MORPHOLOGICAL AND ANATOMICAL INVESTIGATIONS IN DESMODIUM TORTUOSUM (SW.) DC. (FABACEAE): A NEW ADDITION TO THE EGYPTIAN FLORA

The morphological, anatomical and micromorphological features of Desmodium tortuosum (Sw.) DC. were investigated to assist in the identification of the species as a new record for Egypt. Specimens collected from different places in the Egyptian Nubia (Nubian Nile Valley) confirmed the presence of D. tortuosum as a naturalized species in the country. In general, the results obtained from the morphological studies were consistent with the previous studies on this species. However, the present investigation into leaf, petiole and stem anatomy and the seed and the leaf patterns under light and scanning electron microscopes, respectively, is the first such study on the species.


Introduction
Desmodium tortuosum (Sw.)DC. belongs to the family Fabaceae (Leguminosae).The species is common throughout the temperate and tropical regions, with exception of Europe, New Zealand, and the United States's west of the Rocky Mountain (Schubert 1980).The native range of D. tortuosum includes the West Indies; other reports, however, suggest the species also may be native to the Americas (Smith 1889, Hume 1907).Of the 300 species of Desmodium distributed throughout the world, only 39 species occur in Africa, but none has so far been reported from Egypt (Lock 1989, Boulos 2004).
Desmodium tortuosum was once regarded as a useful warm-season crop in the USA, especially important as horse feed and as a crop improving soil structure (Smith 1889, Yaunge et al. 1964).Today, the species is considered among the most troublesome weeds in crop fields of the southeastern coastal plain of the USA (Webster and Cardina 2004), but still regarded as a desirable plant for wildlife.Some anatomical and morphological features of the subfamily Papilionoideae and the genus Desmodium were reported by Heneidak and Shaheen (2007) and Webster and Cardina (2004), respectively.However, so far, there have been no detailed anatomical and morphological studies on D. tortuosum.
In the present study, the morphological, anatomical and ultra-morphological features of fresh samples of D. tortuosum were studied in an attempt to provide more detailed descriptions for this species newly added to the flora of Egypt.The study may also give new characters to assess the identification and the taxonomic position of this species in the future studies.

Materials and Methods
A survey of some herbaria throughout Egypt (CAI: Cairo University Herbarium; CAIM: Agriculture Museum Herbarium; and ASW: Aswan Faculty of Science Herbarium), consultation of all published Egyptian floras and monographs and field exploration in some phyto-geographical regions revealed no report of the genus Desmodium from Egypt.
The author recorded the first population of D. tortuosum in Egypt from the Nubian Nile Valley (Al-Gahfra, latitude 24º24'51" N, longitude 32°57'17" E; 30 km north of Aswan City) in March 2001.The second one was collected from the Nubian Nile Valley (Elephantine Island, latitude 24º05'08" N, longitude 32°53'25" E; 6 km north of Aswan Dam) in November 2001 (Shaheen et al. 2004).Later on plant specimens were collected from different places of the Egyptian Nubian Valley through repeated visits during 2004-2007.Specimens for morphological studies were preserved according to standard herbarium techniques and stored in Aswan Faculty of Science Herbarium (ASW), Egypt.
The taxonomic description of the plant was framed using the stereomicroscope at ASW and compared with that of Webster and Cardina (2004).Anatomical investigations were performed on cross-sections of fresh samples of leaves, petioles and stems at Ain Shams University, Cairo, Egypt.Segments of petiole (distal, medial and proximal), stem and leaf were preserved in 70% alcohol.The cross-and surface-sections were covered with glycerin-gelatin (Vardar 1987) and photographs of the sections were taken using a Leitz Deplane photomicroscope.
Seed coat and leaf surface patterns were first investigated at the 7-14 × magnification using an Olympus stereomicroscope (ASW Herbarium).For more detailed study, seed and leaf were mounted on specimen stubs with conductive material paint.The specimens prepared before being examined and photographed with IVO Stero Scanning Electron Microscope (SEM) at the central laboratory at Qena Faculty of Science, Qena, Egypt.
The findings of morphological studies were generally consistent with the description given by Rodford et al. (1968), Redhead and Polhill (1971), Lock (1989), Brown and Cardina (1992) and Webster and Cardina (2004), especially with those of pod and seed.
Distribution: Northern America (Southeastern and South Central USA) and Southern America (Mesoamerica, Caribbean, Northern South America, Brazil, Western South America and Southern South America) (Schubert 1980).In Africa, the species was collected from three sites of the regional centre of endemism (Zambezian, Sudanian and Somalia-Masai) and one regional transition zones and regional mosaics (Lake Victoria Regional Mosaic).It was also collected from Anthropic Landscape vegetation type (White 1983).
Habitat: Locally, the specimens were collected from Date Palm orchards terrace of the Nubian Nile Valley (latitude 24º8' -24º15'N and longitude 32º32' -33º30' E) which is considered a narrow strip of Nile deposits (Shaheen 1987).The soil comprises a mixture of wadi-fill deposits and Nile alluvium.The surface layer is characterized by the coarse ingredient (67% sand) with smaller amounts of silt (15.9%) and clay (14.4%) (Shaheen 1987).The seasonal range of mean temperature of the region shows that the winter is cooler (December: 16.95°C) and the summer is hotter (July: 33.25°C).The annual mean rainfall in the region is negligible, and the relative humidity suggests the region to be dry.The plant community is basically formed of herbaceous annual and perennial species characterized by grasses in addition to prostrate weedy plants such as dominating Oxalis corniculata L. and Phyla nodiflora (L.) Greene.The main crops cultivated in this terrace are date palm, mango, clover, sorghum and maize.
Phenology: Flowering -February to April; fruiting -March to June.

