Introduction

Tephrosia Pers. comprises nearly 350 species (Schrire 2005) and is one of a number of large genera in the family Leguminosae (Geesink 1984). It displays a variable habit, from subshrub to shrub, with imparipinnate leaves, axillary or terminal pseudoracemes and long, laterally compressed, dehiscent pods containing many seeds. Its geographical distribution is pantropical and also subtropical (Brummitt 1981).

Bentham (1862) divided Tephrosia into two sections: Tephrosia sect. Brissonia (Neck.) DC. and Tephrosia sect. Recueria Benth. The section Brissonia is characterized by a shrubby habit, terminal or axillary (rarely leaf-opposed) inflorescence, by rounded (rarely subulate) calyx teeth and by a glabrous stigma. The section Recueria has terminal inflorescences, axillary flowers, subulate calyx teeth, a glabrous style and penicillate (rarely glabrous) stigma. The most recent infrageneric classification was proposed by Brummitt (1981) who, based on the work of Wood (1949), Forbes (1948), Cronquist (1954) and Gillett (1958), divided Tephrosia into two subgenera: Tephrosia subg. Tephrosia and Tephrosia subg. Barbistyla Brummitt. Subgenus Tephrosia is characterized by a glabrous style with trichomes present on the stigma of some species, while subg. Barbistyla has trichomes present along the style and stigma. Both subgenera occur in South America. Subgenus Tephrosia is represented by T. adunca Benth., T. cinerea (L.) Pers. f. cinerea, T. cinerea f. pseudo-adunca Hassl., T. egregia Sandw., T. leptostachya DC., T. marginata Hassl., T. noctiflora Boj., T. purpurea (L.) Pers. subsp. purpurea, T. rufescens Benth., T. sessiliflora (Poir.) Hassl. and T. senna Kunth and Tephrosia subg. Barbistyla by T. candida DC., T. nitens Benth., T. sinapou (Buc’hoz) A. Chev. and T. vogelii Hook. f. Taxonomic studies of neotropical species of Tephrosia are required because the most recent infrageneric classification of the genus by Brummitt (1981) did not include all 17 of the South American species currently recognized (personal observation, R.T.Q.).

Barthlott (1981) examined the seed surface of 5,000 angiosperm species and 100 gymnosperm species and found that scanning electron microscopy (SEM) revealed new structural details. In his study of dicotyledonous seeds, Corner (1976) included 37 species of Leguminosae, including T. candida DC., and described a number of characters important for the taxonomy of the Leguminosae. Kirkbride et al. (2003) described the fruits and seeds of genera of Faboideae (=Papilionoideae) and produced a dichotomous key based on seed structures. The major diagnostic characters found in the 50 species of Tephrosia analysed by Kirkbride et al. (2003) were the cotyledon with inner face wrinkled, embryonic axis perpendicular to the length of the seed, reticulate testa and hilum within the corona.

Meireles and Tozzi (2008) and Paulino et al. (2010), studying species of Poecilanthe and Indigofera, respectively, used seed morphology to help solve taxonomic problems. Studies of Tephrosia have likewise added new data of taxonomic importance. Bhandari et al. (1985) used seed morphology to delimit nine Indian species of Tephrosia. Subba Rao and Shanmukha Rao (1992) used SEM to study the testa patterns of 12 Indian species of Tephrosia, from which they obtained the following patterns: simple-reticulate, multireticulate and crested. The recent SEM studies of Al-Ghamdi and Al-Zahrani (2010) in eight species from Saudi Arabia revealed two patterns: simple-reticulate and multireticulate. In addition, these authors drew attention to the organization of the reticulum wall: papillate or wavy. However, these studies did not address the utility of these characters to the infrageneric classification of Tephrosia.

The aims of the present study were to describe the macromorphological and micromorphological characters of seeds of neotropical Tephrosia species using SEM, and to assess the taxonomic significance of seed characters with regard to the recognition of subgenera and species.

