Morphology of fruits , seeds , seedlings and saplings of three species of Macrolobium Schreb . ( Leguminosae , Caesalpinioideae ) in the Brazilian Amazon floodplain 1

The evaluation of the post-germination phases of plants allows the recognition of transitional structures that support the relationships among taxa and their establishment. Together with fruit and seeds, seedlings and saplings provide useful characters for identifying many species. This study aimed to describe and characterize the morphology of the fruits, seeds, seedlings and saplings of Macrolobium acaciifolium, M. bifolium and M. pendulum, as well as to evaluate the ecological, taxonomic and phylogenetic value of the structures identified. In this work, the seeds were sown in plastic trays with sterilized sand and sawdust, without pre-germination treatment. The fruits of the three species are woody legumes, dehiscent fruit or indehiscent fruit. The seeds have seed coats with or without venation and variable embryos, with plumules differing among the three species; this trait can be helpful in distinguishing among the species studied. The seedlings are of the cryptocotylar-hypogeal-reserve or phanerocotylar-epigeal-reserve type. Macrolobium bifolium and M. pendulum are similar species, differing in only a few characters. These characters have taxonomic value and aid species identification. The characteristics of M. pendulum are described here for the first time.


Introduction
The morphological aspects of the initial stages of plant development have been poorly investigated in various families, principally when compared with the structure of mature vegetative organs.The study of the initial morphology provides knowledge about transitional or ephemeral structures that can supply relevant insights into phylogenetic relationships (Moreira-Coneglian & Oliveira 2006).Likewise, the morphological characters of fruits and seeds constitute highly reliable criteria for botanical identification, because they normally have little phenotypic plasticity and are therefore good taxonomic characters (Oliveira & Pereira 1984;Oliveira 1999a).In addition, data about seedlings, fruits and seeds can contribute information that is valuable for analyzing the systematics and ecology of various groups of tropical plants (Garwood 1995).
Although Leguminosae is a well-known family, there have been few studies of its representatives in the Amazon, notable exceptions including studies conducted in the Venezuelan Amazon (Ricardi 1996;1999a;1999b) and in the Brazilian Amazon (Silva et al. 1988;Vieira et al. 1996;Aguiar & Mendonça 2000;Gurgel et al. 2002;Ramos & Ferraz 2008;and Rodrigues et al. 2012).There is also a lack of data on seedlings that occur in specific environments of the Amazon, such as the floodplain forests, where species are subjected to periodic flooding (Silva et al. 1988;Moreira & Moreira 1996;Gama et al. 2003;Maia et al. 2005;Schöngart et al. 2005).Such knowledge is important to protecting and understanding the regeneration of these ecosystems.
Macrolobium comprises approximately 80 species and is strictly distributed in the tropics (De la Estrella et al. 2012).Approximately 40 species occur in Brazil, 15 of which are endemic (Martins-da-Silva & Lima 2013).Macrolobium is the second most representative genus of Detarieae s.l (Lewis et al., 2005), being used as an ornamental, for timber, to produce fish poison and for lumber in construction (Gama et al. 2003;Mackinder 2005;Felix-da-Silva et al. 2013).The genus is common in the Brazilian Amazon, mostly in the Negro River Basin, where it constitutes one of the dominant floristic elements of floodplain forests (Ducke 1949).
The phylogenetic relationships of Detarieae s.l. were analyzed by Bruneau et al. (2000;2001), on the basis of trnL nucleotide sequence data, and by Herendeen et al. (2003), who used morphological data merged with the aligned trnL data.Detarieae s.l. has a pantropical distribution, approximately 58% of the species being restricted to Africa, including Madagascar, 20% being endemic in the Neotropics; and 12% being endemic in tropical Asia (Mackinder 2005).The genus Macrolobium is at the base of the "higher" Detarieae s.l., constituting a group together with species of the genera Brownea Jacq., Browneopsis Huber, Elizabetha Schomb.ex Benth., Paloue Aubl.and Ecuadendron D.A. Neill.(Herendeen et al. 2003).This underscores the statement made by Bruneau et al. (2000): that the Macrolobium group (sensu Cowan & Polhill 1981) or complex (sensu Léonard 1957) has a complicated history.Bruneau et al. (2000) also stated that the relationships of the Macrolobium group requires a critical evaluation of morphological and anatomical characters in order to examine the possibility of a close affinity between New World Macrolobium and members of the South American Brownea group.
