Ontogenetic anatomical and histochemical studyof the extrafloral nectaries of Sapium biglandulosum(Euphorbiaceae)

Iacutetalo Antocircnio Cotta CoutinhoA Vacircnia Maria Moreira ValenteA

and Renata Maria Strozi Alves MeiraAB

AUniversidade Federal de Vicosa (UFV) Departamento de Biologia VegetalVicosa 36570-000 Minas Gerais Brazil

BCorresponding author Email rmeiraufvbr

Abstract The present paper aims to confirm the nature of secretory structures found on the petiole and leaf margins ofSapium biglandulosum Muumlell Arg The anatomy ontogenesis and histochemistry were studied by light microscopywhereas mono- and disaccharides in the exudates were detected by high performance liquid chromatography The exudatefrom the petiole had a total sugar concentration of 325 (wv) of which 381 was fructose 437 glucose and 182sucrose The petiolar gland started its development from a group of meristematic cells that underwent asynchronousdivisions At the end of the ontogenesis a well structured vascularised gland made up of a palisade secretory epidermissecretory parenchyma and a secretory pore was observed Leaf-margin glands showed a similar anatomy Histochemicaltests revealed the presence of proteins pectins carbohydrates tannins and anthocyanins On the basis of our results thereis compelling evidence that the studied glands are in fact extrafloral nectaries

Introduction

The genus Sapium (Euphorbiaceae) is characterised by itsarboreal size latescent branch and fruit and leaves that aregenerally glandulous (Muumlller 1895) that is a pair of bulbousglands at the bladendashpetiole junction (Correll and Johnston 1979)Glands are distinct groups of highly specialised cells that worktogether to discharge substances to the exterior or into specialintercellular cavities They are composed of secretory cells andmultiple types of auxiliary cells (Schnepf 1974 Fahn 1979)S biglandulosum is a latescent species with secretory glandson both the petiole and the leaf margins (Muumlller 1895)

These types of glands in Euphorbiaceae are frequentlyrecognised as extrafloral nectaries (Metcalfe and Chalk 1950)that have also been reported for other species of the genusSapium (Rogers et al 2003 So 2004) However these reportswere based only on the topography These glands are welldeveloped structures and have different shapes They arefound on the petiole either associated to stipules orsubstituting them on the leaf blade between the teeth and onfloral parts (Metcalfe and Chalk 1950 Schnepf 1974 Dave andPatel 1975 Fahn 1979 Fiala and Maschwitz 1991 Freitas et al2001 Linsenmair et al 2001)

Glands morphologically similar to extrafloral nectaries havebeen reported as extrafloral nectaries when they were actuallyresin glands (Curtis and Lersten 1978 Durkee et al 1984) Toavoid such a mistake an anatomical description and an analysisof the contents present in the secretion of secretory glands arenecessary to confirm the kind of glands present on a particularplant

Nectaries are specialised glands that exude nectar a sweetsolution composed primarily of glucose fructose and sucroseThey may be found on both vegetative and reproductive organs(Fahn 1979 Bentley and Elias 1983 Roshchina and Roshchina1993 Nicolson and Thornburg 2007) Extrafloral nectariesusually function as a defensive strategy against herbivores(Elias 1972 1980 Curtis and Lersten 1978 Freitas andOliveira 1996 McDade and Turner 1997 Oliveira 1997Madureira and Sobrinho 2002 Almeida and Figueiredo 2003Paiva and Machado 2006)

Therefore the present paper will study the ontogenesis andstructure of the secreting tissues and the chemical nature of theexudates to clarify the nature of the glands present on the petioleand leaf margins of S biglandulosum and to contribute to theunderstanding of the defensive strategies of this species

Materials and methodsTwigs of S biglandulosumwere collected at Siacutetio Bom SucessoVicosa Municipality and Parque Estadual Serra do BrigadeiroAraponga Municipality in the state of Minas Gerais BrazilEight twigs were collected at Siacutetio Bom Sucesso in May 2004and September 2008 In November 2005 and January 2007seven twigs were collected at Parque Estadual Serra doBrigadeiro

The first node considered was the first leaf primordiumwhich comes right after the shoot apex The shoot apexusually contained 8ndash10 leaf primordia (Fig 1A)

The samples were fixed in formalinndashacetic acidndashalcohol(FAA50) for 24 h then stored in 70 ethanol (Johansen 1940)

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These samples were dehydrated through an ethyl alcoholndashxyleneseries and then embedded in paraffin plus 8 wax (Roeser1972) The blocks were sectioned with a rotary microtome(Spencer 820 American Optical Corporation Buffalo NYUS) producing cross- and longitudinal serial sections 7ndash9mmthick For the ontogenetic characterisation sections were stainedwith safranin and astra blue (Roeser 1972) and mounted inresin (Permount Fisher Scientific Bridgewater NJ US) Forthe structural characterisation of mature glands fixed sampleswere embedded in methacrylate (Historesin Leica InstrumentsHeidelberg Germany) and sectioned as described aboveSections were stained with toluidine blue (OrsquoBrien andMcCully 1981) and mounted in resin

