Scanning Electron Microscopic Studies (Euphorbiaceae) *

Original article

Scanning electron microscopic studies on pollen from honey. IV. Surface pattern of pollen of Sapium sebiferum and Euphorbia spp (Euphorbiaceae) *

JH Dustmann, K von der Ohe

Niedersächsisches Landesinstitut für Bienenkunde, Wehlstraße 4a, D-3100 Celle, Germany

(Received 19 March 1992; accepted 14 October 1992)

Summary — Several species of Euphorbia and Sapium plants produce considerable amounts of nectar, which is collected by bees for honey production. In order to identify the nectar source species we used the usual method of melissopalynology (identification of pollen grains by light microscopy) as well as scanning electron microscopic (SEM) studies. Authenticated specimens of pollen were examined in a state of contraction as well as expansion. Four types of exine surface pattern could be distinguished by SEM. E Cœrulescens, E ledienii and E tetragona show the same 3 variations in exine structure (type A, B, C). It was therefore not possible to distinguish between these 3 Euphorbia species. However Sapium sebiferum showed a specific pattern and uniformity of exine structure (type D). pollen / honey / Euphorbiaceae / light microscopy / scanning electron microscopy (SEM)

INTRODUCTION or Ricinus in honeys from different coun- tries (Zander, 1941). The existence of The family Euphorbiaceae is one of the special honey sources with a large largest in the worlk, comprising > 300 gen- amount of nectar from Euphorbia species era (Heywood, 1982). Pollen of some gen- led us to investigate the possibility of iden- era gathered by honeybees are well- tifying pollen of different species within the known in melissopalynology and enable genus Euphorbia. These honeys are com- the botanical origin of honey specimen to monly known as ’noors-honeys’, which be identified. For example, it is possible to can cause acute burning in the mouth and identify pollen of different species of Eu- throat (Sosath et al, 1988). In addition we phorbiaceae such as Croton, Euphorbia wished to determine pollen characters via

* These studies constitute part of a comprehensive research program led by E Hecker, Krebsfors- chungszentrum Heidelberg on several Euphorbiaceae. examination of pollen grains that would found in the LM glycerine jelly preparations, pol- distinguish Euphorbia honey from that of len grains were transferred into filtered honey Sapium sebiferum (Euphorbiaceae). Mi- for 48 h at 35 °C. After using Method I of Dust- mann and Bote (1985) — transfer to a wire as well as elec- croscopy (LM) scanning screen, removal of ’pollenkitt’ by ultrasonics, de- tron was therefore microscopy (SEM) hydration in acetone and a graded ethanol se- used. The melissopalynological study of ries, critical point drying (CPD) — pollen was the Euphorbia honeys was uninformative dusted onto an aluminum stub provided with because of the naturally low content of Eu- double-sided tape, and coated with a 20-nm phorbia pollen grains. Nevertheless these gold layer. All SEM specimens were examined at an acceleration voltage of 10 kV. honeys contained high amounts of Eu- The was used: Photo- phorbia nectar, as confirmed by sensory following equipment III SEM ISI-Super-II; Sput- test and bee botanical observations. Low Microscope (Zeiss); ter-Coater ISI PS-2; Critical Point Drying Appa- pollen content is well-known for some oth- ratus E 3000 (Polaron). Nomenclature for de- er honey sources such as those of lime scriptions was according to Faegri and Iversen tree (Tiliaceae) and false acacia (Legu- (1964). minosae) (Zander, 1935; Louveaux et al, 1970, 1978). Based on these facts, we examined authentic specimens of pollen RESULTS gathered in South Africa (Euphorbia) and Louisiana, USA (Sapium). To compare complete the results of the LM and SEM LM examination examination we used Method I of Dust- mann and Bote (1985) to obtain speci- Expanded pollen grains of Euphorbia coer- mens from similar honey of expanded pol- ulescens, E tetragona and Sapium sebifer- len grains for SEM study thus providing a um are tricolporate and characterized by possibility to transfer our results directly to the same spherical shape (equatorial melissopalynology. view). The ectexine includes the foot layer, columellae and a perforate tectum. The grains lack any supratectal sculpturing. MATERIALS AND METHODS Size varied from 32.5-42.5 μm within each species. In all pollen grains the characteristic contour of the cell could be ob- The Krebsforschungszentrum Heidelberg ob- living served a around the tained the following authenticated pollen sam- forming yard pores ples: 1) Euphorbia coerulescens: Jansenville / which depended on intine thickness, and Caroo, South Africa, 1980; 2) Euphorbia ledie- seen in polar view (fig 1). nii: Kleinpoort / Caroo, South Africa, 1980; 3) Euphorbia tetragona: Keiskama Valley, South Africa, 1980; 4) Sapium sebiferum: Louisiana, SEM examination USA, 1982. After degreasing in ether, pollen grains were prepared for LM and SEM examination. The Contracted pollen grains examined of the common palynological method of embedding in genera Euphorbia and Sapium were visible glycerine jelly (Erdtman, 1952) was used for only in equatorial view depending on their LM. Pollen grains were prepared in 2 ways for prolate-oval shape. Differences in size and SEM: 1) contracted pollen was directly dusted were The character- onto aluminium stubs provided with double- shape non-significant. istic was sided tape, then coated with a 20-nm gold margo along every colpus hardly layer; 2) for the expanded condition at onset of visible as the germination furrows were germination, ie the same condition and shape closed. Parts of the exine or single perfora- tened straight to the colpus margin, the apertural endexine shows a verrucate pattern; the exine structure is tectate-perforate.