The Environmental Protection Area of Combu Island (EPA of Combu Island), in the state of Pará, Brazil, presents a varied floristic structure and composition (Rodrigues et al. 2006).Macrolobium bifolium (Aubl.)Pers. is native to the Amazon region and has been recorded for the Combu Island EPA (Maúes et al. 2011).Macrolobium acaciifolium (Benth.)Benth.has a wide distribution in Brazil, and M. pendulum Willd.ex Vogel is endemic in the country, occurring in the states of Amazonas, Pará and Maranhão (Martins-da-Silva & Lima 2013); both species represent new records for the Combu Island EPA.
The present study describes the morphology of fruits, seeds and the post-germination phases of Macrolobium acaciifolium, M. bifolium and M. pendulum, aiming to aid the identification of native species of Amazon floodplain forests and to provide a foundation for further phylogenetic and ecological studies involving this complex group.

Material and methods
Fruits and seeds of Macrolobium acaciifolium, M. bifolium and M. pendulum were collected from natural populations in the Combu Island EPA (48°25'W; 1°25'S).The area is entirely constituted of flooded forest (alluvial rain forest) and presents Humic Gleysols, with high percentages of silts and clay and a low percentage of sand (Silva & Sampaio 1998).According to the Köppen classification system, the climate of the region is type Am, with a mean annual rainfall of 2500 mm and a mean annual temperature of 27°C (Jardim & Vieira 2001).
Two matrix trees were selected for each Macrolobium species studied.The fruits were collected between January and April 2012, from directly under the trees or with a trimmer.The fruits were packed into polyethylene bags and transported to the Laboratory of Fruit and Seed Morphology of the Museu Paraense Emílio Goeldi for detailed analysis and measurement.Voucher specimens were deposited in the Herbarium of the Museu Paraense Emílio Goeldi, in Belém, Brazil (MG), under the accession numbers MG-204242 (M.acaciifolium), MG-204243 (M.bifolium) and MG-204244 (M.pendulum).
Samples (30 fruits and 50 seeds of each species) were randomly collected and analyzed.The dimensions (length, width and thickness) were measured with an UltraTech™ digital fractional caliper (0.01 mm accuracy; General Tools, New York, NY, USA).The length was considered the distance from the apex to the base; the width was measured from the dorsal to ventral regions, and the thickness between valves was determined.For observation and illustration of fruits and seeds, we used a stereomicroscope (Motic, SMZ-168 series, Vancouver, Canada) and a camera lucida.The terminology was based on Gunn (1981;1991), Barroso et al. (1999) and Oliveira (1999b).
For the description of plumule development, three embryos of each species were selected and samples of the embryo axis were fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer pH 7.2 for 24 h.The samples were then dehydrated in a graded ethanol series, dried in a CO 2 critical point dryer, sputter-coated with gold (Robards 1978) and analyzed in a scanning electron microscope (1450VP; LEO Electron Microscopy Ltd, Cambridge, UK).The morphology of each plumule was described according to Oliveira (1999b), considering the types undifferentiated, rudimentary, moderately differentiated and well differentiated.
For the post-germination study, 30 seeds of each matrix tree, without pre-germination treatment, were sown in Acta bot.bras.28(3): 422-433. 2014.plastic trays (80 × 40 × 20 cm) containing sterilized sawdust and sand (1:1) at a depth of ± 1 cm, maintained at room temperature and in natural light in a greenhouse (average temperature, 26°C).After the expansion of the second eophyll, the saplings were transplanted to polyethylene bags that were filled with a mixture of sand and vermiculite (1:1), after which they were watered once a day.