The samples used in the histochemical tests for detection ofphenolic compounds and fats were fixed in a solution of ferrous

sulfate in formalin (Johansen 1940) and in buffered neutralformalin (Clark 1981) respectively Control sections wereperformed simultaneously For detection of carbohydratesproteins and pectins some of the fixed samples wereembedded in Histosecreg (Merck) Sections varying from 7 to9mm were cut with a rotary microtome (Leica 2155 LeicaDeerfield IL US) For carbohydrate detection slides werestained with periodic acid Schiff (PAS) (Maia 1979) for totalproteins with xylidine pounceau (OrsquoBrien and McCully 1981)and for pectins ruthenium red (Johansen 1940) All slides weremounted in resin (Permount Fisher)

Freehand sections of fresh samples made on an LPC tablemicrotome (Rolemberg and Bhering Comeacutercio e ImportacatildeoLTDA Belo Horizonte Brazil) were used in the followinghistochemical tests sudan IV (70 (vv) ethanol) for detection

(A)

(F ) (H )

(G )

(I ) (J )

(E )

(B) (C) (D )

Fig 1 Developmental stages of the glands of Sapium biglandulosum seen under the stereo microscope (A) Shoot apex formed by nine leaf primordiaNote that one of the two stipules (white arrow) found on the leaf primordia covers the meristematic area where the gland originates (BndashF ) Pair of petiolarglands during the development (B) The first node Note the gland primordium (black arrow) (C) The second node (D) The third node (E ) The sixthnode (F ) The ninth node (GndashJ ) Development of the leaf-margin glands (G) The eleventh node (H ) The twelfth node (I J ) The thirteenth node(I ) side view and (J ) frontal view

Extrafloral nectaries of Sapium biglandulosum Australian Journal of Botany 225

of lipid compounds (Pearse 1980) Nadi reagent for essential oilsand oleoresins (David and Carde 1964) autofluorescence underUV (OrsquoBrien and McCully 1981) and potassium dichromate(Gabe 1968) for phenolic compounds vanillinndashhydrochloricacid for tannins (Mace and Howell 1974) phloroglucinol forlignins (Johansen 1940) Dittmar and Wagner reagents foralkaloids (Furr and Mahlberg 1981) and lugol for starch(Johansen 1940)

Observations and photographic documentation wereperformed with a light microscope (Model AX70TRFOlympus Optical Tokyo Japan) equipped with a U-Photosystem and an epifluorescence HBO 50W mercury vapourlamp and a filter block A (exciter filter BP 340ndash380 dichroicmirror 450 barrier filter LP-430)

Images of the leaves showing gland development weretaken with a SZX7 Olympus stereo microscope equippedwith an EVOLT E-300 Olympus digital camera (OlympusOptical)

Branches kept in a bucket with tap water were kept in thelaboratory for 2 days Samples of exudates from 15 randomlychosen secreting petiolar glands were collected directly withcapillary tubes on both days Because of the small amount ofsecretion it was not possible to collect exudates from leaf-margin glands The secretion was then transferred to anEppendorf tube and stored in a freezer The analysis wascarried out in the Laboratoacuterio de Anaacutelises e Siacutentese deAgroquiacutemicos at the Universidade Federal de Vicosa MinasGerais State Brazil The exudate (454mg) was diluted with5mLof ultra-purewater and analysed by high performance liquidchromatography (HPLC) It was used in a chromatographer(Type LC-6AD Shimadzu Osaka Japan) equipped with arefractive index detector (RID) a data collecting system (TypeClass 5000 Shimadzu Osaka Japan) a Techspherene columnNH2 (250mm 46mm internal diameter (id) 5mm) at 40Cand a Techspherene pre-column NH2 (20mm 46mm id5mm) Deionised waterndashacetonitrile (80 20 vv) at a columnflow of 05mL minndash1 was used as the mobile phase Aliquots of20mL of fructose glucose and sucrose solutions at 00 01 0610 and 20mgmLndash1 concentrations were used to achieve acalibration curve Three 20-mL samples of the diluted exudatewere used

Results

Gland ontogenesis

In the very beginning of gland development it was observedthat the area that would later form the glands was protected byone of the two stipules found on the leaf primordium(Fig 1A B) The stipules dropped off and were not foundon leaf primordia placed after the first node Each gland of thepetiole originated from a group of meristematic cells As thecells started to divide the gland primordia appeared as twocushion-like structures (Fig 1B) on the apex of the petioleThese structures increased in size and a deepening at thecentral area was observed (Fig 1C) As gland developmentwent on the structures continued to emerge (Fig 1D E)Apart from moving to the opposite sides of the petiole(Fig 1F ) the gland primordia from the fourth node upto their mature stage ndash the ninth node ndash did not show any

drastic changes in their morphology The final result is a pairof opposite glands (Fig 1F ) each one bearing a slit-likesecretory opening

For the glands on the leaf margin we observed that around protuberance would form a gland whereas a pointyprotuberance would form a tooth (Fig 1G) Similarly to thepetiolar glands a deepening at the centre of the roundprotuberance appeared As glandular development went onthis deepening transformed into a concavity (Fig 1G H) Bythe end of the gland ontogenesis round glands bearing a redring at the concavity opening (Fig 1J) had formed Theconcavity openings of these glands were aligned with the leafmargin (Fig 1I )

