INTRODUCTION
Tribe Trifolieae is one of the major group belonging to subfamily Papilionoideae, family Leguminosae and considered by Debelle et al.[1] as one of the Leguminous tribes forming the galepoid group. According to Berchtold and Presl[2] and recently Polhill and Raven[3] this tribe includes seven genera; Ononis, Parochetus, Melilotus, Trigonella, Factorovskya, Medicago and Trifolium. These genera are characterized by having stipules adnate to the petiole, except in Parochetus, trifoliate leaves and small seeds with well developed radicle. Schulz[4] found that the ovules within these genera differ and he separated the genera Trifolium, Parochetus and Ononis in a tribe called Trifolieae for having seeds with micropyle supra funiculum. He put the other four genera in another tribe, Trigonelleae, having seeds with micropyle infra funiculum. Hutchinson[5] separated the genus Ononis in a monogeneric tribe, Ononideae, for having reduced leaves and monadelphous dimorphic androecium. Meanwhile, he kept the other six genera in tribe Trifolieae. Hutchinson treatment of the genera has been accepted[6-9]. Small[10], Small et al.[11,12] accepted the separation of genus Ononis in a monogeneric tribe but he regrouped the other six genera in two subtribes; subtribe Trifoliinae with the genus Trifolium only and subtribe Trigonellinae with the rest of the genera. This treatment of the genera has been supported by Lersten[13] who found that genus Ononis has uniseriate, from one to eight cells, suspensor and genus Trifolium has small spherical poorly developed suspensor while the rest of the genera has suspensors containing from one to four series of cells. This treatment of the genera is followed by most of the taxonomists till Chaudhary and Sanjappa[14] when restudied this tribe and suggest the separation of genus Parochetus in a new subtribe Parochetinae. Inspite of that, genera within tribe Trifolieae still in need to more taxonomic findings to assess these opinions. For that the seeds of five genera of them; Ononis, Melilotus, Trigonella, Medicago and Trifolium have been studied as first step to assess the relationships between them.
MATERIALS AND METHODS
Thirty eight species, from the Egyptian flora, are represented in this study as follow: Ononis six species, Melilotus five species, Trigonella eight species, Medicago eleven species and Trifolium eight species. A complete list of the species, their life forms, locality and collector are given in Table 1. Mature seeds from ten individuals belonging to each species are measured and subjected to stereozoom examination. Selected seeds were mounted in silver paste onto standard stubs, coated with gold and examined by JEOL JSM T-20 scanning electron microscope at 15 kv. The terminology used here is that of Barthlott[15]. This study has been carried out during the period of 2000 to 2001.
| Table 1: | Taxa investigated, their life form, locality and collector |
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The arrangement of the genera and species is according to Tackholm
Abbreviations: L.F.= Life Form, Per.= Perennial, Ann.= Annual, Bi.= Biennial, Alex.Univ.= Alexandria University Herbarium, CAI= Cairo University Herbarium, Kew= Kew gardens |
RESULTS AND DISCUSSION
The results are summerised in Table 2 and Fig. 1 to 68. These results revealed that the variation in the seeds of the studied taxa are quite obvious and can be of taxonomic use in the identification of certain taxa. Figure 1-12 show the seeds of the studied Ononis species which are mostly lenticular or global with papillate testa and superficial or sunken micropyle. Figure 23-36 show the variations in the seed shape within Trigonella species. From these Fig. 23-36 it can be observed that both T. maritime (Fig. 23 and 24) and T. occulta (Fig. 25-26) have lenticular seeds, while T. anguina (Fig. 35-36) and T. hamosa (Fig. 31-32) have globular seeds. Meanwhile, T. arabica (Fig. 37) and T. laciniata (Fig. 31-32) have bilateral seeds and T. monspeliaca (Fig. 27-28) has trigonal seeds. Melilotus species are illustrated in Fig. 13-22. From these Fig. 13-22 it can observe the great variations in seed shapes and testa ornamentations within the studied species. Medicago species have mostly uniform seed shape and testa pattern as all of them have kidney shaped seeds with smooth testa and sunken micropyle (Fig. 38-52). The only variable character within the Medicago species is the arrangement of the seed coat cell and their anticlinal walls as illustrated in Fig. 39, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51 and 52. Trifolium species are less uniform than Medicago species (Fig. 53-68) as they vary in shape from oval in T. resupinatum, (Fig. 59) T. tomentosum (Fig. 67), T. procumbens (Fig. 61) and T. scabrum (Fig. 53), to global in the rest of the studied species. The seed coat pattern are also variable from smooth in T. tomentosum (Fig. 68), T. procumbens (Fig. 62), T. scabrum (Fig. 54), T. stellatum (Fig. 56) and T. desvauxii (Fig. 64) to striate in T. frageferum and T. resupinatum (Fig. 60 and 62) or even wrinkelled in T. alexandrenum (Fig. 66).
