Journal of Applied Sciences Research 1(2): 130-136, 2005 © 2005, INSInet Publication

Flavonoids Diversification in Organs of Two Farcta (Banks & Sol.) Eig. (Leguminosea, Mimosoideae) Populations Occurring in the Northeast and the Southeast of Tunisia

1F. Harzallah-Skhiri and 2H. Ben Jannet

1Laboratoire de Biologie Végétale et botanique, Ecole Supérieure d’Horticulture et d’Elevag de Chott-Meriem, 4042 Chott-Meriem, Tunisie. 2 Laboratoire de Chimie des Substances Naturelles et de Synthèse Organique, Faculté des Sciences de Monastir, 5000 Monastir Tunisie

Abstract: The patterns of P. farcta flavonoids were investigated in branches, leaves, roots, flowers and pods without seeds (pericarps) of two populations belonging to this species occurring in the northeast (Nabeul locality) and the southeast (Gabès locality) of Tunisia. Sixteen constant phenolic compounds were identified in P. farcta plant material: Vicenin-2, Apigenin C-glycoside, Iso-orientin (=Luteolin 6-C-glucoside), Myricetin 3-O-glucoside, Vitexin, Luteolin 7-O-glucoside, Isovitexin, Quercetin 3-O-glucoside, Rutin, Quercetin 3-O-galactoside, Chrysoeriol 7-O-glucoside, Kaempferol 3-O-rutonoside, Isorhamnetin 3-O-rutinoside, 5-deoxyluteolin, Caffeic acid derivative and Luteolin, six among them were major at least in one organ. Four unidentified compounds were found diversely distributed in organs. A relative similarity was found in the compounds identified in from the two populations although the occurrence of one unidentified compound only in branches of Gabès plants. In addition, all Nabeul or Gabès plant organs have not the same major flavonoid. The present data support the hypothesis of the occurrence in Tunisia of two varieties for the species P. farcta such as mentioned in literature and permit to specify the chemotype race of each plant populations only by analysing pericarp flavonoids.

Key words: Prosopis farcta, flavonoids, plant organs, locality, chemotype race.

INTRODUCTION Those investigations lead to propose varieties and hybrids.[8] Species belonging to the genus Prosopis L. In several biosystematical and evolutionary studies, (Leguminosae, subfamily Mimosoideae) are multipurpose, secondary compounds like flavonoids have been very providing a range of products including timber, firewood, valuable for obtaining information about relationships and livestock feed, human food and beverages, fibres and the hybrid origin of taxa. [9-13] Nevertheless, Markham et al. tannins. They are also valued for shade, shelter belts, bee [14] reported about intra-individual variation of phenolics forage and as nitrogen-fixing soil improvers. [1] and its implication for chemosystematics. Prosopis was difficult taxonomically because of the Carman [15] has carried out a general study on the limitation of its species, which are often without sharp chromatographic characterisation of different Prosopis morphological discontinuities.[2-5] Hybridization and species. Bragg et al. [7] have reported on the perhaps transitional forms from one species to another chromatographic variation within the P. juliflora seem to be frequent as has been suggested by several complex and Palacios and Bravo [16] have studied the authors.[6] Despite its great polymorphism and its large morphology and flavonoid chromatographies of geographic distribution through-out the drier subtropical Prosopis species and hybrids of the Argentinian and tropical regions of the world taxa within Prosopis Chaquena region. have received various taxonomic treatment based on Because flavonoids are routinely used for systematic interspecific morphologic variation. Species of Prosopis studies [15] and may exhibit ecogeographic variations [17] we specially those from section Algarrobia are examined two ectopically differentiated populations of morphologically variable causing taxonomic difficulty.[7] P. farcta occurring in Tunisia.

