Chemical aspect of floral biology inEuphorbia fragifera

I~URA 0OASSlNI L o ~ ,

V~NZmA~DO MA~RIOH2 a n d L M O POLDINI t

, I n s t i t u t o ed Orto Botanico, UniversitA, Via Valerio 30, Trieste, Italy 2 Institute Tecnica e Legislazione Farmaceutics, 17niverslt/~, Via Cumano 2, Trieste, Italy

Chemical Aspect of Floral Biology in Euphorbia/ragi/era

Keywords

Euphorbiaceae, Euphorbia ]ragi/era, Floral biology Abstract COASSINI LOKARL., MAURICHV. et POLDINI L. (1986): Chemical aspect of floral biology in

Euphorbia ]ragi]era. -- Foils Geobot. Phytotax., Praba, 21: 277--285. -- The composition of volatile oils produced by Euphorbia ]ragifera is reported. The plant samples were collected in two different habitats on the Karst plateau :North of Trieste (North :Eastern Italy). The analysis was performed by capillary GC. Differences between samples from the two habitats have been found in the quantitative composition of the oils. The plants coming from the optimal, xeric habitat produce oils in which aromatic terpenes (mostly carvacrol) are more abundant, whereas the oils of plants growing in shady and moist silos are mostly composed by acyclic compounds (chiefly geraniol). It seems therefore possible to distinguish two chemical variants of Euphorbia ]ragi]era. The number and kind of pollinating insects have been recorded. Terpenoids from the two kinds of essential oil could be responsible of two different pheromones that attract different kinds of pollinators. INTRODUCTION

Euphorbiafragifera JA~ (Euphorbiaceae) is a n l l l y r i a n endemic species ranging from the Trieste K a r s t in N o r t h - E a s t e r n I t a l y (where it occurs from sea level to 600 m) to D a l m a t i a a n d Montenegro in Yugoslavia. I t is a typical c o m p o n e n t of xerophytic v e g e t a t i o n o n limestone, being a high frequency species in the Salvio-Euphorbietum fragiferae ( I ~ u s I et POLDn~I 1962), a garigue d o m i n a t e d b y Labiatae a n d •uphorbiae. This species m a y be also f o u n d outside its o p t i m a l h a b i t a t , within s h a d y mesophytic woods, where its occurrence is sporadic. The chemical composition of the latex of Euphorbia species is well k n o w n ( A ~ D et al. 1977; E v e s 1977; Kr~GHOR~ et EVANS 1975; NI~.LS~N et al. 1979; RODRmtmZ et al. 1976; SCHmDT et EvA,~-S 1976; UPADn~Au et H ~ C K ~ 1975). However, little is k n o w n a b o u t the composition of essential oils in Euphorbia. I n particular, the oils

FOLIA OEOBOTANICA ET PHYTOTAXONOMICA 21, 1988

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of Euphorbiafra~ifera h a v e b e e n n e v e r analyzed. This species a t t r a c t e d o u r a t t e n t i o n because of its strong scent. This is similar t o t h e o n e of Valeriana officinalis L. s. 1., a n d is p r o d u c e d during t h e anthesis. The o d o u r is s t r o n g e r in t h e p r i m a r y s t a t i o n s (Salvio-Euphorbietum ]raffiferae), weaker a n d s u b t l e r in sh ad y a n d m o i s t sites. I n t h e present p a p e r we p r e s e n t t h e results of t h e chemical analysis of essential oils in Euphorbia fraffifera, w i t h p a r t i c u l a r r e g a r d t o differences b e t w e e n specimens growing in xeric a n d in mesic conditions. W e also t e s t w h e t h e r such differences influence t h e kind a n d t h e n u m b e r of visiting insects d u r i n g t h e anthesis. M A T E R I A L S AN D M E T H O D S Sampling

