Taxonomic value of foliar characters in Dahlstedtia Malme-Leguminosae , Papilionoideae , Millettieae 1

(Taxonomic value of foliar characters in Dahlstedtia Malme Leguminosae, Papilionoideae, Millettieae). Dahlstedtia Malme (Leguminosae) is a neotropical genus, native to the Brazilian Atlantic Forest, and comprises two species, D. pinnata (Benth.) Malme and D. pentaphylla (Taub.) Burk., although it has been considered a monotypic genus by some authors. Leaf anatomy was compared to verify the presence of anatomical characters to help delimit species. Foliar primordium, leaflet, petiolule, petiole and pulvinus were collected from cultivated plants (Campinas, SP, Brazil) and from natural populations (Picinguaba, Ubatuba and Caraguatatuba, SP, Brazil D. pinnata; Antonina, PR, Brazil D. pentaphylla). Studies on leaflet surface assessment (Scanning Electron Microscopy), as well as histology and venation analyses were carried out of dehydrated, fresh and fixed material from two species. Leaflet material was macerated for stomatal counts. Histological sections, obtained by free-hand cut or microtome, were stained with Toluidine Blue, Safranin/Alcian Blue, Ferric Chloride, Acid Phloroglucin. Secretory cavities are present in the lamina, petiolule, petiole, pulvinus and leaf primordium in D. pentaphylla, but not in D. pinnata, and can be considered an important character for species diagnosis. Other leaf characters were uninformative in delimiting Dahlstedtia species. There is cambial activity in the petiolule, petiole and pulvinus. This study, associated with other available data, supports the recognition of two species in Dahlstedtia.

Dahlstedtia species have been recorded from Brazil, especially the Atlantic Forest.Their representatives are shrubs or trees, with conspicuous papilionate flowers.The corolla of D. pinnata is pink and of D. pentaphylla reddish, besides the presence of conspicuous lenticels on the floral branches of D. pentaphylla and their absence in D. pinnata (Teixeira & Gabrielli 2000).
The present study compared anatomically the leaves of D. pinnata and D. pentaphylla, to seek characters that can help distinguish between the species.These characters were then related to the other data available for the genus.

Material and methods
Foliar primordium, leaflet, petiolule, petiole and pulvinus of both species were collected from cultivated plants in Campinas, SP, Brazil. Material  The material from herbarium specimens was treated according to Smith & Smith (1942).
Whole leaflets were cleared and stained according to Berlyn & Miksche (1976) modified by Monteiro et al. (1979) for venation studies.
Histological sections from herbarium specimens after treatment and from fresh material were stained with Toluidine Blue or Alcian Blue/Safranin and mounted in glycerin jelly.Fixed material was submitted to the standard techniques of infiltration with paraffin, stained with Safranin and Alcian Blue and the slides mounted in synthetic resin (Gerlach 1969).
To detect phenolic compounds and lignin, free hand sections were treated with Ferric Chloride and Acid Phloroglucin, respectively (Johansen 1940).The chemical composition of crystals was verified by 10% chloric acid, glacial acetic acid and sulfur acid (Chamberlain 1932apud Arduin & Krauss 1997).
Leaflet material was macerated following Jeffrey's method (Johansen 1940;Foster 1949) for stomatal counts.The Kolmogorov-Smirnov test was used (Zar 1996) to evaluate the significance of the difference found in the number of stomata/cm 2 of the foliolar lamina.
Photomicrographs were taken using a Zeiss II fotomicroscope and diagrams using a camera lucida.
Small pieces of leaflets were dehydrate in an ethanol series followed by critical-point drying in a Balzers CPD 030 apparatus; the specimens were mounted on aluminum stubs with colloidal carbon and coated with gold in a Balzers SCD 050 sputter coater for 280 s.The samples were observed with a Jeol JSM 5200 scanning electron microscope at 15 kv, coupled with a Sinar 67 camera.
The terminology of Hickey (1973) was followed to describe the leaf architecture.
Mesophyll is dorsiventral with approximately four layers (Fig. 11).The median layer has conspicuous spongy parenchyma cells (Fig. 11-12), with fewer chloroplasts than the other layers.These cells are horizontally enlarged; in paradermal sections, they are lobed and have conspicuous intercellular spaces (Fig. 12).
Secretory cavities (Fig. 9, 13-14) are only recorded for Dahlstedtia pentaphylla, and are characterized by a lumen surrounded by a one-layered epithelium.They are present in the palisade and bundle parenchyma in the whole leaflet lamina, in the cortical parenchyma of the petiolule and petiole, and in the peripheral parenchyma of the pulvinus (Fig. 14).
Secretory and tector trichomes occur throughout the epidermis at several developmental stages in the foliar primordium.The tector trichomes are uniseriate and falcate.The secretory trichomes (Fig. 13) vary in size, because the head can have four to six cells and the peduncle, two to three cells.The head cells have more evident nuclei, granular content and are more stained than the other trichome cells.No trichomes are present in the adult leaf.
The central bundle shape (Fig. 15-19) and the sclerenchyma structure (Fig. 15-19) vary along the foliar organs (Table 1).A vascular cambium (Fig. 20) is present in the petiolule, petiole and pulvinus (Table 1), originating a secondary structure in the vascular system.The xylem fibres increase in number and the rays are already formed (Fig. 21-22).
Crystalliferous and phenolic idioblasts occur along the leaf (Table 1).The crystals are prismatic (Fig. 23) and of calcium oxalate, and occupy the cell lumen almost completely.Both types of cells are very long, and form chains, when observed in longitudinal sections.Only in Dahlstedtia pinnata crystalliferous idioblasts are located in the medulla of the petiole.