During the first stage of development of petiolar glands it wasobserved that the cells from the ground meristem slightlyincreased their volume which precipitated the formation of aconvex protrusion (Fig 2A) Protodermal cells began dividing bymeans of anticlinal divisions greatly increasing the superficialarea At that stage the protodermal cells were cubical withdense staining cytoplasm and large nuclei with thin wallsbut were not larger than the cells from the ground meristem(Fig 2A)

The divisions occurred at a lower rate at the centre of thegland primordium than at its margin This peculiar way ofcellular proliferation resulted in a deepening of the central areaof the gland primordium (Fig 2B) The cellular proliferation inthe ground meristem started later than in the protodermThese meristematic cells were small polyhedral and containedconspicuous nuclei Anticlinal periclinal and oblique(Fig 2C D) divisions occurred asynchronously

The protodermal cells of the sunken area slowly took thetypical columnar shape of secretory cells Thus they could beclearly distinguished from the cells of the ground meristemAlthough the differentiation began in the protoderm theproliferation process continued (Fig 2E)

During this stage the ground-meristem cells became alittle bigger and continued to divide (Fig 2F ) Procambialcells were observed among the ground-meristem cells near theprotoderm (Fig 2G)

In the stages that followed cellular expansion anddifferentiation were intensified while cell divisions continued(Fig 2H I) This process culminated in the formation of anenlarged structure with a deep central cavity (Fig 2J)Protodermal cells became higher and more columnar-shaped(Fig 3A) characterising a secretory epidermis however thegland was not functional by that time The cells at the cavityaperture were similar to the non-secretory epidermal cells(Fig 3B) The ground meristem was differentiated into asecretory parenchyma (Fig 3A)

A single procambial vascular bundle from the vascular systemof the petiole branched towards the gland (Figs 2J 3A) Suchvenation was later observed surrounding and entering thesecretory parenchyma

Cell divisions were not observed in leaves measuringmore than 50mm in length A quick cellular expansion wasobserved as the leaves expanded

Finally a fully developed vascularised secretory gland madeup of a secretory epidermis and secretory parenchyma wasobserved

226 Australian Journal of Botany Iacute A C Coutinho et al

Gland characterisation

The glands of the petiole contained a single-layered secretoryepidermis lacking stomata with adjoining columnar-shape cellsmaking up a palisade and a very thin cuticle (Fig 3C) The nucleiof these cells were large and located at the bottom of the cells

(Fig 3C) The cytoplasm was more densely stained than thesecretory parenchyma cells (Fig 3A)

The closer the epidermal cells were to the cavity aperturethe less columnar-shape they had until the epidermal cellsabruptly became cubical similar to the other non-secretoryepidermal cells (Fig 3B)

(A) (B) (C)

(E ) (F ) (G)

(J )( I )(H )

(D )

Fig 2 Developmental stages of the petiolar glands of Sapium biglandulosum seen under light microscopy (AndashC G J ) Longitudinal sections (DndashF H I )Cross-sections (A) Gland primordium of a shoot protected by the stipule (B) Gland primordium with the deepened central area (C ) The second node leafprimordium (D E ) The third node (F G) The fourth node showing the very beginning of the gland differentiation process (H I ) The fourth node Note thecavity formed in the central area (J ) The fifth node Note the ramification of the petiolar vascular bundle Ca cavity Dc dividing cell De depressionEp epidermis Gm ground meristem Gp gland primordium Pc procambium Pr protoderm S stipule Spa secretory pore aperture and Va vascularisation

Extrafloral nectaries of Sapium biglandulosum Australian Journal of Botany 227

The secretory parenchymawasmade up of three to nine layersof cells (Fig 3E F) These cells were smaller than thosebelonging to the ground parenchyma Scattered druses werefound in the ground parenchyma (Fig 3D) The walls of thesecretory parenchyma cells were thin These cells contained largenuclei and dark-staining cytoplasm (Fig 3E F)

A vascular bundle branched from the petiolar vascularsystem into the gland The mature extrafloral glands werevascularised by xylem and phloem with phloem moreabundant than xylem The vascular bundle surrounded thesecretory parenchyma and entered among its cells (Fig 3E)

The glands on the leaf margins were in structure similar tothose on the petiole (Fig 3G H) The major difference betweenthe two types of glands was that the petiolar glands had a deepcavity with an aperture (Fig 3A) whereas the leaf-margin glandshad a concavitywith a round aperture (Fig 3H) Such a differenceinfluenced the manner of the exudate-elimination pattern Inthe petiolar glands exudate accumulated for a short period andthen dripped off whereas in the leaf-margin glands exudate waskept within the concavity

The histochemical tests detected carbohydrates (Fig 4A B)pectins (Fig 4C D) and proteins (Fig 4E F ) at the secretory

(A)

(B)

(C) (D)

(E ) (F )

(G ) (H )

Fig 3 Mature glands of Sapium biglandulosum as seen under light microscopy (AndashF ) Petiolar glands in (AndashE ) longitudinal sectionsand (F ) cross-section (G H ) Mature secretory leaf-margin glands in (G ) cross-section and (H ) longitudinal sections (A) Maximisationof the Fig 2J (BndashD) The ninth node (E F ) Mature secretory petiolar glands Note the vascularisation surrounding and entering thegland Xylem (black arrows) Secretion (asterisk) Ep epidermal cells Gp ground parenchyma Sp secretory parenchymaPc procambium Cu cuticle Dr druses Spa secretory pore aperture (Spa) and Va vascularisation