Materials and methods

Seeds of 13 species, one subspecies and one forma of Tephrosia were obtained from specimens collected in the field or from herbarium specimens, or both (Table 1). Newly collected specimens were deposited in herbarium UEC (Holmgren et al. 1990). Specimens were identified by the first author. In our study, T. leptostachya DC. is treated at the specific level, although Brummitt (1968) referred to it as an infraspecific taxon of T. purpurea [T. purpurea subsp. leptostachya (DC.) Brummitt var. leptostachya]. We prepared the seed material for SEM study following the procedure of de Castro (2002), and we only used dry seeds. The seeds of each taxon studied were cleaned and mounted directly on a stub and coated with gold using an evaporator (Balzers SCD 050 BAL-TEC). We used a JSM-5800LV (JEOL, Tokyo, Japan) instrument. Testa pattern terminology is based on that of Lersten (1981): substriate (short parallel ridges), simple-reticulate (meshwork of ridges enclosing single cells), simple-foveolate (single cell surrounded by several grooves), multifoveolate (unit of cells surrounded by several grooves) and lophate or crested (short ridges with irregular sides).

Table 1 Tephrosia species studied with specimen collector, herbarium and collection site
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Results

Seed length of the species studied ranged from 2 to 6 mm and width from 1.2 to 4 mm (Table 2). Most species have seeds 2–4 mm long; longer seeds were found in T. adunca, T. candida, T. sessiliflora and T. vogelii (Figs. 1, 4, 37, 43). The smallest width was found in T. cinerea f. pseudo-adunca and T. marginata (Figs. 10, 19); the highest in T. candida (Fig. 4). Seed shape is oblong (Figs. 1, 7, 10, 16, 22, 28, 40); oval (Figs. 4, 37, 43), rectangular (Figs. 13, 19, 31) or reniform (Figs. 25, 34). Seeds with the lowest length–width ratio occur in T. candida (1.25–1.33) and the highest in T. vogelii (2–2.5) (Table 2).

Table 2 Macromorphological seed characters in Tephrosia species: size, form, texture and testa colour, hilum position and presence or absence of an aril
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Figs. 1–9
figure 1

Seeds of Tephrosia: 1–3 T. adunca (1 whole seed side view, 2 circular hilum shape, 3 simple-reticulate testa ornamentation); 4–6 T. candida (4 whole seed side view, 5 elliptical hilum shape, 6 crested testa ornamentation); 7–9 T. cinerea f. cinerea; (7 whole seed side view, 8 circular hilum, 9 simple-reticulate testa ornamentation). Scale bars Figs. 1, 4 and 7 whole seed view, 1 mm; Figs. 2, 5 and 8 hilum shape, 100 μm; Figs. 3, 6 and 9 testa ornamentation,  10 μm

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Figs. 10–18
figure 2

Seeds of Tephrosia: 10–12 T. cinerea f. pseudo-adunca (10 whole seed side view, 11 circular hilum shape, 12 simple-reticulate testa ornamentation); 13–15 T. egregia (13 whole seed side view, 14 elliptical hilum shape, 15 simple-reticulate testa ornamentation); 16–18 T. leptostachya (16 whole seed side view, 17 circular hilum shape, 18 simple-reticulate testa ornamentation). Scale bars Figs. 10, 13 and 16 whole seed view, 1 mm; Figs. 11, 14 and 17 hilum shape, 100 μm; Figs. 12, 15 and 18 testa ornamentation, 10 μm

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Figs. 19–27
figure 3

Seeds of Tephrosia: 19–21 T. marginata (19 whole seed side view, 20 oval hilum shape, 21 simple-reticulate testa ornamentation), 22–24 T. nitens (22 whole seed side view, 23 elliptical hilum shape, 24 crested testa ornamentation); 25–27 T. noctiflora (25 whole seed side view, 26 circular hilum shape, 27 simple-reticulate testa ornamentation). Scale bars Figs. 19, 22 and 25 whole seed view, 1 mm; Figs. 20, 23 and 26 hilum shape, 100 μm; Figs. 21, 23 and 27 testa ornamentation, 10 μm

