited the flowers in 1988, on averagesignificantly longer time to handle ofthe studied species (Table 5). Most ofthe nonhymen- theflowers than did workers(Smirnov's two-sample test; opteranspecies listed were infrequently observed and were P < 0.00001,data fromboth 1986 and 1988were highly consideredas "tourists."In Monesesand Pyrola, buzzing, significant).In C. umbellata,Bombuspascuorum account- pollen-collectingbumblebees were the most numerous ed for73% of all visitorsand 66% of all visits.None of visitors.In Orthiliaboth buzzingand nectarimbibing thevisiting bees wereobserved to collectpollen from the behaviorswere observed, although the latter dominated. flowers. The late-floweringC. umbellata was visitedfor its nectar Althoughthe Pyrolaceae species we studiedutilized the and was notobserved to be buzzedby any visitor. Bum- same guildof pollinators,different castes of bumblebees blebeescollecting pollen in M. unifloragrasped and vi- dominatedplant species with different flowering phenol- bratedtwo to fourstamens at a time.In P. minorthey ogy(Fig. 11). Monesesuniflora and P. minorwere pol- vibratedthe entireflower, and in 0. secunda,P. rotun- linatedprimarily by queensand workersof B. lucorum; difolia,and P. chloranthathey grasped and vibratedthe 0. secundaby queens and workersof B. pratorumand B. bunchof anthers above the style. Tape recordingsof bum- lucorum;P. rotundifoliaby workers of B. lucorumand B. blebeesbuzzing P. minorrevealed that apparent single hypnorum;P. chloranthaby workersof B. lucorumand buzzesactually were sequences often to 14 shorterbuzzes queensand workersof B. pascuorum;and C. umbellata lasting0.06-0.25 sec each (N = 64 sequences).To collect by workersand malesof B. pascuorum,B. hypnorum,B. nectarin 0. secunda,a givenbee graspeda flowerand pratorum,and B. lucorum. visiting P. minorinserted insertedits proboscis at thecorolla opening and distended theirheads into the corollaopening, probably gleaning itto thebase ofthe petals where nectar had accumulated. leftoverpollen. Individuals of Thricops semicinereus (Syr- In C. umbellata,a givenbee oftenlanded on theoutside phidae)often sat on theinner side ofpetals gleaning the of thecorolla and crawledinto its interiorand immedi- poreareas forpollen grains. August 1993] KNUDSEN AND OLESEN-SEXUAL PATTERNS AND POLLINATION 909

100

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0 rt P 60 -C.i.umb. e n f 0 W 40- e r S

20 -sc

0- 15-jun-88 25-jun-88 5-jul-88 15-jul-88 25-jul-88 Date Fig. 10. Floweringphenology of thePyrolaceae in 1988. Percentageof flowersopen in markedindividuals of M. uni.,Moneses uniflora; 0. sec.,Orthilia secunda; P. min.,Pyrola minor; P. rot.,P. rotundifolia;P. chl.,P. chlorantha;and C. umb.,Chimpahila umbellata.

In 1986, the mostabundant visitor to P. rotundifolia ofpollen-collecting pollinators among co-occurring buzz- was the smallbeetle Kateretes pedicularis (Nitidulidae). flowershas been observedseveral times (e.g., Macior, It chewedthe anthers and ate exposed pollen grains. More 1971; Buchman,Jones, and Colin, 1977; Renner,1986). thanhalf of the number of anthers showed some damage. Hybridshave indeedbeen recorded between Pyrola spe- Thus pollenin poricidalplants is difficultfor nonbuzzing cies(e.g., Knaben, 1944; Bocher, 1961). Pyrola minor and insectsto harvest. 0. secunda,when growing intermingled, may benefit mu- The corbicularpollen load of an individualof B. so- tually.In the Klim locality,the rarer0. secundawas roeensisforaging on M. unifloracontained 89% M. uni- visitedfor its nectarby visitorsto P. minor.The pollen florapollen and 11% fromtwo otherspecies (in 1987). flowersof Pyrola species might also be visitedby mistake Bombussoroeensis foraging on 0. secundahad corbicular by nectar-collectingbumblebees (Knaben, 1944). pollenloads composedof 45% 0. secundapollen and the remainderfrom six otherspecies (in 1986). Bumblebees TABLE 4. Percentageseed set (numberof (seeds/ovule)x 100) in on P. minorhad