Corresponding Author: F. Harzallah-Skhiri, Laboratoire de Biologie Végétale et botanique, Ecole Supérieure d’Horticulture et d’Elevag de Chott-Meriem, 4042 Chott-Meriem, Tunisie,

130 J. App. Sci. Res. 1(2): 130-136, 2005

Prosopis farcta, is a shrub that grows naturally in volume of 10 ml and a sample of each of the extracts was two distinct areas, one in the northeast and the second in analysed for flavonoids content by HPLC. the southeast of Tunisia. [18,19] This species is well adapted Flavonoids of methanol and acetone extracts were to drought and high temperatures and exhibit a high analysed by HPLC using a reversed phase analytical degree to salt tolerance. [20,19] It plays also an essential column (RP18) equipped with detector which measured at ecological role in the protection and improvement of soils UV. Individual components were identified by comparison since its root system grows vertically as well as laterally of their UV spectra with those indicated in literature [24] in the soil. The species provides firewood for local and with those of authentic standards. Importance of each population, livestock feed and bee forage.[19] phenolic compound was estimated by the corresponding Pods and seeds of this plant, collected at different pick area. localities, differ in form, size and color, though those collected at each locality show considerable uniformity. Analysis conditions: HPLC consists of a Waters LC600 Study of root, stems and pollen morphology allows pump and a 996 photodiode array detector which distinguishing also high differences between the two measured at UV. HPLC was fitted with a Merck population.[21,22] So, we suppose the presence in Tunisia (Darmstadt, Germany) LiChrospher 100RP-18 (250x 4.0 mm of the two varieties, previously mentioned by Burkart.[3] i.d.; 5 µm particle size) column using a 20 min linear Here we have used chemosystematic approaches to gradient of 25-100% MeOH in 1% aq. HOAc at 1ml/min. investigate the problems of plant disjunctions.[23] So, Eluting solvents were 2% aq. HOAc and MeOH, HOAc, flavonoid chemistry of all organs of P. farcta plants from H2O in proportion of 18:1:1. The column temperature was two localities was utilized to distinguish between the two 30°C. population. In the present paper, two ecotypically-differentiated RESULTS AND DISCUSSIONS population of Prosopis farcta developed in two distinguished areas were examined for their flavonoids Yield in phenolic compounds within locality and organ: and we report on the intra-specific variation of phenolic Yield in phenolic compounds varied within organ and compounds in each organ of the species. Systematic and locality (Table 1). Most of phenolic compounds were ecological implications of the flavonoids data for the P. extracted with methanol. Flowers from Prosopis farcta farcta complex were discussed. Gabès population showed the highest amount of phenolic compounds found in the methanolic extract (78.6%). MATERIALS AND METHODS Except leaves and branches, the other organs from P. farcta Gabès population gave more phenolic compounds Plant material: Plant materiel used in this investigation both with acetone and methanol. The lowest yields in was obtained from wild populations of P. farcta flowering phenolic compounds were obtained in the case of shrubs developed in two distinct localities in Tunisia: branches and pericarps. Gabès and Nabeul. All voucher specimens are deposited in the Ecole Supérieure d’Horticulture et d’Elevage, Phenolic composition of different P. farcta organs: The Botanic Laboratory Herbarium, Chott-Meriem, Tunisia. data were given in table 2. Composition of the different Mature pods were collected in October (for Gabès) or plant part of P. farcta shows a relative similarity between November (for Nabeul). the two localities. Sixteen constant phenolic 500 g of adult branches, leaves, roots, flowers and compounds were identified in P. farcta plant pods without seeds (pericarps) were air dried slightly material: Vicenin-2, Apigenin C-glycoside, Iso-orientin above room temperature, grounded with a mortar and (=Luteolin 6-C-glucoside), Myricetin 3-O-glucoside, weighed. Vitexin, Luteolin 7-O-glucoside, Isovitexin, Quercetin 3-O-glucoside, Rutin (= Quercetin glycoside I), Quercetin Extraction and analysis of flavonoid compounds: Powder 3-O-galactoside (= Quercetin glycoside II), Chrysoeriol of each plant part was placed in a Soxhlet apparatus and 7-O-glucoside, Kaempferol 3-O-rutonoside, Isorhamnetin extracted at room temperature by petroleum ether. Until 3-O-rutinoside, 5-deoxyluteolin, Caffeic acid derivative 12h, waxes and pigments were eliminated. The plant and Luteolin, six among them were major at least in one materiel was re-extracted successively with acetone and organ. Four unidentified compounds were diversely methanol. All extracts were concentrated by rotary distributed in organs: C1 in branches of Gabès plant evaporation at 40°C. The concentrated extracts were population, C2 and C3 in branches and flowers and C4 in resuspended in 80% aq. acetone or MeOH to a total pericarps of Nabeul and Gabès plant populations.