Also, it can observe that the arrangements of the seed coat cells has a considerable weight in the sumscription of the genera, then the micropyle position.
| Table 2: | Seed characters as investigated by both stereo- and scanning electron microscopes |
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Characters investigated: 1-Seed colour; 1-Yellowish-green 2-Light brown 3-Dark brown 2-Seed size in mm. (L.=Length, W.=Width), average of 10 readings; lowest-highest (mean) 3-Seed shape; 1-Lenticular 2-oval 3-global 4-trigonal 5-kidney-shaped 6-bilateral 4-seed -coat pattern; 1-smooth 2-wrinkled 3-striate 4-papillate 5-granulate 5-micropyle position; 1-superficial 2-sunken 3-grooved 6-arrangement of seed coat cells; 1-with elevated cells 2-with supercellular net-like pattern 3- with flat cells 7- outlines of cells; 1-isodiametric 2-elongated 3-irregular 4-rectangular 5-others 8-anticlinal wall; 1-straight 2-irregular 3-curved 4-sinuate 9-relief of cell boundary; 1-chanelled 2-superficial 3-raised 10-thickness of cell boundary; 1-thin 2-moderately thick 3- considerably thick 11-curvature of outer periclinal wall; 1-flat 2-concave 3-convex 12-secondary sculpture; 1-smooth 2-striate 3-wrinkeled 4-retticulate 5-micropapillate 13-epicuticular secretion; 1-present 2-absent |
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| Fig. 1and 2: |
Ononis serrata (Show the variations of characters) |
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| Fig. 4: |
Ononis sicula (show the papillae) |
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| Fig. 6: |
Ononis reclinata (show the papillae) |
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| Fig. 8: |
Ononis pubescens (arrengement of seed coat cells). |
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| Fig. 9 and 10: |
Ononis natrix (Fig. 10 show arrengement of seed coat cells). |
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| Fig. 11 and 12: |
Ononis vaginalis (Fig. 12 show arrengement of seed coat cells). |
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| Fig. 13 and 14: |
Melilotus sulcata (Fig. 14 show part of the testa) |
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| Fig. 15 and 16: |
Melilotus alba (Fig. 16 show part of the testa). |
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| Fig. 17 and 18: |
Melilotus elegans (Fig. 18 show part of the testa). |
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| Fig. 19 and 20: |
Melilotus indica (Fig. 20 show part of the testa) |
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| Fig. 21 and 22: |
Melilotus sicula (Fig. 22 show seed-coat cells). |
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| Fig. 23 and 24: |
Trigonella maritime (Fig. 22 show seed-coat cells). |
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| Fig. 25 and 26: |
Trigonella occulta (Fig. 26 show seed-coat cells). |
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| Fig. 27 and 28: |
Trigonella monspeliaca (Fig. 28 show the seed coat cells) |
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| Fig. 29 and 30: |
Trigonella laciniata (Fig. 30 show the seed coat cells) |
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| Fig. 31 and 32: |
Trigonella hamosa (Fig. 32 show the seed-coat cells). |
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| Fig. 33 and 34: |
Trigonella anguina (Fig. 34 show seed coat cells) |
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| Fig. 35 and 36: |
Trigonella stellata (Fig. 36 show seed coat cells) |
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| Fig. 37: |
Trigonella arabica |
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| Fig. 38 and 39: |
Medicago marina (Fig. 39 show seed coat cells with bar = 5 Um and Fig. 38 with bar = 0.5 mm) |
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| Fig. 40 and 41: |
Medicago intertexta (Fig. 40 show seed coat cells with bar = 0.5 mm and Fig. 41 with bar = 5 Um) |
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| Fig. 42: |
Medicago truncatula |
Fig. 43: |