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Figs. 28–36
figure 4

Seeds of Tephrosia: 28–30 T. purpurea subsp. purpurea (28 whole seed side view, 29 circular hilum shape, 30 simple-reticulate testa ornamentation); 31–33 T. rufescens (31 whole seed side view, 32 circular hilum shape, 33 simple-reticulate testa ornamentation); 34–36 T. senna (34 whole seed side view, 35 circular hilum shape, 36 simple-reticulate testa ornamentation). Scale bars Figs. 28, 31 and 34 whole seed view, 1 mm; Figs. 29, 32 and 35 hilum shape, 100 μm; Figs. 30, 33 and 36 testa ornamentation, 10 μm

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Figs. 37–45
figure 5

Seeds of Tephrosia: 37–39 T. sessiliflora (37 whole seed side view, 38 elliptical hilum shape, 39 multifoveolate testa ornamentation); 40–42 T. sinapou (40 whole seed side view, 41 circular hilum shape, 42 crested testa ornamentation); 43–45 T. vogelii (43 whole seed side view, 44 elliptical hilum shape, 45 foveolate testa ornamentation). Scale bars Figs. 37, 40, 43 whole seed view, 1 mm; Figs. 38, 41, 44 hilum shape, 100 μm; Figs. 39, 42, 45 testa ornamentation, 10 μm

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Seed colour is brown, rust coloured or ochre, all with black speckling, or totally black without speckling. Black seeds are found in T. sessiliflora and T. vogelii. The hilum is located in a central or subcentral position and is circular (Figs. 2, 8, 11, 17, 26, 29, 32, 35, 41), elliptical (Figs. 5, 14, 23, 36, 44) or oval (Fig. 20). An aril is present in the seeds of only four species: T. candida, T. nitens, T. sessiliflora (Fig. 37) and T. vogelii (Table 1).

The seed testa is smooth, except in T. noctiflora, in which it is creased (Fig. 25; Table 2). Micromorphologically, crested (Figs. 6, 24, 42), foveolar (Fig. 45), multifoveolar (Fig. 39), simple-reticulate (Figs. 3, 9, 12, 15, 18, 21, 27, 30, 36) and substriate (Fig. 33) testa patterns are found, with a smooth (Figs. 9, 21, 30, 36), papillate (Figs. 3, 12, 15), wavy (Fig. 27) or uniformly reticulate (Figs. 9, 21, 30, 36) anticlinal wall, with the boundaries of the anticlinal walls elevated (Figs. 3, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36) or channelled (Figs. 35, 45) (Table 3).

Table 3 Micromorphological seed characters in species of Tephrosia Pers.: testa pattern, reticulum anticlinal wall, boundaries of anticlinal wall and hilum form
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Discussion

Although the size and shape of seeds are relatively consistent among the species studied, some characters are diagnostic, e.g. seeds in T. candida and T. vogelii exceed 4 mm in length (Figs. 4, 43), seeds in T. candida exceed 3 mm in width, and seed length in T. vogelii is more than double the width, while the ratio is lower in the other species. The substriate testa pattern in T. rufescens (Fig. 33), the foveolar pattern in T. vogelii (Fig. 45) and the multifoveolar pattern in T. sessiliflora (Fig. 39) are new characters for the genus. The presence of a substriate testa pattern in T. rufescens is important because this species is morphologically very similar to T. adunca (Fig. 3) and has a similar geographical distribution. The characters listed by Bentham (1862) to separate these two species are essentially limited to the difference in plant stature, the differing number of leaflets, the different plant indumentum and the variation in flower size. In fact, Hassler (1919) considered T. rufescens as a variety of T. adunca. Our study provides two more characters to differentiate these species: testa pattern (simple-reticulate in T. adunca, substriate in T. rufescens) and reticulum wall (papillate in T. adunca, Fig. 3; smooth and uniform in T. rufescens, Fig. 33).