131 J. App. Sci. Res. 1(2): 130-136, 2005

Table 1: Yield in phenolic compounds within locality and organ. Acetone extract Yields % Organs Nabeul locality Gabès locality Branches 0.9 0.6 ------Leaves 4.2 2.5 ------Roots 0.6 2.2 ------Flowers 1.7 2.4 ------Pericarps 0.4 1.3 Methanol extract organs Nabeul locality Gabès locality Branches 6.1 5.0 ------Leaves 32.8 12.9 ------Roots 9.7 12.8 ------Flowers 13.0 78.6 ------Pericarps 3.1 3.9

Composition of branches: Similarity was reported between 3). Isovitexin, Rutin, Quercetin 3-O-galactoside and the phenolic compounds identified in those plant parts but unknown compound C3 were in an appreciate amount for with some differences in their relative proportions. The the 2 localities, the rest of products was minor. Saponin unknown compound C1 was found only in branches of flavonoids were almost four times more abundant than Gabès plant population. Quercetin 3-methyl ether was free flavonoids and other components. specific to branches of the two plant populations. The Composition of pericarps: Appreciate differences in overvalue phenolic product found in Nabeul branches phenolic compounds quantities were found between was an heteroside: Quercetin glycoside I (= Rutin) (Table plants. Pericarps from the two localities do not have the 3). In the case of Gabès, the two major products were same majority product (Table 3). In case of Nabeul, it was Apigenin C-glycoside and 5-deoxyluteolin. The remaining the Isorhamnetin 3-O-rutinoside, the other products compounds were minor or in appreciate amount. Myricetin 3-O-glucoside, Rutin, Quercetin 3-O- Moreover, chromatogram analysis shows that saponin galactoside, the unknown C4, Chrysoeriol 7-O-glucoside flavonoids were nearly twice more abundant than all other were in appreciate amounts. products (tannins and free flavonoids). In the case of Gabès the major compound was caffeic acid derivative. All the other products were in appreciate Composition of roots: The same phenolic composition amounts. Saponin flavonoids in the case of Nabeul were was found in the roots from the two localities but with a found more abundant than tannins and free flavonoids. difference in the amounts of Caffeic acid derivative (R.T =17.82 min) and Luteolin. The 5-deoxyluteolin was major Comparison of Nabeul and Gabès flavonoid organ in roots of Nabeul plant population (Table 3). Tannins composition: Repartition pattern of the six major were specially found in great quantity. Phenolic products flavonoids within organs and locality of P. farcta plants were all free flavonoids. were reported in table 3. Myricetin 3-O-glucoside was major in flowers and abundant in pericarps of Nabeul and Composition of leaves: Leaves from the two plant Gabès plant populations. Isovitexin was major in leaves localities have practically the same phenolic compounds and abundant in flowers of plants from the two localities. with Isovitexin as the same major product (Table 3). Rutin and Isorhamnetin-O-rutinoside were major Vitexin was in an appreciate amount in the two localities. respectively in branches and pericarps of Nabeul plant Saponin flavonoids were more abundant than free populations, whereas roots of Gabès plant populations flavonoïds and tannins. were rich in 5-deoxyluteolin. Caffeic acid derivative was identified as the major phenolic compound in pericarps of Composition of flowers: Flowers from the two plant plants from the same locality (Gabès). localities have also the same phenolic compounds with A relative similarity was found in the compounds the same major product: Myricetin 3-O-glucoside (Table identified in plants from the two populations and all