of plants

and

pollinator

insects

Plant material has boon collected during the flowering phase in the two habltats (A = Salvioand moist sites). Collection was carried out from April 15th to May 15th in the years 1981 and 1982, at five days' intervals. In each habitat 50 specimens were collected by random sampling within 8 grid-shaped transects. The 50 specimens represent an average sample. Both of the habitats had limestone as geological substrate. No significant morphological differences exist between the populations of the A-habitat and t h o s e of the B-habitat. Duncan's test performed on the basis of several morphological characters gave a similarity ratio P < 0.001. Insects have been captured during the period of anthesis, from 9 am to 7 pm, at one hour intervals for several days. The captures have boon made both with optimal weather conditions (lack of wind, clear sky, temperatures within the seasonal range) and with non optimal conditions (wind, cloudy sky, temperatures and air humidity far from the seasonal range). The insects have been identified in the laboratory, t) Plant specimens of dry and mesic habitats a r e deposited in the tIerbarium Universitatis Torgostinae (TSB).

Euphorbietum ]ragiferae; B -~--shady

Methods The plant samples were carried to the laboratory and conserved in a freezer. The infloreseencos were treated witil liquid N2, finely ground (60 mesh), weighed (50 g. dry weight estimated on another sample). The extraction was carried out with steam current (Markuson's device) to exhaustion. The essential oil, extracted by ether, has been analyzed by GLC (Fraetovap 2450 C. Erba) with double stainless column (2 m • 4/2 ram), 10 % Carbowax 20 M on Chromosorb n (60--80 mesh), flame ionization detector, digital integrator; 1 ~t sample; injection and detector temperatures 260 ~ linear temperature programmer 60--220 ~ (4 ~ N2 flow rate 30 ml/m~n. Compound identities were confirmed by co-chromatography with authentic compounds, and by the technique of single fractions enrichment. A contingency table has boon obtained on the basis of the number of visiting insects in each habitat. Such table has been analyzed by the chi square analysis with the program CLACOMP (FEOLI, Laoo:~v.~Ir et ORLOC~ 1984). RESULTS Most of t h e essential oil is p r o d u c e d b y Euphorbia fragifera d u r i n g t h e flowering phase corresponding to full anthesis. Samples f r o m t h e A - h a b i t a t a l w a y s c o n t a i n higher q u a n t i t i e s of oils (0.20 % ) t h a n those f r o m t h e B - h a b i t a t (0.12 %) . 1) We thank Dr. P. BRA.N'D~AYR,of the Zoological Institute of Trieste University for his help in identifying the visiting insects, and Dr. P. G.,~IS, of the Botanical Institute of Trieste University, for her collaboration in data processing.

COA$INI :LOKAR, I~AURICII AI~D POLDINI: EUPHORBIA FRAGIFERA

279

No significant differences in the composition of the essential oils have been found between the samples coming from the transects of the A- and the B-habitat. Both are rich in compounds (36 compounds, of which the most important have been identified). They contain acyclic compounds (geraniol, linalbol), monocyclic compounds (T-terpinene, carvon, limonene, menthol), bicyclic compounds (thujone, borneol, bornylacetate) and sesquiterpenic compounds (:Figs. ], 2). Tab.

1. Percent composition of essential oils extracted from samples of Euphorbia ]ragifera collected in the t w o habitats (A and B)

number

compounds

A habitat

1

Thujone

0.79

2

--

tr

linalool -bornylacetate ~-caryophylleno borneol earvon

0.46 tr 1.81 5.80 tr 3.86 9.22

3 4 5 6 7 8 9 10

--

--

2.11

I 1

-

0.22

12

-

--

B habitat 1.29 tr -tr --

9.05 --

1.08 1.65 15.85 -tr

13 14

geraniol

1.24

--

tr

--

15

--

3.03

4.31

16 17

---

0.31 --

tr

18

--

0.90

19 20

-

5.25 0.77

21

--

22 23 24 25

--thymol carvaerol

--

tr 0.30 1.88 61.55

59.65 --

52 0.45 0.78 tr 0.60 1.42 1.21 3.36 O.