We defined the seedling phase as that occurring between the protrusion of the primary root and the full expansion of the eophylls of the first node (sensu Oliveira 2001).Voucher specimens of Macrolobium saplings collected at 40 days after germination were prepared and deposited in MG under the accession numbers MG-204245 (M.acaciifolium), MG-204246 (M.bifolium) and MG-204247 (M.pendulum).Some specimens were preserved in 70% ethanol to show the different stages of post-germination development.
To describe the morphology of seedlings and saplings, we selected 20 samples of each species and we photographed each sample with a digital camera (FinePix S4500; FUJIFILM do Brazil, São Paulo, Brazil).The structure of the hypocotyl, epicotyl, cotyledons, cataphylls and eophylls was described.The terminology for the seedling morphological types-cryptocotylar-hypogeal-reserve (CHR) and phanerocotylar-epigealreserve (PER)-followed Garwood (1996), and descriptive terms were based on Radford et al. (1974) and Harris & Harris (2001).We also employed the terminology established by Camargo et al. (2008), describing seedlings according to the unipolar or bipolar emergence of the root and epicotyl.The saplings were evaluated until the complete expansion of the third eophyll, as adopted for other Leguminosae species by Rodrigues & Tozzi (2008) and Rodrigues et al. (2012).

Results
The quantitative and qualitative data related to fruits, seeds and seedlings are summarized in Tab. 1 and 2, respectively.The fruits of Macrolobium acaciifolium (Fig. 1 and 2), M. bifolium (Fig. 3 and 4) and M. pendulum (Fig. 5  and 6) are woody legumes, dehiscent fruit or indehiscent fruit (Tab.2), all of which are subapiculate and substipitate.The funiculus is flattened, and the seeds are transversally disposed (Fig. 2, 4 and 6).
The seeds are brownish and laterally compressed, variable in form (Fig. 7-9, Tab. 2), with or without conspicuous venation; the micropyle is inconspicuous and the hilum is basal and elliptical.The endosperm is absent, and the cotyledons are storage organs that are juxtaposed, symmetric or asymmetric, and plano-convex, the abaxial surface being sinuate or entire (Fig. 7-9, Tab. 2).The embryos are investing, and the embryo axis is straight or oblique, oblong or conical (Fig. 10-12, Tab. 2).Among the three species, plumules vary from well differentiated, to moderately differentiated and undifferentiated (Fig. 13-15).
All of the seeds we tested germinated.The morphological aspects of seedlings and saplings of Macrolobium acaciifolium, M. bifolium and M. pendulum can be seen in Fig. [16][17][18][19][20][21], respectively.The germination of all species began with the rupture of the seed coat in the hilum-micropyle region and the emergence of the primary root, which is whitish, non-tuberose and cylindrical (Fig. 16, 22 and 30).The hypocotyl is cylindrical or quadrangular (Fig. 28).The seedlings are type CHR (Fig. [16][17][18][19] or PER .The emergence of the primary root and epicotyl is unipolar.The vegetative axis is lateral to the cotyledons (Fig. 17) or between them (Fig. 24 and 32).All three species presented an alternate arrangement of the eophylls (Fig. 21, 29 and 35).In all three species, the eophylls of the three first nodes were very similar, occurring as even-pinnate (Fig. 20 and 21) or bifoliolate (Fig. 27, 29 and 35) and variable in form.In young eophylls, we observed pairs of stipules that are axillary, filiform and free between them; the stipules are caducous (Fig. 21 and 29) or persistent (Fig. 35).Nyctinastic movement is observed in all species, in which the leaflets are "forward-folding".
