Comparative Leaf Anatomy and Systematics in Dendrobium, Sections Aporum and Rhizobium (Orchidaceae) Barbara S

Eastern Illinois University The Keep

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January 1997 Comparative leaf anatomy and systematics in Dendrobium, sections Aporum and Rhizobium (Orchidaceae) Barbara S. Carlsward Eastern Illinois University, [email protected]

William Lois Stern University of Florida

Walter S. Judd University of Florida

Terence W. Lucansky University of Florida

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Recommended Citation Carlsward, Barbara S.; Stern, William Lois; Judd, Walter S.; and Lucansky, Terence W., "Comparative leaf anatomy and systematics in Dendrobium, sections Aporum and Rhizobium (Orchidaceae)" (1997). Faculty Research & Creative Activity. 260. http://thekeep.eiu.edu/bio_fac/260

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COMPARATIVELEAF ANATOMY AND SYSTEMATICS IN DENDROBIUM, SECTIONSAPORUM AND RHIZOBIUM (ORCHIDACEAE)

BARBARA SUE CARLSWARD,* WILLIAM LOUIS STERN,I't WALTER S. JUDD,t AND TERENCE W. LUCANSKYt *Departmentof Ecology, Evolution and Marine Biology, Universityof California,Santa Barbara, California93106-9610; and tDepartmentof Botany,University of Florida, Gainesville, Florida 32611-8526

The specialized leaf anatomyfor species of Dendrobiumwithin section Aporum is similarto thatof species in section Rhizobium.In both sectionsleaves are characterizedby a unifacialor nearlyunifacial surface where the exposed surface is abaxial. However, leaves in section Rhizobiumalso featurea lacuna submergedin the mesophylland surroundedby an adaxial epidermis.In contrast,leaves in sectionAporum merely present an internalsuture that divides the leaf into bilateralhalves. These two sections of Dendrobium are hypothesizedto be sistertaxa because of synapomorphiesin theirfoliar anatomy.A cladistic analysis performedwith various anatomical charactersof the leaf demonstratesthat both groups are monophyletic.Little resolutionwas found withinthe anatomicallyand morphologicallydistinctive Aporumclade.

Introduction Indonesian islands, Papua New Guinea, Andaman Is- lands, Thailand,Burma, Laos, Vietnam,and regionsof Dendrobium Swartz, a genus withintribe Dendro- northeasternIndia and southernChina. Plants of Apo- bieae, subtribeDendrobiinae (Orchidaceae), comprises rum are characterizedby shortrhizomes that bear fas- about 900 species (Bechtel et al. 1992; Mabberley cicles of uprightor pendentslender stems,sometimes 1993) that exhibit a wide range of vegetative mor- obscured by overlapping leaves. Leaves are coria- phology and anatomy. Dendrobium is distributed ceous, equitant,and laterally flattened;they vary in throughouttropical Asia, some Pacific islands, and outlinefrom scalpelliform (fig. 1), lanceolate, and fal- Australasia (Dressler 1993). Plants may be epiphytic, cate (fig. 2), to triangular.The laminae arise from a lithophytic,or terrestrialwith slender to pseudobul- sheaththat is obliquely insertedon the stem (Kranzlin bous stems composed of one to several nodes. Leaves [1910] 1957). may be equitantor more or less widely separateddis- Leaf anatomy of members of section Aporum has tichouslyalong the stem, conduplicate,laterally flat- ex- tened,or cylindrical. not been studiedextensively and was only briefly Schlechter ([1912] 1982) is the most widely cited amined by Morris et al. (1996) in theirtreatment of authorityin the classificationof Dendrobiumbased on subtribeDendrobiinae and by Solereder and Meyer his work in the orchid floraof German New Guinea. (1930) in their survey of the vegetative anatomy of We used Kranzlin's ([1910] 1957) classification,even Orchidaceae. Solereder and Meyer reported on the thoughit is not favoredby currentworkers, because it anatomy of Orchidaceae but made no attemptto use contains a more comprehensivetreatment of section this informationsystematically. They mentionedonly Aporum Blume. Kranzlin treatsforty-nine species of one species of Aporum, Dendrobium aloifolium,the which we had ten for studyin additionto Dendrobium leaves of which were characterizedas having: (1) eq- brevimentum,more recentlydescribed by Seidenfaden uitant arrangement,unifacial orientation,and an ex- (1985). posed abaxial epidermis; (2) glandular,sunken, mul- Kranzlin divided section Aporum (as a subgenus) ticellular hairs; (3) peripheral "bast" fiber bundles intotwo subgroups,Hemiphylla and Holophylla,based below the subepidermal cell layer ("nur periphere upon the distributionof leaves along the stem. Plants Bastfaserbtindel,zumeist unter der subepidermalen in sectionHemiphylla possess leaves thatonly partially Zellschicht"; Solereder and Meyer 1930, p. 118); (4) cover the stem, whereas leaves in section Holophylla vascular bundles arranged in two symmetricalrows completelycover the stem. within the mesophyll; and (5) stegmata containing Species of sectionAporum are centeredaround Bur- spherical silica bodies. In addition,they cited, as gen- ma, but they also occur in Malaysia, throughoutthe eral characteristicsof the Dendrobium leaf, calcium- oxalate raphidesand druse-likestructures and "antho- 'Authorfor correspondenceand reprints. cyanin pigments" (anthocyanartige Farbstoffe) in Manuscript received June 1996; revised manuscriptreceived De- solutionwithin the cell vacuoles. They describedfiber cember 1996. bundles, variouslythickened walls of cells comprising

