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5-1991

Osmophores, Floral Features, and Systematics of ()

Kenneth J. Curry University of Southern Mississippi, [email protected]

Lorraine M. McDowell University of Florida

Walter S. Judd University of Florida, [email protected]

William Louis Stern University of Florida

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Recommended Citation Curry, K. J., McDowell, L. M., Judd, W. S., Stern, W. L. (1991). Osmophores, Floral Features, and Systematics of Stanhopea (Orchidaceae). American Journal of Botany, 78(5), 610-623. Available at: https://aquila.usm.edu/fac_pubs/9

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Osmophores, Floral Features, and Systematics of Stanhopea (Orchidaceae) Author(s): Kenneth J. Curry, Lorraine M. McDowell, Walter S. Judd, William Louis Stern Reviewed work(s): Source: American Journal of Botany, Vol. 78, No. 5 (May, 1991), pp. 610-623 Published by: Botanical Society of America Stable URL: http://www.jstor.org/stable/2445082 . Accessed: 14/11/2011 09:49

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http://www.jstor.org American Journalof Botany 78(5): 610-623. 1991.

OSMOPHORES, FLORAL FEATURES, AND SYSTEMATICS OF STANHOPEA (ORCHIDACEAE)1

KENNETH J. CURRY,2 LORRAINE M. MCDOWELL,3 WALTER S. JUDD,3 AND WILLIAM Louis STERN3,4 2Departmentof Biological Sciences, University of Southern Mississippi, Hattiesburg,Mississippi 39406-5018; and 3Departmentof Botany, University of Florida, Gainesville, Florida 32611-2009

The floralfragrance glands (osmophores) of 18 speciesof Stanhopeaand Sievekingiawere examinedthrough a seriesof developmentalstudies at lightand electronmicroscope levels includinglate bud stagesthrough postanthesis. Various characters were identified to be of potentialsystematic value and were recorded for each species. These characters included: texture ofthe osmophore surface, number of distinct cell layerscomprising the osmophore, nature of lipidinclusions in osmophorecells, and presenceor absenceof plastoglobuliin osmophore amyloplasts.These characters were combined with traditional features of floral lip morphology forcladistic analysis. was thepostulated outgroup. Stanhopea ecornuta showed the largestnumber of plesiomorphic characters. Stanhopea pulla, S. annulata,and S. candidawere onlyslightly more derived. Stanhopea anfracta, S. gibbosa,S. martiana,S. oculata,S. radiosa, S. ruckeri,S. saccata,S. shuttleworthii,S. tigrina, S. vasquezii,and S. wardiiform a monophyletic groupthat can be recognized by a labellumwith an articulated epichile and a bicornuatemesochile (or hypochile). may be a hybridbetween a speciesof Sievekingiaand Stanhopea.

StanhopeaFrost ex Hookeris thetype genus The genushas not been monographed,but of subtribeStanhopeinae which are all Neo- Dodson and Frymire(1961; hereinafterre- tropicalepiphytes pollinated exclusively by ferredto withoutyear) suggestedan evolu- male euglossinebees. Stanhopea,with about tionaryscheme (Fig. 1) thatincluded many of 50 species,is amongthe largest genera in the thespecies, and thegenus Sievekingia Reichb. subtribeand is characterizedby ribbedpseu- f. was suggestedas a possibleancestor from dobulbs,each bearing a single,plicate . The whichStanhopea was derived.Dodson (1963, inflorescenceis directeddownward from the 1975a, b) has since publishedadditional in- base of thepseudobulb. There are two to 15 formationconcerning individual species of simultaneouslyopening that last 1 or Stanhopea. Dressler(1981) reproducedthe 2 daysand are intenselyaromatic. The aroma originalevolutionary scheme (Dodson and is a combinationof terpenesand aromatics Frymire)with little basic change to incorporate (Williamsand Whitten,1983) produced by se- newdata on -pollinatorrelationships. In cretorytissue called the osmophore(Vogel, a recentpersonal communication, Dodson in- 1963). dicatedthe placement in his originalscheme Pollinationof plantsin thissubtribe is un- of some newlydescribed species. usual in thatthere is no foodreward for the Flowersof Sievekingiaare less specialized pollinator.Male euglossinebees are attracted thaneven the least specialized of Stan- to plantsby thefragrance produced in theos- hopea.A labellummay be distinguishedfrom mophore.Apparently, visual cues are second- the othertwo petalsby its morphologyand ary. collectthe fragrance which they use thepresence of a callus,but it is notsufficiently presumablyas a precursorfor a sexpheromone differentiatedto definea hypochile,meso- (Dressler,1982). chile,or epichile,as is generallytrue in Stan- hopea.The proximalportion of thelabellum 1Received for publication 13 July1990; revisionac- behindthe callus forms a shallowpouch rem- cepted3 January1991. iniscentof the pouch in thehypochile of Stan- The authorsthank Henry C. Aldrichfor use ofthe Elec- hopea.The floorof this shallow pouch in the tronMicroscope Core Laboratory, University of Florida, labellumcontains the osmophoreof Sievek- andVicki A. Funk,Smithsonian Institution, for her review (Fig.-2). whichhelped us avoidcertain embarrassing discrepancies. ingia This studywas supportedin partby NSF grantsDEB 82- Classificationof Stanhopea is based almost 19120and BSR-8607212to WLS. exclusivelyon characteristicsof the labellum. 4Author forcorrespondence. In some speciesthe labellum has become so 610 May 1991] CURRY ET AL. -OSMOPHORES AND SYSTEMATICS IN STANHOPEA 611