132 J. App. Sci. Res. 1(2): 130-136, 2005

Table 2: Flavonoids and other phenolic compounds and their time retention identified in different organs of Prosopis farcta from two localities (Nabeul and Gabès) (branches, leaves, roots, flowers and pericarps). Organs Phenolic compounds R.T (min) Branches Tannins 2.37 ------Vicenin-2 8.88 ------5-deoxyflavone glycoside 10.45 ------Apigenin C-glycoside 10.90 ------Iso-orientin (= Luteolin 6-C-glucoside ) 11.25 ------Myricetin 3- O-glucoside 12.11 ------Vitexin (=Apigenin 8-C-glucoside) 12.58 ------C1a (found only in Plants from Gabès locality) b 12.66 ------Luteolin 7-O-glucoside 13.13 ------Isovitexin 13.38 ------Quercetin 3-O-glucoside (found only in branches of Plants from Gabès locality) 13.60 ------Quercetin Glycoside I = Rutine 13.95 ------Quercetin Glycoside II = (Quercetin 3-O-galactoside) 14.13 ------Mixture of Tricine and Chrysoeriol 7-O-glucoside 14.83 ------Mixture of Kaempferol 3-O-glucoside and Isorahmnetin 3-O-glucoside 15.08 ------Chrysoeriol 7-O-glucoside 15.35 ------Kaempferol 3-O-rutinoside 15.50 ------Isorhamnetin 3-O-rutinoside 15.72 ------5-deoxyluteolin 16.11 ------C2 17.40 ------Caffeic acid derivative 17.80 ------C3 18.00 ------Luteolin 18.44 ------Quercetin 3-methyl ether 18.99 Leaves Tannins 2.77 ------Galloyl tannin 5.75 ------Vicenin-2 (= apigenin 6,8-diC-glucoside) 8.80 ------Luteolin C-glycoside 10.50 ------Apigenin C-glycoside 11.01 ------Iso-orientin (= Luteolin 6-C-glucoside ) 11.20 ------Myricetin 3-O-glucoside 12.08 ------Vitexin (= Apigenin 8-C-glycoside) 12.53 ------Caffeic acid derivative 13.07 ------Isovitexin (= Apigenin 6-C-glycoside) 13.37 ------Rutin (= Quercetin glycoside I) 13.88 ------Quercetin 3-O-galactoside = Quercetin glycoside II 14.03 ------

133 J. App. Sci. Res. 1(2): 130-136, 2005

Table 2: Continued. Tannins 14.22 ------Mixture of Kaempferol 3-O-glucoside and Isorahmnetin 3-O-glucoside 15.11 ------Chrysoeriol 7-O-glucoside 15.34 ------Kaempferol 3-O-rutinoside 15.50 ------Isorhamnetin 3-O-rutinoside 15.70 Roots Tannins 2.40 ------5-deoxyluteolin 16.11 ------Caffeic acid derivative 17.82 ------Luteolin 18.42 Flowers Tannins 2.37 ------Caffeic acid derivative 6.14 ------Vicenin-2 (= Apigenin 6,8 diC-glucoside) 8.78 ------Apigenin C-glycoside 10.82 ------Iso-orientin (= Luteolin 6-C-glucoside ) 11.20 ------Myricetin 3- O-glucoside 12.10 ------Vitexin 12.52 ------Caffeic acid derivative 13.13 ------Isovitexin (=Apigenin 6-C-glucoside) 13.37 ------Rutin (= Quercetine glycoside I) 13.94 ------Quercetin Glycoside II (= Quercetin 3-O-galactoside) 14.09 ------Mixture of Isorhamnetin and Kaempferol 3-O-rutinoside 15.55 ------Isorhamnetin 3-O- rutinoside 15.71 ------C2 17.40 ------C3 18.09 Pericarps Tannins 2.37 ------Vicenine-2 8.30 ------Apigenin C-glycoside 10.90 ------Iso-orientin (= Luteolin 6-C-glucoside ) 11.20 ------p-coumarinic acid derivative 11.92 ------Myricetin 3-O-glucoside 12.11 ------Caffeic acid derivative 12.50 ------Luteolin 7-O-glucoside 13.19 ------Isovitexin 13.37 ------Quercetin 3-O-glucoside 13.60 ------Rutin (=Quercetine glycoside I) 13.95 ------Quercetin glycoside II (= Quercetin 3-O-galactoside) 14.10 ------Tannins 14.24 ------Mixture of Kaempferol 3-O-glucoside and Isorhamnetin 3-O-glucoside 15.17 ------

134 J. App. Sci. Res. 1(2): 130-136, 2005

Table 2: Continued. Chrysoeriol 7-O-glucoside 15.30 ------Mixture of Isorhamnetin and Kaempferol 3-O-rutinoside 15.53 ------Isorhamnetin 3-O-rutinoside 15.73 ------5-deoxyluteolin 16.12 ------Caffeic acid derivative 17.82 ------C4 18.20 ------Luteolin 18.46 a : Ci : unidentified compound b: Italic : Compound specific to a one plant locality.