Figs.1-5 Leaves of Dendrobium section Aporum. Bars = 10 ,um.Figs. 1 and 2 show leafy stems. Fig. 1, D. leonis with scalpelliform, equitant,closely overlappingleaves; x 0.40. Fig. 2, D. acinaciformewith falcate,equitant, widely separatedleaves; x 0.5. Figs. 3-5 show cross sections of leaves. Fig. 3, D. acinaciformewith reniformsuberin-like bodies (arrow) in adaxial epidermalcells; X 1360. Fig. 4, D. distichumunder polarized light showing thickenedadaxial epidermalcell walls (arrow) impregnatedwith a suberin-likesubstance; X 1360. Fig. 5, D. acinaciformewith heterogeneousfiber bundles; x 2200.

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Table1 Table2

SOURCES OF SPECIMENS CHARACTERS USED IN CLADISTIC ANALYSIS OF SECTIONS APORUM AND RHIzOBIuM Taxon Sourcea 1. Cells surroundinghair base: thin- and thick-walled(0), thin- D. acinaciformeRoxb ...... WLS hort.FLAS walled only (1). D. aloifolium(Bi.) Rchb. f ...... K s.n. 2. Stomatal apparatus:paracytic (0), tetracyticand cyclocytic(1), D. anceps Sw...... WLS hort.FLAS mainlyparacytic with some anomocytic(2) [unordered]. D. brevimentumSeidenf ...... WLS hort.FLAS 3. Cells surroundingsubstomatal chamber: thin-walled(0), thin- D. distichum(Presl) Rchb. f...... SEL 1987-0062A and thick-walled(1). D. indivisum . K (Bi.) Miq ...... 1984-3270 4. Abaxial cuticle: thin,less than 3 pm (0); thick,more than 3.5 D. leonis (Lindl.) Rchb. f...... WLS hort.FLAS PAm(1). D. linguiformeSw ...... WLS hort. 5. Epidermal cell shape (x-s): rounded, width:length ratio less D. mannii Ridl...... K 1986-1031 than 46 (0); elongate,width: length ratio greaterthan 54 (1). D. nathanieleRchb. f...... K 1986-5692 6. Fiber bundles: absent (0), presentwith heterogeneous cell com- D. rigidumR. Br...... WLS hort.FLAS position (1). D. rosellumRidl ...... SEL 1978-0200A 7. Water-storagecells: absent (0), presentwith banded thickenings D. salaccense (Blume) Lindl.i...... WLS hort.FLAS (1), presentwith pleats (2) [unordered]. D. toressae (F M. Bailey) Dockrill...... WLS hort. 8. Distributionof stegmata: presentalong vascular bundles only (0), absent (1), presentalong vascular bundles and fiberbundles a = = WLS William Louis Stern,private collection; K Royal Bo- (2), presentalong fiberbundles only (3) [unordered]. tanic Gardens, Kew; SEL = The Marie Selby Botanical Gardens. 9. Leaf orientation:bifacial (0), primarilyabaxial epidermis ex- Abbreviationsof herbariaare according to Holmgrenet al. (1990), posed with lacuna or incipientlacuna (adaxial groove) present and those of authorsfollow Brummittand Powell (1992). (1), only abaxial epidermisexposed and no lacuna (2) [unordered]. 10. Hypodermis: absent (0), composed of thick- and thin-walled hypodermal tissue, sunken trichomes, and guard cells banded cells (1), composed of thin-walledbanded and unbanded with small lumens and thickened walls with protruding cells (2), composed of thin-walledunbanded cells (3) [unorder- outer ledges. ed]. Plants in section Rhizobium (Dendrobium) possess 11. Leaf width:length ratio: narrow,0-4 (0); wide, 43-70 (1); in- termediate,10-38 (2) [unordered]. some vegetative characteristics similar to those of Apo- 12. Mesophyll canal: absent (0), present(1). rum, such as the unifacial leaf, and it has been sug- 13. Basal leaf sheath: present(0), absent (1). gested that section Rhizobium is the sister taxon of 14. Suturewithin mesophyll: absent (0), present(1). section Aporum (Stern et al. 1994). We have used fo- 15. Suberin-likesubstance associated with adaxial epidermalcells: absent (0), presentinside the epidermalcells (1), presentin the liar anatomy and morphology to derive characters that epidermalcell walls (2) [unordered]. can be used in cladistic analyses of species within sec- 16. Major vascular bundle at leaf pole (x-s): absent (0), present(1). tion Aporum and to provide a basis for comparison with leaves of section Rhizobium. Dendrobium "has caused more problems for the ranin (Stem et al. 1994). Differentiationand dehydration taxonomist than almost any other genus of orchids." were carried out in a graduatedethanol series followed by (Bechtel et al. 1992, p. 145). Traditional taxonomic clearingin limonene(Hemo-De, FisherScientific Company). studies have utilized floral characters almost exclu- Leaf scrapingswere made followingCutler (1978), stained sively to classify taxa, and Dendrobium is no excep- with a 1% solution of safraninin 50% ethanol,dehydrated tion. This study was undertaken with the hope that with ethanol,and cleared in limonene. Surface observations additional features derived from vegetative structure of leaves were made fromthese scrapings.All sections and would help us to align putatively related groups. The scrapingswere mountedon microscope slides with Canada anatomical information will be applied to assess pos- balsam and photographedwith a Nikon HFX-DX photomi- crographicsystem. sible evolutionary relationships between sections Apo- Lengths and widthsof 10 guard-cellpairs were measured rum and Rhizobium and to assist us in the evaluation fromone leaf of each species. Because of the limitedplant of Kranzlin's classification of section Aporum. material,other observations were based on several sections Materialand methods fromone or two leaves of the same plant. We used "hypodermis" as a nonspecificterm to identifythe distinctivecell Plant materialused in this study was obtained fromthe layers beneath the epidermis in the sense of Esau (1960, Marie Selby Botanical Gardens, Royal Botanic Gardens, 1977). Centricleaf, as described by Metcalfe (1979), refers Kew, and the personal collection of William Louis Stern. to one completelycovered by an abaxial surface while the Table 1 containsa list of binomialswith authorities for plants adaxial surfacehas been completelyeliminated. In the cen- examined. Fresh plant materialwas preservedin FAA forat tricleaf, bilaterallyarranged vascular bundles are in opposite least 48 h and storedin 70% ethanol.Cross sections (50-60 pairs withxylem regions facing the median longitudinalaxis ,um) of unembedded leaves at the midpointof the lamina of the leaf section. Wall outlines of water-storagecells ap- and at the leaf sheath were made with a sliding microtome. pear pleated in sectional view and are referredto as pleated Leaves were also embedded in paraffin,and cross sections water-storagecells. Adaxial featureswere recordedfrom tis- (10 ,im) of the lamina and leaf sheath were made with the sues exposed withinthe leaf sheath. rotarymicrotome. For study,thin and thick cross sections Features of foliaranatomy and morphology(table 2) were provide a more complete, three-dimensional structural used to constructthe charactermatrix (table 3) employed in frameworkthan is possible withthin sections alone. All sec- the cladistic analysis of sectionsAporum and Rhizobium.As tions were stained with Heidenhain's hematoxylinand saf- discussed by Seidenfaden and Wood (1992), leaf morphol-