St. oculata St. tigrina St. martiana

i St. bucephalus St. saccata

St. wardii

St insignis

- - - - ~~~~~~St.grandiflora St. rodrigasiana St. connata /

St. Iewisae

( - ~~St. reichenbachiana St. cirrhata St. tricornis /

St. ecornuta

\\ ~~I _ I - __-~- St. pulla

Si. trollii Si. suavis

Si. shepheardii

Fig.1. RepresentationofDodson and Frymire's (1961) diagram of labella and columns of Stanhopea and Sievekingia suggestingrelationships among the species. specializedthat it may be divided forcon- withpollination. The labellumhas becomea venienceof reference and discussion into three centralfocus for Stanhopea because parts:the proximalhypochile that includes itsvariability and structuralcomplexity allow theosmophore, the central mesochile (which theidentification of numerousfairly constant may be indistinguishablein some speciesof charactersthat may be used in speciesdelim- Stanhopea),and thedistal epichile. The more itation. specializedlabella have a mesochilebearing We considerthe evolutionary scheme (Fig. a pair of forward-projectinghorns. The epi- 1) proposedby Dodson and Frymire, including chileis articulatedto themesochile when that thepossible ancestral relationship with Sieve- structureis present.Petals of Stanhopea flow- kingia,to be a usefulworking hypothesis. Al- ers (excludingthe labellum)are stronglyre- thoughthey did not addressthe issue, their flexedin all butone species.The columnarch- evolutionaryscheme, if accepted,would lead es parallelalong the labellum, and its terminal to the considerationof Sievekingiaas likely antherlies in close proximityto theepichile. paraphyletic,with Stanhopea being the sister Dodson and Frymiresuggested that floral groupof a species(or clade) within Sievekingia. charactersintimately associated with pollina- The questionof themonophyly or paraphyly tionwould be understrong selective pressure, of Sievekingiais not addressedin our study, whereascharacters less importantto pollina- and wouldrequire the inclusion of additional tionwould be morevariable within a species. speciesof this genus, as wellas relatedgenera, Theycited high variability in coloras an ex- in thecladistic analyses. We have examined, ampleof a characternot intimately associated throughdevelopmental studies, the osmo- 612 AMERICAN JOURNAL OF BOTANY [Vol. 78

I ~~~~~~~~~~~St.vasquezii St. oculata

St. tigrina

St. tricornis .

\\ /t X St. saccata

. e 5S I ' / pulla

Si. suavis Si. marsupialis

Fig. 2. Representativelabella in top and sagittalviews organized to depictour approximation of the evolutionary schemeproposed by Dodson and Frymire(1961). Sievekingia suavis is postulatedas closeto theancestor of Stanhopea. Stanhopeapulla typifiesprimitive members of thegenus. and S. tigrinarepresent the "insignis" complex,and Stanhopeaoculata and S. vasqueziirepresent the "oculata" complex. Stanhopea tricornis is thoughtto be a hybridbetween Stanhopea and Sievekingia.Top and sagittalviews of labella for each speciesare drawnto scale; however,labella of the different species are notreproduced proportionately. Si = Sievekingia;St = Stanhopea. phoresof many species of Stanhopea and two MATERIALS AND METHODS species of Sievekingia.Herein we describe characteristicsof osmophoresof these species Plantsof Stanhopea and Sievekingiaused in and applya cladisticanalysis to thecombined thisstudy are maintainedin thegreenhouses osmophoreand otherfloral characteristics to of the Departmentof Botany,University of clarifyfurther phylogenetic relationships with- Florida.The speciesrepresented by these in Stanhopea,and to testDodson and.Fry- werethe only ones available to us and forthis mire'soriginal scheme. reasonwe wereunable to studyall thoseused May 1991] CURRY ET AL. -OSMOPHORES AND SYSTEMATICS IN STANHOPEA 613

TABLE 1. Characterstates of taxa usedin cladisticanalysis of Stanhopea and Sievekingiaa

