Biochemical Systematics. 1973. VoL 1. pp. 45 to 49. Pergamon Press. Printed in England.

Chemotaxonomic and Ecological Implications of Anthocyanins in Elythranthera*

ALEX GEORGE Western Australia Herbarium. Department of Agriculture, Perth, Western Australia

and

CHRIS GONZALESt, MICHAEL S. STRAUSS and JOSEPH ARDITTIt Department of Developmental and Cell Biology, University of California. Irvine, CA 92664, USA

Key Word Index--Elythranthera brunonis; Elythranthera emarginata; Glossodia; Caladenia; Sebacina vermifera; Orchidaceae; Angiospermae; orchids; anthocyanins; cyanidin-3.5-diglucoside; delphinidin-3,5-diglucoside; pollination mechanisms. Abstraot--Elythranthera emarginata (Lindl.) A. S. George and E. brunonis (Endl.) A. S. George both contain cyanidin-3,5-diglucoside and delphinidin-3.5-diglucoside. Pigment concentrations in E. marginata are 2.5 times those of E. brunonis. Cyanidin/delphinidin ratios in both species are essentially the same. These findings are discussed in relation to the taxonomy, color, and the life cycle of Elythranthera.

I n t r o d u c t i o n

Our interest in the colors of Australian orchids was stimulated by the relative abundance of blue or purple species in the area around Perth, Western Australia. Elythranthera was selected for study because it consists of only two species: E. brunonis (Endl.) A. S. George, the Purple Enamel Orchid (actually its color is a purplish blue), and E. emarginata (Lindl.) A. S. George, the Pink Enamel Orchid. 1-s The genus is closely related to the genera Glossodia and Caladenia. s Glossodia, not found in Western Australia," also con- tains only two species, both purplish blue, G. major and G. minor. On the other hand, Caladenia is a much larger genus of seventy or more species whose flowers may be yellow, pink, purple, red or a combination of these s (44 species are found in Western Australia4). For a long time the two Elythranthera species were included in Glossodia as G. intermedia,

G. emarginata and G. brunonis. 4 Both species were also placed in Caladenia, but this arrangement was not accepted. 4.e

Elythranthera emarginata (the specific epi- thet refers to the notched labellum) bears flowers, 3 -4 cm in diameter, whose petals and sepals are of a glossy (enamel-like) pink color (hence the common name). The outer surface of the perianth is unglazed, but spotted pink and white. Plants are 15-30 cm tall, f inely glandular-hirsute, wi th an oblong lanceolate leaf 5-10 cm long, green on both sides. There are usually 1 or 2 flowers, oc- casionally 3 or 4. The labellum is white, membranous, broadly strap-shaped, and sig- mold towards the emarginate apex. There are two linear calli on the base of the label- lum, longer than it, purple and yellow. The column is winged, wi th a cucullate hood over the anther and stigma (hence the generic name, which means 'protected anther'). Eo

*Part II in the series "Orchid Anthocyanins". For Part I see Arditti, J. (1969) Am. J. Botany 56, 59, 21 and for a review see Arditti, J. and Ernst, R. (1971 ) Proc. 6th World Orchid Conference p. 203, Sydney. ~ tState of California SER Trainee. :J:Address for reprint requests. 1Cady, I_ and Rotherham, E. R. (1970) Australian Orchids in Color, A. H. ~ A. W. Reed, Sydney. 2Clyne, D. (1970) Australian Ground Orchids, Periwinkle Books, Melbourne. 3George, A. S. (1963) W. Austral. Nat. 9, 6. 4George, A. S. (1971) Nuytsia 1,180. SGeorge, A. S. and Foote. H. E. (1927) Orchids of Western Australia, Westways. Australia. 6Nicholls, W. H. (1969) Orchids of Australia, Thomas Nelson, Melbourne.

(Received 23 August 1972. Accepted 23 November 1972)

45

46 ALEX GEORGE, CHRIS GONZALES, MICHAEL S. STRAUSS AND JOSEPH ARDITII

emarginata can be found in clay and granite soils of the open forest, or swamp areas of the coastal plain, in South-Western Australia. 6 It extends from the Moore River some 100 km north of Perth on the lower west coast to Esperance on the central south coast.

