Morphology of seeds and seedlings of four species of Vigna Savi ( Leguminosae , Phaseolinae )

Four neotropical species of Vigna Savi (Leguminosae, Phaseolinae) have potential value as forage crops or ornamentals and could be cultivated in tropical or subtropical areas, even on floodplains. In order to obtain useful data for their culture and taxonomy, the seed morphology, germination pattern (hypogeal or epigeal) and seedling development were studied. The studied species belong to different sections of the genus: V. adenantha (G.F.W. Meyer) Maréchal, Mascherpa & Stainier (Sect. Leptospron); V. candida (Vell.) Maréchal, Mascherpa & Stainier (Sect. Sigmoidotropis); V. caracalla (L.) Verdc. (Sect. Caracallae) and V. luteola (Jacq.) Benth. (Sect. Vigna). The seeds were collected during fieldwork conducted in northwestern and northeastern Argentina. The qualitative and quantitative characters of the seeds were registered, after which they were sown. The development of the emerged seedlings was followed, first in a greenhouse and thereafter in open field. We recorded the type of germination, the thigmotropic movements of the hypocotyl and of the stem, seedling architecture and plant longevity. These traits allowed us to differentiate the species and construct an identification key that could be useful for agronomic or floricultural purposes. The data obtained partially support the current taxonomic treatment of the genus.


Introduction
In Argentina, the genus Vigna Savi (Leguminosae) is represented by eight species (Palacios & Hoc 2001).The populations grow throughout the riparian forests from Misiones to Buenos Aires in the east and on the lower slopes of the subtropical cloud forests in the northwest; the former within the Paranaense Province and the latter within the Yungas Province, according to the phytogeographic regions defined by Cabrera (1976).In addition, populations of V. luteola have been recorded for disturbed sites from Salta to Córdoba, i.e., from northwestern to central Argentina (Hoc et al. 2007).Four species are economically important: V. adenantha and V. luteola are promising forage crops in subtropical or tropical areas (Fernández et al. 1988), even on floodplains, where their nitrifying properties are advantageous; and V. adenantha, V. candida and V. caracalla have potential value as ornamentals (Hoc & Ojeda 2013;Etcheverry, unpublished data).
Considering the economical potential, knowledge of the seed morphology, germination process, seedling development and plant longevity is necessary in order to cultivate these species.Such knowledge could also contribute to clarifying the taxonomy of the group.Anatomical and morphological characters are useful for distinguishing among species at the seed stage (Corner 1976;Alsina 1988;Juan et al. 1998).The same is valid for seedlings, for various taxonomic levels, down to the level of subfamily (Silva & Scatena 2011), genera (Rodriguez & Tozzi 2008) or species (Dottori et al. 2000;Perícola et al. 2004;Mendiondo & Amela García 2005).These characters can be employed to devise keys, such as those designed to categorize seeds or seedlings (Duke 1965(Duke , 1969;;Csapody 1968) by family (Sánchez Sánchez & Hernández Cepeda 2004), subfamily (Kirkbride et al. 2003) or genus (Deginani 2001), as well as those designed to differentiate among plants that grow in a certain region (Marino et al. 2008;Zamora-Cornelio et al. 2010).
Various authors have emphasized the taxonomic relevance of the morphology and anatomy of the seeds of legumes.De Candolle (1825) distinguished the systematic value of the curvature of the embryo.Capitaine (1912) differentiated the three subfamilies by morphological features, and Corner (1951) underscored those findings with anatomical studies.Recently, Kirkbride et al. (2003) and Ubiergo & Lapp (2007) demonstrated the diagnostic value of seed characters at the generic and specific level.The seeds of Papilionoideae species have been described in many reports (Corner 1951(Corner , 1976;;Gunn 1981;Alsina 1988;Kirkbride et al. 2003;Ubiergo & Lapp 2007).Gunn (1981) differentiated the type of arils among this group.Despite these numerous contributions, the seeds of only two species of Vigna have been analyzed (Kirkbride et al. 2003), neither of which was from the neotropics.
Seedlings of legumes have been described by several authors.Compton (1912) related structure, size, form, habit and morphology within each species.Duke & Polhill (1981) reviewed matters of ecology and systematics.Cotyledons of legumes can vary by morphology and function as well as in terms of their exposure at germination (Duke & Polhill 1981).Smith (1981) assigned seedlings to different tribes within the Papilionoideae by cotyledon venation pattern.According to Ye (1983), the seedlings of Papilionoideae can be categorized by morphological type (Cinnamomum, Chimonanthus or Sophora).However, there is as yet little knowledge of the germination and seedling morphology of Vigna spp.The characters reported to date are the display of two opposite unifoliate primary leaves (Maréchal et al. 1978) and the hypogeal cotyledons (Maréchal et al. 1981).Beyra & Reyes Artiles (2004) pointed out that few characters can be used to distinguish the species within the genus Vigna; neither of them involved the seed nor the seedling in detail.The aim of the present work was to analyze the seed morphology, germination type, seedling development and plant longevity of four native species of Vigna in order to identify characters with taxonomic value.