Species lb 2 3 4 5 6 7 8 9 10 11 12 13 1.S. annulata 1 0 0 0 1 0 1 1 1 0 0 0 0 2. S. ecornuta 0 0 0 0 1 0 1 1 1 0 0 0 0 3. S. pulla 0 0 0 0 1 1 1 1 1 0 0 0 0 4. S. candida 1 0 0 0 1 1 1 1 1 0 0 0 0 5. S. tricornis 1 0 1 0 0 1 1 0 1 0 1 1 0 6. S. martiana 1 1 1 0 0 0 1 1 1 0 1 1 1 7. S. tigrina 1 0 0 0 0 0 1 1 1 1 1 1 1 8. S. radiosa 1 1 1 1 0 0 1 1 1 0 1 1 1 8. S. saccata 1 1 1 1 0 0 1 1 1 0 1 1 1 9. S. gibbosa 1 0 0 0 1 1 0 1 1 1 1 1 0 1. S. vasquezii 0 0 1 0 0 0 0 1 1 1 1 1 0 11. S. anfracta 1 0 0 0 0 0 0 1 1 1 1 1 0 11. S. oculata 1 0 0 0 0 0 0 1 1 1 1 1 0 11. S. ruckeri 1 0 0 0 0 0 0 1 1 1 1 1 0 11. S. shuttle. 1 0 0 0 0 0 0 1 1 1 1 1 0 11. S. wardii 1 0 0 0 0 0 0 1 1 1 1 1 0 12. Sieve. mars. 0 0 1 0 0 0 0 0 0 n/a 0 0 0 12. Sieve. suavis 0 0 0 0 0 0 0 0 0 n/a 0 0 0 a Species 8 and 11 are members of the "insignis" and "oculata" groups,respectively. They are treatedas units for cladisticpurposes because theyare identicalfor the characterset we used. S. shuttle.= S. shuttleworthii.Sieve. mars.= Sievekingiamarsupialis. See textfor explanation of plesiomorphic(scored 0) and apomorphic(scored 1) conditions. b Numbersheading columns pertain to similarlynumbered characters polarized in Table 2. by Dodson and Frymirein theirscheme (Fig. inSato's or Reynolds' lead citrate (Hayat, 1981). 1). However,we studiedother species not con- Dehydratedmaterial for scanning electron mi- sideredby them. In mostcases, several plants croscopy(SEM) was critical-pointdried and ofeach specieswere examined. Abbreviations gold-coated. ofnames for authors of binomials follow those recommendedby Meikle(1980). Cladistics-A preliminaryanalysis of the cladisticrelationships of selectedspecies of Microscopy-Tissuesfor studywere sam- Stanhopeaand Sievekingiawas conductedus- pled,where possible, at severaldevelopmental ing the branch-and-boundalgorithm (Hendy stagesof the beginning with a bud and Penny,1982) as employedin the PAUP stagethe day before the flowers open and pro- (PhylogeneticAnalysis Using Parsimony, Ver- ceedingthrough postanthesis. Flowers reached sion 2.4) computerprogram developed by postanthesisin 1 to 3 daysdepending on spe- Swofford(1985). cies. Twelvetaxa were used in the analyses (Table All osmophoretissue was fixedfor 2 hr in 1), based on a surveyof morphologicaland 2% (w/v)formaldehyde (freshly made from ultrastructuralvariation in 18 speciesof Stan- paraformaldehyde),2.5% (v/v) glutaralde- hopeaand Sievekingia.Species showing iden- hyde,and 2 mm CaCl2 in 0.1 M cacodylate ticalcharacter states for the subset of characters buffermade to pH 7.2. Materialwas thenbuff- we used weregrouped into operationaltaxa. er-rinsed(0.1 M cacodylate,pH 7.2), postfixed Thus,Stanhopea radiosa Lemaire and S. sac- for45 min in 1% (w/v)osmium tetroxide in cata Bateman,part of Dodson and Frymire's 0.1 M cacodylatebuffer (pH 7.2), buffer-rinsed"insignis" group,and Stanhopea anfracta (0.1 M cacodylate,pH 7.2), and dehydrated Rolfe,S. oculata(C. Lodd.) Lindley,S. ruckeri throughan ethanolseries (50%, 70%, 85%, 95%, Lindley,S. shuttleworthiiReichb. f., and S. 100%) followedby 100% acetone. wardiiC. Lodd. ex Lindley,included in Dod- Tissuesfor light microscopy (LM) andtrans- sonand Frymire's"oculata" group, were com- missionelectron microscopy (TEM) wereem- binedinto two respective groups. beddedin epoxy resin ERL 4206 (Spurr,1969). Thirteencharacters were delimited (Table 1) Materialfor light microscopy was thick-sec- and assignedplesiomorphic (ancestral; scored tioned(ca 1 gAm)and stainedwith 0.1% (w/v) as 0) and apomorphic(derived; scored as 1) toluidineblue 0 in 1% (w/v)sodium borate. states.Plesiomorphic and apomorphic features Materialfor TEM was thin-sectioned(ca 90- usedin thecomputer analysis are listedin Ta- 100 nm),stained 12-30 min in 1% (w/v)aque- ble 2, and theirassigned numbers correspond ous uranylacetate, and poststained7-10 min withthose heading the columnsin Table 1. 614 AMERICAN JOURNAL OF BOTANY [Vol. 78

TABLE 2. Charactersused in cladisticanalysis of Stan- andMcDowell (1 9 8 8). We viewthe acquisition hopeaand Sievekingia ofpapillae and rugaeas a singlephenomenon involvingclusters of cells and have made no Plesiomorphic(ancestral) Apomorphic (derived) distinctionbetween papillae and rugaeand the 1. Osmophore surface Osmophoresurface ru- relativedegree of surfaceconvolution. smooth,individual cells gose/papillate 2. These trichomesare not consideredho- bullate 2. Osmophoresurface Osmophoresurface with mologous with the large, multicellulartri- lackingunicellular tri- unicellulartrichomes chomesthat may be foundon theosmophore chomes surfaceof Sievekingia marsupialis (a probable 3. Epidermalcells of os- Epidermalcells of osmo- autapomorphy). mophoreanatomically phoreanatomically dif- 3. See relateddiscussion in Curry,Stern, similarto subjacent ferentfrom subjacent and McDowell (1988). Because Sievekingia cells;tissue homoge- cells;tissue bilayered neous suavis has homogeneousosmophore layers 4. Cytoplasmiclipid in- Cytoplasmiclipid inclu- whilethose in S. marsupialisare bilayered, this clusionsof one pat- sionsof two patterns, featurewas not polarized in the computer anal- tern,either uniformly someuniformly elec- yses.The polarityassessment given in Table electrondense or mac- trondense and others 2 is based on the mostparsimonious distri- ulatea witha darkperipheral ofcharacter states suggested by the dis- halo surroundinga less bution electron-densecenter coveredcladograms. 5. Cytoplasmiclipid in- Cytoplasmiclipid inclu- 7. Delimitationof the statesof thisquan- clusionsspherical only sionsspherical to highly titativecharacter is somewhatarbitrary. See (eitherall electron irregular(all maculate) Almeidaand Bisby(1984) fora discussionof denseor all witha problemsin delimitingstates in continuously darkperipheral halo varyingmeasurement data. surroundinga less densecenter) 10. SinceSievekingia lacks a distincthypo- 6. Plastoglobulipresent Plastoglobuliabsent chilethe ancestral state was scoredas "miss- 7. Inflorescenceswith 4 Inflorescenceswith 1 to 3 ing" and thecharacter was consideredunpo- or moreflowers flowers larizedin thecladistic analyses. The polarity 8. Petalserect Petalsreflexed presentedhere is based on the most parsi- 9. Distincthypochile Distincthypochile present moniousdistribution of characterstates sug- lacking 10. Hypochileglobose Hypochilerectangular gestedby thediscovered cladograms. 11. Labellumecornuate Labellumbicornuate 13. The singleapiculation of the distal mar- 12. Articulatedepichile Articulatedepichile pres- gin of the labellumof Sievekingiais compa- absent ent rable to the singleapiculation on the distal 13. Apexof labellum uni- Apexof labellum tri-apic- marginof the epichile of some species of Stan- apiculate - ulate hopea,and thesestructures are consideredto a Maculate:spots of electron-dense matter in a lesselec- be homologous.The numberof apiculations tron-densematrix. is,therefore, used in the cladistic analyses with- outregard to thepresence or absenceof a dis- Characterswere polarized by outgroup analysis tinctepichile. (Stevens,1980, 1981; Wheeler,1981; Wiley, 1981; Maddison,Donoghue, and Maddison, RESULTS 1984) usingSievekingia suavis Reichb. f. and S. marsupialisDodson. Two characters(num- Developmentalstudies of osmophoresof bers 3 and 10 in Table 1) are eithervariable Stanhopeaand Sievekingialisted in Table 1 ornot applicable in these species and are there- werepursued wherever possible to establish foreunpolarized in thecladistic analyses. Au- thesequence of cellular events in thissecretory tapomorphicand uniformcharacters were not tissue.Each studyconsisted of a dailysample includedin the analysisbecause theydo not of tissuefrom a singleflower from a late bud elucidatephylogenetic relationships. Howev- stagethrough postanthesis. The plantsusually er,character 4 (Table 1) was includedbecause bloomannually and produce only a smallnum- thisfeature is a synapomorphyfor S. saccata ber of flowers,so multipleplants of a species and S. radiosa(the two species comprising the were used to establisha completedevelop- "insignis"group). The charactersused to an- mentalsequence. Stages of development were alyzecladistic relationships of Stanhopeaand corroboratedwith additionalfloral samples Sievekingiaare listedin Table 2 followedby wherepossible, but, in somecases, a complete explanatorynotes in numericalsequence re- studywas notpossible. Data reportedin Table latingto our interpretationsof features. 1 are,to everyextent possible, representative 1. See relateddiscussions in Curry,Stem, ofosmophore tissue in a flowerduring anthesis. May 1991] CURRY ET AL. -OSMOPHORES AND SYSTEMATICS IN STANHOPEA 615