Type A

The exine pattern is regular tectate- perforate, the luminae are small and circular. The diameter is frequently smaller (up to 0.2 μm) than the width of the muri (up to 0.7 μm). The muri are plain or slightly vaulted (figs 2a, b; 3a, b)

Type B

The exine pattern is regular tectate- perforate. The luminae vary in size (up to 0.7 μm) and shape (circular to oval). The muri are vaulted, with a width ≤ diameter of the luminae. The tectum pattern mainly at the intercolpium is partially striate- reticulate (figs 2c, d; 3d, c).

Type C tions (luminae) of the tectum were covered with ’pollen kitt’. The exine pattern is irregular tectate- Expanded pollen grains of all speci- perforate. Most of the luminae are small mens allowed a more detailed study in the (≈ 0.2 μm), with irregular sometimes elon- polar as well as in the equatorial view. In E gated dimensions this possibly being de- coerulescens, E ledienii and E tetragona termined by compressed and partially in- we found the same 3 variations in exine complete muri. The width of the high pattern, designated as type A, B and C. vaulted muri measures up to 0.7 μm (figs Only Sapium sebiferum showed uniformity 2e, f; 3e, f). in pollen exine pattern, which as it differed from type A, B and C was termed type D. Type D

exine Description of pollen pattern The exine pattern is tectate- in the different regular types perforate characterized by small luminae (diameter ≈ 0.2 μm). The muri are wide (up All pollen types are tricolporate; colpi are to 1.2 μm) and generally plain (figs 2g,h; surrounded by a smooth, wide margo, flat- 3g,h).

