Annals of Plant Sciences ISSN: 2287-688X www.annalsofplantsciences.com OPEN ACCESS Original Research Article The Phylogenetic Relations and Biogeography of Three Indian and Two African Species of Abrus Adanson Amal Kumar Mondal1* and Sanjukta Mondal Parui2 Taxonomy, Biosystematics and Molecular Taxonomy Laboratory, UGC-DRS-SAP Department, Department of Botany & Forestry, Vidyasagar University, Midnapore-721102, West Bengal, India 2 Department of Zoology, Lady Brabourne College, Kolkata-700 017, West Bengal, India 1 Plant Received for publication: April 21, 2014; Accepted: May 17, 2014 Abstract: Phylogenetic interrelationship between five species of Abrus Adanson was studied based on SDS-PAGE protein profile of their seeds. These included Abrus precatorius Linn. and A. pulchellus Thw, two circumtropical species from India and A. canensis Welw. ex Bak. and A. fruticulosus W. & A., two species from continental Africa. The fifth species was also A. precatorius but with white seeds. Pairing affinity values of this species was highest with A. precatorius having red black seeds but varied considerably in their protein profile. A. precatorius was also found to be more related to the other Indian species A. pulchellus. On the other hand A. pulchellus which has been placed as a separate species by Verdcourt but considered to be a subspecies of A. fruticulosus by Breteler, were found to be two separate species. A. precatorius and A. fruticulosus are the most distantly related species. Key Words: Phylogenetic interrelationship, Abrus, SDS-PAGE, protein profile, seeds, Indian species and African species Introduction Abrus Adanson is a pantropical genus and is a member belonging to the tribe Vicieae in the sub-family Papilionaceae of the Leguminosae. As a genus it was first described by Adanson in 1763 who based it on Glycine abrus L. (Breteler, 1960). This genus is well distinguished by the presence of 9 connate stamens. It is often placed in its own tribe Abreae (Polhill, 1981). Morphologically, it has affinities with the Vicieae because of the presence of twining stems and paripinnate leaves ending in a bristle (Hutchinson, 1964). Based on the general appearance, it has affinities with Dalbergieae and Phaseoleae (Baillon, 1876) and also with the African genus Millettia because of its tendency to twine and its pseudoracemes (Pohill, 1981). Recent studies on chloroplast trm K/mat K sequences (Hu et al., 2000) and rbcL data (Doyle et al., 1997) which have been used successfully in phylogenetic studies at the generic level places Abrus as a sister group to the core Millettieae plus Galactia. According to Hu et al. (2000), Abrus shows affinity with core Millettieae members is having a pseudoraceme inflorescence, an absence of canavanine (except in the Philonoptera clade) and chromosome number of x=11. There still prevails a controversy regarding the taxonomy of this genus as there are conflicting reports of the exact number of species in this genus. The first worldwide revision of this genus was done by Bretler (1960). He recorded four species in the genus viz. Abrus precatorious L., A. canescens Welw. ex Baker, A. diversifoliolatus Breteler nom. nov. and A. fruticulosus Wall. ex W. & A. A. canescens is confined to continental Africa, A. diversifoliolatus is found in Madagascar and the remaining two have a more or less circumtropical distribution and are found in Africa, America and Asia. It is quite amazing and unacceptable to see the number of variation which he classified under the name A. fruticulosus (Table 1). According to Breteler’s revision later (Breteler, 1994), he felt that such variation is not well described and insufficiently illustrated which is a minus point to its acceptability. He felt that this group consisting of numerous species appears to be morphologically complex and it is debatable whether this confused areas of specimens needing taxonomic order, represents a pluriform species or *Corresponding