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Comparison of secondary compounds in Heterozostera tasmanica from Australia and Chile
Aquatic Botany, 15 (1983) 205--207 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
205
Short Communication COMPARISON OF SECONDARY COMPOUNDS IN HETEROZOSTERA TASMANICA FROM AUSTRALIA AND CHILE
CALVIN McMILLAN
Department of Botany and Plant Ecology Research Laboratory, University of Texas, Austin, TX 78712 (U.S.A.) (Accepted for publication 7 June 1982)
ABSTRACT
McMillan, C., 1983. Comparison of secondary compounds in Heterozostera tasmanica from Australia and Chile. Aquat. Bot., 15: 205--207. Leaves of Heterozostera tasmanica (Martens ex Aschers.) den Hartog from both Australia and Chile lack the sulfated flavones that are characteristic of Zostera. Similar phenolic compounds are present in leaves of both Australian and Chilean plants of Heterozostera, but none of the numerous flavonoids (sulfated or non-sulfated) that occur in Zostera were detected in two-directional chromatograms of the Heterozostera leaves. The nearly identical patterns of secondary compounds in leaves from Australia and Chile support their position in the same species.
INTRODUCTION R e c e n t l y , Phillips et al. ( 1 9 8 2 ) c o n f i r m e d t h e p r e s e n c e o f Heterozostera tasmanica ( M a r t e n s ex Aschers.) d e n H a r t o g in Chile. T h e y r e p o r t e d its o c c u r r e n c e a t Aldea, B a h i a T o n g o y , 16 k m s o u t h o f C o q u i m b o . D e n H a r t o g ( 1 9 7 0 ) h a d r e p o r t e d a c o l l e c t i o n f r o m C o q u i m b o ( H e r r a d u r a B a y ) in 1 8 6 4 a n d h a d c o m p a r e d it w i t h c o l l e c t i o n s f r o m A u s t r a l i a . All c h a r a c t e r s a p p e a r e d t o b e identical, a n d , t h e r e f o r e , d e n H a r t o g c o n c l u d e d t h a t all Heterozostera m a t e r i a l s h o u l d be i n c l u d e d in t h e s a m e species. Heterozostera f r o m A u s t r a l i a was c o m p a r e d w i t h v a r i o u s species o f Zostera a n d Phyllospadix in a s u r v e y o f s u l f a t e d p h e n o l i c c o m p o u n d s (McMillan et al., 1 9 8 0 ) . All o f t h e species t h a t w e r e s t u d i e d f o r e i t h e r Zostera or Phyllospadix c o n t a i n e d s u l f a t e d flavones, b u t Heterozostera c o l l e c t i o n s t h a t w e r e c o m p a r e d f o r b o t h V i c t o r i a a n d S o u t h A u s t r a l i a did n o t c o n t a i n s u l f a t e d f l a v o n o i d s . T h e r e c e n t c o l l e c t i o n in Chile p r o v i d e d m a t e r i a l f o r a s t u d y o f t h e s e c o n d a r y c o m p o u n d s a n d f o r a c o m p a r i s o n w i t h p l a n t s o f Australia.