Tephrosia leptostachya (Figs. 16, 17, 18) and T. purpurea subsp. purpurea (Figs. 28, 29, 30) are part of a species complex. Bentham (1862) was the first botanist to highlight the similarity between these two species in Brazil. Brummitt (1968) considered both to belong to one species and recognized subspecies and varieties within T. purpurea. In this paper, we treat the two as distinct species, since, notwithstanding the phenotypic similarity in habit, and morphology and colour of the flowers, they are morphologically separable by leaflet form and inflorescence length. The seeds of T. leptostachya and T. purpurea subsp. purpurea are very similar (Figs. 16, 28) but in T. leptostachya the reticulum wall is wavy (Fig. 18), while in T. purpurea subsp. purpurea the wall is uniform (Fig. 30).

Hassler (1919) recognized the taxon T. cinerea f. pseudo-adunca, differentiated from typical T. cinerea by habit, form and size of leaflets, type of indumentum and form of calyx teeth. Apart from the distinct morphology, the typical form has a wide geographical distribution throughout Latin America, while T. cinerea f. pseudo-adunca has a narrower range in the Chaco in Argentina, Bolivia, Brazil, Paraguay and Uruguay. The testa pattern found in the seeds of these two taxa is the same: simple-reticulate, but forma pseudo-adunca has a papillate reticulum wall (Fig. 12), which is not found in T. cinerea f. cinerea (Fig. 9).

The testa patterns of T. candida and T. purpurea were studied by Subba Rao and Shanmukha Rao (1992). Our results (namely: oblong seeds with a crested testa pattern, in T. candida and reniform seeds, with a simple-reticulate testa pattern, in T. purpurea) corroborate their results and indicate that the character states are stable across the geographical range of the two species.

As in the study of Bhandari et al. (1985), we confirm that size, texture, colour, presence or absence of an aril, and seed ornamentation type assist in identifying species. Of the 15 taxa studied by us, T. adunca, T. cinerea f. cinerea, T. cinerea f. pseudo-adunca, T. egregia, T. leptostachya, T. marginata, T. noctiflora, T. purpurea subsp. purpurea, T. rufescens, T. sessiliflora and T. senna have a glabrous style, while in T. candida, T. nitens, T. sinapou and T. vogelii it is pilose. In the classification of Brummitt (1981), subgenus Tephrosia included T. noctiflora and T. sessiliflora, in addition to the species of section Recueria Benth. with glabrous styles, while Tephrosia subg. Barbistyla included only those species with trichomes present on the style (T. candida, T. nitens, T. vogelii and T. sinapou). In section Brissonia (Neck.) DC. recognized by Bentham (1862) there are unique seed characteristics, including oblong shape and crested testa pattern in T. nitens and T. sinapou (Figs. 22, 24, 40, 42), testa pattern lacking speckling, multifoveolate in T. sessiliflora (Fig. 39), testa with boundaries of anticlinal wall corrugated, and an aril present in T. nitens and T. sessiliflora (Figs. 22, 37). In Tephrosia sect. Recueria, on the other hand, we found the peculiar characteristics of a substriate testa pattern in T. rufescens (Fig. 33), a simple-reticulate testa pattern in T. adunca, T. cinerea and T. leptostachya (Figs. 3, 9, 18), and a papillate testa pattern in T. adunca (Fig. 3), a uniform reticulum anticlinal wall in T. cinerea (Fig. 9), a wavy reticulum anticlinal wall in T. leptostachya (Fig. 18) and a central hilum position in all but one species studied (Tables 2, 3).