228 Australian Journal of Botany Iacute A C Coutinho et al

epidermis and secretory parenchyma of both types of glandsThe UV light detected phenolic compounds only at the outer cellwalls of the secretory epidermal cells for both the petiolar(Fig 4G) and the leaf-margin glands However the phenoliccompounds in the petiolar glands were identified as tannins asthey reacted positivelywith vanillinndashhydrochloric acid (Fig 4H)whereas the phenolic compounds in the leaf-margin glandswere identified as anthocyanin Anthocyanin formed a roundring at the borders of the leaf-margin glands (Fig 1J) UV lightalso showed the xylem that reacted positively to the test forlignins (Fig 4I ) The other histochemical tests returned negativeresults

Exudate and exudate secretion

The total concentration of sugars in the secretion was 325(mgmLndash1) Of this 381 was fructose 437 glucose and182 sucrose The disaccharidendashmonosaccharide ratio was022 (sucrose(fructose + glucose))

Exudate secretion in S biglandulosum occurred before thecomplete leaf expansion starting while the leaf margins werestill coiled The exudate from the petiolar glands was secreted

and flowed to the outer surface from where it later dropped offwhereas the exudate produced by the leaf-margin glands wasaccumulated into the concavity

During the collections of S biglandulosum insects such asants flies and bugs were observed collecting exudate in the earlymorning and late afternoonGlandswere observed secreting evenwhen they had been partially wounded

Discussion

The structural features of the secreting tissues the position andthe detection of glucose fructose and sucrose in the exudatesallow us to characterise these glands as extrafloral nectaries

The pattern of development observed in the nectaries ofS biglandulosum was similar to what has been reported forextrafloral nectaries of other plant families (Arbo 1972 Daveand Patel 1975 Roshchina and Roshchina 1993 Silva andMachado 1999 Leitatildeo et al 2002 Paiva and Machado 2006Thadeo et al 2008) The asynchronous cellular divisions duringthe nectary ontogenesis are an ordinary process as described forPedilanthus tithymaloides Euphorbiaceae (Dave and Patel1975) and Triumfetta semitriloba Tiliaceae (Leitatildeo et al

(A) (B) (C)

(D) (E ) (F )

(G ) (I )(H )

Fig 4 (AndashF H I ) Histochemical tests and (G) UV-light autofluorescence of the (A C F I ) leaf margin and (B D E G H ) petiolar glands (A B )PAS reaction The maximisation on the upper right side in B shows the sugar grains formed outside the secretory epidermis (C D ) Ruthenium red(E F ) Xylidine pounceau (GndashI ) Phenolic compounds (H ) Vanillinndashhydrochloric acid (I ) Phloroglucinol Ep epidermal cells Co concavity Gpground parenchyma Sp secretory parenchyma pp palisade parenchyma and Va vascularisation bundle Black arrow indicates xylem White arrowindicates epidermal cells with phenolic compounds

Extrafloral nectaries of Sapium biglandulosum Australian Journal of Botany 229

The secretory parenchymawasmade up of three to nine layersof cells (Fig 3E F) These cells were smaller than thosebelonging to the ground parenchyma Scattered druses werefound in the ground parenchyma (Fig 3D) The walls of thesecretory parenchyma cells were thin These cells contained largenuclei and dark-staining cytoplasm (Fig 3E F)

A vascular bundle branched from the petiolar vascularsystem into the gland The mature extrafloral glands werevascularised by xylem and phloem with phloem moreabundant than xylem The vascular bundle surrounded thesecretory parenchyma and entered among its cells (Fig 3E)

The glands on the leaf margins were in structure similar tothose on the petiole (Fig 3G H) The major difference betweenthe two types of glands was that the petiolar glands had a deepcavity with an aperture (Fig 3A) whereas the leaf-margin glandshad a concavitywith a round aperture (Fig 3H) Such a differenceinfluenced the manner of the exudate-elimination pattern Inthe petiolar glands exudate accumulated for a short period andthen dripped off whereas in the leaf-margin glands exudate waskept within the concavity

The histochemical tests detected carbohydrates (Fig 4A B)pectins (Fig 4C D) and proteins (Fig 4E F ) at the secretory

(A)

(B)

(C) (D)

(E ) (F )

(G ) (H )

Fig 3 Mature glands of Sapium biglandulosum as seen under light microscopy (AndashF ) Petiolar glands in (AndashE ) longitudinal sectionsand (F ) cross-section (G H ) Mature secretory leaf-margin glands in (G ) cross-section and (H ) longitudinal sections (A) Maximisationof the Fig 2J (BndashD) The ninth node (E F ) Mature secretory petiolar glands Note the vascularisation surrounding and entering thegland Xylem (black arrows) Secretion (asterisk) Ep epidermal cells Gp ground parenchyma Sp secretory parenchymaPc procambium Cu cuticle Dr druses Spa secretory pore aperture (Spa) and Va vascularisation

