REINWARDTIA Vol. 10, Part 3, pp. 271 - 280 (1987) MORPHOLOGICAL, ANATOMICAL AND CHEMICAL ANALYSES OF AMORPHOPHALLUS PAEONIIFOLIUS AND RELATED TAXA ELIZABETH A. WIDJAJA Herbarium Bogoriense, Bogor, Indonesia & R.N. LESTER Plant Biology Department, Birmingham University, Birmingham, England ABSTRACT A study on the affinity of undescribed taxa of Amorphophallus with A. paeoniifolius and its related species was undertaken using numerical ana- lyses of data derived from preserved and living specimens. The results ob- tained were compared with the system of classification produced by classi- cal taxonomic methods. The Javanese wild and cultivated forms of Amor- phophallus paeoniifolius respectively known locally as "Amorphophallus campanulatus f. hortensis" and "Amorphophallus campanulatus f. sylves- tris" were confirmed as two distinct but closely related taxa. ABSTRAK Penelitian hubungan takson-takson Amorphophallus yang baru untuk ilmu pengetahuan dengan Amorphophallus paeoniifolius dan jenis kerabat- nya dilakukan dengan menggunakan analisis numeris yang datanya diambil dari spesimen hidup dan mati. Hasil yang diperoleh diperbandingkan de- ngan sistem klasifikasi taksonomi klasik. Bentuk liar dan kultivar A. paeo- niifolius yang berturut-turut dikenal sebagai A. campanulatus f. hortensis dan A. campanulatus f. sylvestris dalam kepustakaan setempat dinyatakan sebagai dua takson yang berbeda tapi berkerabat dekat. INTRODUCTION Amorphophallus Bl. is one of several important genera in the family Araceae grown for human consumption as well as for animal feed, medicine and other industrial purposes. The elephant yam (Amorphophallus paeonii- folius (Denns.) Nicolson), devil's tongue (A. konjac C. Koch) and A. onco- phyllus Prain ex Hook.f. are among the approximately one hundred species 2 7 1 272 REINWARDTIA [VOL. 10 which make up this genus, which is widely spread throughout the tropics of Asia, Africa, the Pacific Islands and some other areas in the subtropics. The main characteristics of the genus is that the tuber produces an evil- smelling inflorescence with a terminal sterile spongy phallic appendage en- closed in a spathe. Male flowers are on the upper part of the sterile zone of the spadix and female ones below. It has a single petiole and a compound leaf which dies down before the plant flowers. The discovery of the recently described Amorphophallus lambii Mayo & Widjaja (1983) as well as an undescribed cultivar from Timor (here- after referred to as Amorphophallus EAW 1177) and two other taxa from Thailand (Amorphophallus NG 2 and Amorphophallus RBG 1) which in some respects resembled Amorphophallus paeoniifolius, made it necessary to study their taxonomic relationships to other described species of Amor- phophallus. Since not all of these undescribed taxa produced inflorescences, all available data which might be useful as taxonomic evidences have been employed. Pending their formal descriptions as new taxa, the present study was designed to investigate the affinity of these undescribed taxa with A. paeoniifolius, A. oncophyllus, A. bulbifer (Roxb.) Bl. and A. variabilis Bl. using data derived from vegetative morphology, anatomy and chemotaxo- nomy. MATERIALS AND METHODS Both living plants and herbarium specimens were used in this study. A total of 30 accessions of living plants were grown, including four accessions of cultivated A. paeoniifolius, six of wild A, paeoniifolius, seven of A. varia- bilis, eight of A. oncophyllus and one accessions each of A. bulbifer, A. lam- bii, Amorphophallus EAW 1177, Amorphophallus NG 2 and Amorphophallus RBG 1 (Widjaja 1981). Twenty-three of these living accessions had been collected in various parts of Indonesia. Living materials were brought to Birmingham (England) in August 1980 and the tubers were planted in John Innes No. 2 compost in pots and grown in a glass house at about 25°C. Automatic watering was used and mercury vapour provided supplementary lighting, especially during the winter period. It was difficult to obtain leaf material under standard condi- tions and impossible to obtain replicate leaves for any one accessions, since only one tuber was available of most accessions and the leaves were produced singly and erratically, independent of any season. One accession of cultivated A. paeoniifolius was bought from an Indian