Seed characters
A quantitative-qualitative analysis of the seeds was performed before a germination experiment.
Quantitative characters.The length and width of thirty seeds of each species were measured under a stereoscopic microscope, with a metric scale; the thickness was measured with a Vernier caliper at the macroscopic scale.
For each quantitative character, ANOVA was performed using the GraphPad Prism 5 software.
Qualitative characters.The shape of the seeds, the hilum and the rim aril were described.The color was determined using the color chart devised by Kelly (1964).
To study the seed surface, the hilum and the rim aril were examined under scanning electron microscopy (SEM), the seeds were dehydrated in a graded ethanol series (50, 60, 70, 80, 90 and 100%), mounted on stubs, coated with goldpalladium alloy and observed with a field emission scanning electron microscope (Supra 40; Carl Zeiss, Oberkochen, Germany).The terminology employed to describe the seed surface follows Barthlott et al. (1998).

Seedling characters
Under laboratory conditions, we scarified the seeds by making a cut in the integument, placed them in Petri dishes previously prepared with a base of cotton and Whatman paper wetted with distilled water.After germination, the seedlings were transferred to pots containing 500 mm 3 of soil perlite at 3:1.Seedling stage was set based on the appearance of the leaves.Seedling development was recorded until the first (trifoliate) second leaf expanded and the lateral branches of the main axis began to grow (after approximately one month).We recorded the emergency of the cotyledons, as well as the growth pattern and thigmotropic movements of the hypocotyl and the stem.The length of the epicotyl and the length and width of the primary leaves (pair of opposite unifoliate leaves) were measured with a metric bar made of graph paper.The branching system was described after the development of the first axillary bud.The plants were then transferred to the field and their growth habit was studied for three consecutive years to assess architecture and longevity.

Seed characters
The significant differences among the species, in terms of the quantitative characters of seeds, are shown in Tab. 1.
SEM: The cells of the hilum are disposed in moniliform uniseriate to triseriate interwoven filaments (Fig. 22).In the seed (Fig. 21) the elements of the reticulum are fused in a strictly striate pattern towards the rim aril (Fig. 23).Reticulate-striate testa (Fig. 24).

Discussion
The seeds of all four species studied, except those of V. luteola, have an expansion of the rim aril, which corresponds to the dry tongue aril typified by Gunn (1981) for some Papilionoideae taxa.
The morphological characters of the seedlings of the studied species correspond to the usual ones in Phaseoleae enumerated by Duke & Polhill (1981).The seedlings of V. caracalla and V. luteola can be assigned to the Cinnamomum type; and those of V. adenantha and V. candida can be assigned to the Sophora type (Ye 1983).Vigna luteola is assigned to a different subgenus than are the rest of the species, so these characters do not fit the actual infrageneric categories.
Occasional variation in the blade division of the primary leaves has also been observed for several species of Sesbania (Veasey et al. 1999).This could be a teratological case.Future studies will allow to corroborate or disallow this assumption.Maréchal et al. (1981) mentioned that the germination type in the Phaseolus-Vigna complex allows the differentiation of the genera included in the group (epigeal in Phaseolus and hypogeal in Vigna).In contrast, in the present study, we identified both phanero-epigeal and crypto-hypogeal germination within Vigna.Duke (1969) stated that many genera have species with both germination types.Two of the species studied here, included in the subgenus Sigmoidotropis, exhibited phanero-epigeal germination, whereas the other two, included in the subgenera Sigmoidotropis and Vigna, exhibited crypto-hypogeal germination.Therefore, Acta bot.bras.27(3): 483-489.2013.germination type is not useful for distinguishing between Vigna and Phaseolus, not even for distinguishing between subgenera within Vigna.
Seed size and shape, as well as hilum shape, had diagnostic value at the species level, in accordance with the findings of Alsina (1988) and Paulino et al. (2010) for other genera of legumes, underscoring the importance of these characters for taxonomy in the family Fabaceae.Although the testa of all the species consists of crusts corresponding to the classification of Barthlott et al. (1998), its varied ornamentation was useful for differentiating among species, as in other Papilionoideae (Alsina 1988).
The seed characters that contributed to the differentiation of the studied Vigna species were the width, thickness, shape, as well as the presence and shape of the rim aril expansion and of the coat sculpturing.Although the coloration pattern also differed among species, this feature should be confirmed through studies of seeds from additional populations, as variation in this trait can occur within a single species, exemplified in various genera of legumes (Werker, 1997).Our SEM studies allowed us to describe the morphology of the testa in detail, as well as the transition from the seed coat to the rim aril.The seed characters described may be useful for agronomic purposes, such as seed recognition and quality analysis.Plant longevity also differed among species belonging to different subgenera, V. luteola (subgen.Vigna) being annual, whereas the other three species (subgen.Sigmoidotropis) were pluriannual.Further studies of this trait in more Vigna spp.are warranted in order to determine whether the genus merits subdivision.The germination type, the growth pattern of the seedlings during the first year of development and the (previously unknown) plant longevity are also relevant for future taxonomic, systematic and agronomic treatments.

Table 1 .
ANOVA and Tukey's multiple comparison test of morphometric data related to the seeds of four Vigna species.