Tissuein eachstudy was examinedby LM and Sievekingiaspecies. Cytoplasmic lipid inclu- by bothSEM and TEM. sionsand plastoglobuliin amyloplastsare two Selectedmorphological characters used by featurespresumably associated with fragrance Dodson and Frymireto establishtheir sug- production(Curry, 1987; Stem, Curry,and gestedphylogenetic relationships within the Pridgeon,1987; Curry,Stem, and McDowell, genusStanhopea, and betweenStanhopea and 1988) thatshowed sufficient variation for in- Sievekingia,were examined in our specimens clusionhere as potentiallyuseful characters. and are reportedin Table 1. Labellummor- Plastoglobuliare present in theamyloplasts of phologyis shownin Fig. 2 and is organizedto bothspecies of Sievekingia and most,but not approximatethe evolutionary scheme (Fig. 1) all, species of Stanhopea(Figs. 13, 15, 16). ofDodson and Frymire.Data fornumbers of Lipid inclusionswere observed in unstained flowersper inflorescenceare fromour obser- sectionsto verifythat they were osmiophilic vations and frompreviously published ac- and,therefore, probably lipid, rather than some countsand descriptions(e.g., Dodson, 1963, othercell inclusionthat accepted the uranyl 1975a,b; Dodson and Frymire). acetateor lead citratepoststains. Three differ- ent variationsof lipid inclusionswere ob- Osmophorecharacters-The osmophore of served.Some of the inclusionstended to be Stanhopeais locatedin a pouchat the proximal sphericalto somewhatirregular and uniformly endof the labellum as illustratedin Stem,Cur- electrondense (Figs. 13, 16). Stanhopeasac- ry,and Pridgeon (1987), Curry, Stem, and Mc- cata (Fig. 15) and S. radiosahave, in addition Dowell (1988), and as shownat thearrow in to theaforementioned lipid type, another lipid Fig. 2. The osmophoreof Sievekingiais also typecharacterized by a darkring surrounding locatedat the proximalend of the labellum, a lesselectron-dense center. Both of these lipid butthere is no sharplydefined pouch. The os- typesare present in eachosmophore cell of the mophoresurface was examinedin each spec- above-mentionedspecies. A thirdvariation of imenusing SEM. Sievekingiasuavis and sev- lipidinclusion consists of structures that range eral speciesof Stanhopeahave flatto bullate fromspherical to highlyirregular. These occur osmophoresurfaces (Figs. 3, 4). Sievekingia independentlyof thetwo previously described marsupialishas a flatsurface with occasional types,and are characterizedby smallpatches multicellulartrichomes. Some speciesof Stan- of electron-densematerial in a less electron- hopea have osmophoresurfaces with multi- densestructure (Fig. 14). We referto thiscon- cellularpapillae (Fig. 5). The remainingspecies ditionas maculate. have an osmophoresurface convoluted by ru- gae and papillae(Fig. 6). Some epidermalcells Cladistics-Initialcomputer runs using the on the papillae of S. saccata (Fig. 7) and S. entiredata set forthe 12 taxa resultedin the radiosa formdistinct, unicellular trichomes. discoveryof 100+ equallyparsimonious trees Comparable, but smaller, unicellular tri- (butonly 18 differenttopologies when multi- chomesare present on therugae of S. martiana furcationsare takeninto account),all witha Batemanex Lindley(Fig. 8). lengthof 23 stepsand a consistencyindex of Allosmophores of Stanhopea and Sieveking- 0.565. Thesecladograms are diversein topol- ia were identifiedas secretorytissue by the ogy,and the strict consensus tree resulting from presenceof cells with dense cytoplasm (Schnepf, thisinitial analysis is merelya largepolytomy, 1969;Fahn, 1979) and numerous starch grains. indicatingonly that the "insignis"group (S. Secretorytissue grades imperceptiblyinto saccataand S. radiosa)is mostclosely related groundparenchyma. Examination of the anat- to S. martiana,based on thepresence of uni- omyof osmophore tissue in sectionat thelight cellularhairs on theosmophore and three teeth microscopelevel indicatedthat some osmo- at the apex of the labellumin thesespecies, phoreshave epidermalcells that are morpho- andthat S. tricornisLindley may link with this logicallydifferent from the subjacent cells (Figs. cladeowing to itsbilayered osmophore tissue. 9, 10). In otherspecies the osmophorecom- In 14 trees,S. ecornutaLemaire, S. pulla prisedhomogeneous layers of cells (Figs. 11, Reichb.f., S. annulataMansf., and S. candida 12). Barb.Rodr. comprise a paraphyleticor mono- We have previouslyexamined the ultra- phyleticgroup near the base of the tree(so structureof osmophorecells in Stanhopeain placedbecause these species lack bothmeso- somedetail (Stem, Curry, and Pridgeon,1987; chilehorns and an articulatedepichile), while Curry,Stem, and McDowell, 1988) and have infour trees these species form a monophyletic continuedour investigationsfor this study. groupnear the apex (based on globosehypo- Thereis considerableuniformity ofultrastruc- chile shape, a loss ofmesochile horns, and loss turein osmophorecells of both Stanhopea and of an articulatedepichile). The reversalof an 616 AMERICAN JOURNAL OF BOTANY [Vol. 78