food shop in Smethick near Birmingham. Herbarium specimens, living collections and paintings were studied at the Royal Botanic Gardens, Kew. These were particularly valuable for pro- 1887) WIDJAJA & LESTER : Numerical taxonomy of Amorphophallui 273 viding data on inflorescence characters since only A. variabilis flowered in Birmingham. Morphological observation Thirty-five morphological characters of tubers, petioles and leaves were recorded for 15 accessions of living plants of four species. Twenty-five cha- racters were recorded for all 15 accessions but some data were unobtainable for some accessions of A. oncophyllus and in this case the average value of the appropriate characters from the other accessions was used (Widjaja 1981). Chemotaxonomic study Chromatography of phenolic compounds Fully mature top folioles were collected from 25 plants and dried in a forced ventilation oven at 45°C for 96 hours. Half a gram of dried leaves were ground and extracted with sufficient 3 ml 0.5% v/v hydrochloric acid in methanol kept overnight in darkness. Leaf extracts were applied to a 1.5 cm diameter circle 4.5 cm from the corner of Whatman No. 1 filter paper. The papers were clipped into a Shandon Universal Holder and run in 250 ml of the first solvent (butan-1-ol : acetic acid : water = 3:1:1) for about 12 hours; the papers were dried then run in the second solvent (acetic acid : water = 15% v/v) for 4 hours. After drying, the papers were analysed under an ultra violet lamp. Any spots and their colours were recorded and their distribution was examined. Electrophoresis of proteins Electrophoresis of proteins was done by polyacrylamide gel methods following the method of Davis (1964). About 10 grams of good fresh tubers and 7.5 grams of fresh leaves were sampled from each of 14 accessions. After washing with tap water and cleaning from soil, tubers and leaves were kept in polythene bags in the deep freeze for at least 4 hours. Tubers and leaves were thawed to ± —1°C and then crushed in the polythene bag by hand at room temperatures. The juices were collected, spun in a centrifuge for about 6 minutes at 15,000 g and then kept in the freeze at ± —3°C. Samples of 0.1 — 0.2 ml of protein extract were analysed and after electrophoresis the gels were stained with 0.1% w/v naphthalene black in 7% v/v acetic acid for 1 — 2 days and stored in the dark. Examinations were made over fluorescent light and the distribution of bands of proteins from each sample were recorded. 274 REINWARDTIA [Vol. 10 Anatomical observations Only the forms of raphide were investigated during the course of this anatomical study. Raphides were prepared by crushing the tubers in a mortar with some distilled water and allowed to settle or filtered by filter paper and dried. A pinch of sample was taken and placed on a stub and coated with gold. Examinations were conducted under a light microscope and a scanning elec- tron microscope (Cambridge Stereoscan 600). Data analyses Data were analysed numerically using Principal Components Analysis, and clustering techniques by Euclidean Distance and Ward's method using Clustan l.C programmes. The chromatographic and electrophoresis data were also analysed by Jaccard's Coefficient and UPGMA using Clustan l.C prog- ramme (Wishart 1978). RESULT AND DISCUSSIONS MORPHOLOGY Cluster analyses based on all 35 morphological characters of tubers, petioles and leaves of all 15 accessions indicated that A. oncophyllus was very distinct and clearly separated from the other species in Principal Components Analysis by the first factor. This distinction was maintained even when only 25 characters recorded from all four accessions of A. oncophyllus were used. Because A. oncophyllus was so distinct, further analyses were made of the remaining 11 accessions using all 35 characters. Principal Components Analysis of four accessions of the wild A. paeoniifolius showed that they formed a very distinct group. However, one accession was shown to be dis- tinct from the other accessions of the wild A. paeoniifolius and rather similar to the undescribed Amorphophallus EAW 1177 and three accessions of the cultivated A. paeoniifolius (Fig. 1).. The cluster analysis also showed that the two accessions of A. variabilis were similar to each other. CHEMOTAXONOMY Chromatography Chromatographically speaking