228 Australian Journal of Botany Iacute A C Coutinho et al

epidermis and secretory parenchyma of both types of glandsThe UV light detected phenolic compounds only at the outer cellwalls of the secretory epidermal cells for both the petiolar(Fig 4G) and the leaf-margin glands However the phenoliccompounds in the petiolar glands were identified as tannins asthey reacted positivelywith vanillinndashhydrochloric acid (Fig 4H)whereas the phenolic compounds in the leaf-margin glandswere identified as anthocyanin Anthocyanin formed a roundring at the borders of the leaf-margin glands (Fig 1J) UV lightalso showed the xylem that reacted positively to the test forlignins (Fig 4I ) The other histochemical tests returned negativeresults

Exudate and exudate secretion

The total concentration of sugars in the secretion was 325(mgmLndash1) Of this 381 was fructose 437 glucose and182 sucrose The disaccharidendashmonosaccharide ratio was022 (sucrose(fructose + glucose))

Exudate secretion in S biglandulosum occurred before thecomplete leaf expansion starting while the leaf margins werestill coiled The exudate from the petiolar glands was secreted

and flowed to the outer surface from where it later dropped offwhereas the exudate produced by the leaf-margin glands wasaccumulated into the concavity

During the collections of S biglandulosum insects such asants flies and bugs were observed collecting exudate in the earlymorning and late afternoonGlandswere observed secreting evenwhen they had been partially wounded

Discussion

The structural features of the secreting tissues the position andthe detection of glucose fructose and sucrose in the exudatesallow us to characterise these glands as extrafloral nectaries

The pattern of development observed in the nectaries ofS biglandulosum was similar to what has been reported forextrafloral nectaries of other plant families (Arbo 1972 Daveand Patel 1975 Roshchina and Roshchina 1993 Silva andMachado 1999 Leitatildeo et al 2002 Paiva and Machado 2006Thadeo et al 2008) The asynchronous cellular divisions duringthe nectary ontogenesis are an ordinary process as described forPedilanthus tithymaloides Euphorbiaceae (Dave and Patel1975) and Triumfetta semitriloba Tiliaceae (Leitatildeo et al

(A) (B) (C)

(D) (E ) (F )

(G ) (I )(H )

Fig 4 (AndashF H I ) Histochemical tests and (G) UV-light autofluorescence of the (A C F I ) leaf margin and (B D E G H ) petiolar glands (A B )PAS reaction The maximisation on the upper right side in B shows the sugar grains formed outside the secretory epidermis (C D ) Ruthenium red(E F ) Xylidine pounceau (GndashI ) Phenolic compounds (H ) Vanillinndashhydrochloric acid (I ) Phloroglucinol Ep epidermal cells Co concavity Gpground parenchyma Sp secretory parenchyma pp palisade parenchyma and Va vascularisation bundle Black arrow indicates xylem White arrowindicates epidermal cells with phenolic compounds

Extrafloral nectaries of Sapium biglandulosum Australian Journal of Botany 229

epidermis and secretory parenchyma of both types of glandsThe UV light detected phenolic compounds only at the outer cellwalls of the secretory epidermal cells for both the petiolar(Fig 4G) and the leaf-margin glands However the phenoliccompounds in the petiolar glands were identified as tannins asthey reacted positivelywith vanillinndashhydrochloric acid (Fig 4H)whereas the phenolic compounds in the leaf-margin glandswere identified as anthocyanin Anthocyanin formed a roundring at the borders of the leaf-margin glands (Fig 1J) UV lightalso showed the xylem that reacted positively to the test forlignins (Fig 4I ) The other histochemical tests returned negativeresults

Exudate and exudate secretion

The total concentration of sugars in the secretion was 325(mgmLndash1) Of this 381 was fructose 437 glucose and182 sucrose The disaccharidendashmonosaccharide ratio was022 (sucrose(fructose + glucose))

Exudate secretion in S biglandulosum occurred before thecomplete leaf expansion starting while the leaf margins werestill coiled The exudate from the petiolar glands was secreted

and flowed to the outer surface from where it later dropped offwhereas the exudate produced by the leaf-margin glands wasaccumulated into the concavity

During the collections of S biglandulosum insects such asants flies and bugs were observed collecting exudate in the earlymorning and late afternoonGlandswere observed secreting evenwhen they had been partially wounded

Discussion

The structural features of the secreting tissues the position andthe detection of glucose fructose and sucrose in the exudatesallow us to characterise these glands as extrafloral nectaries

The pattern of development observed in the nectaries ofS biglandulosum was similar to what has been reported forextrafloral nectaries of other plant families (Arbo 1972 Daveand Patel 1975 Roshchina and Roshchina 1993 Silva andMachado 1999 Leitatildeo et al 2002 Paiva and Machado 2006Thadeo et al 2008) The asynchronous cellular divisions duringthe nectary ontogenesis are an ordinary process as described forPedilanthus tithymaloides Euphorbiaceae (Dave and Patel1975) and Triumfetta semitriloba Tiliaceae (Leitatildeo et al

(A) (B) (C)

(D) (E ) (F )

(G ) (I )(H )