Author: Dr. Amal Kumar Mondal, Associate Professor in Botany & Deputy Coordinator of UGC-DRS-SAP DRS-SAP Department, Department of Botany & Forestry, Plant Taxonomy, Biosystematics and Molecular Taxonomy Laboratory Vidyasagar University, Midnapore-721 102 West Bengal, India. Page| 726 Amal Kumar Mondal and Sanjukta Mondal Parui, a complex of perhaps closely allied species or just infraspecific taxa. Verdcourt (1970) however reported thirteen species (A. canescens Welv ex Baker, A. diversifoliolatus Breteler, A. fruticulosus Wall ex. W. & A., A. grandiflorus Vig., A. parvifolius (Vig) Verdc., A. sambiranensis Vig., A. schimperi Bak., A. bottae Defl., A. mandagascariensis Vig., A. laevigatus Mey., A. pulchellus Thw., A. aureus Vig. and A. precatorius L.). He further recorded three subspecies under A. schimperi, five under A. pulchellus, two subspecies under A. aureus and two subspecies under A. precatorius (Table 1). Unlike Breteler, Verdout described the variation in detail and hence proposed many subspecies to accommodate the specimens examined by him. However his Annals of Plant Sciences, 2014, 3 (06), 726-733 description of morphological characters, often derived from the leaves, are sometimes not consistently, combined with geographical separation. Hutchinson and Dalziel (1958) reported three species of Abrus, A. precatorious L., A. pulchellus Wall. and A. canescens Welw. ex Baker in tropical West Africa. Labat (1991) reported a new species A. longibracteatus Labat from Laos and Vietnam, while Thulin (1994) published A. baladensis Thulin and A. gawwenensis from Somalia which was species nova. Thus a wide disagreement about the classification of a plant group can be very problematic for those working with this group of plants. Table 1: Taxonomy of Abrus Adanson (Breteler, 1994) Breteler (1960) Species Distribution A. canescens Welw. Africa ex. Bak. A. diversifoliolatus Africa Bret. (Madagascar) A. fruticulosus W. & Africa, America, A. Asia Species Verdcourt (1970) Subspecies A. canescens Africa A. diversifoliolatus Africa A. fruticulosus s.s Asia (India) A. grandiflorus Vig. A. parvifolius (Vig.) Verdc. A. sambiranensis Vig. A. schimperi Bak. Africa(Ma dagascar) Africa (Madagascar) Africa (Madagascar) ssp. schimperi A. bottae Defl. A. madagascariensis Vig. A. laevigatus Mey. A. pulchellus Thw. ssp. africanus (Vatke) Verdc. ssp. oblongus Verdc. ssp. pulchellus ssp. cantoniensis (Hence) Verdc. ssp. suffruticosus (Boutique) Verdc. ssp. tenuiflorus (Benth.) Verdc. Circumtropical A. aureus Vig. A. precatorius Sudan, Ethiopia, Tanzania, Zambia, Africa (Malawi, Zimbabwe) Asia (Arabia) Africa (S. Africa, Mozambique) Africa, America, Asia Asia (India, China, Malesia) Asia (Thailand, China) Asia (Assam, Malay Peninsula, Java, Vietnam, Thailand, China, Philippines, Sarawak, Borneo, Papua) Africa (Sierra Leone, N. Nigeria, R.C.A. Zaire, Tanzania, Zambia, Angola) America, Africa (from Senegal to Sudan, Ango la and Mozambique) Africa ssp. aureus ssp. littoralis (Vig.) Verdc. ssp. precatorius ssp. Verdc. www.annalsofplantsciences.com Africa Africa (R.C.A., Uganda) Africa (Kenya, Zimbabwe) Africa (Madagascar) ssp. mollis (Hance) Verdc. A. precatorius Linm. s.l. Distribution africanus Africa (Madagascar) Africa (Madagascar) Asia Africa (including Seychelles, Mauritius, America & Australia Madagascar), Introduced in Page | 727 Amal Kumar Mondal and Sanjukta Mondal Parui, Seed protein studies have proved to be an excellent parameter to resolve the problems of identification of critical taxa and to understand their relationships and taxonomic status (Esen and Hilu, 1991; Khan, 1992; Sanchez-Yelano et. al., 1992). Seed protein patterns are highly stable and are unaffected by environmental conditions (Harborne and Turner, 1984). The present paper reports the study of phylogenetic interrelationship between five species of Abrus Adanson based on SDS-PAGE protein profile of their seeds. These included Abrus precatorius Linn. and