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Table3

CHARACTER MATRIX USED IN CLADISTIC ANALYSIS OF SECTIONS APORUM AND RHIZOBIUM Taxon (section) 12345 67890 12345 6 Dendrobiumsalaccense (Grastidium)...... 00000 00000 00000 0 D. linguiforme(Rhizobium) ...... 01010 02011 11100 0 D. rigidum(Rhizobium) ...... 01010 02111 10100 0 D. toressae (Rhizobium)...... 01010 01012 10100 0 D. acinaciforme(Aporum) ...... 12000 22323 10011 1 D. aloifolium(Aporum) ...... 10001 12223 20000 1 D. anceps (Aporum) ...... 12001 12223 20000 1 D. brevimentum(Aporum) ...... 10001 12223 20010 1 D. distichum(Aporum) ...... 02010 12223 20002 1 D. indivisum(Aporum) ...... 10011 12223 20010 1 D. leonis (Aporum)...... 02110 12223 20010 1 D. mannii (Aporum) ...... 12001 12223 20011 1 D. nathaniele (Aporum) ...... 12010 12223 20010 1 D. rosellum(Aporum) ...... 12001 12223 20002 1 ogy is variable withineach species of sectionAporum; there- ric, average width:length ratio = 33 ,um:50 ,um.Ad- fore we used it sparinglyin this analysis to preventmisin- axial-cells tangentially elongate, average width: terpretation of data. We did not use guard-cell pair lengthratio = 35 ,um:143 ,um. dimensionsin the cladistic analysis because theywere con- tinuous,and discretestates could not be delimited(table 4). Variabilityin stomataland trichomedensities, presumably a STOMATA. Abaxial only, paracytic in Denrobium response to environmentalconditions, precluded their use in aloifolium,D. indivisum,and D. brevimentum;mostly our analysis. We used Dendrobium salaccense, a species in paracyticwith few anomocyticin all otherspecies ex- section Grastidium,as the outgroup(Maddison et al. 1984) amined. Average stomatallengths range from 25.9 ,um followingthe analysis of Morris et al. (1996). In thatwork to 37.8 ,um;average stomatalwidths range from20.4 D. salaccense was chosen as thefunctional outgroup because ,umto 31.7 ,um(table 4). it was hypothesizedto be a paraphyleticrelative to the re- mainingtaxa in Dendrobiinae and because it exhibiteda rel- LEAF SECTION atively simple vegetative structurepresumed to be plesio- CUTICLE. Abaxial-smooth, thickin D. leonis, D. morphic. nathaniele,and D. = We used the computerprogram Hennig86 (Farris 1988) to distichum(average 3.9 ,um);mi- performa branch-and-boundanalysis (implicitenumeration, nutelyand irregularlyverrucose, thick (2.7-6.7 ,um)in ie), with extended branch swapping (bb*). All characters D. indivisum;smooth, thin for all other species (av- were unordered.ACCTRAN optimizationwas used to situ- erage = 2.8 ,um). Adaxial-smooth, thin (average = ate charactersthat were equivocally placed fromthe stand- 0.9 ,um). point of parsimony(Wiley et al. 1991). Results HAIRS. Uniseriate, bicellular, glandular, sunken, abaxial, and adaxial; cells surroundingbase typically LEAF SURFACE thin-walled,except in D. leonis and D. distichum, EPIDERMIS. Parenchyma cells thin-walled with an- where both thick- and thin-walledcells occur. Hairs ticlinal walls varying from straight-sided to curvilinear sometimesappear raised owing to exudate but are typ- for all species. Abaxial-cells more or less isodiamet- ically even with otherepidermal cell surfaces.