Fig 4 (AndashF H I ) Histochemical tests and (G) UV-light autofluorescence of the (A C F I ) leaf margin and (B D E G H ) petiolar glands (A B )PAS reaction The maximisation on the upper right side in B shows the sugar grains formed outside the secretory epidermis (C D ) Ruthenium red(E F ) Xylidine pounceau (GndashI ) Phenolic compounds (H ) Vanillinndashhydrochloric acid (I ) Phloroglucinol Ep epidermal cells Co concavity Gpground parenchyma Sp secretory parenchyma pp palisade parenchyma and Va vascularisation bundle Black arrow indicates xylem White arrowindicates epidermal cells with phenolic compounds

Extrafloral nectaries of Sapium biglandulosum Australian Journal of Botany 229

2002) Arbo (1972) described such asynchrony for theontogenesis of the nectaries of Byttneria However suchasynchronous divisions caused a deepening of the nectaryitself instead of its emergence as shown for S biglandulosum

The extrafloral nectaries of S biglandulosum can be classifiedas structural nectaries according to Fahn (1979) These structuresoriginate from a small group of meristematic cells in the petiolewhich are covered by two stipules It is believed that the stipulesact on the protection of the structure being formed becausemeristematic cells are fragile

Because the epidermis is secretory has a thin cuticle and nostomata it is believed that the secretion is released through thecuticle as reported for the extrafloral nectaries of Crotonsarcopetalus Euphorbiaceae (Freitas et al 2001) where thesecretion is exudated by channels In species belonging toother botanical families (Elias et al 1975 Bentley and Elias1983 Roshchina and Roshchina 1993 McDade and Turner1997) cuticular pores have been reported as a way to exudatethe nectar In S biglandulosum nectar may or may notaccumulate For the smaller marginal nectaries the nectar wasfound to accumulate in the concavity This may be an advantagebecause it reduces the evaporative loss of nectar thus increasingthevolumeof available nectar (Keeler andKaul1979Leitatildeo et al2005) In contrast the petiole nectaries are larger with nectaraccumulating for a short period and then dropping off

Nectaries are closely related to the vascular system oftenhaving their own vascular bundle and sometimes an underlyingnectary parenchyma (Bentley and Elias 1983 Contreras andLersten 1984 Roshchina and Roshchina 1993 Leitatildeo et al2005 Paiva and Machado 2006 Nepi 2007) This pattern oforganisation was observed in S biglandulosum The continuousflow of organic matter from the phloem is necessary for nectarproduction in S biglandulosum because chloroplasts are notfound in the nectaries

Nectar was classified as hexose-rich according to Baker andBaker (1983) Such a sugar profile may be related to visitors(Baker and Baker 1983 Nicolson 2007) Freitas et al (2001) alsofound the sugars in the extrafloral nectar of Croton sarcopetalus(Euphorbiaceae) to be primarily monosaccharides with apredominance of glucose over fructose Crop load increaseswith increasing sucrose concentration however when sucroseconcentration is higher than 43 it diminishes because of nectarviscosity (Nicolson 2007) Experimental data demonstrated thatnectar-feeding ants normally forage for sucrose-poor nectarbecause high sucrose concentrations increase viscosity andants are not able to carry such heavy loads (Nicolson 2007)Heil et al (2005) reported that for the extrafloral nectaries ofmyrmecophyticAcacia speciesmutualistic ants prefer extrafloralnectars that have glucose and fructose in their profile whilenonsymbiotic ants prefer extrafloral nectars that have sucroseOnce extrafloral nectar of S biglandulosum has sucrose thatattracts patrolling ants that may play a defensive role such antsseem to be generalists that is they randomly look for extrafloralnectars available instead of having a close relationship withS biglandulosum

Although S biglandulosum has two large nectaries on thepetiole smaller nectaries are also found on the leaf marginthus increasing the area patrolled by ants and allowing forprotection of the whole leaf Elias and Gelband (1976)

considered the presence of many small nectaries at a particularsite as a form of specialisation In support of their conclusionElias and Gelband (1976) stated that malfunction or damagefromeither internal or external factors in oneor twoof thesemanysmall nectaries will not eliminate that site as an ant-attractinglocation This nectary-distribution pattern may be considered anecological advancement compared with a single large nectary(McDade and Turner 1997 Leitatildeo et al 2005)

The premature secretion of nectar in S biglandulosum isimportant for attracting ants which then protect the budsbecause during development the buds are delicate structuresand subject to injuries Additionally ants visit the leaf bladethus protecting the photosynthetic sites The antndashextrafloralnectaries mutualistic interactions can reduce the damagecaused by herbivores to the host plant (Elias 1972 1980 Eliaset al 1975 Keeler 1977 Curtis and Lersten 1978 Anderson andSymon 1985 McDade and Turner 1997 Oliveira 1997 Kopturet al 1998 Madureira and Sobrinho 2002 Almeida andFigueiredo 2003 Paiva and Machado 2006) Anderson andSymon (1985) demonstrated that ant activity was higherduring the younger leaf stages which matches the period ofhigher activity of the extrafloral nectaries of Solanum Andersonand Symon (1985) also noted the protection afforded by insectsduring the early leaf stages