Table 3: Repartition of the major phenolic compounds within P. farcta organs and plant populations locality Phenolic compound Branches Roots Leaves Flowers Pericarps Myricetin 3-O-glucoside +C - + Major (Gabès and Nabeul) +++ ------Isovitexin + - Major (Gabès and Nabeul) +++ + ------Rutin =Quercetin glycoside I Major (Nabeul) - + +++ + ------Isorhamentin 3-O-rutinoside ++ - + + Major (Nabeul) ------5-deoxyluteolin ++ Major (Gabès) - - +++ ------Caffeic acid derivative + ++ - - Major (Gabès) ------(R.T : 17.82 min) c : ( +) minor ; (++) appreciate ; (+++) abundant; (-) absent

Nabeul or Gabès plant organs have not the same major their chemistry and leaf morphology. Naranjo et al.[27] flavonoid. Sixteen constant phenolic compounds were proposed two chromatographic patterns to P. alba identified in P. farcta plant material: Buckingham [25] probably related with geographical distribution reported that 3 flavonoids; Patulitrin, Patuletin and Rutin (chemovarieties). have been identified in pods and seeds of P. farcta. Bragg Nevertheless, this investigation lets us to identify the et al. [7] have reported that five species in the Prosopis specific chemotype race of P. farcta plants from Gabès juliflora complex, as well as, two varieties and and those from Nabeul. In fact, analysis of methanolic putative hybrid, were found to have the similar flavonoid extract flavonoids of the pericarps can discern between patterns. In addition to the considerable plants from the two localities, Isorhamnetin-O-rutinoside morphological variation previously reported was major in the case of Nabeul plants but for plants from between the two plant populations[21,22] flavonoid Gabès locality Caffeic acid derivative was the major one. chemistry investigation shows also a relatively Since several authors [7] reported that no flavonoid difference between those plants. The most notable correlation with ecotypes was observed so our results variation concerns unknown compound C1 present only widen the range of phyto-constituents known in this in branches of Gabès population. Harzallah-Skhiri et al. [26] species and allow us to justify the occurrence of two reported also differences in the volatile compounds varieties for P. farcta in Tunisia such as proposed in composition between populations from those two previous works [22,26]: P. farcta var. farcta for those from localities. Nabeul, and P. farcta var. glabra for those from Gabès It is interesting to find that P. farcta plants occurring locality. in two distinct areas with ecotypic differentiation as well as morphological diversity were moderately uniform in ACKNOWLEDGMENTS their flavonoid patterns. On the other hand, Carman and Mabry [23] detected seventeen flavonoids and nine major The authors express their gratitude to Professor ones were identified in several populations of P. reptans Monique S.J. Simmonds and Rannee J. Grayer for their collected along the Texas Gulf coast. Authors established help in the analysis and identification of flavonoids with the presence of a single chemical race since all specimens HPLC in the Biochemistry section of Jodrell Laboratory, from the Texas Gulf coast populations were uniform in Royal Botanic Garden, Kew, England.