EPIDERMIS. Abaxial-cells thin-walled, outer Table4 walls variably thickened,polygonal, and tangentially DIMENSIONS OF GUARD-CELL PAIRS FOR SPECIES OF SECTION APORUM elongate in D. aloifolium, D. mannii, D. indivisum, Range (pUm) Mean (Arm) D. brevimentum,D. rosellum, and D. anceps; polygonal and rounded in D. acinaciforme,D. nathaniele, Taxa Length Width Length Width D. distichum, and D. leonis. Adaxial-cells thin- D. acinaciforme.... 27-31 24-31 28 26 walled, polygonal, and tangentiallyelongate, except D. aloifolium...... 23-28 19-23 26 20 in D. acinaciforme and D. indivisum,where the cells D. anceps ...... 29-33 24-31 31 28 D. brevimentum.... 27-29 21-29 28 25 are more or less isodiametric;those of D. mannii and D. distichum...... 33-43 29-34 38 32 D. acinaciforme enclose reniform,suberin-like bod- D. indivisum...... 32-37 25-31 35 29 ies (fig. 3). Cell walls of D. distichumand D. rosel- D. leonis ...... 25-28 23-28 27 25 lum are impregnated with a suberin-like substance D. mannii ...... 27-35 27-31 31 29 where the D. nathaniele...... 31-37 29-32 33 31 outer wall is thickerthan the inner (fig. D. rosellum...... 29-35 28-36 33 32 4). Suberin-like substances are absent in other species studied.

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Ftgs.6, 7 Cross sections, leaves of Dendrobium section Aporum.Bar 10 ,um.Fig. 6, D. rosellumshowing undifferentiatedmesophyll composed of water-storagecells and assimilatorycells; X 940. Fig. 7, D. rosellummesophyll with pleated water-storagecell; X 1500. a = assimilatorycell, w = water-storagecell.

STOMATA. Abaxial only: superficial, cells sur- nathaniele,D. mannii,D. indivisum,D. brevimentum, rounding substomatalchamber thin-walledand chlo- and D. leonis, water-storagecells adjacent to the mid- renchymatous,except in D. leonis, where both thin- axis or sutureare noticeablyelongated and cell walls and thick-walledchlorenchymatous cells occur. Outer are pleated (fig. 8); thereis no discerniblesuture in D. ledges prominent,inner ledges inconspicuous. anceps, D. aloifolium,D. indivisum,D. brevimentum, D. distichum,and D. rosellum,and all water-storage HYPODERMIS. Abaxial only, two- or three-layered, cells are rounded (fig. 9). Starch grains absent in all cells thin-walled,chlorenchymatous, rounded, smaller, species studied. Intercellularspaces small and numer- and more densely stainingthan those of themesophyll; ous. Raphides present in saccate idioblasts scattered intercellularspaces small and numerous. infrequentlythroughout the mesophyll.