t r - - traces. Significant differences have been found in the quantitative composition of the extract between samples from the A- and the B-habitats. The essential oil of samples collected in the optimal habitat (A) is characterized by a high percentage of carvacrol (61.55 %), followed by earvon (9.22 %), borneol (3.86 %), ~-caryophyllene (5.80 %}, thymol (1.88 %), and bornylacetate (1.81%). The principal component of the oil extracted from samples collected in the B-habitat is geraniol (59.64 %), followed by ~-caryophyllene (9.05 %), carvacrol (3.36 %), carvon (1.65 %) and borneol (1.08 %) (Tab. 1). I t follows that in the optimal A-habitat the essential oil of Euphorbia fraqifera is richer in aromatic terpenes (chiefly carvacrol), monocyclic and bicyelic terpenes, while in the B-habitat the oil has a higher content in acyclic terpenes of alcoholic nature and in sesquiterpenes (Tab. 2).

280

FOLIA GEOBOTANICA ET PHYTOTAXONOI~IICA 21, 1986

25

fl

19

22 23/ 19 2 4 / / ' 15

25 20

10

1

1

Fig. 1. Gas-chromatogram of the essential oil of Euphorbia ]ragi]era, xeric habitat (A).

13

5

Fig. 2. Gas-chromatogram of t h e essential oil of Euphorbia ]ragi]era, shady habitat (B).

Tab. 2. Percentages of terpene classes in the essential oils extracted from samples of Euphorbia fragifera collected in t h e t w o habitats (A and B) Percent composition

acyclic terpenes 1) monocyclic terpenes:) bieyelic terpenes 3) aromatic terpenes 4) sesquiterpenes s)

habitats A

B

1.70 9.22 6.46 63.43 5.80

59.64 1.65 2.37 4.57 9.05

x) geraniol, linalool, 2) carvon~ 3) thujone, bornylacetate, borneol, 4) thymol, carvacrol, 5} ~-earyophyUene.

Since we do not know whether this chemical variation is due to genetical (chemotypes) or ecological reasons, in the following we shall use the less binding terms of "chemical variant 1 (carvaerol)" for the samples coming from the A-habitat, and "chemical variant 2 (geraniol)" for those coming from the B-habitat. The evident relation between the ecological characterization of the two habitats and the different kinds of scent produced by Euphorbiafragifera, suggests that differences in-the scent could be related to different visiting and/or pollinator insects present in the two habitats.

COASINI LOKAR, MAURICH AND POLDINI: EUPHORBIA FRAGIFERA

281

Enthomogamous plant species produce scents, called Pheromones, that are mixtures of chemicals carrying biological messages to the pollinating insects. The most important receptors for plant pheromones ( = releaser pheromones) are the antennae of the insects, that may perceive odours thanks to sensory cells (sensillae). The difference in electric potential created between the base and the top of the antennae is transmitted to the central nervous system and transformed into a biochemical impulse. Euphorbia fragifera, like other non self-compatible species, requires insects for impollination. This is the main reason for the production of the scent, that is perceivable also at a certain distance.

2O

-

]01'11'21'31~4]'5161'7181~)

hours

Fig. 3. Number of insects visiting Euphorbia/ragi/era in the A-habitat during the day (optimal weather conditions). 9 Hymenoptera (ram.: Formicidae). o Coleoptera (fam. : Mordellidae, 1Vitidulidae, Buprestidae). D Diptera (faro.: Syrphidae). 9 Heteroptera (faro.: Pentatomidae).