DISCUSSION um sebiferum exhibited only 1 type of characteristic tectate pattern, type D, which was not found in any of the Euphorbia Pollen grains of Euphorbia coerulescens, species. E ledienii, E tetragona and Sapium sebiferum exhibit similarities in size, overall The results of the present study show shape and exine structural pattern. Using that there are no microscopic characters of the 3 LM we were able to determine shape, con- that distinguish pollen grains spe- struction of the exine and contour of the cies of Euphorbia via analysis of the exine living cell. As volume of pollen grains structural pattern. However, it is quite pos- changes in particular and possibly also sible to find specific identifying marks in shape and surface pattern hydration of the different general of Euphorbiaceae via plasm (Blackmore and Barnes, 1985; SEM. Dustmann and Bote, 1987; von der Ohe and Dustmann, 1990) size cannot be a cri- terion in determining specific differences. ACKNOWLEDGMENTS The study of structural pattern of the ectexine was not satisfactory due to the limit- We are very grateful to E Hecker and S Sosath ed depth of focus of the LM. SEM studies (Krebsforschungszentrum, Heidelberg) for sup- were carried out without acetolysis, a plying us with samples of honey and pollen from method already used in identifying ultra- the different Euphorbiaceae. structures in the pollen surface of apple varieties (Dustmann and Bote, 1987; von Résumé — Étude en élec- der Ohe, 1991). microscopie tronique à balayage du pollen du miel. Besides this, the acetolysis method IV. Sculpture de l’exine des pollens de (Erdmann, 1960) causes damage and loss diverses espèces d’Euphorbiacées. De of single pollen grains. This would not al- nombreuses plantes de la famille des Eu- low the SEM method to be used for Eu- phorbiacées produisent des quantités which shows an ex- phorbiceae honey, considérables de nectar, que les abeilles treme of under-representation pollen. utilisent pour le miel. L’existence des The differences in structural pattern «miels de noors» ayant un fort pourcen- formed by a perforate tectum observed tage de nectar d’Euphorbia et de Sapium via SEM do not allow differentiation of the nous a permis de rechercher des moyens 3 Euphorbia species. Although 3 structural d’identification de ces pollens, afin de dé- pattern types (type A, B and C) were terminer sans ambiguïté l’origine botanique recognized, a reference to any species is des miels. Pour ce faire, des échantillons not possible: all 3 types were found in de pollen d’Euphorbia coerulescens, E le- each specimen of Euphorbia coerules- dienii, E tetragona et Sapium sebiferum cens, E ledienii and E tetragona. The ont été observés en microscopie optique et pattern differs in shape and diameter of en microscopie électronique à balayage the luminae, which are sometimes elon- (MEB). L’analyse en microscopie optique gated and formed by incomplete muri, as n’a permis aucune différenciation : les pol- well as in the width and height of the muri. lens d’Euphorbia aussi bien que de Sa- As all pollen grains were prepared in the pium sont tricolporés, avec une marge, et same manner, we conclude that this varia- montrent en vue polaire le contour typique tion is characteristic of these species of du cytoplasme (fig 1). Il n’est pas possible Euphorbia. In comparison, pollen of Sapi- de mettre en évidence des différences dans la structure de l’exine en raison de la Hierzu wurden authentische Pollenpro- faible profondeur de champ du microscope ben von Euphorbia coerulescens, E ledie- optique. Pour les études au MEB, on a fait nii, E tetragona und Sapium sebiferum passer des pollens en état de contraction à sowohl mit dem Lichtmikroskop als auch l’état d’expansion, en les mettant dans du mit dem Raster-Elektronenmikroskop miel filtré. Après les avoir isolés, on les a (REM) untersucht. Lichtmikroskopische transférés sur un crible métallique (Dust- Analysen ermöglichten keine Differenzie- mann et Bote, 1985). rung: Sowohl Euphorbia- als auch Sapium- Pollen sind und Tous les pollens étudiés ont montré une tricolporat, marginat in der die Kontur structure de l’exine tecto-perforée. Quatre zeigen Pollage typische des Plasmas types de structures ont été trouvés : type A (Abb 1). Unterscheidungen der Exine-Struktur waren der (fig 2a, b; 3a, b), type B (fig 2c, d; 3c, d), aufgrund ge- Tiefenschärfe des Lichtmikroskopes type C (fig 2e, f; 3e, f) et type D (fig 2g, h; ringen nicht Für die REM- 3g, h). Les sculptures se différencient par möglich. wurden kontrahierte la disposition et la taille des perforations et Untersuchungen Pollen zur Expansion in Honig par l’architecture des muri. Les types A, B pollenfreien überführt und nach Isolierung in ein Edel- et C se rencontrent chez toutes les espè- stahlnetz präpariert (Dustmann und Bote, ces d’Euphorbia et ne permettent donc pas Alle untersuchten Pollen de différencier les 3 espèces coerules- 1985). zeigen eine tectat-perforate Exine-Struktur. Insge- cens, ledienii et tetragona. Par contre Sa- samt wurden 4 verschiedene Struktur- pium sebiferum présente une sculpture Typen gefunden: Typ A (Abb 2a, b; 3a, b), spécifique et une uniformité de la structure Typ B (Abb 2c, d; 3c, d), Typ C (Abb 2e, f; de l’exine (type D) qui, par ses muri extrê- 3e, f) und Typ D (Abb 2g, h; 3g, h). Die mement et plats, se distingue de larges Oberflächenmuster unterscheiden sich in tous les autres Il est donc possible types. und Größe der Perforationen de différencier les miels d’Euphorbia de Anordnung sowie im Aufbau der Muri. Typ A, B und C ceux de Sapium sebiferum. treten bei allen Euphorbia-Arten auf und lassen somit eine von E pollen / miel / Euphorbiacea / microsco- Differenzierung coerulescens, E ledienii, und E pie optique / microscopie électronique tetragona nicht zu. à balayage Sapium sebiferum zeigt jedoch ein spe- zifisches Muster und eine einheitliche