The detection of polysaccharides by PAS and ruthenium redin the secretory cells of the nectaries confirmed the chemicalresults found for disaccharides and monosaccharides Similarreports are found in studies of other Euphorbiaceae species andother botanical families (Rocha et al 2002 Leitatildeo et al 2005Thadeo et al 2008) The detection of protein by xylidinepounceau indicates that proteins may be found in the nectarThese results showed that the nectar of the S biglandulosumis nutritionally rich The presence of proteins sugarspolysaccharides and pectin is common among several kindsof nectary glands (Bentley and Elias 1983 Roshchina andRoshchina 1993 Leitatildeo et al 2002 Nicolson and Thornburg2007)

The characteristics of the secretory epidermis andnectariferous tissue observed in the nectary ofS biglandulosum match the description of structural extrafloralnectaries in other species as reported by several authors (DaveandPatel 1975Elias andGelband1976Fahn1979Metcalfe andChalk 1979 Bentley andElias 1983Anderson and Symon 1985Gaffal et al 1998 Freitas et al 2001 Paiva et al 2001 Leitatildeoet al 2002 Rocha et al 2002 Thadeo et al 2008)

As observed by other authors studying different nectariesdruses are scattered among the ground parenchyma (Arbo 1972Elias et al 1975 Metcalfe and Chalk 1979 Bentley andElias 1983 Thadeo et al 2008) and may be related to calciumimmobilisation in the nectary region where active sugar transportis presumably occurring (Vezza et al 2006) Because sucrosetransport in plants involvesATPase activity (Giaquinta 1979) andCa2+ inhibits plasma membrane ATPase (Leonard and Hodges1980) this immobilisation may be a way to guarantee thecontinuous transport of sucrose

Tannins alkaloids and phenolic compounds are known tobe important substances acting on the anti-herbivore system ofthe plants (Croteau et al 2000 Dyer et al 2001 Almeida andFigueiredo 2003 Nicolson and Thornburg 2007) However

230 Australian Journal of Botany Iacute A C Coutinho et al

because the secreting cells are hidden within the secretory poresof this species producing these chemicals to help with nectaryprotection could be a waste of energy Because producingnectar is already a high-cost investment for the plant the lackof these compounds could be considered an adaptation to saveenergy Moreover as tannins were found at low concentrationsand ants were observed on the nectaries a shift in which theprotective chemicals are substituted by the patrolling ants couldbe possible Such a hypothesis concurs with what Elias (1980)had earlier hypothesised while studying Leonardoxa africana

Conclusion

On the basis of the ontogenetic process of the secretory structuresof Sapium biglandulosum anatomical description histochemicaltests and disaccharide and monosaccharide analysis of theexudate it can be concluded that the studied glands are in factextrafloral nectaries Although the presence of ants on themeristematic areas may be important to the plantrsquos survival aspecific study on the role of extrafloral nectaries inS biglandulosum is required for further conclusions

Acknowledgements

We thank FAPEMIG (Fundacatildeo de Amparo a Pesquisa de Minas Gerais) forthe financial support

References

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Clark G (1981) lsquoStaining proceduresrsquo (Williams ampWilkins BaltimoreMD)Contreras LS Lersten NR (1984) Extrafloral nectaries in Ebenaceae

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Elias TS (1980) Foliar nectaries of unusual structure in Leonardoxa africana(Leguminosae) an African obligate myrmecophyte American Journalof Botany 67 423ndash425 doi1023072442352

Elias TS Gelband H (1976) Morphology and anatomy of floral andextrafloral nectaries of Campsis (Bignoniaceae) American Journal ofBotany 63 1349ndash1353 doi1023072441843

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Fiala B Maschwitz U (1991) Extrafloral nectaries in the genus Macaranga(Euphorbiaceae) in Malaysia comparative studies of their possiblesignificance as predispositions for myrmecophytism BiologicalJournal of the Linnean Society Linnean Society of London 44 287ndash305doi101111j1095-83121991tb00621x

Freitas AVL Oliveira PS (1996) Ants as selective agents on herbivorebiology effects on the behaviour of a non-myrmecophilous butterflyJournal of Animal Ecology 65 205ndash210 doi1023075723

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Gabe M (1968) lsquoTechniques histologiquesrsquo (Masson et Cie Paris)Gaffal KP Heimler W El-Gamml S (1998) The floral nectary of Digitalis

purpureaL structure andnectar secretionAnnalsofBotany81 251ndash262doi101006anbo19970546

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Heil M Rattke J Boland W (2005) Postsecretory hydrolysis of nectarsucrose and specialization in antplant mutualism Science 308560ndash563 doi101126science1107536

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Keeler KH (1977) The extrafloral nectaries of Ipomoea carnea(Convolvulaceae) American Journal of Botany 64 1182ndash1188doi1023072442480

Keeler KH Kaul RB (1979) Morphology and distribution of petiolarnectaries in Ipomea (Convolvulaceae) American Journal of Botany 66946ndash952 doi1023072442236

Koptur S Rico-Gray V Placios-Rios M (1998) Ant protection of thenectaried fern Polypodium plebeium in central Mexico AmericanJournal of Botany 85 736ndash739 doi1023072446544

Leitatildeo CAE Meira RMSA Azevedo AA Arauacutejo JM (2002) Ontogenia dosnectaacuterios extraflorais de Triumfetta semitriloba (Tiliaceae) PlantaDaninha 20 343ndash351