corbicularpollen loads composed of 46% thePyrolaceae after crossing experiments P. minorpollen, 25% 0. secundapollen, and 28% from one to threeother species (in 1986).Bumblebees foraging 4) Bagged emascu- 5) Open on P. rotundifoliahad corbicularpollen loads composed 1) Bagged 3) Bagged lated poili- untreated emascu- unpolli- nated of 38% P. rotundifoliapollen and 62% fromtwo to seven flowers 2) Bagged lated nated flowers (auto depo- selfed crossed flowers (con- otherspecies (in 1986).None ofthe pollen loads ofbum- Species sition) flowers flowers (apomixis) trols) blebeescaught foraging at otherco-occurring species con- tainedany pollenof Pyrolaceae. M. uniflora 0.2 - I100a - I100a 0.1 100.0 0. secunda 0.5 b b b 84.4 P. minor 44.1 b b b 65.6 DISCUSSION P. rotundifolia 2.4 25.1 28.9 0.1 59.7 P. chlorantha 5.7 71.5 61.8 0.1 85.9 Generally,the habitatsin whichPyrolaceae grow are C. umbellata 1.9 51.6 41.6 0.4 37.8 poorin entomophilousplant species. However, it is com- a Degreeof compatibility estimated from pollen tube germination, all mon to findseveral species of Pyrolaceaegrowing to- pollengrains on thestigma seemed to havegerminated and grown. gether,as previouslynoted by Copeland(1947). Sharing b No experimentaldata. 910 AMERICANJOURNAL OF BOTANY [Vol. 80

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Speciesof the Pyrolaceae often grow under reduced light 100 Queens conditions,and thewhitish flowers and theirfloral scent mightenhance their conspicuousness to visitinginsects. 80 E Workers The whiteflowers were almost UV-dark as observedin mostwhite-flowered species (Kevan, 1983). Orthiliase- Males cunda and P. chloranthawere exceptions. They had no ~60-~ perceivablescent, and thearea aroundthe pore openings of theanthers in thecorolla opening of 0. secundaand ~40- thewhole staminal bunch in P. chloranthacontrasted with thegreen corolla by reflecting UV-light. The scent-active 20 areaaround the anther pore openings and the anther tubes indicatedby neutralred stainingmight serve as signals to pollen-collectingbees. In M. unifloraa quantitative 18June 22 June29 June 1 July 2 July 19July and in P. rotundifoliaa qualitative differentiation in floral scentis foundbetween and petals,and thisdif- Peakflowering ferentiationis regardedas a close-rangeorientation cue Fig. 11. Frequencyof castes of visiting bumblebees at peakflowering ofMoneses uniflora, Pyrola chlorantha, Orthilia secunda, P. rotundifolia, (Knudsenand Tollsten,1991). Bolwig(1938) and Dob- P. minor,and Chimaphilaumbellata. son,Bergstrom, and Groth(1990) amongothers also re- gardeddifferences in the scentprofile of differentfloral partsas orientationor rewardlevel cues. data). In Denmark,the nonporicidal flowers of Rosa ru- The prolongedflower longevity of unpollinated flowers gosa are mainlybuzzed between 09 and 14 hr,and prob- ofM. unifloraand C. umbellata,and therather long lon- ably also ceases as a responseto shortageof pollen in gevityof individual flowers for all species(Table 1) may openanthers (J. M. Olesen,unpublished data). Buzz-pol- functionto enhancethe conspicuousnessof the flowers linationmay thus at higher latitudes show diurnal patterns to visitinginsects and ultimately enhance pollination suc- relatedto rewardlevels. However, Cane and Buchman cess. (1989) also reportedceasing visitation of buzzingbees Pollenis an importantreward to visitingbees in buzz- becauseof pollen shortage in Solanumelaeagnifolium at pollinatedflowers (Vogel, 1978; Buchmann, 1983; Buch- lowerlatitudes. mannand Cane, 1989).Vogel (1978) definespollen flow- The valuesof P:O ratioswere low in all species,indi- ers as flowersthat offer a surplusof pollenas the only catingefficient pollen transfer (Cruden and Miller-Ward, rewardto theirinsect visitors. The flowersof thePyro- 1981). Improvedpollen transfermay be achieved by laceae produceda largeamount of pollenand belongto switchingto autogamy(Cruden, 1977), specializing on a the Solanum-type(sensu Vogel, 1978); zygomorphic, restrictedgroup