0304-3770/83/0000--0000/$03.00
© 1983 Elsevier Scientific Publishing Company
206 MATERIALS AND METHODS
Leaves collected at Bahia Tongoy by R.C. Phillips on 1 May 1981 were air dried and s u b s e q u e n t l y compared with leaves that were collected at Victor Harbor, S o u t h Australia, by C. McMillan on 18 N o v e m b e r 1979. Leaves from Port Stanvae, South Australia, that were collected in July 1979 and provided b y H.B.S. Womersley were also used in the comparison. The dried leaves were evaluated for secondary c o m p o u n d s by the standard procedures outlined in McMillan et al. (1980). A p p r o x i m a t e l y 0.5 g o f leaf tissue was extracted in 10 ml 85% methanol on a mechanical shaker for 24 h. The extracts were concentrated on chromatographic paper and were subjected to two-directional paper chromatography in TBA (t-butanol:acetic acid:water; 3:1:1) for 24 h and 15% acetic acid for 4 h. After the papers were dried, the c o m p o u n d s were detected b y changes in fluorescence and color under u.v. light (360 nm) in the absence and presence of ammonia fumes. RESULTS Neither the Chile nor the Australia leaves contained any secondary compounds with flavonoid characteristics. These c o m p o u n d s would be detectable b y their fluorescence and purplish coloration under u.v. light. There were no purple spots on the chromatograms of either the Chile or Australia plants. Leaves of b o t h the Bahia T o n g o y , Chile, and the Victor Harbor, South Australia, collections had an almost identical display of other phenolic constituents. In each two-directional chromatogram, the 7--8 c o m p o u n d s with white to blue fluorescence had similar R f values for both the Chile and Australia plants. DISCUSSION
The nearly identical patterns o f secondary c o m p o u n d s in leaves of plants from Chile and Australia s u p p o r t the position that den Hartog (1970) t o o k in placing them within the same species. The lack of flavonoids and the presence of 7--8 other phenolic c o m p o u n d s show the similarity o f these collections. The pattern of the phenolic c o m p o u n d s in Heterozostera tasmanica (Table I) is unlike that o f any o f the species o f Zostera that was examined by McMillan et al. (1980). N o t only do the Heterozostera leaves lack the sulfated flavones that are characteristic of each of the 10 Zostera species that were examined, b u t the Heterozostera collections also lack non-sulfated flavonoids that are present in each of the Zostera species (C. McMillan, unpublished data, 1982). Isozyme data (McMillan, 1982) also show different patterns for Heterozostera and Zostera. Although only Australian plants of Heterozostera were used in the isozyme comparison, the collection of H. tasrnanica from Victor Harbor, South Australia, had different isozymes of 7 enzymes from those of
207 TABLE I Comparison of phenolic compounds in H e t e r o z o s t e r a , Z o s t e r a and P h y l l o s p a d i x * Genus
He t e r o z o s t e ra Zostera Phyllospadix
Presence or absence of phenolic compounds Sulfated flavonoids
Non-sulfated flavonoids
Other phenolics
-+ +
-+ +
+ + +
*Collections of H e t e r o z o s t e r a t a s m a n i c a from both Chile and Australia lacked flavonoids. The ten species examined by McMillan et al. (1980) in Z o s t e r a contained both sulfated and non-sulfated flavonoids. Each of the five species in P h y l l o s p a d i x contained both sulfated and non-sulfated flavonoids. The various Z o s t e r a species contained 2--5 sulfated flavonoids and the P h y l l o s p a d i x species contained 5--7 sulfated flavonoids.
L . , Z . c a p e n s i s Setchell, Z . c a p r i c o r n i Aschers., Z . m u e l l e r i Irmisch ex Aschers. and Z . n o v a z e l a n d i c a Setchell. The data from t he comparisons of secondary c o m p o u n d s and isozymes s u p p o r t t he separation o f H e t e r o z o s t e r a fr o m Z o s t e r a as p r o p o s e d by den Hartog (1970).
Z. marina
ACKNOWLEDGEMENTS
This research was f u n d e d , in part, by National Science F o u n d a t i o n grant OCE 77-26399. The a u t h o r thanks R.C. Phillips for t he Chile collection, H.B.S. Womersley for t h e Port Stanvae, Sout h Australia, collection, S. Beck for technical assistance and T.J. Mabry f or use o f his p h y t o c h e m i c a l facilities.