Leitatildeo CAE Meira RMSA Azevedo AA Arauacutejo JM Silva KLFCollevatti RG (2005) Anatomy of the floral bract and foliarnectaries of Triumfetta semitriloba (Tiliaceae) Canadian Journal ofBotany 83 279ndash286 doi101139b05-001

Extrafloral nectaries of Sapium biglandulosum Australian Journal of Botany 231

Leonard RT Hodges TK (1980) The plasma membrane In lsquoThebiochemistry of plants Vol 1rsquo 1st edn (Ed NE Tolbert) pp 163ndash182(Academic Press New York)

Linsenmair KE Heil M Kaiser WM Fiala B Koch T Boland W (2001)Adaptation to biotic and abiotic stress Macaranga-ant plants optimizeinvestment in biotic defense Journal of Experimental Botany 522057ndash2065 doi101093jexbot523632057

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Madureira M Sobrinho TG (2002) Evidecircncia de mutualismo entre Qualeacordata (Vochysiaceae) e Cephalotes sp (Hymenoptera Formicidae)Academia Insecta 2 1ndash4 Available at wwwinsectaufvbracademiainsectavol2(1)201-4pdf [verified 18 August 2009]

Maia V (1979) lsquoTeacutecnica histoloacutegicarsquo (Atheneu Satildeo Paulo Brazil)McDade LA Turner MD (1997) Structure and development of bracteal

nectary glands in Aphelandra (Acanthaceae) American Journal ofBotany 84 1ndash15 doi1023072445877

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Metcalfe CR Chalk L (1979) lsquoAnatomy of the dicotyledons Systematicanatomy of leaf and stem with a brief history of that subject Vol 1rsquo2nd edn pp 124ndash131 (Claredon Press Oxford UK)

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NepiM (2007) Nectary structure and ultrastructure In lsquoNectaries and nectarrsquo1st edn (Eds SW Nicolson M Nepi E Pacini) pp 129ndash166 (SpringerDordrecht The Netherlands)

Nicolson SW (2007) Nectar consumers In lsquoNectaries and nectarrsquo 1st edn(Eds SWNicolsonMNepi E Pacini) pp 289ndash342 (Springer DordrechtThe Netherlands)

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Oliveira PS (1997) The ecological function of extrafloral nectariesherbivore deterrence by visiting ants and reproductive output inCaryocar brasiliense (Caryocaraceae)Functional Ecology 11 223ndash330doi101046j1365-2435199700087x

Paiva EAS Machado RS (2006) Ontogecircnese anatomia e ultra-estrutura dosnectaacuterios extraflorais de Hymenaea stigonocarpa Mart ex Hayne(Fabaceae ndash Caesalpinioideae) Acta Botacircnica Brasileira 20 471ndash482

Paiva EAS Morais HC Isaias RMS Rocha DM Oliveira PE (2001)Occurrence and structure of extrafloral nectaries in Pterodonpubescens Benth and Pterodon polygalaeflorus Benth PesquisaAgropecuaria Brasileira 36 219ndash224doi101590S0100-204X2001000200002

Pearse AGE (1980) lsquoHistochemistry theoretical and applied Vol 2rsquo4th edn (Churchill Livingston Edinburgh UK)

Rocha JF Neves LJ Pace LB (2002) Estruturas secretoras em folhas deHibiscus tiliaceus L E Hibiscus pernambucensis ARRUDAUniversidade Rural Seacuterie Ciecircncias da Vida 22 43ndash55

Roeser KR (1972) Die Nadel der Schwarzkiefer Massenprodukt undKunstwerk der Natur Mikrokosmos 61 33ndash36

Rogers WE Siemann E Lankau RA (2003) Damage induced productionof extrafloral nectaries in native and invasive seedlings of Chinesetallow tree (Sapium sebiferum) American Midland Naturalist 149413ndash417 doi1016740003-0031(2003)149[0413DIPOEN]20CO2

Roshchina VV Roshchina VD (1993) lsquoThe secretory function of higherplantsrsquo (Springer-Verlag Berlin)

Schnepf E (1974) Gland cells In lsquoDynamic aspects of plant ultrastructurersquo(Ed AW Robards) pp 331ndash357 (McGraw-Hill London)

Silva EMJ Machado SR (1999) Estrutura e desenvolvimento dos tricomassecretores em folhas de Piper regnellii (Miq) CDC var regnellii(Piperaceae) Revista Brasileira de Botacircnica 22 117ndash124

So ML (2004) The occurrence of extrafloral nectaries in Hong Kong plantsBotanical Bulletin of Academia Sinica 45 237ndash245

Thadeo M Cassino MF Vitarelli NC Azevedo AA Arauacutejo JM ValenteVMM Meira RMSA (2008) Anatomical and histochemicalcharacterization of extrafloral nectaries of Prockia crucis (Salicaceae)American Journal of Botany 95 1515ndash1522 doi103732ajb0800120

Vezza M Nepi M Guarnieri M Artese D Rascio N Pacini E (2006) Ivy(Hedera helix L) flower nectar and nectary ecophysiology InternationalJournal of Plant Sciences 167 519ndash527 doi101086501140

Manuscript received 6 November 2009 accepted 5 February 2010

232 Australian Journal of Botany Iacute A C Coutinho et al

httpwwwpublishcsiroaujournalsajb