A. pulchellus Thw, two circumtropical species from India and A. canensis Welw. ex Bak. and A. fruticulosus W. & A., two species from continental Africa. The fifth species was also A. precatorius but with white seeds. Materials and Methods Collection of seeds Seeds of Abrus precatorius Linn. were collected from mature pods from plants growing in Santiniketan, West Bengal, India while the seeds of A. pulchellus Thw were collected from plants growing in Kanyakumari, Tamil Nadu, India. The seeds of A. fruticulosus W. & A. were collected from Usambara (North-East Tanzania), south-east Africa and Ivory Coast or Côte d'Ivoire (near Tiassate), west Africa while those of A. canensis Welw. ex Bak. was collected from Burundi, Bujumbura Province, south east Africa and Liberia, East of Ganta, road to Buchanan, west Africa. The seeds were sterilized in 10% (v/v) chlorox – 0.1% (v/v) Tween 20 for 5 min (Mondal et al., 1998). After rinsing in sterilized distilled water for 30 min, the seeds were immersed in sterilized distilled water overnight and used for protein extraction. Protein extraction from seeds For the extraction of protein from the seeds, a modification of Gifford's method (Gifford et al., 1982) was used. Protein was extracted from seed endosperm plus embryo in Tris-glycine buffer (0.01 M Tris; 0.08 M glycine), pH 8.2 [TGP buffer] containing 2% NaCl and clarified by centrifuging at 19,000 g for 20 min at 4°C. The supernatant containing the soluble proteins was pooled off while the pellet containing the insoluble storage proteins was suspended in the same TGP buffer and an equal volu me of 62 mM Tris-HCl (pH 6.8) buffer with 3.05% (w/v) SDS and 10.7% (w/v) glycerol and boiled for 8 min (Jenson and www.annalsofplantsciences.com Annals of Plant Sciences, 2014, 3 (06), 726-733 Lixue, 1991). After centrifugation, the supernatant was used for SDS-PAGE. Gel electrophoresis Protein electrophoresis by SDS-PAGE was carried out according to the method of Laemmli (1970) with samples containing 80 µg of protein each. The samples were boiled for 3 min with equal amount of sample buffer (0.6 M Tris HCl (pH 6.8), 1% SDS, 10% sucrose, 0.5% mercaptoethanol, 0.01% bromophenol blue] at 100°C and applied to each well of a mini-gel (8X7 cm gel). The acrylamide concentration in the gel was 15% and electrophoresis was performed with electrode running buffer, pH 8.4 (0.05 M Tris, 0.192 M Glycine, 0.1% SDS). A constant current of 50 mA was supplied until bromophenol blue entered the separating gel and then the current was increased to 60 mA until the dye moved to the bottom of the gel. After electrophoresis the gel was stained with 0.1% Coomassie Brilliant Blue R250 and destained with methanol: acetic acid: water (4:1:5) mixture. Numerical analysis Pairing affinity or similarity index was calculated by the method described by Sokal and Sneath (1963) and Romero Lopes et al., (1979). Based on the results of electrophoretic analysis, the degree of pairing affinity (PA) was calculated by the following formula: P. A. = Bands common to species A and B Total bands in A and B x 100 Dendogram expressing the average linkage was computed using the cluster method of Unweighted Pair Group Method with averages (UPGMA relationship) [Sneath and Sokal, 1974]. NTSYSpc.2.2 software was used to compute the dendogram. Re sults an d Discussion Abrus is a genus where the characters of the inflorescence and pod proved to be more useful in distinguishing between species as flowers of the species do not show any character leading to specific segregation. The morphological appearances of the four species are presented in Table 2. The results showed that they are four distinct species of Abrus. However the fifth species (A. precatorius but with white seeds) did not show any morphological variation from A. precatorius with scarlet seeds having a black spot around the hilum. Page | 728 Amal Kumar Mondal and Sanjukta Mondal Parui, Annals of Plant Sciences, 2014, 