Smaller flowered than E. emarginata, but more common, E. brunonis (named after Robert Brown) produces flowers which are about 2-3 cm in diameter 6 and glazed pur- plish-blue in color (hence the common name). On the outside the perianth is spotted blue-purple and white in a pattern identical with that of E. emarginata. Plants are 15-35 cm tall, glandular-hirsute,with a single narrow- oblong to lanceolate leaf 5-10 cm long which is dark red on the lower surface. There are 1-3 flowers. The labellum is white, mem- branous, narrowly strap-shaped, and re-curved below the obtuse apex. Two fleshy calli on short claws at the base of the labellum, are about as long as it, purple and white. The column is very broadly winged and cucullate over the anther and stigma. E. brunonis is especially common on the sand plains of South-Western Australia, extending from Watheroo, 200 km north of Perth, to the Thomas River, 100 km east of Esperance on the south coast. In some places the two species are sympatric and flower at similar times, though the flowering period of E. brunonis is late August to mid-October, while tha{ of

E. emarginata is late September to early November.

Despite being sympatric and of similar shapes the two species do not interbreed in nature. This is an indication of very effective isolation. No scents (at least none that the human nose can detect) appear to be pro- duced by either species. Therefore, it is possible that pollinating vectors may be attracted by flower color, floral morphology, or both. For this reason, identification of their anthocyanins is of particular interest. Further, we wanted to determine whether the antho- cyanins of E/ythranthera were similar to those

OH

~ Ogic

g(cO

( I ) R=H C yan id in -3 ,5 - diglucoside (11) R=OH Delph in id in -5 ,5-d ig lucos tde

OMe

H O ~ O ~ ~ H

HO [ I I I )R=H Petunidin ( IV)R=OH Malvid[~

TABLE 1. ANTHOCYANINS OF ELYTHRANTHERA BRUNON/S (ENDL.) A. S. GEORGE AND E. EM,4RGINATA (LINDL.) A. S. GEORGE

Band PC, Rf x 100" Speetrophotometry in 001% metholic HCI

BAW BuHCI 1% HCI HOAc-HCI x. With max aluminum,

(nm) bathochromic Species shift (nm) Identity:[: _

Elythranthera 1 21 6 12 58 526 43 Cyanidin-3,5- brunonsL~ diglucoside (I) (Endl.) A. S. George 2 14 3 8 34 533 38 Delphinidin-3,5-

diglucoside (11) E. emarg~nata 1 21 6 12 58 526 43 Cyanidin-3,5- (Lindl.) A. S. George diglucoside (I)

2 14 3 8 34 533 43 Delphinidin-3,5- Standards diglucoside (11)

Cyanidin-3.5- 21 5 12 40 526 diglucoside

Delphidin-3,5- diglucosidet 15 3 8 32 533-535

°BAW, n-butanol-aceticacid-water (4 : 1 : 5, upper, used within 1-2 hr after mixing); BuHCI, n-butanol-2N HCI (1 : 1, paper equili- brated for 24 hr in tank over lower layer); 1% HCI, water-conc. HCI (97 : 3); HOAc-HCI, acetic acid-conc. HCI-H20 (15 : 3 ; 82). all on Whatman No. 1 paper. tFrom the literature. ,tThe aglycones cyanidin and delphinidin were obtained from bands 1 and 2 respectively and identified by spectra and by PC. GIucose was the only sugar in each case identified by PC (4 systems) and TLC (2 systems).

ANTHOCYANINS OF ELYTHRANTHERA 47

of Glossodia, and Caladenia as well as the bluish purple Thelymitra ixiodes and T. venosa. 7

Results Two anthocyanin bands were obtained from each species (Table 1). On hydrolysis these bands yielded the two anthocyanidins, cyani- din and delphinidin respectively, and glucose. The anthocyanins were thus identified as cyanidin-3,5-diglucoside (I) and delphinidin- 3,5-diglucoside (11). Anthocyanin concen- tration in E. brunonis is lower than that of E. emarginata (Table 2). For both cyanidin-3,5- diglucoside and delphinidin-3,5-diglucoside the E. emarginata/E, brunonis ratio is 2.5. It is interesting to note that 1.19% of dry tissue weight in E. brunonis consists of anthocyanins whereas in E. emar,qinata the figure is 2.93%.

TABLE 2. ANTHOCYANIN CONTENT OF ELYTHRANTHERA EMARGINATA AND E. BRUNONIS

Species

Cyanidin-3,5- Delphinidin-3,5- diglucoside diglucoside

IJmol/g % of I~mol/g % of Cyanidin/ tissue tissue tissue tissue delphinidin

wt wt molar ratio Elythranthera

emarginata 31.01 1.89 16-53 1.04 1.87 E. brunonis 12.54 0.77 6-76 0.42 1,84 E. marginata/

E. brunonis ratio 2.47 2.45

Discussion Elythranthera differs from Caladenia and Glos- sodia in the glazed inner surface of the perianth which is spotted outside, in the membranous strap-like labellum which bears 2 hinged call/0 and in the column-hood above the anther and stigma. The rich purple-blue color of E. brunonis does not occur in Cala- den/a, but the pink of E. emarginata is seen in such species as Ca/adenia reptans Lindl., and C. nana Endl., while many other colors also occur in Caladenia. Elythranthera is endemic in South-Western Australia while Caladenia occurs across Southern Australia, with a few species in Malaysia, New Caledonia and New Zealand. Presently available information indi- cates that the two genera share one antho-

cyanidin, cyanidin, but probably not any of its glycoside@ (Table 3). One supposed hybrid between these two genera has been found near Albany on the south coast of Western Australia. The plants appear quite inter- mediate between Elythranthera brunonis and Caladenia sericea Lindl., both of which occur in the vicinity and flower at the same time. This conclusion is based on their mOrphology, and the cross has not been attempted experi- mentally.