REFERENCES Den Hartog, C., 1970. The Sea-Grasses of the World. North-Holland, Amsterdam, 275 pp. McMillan, C., 1982. Isozymes in seagrasses. Aquat. Bot., 14: 231--243. McMillan, C., Zapata, O. and Escobar, L., 1980. Sulphated phenolic compounds in seagrasses. Aquat. Bot., 8: 267--278. Phillips, R.C., Santelices, B., Bravo, R. and M c Roy, C.P., 1982. Heterozostera tasmanica (Martens ex Aschers.) den Hartog in Chile. Aquat. Bot., 15: 195--200. J
205
Short Communication COMPARISON OF SECONDARY COMPOUNDS IN HETEROZOSTERA TASMANICA FROM AUSTRALIA AND CHILE
CALVIN McMILLAN
Department of Botany and Plant Ecology Research Laboratory, University of Texas, Austin, TX 78712 (U.S.A.) (Accepted for publication 7 June 1982)
ABSTRACT
McMillan, C., 1983. Comparison of secondary compounds in Heterozostera tasmanica from Australia and Chile. Aquat. Bot., 15: 205--207. Leaves of Heterozostera tasmanica (Martens ex Aschers.) den Hartog from both Australia and Chile lack the sulfated flavones that are characteristic of Zostera. Similar phenolic compounds are present in leaves of both Australian and Chilean plants of Heterozostera, but none of the numerous flavonoids (sulfated or non-sulfated) that occur in Zostera were detected in two-directional chromatograms of the Heterozostera leaves. The nearly identical patterns of secondary compounds in leaves from Australia and Chile support their position in the same species.
INTRODUCTION R e c e n t l y , Phillips et al. ( 1 9 8 2 ) c o n f i r m e d t h e p r e s e n c e o f Heterozostera tasmanica ( M a r t e n s ex Aschers.) d e n H a r t o g in Chile. T h e y r e p o r t e d its o c c u r r e n c e a t Aldea, B a h i a T o n g o y , 16 k m s o u t h o f C o q u i m b o . D e n H a r t o g ( 1 9 7 0 ) h a d r e p o r t e d a c o l l e c t i o n f r o m C o q u i m b o ( H e r r a d u r a B a y ) in 1 8 6 4 a n d h a d c o m p a r e d it w i t h c o l l e c t i o n s f r o m A u s t r a l i a . All c h a r a c t e r s a p p e a r e d t o b e identical, a n d , t h e r e f o r e , d e n H a r t o g c o n c l u d e d t h a t all Heterozostera m a t e r i a l s h o u l d be i n c l u d e d in t h e s a m e species. Heterozostera f r o m A u s t r a l i a was c o m p a r e d w i t h v a r i o u s species o f Zostera a n d Phyllospadix in a s u r v e y o f s u l f a t e d p h e n o l i c c o m p o u n d s (McMillan et al., 1 9 8 0 ) . All o f t h e species t h a t w e r e s t u d i e d f o r e i t h e r Zostera or Phyllospadix c o n t a i n e d s u l f a t e d flavones, b u t Heterozostera c o l l e c t i o n s t h a t w e r e c o m p a r e d f o r b o t h V i c t o r i a a n d S o u t h A u s t r a l i a did n o t c o n t a i n s u l f a t e d f l a v o n o i d s . T h e r e c e n t c o l l e c t i o n in Chile p r o v i d e d m a t e r i a l f o r a s t u d y o f t h e s e c o n d a r y c o m p o u n d s a n d f o r a c o m p a r i s o n w i t h p l a n t s o f Australia.