Medicago arabica |
| (Fig. 42 and 43 show seed coat cells with bar = 5 Um) |
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| Fig. 44: |
Medicago lupulina |
Fig. 45: |
Medicago rigidula |
| (Fig. 44 and 45 show seed coat cells with bar 5 Um) |
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| Fig. 46 and 47: |
Medicago aschersoniana (In Fig. 46 bar = 0.5 mm and in Fig. 47 bar = 5 Um) |
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| Fig. 48 and 49: |
Medicago minima (In Fig. 48 bar = 0.5 mm and in Fig. 49 bar = 5 Um) |
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| Fig. 50: |
Medicago sative (bar = 5 Um) |
Fig. 51: |
Medicago coronata (bar = 5 Um) |
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| Fig. 52: |
Medicago orbicularis (bar = 5 Um) |
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| Fig. 53 and 54: |
Trifolium scabrum |
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| Fig. 55 and 56: |
Trifolium stellatum |
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| Fig. 57 and 58: |
Trifolium fragiferum |
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| Fig. 59 and 60: |
Trifolium resupinatum |
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| Fig. 61 and 62: |
Trifolium procumbens |
Fig. 63 and 64: |
Trifolium desvauxii |
| Fig. 65 and 66: |
Trifolium alexandrinum |
Fig. 67 and 68: |
Trifolium tomentosum |
These results are in accordance with Gutterman and Heydecker[16] and La Sota[17] who pointed to the importance of seed characters in the identification of species of Ononis. It can be observed that seed colour was of limited use as all the studied taxa have brown seeds except O. vaginalis which has greenish-yellow seeds (Table 2). The seed shape were mostly lenticular, oval or global except in Trigonella monspeliaca (Fig. 27) which is trigonal and in both Trigonella laciniata and T. arabica (Fig. 29 and 37) the seeds are bilateral or winged. All the studied Medicago species have kidney-shaped seeds which varies in size and seed-coat pattern only. The sizes of the seeds are almost small except in both Trigonella occulta and Melilotus sicula the seeds are large. Seed-coat pattern or the micro-ornamentation on the surface of the outer cell wall can be considered of high taxonomic value in the identification of the species as indicated by Duke[18], Dahlgren and Clifford[19], Berthlott and Frolich[20], Berchtold and Presol[2] and Taia[21]. According to this character it can easily distinguish the papillate seeds of Ononis (Fig. 1, 3 and 5) and the striate seeds in both Melilotus elegans and M. indicus (Fig. 17 and 19). Medicago species have almost smooth seeds which varies in the microornamentation of the outer cell wall surface (Fig. 38 to 52). Accordingly, it will be limited to use the seed characters in the circumscription of the Medicago species as previously mentioned by Ahmed
and Taia[22]. Species of Trifolium have smooth seeds, as well, except both T. alexandrinum which has faintly striate seeds (Fig. 65) and T. scabrum which has granulate seeds (Fig. 53). The epicuticular secretions such as waxes and related substances were used in taxonomy by Barthlott and Frolich[20], but here it is restricted to few species, Trigonella laciniata, T. hamosa, Melilotus elegans, M. indica, Trifolium procumbens, T. alexandrinum and T. desvauxii.
Thus, the results obtained from the seed characters support the separation of the genus Ononis in a monogeneric tribe Ononideae as proposed by Hutchinson[5] and Larsten[13]. The genera Trigonella and Melilotus have different seed shapes and ornamentations which can be used in the identification of their species. These variations do not reach to separate either of them in a separate subtribe. The genus Medicago has completely unique seed shape in the group which can be separated in a monogeneric subtribe, Medicagoneae, if this results supported by the other taxonomic tools. The genus Trifolium has characteristic seeds which can support[10-12] in putting this genus in a separate subtrbe Trifoliinae.