TABLE 3. ANTHOCYANINS OF ELYTHRANTHERA AND RELATED GENERA*

Species Anthocyanint Caladenia carnea

C. paterson/ Elythranthera brunonis

E. emarginata

Glosodia major (New South Wales)

G. major (Victoria) G. minor Thelymitra ix/odes T. venosa

Cyanidin and some malvidin (IV) bioside Malvidin (IV) bioside Cyanidin-3.5-diglucoside (I) Delphinidin- 3,5-diglucoside (11) Cyanidin-3.5-diglucoside (I) Delphinidin- 3.5-diglucoside (11)

Delphinidin dimonoside Delphinidin bioside Petunidin (111) dimonoside Cyanidin dimonoside Cyanidin monoside

*From Arditti and Ernst. ~ l'Structures based on I-IV. Note that the bioside or climonoside imply attachment of a disaccharide or two monosaccharides to the aglycone but without stating the exact hydroxyl group to which they are attached.

Glossodia occurs only in eastern Australia including Tasmania. 6 It differs from Caladenia principally in having only a single callus on the labellum, clavate in Go major and deeply bifid in G. minor. Flowers of Glossodia major may be bluish, mauve, pink and rarely white. 6 Those of G. minor are usually bluish-mauve and seldom white. Occasional white flowers occur in both Elythranthera species. Such albinos are common in the Orchidaceae and are accorded varietal status in many species as var. "alba" "a/bus" or "concolor "7

Elythranthera and Glossodia share one anthocyanidin, delphinidin, and possibly one of its glucosides e (Tables 1 and 3). Caladenia and Elythranthera have one anthocyanidin, but no glycosides in common. Insofar as known, Glossodia and Caladenia do not share any anthocyanidins or anthocyanins (Table 3). 7 This, despite the fact that morpho- logically these two genera are much closer

~Arditt i, J. and Ernst. R. (1971 ) in Proc. 6th Wor ld Orchid Conference (Corr igan, M. J. G., ed.), p. 203. Sydney. eGascoigne, R. M., Ritchie, E. and White, D. E. (1959) Proc. Roy. Soc. N.S.W. 82, 44.

48 ALEX GEORGE,

to each other than either is to E/ythranthera. Of course, if more chemical constituents, not just anthocyanins, of the three genera were known, it is reasonable to expect that morpho- logical similarities and classical taxonomy would be reflected phytochemically. That these genera are similar is evident from the fact that they share a mycorrhizal fungus, Sebacina vermifera. 9

E/ythranthera brunonis and E. emarginata are two fairly similar species. This is not reflected in their anthocyanin content (Table 2), E. brunonis, containing much less cyani- din-3,5-diglucoside and delphinidin-3,5-di- glucoside.

Both cyanidin and delphinidin shift to- wards the blue in the presence of aluminium ions. lo Cyanidin-3-glucoside forms a blue complex wi th chlorogenic acid in the presence of aluminium salts. 11 This may be responsible for the blue color of Hydrangea macro- phy//a. 12.13 In Rosa cv. Better Times a copigment complex of cyanidin-3,5-digluco- side and quercetin or kaempferol glycosides turns blue as the tissue ages and its pH increases from 3.70-4.15 to 4'40-4-50.14 Similar factors may well be responsible for the bluer color of E/ythranthera brunonis.

Except for their different colors, Elythranthera emarginata and E. brunonis appear similar to the human eye. Neither produces a detectable scent, both shine equally because of their glazing and have the same 'polka-dot' pattern on petal and sepal undersides. The 2 species are sometimes sympatric. In some areas they grow side by side and f lower simultaneously. Despite all this no natural hybrids have been found and there are no man-made ones. This points to a very effective breeding isolation and suggests that each species may be pollinated

CHRIS GONZALES. MICHAEL S. STRAUSS AND JOSEPH ARDITTI

by a different vector. If so, it would seem that the vectors are probably attracted solely by color as is the case with the fly Bobylius fu/iginosus and the blue-purple colored grape-hyacinth Muscari comosum (Lilia- ceae). 1~ Interestingly The/ymitra media, a blue Australian orchid, is visited often by a hover fly, Eristafis tenax w h i c h ' . . , could be possible pollinating agents' although 'No confirmatory observation was made'. 16 Two species of The/ymitra share one anthocyanin, cyanidin. with E/ythranthera (Table 3).