Zusammenfassung — Raster- Exine-Struktur (Typ D), die sich aufgrund elektronen-mikroskopische Studien an der extrem breiten, flachen Muri von allen anderen absetzt. Pollen aus Bienenhonig. IV. Oberflä- Typen chenmuster von Pollen aus verschiede- Die Ergebnisse zeigen, daß mit Hilfe nen Euphorbiaceae Arten. Zahlreiche des Raster-Elektronenmikroskopes die Pflanzen aus der Familie der Euphorbiace- hier untersuchten Pollen der Gattung Eu- ae produzieren beträchtliche Mengen an phorbia nicht untereinander aber von Nektar, der von Honigbienen eingetragen Sapium sebiferum unterschieden werden wird. Das Auftreten sog "Noors-Honige" mit können und somit eine Differenzierung der hohen Anteilen an Euphorbia- und entsprechenden Honige möglich ist. Sapium-Nektar veranlaßte uns, eine Mög- lichkeit zu finden, diese Pollen zu identifi- Pollen / Honig / Euphorbiaceae / Licht- zieren, um die botanische Herkunft der mikroskopie / Raster-Elektronen- Honige sicherzustellen. mikroskopie REFERENCES der IUBS — Methodik der Melissopalynologie. Apidologie 1 (2), 193-209 Blackmore S, Barnes SH (1985) Harmomegath- Louveaux J, Maurizio A, Vorwohl G (1978) ic mechanisms in pollen grains. In: Pollen Methods of melissopalynology. Bee World 59 and Spores: Form and Function: No 12 (4), 139-157 (Blackmore S, Ferguson IK, eds), London Sosath S, Ott HH, Hecker E (1988) Irritant prin- 173-149 ciples of the spurge family (Euphorbiaceae) Dustmann JH, Bote K (1985) Raster-elektronen- XIII. Oligocyclic and macrocyclic diterpene esters from lattices of some mikroskopische Studien an Pollen aus Bie- Euphorbia spe- nenhonig. I. Vergleich verschiedener cies utilized as source plants of honey. J Nat Präparationsmethoden. Apidologie 16 (3), Prod 51, 1062-1074 331-340 Ohe K von der, Dustmann JH (1990) Raster- Studien an Pollen Dustmann JH, Bote K (1987) Raster-elektronen- elektronenmikroskopische aus III. Der mikroskopische Studien an Pollen aus Bie- Bienenhonig. Harmomegathie- nenhonig. II. Vergleich der Exine-Strukturen Mechanismus und seine Auswirkungen auf trockener und in Honig expandierter Pollen die Exine-Strukturen am Beispiel unter- bei verschiedenen Apfel- bzw. Zierapfelsort- schiedlicher Pollentypen. Apidologie 21, 293- 302 en. Gartenbauwissenschaft52, 271-278 Erdtman G (1952) Pollen Morphology and Plant Ohe K von der (1991) Scanning electron micro- Taxonomy. Almquist and Wik- scopic studies of pollen from apple varieties. Angiosperms. 6th Pollination Acta Hortic 405- sells, Uppsala, 539 pp Symposium. 409 Erdtman G (1960) The acetolysis method. Zander E zur Herkunftsbestim- Svensk Bot Tidsskr 54 (4), 561-564 (1935) Beiträge mung bei Honig. I. Pollengestaltung und Her- Faegri K, Iversen J (1964) Textbook of Pollen kunftsbestimmung bei Blütenhonig. Verlag Analysis. Munksgaard, Copenhagen, 237 pp der Reichsfachgruppe Imker eV, Berlin VH der Welt. Heywood (1982) Blütenpflanzen Zander E (1941) Beiträge zur Herkunftsbestim- Birkhäuser 335 Verlag, Basel, pp mung bei Honig. III. Pollengestaltung und Louveaux J, Maurizio A, Vorwohl G (1970) In- Herkunftsbestimmung bei Blütenhonig. Ver- ternationale Kommission für Bienenbotanik lag Liedloff, Loth und Michaelis, Leipzig