We also assessed the classification of Brummitt (1981). In Tephrosia subg. Tephrosia the testa sculpture is predominantly simple-reticulate in T. adunca, T. cinerea f. cinerea, T. cinerea f. pseudo-adunca, T. egregia, T. leptostachya, T. marginata, T. noctiflora, T. purpurea subsp. purpurea and T. senna (Figs. 3, 9, 12, 15, 18, 21, 27, 30 36). Seeds have the unique characteristics of rectangular shape in T. egregia (Fig. 13) or oblong in T. adunca, T. cinerea f. cinerea, T. cinerea f. pseudo-adunca and T. purpurea subsp. purpurea (Figs. 1, 7, 10, 28). The testa pattern is multifoveolate in T. sessiliflora (Fig. 39), multireticulate or substriate in T. rufescens (Fig. 33), and the anticlinal wall is reticulate and papillate in T. adunca, T. cinerea f. pseudo-adunca and T. egregia (Figs. 3, 12, 15) or uniform in T. cinerea f. cinerea, T. marginata, T. purpurea subsp. purpurea and T. senna (Figs. 9, 21, 30, 36), with a circular or oval hilum in T. marginata. Unique to Tephrosia subg. Barbistyla is the foveolar testa pattern in T. vogelii (Fig. 45) and crested pattern in T. candida, T. nitens and T. sinapou (Figs. 6, 24, 42) (Tables 2, 3).

Subba Rao and Shanmukha Rao (1992) were the first authors to study the micromorphology of Tephrosia seeds in 12 species occurring in India. They recorded crested, simple-reticulate, and multireticulate testa patterns (Table 4). Al-Ghamdi and Al-Zahrani (2010) studied seed micromorphology in eight taxa from Saudi Arabia and also observed the multireticulate and simple-reticulate testa patterns (Table 4).

Table 4 Seed testa pattern in species of Tephrosia
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From Table 4, which summarizes data of species from Argentina, Brazil, India and Saudi Arabia, we can see that the characteristics of the testa support previous infrageneric classifications. The two subgenera display fairly characteristic testas, i.e. in Tephrosia subg. Tephrosia the simple-reticulate ornamentation is predominant, whereas most of the species of Tephrosia subg. Barbistyla and Tephrosia sect. Brissonia have a testa with a crested pattern. Based only on the character of the testa pattern, the placement of T. noctiflora fits better in Tephrosia subg. Tephrosia according to the classification proposed by Brummitt (1981), whereas T. sessiliflora remains uncertain because of its unique pattern (multifoveolate).

The results provided macromorphological and micromorphological seed characters which aid identification of the Tephrosia species occurring in Latin America.

Key to the identification of seeds of Tephrosia in the neotropics

  • 1. Seed testa with substriate pattern ….. T. rufescens

  • 1. Seed testa with a simple-reticulate, foveolate or multifoveolate pattern

  •  2. Seed testa pattern foveolate

  •   3. Seed testa pattern simple-foveolate ….. T. vogelii

  •   3. Seed testa pattern multifoveolar ….. T. sessiliflora

  •  2. Seed testa pattern reticulate or crested

  •   4. Seed testa pattern crested

  •    5. Seed without aril ….. T. sinapou

  •    5. Seed with aril

  •     6. Wavy anticlinal wall ….. T. nitens

  •     6. Straight anticlinal wall ….. T. candida

  •   4. Seed testa pattern simple-reticulate

  •  7. Papillate reticulum

  •   8. Elliptical hilum in central position ….. T. egregia

  •   8. Circular or oval hilum in subcentral position

  •    9. Rectangular–reniform seed, oval hilum ….. T. adunca

  •    9. Oblong seed, circular hilum ….. T. cinerea f. pseudo-adunca

  •  7. Smooth reticulum

  •   10. Reticulum simple, undulating

  •    11. Seed rugose, brown, without speckling ….. T. noctiflora

  •    11. Seed smooth, ochre coloured, with black speckling ….. T. leptostachya

  •  10. Reticulum uniform, plain

  •   12. Hilum oval ….. T. marginata

  •   12. Hilum circular

  •    14. Subcentral hilum ….. T. cinerea f. cinerea

  •    14. Central hilum

  •     15. Oblong seeds, length 2.9–3.2 cm ….. T. purpurea subsp. purpurea

  •     15. Reniform seeds, length 3.5–4.0 mm ….. T. senna