Corybas rivularis (flowers green with pur- plish red spots) and C. macrantha (similarly colored), two New Zealand orchids, are also fly pollinated. 1 Elsewhere, Bu/bophyllumgraci/- //mum ~ 6 (Cirrhopetalurn psittacoides, Malaya), Disa /ugens and D. comuta (So. Africa) and Masdevallia fractif/exa all have flowers which are bluish, crimson, purplish or spotted with these colors and are fly pollinated. 17 Notably the vision of many flies is good in the blue region of the spectrum. ~8 Perhaps this may explain the lower pigment levels in E. brunonis (Table 2). It is possible that at least E/ythran- thera brunonis may be fly pollinated. The great abundance of very active flies of all kinds (which never fail to plague orchid collectors) in the area also points in this direction and the possibility clearly bears further investigation.

On the whole blue flowers are not very common in nature. When they occur, their anthocyanins tend to be cyanidin, petunidin and delphinidin glycosides. 9 Examples are Lycoris sperengeri, ~9 blue pansies, 2° a 'blue' rose, 21 Hyacinthus orientalis, lo Jacaranda acutifolia 1° and Delphinium. 1° That the same appears to be true for orchids (Table 3) ~ is not surprising.

9Warcup, J. H. (1971 ) New Phytologist 70, 41. ~OHarborne, J. B. (1967) Comparative Biochemistry of the Flavonoids. Academic Press, New York. ~lJurd, L. and Asen, S. (1966) Phytochemistry 5, 1263. 12Asen, S. and Siegelman, H. W. (1957) Proc. Am, Soc. Hort. ScL 70, 478. 13Asen, S., Siegelman. H, W. and Stuart, N. (1957) Proc. Am, Soc. Holt. Sci. 69, 561. 14Asen, S., Norris, K. H. and Stewart, R. N. (1971) J. Am. Soc, Hort. ScL 96, 770. l SMeeuse, B. J. D. (1961 ) The Story of Pollination, Ronald Press, New York. ~6Thomson, G. M. (1927) Trans. Proc. Roy. Soc. NewZeaL 57, 106. ~van der Pijl, L. and Dodson, C. H. (1966) Orchid Flowers, Their Pollination and Evolution, University of Miami

Press, Coral Gables, Florida. ~SKoopowitz, H., personal communication. University of California, Irvine. 9Arisumi, K. (1971 ) Bull. Fac. Agr. Yamaguti Univ. 22, 171.

2oOotani, S., Hsieh, K. C. and Kondo, N. (1966) Mem. Tokyo Univ. Agr. 10, 1. 2~Arisumi, K. (1968) Bull. Fac. Agr. YamagutiUniv. 19, 1173.

ANTHOCYANINS OF ELYTHRANTHERA 49

Experimental Plant material. Flowers of Elythranthera brunonis

(Endl.) A. S. George and E. emarginata were collected in Western Australia, near Perth. Voucher specimens are stored in Western Australia Herbarium, Department of Agriculture, Perth, W.A. They were dried for 3 days in a 50 ° oven and extracted.

Extraction purification and identification procedures. Anthocyanins were extracted, purified and identified by the usual methods we have used previously in work with orchid anthocyanins 22 and sugar content of nectars. 23

Estimation of anthocyanin concentrations. Absorp- tivities were determined in 0"01% HCI in MeOH at 526 and 533 nm. 24.2s Cyanidin-3,5-diglucoside con- tent was calculated using 1.25 x 104 as molecular

22Arditti, J. (1969) Am. J. Botany 56, 59,

extinction coefficient. 26 The extinction coefficient for delphinidin, 2.32 x 104 was extrapolated from avail- able information2 e (The slope of the line joining the extinction coefficients 2e of delphinidin and delphinidin- 3-glucoside is parallel to that obtained for cyanidin and cyanidin-3,5-diglucoside. The extinction coefficient for delphinidin-3,5-diglucoside was obtained by extra- polation.)

Acknowledgements--Supported in part by grants from the National Science Foundation (GB-1 3417) and the Population Council. We thank Drs. K. Tewari and Michael White, Department of Molecular Biology and Biochemistry for the use of their Cary Spectrophoto- meter; Mr. Robert Ernst, President, Textilana Co- potation for donating most of the solvents used in this work; and Brigitta H. Flick for technical assistance.

23Jeffrey, D. C., Arditti, J. and Ernst, R. (1969) J. Chromatog. 41,475. 24Furuya, M. and Thimann, K. V. (1964) Arch. Biochem. Biophys. 108, 109. 2SArditti, J. and Knauft, R. L. (1969) Am. J. Botany 56, 620. 26Fuleki, T. and Francis, F. J. (1968) J. FoodSci. 33, 72.