FIBER BUNDLES. Abaxial only: heterogeneousbun- VASCULARBUNDLES. Collateral, primary phloem dles of both small, thick-walledfibers and large, thin- abaxial, primaryxylem oriented toward leaf axis. A walled fibersalternate with small homogeneous bun- suture separates symmetricallyarranged, regularly dles composed of eitherthick- or thin-walledfibers in spaced small and large vascular bundles in D. acina- all species (fig. 5). Thick- and thin-walledcells vary ciforme,D. leonis, D. nathaniele,D. mannii,D. indi- fromrounded to polygonal in both heterogeneousand visum,and D. brevimentum(fig. 8). The leaf is divided homogeneousbundles. Stegmatacontaining rough-sur- bilaterally,regardless of the presence of a suture,by faced, spherical silica bodies are associated withinner one row of vascular bundles on eitherside of the mid- and outersurfaces of heterogeneousand homogeneous line in D. brevimentum,D. aloifolium,D. anceps, D. fiberbundles in all species. distichum,D. indivisum,D. leonis, D. mannii,and D. nathaniele (fig. 9); D. acinaciforme and D. rosellum MESOPHYLL. Undifferentiated,composed of small- possess two or threerows of vascular bundles. All spe- er assimilatorycells and scattered,larger water-storage cies have a single, major vascular bundle at only one cells (fig. 6); assimilatory cells living, thin-walled, pole of the leaf section (fig. 10). Sclerenchymapre- rounded with conspicuous primarypit fields,chloren- dominatesat phloem regions of vascular bundles; it is chymatous;water-storage cells nonliving,thin-walled, poorly developed at xylem regions of the larger vas- rounded with conspicuous primarypit fields. Water- cular bundles and absent next to the xylem of the storage cells with smooth or pleated (fig. 7) walls smaller vascular bundles. Stegmata containingrough- grade in size from small to large fromthe periphery surfaced, spherical silica bodies are associated with toward the centerof the leaf. In D. acinaciforme,D. phloic sclerenchymaof the largerbundles of D. leonis,

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Figs.8, 9 Cross sections,leaves of Dendrobiumsection Aporum. Bar = 10 SLm.Fig. 8, D. leonis showingsuture (parentheses) in mesophyll surroundedby symmetricallyarranged vascular bundles (arrows); X 216. Fig. 9, D. anceps showing mesophylllacking sutureand symmetrically arrangedvascular bundles (arrows); X( 216.

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Fig.10 Cross section, leaf of Dendrobium section Aporum. Bar = 10 ,um.Dendrobium acinaciformeshowing major vascular bundle at one pole (arrowhead); X 192.

D. anceps, D. brevimentum,D. aloifolium,D. disti- ence of thick-walled,chlorenchymatous cells thatsur- chum, D. indivisum,D. mannii,D. rosellum,and D. roundthe substomatalchamber. The remainingspecies nathaniele; theyare absent along vascular-bundlescle- thatwere studied formeda clade on the basis of thin- renchyma in D. acinaciforme. Bundle sheath walled chlorenchymatouscells that surroundthe hair uniseriate, composed of thin-walled chlorenchyma bases. Configurationof the stomatalapparatus, abaxial cells in all species. cuticle thickness,abaxial epidermal cell shape, pres- Discussion ence of a suturewithin the mesophyll,and form/presence of a suberin-likesubstance were homoplaseous CLADISTICS characterswithin this clade, here referredto as the D. Features of leaf anatomy were used to constructa nathaniele-D. brevimentumclade. Apomorphies for charactermatrix (table 3) as the basis for a cladistic the clade comprisingsections Rhizobiumand Aporum analysis of species withinKranzlin's subgroupsHem- were the presence of pleated water-storagecells and a iphyllaand Holophylla (section Aporum) and between thickcuticle. Orderingthe characterstates of leaf ori- sectionsRhizobium and Aporum(table 2). The branch- entationwould resultin the unifacialleaf being an ad- and-bound analysis resulted in the discovery of 68 ditional synapomorphyfor the two clades. Characters supportingthe equally parsimonioustrees (length = 33, consistency monophylyof sectionAporum and section index = 0.75, and retentionindex = 0.81). A strict Rhizobium are provided in figure 12 (see also consensus tree is shown in figure11. "Comparative anatomy" below). One of the 68 equal- The strictconsensus of the 68 trees showed little ly parsimonioustrees is presentedin figure12. Using resolution among the species of section Aporum, al- this tree,more resolutionis evident withinthe D. na- though Dendrobium distichum and D. leonis were thaniele-D. brevimentumclade, but these subclades are placed basally withinsection Aporum in all generated supportedby very homoplaseous and/orsingle char- cladograms (fig. 11). Dendrobium distichumis sepa- acters and are not supportedin other equally parsi- rated fromthe rest of Aporumbecause of the suberin- monious trees. Note thatthe monophylyof neitherof like substance that impregnatesthe cell wall in this Kranzlin's subgroupsof Aporum is supported. species. The remainingspecies formeda clade based COMPARATIVE ANATOMY on the shared presence of a sutureembedded within the mesophyll,a characterthat reversed in some spe- This analysis, combined with the investigationof cies. Dendrobiumleonis was distinguishedby thepres- Morriset al. (1996), providesconvincing evidence that