4p ~ ~ ~ ~ 4-

WO ~ ~~~~N

--7 A-

-~ 1.~ ~ - -Ok- May 1991] CURRY ET AL. -OSMOPHORES AND SYSTEMATICS IN STANHOPEA 617 articulatedepichile to a nonarticulatedepichile hairson theosmophore surface (2) and(in some (witha loss of hornson labellum)does not trees,Fig. 17) by the additionalfeature of a seem to be structurallyor biologicallymean- labellumapex with three small apiculations (13). ingful.Thus, an additionalcladistic analysis This unicellular-haired-osmophoreclade then was conductedin whichcharacter 12 (articu- linkswith S. tricornison thebasis of a bilayered latedepichile) was weighted(by two) in order osmophoretissue (3), althoughit is notedthat to inhibithomoplasy in thisdevelopmentally this character is homoplasious(see S. vasquezii, complexfeature. The secondanalysis gener- Fig. 17). In fourcladograms, the clade char- ated 84 equallyparsimonious trees (but com- acterizedby bilayered osmophore tissue is po- prisingonly 14 differenttree topologies) Qf 24 sitionedas thesister group of S. tigrina,the two stepsand witha consistencyindex of 0.583. beinglinked by the synapomorphy ofa labellum The strictconsensus tree resulting from this apex withthree small apiculations (13). How- analysisalong with a representativecladogram ever,in the remaining trees S. tigrinais isolated showingdistribution of character states is pre- or linkedwith S. vasquezii,S. gibbosa,and sentedin Figs. 17, 18. membersof the "oculata" group, because of its All treesdiscovered in the secondcladistic rectangularhypochile (10). analysissupport the hypothesis that Stanhopea is monophyletic,based on thesynapomorphies DISCUSSION ofthe presence of a distincthypochile (9) and flowerswith reflexed petals (8). The latterchar- We haveselected as thebasic framework for actershows a reversalin S. tricornis.In all ourdiscussion of the systematics of the genus cladograms,S. pulla,S. ecornuta,S. annulata, Stanhopea,the scheme(Fig. 1) publishedby and S. candidaare positionednear the base; Dodson and Frymire,and have comparedour thesespecies show many plesiomorphic char- cladogramswith this influential classification. acters.In addition,in most cladograms,S. Severalspecies of Stanhopeathat can be in- ecornutais cladisticallybasal, a positionsup- sertedat appropriatepoints in the original portedby its smoothosmophore surface (as schemehave been describedsince that publi- well as its lack of otherdistinctive apomor- cation(Dressler, 1981; Dodson, personal com- phies;Figs. 17, 18); mostspecies of Stanhopea munication).Dodson and Frymire'sscheme includedin the cladistic analysis (Fig. 17)likely includestwo named complexes of species, two forma monophyleticgroup on thebasis of the unnamedcomplexes, and severalindividuals synapomorphyof a rugose/papillateosmo- withunusual characteristics that set them apart phoresurface (1), althoughthe osmophore sur- fromany complex. As mentionedabove, Sieve- facehas revertedto the smoothcondition in kingiais consideredby Dodson and Frymire S. vasqueziiDodson. (Fig. 1) as theancestor of Stanhopea. Stanhopeatigrina Bateman ex Lindley,S. Accordingto Dodson and Frymire,only martiana,S. tricornis,S. vasquezii,S. gibbosa those partsof the flowerdirectly associated Reichb.f., and membersof the "insignis" com- withpollination will be understrong selective plex(S. saccataand S. radiosa)and the"ocu- pressure.Other parts of theflower can be ex- lata"group (S. wardii,S. shuttleworthii,S. ruck- pectedto showmore variation than those parts eri, S. oculata,and S. anfracta)all forma intimatelyassociated with pollination. Evo- monophyleticcluster, which can be recognized lutionarypressure on selectedparts of orchid bythe labellum with an articulatedepichile (12) flowershas also been cited by Ackerman (1983). and a bicornuatemesochile (or hypochile;11). In Stanhopea,the labellum and itsosmophore Thesecharacters are morphologically (and like- are closelyassociated with the attraction and lydevelopmentally) complex. Species relation- orientationof the pollinatorbringing that shipswithin this group are poorlyresolved as structureunder strong selective pressure. The canbe seenin the consensus tree (Fig. 18). How- labellumhas been the majorfeature used by ever,the two species of the "insignis" group are Dodsonand Frymire in constructingtheir evo- linkedby the presence of cytoplasmic lipid in- lutionaryscheme for Stanhopea. Vegetative clusionsof two types (4), and bothspecies then characteristics,since they are so uniform,have linkto S. martianaon thebasis of unicellular notbeen used.