Discussion
In comparison with the vegetative axis (Teixeira & Gabrielli 2000), more useful characters are provided from leaves to distinguish between the two species of Dahlstedtia.The presence of secretory cavities in D. pentaphylla and their absence in D. pinnata was an important character for diagnosis of the species (Table 2).Secretory cavities were similarly located in all the examined representatives of D. pentaphylla, and they are an easily observed character for species identification with the use of free hand sections.Clarified leaflets should be used carefully, because the presence of conspicuous spaces in the mesophyll of D. pinnata may be confused with glandular punctuations, as in Brazilian Lonchocarpus Kunth.species (Teixeira et al. 2000).
Other leaf characters deserve comments: the number of stomata and the distribution of crystalliferous idioblasts.The differences found in the stomata number/cm 2 between the two species of Dahlstedtia were significant (Kolmogorov-Smirnov test).Despite the similarity of the morphology and type of crystal between species, there was difference in the distribution of crystalliferous idioblasts in the petiole (Table 2).Therefore, these characters may also be informative for the delimitation of these species.
The presence of glandular trichomes and idioblasts containing phenolic compounds is a similarity in both    16) fibres (Fig. 16) in the perivascular tissue and central parenchyma cells Petiole closed (Fig. 17) grooves of + among the collenchyma among the phloem and lignified fibres (Fig. 17  species as are the characteristics of dorsiventral mesophyll, hypostomatic leaflet and paracytic stomata. The vascular system of the pulvinus, the petiole, the petiolule and the leaf lamina (see Fig. 15-19) are similar in D. pinnata and D. pentaphylla, although the vascular system patterns of the petiole in Leguminosae are variable at tribal, generic or specific level (Watari 1934;Dormer 1945).The petiole of Dahlstedtia is hold by the medium and lateral traces, while the stipules are hold by the lateral ones.
The median layer of the mesophyll observed in Dahlstedtia is similar to a very developed spongy parenchyma.Morphologically, this tissue can be compared to the paraveinal mesophyll (Fisher 1967) or extended bundle sheath system, the most recent designation (Kevekordes et al. 1988).The extended bundle sheath system has already been described in 52 legume species (Kevekordes et al. 1988) and characterized as one-layered, horizontally displayed and with lobed cells that occur between the spongy and palisade parenchyma (Solereder 1908;Metcalfe & Chalk 1950;Fisher 1967), of procambial origin (Weston & Cass 1973).Despite the morphological similarity, this tissue deserves developmental studies to clarify its origin in Dahlstedtia.
Cambial activity in the petiolule, petiole and pulvinus of Dahlstedtia species was confirmed by the presence of a radial structure observed in radial sections.Most reports of cambial activity in leaves refer to Gymnosperms (Mauseth 1988).The few dicotyledon species presenting cambial activity in the leaves, Laurocerasus officinalis Roem., Ligustrum vulgare L., Ulmus montana With.and Viburnum rhythidophyllum Hemsl., were studied by Shtromberg (1959).
This leaf anatomical study, associated to other available data (see table 2), agrees with Burkart's taxonomic position (1957), according to which Dahlstedtia comprises at least two species: D. pinnata and D. pentaphylla.
of Dahlstedtia pinnata from natural populations was collected at "the State Park of Serra do Mar", Picinguaba, Ubatuba and Caraguatatuba, State of São Paulo, Brazil and of D. pentaphylla at "Serra da Graciosa", Antonina, State of Paraná, Brazil.The vouchers are deposited in the Herbarium of Universidade Estadual de Campinas (UEC), São Paulo State, Brazil, under the numbers 28637, 28746, 300 and 17936.

Table 1 .
A comparison of anatomic characteristics in the leaf lamina, petiolule, petiole and pulvinus of Dahlstedtia Malme species.

Table 2 .
Leaf and other available data of the Dahlstedtia Malme species.