135 J. App. Sci. Res. 1(2): 130-136, 2005

REFERENCES 14. Markham, K.R., N.A. Moore and L.J. Porter, 1978. Changeover in flavonoid pattern accompanying 1. Harris, P.J.C., N.M. Pasiecznik, M. Bradbury and L. reproductive structure formation in a Bryophyte. Ramfrez, 1998. Problems and potentials of Prosopis, Phytochemistry, 17:911-913. in: Prendergast, H.D.V., N.L. Etkin, D.R. Harris, P.J. 15. Carman, N.J., 1973. Systematic and ecological Houghton (Eds.), Plants for food and medicine, Royal investigations in the genus Prosopis (Mimosoceae) emphasizing the natural products chemistry. 220 p. Botanical Gardens, Kew, UK, pp: 277-293. Ph D Thesis the University of Texas at Austin, 2. Burkart, A., 1937. Estudios morfologicos y Austin Texas. ecologicos en el genero Prosopis, Darwiniana 3 (1): 16. Palacios R.A. and L.D. Bravo, 1981. Hibridacion 27-59. natural de Prosopis (Leguminosae) en la region 3. Burkart, A., 1976. A monography of the genus chaqueta argentina. Evidenciae morfologicas y Prosopis (Leguninosea Subfam; Mimosoideae). cromatograficas. Drawiniana, 23 :3-35. Journal of the Arnold arboretum, 57(3): 219-249 17. Bate-Smith, E.C. and R.H. Richens, 1973. Flavonoid 450-525 chemistry and in Ulmus. Biochem. Syst. 4. Burkart, A. and B.B. Simpson, 1977. The genus Ecol. 1(3): 141-146. Prosopis and annoted key to the species of the 18. Pottier-Alapetite, G.,1979. Flore de la Tunisie. world. In: Mesquite. Its biology in two desert Angiospermes-Dicotylédones, Apétales ecosystems. US/IBP synthesis ¼. Dowden Dialypétales. Publications Scientifiques tunisiennes, pp: 293. Hutchinson and Rose Inc. 255 p. 19. Harzallah-Skhiri, F., 2003. Thèse d’Etat, Faculté des 5. Ffolliot, P.F. and J.L. Thames, 1983. Manuel de Sciences de Tunis, Tunisie, 2 juillet 2003, 268 p. taxonomie de Prosopis au Mexique, Pérou, Chili. 20. Dafni, A. and M. Negbi, 1980. Variability in Prosopis F.A.O. 33 p. farcta in Israel: fertility and seed production in 6. Hunziker, J.H., B.O. Saidman, C.A. Naranjo, R.A. population from different habitats. Acta Oecologica. Palacios, L. Poggio and A.D. Burghart, 1986. Oecologia Plantarum , 1(15), 4 : 335-344 Hybridization and genetic variation in argentine 21. Harzallah-Skhiri, F., 1999. Caractérisation pollinique species of Prosopis. Forest Ecology and de deux provenances de Prosopis facrta en Tunisie. Management, 16: 301-315. Geo-Eco-Trop. Proceeding of the 4th symposium on 7. Bragg, L. H., A.D. Bacon, Mc-Millan Calvin, and T.J. African Palynology, Sousse (Tunisie), 25-30/04/1999. Mabry, 1978. Flavonoid patterns in the Prosopis Spécial numéro 22:217-226. juliflora complex. Biomedical Systematics and 22. Harzallah-Skhiri, F., 2003. Caractérisation Ecology. 6:113-116. morphologique et anatomique de deux provenances de Prosopis farcta (Banks & Sol.) Eig. (, 8. Burkart, A., 1940. Materiales para una monografia del ) par leurs folioles, fleurs, gousses, graines genero Prosopis (Leguminosae). Darwiniana, 4 et grains de pollen. Geo-Eco-Trop. 27 (1-2) :63-76. (1):57-128 23. Carman, N.J. and T.J. Mabry, 1975. Disjunction of 9. Alston, R.E. and B.L. Turner, 1962. News techniques Prosopis reptans and the origin of the north in analysis of complex natural hybridization. Nat. American populations. Biochemical systematics. Acad. Sc. 48:130-137. 3 :19-23 10. Alston, R.E., 1965. Flavonoid chemistry of Baptisia: 24. Mabry, T.J., K.R. Markham and M.B. Thomas, a current evaluation of chemical methods in the 1970.The systematic identification of flavonoids. analysis of inter-specific hybridization. Taxon Springer – Verlag, Berlin-Heiderbelrg, New York. 14: 268-274. pp: 1-102. 11. Levin, D.A., 1966. Chromatographic evidence of 25. Buckingham, J., 1994. Phytochemical Dictionary of hybridization and evolution in Phlox maculate. the Leguminosae. Chapman and Hall edits. London. 567 p. Amer. J. Bot. 53:238-245. 26. Harzallah-Skhiri, F., H. Ben Jannet, S. Hammami, and 12. Hsiao Ju-Ying., 1973. A numerical taxonomic study of Z. Mighri. Variation of volatile compounds in two the genus Platanus based on morphological and Prosopis farcta (Banks & Sol.) Eig. (Fabales, phenolic characters. Amer. J. Bot. 60: 678-684. Fabaceae = Leguminosare) population. In press. 13. Hunziker, J.H., A.G. Palacios de Valesi and 27. Naranjo, C.A., L. Poggio and S. Enus Zeiger, 1984. L. Poggio, 1978. Hybridization in Larrea Phenol chromatography, morphology and (Zygophyllaceae). A morphological, cytogenetic and cytogenetics in three species and natural hybrids of chemosystematic study. Bol. Acad. Nac.Cs. Cordoba, Prosopis (Leguminosae-Mimosodeae). Pl. Styt. Evol. Argentina, 52: 281-314. 144 :257-276.

136