Macrolobium acaciifolium (Benth.)Benth.-Thefruit is indehiscent, woody, oblong to oval, flattened, sparsely pilose at the margins, without constrictions, substipitate, with an oblique base, apex obtuse and rounded with a short apiculus (Fig. 1-2).The pericarp is externally shiny and slightly rough, with red-brown to brown dots, and internally spongy and brown.The seed is oblong to elliptical (Fig. 2), laterally compressed (Fig. 7), red-brown, with smooth surface (Fig. 2 and 7), radicular lobe poorly pronounced, without seed appendages; micropyle, lens and raphe inconspicuous.The embryo is cream-colored, with symmetrical storage cotyledons that are sinuate, juxtaposed (Fig. 7) and strongly adhered to each other.There is no endosperm; only residues can be seen around embryo axis, which is conical, two-thirds exposed and oblique to the cotyledons (Fig. 10).The plumule is well differentiated (Fig. 13), and the seedling is type CHR (Fig. 16-20).The germination is unipolar, and the vegetative axis is lateral to the cotyledons (Fig. 17).The hypocotyl is cylindrical, whitish and glabrous.The epicotyl is cylindrical, light green and glabrous.The axillary buds are inconspicuous; the first three eophylls are alternate, even-pinnate, with glabrous pulvini, petioles and rachises.Leaflets are oblong, with oblique bases, retuse to emarginate apices, entire margin; a small pulvinule occurs at the base of each leaflet.
The endosperm is absent and the embryo axis is oblong and straight (Fig. 11).The plumule is moderately differentiated (Fig. 14).The seedling is type PER (Fig. 22-26), and germination is unipolar with the vegetative axis between the cotyledons (Fig. 24).The hypocotyl is quadrangular (Fig. 28) and glabrous.The epicotyl is cylindrical, dark-green and glabrous.The axillary buds are inconspicuous.The three first eophylls are alternate (Fig. 26-27 and 29), bifoliolate, with oblong to falcate leaflets (Fig. 27), with oblique bases, acute to acuminate apices and entire margins, showing glabrous pulvini and petioles; a small pulvinule occurs at the base of each leaflet.
Macrolobium pendulum Willd.ex Vogel.-Thefruit is dehiscent, woody, D-shaped (Fig. 5), flattened, glabrous, without constrictions, substipitate, with oblique base and obtuse apex (Fig. 5 and 6).The pericarp is externally shiny and smooth, without apparent venation, and dark-brown; internally, it is light brown.The seed is oval to reniform (Fig. 6), laterally compressed (Fig. 9), dark brown and asymmetrical, with evident seed coat venation; without seed appendages; funicular residue is adhered to the hilum region; the micropyle, lens and raphe are inconspicuous.The embryo is light brown with two storage cotyledons, plano-convex and asymmetrical (Fig. 9).The endosperm is absent, and the embryo axis is oblong and straight (Fig. 12).The plumule is undifferentiated (Fig. 15).The seedling is type PER, and the germination is unipolar with the vegetative axis  between cotyledons (Fig. 32).The hypocotyl is cylindrical and glabrous.The epicotyl is cylindrical, dark-green and glabrous.The axillary buds are inconspicuous.The three first eophylls are alternate and bifoliolate, showing oblong to falcate leaflets, with oblique bases, obtuse to attenuate apices and entire margins; the pulvini and petioles are glabrous, and a pair of persistent stipules can be seen at the leaf base; a small pulvinule occurs at the base of each leaflet.

Discussion
According to Cowan (1953), the species of Macrolobium can present dehiscent fruits, as in M. acaciifolium, or indehiscent ones, as in M. bifolium and M. pendulum.The fruits of this genus are lateral flattened legumes, varying from suborbicular to oblong or spatulate (Cowan 1953).These forms were found for the fruits of M. acaciifolium  and M. bifolium, but not for M. pendulum, in which fruits were D-shaped, as was also reported by Kirkbride et al. (2003).The dehiscence, shape and pericarp traits, as well as other fruit characters, can help recognize the groups in Caesalpinioideae (Gunn 1991, Barroso et al. 1999).