0304-3770/83/0000--0000/$03.00
© 1983 Elsevier Scientific Publishing Company
206 MATERIALS AND METHODS
Leaves collected at Bahia Tongoy by R.C. Phillips on 1 May 1981 were air dried and s u b s e q u e n t l y compared with leaves that were collected at Victor Harbor, S o u t h Australia, by C. McMillan on 18 N o v e m b e r 1979. Leaves from Port Stanvae, South Australia, that were collected in July 1979 and provided b y H.B.S. Womersley were also used in the comparison. The dried leaves were evaluated for secondary c o m p o u n d s by the standard procedures outlined in McMillan et al. (1980). A p p r o x i m a t e l y 0.5 g o f leaf tissue was extracted in 10 ml 85% methanol on a mechanical shaker for 24 h. The extracts were concentrated on chromatographic paper and were subjected to two-directional paper chromatography in TBA (t-butanol:acetic acid:water; 3:1:1) for 24 h and 15% acetic acid for 4 h. After the papers were dried, the c o m p o u n d s were detected b y changes in fluorescence and color under u.v. light (360 nm) in the absence and presence of ammonia fumes. RESULTS Neither the Chile nor the Australia leaves contained any secondary compounds with flavonoid characteristics. These c o m p o u n d s would be detectable b y their fluorescence and purplish coloration under u.v. light. There were no purple spots on the chromatograms of either the Chile or Australia plants. Leaves of b o t h the Bahia T o n g o y , Chile, and the Victor Harbor, South Australia, collections had an almost identical display of other phenolic constituents. In each two-directional chromatogram, the 7--8 c o m p o u n d s with white to blue fluorescence had similar R f values for both the Chile and Australia plants. DISCUSSION
The nearly identical patterns o f secondary c o m p o u n d s in leaves of plants from Chile and Australia s u p p o r t the position that den Hartog (1970) t o o k in placing them within the same species. The lack of flavonoids and the presence of 7--8 other phenolic c o m p o u n d s show the similarity o f these collections. The pattern of the phenolic c o m p o u n d s in Heterozostera tasmanica (Table I) is unlike that o f any o f the species o f Zostera that was examined by McMillan et al. (1980). N o t only do the Heterozostera leaves lack the sulfated flavones that are characteristic of each of the 10 Zostera species that were examined, b u t the Heterozostera collections also lack non-sulfated flavonoids that are present in each of the Zostera species (C. McMillan, unpublished data, 1982). Isozyme data (McMillan, 1982) also show different patterns for Heterozostera and Zostera. Although only Australian plants of Heterozostera were used in the isozyme comparison, the collection of H. tasrnanica from Victor Harbor, South Australia, had different isozymes of 7 enzymes from those of
207 TABLE I Comparison of phenolic compounds in H e t e r o z o s t e r a , Z o s t e r a and P h y l l o s p a d i x * Genus
He t e r o z o s t e ra Zostera Phyllospadix
Presence or absence of phenolic compounds Sulfated flavonoids
Non-sulfated flavonoids
Other phenolics
-+ +
-+ +
+ + +
*Collections of H e t e r o z o s t e r a t a s m a n i c a from both Chile and Australia lacked flavonoids. The ten species examined by McMillan et al. (1980) in Z o s t e r a contained both sulfated and non-sulfated flavonoids. Each of the five species in P h y l l o s p a d i x contained both sulfated and non-sulfated flavonoids. The various Z o s t e r a species contained 2--5 sulfated flavonoids and the P h y l l o s p a d i x species contained 5--7 sulfated flavonoids.
L . , Z . c a p e n s i s Setchell, Z . c a p r i c o r n i Aschers., Z . m u e l l e r i Irmisch ex Aschers. and Z . n o v a z e l a n d i c a Setchell. The data from t he comparisons of secondary c o m p o u n d s and isozymes s u p p o r t t he separation o f H e t e r o z o s t e r a fr o m Z o s t e r a as p r o p o s e d by den Hartog (1970).
Z. marina
ACKNOWLEDGEMENTS
This research was f u n d e d , in part, by National Science F o u n d a t i o n grant OCE 77-26399. The a u t h o r thanks R.C. Phillips for t he Chile collection, H.B.S. Womersley for t h e Port Stanvae, Sout h Australia, collection, S. Beck for technical assistance and T.J. Mabry f or use o f his p h y t o c h e m i c a l facilities.
REFERENCES Den Hartog, C., 1970. The Sea-Grasses of the World. North-Holland, Amsterdam, 275 pp. McMillan, C., 1982. Isozymes in seagrasses. Aquat. Bot., 14: 231--243. McMillan, C., Zapata, O. and Escobar, L., 1980. Sulphated phenolic compounds in seagrasses. Aquat. Bot., 8: 267--278. Phillips, R.C., Santelices, B., Bravo, R. and M c Roy, C.P., 1982. Heterozostera tasmanica (Martens ex Aschers.) den Hartog in Chile. Aquat. Bot., 15: 195--200. J