Visitors of Euphorbiafrayifera's inflorescences have been recorded during the whole day in the period from April 15th and May 15th. Recorded data refer to number of visitors, zoological taxa and behaviour. The data are in the form of averages of observations in the units of the transects located in the A- and B-habitats.: in each of them an equal number of plants has been considered. The selected plants had the same number of inflorescences (5). The observations have been carried out for 70 hours. The number of visits by different insect species was strongly influenced by the flowering ratio; in the periods before and after full anthesis the number was reduced to less than 40 % of that during full anthesis. In the A-habitat, Euphorbia fraqifera has been visited by a great number of Coleoptera (families: Mordellidae, Nitidulidae, Buprestidae; total: 71), b y Diptera (family: Syrphidae; total: 35) and by few Heteroptera (family: Pentatomidae; total: 6). The visits by Coleoptera are more frequent from 1.00 to 6.00 pro, with two peaks at 3.00 and 6.00. Hymenoptera are most frequent at 1.00 and 2.00 pm, with a little number of visits in other periods of the day. Diptera are most frequent in early morning (from 9.00 to 11.00 am). Heteroptera seem to be casual visitors (:Fig. 3). The highest number of visitors, independently from their taxonomical status, is observed from l l . 0 0 a m to 2.00pro, the lowest from 6.00 to 7.00pro. Visitors belonging to other taxa than the ones listed above have not been taken into consideration (e.g. Orthoptera, family Tettiqonidae), because they appeared once or twice in a day,

282

FOLIA GEOBOTANICA ET PHYTOTAXONO~IICA 21, 1986

and because they had too large a size to contribute to pollination. The scent of the chemical variant 1 (earvacrol) selectively attracts insects belonging to four different Orders. They are all of small size (0.5--0.8 cm), with long and remarkably complex antennae. The chemical variant 2 (geraniol), occurring in the B-habitat, produces a type of oil that selectively attracts Diptera (suborder: Nematocera; 37 in total) and Hymenoptera (faro.: Formicidae, Ichneumonidae; total: 119). The latter are most frequent from 1.00 to 2.00 and from 5.00 to 7.00 pm. Visits of Nematacera are evenly distributed throughout the day, with a sl:ght max:mum from noon to 1.00 pm and two minima in early morning and late afternoon (Fig. 4).

3025t

25

20

~o

1

g2o ]

>

; Io~

=

5t 0E

cI0

1011|213141516 #1~319hours

Fig. 4. Number of insects visiting Euphorbia/ragi]era in the B-habitat during the d a y (optimal weather conditions) 9 Hymenoptera (faro.: Formicidae, Ichneumonidae). a Diptera (subord.: Nematocera).

5

1011121~l~lf51~l"/f81'hours 9

Fig. 5. Comparison of cumulative insect visits in the two habitats. (A 9 and B u).

A considerable number of small Apoidea (Hymenoptera) was also captured (some genera were attracted by geraniol), but their contribution to pollination seems doubtful, considering their size, that is much larger than that of the other pollinators. The total number of visits (Fig. 5), subdivided according to different periods of the day, is always smaller in the B-than in the A-habitat. It seems that the scent produced by the chemical variant 2 (geraniol) is particularly attractive for Hymenoptera. These insects probably have a smaller receptivity for pollens and a lower probability of successful pollination, since their antennae are very small and only slightly differentiated.

COASINI LOKAR, I~fAURICH AND POLDINI:

EUPHORBIA

Tab. 3. Contingency table of insects visiting Euphrobia fragifera in Xz analysis (9 degree of freedom) Visitators

Coleoptera

ltfordellidae N/r

Bupre,stidae Formicidae Hymenoptera-- = lchneumonidae Apoidea Diptera _ [= Syrphidae Nematocera Heteroptera Pentatomidae Orthoptera Tettigonidae HomoTtera Aphidiidae

283

FRAGIFERA

the two

habitats (A and B),

A habitat

B habitat

X2probability • 100

48 10 13 63 1 6

3 0 0 5 114 14

100.0 100.0 100.0 100.0 100.0 92.9

35 0 6 3 1

14 37 0 3 I

99.8 100.0 99.6 0.6 0.3

The chi square analysis of the contingency table visitors-habitats gave the results presented in Tab. 3. Statistical differences in the visitation patterns are significant at Z2 > 99.0, only as far as Coleoptera, Hymenoptera and Diptera are concerned. The number and the types of visitors depend on the chemical composition of the essential oil. Ichneumonidae (Hymenoptera) seem to play an important role in pollination only within the B-habitat, whereas Coleoptera and Diptera are important pollinators in the A-habitat. This could affect the distribution of the species in the two habitats, with a possible positive selection towards the Salvio-Euphorbietum fragiferae.