In the present study, the measurements of fruits were consistent with those described for Macrolobium by Gunn (1991), Barroso et al. (1999) and Kirkbride et al. (2003).The analysis of the dimensions of the fruits demonstrated that M. bifolium had the largest fruits and M. acaciifolium had the smallest fruits.
Although the size and shape of the seeds were similar among the species studied, the values of seed length and thickness were highest for Macrolobium bifolium, as were the dimensions of the embryo axis.In M. bifolium and M. pendulum, the venation of seed coat is a notable trait that can be useful in identifying certain Macrolobium species; this feature was previously reported by Cowan (1953) in the last revision of the genus.Apparently, there are different patterns of seed coat venation, although only anatomical studies could describe and discuss those.For the seeds of M. acaciifolium, our data corroborate those of Cowan (1935), who described the crustose testa, and of Gunn (1991), who reported the intrusion of the testa into the corrugate cotyledons.Gunn (1991) stated that the embryo axis is straight in 98% of Caesalpinioideae genera.Macrolobium acaciifolium is one of the exceptions in the subfamily, presenting a clearly oblique embryo axis, as documented in the present work.The asymmetry of the cotyledons of M. pendulum, a state not shared by the other species studied, is noteworthy.Cowan (1953) and Gunn (1991) did not highlight this trait for Macrolobium species.According to Smith (1983) and Duke & Polhill (1981), all of the Amherstieae (sensu Cowan & Polhill 1981) have storage cotyledons, constituting exalbuminous seeds.Our data support that statement, because all three of the species studied showed fleshy cotyledons and only remains of the endosperm were found.
The evaluation of embryo structure of the studied species revealed many distinctive characters (see Tab. 2).Considering the embryo axis of Leguminosae species, the plumule is recognized as one of the most variable traits.In fact, the plumule development was a determinant in the separation of some groups in this family, such as Poecilanthe (Meireles & Tozzi, 2008) and Copaifera (Gurgel, 2009), at the infrageneric and interspecific levels respectively.Caesalpinioideae presents all types of plumules, as observed in the work of Oliveira (1999b), in which the plumule was found to be well differentiated in Caesalpinia leiostachya (Benth.)Ducke; moderately differentiated in Cassia ferruginea (Schrad.)Schrad.ex DC. and Peltophorum dubium (Spreng.)Taub.e; rudimentary in Dimorphandra mollis Benth.; and undifferentiated in Senna multijuga (Rich.)Irwin & Barn.Gunn (1991) observed none of the variation reported here, reporting that the plumule of Macrolobium is rudimentary.In our results, the three Macrolobium species evaluated presented distinct patterns of plumule development, showing that detailed studies of this character can be relevant for species identification.These findings underscore the need for further studies of Macrolobium species in order to clarify aspects of circumscription and phylogeny, exploring the morphological diversity of the genus (Bruneau et al. 2001;Herendeen et al. 2003;Mackinder 2005).
Hypogeal germination, as we observed in Macrolobium acaciifolium, is considered a derived condition for Leguminosae (Moreira & Moreira 1996).The CHR morphological type exhibited by M. acaciifolium was also reported by Silva et al. (1988) and Moreira & Moreira (1996).Although Maia et al. (2005) classified the seedlings of this species as the phanerocotylar-hypogeal-reserve type, there seems to be some discrepancy regarding this point.Analyzing the figure of M. acaciifolium in Maia et al. (2005), we see that seeds were germinated within the indehiscent fruits, which opened during the process, thus inducing the misconception; in the referred figure, authors also drew the typical rugose seed coat of M. acaciifolium, confirming our interpretation.In the present study, Macrolobium bifolium and M. pendulum showed the PER morphological type, in agreement with the results of Gates (1951).Léonard (1957)  seedling type of M. bifolium, which is also in agreement with our data.Some connections can be established between Leguminosae subfamilies and seedling or germination types.According to Gates (1951), seedlings of Caesalpinioideae and Mimosoideae are usually epigeal, differing from Papilionoideae, in which they are cryptocotylar or phanerocotylar.Duke & Polhill (1981) stated that, in Caesalpinioideae alone, there are three groups with respect to the seedling type, in which the consistency and exposure of the cotyledons, as well as the structure of eophylls and metaphylls, can be variable.Gurgel et al. (2012) stated that Caesalpinioideae and Mimosoideae seedlings are considered epigeal with foliar cotyledons, and that hypogeal species with storage cotyledons predominate in the Papilionoideae.Oliveira (1999a) presented data for 30 leguminous tree species that corroborated the statements of Gates (1951), Duke & Polhill (1981) and Gurgel et al. (2012).Examples of hypogeal species in Caesalpinioideae are very rare, and the results of the present study illustrate this pattern in Macrolobium acaciifolium.