of highly constant pollinators, developing whitishflowers, few stamens, and enlarged,yellow, and viscinthreads keeping pollen grains together in larger units oftenporicidal anthers. Pollen grains are probablynutri- (Crudenand Jensen,1979), or packingpollen into tetrads tiousto visitingbees as indicatedby theirlipid content. polyads,and pollinia.PU size increasedwith ovule num- Bakerand Baker(1990) showedthat nectar dominated ber per flower,seed size, and a decreasingflower pro- by sucroseis significantlyrelated to big bee-pollination, ductionper plant. Ovule number increased with a reduced and thegeneralization also holdsfor 0. secundaand C. P:O ratio.Resources seemed to be allocatedbetween the umbellata. numberof flowersper inflorescence on one side and the The overallpattern of floraltraits in thePyrolaceae is numberof pollen grains and ovules,and flowerand seed in accordancewith most otherbuzz-pollinated plants. size on the otherside. The reductionin P:O ratiowith Chimaphilaumbellata is an exceptionbecause of its type increasingPU size in Pyrolaceaeis also exemplifiedin ofpollen and highlyconcentrated nectar, and no buzzing, xenogamousspecies (Cruden and Miller-Ward, 1981) and pollen-collectingbees wereobserved at its flowers.We seemsto be accompaniedby a simultaneousreduction in couldnot confirm Buchmann's (personal communication) pollen numberand an increasein ovule number.The observationof buzzingof thisspecies. positivecorrelation between PU size and a highnumber It is notknown if any of the observed syrphid visitors of ovulesper ovaryfound here conforms with Walker's to speciesof Pyrolaceae buzzed the anthers. This has been (1971) findings.IfPU sizeincreases, fertilization and seed- observedby Buchmann, Jones, and Colin(1977) forVol- setare hypothesizedto be improved.This was,however, ucellamexicana (Syrphidae) on Solanumflowers, and for notdocumented here. Ornidia(Syrphidae) on buzz flowersin Panama (Buch- The speciesgroup studied showed at leastfour trends mann,personal communication). towardconservation of pollen: 1) poricidalanthers, 2) Generally,as shownby Heinrich(1972), Buchmann, pollenunits of increasing size, 3) developmentof anther Jones,and Colin (1977), and Renner(1989), buzzingis tubes,and 4) an increasein anthertube length.Larger an earlymorning behavior, perhaps related to the high pollenunit sizes mayresult in a reducedpollen wastage expendituresof energy for the activity resulting in exces- as suggestedin theOrchidaceae and Asclepiadaceae (Cru- sivebody heating, which is a problemto getrid of during den,1977). No significantcorrelation was, however, found thewarmer part of theday. These studiesare, however, forthe studied Pyrolaceae species. all carriedout at lowerlatitudes than the present one. For Thedevelopment ofpolyads in C. umbellatamight have example,buzzing was observedthroughout the entire day reducedthe number of gamete-donatingmales and thus inP. minor,although it diminished late in the day, perhaps theimportance of femalechoice. This trendcould have becauseof shortage of pollen (J. T. Knudsen,unpublished been counteractedby a selectionin thefemale for larger 912 AMERICAN JOURNAL OF BOTANY [Vol. 80 stigmas,as observedin C. umbellata.The late flowering linationsystem. The single-floweredinflorescence in M. of C. umbellatacoincides with the appearanceof male unifloramay represent an adaptationto reducegeitonog- bumblebees.They frequently visit flowers of C. umbellata amy.In P. minorthe high rate of auto-deposition of pollen to foragefor nectar. Their prolonged handling time com- and the reportof individualswith only cleistogamous paredto workersprobably reduced pollen waste and en- flowers(Andres, 1910) indicatea highdegree of self-pol- hancedpollen transfer. linationin thisspecies. However, according to Takahashi Willson(1979) suggestedthat a largePU or pollenload (1986a) thepollen of P. minorrepresents an ancestraltype may covera largerpart of a stigmaticsurface