A. oncophyllus appeared as a distinct group, with two spots appeared only in this species. The chromatographj also showed that A. bulbifer and Amorphophallus RBG 1 were separatee from each other as well as from the other species. All five accessions of the wild A. paeoniifolius clustered together. Amor- phophallus EAW 1177 was included in this cluster and two accessions of A. 276 REINWARDTIA [VOL. 10 variabilis were close to it. However, the remaining accessions of A. variabilis clustered together outside these groups. The three accessions of the cultivated A. paeoniifolius occured in two clusters together with accessions of A. lambii and Amorphophallus NG 2. These last two taxa biochemically are rather similar to the cultivated A. pae- oniifolius although the latter has more prickles on their petioles. Based on the above data, it seemed that the wild A. paeoniifolius is re- lated to the cultivated one and both taxa are closer to A. variabilis than to A. oncophyllus. Amorphophallus RBG 1 and A. bulbifer are clearly distinct from A. paeoniifolius and A. variabilis. Electrophoresis Leaf proteins From the electrophoresis of leaf proteins a total of 25 protein bands were recognised, of which as many as 11 were detected in one accession but as few as three in another. There were bands which were observed in many taxa (e.g. band 22) but others could be detected only in one species. Consequent- ly band 12 was found in A. paeoniifolius but not in any other species, so that it can be used as a diagnostic character for this species. Band no. 4, 14 and 18 may be considered as diagnostic characteristic for the cultivated A. paeoniifolius and band 17 for the wild one. Both dendrograms derived from Jaccard or Euclidean approaches agreed in placing all four accessions of the wild A. paeoniifolius in a single cluster. A. oncophyllus, A. lambii and Amorphophallus NG 2 were fairly similar to each other, but Amorphophallus EAW 1177 showed little similarity to any of them either by Jaccard's or Euclidean Coefficient. Tuber proteins The result of tuber electrophoresis of 23 accessions of Amorphophallus showed a total of 18 protein bands, with as many as 11 bands in one acces- sion but as few as one in another. The most universal protein was band 15 which appeared in all accessions of Amorphophallus. Band 2 and 3 were found in A. variabilis but not in other species. All accessions of the wild A. paeoniifolius formed a single cluster. Similarly all accessions of A. variabilis also formed a single tight cluster. A. oncophyllus and the cultivated A. paeoniifolius formed two distinct but related clusters by Euclidean Distance, but were more intermingled by Jac- card's Coefficient. Amorphophallus EAW 1177 was similar to A. paeoniifolius accession 7D. The difference between accession 7D and 7Y is noteworthy since the former was sampled from the main tuber and the latter was sampled from a young tuber which grew from the main tuber (Fig. 2). 278 REINWARDTIA [Vol. 10 Based on the tuber electrophoresis it can be concluded that the wild A. paeoniifolius was slightly distinct from the cultivated one and was distant- ly related to A. variabilis. On the.other hand the cultivated A paeoniifolius was somewhat related to A. oncophyllus. ANATOMY Two kinds of raphides, one small and the other one much larger, were observed in the wild and cultivated A. paeoniifolius, A. oncophyllus, A. varia- bilis and Amorphophallus EAW 1177. The length of the large raphides of A. variabilis was 0.0112 —0.0128 mm and the small ones was± 0.0004 mm. In Amorphophallus EAW 1177 the large raphides measured ± 0.0136 mm and the small one ± 0.0032 mm in length. The large raphides of A. paeonii- folius may reach 0.0144 mm long and the small one ± 0.0004 mm. It is difficult to distinguish the two forms of this species by the length of their large raphides but under a light microscope it could be seen that the small raphides in the wild A. paeoniifolius were more abundant than in the culti- vated one. This result is contrary to Kirtikar's (1954) findings who reported that there were two kinds of raphides in the wild variety while the cultivated one showed only one kind. Besides the size, the raphides of the species investigated could also be distinguished by their grooves and their surface ornaments. The tip ends of the large raphides of