3 (06), 726-733 Table 2: Comparative account of the 4 species of Abrus A. precatorius Linn. Winding or trailing woody vine or climber, stem slender, with slender herbaceous much branches, branched with glabrescent mostly greenish yellow young branches, sparsely white strigose Leaves 8-16 jugate, petiole 5-15mm long Plant Leaves Leaflets 6-25mm long, 3-10mm wide, ovate, obovate or oblong, top obtuse or acuminate, base rounded or subcordate, upper surface glabrous or pubescent, lower surface densely or sparsely appressedpubescent Leaflets Inflorescence Pods Seeds Inflorescence rigid, thick, strongly falcate, bracts and bracteoles 0.5-1mm long, flowers crowded, subsessile, calyx 3-4mm long, pubescent, corolla 3-4 times as long as the calyx, pale purple to yellowish Pods roughly rectangular, bulgy, 2-4 cm long, 1-1.5 cm wide, densely warty, tomentose, beak hook shaped, reflexed, 3-7 seeded Seeds ovoid, 5-7mm long, 4-5mm broad, scarlet, with a black spot around the hilum, glossy A. canensis Welw. ex Bak. A. pulchellus Thw. Lianas, climbing, stem slender, sparsely yellow strigose or villous Winding climber with woody pubescent branches, finally glabrescent Leaves paripinnate, petiole short, Leaves 10-14 jugate, petiole short alternate, A. fruticulosus W. & A. A climber or a diffuse creeping fastigiated or straggling shrub or undershrub, generally 3-5 m in length, young branches pubescent, finally glabrescent Leaves 5-20 jugate Leaflets 6-10-paired, opposite, 620mm long, 3-12mm wide, suboblong, oblong, or obovateoblong, , top truncate, base rounded or subcordate, membranous, upper surface sparsely white strigose or densely white villous, lower surface glabrous or pilose Leaflets 6-20mm long, 2-7mm wide, rectangular, white or grey pubescent on both sides Leaflets 3-46mm long, 215mm wide, varying in shape and pubescent, ovate, obovate or oblong, top acute, rounded, obtuse or truncate-emarginate, base cordate, rounded, or cuneate, often unequalsided, upper surface pubescent, glabrescent, lower surface densely or sparsely appressedpubescent Inflorescence racemes axillary, flowers dense; calyx campanulate, 4-toothed, white strigose or densely gray villous, 35mm long, corolla 4-9 mm long pink, purple, or purple-red Inflorescence terminal; flowers subsessile, usually in separated fascicles, bracts and bracteoles as long as calyx,3-6mm long, corolla 10-13mm long, purple Inflorescence terminal, lateral or axillary; flowers in groups, bracts and bracteoles half as long as calyx, corolla 3-6 times as long as calyx, pale purple to yellowish Pods broad, linear, nearly straight, 36cm long, 8-12mm wide, beak hook shaped, recurved, pubescent, 6-9 seeded Pods oblong, 2-10 cm long, 0.5-1.5cm wide, flattened, broadly rounded or cuneate at base, rounded at apex, pubescent or glabrescent, 412 seeded Seeds ovate, 4-6mm long, 3-4mm broad, light brown black, glossy Seeds oval, laterally compressed, 3-6mm long, 25mm broad, brown black, mostly glossy Pods oblong, 3-6.5 cm long, 0.81.5 cm wide, densely white hairy, dehiscent, 4-12 seeded Seeds elliptic or ovoid, compressed, 3-6mm long, 2-5 mm broad, blackbrown or black, glossy to slightly glossy Table 3: Pairing affinity values (%) of the seed protein of Abrus sp. based on electrophoretic patterns A. canensis A.pulchellus A. fruticulosus A. precatorius canensis pulchellus fruticulosus precatorius 100 52.9 53.3 43.8 100 58.8 42.1 100 38.9 100 A. precatorius (white seeds) 60.0 68.8 47.1 78.6 A. A. A. A. Study of the phylogenetic interrelationship between the five species of Abrus based on SDS-PAGE protein profile of their seeds and numerical analysis of the data obtained (pairing affinity values) shows that the two Indian species A. precatorius and A. pulchellus are more related (Table 3) having the affinity of 68.8%. However the two species of A. precatorius which did not differ morphologically however showed a variation www.annalsofplantsciences.com A. precatorius (white seeds) 100 in