GENERAL

CONCLUSIONS

The above discussed results point to a strong correlation between environmental conditions and quantitative composition of the terpenes in the essential oil produced by Euphorbia fragifera. Many cases are known in which the variability of monoterpenes can be interpreted in biosystematical, physiological or evolutionistic terms. In the case of Euphorbia fragifera, it seems that the production of these compounds has an ecological meaning. This species is linked to the mediterranean vegetation belt of the Dalmatian coast (Yugoslavia), where it grows on warm, very arid rock outcrops. Such a kind of environment has its last offshoot along the coastal strip cf the Karst plateau North of the town of Trieste (North-Eastern Italy). The species has a marked chemical polymorphism induced by the environment. In its optimal habitat (Salvio-Euphorbietum fragiferae), the content of carvacrol may be 49 times higher than in the mesic habitat of occasional occurrence, and the content of geraniol is 23 times higher in the latter than in the former. I t is therefore possible to distinguish two chemical variants of Euphorbia fragifera, according to the chemical composition of the essential oil.

284

FOLIA GEOBOTANICA ET PHYTOTAXOhrO,~fICA 21. 1986

The influence of some e n v i r o n m e n t a l factors (like moisture, t e m p e r a t u r e , light i n t e n s i t y , insolation ratio) o n t h e biosynthesis of m o n o t e r p e n o i d s has been d e m o n s t r a t e d b y some authors (GERsH~ZO~ et al. 1978; Ln~COLN et al. 1978, 1979) a n d it has b e e n confirmed b y us in some studies on the c o m p o s i t i o n of essential oil of S a t u r e ] a species ( P o L D I ~ et S T ~ c m ~ 1969; CoAssr~I L o K ~ et al. 1983) a n d of A r t e m i s i a e l b a TURR~ (CoAssI~I L o ~ et al. in p r e s s ) . I n general, high t e m p e r a t u r e a n d strong i n s o l a t i o n favour the biosynthesis of byciclic a n d aromatic terpenes, whereas low ~ m p e r a t u r e s a n d weak i n s o l a t i o n are more favourable for the synthesis of acyclic a n d monocyelic terpenes. The differences in the q u a n t i t a t i v e composition of the essential oil, t h a t are reflected i n the different k i n d of scent produced b y p l a n t s growing in different ecological conditions, have been discussed in relation with their a t t r a c t i v e power t o w a r d s pollinators. Some of the c o m p o u n d s t h a t are present in the oil p r o d u c e d b y p l a n t s growing in v e r y s u n n y sites (A-habitat) have a great a t t r a c t i v e power towards visiting insects. These are a t t r a c t e d i n great n u m b e r , a n d several species are involved. The great d i v e r s i t y in size, a n d i n the shape a n d s t r u c t u r e of the sucker a p p a r a t u s c o n t i b u t e s to a more frequent a n d effective pollination. I n moist a n d shady sites (B-habitat), the principal c o m p o n e n t of the essential oil (geraniol) t e n d s to a t t r a c t D i p t e r a a n d H y m e n o p t e r a . These insects c o n t r i b u t e less to effective pollination, because of their very small a n d simple a n t e n n a e . LITERATURE CITED. A ~ A D S., SELIOMAN~rO. et HUSSAX• G. (1977): Caudicifolin, a new diterpene from E. caudicr /olia. -- Phytochern. 16: 1844. COASSINI LOKAR L., MA~'RIC" V. et POLDI~ L. (1983): Indagine fitochimica comparatlva dolIolio essenziale di Satureja variegata HOSTe della congenoro ~'. subsp/cata VIa. -- Wobbia 37 (1): 000--000. CoAssI•i LoxAR L., MAURIC~ V. et POLDINI L. (1986): Variation in terpene composition of Artemisia cdba (Compositae) in relation with environmental conditions. Biochem. System. Ecol. (in press). DOBSON C. I-L, DaESSLER R. L., HILLS H. G., ADAMSn . M. et W~LLIAMSN. H. (1969): Biologically active compounds in orchid fragrances. -- Science; 164: 1243--1249. EvA.'cs F. J. (1977): A new phorbol triesther from the latices of E . ]ranbiana and E . coerulescens. -- Phytochem., 16: 395--396. FAEGRIK. et vArr DER PIJL L. (1979): Principles of pollinatlon ecology. -- 3rclrev., ed. Pergamon Press, Oxford. FEOLI E., LAaONEGRO M. et ORLOCI L. (1984): Information analysis of vegetation data. -In: H. Licth et H. Mooney (eds.). Tasks for Vegetation Science. Junk. The Hague, Boston FOa~CASrEI~OV., GWOTTOA., CAPO~ALEG., BACC~CHE~"TIF. et MusA~o L. (1969): IdentificazJone della sostanza attrattiva per i maschi della Ceratitia capitata, contenuta nell'olio essenzialo dei semi di Angelica archangelica. -- Gazz. Chim. Ital., 99:700--710 G~BSrrENZO_WJ., LI~COL~r D. E. et LA,',-G~E~-rT~IMJ. H. (1978): The effect of moisture stress on nmnoterpenoid yield and composition in Sature]a d o u g l a s i i . - Bioch. System. and Ecol., 6:33--43 KI~GHORI~A. D. et Ev.~-s F. J. (1975): Isolation of phorbol from E. ]rancbiana. -- Phytochem., 14:585--586 LAL'SI D. et POLDI~I L. (1962): I1 paesaggio vegetalo della costlera triestina. -- Boll. Soc. Adr. Scienze, Trieste, Italia, LII: 1--63 LI~coT.N D. E. et LA~'~OENH~r~J. H. (1978): Effect of light and temperature on monoterpenoid yield and composition In Sa~ure]a douglasii. -- Biochem. System. and Ecol., 6 : 2 1 - - 3 2 -