A. oncophyllus were acute and tapering. There are canaliculate grooves on the side, of this large raphide and its surface is spar- sely ornamented with thin but broad protuberances. The large raphides of A. variabilis were also provided with tapering and acute ends and canalicu- lately grooved, but unlike those of A. oncophyllus the surface is ornamented with sharp barb-like protuberances. In both the wild and cultivated forms of A. paeoniifolius the large raphides appear to have interrupted canaliculate grooves. In this species the barbs occur on the tip ends, whereas in Amor- phophallus EAW 1177 they could not be found at the tip ends of the large raphides. The sharpness and the position of the barbs on the surface of the raphides as well as the abundance of the small raphides might be used as characteristics to distinguish the forms of A. paeoniifolius although it was difficult to see. CONCLUSIONS As a general conclusion of this study it can be stated that within the genus Amorphophallus, A. oncophyllus is very distinct on morphological, chromatographic as well as tuber electrophoretic grounds. A. bulbifer is also distinct, although it clustered with A. oncophyllus in the chromatographic studies. The results of leaf electrophoresis, however, showed A. bulbifer 1987] WIDJAJA & LESTER : Numerical taxonomy of Amorphophallus 2 7 9 to be different from A. oncophyllus. In Engler's (1911) classification A. bulbifer and A. oncophyllfis were placed as neighboring species in section Conophallus, a conclusion which is supported by the present study. It ap- pears that A. variabilis is closer to A. paeoniifolius than to A. oncophyllus; this is contrary to Engler's contention who placed A. variabilis in the section Conophallus as well. The wild A., paeoniifolius stands out as a distinct taxon based on morphological, electrophoretic as well as the chromatographic studies. It is linked with the cultivated A. paeoniifolius and A. variabilis rather than with A. oncophyllus. The smooth petioled Amorphophallus EAW 1177 which was collected in Timor Island as a cultivated plant in the kitchen garden is very similar to the cultivated A. paeoniifolius in morphological and tuber electrophoretic characteristics. Its leaf chromatography, however, points to a relationship with the wild A. paeoniifolius. In Javanese literature the wild and cultivated forms of A. paeoniifolius have been known respectively as A. campanulatus f. sylvestris Backer and A. campanulatus f. hortensis Backer (Backer 1920, Backer & Bakhuizen van den Brink f. 1968, Sastrapradja et al. 1977). Their nomenclature is being worked out in the light of the present study. On vegetative morphological grounds Amorphophallus NG 2 and Amorphophallus RBG 1 appear related also to these last complex of taxa but in the absence of the inflorescence their true relationships cannot as yet be ascertained. Electrophoretically they are related to A. oncophyllus and A. bulbifer, as is the case with A. lambii. ACKNOWLEDGEMENT This paper is adapted from an M.Sc. thesis submitted in 1981 by one of us (EAW) to the University of Birmingham. We are very grateful to Prof. J.G. Hawkes, the then Head of the Department of Plant Biology, University of Birmingham, for allowing us to use the research facilities in the Department. We would like also to thank the Director of National Biological Institute Bogor for her interest and for sending the living specimens used in this study, and to the Director of Royal Botanic Gardens Kew for the permission to examine the herbarium specimens as well as the living collections under his keepings. We are indebted to Dr. Simon Mayo (Kew) for his hospitality and fruitful discussions during the course of this study, and to the British Council (The Colombo Plan Technical Assistance Co- operation Scheme) for a maintenance grant to one of us (EAW). 280 REINWARDTIA [Vol. 10 REFERENCES BACKER, C.A. 1920. Determinatie-tabel voor de Javaansche soorten van Amorpho- phallus Bl. In Tropische Natuur 9 : 21 - 32. BACKER, C.A. &BAKHUIZEN VAN DEN BRINK Jr., R.C. 1968. Flora of Java 3. Wolters- Noordhoff, Groningen. DAVIS, B.J. 1964. Disc-electrophoresis II. Methods and application to human serum proteins. . In Ann. New York Acad. Sci. 121 : 404 - 427. ENGLER, A. 1911. Araceae-Lasiodeae. 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