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Dendrobium salaccense (G) in stegmata; and (5) vascular bundles symmetrically 12. toressae (R) EZZZZZ D. rigidum (R) arrangedwithin the mesophyll.Many membersof sec- [JiZZZiZ~ 12. 1Qlingiforme R) tion Aporum exhibita bilaterallysymmetrical leaf di- 12. distichum,(A) vided by a suturedistinguished by a series of elongate ZI l D. leonis (A ) H water-storagecells parallel with the median longitu- 12.n acinacifo S e (A') 1.anceps (A)me dinal axis of the leaf section. In contrast,advanced aloifolium 12. le (AH;) membersof section have a lacuna embed- 1D. nathanie (AI Rhizobium D. mannii (A ded withinthe mesophylldividing the leaf bilaterally, D. indivisum (A D. brevimentum-(A much as the suturedoes in membersof section Apo- ElI 12. rosellum (A') rum. The lacuna representsa canal thatruns through the leaf fromits pointof attachmentto the stem.Basal 2. salaccense (G) 2. toressae (R) membersof Rhizobiumexhibit what Sternet al. (1994) interpretedas an incipientlacuna, a groove on the up- _ l e 1 | -- ~~~~~~D.rigidu (R) ck per surfaceof the leaf characterizedby an adaxial epi- D. linguiforme (R) a 1H dermis. r. distichum (AN?) Formationof the unifacial leaf and internalsuture D. leonis (AHo characteristicof species in sectionAporum might have b en d. nathaniele (Al) resulted evolutionarilyfollowing the steps suggested below. Orderingthe characterstates that correspond to e~~~~~~~~~~~~~~H mannii ee [1 o - - D. (AHe ~~~~~~~~~~~~~~~~~He)leaf orientation would result in a morphocline,in I. acinaciforme (A which the hypotheticalbifacial leaf would fold inward q He 2. rosellum (A along the midvein, bringingthe adaxial surfaces to- No P. anceRs (A getherto forma nearlyunifacial leaf characteristicof the hypotheticalancestor for Rhizobium and Aporum. . aloifolium (AHe) The g . adaxial epidermis mighthave been reduced further while still maintainingan incipientlacuna as a h . indivisma (AHO) groove on the upper surface. The incipient lacuna, No .. brevimentum (A characteristicof basal Rhizobiummembers, by contin- ued infoldingof the leaf margins,would resultin the Figs. 11, 12 Cladograms of Dendrobium sections Aporum and Rhizobium.Fig. 11, Strictconsensus of 68 trees.Fig. 12, One of 68 unifacial leaf characteristicof advanced members of equally parsimoniouscladograms. Synapomorphiessupporting each Rhizobium,in which thereis a true lacuna embedded clade include: a = 4(1), 7(2); b = 2(2), 6(2), 8(2), 9(2), 10(3), 11(2), in the mesophyllsurrounded by an adaxial epidermis. 16(1); d = 14(1); e = 1(1);f= 4(0), 5(1); g = 14(0); h = 2(0); i The unifacial leaf of Aporum may have evolved = 14(l); j = 10(2), 7(1); k = 8(1); 1 = 12(1); m = 15(2); n = 3(1); o = 5(0); p = 15(1); q = 8(3), 11(1); r = 15(2); s = 4(1). (G) = fromthe same generalized unifacial leaf of the hypo- section Grastidium,(R) = section Rhizobium,(AHO) = sectionApo- thetical ancestor for Rhizobium (fig. 13), or the two rum,subgroup Holophylla, (AHe) = sectionAporum, subgroup Hem- differentunifacial leaves characteristicof sections iphylla.The superscriptspertain to these subgroups. Aporumand Rhizobiummay have evolved in parallel. It is unlikelythat unifacial leaves of Aporum evolved via the degenerationof the lacuna seen in leaves of sectionsAporum and Rhizobiumare monophyleticsis- section Rhizobium because such a hypothesiswould ter taxa. The anatomical studyof Stern et al. (1994), require Rhizobium to be paraphyletic.Paraphyly of based on foliar anatomy of section Rhizobium,sup- section Rhizobium is not supportedby this cladistic portsthe monophylyof thatgroup. analysis or the analyses performedby Stern et al. Synapomorphiesfound in this studyto supportthe (1994) and Morris et al. (1996). The suturepresent in monophylyof the Aporum clade include the presence some studied species of section Aporum mightrepre- of: (1) fiberbundles with heterogeneouscell compo- sent relictualevidence of the adaxial infoldingin the sition;(2) mainlyparacytic stomatal apparatus; (3) hy- same bifacial ancestralgroup hypothesizedfor Rhizo- podermis of small, thin-walled,chlorenchyma cells; bium. A major vascular bundle at one pole of the leaf (4) intermediateleaf width:length ratio = 10-38; (5) section could symbolize the ancestralmidvein. distributionof stegmataalong vascular sclerenchyma Metcalfe (1971) discussed a similarspeculative de- and fiberbundles; (6) major vascular bundle at leaf velopmental concept in leaves of Cyperaceae using pole; and (7) unifacial leaf lacking a lacuna or evi- certaintaxa as hypotheticalmorphological stages. He dence of a lacuna. noted that the conditionin which the area of adaxial We chose anatomicaland morphologicalfeatures of surfaceis reduced compared with the abaxial surface the leaf for a phylogeneticcomparison between sec- resultsin an "isobilateral" leaf. The "isobilateral" leaf tionsAporum and Rhizobium.Some of the similarities has vascular bundles in opposite pairs. There are two of these hypothesizedsister taxa (Morris et al. 1996) "pseudo-margins," one representedby the putative are: (1) leaves unifacial,or nearly so; (2) presence of midribat one pole, the otherby two fused "true mar- an abaxial hypodermis;(3) water-storagecells in the gins" at the opposite pole. The "centric" conditionis mesophyll;(4) rough-surfaced,spherical silica bodies an extremedevelopment from the isobilateralconfig-