Figs.3-8. Scanningelectron micrographs of osmophoresurfaces of speciesof Stanhopeaand Sievekingia.Bar = 100,um. 3. Flatto bullatesurface of Sievekingia suavis. 4. Flatto bullatesurface of Stanhopea pulla. 5. Papillatesurface of Stanhopeacandida. 6. Papillateto rugatesurface of Stanhopeatigrina. 7. Papillaewith unicellular trichomes of Stanhopeasaccata. 8. Papillaewith short, unicellular trichomes of Stanhopea martiana. 618 AMERICAN JOURNAL OF BOTANY [Vol. 78

9 10

,a

Figs.9-12. Lightmicrographs of osmophoresof Sievekingia and Stanhopea. Bar = 100,um. 9. Bilayeredosmophore of Stanhopeatigrina. 10. Bilayeredosmophore of Stanhopeaoculata. 11. Homogeneousosmophore of Sievekingia suavis.12. Homogeneousosmophore of Stanhopea pulla.

Dodson and Frymirepostulated a groupof study);the "insignis"complex including S. primitivestanhopeas derived from a species insignisFrost ex Hook.,S. tigrina,S. saccata, ofSievekingia, S. shepheardiiRolfe being the S. radiosa,and S. martiana;and the "oculata" mostprimitive known species with respect to complexwith S. oculata, S. wardii,S. vas- the slightlylobed, truncatelip. Sievekingia quezii,S. gibbosa,S. shuttleworthii,S. ruckeri, suavisReichb. f., and S. peruvianaRolfe have and S. anfracta. more complexlips thanS. shepheardiiwith Stanhopeatricornis, an isolatedspecies in erectside lobes forminga concavehypochile the Dodson/Frymirescheme, shares some at the base. From these species,"the close characteristicswith members ofthe "insignis" relationshipsof the more primitiveStanho- complexexcept that its petals extend forward peas are obvious," they attested.Figure 1 insteadof beingreflexed, and thehorns arise shows theirscheme clearlywith Stanhopea fromthe hypochile instead of themesochile. cirrhataLindley and S. lewisaeAmes & Cor- This is the only species of Stanhopea with rell(neither species of which was availableto anteriorlyextended petals, and, because the us forstudy) linking the threemore derived petalsin thisspecies have a directinfluence complexes: S. reichenbachiana Roezl ex on themanner in whichpollination is accom- Reichb.f., S. grandiflora(C. Lodd.) Lindley, plished,Dodson and Frymiredid not assign and S. candida (the onlyspecies we had for S. tricornisto one of theirspecies clusters. May 1991] CURRY ET AL. -OSMOPHORES AND SYSTEMATICS IN STANHOPEA 619

az

Figs. 13-16. Osmophorecells of Sievekingiasuavis and speciesof Stanhopea.a = amyloplast,1 = lipidglobule, arrow= plastoglobulus.Bar = 0.1 Mm.13. Sievekingiasuavis showing plastoglobuli and uniformlydense, spherical lipidinclusions. 14. Stanhopeapulla showingmaculate, irregular lipid inclusions. Plastoglobuli were not observed. 15. Stanhopeasaccata showing plastoglobuli and twodifferent lipid inclusions (1 = darkperiphery surrounding less dense center;1' =uniformlyelectron dense). 16. Stanhopeaoculata showing plastoglobuli and uniformlydense, spherical lipidinclusions.

Osmophorecharacters -The relativelyflat the cladisticinvestigation to assess theirtax- osmophoresurface found in Sievekingiaand onomicutility. in some stanhopeasoffers less area for fra- We attemptedto draw parallelsbetween grancedispersal than the more convoluted sur- publisheddata on individualcomponents of facescharacterized by papillae, rugae, and tri- fragrances(Williams and Whitten,1983) and chomesobserved in flowers ofthe more derived ourcharacter set, but without success, and agree stanhopeas.Anatomical differences between withthe comments by Williamsand Whitten epidermalcells and subjacentcells mayhave thatmore detailed information is neededbe- involvedgeneral cell shape or the modification forethis line of inquiry will prove fruitful. We of the surfacewall to forma trichome.The suggestthat the morphological distinctions we functionalsignificance of trichomesis uncer- have notedin lipidinclusions represent rough tain.Previously we postulatedthat plastoglob- aggregationsof fragranceor fragranceprecur- uli and cytoplasmiclipid inclusionswere as- sors.No specificcorrelations can be made be- sociated with fragranceproduction (Curry, tween lipid inclusionsand individual fra- 1987;Stem, Curry, and Pridgeon, 1987; Curry, grancesor groupsof fragrances.As withlipid Stem,and McDowell,1988). The quantityand inclusions,we viewthe presence or absenceof qualityof fragranceproduced is directlyand plastoglobulias a crudemeasure of some aspect stronglyrelated to the effectivenessof polli- offragrance production. The maindifficulty in nation.All thesecharacters were included in assessingthis character is in provingthe ab- 620 AMERICAN JOURNAL OF BOTANY [Vol. 78