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FRAGIFERA

285

LINCOLN D. E. e t LANOENHEIM J . H . (1979): V a r i a t i o n o f Sature]a dauglasli m o n o t e r p e n o i d s in r e l a t i o n t o l i g h t i n t e n s i t y a n d h o r b i v o r y . -- Bioch. S y s t e m . a n d Ecol., 7 : 2 8 9 - - 2 9 8 NIELSEN" l :), E., I~ISHIMURA I'I., LIANO Yo et CALVI~ M. (1979): S t e r o i d s f r o m Euphorbia a n d o t h e r l a t e x - b e a r i n g p l a n t s . -- P h y t o c h e m . , 1 8 : 2 2 1 7 t)OLDINI L. et STANCHER B. (1969): Gli eli e s s e n z i a l i di Sature]a variegata H o s t (-~ •. montana a u c t . pl. n o n L.) nel C a r s o t r i e s t i n o . -- Giorn. B e t . I t a l . , 1 0 3 : 6 5 - - 7 7 RODRIOUEZ E., TOWERS G. H . ~T. et MITCHELL J. G. (1976): Biological a c t i v i t i e s o f s e s q u i t e r p e n e l a c t o n s s . -- P h y t o c h e m . , 1 5 : 1 5 7 3 - - 1 7 7 9 RUDINSKI J . A. (1966): S c o t y t i d b e e t l e s a s s o c i a t e d w i t h D o u g l a s F i r : r e s p o n s e to t e r p e n e s . -Science, 1 5 2 : 2 1 8 - - 2 1 9 SCH:~IDT R . J. e t EVANS F. J. (1976): A n e w a r o m a t i c ester diterpene from Euphorbia poisonii.- Phytochem., 15:1778--1779 UPADHYAY R . l:~. et I-I~CKER E. (1975): D i t e r p e n e e s t e r s o f t h e i r r i t a n t a n d c o c a r c i n o g e n i c l a t e x o f Euphorbia lactea. -- P h y t o c h e m . , 1 4 : 2 5 1 4 - - 2 5 1 5 R e c e i v e d 30 J u n e 1983