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and Rhizobiumdiffers in cellular composition.In the Aporum group it comprises thin-walled,chlorenchymatous cells, while in sectionRhizobium the hypodermis consistsof eitherthick-and/or thin-walled unbanded cells or thin-walledbanded and unbanded cells. Thin-walled,banded cells of the hypodermisin Rhi- zobium may serve as additional water-storagetissue, while thick-walled,unbanded cells may functionin support. Unbanded water-storagecells of the mesophyllare pleated in section Aporum,and in Rhizobium,water- storage cells typically are pleated and lack banded thickenings or, infrequently,they may have these A~~~~~~~A (Stem et al. 1994). With some exceptions, stegmata are found throughoutthe genus Dendrobium (Soler- eder and Meyer 1930; Morris et al. 1996) in various Section Rhizobium distributionalpatterns. These silica-containingcells are Section Aporum associated with the fiberbundles and vascular-bundle sclerenchymaof leaves in section Aporum,but they only occur along the vascular-bundlesclerenchyma of leaves in section Rhizobiumbecause fiberbundles are absent in this group. Silica-body-containingstegmata, Hypothes Ized ancestor as well as the fiberbundles and vascular-bundlesclerenchyma,may serve as additionalstructural reinforce- mentto hold the leaves erect on stems duringperiods of water stress in plants of sections Rhizobium and Aporum.The elongatedraphide-containing cells of the mesophyll noted in section Aporum are of wide dis- tributionin Orchidaceae and, as such, are of littleor no systematicvalue. Outgroup condition The most obvious unifyingcharacteristic of sections Fig. 13 Hypothesis of leaf infoldingfor Dendrobium sections Rhizobiumand Aporum is the unifacial orientationof Aporumand Rhizobium.Adaxial surfaceblack, abaxial surfacegrey, the leaf surface,where only the abaxial epidermalsur- and dottedline representssuture. face is exposed. Unifacial leaves are not unique to these sections of Dendrobiumbut occur in othergenera of orchids, such as Epidendrum and Maxillaria urationin which the adaxial surfaceis eliminatedal- (Solereder and Meyer 1930). The unifacial condition together (Metcalfe 1979). Metcalfe also speculated might be considered ecologically analogous to am- about the occurrenceof this phenomenonwithin Or- phistomaty,in which all leaf surfaces are covered by chidaceae. His interpretationsappear analogous to our stomata. Mott et al. (1982) have suggested that am- proposal for the formationof unifacial leaves in sec- phistomatymight be correlatedwith thick,sun leaves tion Aporum. as an adaptationto high leaf conductanceand therefore Stern et al. (1994) discussed the formationof uni- high photosyntheticpotential. This hypothesismight facial leaves in section Rhizobiumand suggestedthat also be applicable to the thick,unifacial leaves of sec- those members with an internallacuna evolved in a tions Aporum and Rhizobium.Amphistomaty and un- mannersimilar to the hypothesizedinfolding of leaves ifacial leaves are foundin othergroups of plants,such in section Aporum supportedhere. Such a develop- as succulent CAM (crassulacean acid metabolism) mentalseries could resultin a unifacialleaf and would plants (Kluge and Ting 1978) and are correlatedwith thereforeconstitute a synapomorphyfor sectionsRhi- high lightlevels (Mott et al. 1982). zobium and Aporum.Unifacial leaves in sectionsRhi- Salisbury (1927) suggested that amphistomaty zobium and Aporum are differentlyinserted along the mightbe an adaptationto dry habitats,as the coria- stem and vary in overall form.Therefore, we did not ceous conditionof the leaves he observed seemed to assume, a priori,that the unifacial conditionin these indicate.Coriaceousness, the presence of fiberbundles, two groups was homologous. Nevertheless,the results and wall thickeningsof cells surroundingthe substo- of cladistic analyses do suggest thatthe common an- matal chamberin D. leonis and hair bases in D. leonis cestorof plantsin sectionsRhizobium and Aporumhad and D. distichum,as well as the suberin-likesubstance unifacial leaves. that occurs in adaxial epidermal cells of some mem- Members of section Rhizobium contain symmetri- bers of Aporum,could also be adaptationsto xeric con- cally arrangedfoliar vascular bundles,a conditionalso ditions.The walls of adaxial epidermalcells in D. dis- found in sectionAporum. The hypodermisin Aporum tichum and D. rosellum are impregnatedwith this