the protein profile and instead of showing 100% affinity showed a pairing affinity of 78.6%. According to Breteler (1960), the seeds of A. precatorius sometimes appear to be entirely black or white. He explained that this is accidental and is not correlated with any morphological character. This seems to be contradictory to the results of the present study. On the other hand A. pulchellus which has been placed as a separate species by Page | 729 Amal Kumar Mondal and Sanjukta Mondal Parui, Verdcourt but considered to be a subspecies of A. fruticulosus by Breteler, were found to be two separate species showing 58.8% affinity. Thus the taxonomy of A. fruticulosus which has long remained debated and has been reduced as a synonym of A. pulchellus and A. mollis and others by Breteler (1960) needs to be treated separately now as proposed by Verdcourt (1970). A. fruticulosus has also been reported from India but as sparse in some parts of its range in India particularly coastal forest in Tamil Nadu. It is believed to have suffered a population decline over the last 10 years here, however, this is not thought to be greater than 30%, and there are possibly more than 10,000 mature individuals (Sanjappa pers comm., 2011). The other species A. canensis was more related to A. fruticulosus (53.3%) followed by A. pulchellus (52.9%) and A. precatorius (43.8%). A. precatorius and A. fruticulosus are the most distantly related species with only 38.9% affinity. Con clu sion The results of the present investigation highlights the usefulness of seed proteins as an excellent parameter to resolve the problems of identification of critical taxa and shows the demerit of over dependence on morphological and especially morphometric studies for taxonomic elucidation of species. A. precatorius with white seeds and A. precatorius with scarlet seeds having a black spot around the hilum, which did not show any morphological variation proved to be chemotaxonomically different and needs to be treated separately. A. precatorius with white seeds may be placed as a subspecies under A. precatorius. This however needs further investigations and may be proved through molecular studies particularly DNA. A. precatorius and A. pulchellus are more related while A. precatorius and A. fruticulosus are the most distantly related species. A. pulchellus which has been placed as a subspecies of A. fruticulosus by Breteler needs to be treated as a separate species and this is in confirmation with the placement of the species by Verdcourt. A. canensis is more related to A. fruticulosus followed by A. pulchellus and A. precatorius. www.annalsofplantsciences.com Annals of Plant Sciences, 2014, 3 (06), 726-733 a) b) Fig. 1: a) A twig of Abrus precatorius with pods b) The seeds of A. precatorius. a) b) Fig. 2: a) Pod of Abrus precatorius with white seeds b) The seeds of A. precatorius (white seeds). Page | 730 Amal Kumar Mondal and Sanjukta Mondal Parui, a) Annals of Plant Sciences, 2014, 3 (06), 726-733 b) Fig. 5: The seeds of A. fruticulosus collected from a) Usambara (North-East Tanzania), south-east Africa and b) Ivory Coast or Côte d'Ivoire (near Tiassate), West Africa. b) Fig. 3: a) A flowering twig of Abrus pulchellus b) The seeds of A. pulchellus. a) Fig. 4: A flowering twig of Abrus fruticulosus collected from Kanyakumari, Tamil Nadu. b) Fig. 6: The seeds of A. canensis collected from a) Burundi, Bujumbura Province, south east Africa and b) Liberia, East of Ganta, road to Buchanan, West Africa. a) www.annalsofplantsciences.com Page | 731 Amal Kumar Mondal and Sanjukta Mondal Parui, Annals of Plant Sciences, 2014, 3 (06), 726-733 Netherlands for sending the seeds of A. canensis and A. fruticulosus. a b c d e f g h Fig. 7: SDS-PAGE protein profile of the seeds of Abrus a) Marker proteins, b) A. canensis, c&d) A. pulchellus, e) A. fruticulosus, f) A. precatorius, g) A. precatorius (white seeds), h) 66kDa protein (BSA). Fig. 8: Diagramatic representation of the SDSPAGE protein profile of the seeds of Abrus. Referen ce s 1. Polhill RM, Raven PH, Papilionoideae, In: Advances in legume systematics, Part 1 Ed, Royal Botanic Gardens, Kew, UK, 1981, 191-208. 