and thus withinthe Pyrola. reducethe competitive ability of other males, the polyad Regardingfloral scent composition, Moneses uniflora beingthe botanical analogue to a copulatoryplug in the appearsto have a uniqueposition within the family. The animal kingdom.Larger PU size increasesthe genetic floralscent of thisspecies is dominatedby compounds relatednessof seedsin a fruit,and thusprobably reduces of isoprenoidorigin, whereas species of Pyrolamainly offspringor siblingconflict. Furthermore, reduced off- have compoundsof aromatic origin (Knudsen and Toll- springconflict should reducevariance in investedre- sten,1991). Floral scent of P. minoris weak,but contains sourcesamong the differentsibs in a fruitand thereby smallamounts of some aromatic compounds (J. T. Knud- probablydiminish abortion. Consequently an increased sen,unpublished data) also presentin thefloral scent of PU size mayincrease seed set,not by improvingpollen P. rotundifolia.Thus a declinein scentproduction may transferbut by reducing abortion and wasteof resources. have accompanieda greaterreliance on self-pollination Seed set is thusenhanced as therelatedness among sibs in P. minor.Knaben (1944) describeda seriesof reduc- increases(Uma-Shaanker, Ganeshaiah, and Bawa, 1988). tionsin thelarge-flowered Pyrola species, P. rotundifolia, An increasein seed set and a lowerabortion rate may P. norvegica,and P. grandiflora,e.g., reduction in flower selectfor higher ovule production.This mayexplain the number,style and stamenlength, elimination of anther occurrencewithin the Pyrolaceae of highovule numbers tubes,and an incrementof pore size. These reductions per ovaryas also is foundin othergroups with polyads, may also have accompanieda greaterreliance on self- e.g.,the Asclepiadaceae and Orchidaceae. pollination. Seed parameterswere not measured in thisstudy. Sal- Chimaphilaumbellata is visitedonly by nectar collec- isbury(1942) and Pyykko(1.968) give figures for seed size torsand appearsto be theleast specialized of the species (Table 1). Seedsof Pyrolaceae are the size oforchid seeds studied.Orthilia secunda was visitedby both nectar- and or even smaller(Table 1). The positivecorrelation be- buzzingpollen-collectors. Thus, the pollination system of tweenseed size and numberof ovules was significant 0. secundamay be regardedas intermediatebetween that (Table 3). Generally,investment in seeds is muchmore of C. umbellataon one side and thatof the exclusively expensivethan that in pollen(Bertin, 1988). However, in buzz-pollinatedtaxa, Moneses and speciesof Pyrolaon Pyrolaceaethere is only a ca. 30-50-folddifference in the otherside. Vogel (1978) suggestedthat many Sola- lengthbetween a pollengrain and a seed. If largerpollen num-typepollen flowers may be derivedfrom a nectar unitsize and smallerpollen production reduce waste of flowertype with few stamens. Most membersof theEr- resourcesby abortions,then these resources may be al- icaceae,in whichthe Pyrolaceaesometimes is included locatedand spentby the hermaphroditicflowers on an as a subfamily,have poricidaldehiscent anthers and se- increasein thefemale fitness component such as an en- cretenectar (Henderson, 1919; Stevens,1971). Thus in hancednumber and size ofovules. In thePyrolaceae, this thePyrolaceae, we suggestthat nectar secretion is an an- was observedas an increasein bothovule numberand cientattractant and thatbuzz-pollination is an advanced seed size. Thus the usual size-numbertrade-off as our system.In a studyof Boraginaceae,Dukas and Dafni data indicatemight not be expected. (1990) presentedthe same scenario. Henderson(1919) recognizeda gradualincrease in sap- rophytismand a decreasein amount of wood formed from Ericaceaevia Chimaphila,Orthilia, and furtherto Pyrola LITERATURE CITED and Moneses,and finallyto Monotropaceae.Based on that,she suggested that Chimaphila is themost primitive AN1DERSEN,A. 1943. Pyrolaceernesog Plumbaginaceernesudbredelse memberof the Pyrolaceae. Based on anatomyand form, i Danmark. Botanisk Tidsskrift47: 123-142. Copeland(1947) consideredChimaphila and Orthiliato ANDREs,H. 1910. Die Pirolaceen des Rheinischen Schiefergebirges, der angrenzendenTieflainder des Rheins und des Mainzer Beckens. be primitivein manyrespects compared to theremaining Verhandlungendes NaturhistorischenVereins der preussischen generaof thefamily. However, he regardedChimaphila Rheinlandeund Westfalens,Bonn 66 (1909): 99-151. to be morespecialized than Orthilia. Generally, polyads BAKER, H. G.,AND I. BAKER. 