When the ecological aspects are considered, the current knowledge about seedlings allows interesting evaluations to be performed.According to Lima (1989-90), the CHR morphofunctional type is normally found for species occurring in periodically flooded environments.This was confirmed by the Ressel et al. (2004), who identified CHR seedlings in species occurring in temporarily flooded areas; the authors also correlated CHR seedlings with the climax species.According to Felix-da- Silva et al. (2013), Macrolobium bifolium and M. pendulum (both having PER seedlings) occur only in flooded environments, as is the case for M. acaciifolium (which has CHR seedlings).It is possible that the occurrence in flooded environments was related to the presence of fleshy cotyledons, which supply the initial requirement of reserves for the establishment of the plant.It is also necessary to emphasize that, although producing phanerocotylar seedlings, M. bifolium and M. pendulum have seed coats that remain adhered to the abaxial face of the cotyledons, exposing only their adaxial face.Thus, the seed coat is partially responsible for the protection of cryptocotylar seedlings, exposing only part of the cotyledon surface.
The stipules observed for the studied taxa were persistent only in Macrolobium pendulum.Cowan (1953) stated that stipules are more frequently caducous in Macrolobium species but are persistent, which is a useful trait, in M. huberianum Ducke and M. pendulum.According to Cowan (1953), the persistence of stipules in M. pendulum is an important state distinguishing M. pendulum from its relatives M. palustre Ducke and M. savannarum R.S.Cowan when specimens are adults and not in the reproductive phase.Duke (1965) demonstrated that the presence or absence of stipules is a useful criterion for interspecific differentiation within a given genus.The occurrence of stipules in seedlings has also been described in other species of Caesalpinioideae, such as Apuleia leiocarpa (Vog.)Macbr., Senna macranthera (Collad.)H.S. Irwin & Barneby (Finger et al. 1979) and Senna multijuga var.lindleyana (Amorim et al. 2008).
Typical of the legumes, the foliar nyctinasty is still scarcely described for seedlings of the Leguminosae family.The "forward-folding" nyctinasty found in the present study for the leaves of the Macrolobium species is probably related to the asymmetrical leave base, similar to what occurs in the Dalbergia clade (Lavin et al. 2001) and in the genus Acosmium (Rodrigues & Tozzi, 2008).
As can be inferred from the data shown in Tab. 2, Macrolobium bifolium and M. pendulum are closely related species, sharing 10 character states, compared with only three shared by M. acaciifolium and M. bifolium, whereas M. acaciifolium and M. pendulum have no character states in common.These data corroborate the relationships within the genus Macrolobium described by Cowan (1953).According to that author, despite being in the same section (Vouapa), M. bifolium and M. pendulum are more closely related to each other than to M. acaciifolium, a species with more ancestral traits.
The data presented in this work underscore the relevance of detailed morphological studies (especially of seeds and seedlings) to understanding the ecological and phylogenetic aspects of leguminous species.Structural information about Macrolobium pendulum is provided here for the first time, contributing interesting knowledge for recognizing this species in the natural environment.
*All measurements are expressed in millimeters.