2. Hu Z, Bonifas JM, Beech J, Bench G, Shigihara T, Ogawa H, Ikeda S, Mauro T, Epstein EH, Jr, Mutations in ATP2C1, encoding a calcium pump, cause Hailey-Hailey disease, Nature Genet, 2000, 24, 61-65. 3. Bretler FJ, Revision of Abrus Adanson (Pap.) with special reference to Africa, Blumea, 1960, 10, 607-624. 4. Breteler FJ, The use of the subspecies concept in African plant taxonomy with particular reference to Abrus (Leguminosae – Papilionoideae). Seyani, JH and Chikuni, AC Proc. XIIIth Plenary Meeting AETFAT, Malawi, 1994, 1, 309-314. 5. Harborne JB, and Turner BL, Plant chemosystematics. Academic Press, London, 1984. 6. Hu JM, Lavin M, Wo jciechowski MF, Sanderson MJ, Phylogenetic systematics of the tribe Millettieae (Leguminosae) based on chloroplast trnK/matK sequences and its implications for evolutionary patterns in Papilionoideae, Am J Bot, 2000, 87, 418-430. 7. Doyle MP, Zhao T, Meng J, Zhao S, Escherichia coli O157: H7, In: Foo d Microbiology: Fundamentals and Frontiers (Doyle, MP, Beuchat, LR, Montville, TJ Eds, ASM Press, Washington, DC, 1997, 171–191. 8. Esen A, Hilu KW, Electrophoretic and immunological studies of prolamines in the Poaceae – II, Phylogenetic affinities of the Aristideae, Taxon, 1991, 40, 5-17. 9. Gifford DJ, Greenwood JS, Bewley JD, Deposition of matrix and crystalloid storage proteins during protein body development in the endosperm of Ricinus communis L. ev. Hale seeds, PI. Physiol, (Lancaster), 1982, 69, 1471-1478. Fig. 9: Dendogram representing the “average linkage” relationship between the 5 species of Abrus as revealed by SDS-PAGE of seed protein. Ackn owle dge men t The authors would like to thank Professor F.J. Breteler of the Department of Plant Taxonomy, Laboratory of Plant Taxonomy and Plant Geography, Herbarium Vadense, Agricultural University, Foulkesweg 37, Wageningen, The www.annalsofplantsciences.com 10. Hutchinson J, Dalziel JM, Flora of West Tropical Africa, Part 2, Crown Agents for Oversea Governments and Administrations, London, 1958. 11. Jensen U, Lixue C, Abies seed protein profile divergent from other Pinaceae, Taxon, 1991, 40, 435-44. 12. Khan MA, Seed-protein electrophoretic pattern in Brachypodium P. Beauv. Species, Ann of Bot, 1992, 70, 61-68. Page | 732 Amal Kumar Mondal and Sanjukta Mondal Parui, 13. Labat JN, Abrus longibracteatus, une espece nouvelle de Lguminosae – Papilionoideae du Laos et du Vietnam, Bull, Mus, Nation, Hist, Nat, sect, B, Adansonia, 1991, 13(3-4), 167171. 14. Laemmli VK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, 227, 681685. 15. Mondal AK, Parui S, Nandi JB, Mandal S, Studies on the effect of temperature on the germination of seeds, Indian J Plant Physiol, 1998, 3(2), 168-171. 16. Romero Lopes C, De Lucca EJ, De Andrade AM, Jacoia Faulin CM, A phylogenetic interpretation of chromosomal and electrophoretic data in Columbiformes, Cytologia, 1979, 44, 39-47. 17. Sanchez-Yelamo MD, Ortiz JM, Gogorcena Y, Comparative electrophoretic studies of seed Annals of Plant Sciences, 2014, 3 (06), 726-733 proteins in some species of the genus Diplotaxis, Erucastrum and Brassica (Cruciferae: Brassiceae), Taxon, 1992, 41, 477-483. 18. Sneath PHA, Sokal RR, Numerical Taxonomy, WH, Freeman and Company, San Francisco, USA, 1973. 19. Sokal RR, Sneath PHA, Principles of numerical taxonomy, W,H, Freeman and Company, San Francisco, USA, 1963. 20. Thulin M, Two new species of Abrus (Leguminosae – Papilionoideae) from Somalia, Nord J Bot, 1994, 14, 55-58. 21. Verdcourt B, Studies in the Leguminosae – Papilionoideae for the Flora of Tropical East Africa: Abreae. A reappraisal of the species of the genus Abrus Adans Kew Bull, 1970, 24, 235-253. Source of support: Nil Conflict of interest: None Declared www.annalsofplantsciences.com Page | 733