1979a. Starchin angiosperm pollen grains areregarded as moreadvanced than tetrads, but according and its evolutionarysignificance. American Journal of Botany 66: to Walkerand Doyle (1975) some linesof polyadsmay 591-600. secondarilyhave givenrise to monads.Thus the monads ,1AND .1979b. Sugar ratios in nectar. Phytochemical Bulletin 12: 43-45. ofOrthilia may be derivedfrom either polyads or tetrads, , AND . 1983. Some evolutionaryand taxonomic impli- and thefew dyads found support this. Psilate sculpturing cations of variation in the chemical reserves of pollen. In D. L. as foundin C. umbellatais generallyregarded as a prim- Mulcahy and E. Ottaviano [eds.], Pollen: biology and implication itivefeature (Walker and Doyle, 1975). Movingtoward forplant breeding,43-52. Elsevier Scientific,New York, NY. a morespecific pollinator group by developingporicidal , AND . 1990. The predictivevalue of nectarchemistry anthersmay be a prerequisiteto anyevolution of tetrads to the recognitionof pollinatortypes. Israel Journalof Botany 39: 157-L66. and polyads. BERTIN, R. I. 1988. Paternityin plants. In J. Lovett Doust and L. Amongthe buzz-pollinatedspecies studied, Moneses Lovett Doust [eds.], Plant reproductiveecology. Patterns and strat- unifloraand Pyrolaminor may have a morederived pol- egies, 30-59. Oxford UniversityPress, New York, NY. August 19931 KNUDSEN AND OLESEN-SEXUAL PATTERNS AND POLLINATION 913

BOCHER,T. W. 1961. Studies in Pyrolaceae-two interestingwinter- KNUTH, P. 1905. Handbuch der Bliitenbiologie.W. Engelmann,Leip- greensfrom West Greenland. Botanisk Tidsskrift57: 28-37. zig. BOLWIG,N. 1938. Nogle eksperimentelleUnders0gelser over den LOVELL,H. B., AND J. H. LOVELL. 1936. Pollination-ofthe : Tiltrekning Blomsternes Saftmerker ud0ver paa Honningbien. IV. Ledumand Pyrola.Rhodora 38: 90-94. Sa?rtryk,Entomologiske Meddelelser 20. MACIOR, L. W. 1971. Co-evolution of plants and animals-systematic BucHMANN,S. L. 1974. Buzz pollination of Cassia quiedondilla (Le- insightsfrom plant-insect interactions. Taxon 20: 17-28. guminosae) by bees of the genera Centrisand Melipona. Bulletin MATTSSON,0. 1968. Vejledning i mikroskopiskteknik til brug ved ofSouthern California Academy of Science 73: 171-173. 0velsernei botanisk cytologiskteknik. Institut for Plante Anatomi 1983. Buzz pollinationin angiosperms.In C. E. Jonesand R. og Cytologi,Copenhagen UniversityPress, Copenhagen. J. Little [eds.], Handbook of experimentalpollination biology, 73- MICHENER, C. D. 1962. An interestingmethod of pollen collectingby 113. Van Nostrand Reinhold, New York, NY. bees fromflowers with tubular anthers.Revista Biolog(a Tropical , AND J. H. CANE. 1989. Bees assess pollen returnswhile son- 10: 167-175. icatingSolanum flowers.Oecologia 81: 289-294. MOLLER, H. 1881. Die Alpenblumen,ihre Befruchtung durch Insekten , C. E. JoNEs,AND L. J. CoLIN. 1977. Vibratile pollination of und ihre Anpassungenan dieselben. W. Engelmann,Leipzig. Solanum douglasiiand S. xanti (Solanaceae) in southernCalifornia. OLDF1ELD, F. 1959. The pollen morphologyof some of the West Eu- WasmannJournal of Biology 35: 1-25. ropean . Pollen et Spores 1: 19-58. CANE, J. H., AND S. L. BUCHMANN. 1989. Novel pollen-harvesting POOLE,R. W., AND B. J. RATHCKE. 1979. Regularity,randomness, and behavior by thebee Protandrenamexicanorum (Hymenoptera: An- aggregationin floweringphenologies. Science 203: 470-471. drenidae). 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