Anatomical features
Petiole anatomy: In the proximal region, the leaf trace has a less irregular shape, the adaxial and the abaxial bundles have fussed into a more or less complete vascular cylinder with a continuous layer of pericyclic fibres (Figs 2A-C).The secondary bundles and cortical sclerenchyma are completely absent.Dense solitary crystals are present in this part of the petiole.Dense unicellular and multicellular trichomes are present.
In the medial region, the petiole trace has a more irregular shape; it is already divided clearly into three adaxial and 13 small abaxial bundles.In addition to the main bundles, there is an arc of accessory bundle lie within the core of the trace under the adaxial bundles; it differentiated from the abaxial and adaxial bundles by being concentric one.Pericyclic fibres are present in separate regions outside the phloem of each of these bundles.There are no secondary bundles.The cortical sclerenchyma and crystals are absent.The unicellular trichomes are sparse.
In the distal region, the leaf trace becomes more irregular in shape; it is clearly divided into one large adaxial (weakly divided) and eight abaxial bundles forming the main trace, above which lie laterally a pair of secondary bundles.Pericyclic fibres are present as a separate layer above the phloem of each bundle of the main trace only (adaxial and abaxial bundles) while each secondary bundle has its own separate fibre cap.Cortical sclerenchyma and crystals are absent.Dense unicellular trichomes are present.
The anatomical analysis given in these studies provides the first detailed description of D. tortuosum.Analysis of the petiole trace structure in cross-sections (proximal-distal) shows that the petiole trace of this species has a major change in the anatomy structure throughout its petiole.The disappearing of the secondary bundles in the proximal and medial regions is also documented in this study.In addition, there is also an abnormality in the leaf trace structure of the medial region (having an accessory bundles in the core of the trace).These results are consistent with the description given by Heneidak and Shaheen (2007) in their investigation of the petioles of some papilionoid species.In this connection, Shaheen (2006Shaheen ( , 2007) ) reported the usefulness of anatomy of stem-leaf transitional zone in the identification of some mimosoid and caesalpinioid species.Leaf anatomy: The upper and lower leaf epidermis layers are composed of uniseriate, large elongated cells, and thick lateral walls (Fig. 2D).Both epidermal layers are covered with thick cuticle.Unicellular and multicellular hooked trichomes are very dense on both surfaces.There are some glandular trichomes as well.The stomata type is paracytic (rubiaceous) and they occur on the surface of both sides, being more abundant on the upper surface.They are in sunken position with the epidermal cells as mentioned in the micromorphology section (Fig. 3C).The mesophyll is composed of two layers of palisade tissue.Palisade tissue has solitary crystals.The midrib is well developed.The xylem and phloem are in the normal position; the xylem (three arches) is towards the upper side, while the phloem is on the lower side.These present findings are in agreement with those of Shaheen (1995) who reported the distribution of stomata on both sides of the leaf in some species of Egyptian and Australian Acacia.In general, these anatomical features observed on the leaves are consistent with those of Metcalf and Chalk (1950) and Philipson (1963) for the description of leaf anatomy of Leguminosae (Fabaceae).

Stem anatomy:
The epidermis is composed of almost elongated cells, with compactly arranged cells and bears no stomata (Fig. 2E).The epidermis is covered with a relatively thick cuticle and contains dense uni-and multicellular trichomes.The cortex is 4-5layered consisting of irregular parenchyma with chloroplasts and patches of collenchyma cells.The single-layered endodermis consists of elongated cells.The pericycle is wide consisting of 5-6-layered sclerenchyma cells.The phloem is 2-3-layered consisting of irregular cells.The fascicular and interfascicular cambium is distinguishable (6-layered cells).Xylem is composed of vessels and the phloem contains resin ducts.The pith consists of large orbicular pentahedral parenchymatous cells.These cells underlie the xylem and are thin-walled.
A sclernchymatous ring with varying width found in different genera and species of papilionoid species also characterizes the pericyclic of D. tortuosum.The xylem and phloem also form a continuous ring in the studied species as well as in certain species of Desmodium (Devadas and Brck 1972).In addition, there were no calcium oxalate crystals in their stems.These results are in agreement with Ataslar (2004) who reported the absence of calcium in the stem of Saponaria kotschyi Boiss.

Micromorphology
Micromorphological studies of the leaf surface pattern by Scanning Electron Microscope (SEM) show that the leaflet has clearly defined epidermal cells with fine crystals and the stomata are paracytic type and present on both top and bottom surfaces of the leaf .Stomata are at the lower level than other epidermal cells that is in a sunken position.In addition, study of the seed surface under SEM shows an irregularly rippled pattern (Figs 3E-H).
There has been no report on micromorphology of D. tortuosum.Nonetheless, the results agree with those of Shaheen (1995) who reported the usefulness of sunken stomata on leaf in identification of Acacia ehrenbergiana Hayne.Irregularly rippled pattern on seeds has also been documented in some species of spinescent Acacia (Shaheen 1995).In this connection, valuable taxonomic evidence has been obtained from studying seed characteristics under SEM in some species of Cassia and Senna as well as some species of Caesalpinioideae (Hussein et al. 2002a, b).
The micromorphological as well as the anatomical investigations into Desmodium tortuosum recorded here provide the first detailed description for this species.It can be concluded that, the species has some diagnostic morphological and anatomical features with taxonomic values, especially those of the seed and pod and also the abnormality in the vascular trace structure of its petiole.