This content downloaded from 139.67.65.234 on Wed, 14 May 2014 15:29:04 PM All use subject to JSTOR Terms and Conditions CARLSWARD ET AL.-LEAF ANATOMY DENDROBIUM SECTION APORUM 341 suberin-likesubstance. Esau (1965) and Fahn (1990) Conclusions noted that suberinmay occur in the cell wall matrix, Althoughour resultsfrom foliar anatomyillustrate and Metcalfe (1960) described suberindeposits in cell thatsections Rhizobium and Aporumare monophyletic walls of leaves in Gramineae. Exudate produced by sister taxa, supportingthe analysis of Morris et al. the glandularhairs in sectionAporum could be an ad- (1996), therewas very littleresolution within section aptationto a xeric habitat,as hypothesizedby Ehler- Aporum (fig. 11). The only consistent resolution inger (1984), but it also mightserve as a deterrentto among the species studied in the Aporum clade is the herbivory,as pointed out by Kelsey et al. (1984). Ex- basal placement of Dendrobium leonis and D. disti- udations may be composed of "oils" (Solereder and chum.The absence of resolutionprobably results from Meyer 1930) or substancesthat might prevent herbiv- the relativelysmall numberof charactersused, as well ory, such as terpenes and phenolics (Kelsey et al. as the homoplaseous natureof some of these charac- 1984). Sunken, glandular hairs occur throughoutthe ters. Our results indicate that neither of Kranzlin's genus Dendrobium as well as in othergenera of Or- groups,i.e., Holophylla and Hemiphylla,can be con- chidaceae, such as Bulbophyllum,Epidendrum, and sidered monophyletic.Some of the members within Maxillaria (Solereder and Meyer 1930). Holophylla are nestedwithin the unresolved clade con- Although all members of section Aporum are epi- sistingprimarily of Heterophyllaspecies (figs. 11, 12). phytic and thereforesubjected to water stress (Carl- Furtherstudies involving floral morphology, as well as quist [1975] disputesthis), the habitatsof plantsmen- additional research on vegetative anatomy and mor- tioned by Seidenfadenand Wood (1992) indicate wet, phology incorporatingadditional taxa, and molecular shaded conditions.In this humid environment,the ab- investigationscould possibly provide data for further axial stomata of unifacial leaves could enhance gas resolutionof the Aporumclade. exchange by providinga greatersurface for diffusion Acknowledgments of carbon dioxide into the leaf and transpirationof excess water vapor fromthe leaf in a moisture-laden We are gratefulto Robert L. Dressler,Florida Mu- atmospherethan would occur in bifacial leaves. Still, seum of Natural History,for help in identifyingDen- the ecological significanceof the hypothesizedxero- drobiumbrevimentum. We thankSandra Bell, curator morphicadaptations in leaves of sectionAporum -lacks of Living Collections, Royal Botanic Gardens, Kew, an unequivocally supportableexplanation. for sending preserved materials of some specimens Sternet al. (1994) also questionedthe purported sig- used in this study. The plant of Dendrobium brevi- nificanceof the xeromorphicfeatures they observed in mentumwas suppliedby Fuchs Orchids,Naranja, Flor- leaves of sectionRhizobium, vis-'a-vis the environmen- ida. We appreciatespecimens provided by JohnT At- tal conditionsunder which many of these species live. wood, Marie Selby Botanical Gardens, Sarasota, Florida. Scott Whittaker,of the InterdisciplinaryCen- A plausible hypothesisaccounting for these apparent ter for Biotechnology Research at the Universityof discrepancieswould be that the common ancestor of Florida, helped with photographicprocessing. Laurie the Rhizobium and Aporum groups developed a uni- Walz illustratedour phylogeneticinterpretation of leaf facial leaf throughstages similar to the ones hypoth- infolding. esized by Metcalfe (1971) in response to an ancestral, Portionsof thisstudy were carriedout withfinancial xerophyticenvironment. Because the xeromorphicfo- assistance fromthe American Orchid Society (Amer- liar conditions were not disadvantageous to present- ican Orchid Society 11th World Orchid Conference day plants in sections Rhizobium and Aporum, they Fellowship) throughThomas J. Sheehan, Universityof have been retained.This scenario mightalso explain Florida, and the Lewis and Varina Vaughn Graduate why the basal membersof Aporum,i.e., D. leonis and Fellowship in OrchidBiology throughDavid A. Jones, D. distichum,exhibit several unique xeromorphicad- chairmanof the Departmentof Botany, Universityof aptations. Florida. Literaturecited

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