41~~~~~~~~~~

13 6~~~~~~~~~~ Va~~~~~~~~~~ 8*X *1e 7* 66

* REVERSAL

Fig. 17. Representativetree resulting from analysis in whichcharacter 12 was weighted(see discussionin text). Taxa indicatedby specific epithet (or nameof species complex); characters as in Tables 1, 2, and text. sence of plastoglobuli.Plastoglobuli may be fewflowers may enhancethe long-rangeat- producedinfrequently in the amyloplastsof tractionof pollinatorsthrough increased fra- cellswhere we reportno production.Thus, our granceproduction. Species of Stanhopea in our data mayrepresent relative production rather greenhousegenerally produced flowers in pairs thanabsolute production. or in clustersof five or more.We dividedthe speciesinto those that produce three or fewer Otherfloral characters-Visual cues are ap- flowersper inflorescenceand thosethat pro- parentlyunimportant in thepollination system duce fouror moreper inflorescence.The an- of Stanhopea(Dodson and Frymire),so the cestralstate in Sievekingiaseems to be thepro- massingof flowers for visual display may like- ductionof a largenumber of flowersleading wisebe consideredunimportant. However, the to a reductionin floralnumber in ancestral possessionof with numerous vs. stanhopeas.The more elaboratepouch in

Fig.18. Strictconsensus tree of 84 equallyparsimonious 24-step cladograms discovered in above-mentioned analysis. May 1991] CURRY ET AL. -OSMOPHORES AND SYSTEMATICS IN STANHOPEA 621

"primitive"stanhopeas probably increases of the osmophoreare bilayeredor homoge- theircapacity for fragrance production over neous. The globosehypochile in conjunction thatin Sievekingia.After these events, selec- withmesochile horns is alwaysassociated with tive pressureseems to have favoredthe de- a bilayeredosmophore, and a rectangularhy- velopmentof more elaborate osmophores and, pochileis associatedwith a homogeneousos- in some species,decreased floral number. mophorein everyspecimen examined except One ofthe most obvious advances from the S. vasquezii.Curiously, S. vasqueziiis theonly ancestralstanhopeas is thedevelopment of a memberof the "oculata" complexof species homedmesochile and an articulatedepichile. thathas a smoothosmophore surface. The sig- This relatesto the"slide-fall" mechanism by nificanceof these observationsremains ob- whichStanhopea is pollinated(Pohl, 1927).A scure. euglossinebee entersthe flowerfrom either Correlationbetween the various characters side landingon thelabellum. In thecourse of discussedabove and specificpollinators was collectingfragrance (there is no foodreward), attemptedwithout success. Too littleis known thebee apparentlybecomes disoriented by the about the pollinationof specificspecies of fragranceand fallsout of the flowerbetween Stanhopea(Williams, 1983) to derivemean- theepichile of the labellum and thetip of the ingfulcorrelations. Dressler (1981) has de- columnwhere the pollinia are located. The scribedgeneral associations in Stanhopeabe- polliniaare attached to thebee bya stickypad, tweenpollinator size and flowersize. theviscidium, as thebee exitsthe flower. The stigma,located just behind the anther,be- Cladistics-The cladogrampresented in Fig. comesreceptive to thepollen after the pollinia 17 (based on floralmorphology as wellas ul- are removedso thata secondvisit by a trastructuraland micromorphologicalfeatures carryingpollinia may resultin pollinationas ofthe osmophore) is representativeofmost of thesecond bee fallsthrough the flower leaving the discoveredtrees, and supportsthe tradi- itspollinia on thesticky stigma. tional(i.e., evolutionarytaxonomic) classifi- The developmentof mesochile horns estab- cationproposed by Dodson and Frymire.In lisheda mechanismto ensurethat bees would theirevolutionary tree, Stanhopea ecornuta and fallstraight from the fragrance-producingre- S. pullaare positioned at thebase ofthe genus, gionof the hypochile, and past the pollinia and and the specieswith horns on the labellum stigma,not out to one side,thus missing the forma monophyleticgroup (except for S. tri- pollinationtarget. The articulatedepichile, cornis).Within the group possessing mesochile whichis associatedwith mesochile horns (hy- horns,a close connectionis seen betweenS. pochilehorns in S. tricornis),acts as a spring insignis,S. martiana,and S. tigrina,on one betweenthe epichile and columntip. That dis- hand, and S. wardiiand S. oculata,on the tanceis criticalto effectivepollination. A spring other.Our cladograms,in largepart, support mechanismallows a greatdegree of tolerable these evolutionaryhypotheses. Dodson and variationin both the size of the individual Frymire's"insignis" complex may be mono- pollinatorand the distancebetween the epi- phyletic(although placement of S. tigrinais chileand columntip while still maintaining a problematic),and themembers of their "ocu- highsuccess rate in effectivepollination. lata" complex formeither a monophyletic The reflexedpetals, of course,are an inte- group(Fig. 17, supportedby ten trees)or a gratedpart of "slide-fall"pollination. Stan- paraphyleticgroup (supported by fourtrees, hopea tricornisand Sievekingiaspecies, with notshown) in our analysis. The strict consensus theirpetals directedforward, preclude entry tree(Fig. 18) indicatesthe branchingpoints intothe flower from the side. Instead, the pol- supportedby all discoveredcladograms. It is linatormust enter from the front and backout evidentthat all treessupport the monophyly of the flowerthe way it came in. Stanhopea of Stanhopea,as well as the distinctlybasal tricornishas hornsarising from the hypochile positionof S. ecornutaand S. pulla relativeto to guidea fallingbee. The distancebetween themonophyletic group of specieswith artic- thecolumn tip and theepichile is stillcritical ulatedepichiles and horns.This broadagree- to pollination,but thebee need not fallfrom mentis perhapsto be expected,since both our theflower. cladogramsand Dodson and Frymire'sevo- The shape of the hypochile(rectangular or lutionarytree employed many of the same flo- globose)and the numberof apiculationsor ral characters,and both assumedthat Siev- lobes on theepichile are arcanecharacters as ekingiais theancestor of Stanhopea. However, faras presentdata are concerned. Interestingly, it is noteworthythat several ultrastructural/ thereis a strongcorrelation in Stanhopeabe- micromorphologicalcharacters, e.g., osmop- tweenhypochile shape and whetherthe cells horesurface texture (smooth vs. rugose/papil- 622 AMERICAN JOURNAL OF BOTANY [Vol. 78 lose), osmophoreindumentum (presence vs. speciesof the articulatedepichile clade and absenceof unicellular trichomes), osmophore Sievekingia)would provide an explanationof tissuedifferentiation (homogeneous vs. bilay- thereversal to erectpetals seen in thisspecies. ered), and cytoplasmiclipid inclusions,are Positioningof the labellum horns on thehy- usefulin discerningphylogenetic relationships pochilein S. tricornismay relate to theinter- and supportgroupings established on floral playamong the divergent developmental path- morphologicalfeatures (Fig. 17). waysof the labellumin the parentalspecies. A significantdifference between the clado- Additionalbiosystematic investigations are gramspresented here (Figs. 17, 18) and Dodson clearlyneeded to clarifythis situation, but at and Frymire'stree is seen in the positionof presentwe preferthe hypothesisthat S. tri- Stanhopea tricornis.Dodson and Frymire cornisis ofhybrid ancestry. Such a hypothesis placedthis species in an isolatedposition and is muchmore parsimonious than one thatde- derivedit from ancestors similar to Sievekingia rivesthis species as a separatelineage (evolving trolliMansf. Thus, they hypothesized that its Stanhopea-likefeatures in parallel). articulatedepichile and labellumhorns origi- natedindependently from those of the other LITERATURE CITED speciesof Stanhopea.This evolutionaryhy- pothesiswould make the genusStanhopea a AcKERmAN,J.D. 1983. Specificityandmutualdependen- polyphyleticevolutionary grade. One could cyof the orchid-euglossine bee interaction.Biological evenargue that the positioning of the labellum Journalof the Linnean Society 20: 301-314. ALMEIDA,M. T. ANDF. A. BISBY. 1984. A simplemethod hornson thehypochile (instead of themeso- forestablishing taxonomic characters from measure- chile)in S. tricornismay indicate that the horns mentdata. Taxon33: 405-409. ofthis species are nothomologous with those CURRY,K. J. 1987. Initiationof terpenoidsynthesis in of otherspecies of Stanhopea,although their osmophoresof Stanhopeaanfracta (Orchidaceae). structureis very similar. However, in theirdis- AmericanJournal of Botany 74: 1332-1338. cussionthey suggest an alternativehypothesis, , W. L. STERN, AND L. M. McDowELL. 1988. Os- mophoredevelopment in Stanhopeaanfracta and S. that"S. tricornismay have been derivedas a pulla (Orchidaceae).Lindleyana 3: 212-220. resultof hybridizationbetween Sievekingia DODSON, C. H. 1963. The Mexicanstanhopeas. Amer- trolliior a similarform and some Stanhopea ican OrchidSociety Bulletin 32: 115-129. sympatricin distribution"(Dodson and Fry- * 1975a. Clarificationofsome nomenclature inthe mire). They suggestStanhopea bucephalus genusStanhopea (Orchidaceae). Selbyana 1: 46-55. Lindleyas a possibleparent and statethat "if * 1975b. OrchidsofEcuador:Stanhopea.Selbyana 1: 114-129. chancepollination of Stanhopea bucephalus by ,tANDG. P. FRYMIRE. 1961. Preliminarystudies theSievekingia should occur the resulting in- in thegenus Stanhopea (Orchidaceae). Annals of the termediatehybrid might well be visitedby a MissouriBotanical Garden 48: 137-173. pollinatornot utilizedby eitherparental spe- DRESSLER,R. L. 1981. The orchids:natural history and cies and therebybe stabilizedas a species."If classification.Harvard University Press, Cambridge, thishypothesis is accepted,then Dodson and MA. 1982. Biologyof the orchidbees (). Frymire'sevolutionary tree can be viewedas AnnualReview of Ecology and Systematics13: 373- supportingthe monophyly of Stanhopea. 394. Our cladogramssupport their second hy- FAHN,A. 1979. Secretorytissues in plants.Academic pothesis,i.e., the view thatS. tricornisis of Press,New York. hybridancestry. Stanhopea tricornis has been HAYAT,M. A. 1981. Principlesand techniques ofelectron consistentlyplaced among those species of microscopy:biological applications, 2d ed., vol. 1. UniversityPark Press, Baltimore. Stanhopeawith an articulatedepichile and a HENDY,M. D., ANDD. PENNY.1982. Branchand bound homedlabellum. This placementis strength- algorithmsto determineminimal evolutionary trees. ened by the factthat S. tricornisshows a bi- MathematicalBiosciences 59: 277-290. layeredosmophore. However, the species ex- MADDISON, W.R., M. J.DONOGHUE, AND D. R. MADDISON. hibitsa reversalfrom reflexed to erectpetals, 1984. Outgroupanalysis and parsimony.Systematic a characteristicofSievekingia, and shows other Zoology33: 83-103. MEIKLE,R. D. 1980. Draftindex of author abbreviations similaritieswith Sievekingia not includedin compiledat theHerbarium, Royal Botanic Gardens, ouranalysis but discussed by Dodson and Fry- Kew. Her Majesty'sStationery Office, London. mire. POHL,F. 1927. Die anatomischenGrundlagen fur die The consistentlinking of S. tricorniswith GleitfallenfunktionderBliuten von Stanhopeatigrina thespecies of Stanhopea possessing articulated und St. oculata.Jahrbiicher fur wissenschaftliche Bo- is not one would it tanik66: 556-577. epichiles surprising; expect SCHNEPF, E. 1969. Glandcells. In A. W. Robards[ed.], to join withthe parentpossessing the most Dynamic aspects of plant ultrastructure,331-357. apomorphiccharacters. Hypothesizing a hy- McGraw-Hill,New York. brid ancestryfor S. tricornis(i.e., betweena SpuRR,A. R. 1969. A low-viscosityepoxy resin embed- May 1991] CURRY ET AL. -OSMOPHORES AND SYSTEMATICS IN STANHOPEA 623

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