Seedling structure of Euphorbia L . and Chamaesyce Gray species

Chamaesyce prostrata (Aiton) Small, Euphorbia heterophylla L. and Euphorbia graminea Jacq. are weedy species that occur with relative frequency in the region of Maringá, Paraná State, Brazil. Data about the morphology and anatomy of seedlings of these species were obtained, with emphasis on significant structural differences of seedlings between species of Euphorbia and Chamaesyce. Seedlings are phanerocotylar and epigeal. Root is axial and triarch. Cotyledons are leaf-like and dorsiventral. Eophylls and prophylls are simple and dorsiventral. Morphological variations occur in the cotyledon shape of Euphorbia seedlings. It is remarkable the Kranz structure of leaves that occurs only in Chamaesyce.


Introduction
The subtribe Euphorbiinae contains genera that are universally accepted, as Euphorbia L., Endadenium L.C. Leach, Monadenium Pax, Synadenium Boiss., Pedilanthus Neck.ex Poit., and Cubanthus (DC.) Millsp., and genera that are frequently less accepted, as Chamaesyce Gray, Elaeophorbia Stapf, and Poinsettia Graham.Euphorbiinae with approximately 2000 species is dominated by Euphorbia, which accounts for around 80% of species and occurs throughout the geographic range of the subtribe.With about 300 species, Chamaesyce is the largest segregate genus from Euphorbia, and has a wide distribution, but most species are confined to the New World (STEINMANN;PORTER, 2002).
A study on seedling structure of Euphorbia and Chamaesyce species would help in the identification of these species and is also likely to be useful in the separation or not of both genera.Therefore, this research deals with morphology and anatomy of seedlings of the weedy species Chamaesyce prostrata (Aiton) Small, Euphorbia heterophylla L. and E. graminea Jacq., which occur with relative frequency in the region of Maringá, Paraná State, Brazil.In this analysis it was emphasized significant structural differences of seedlings between species of Euphorbia and Chamaesyce.Seeds were washed in solution of sodium hypochlorite and distilled water and finally air-dried.Seeds were left to germinate on moist filter paper in Petri dishes, which were placed in germinator TE 400 Tecnal with controlled light and temperature.Germinated seeds were sown in soil (mixture of soil and organic substratum in equal proportion) contained in plastic sacs in a greenhouse.
Photographs were taken with the stereomicroscope Leica EZ4D and microscope Olympus BX50 with digital camera Canon Power Shot A95, and subsequently prepared using the software Zoom Browser EX 4.6.All samples were prepared on the same micrometric scale.

Seedling morphology
Seedlings of the weedy species (Figure 1) are phanerocotylar and epigeal.Root system is axial and the hypocotyl is long, cylindrical, and green/red.Cotyledons (Figure 1) are thin, green (purple in C. prostrata), petiolate and leaf-like.Cotyledons have different shape among species: oblong with obtuse apex and base, entire margin in C. prostrate (Figure 1A); orbicular with rotundate apex and base, entire margin in E. graminea (Figure 1B); and broadly lanceolate with acute apex and base, of entire margin in E. heterophylla (Figure 1C).
Seedlings of Euphorbiaceae tropical trees are usually phanerocotylar with long-petiolate cotyledons (DUKE, 1969); these characters may be extended to the studied weed species with herbaceous habit.Leaf-like and photosynthetic cotyledons at least in a number of exemplars are considered by Vogel (1980) as homologous with the lowermost stem leaves, and not directly with haustorial or foodstoring cotyledons.In still agreement with this author, it is better to avoid the term cotyledon for this sort of primary seedling leaves, and apply another term: paracotyledons.
Absence of epicotyl was recorded in seedlings of C. prostrata, in which two buds are present in the cotyledonary axils, from the two non-equivalent shoots developed (Figure 1A).The abortion of the main shoot and subsequent sympodial growth in the adult plant was registered by Koutnik (1984) as a unusual synapomorphy.In the other two studied species the epicotyl is green and cylindrical, with edges only in E. graminea.
Seedlings of E. graminea and E. heterophylla present eophylls, while C. prostrata do not.Eophylls (Figure 1B and C) of two species have stipules, petiole, and the leaf blade shows lanceolate-ovate shape, of acute apex, acute or obtuse base and entire margin.The C. prostrata prophylls (Figure 1A) of two shoots that develop in the cotyledonary axils are not stipulate; they have green or dark red color, with obovate shape, obtuse or retuse apex, acute base, and entire and crenate margin only in the apical region.
Seedlings of C. prostrata, E. graminea and E. heterophylla may be included into the Macaranga type which was formulated by Vogel (1980).In agreement with this author, the Macaranga morphological characters are epigeal germination, phanerocotylar seedlings, thin and leaf-like cotyledons and the first leaves are all spirally arranged.Seedlings of C. prostrata, E. graminea and E. heterophylla may also be classified as PEF (Phanerocotylar, Epigeal, Foliaceous) (GARWOOD, 1996), considering the persistent foliaceous cotyledons and the pioneering behavior of weeds (LORENZI, 2008).

Seedling anatomy
Primary root is triarch (Figure 2A) and the structure quite similar among the three species.The epidermis is uniseriate with thin-walled cells and root hairs (Figure 2A and B).The cortex (Figure 2A and B) consists of exodermis that may be collapsed in C. prostrata and E. graminea, pluriseriate parenchyma and endodermis with Casparian strips.Central cylinder is composed of uniseriate parenchymatous pericycle and three strands of primary xylem and phloem (Figure 2A and B).A vascular cambium arises very early in the seedling.
The vascular cylinder of the primary root of seedlings is commonly diarch or tetrarch; other types seem to represent modifications of these basic types (EAMES, 1961).The triarchy observed in Chamaesyce and Euphorbia species seem to be slightly frequent in dicotyledon according to the author.The literature registers triarch roots in Ormosia Jacks.(Fabaceae) (GURSKI et al., 2012), Arrabidaea mutabilis Bureau and K. Schum.(Bignoniaceae) and Vismia guianensis (Aubl.)Pers.(Hypericaceae) (SOUZA, 2009).
The root/stem transition region of the three species (Figure 2C) is short and starts in the base of the root.In the base of the hypocotyl occurs the change of the exarch, typical condition of the root to the endarch one, and the arrangement of xylem and phloem assumes the collateral position (Figure 2D  and E).In E. graminea and E. heterophylla three strands of primary xylem and phloem coming from the root separate into six collateral vascular bundles in the hypocotyl.Similar to the other two species, Chamaesyce prostrata has the same hypocotyledonary vascular structure, but may present three or four collateral bundles.
The level of the transition from the exarch to the endarch condition of the three Euphorbiaceae species is usually low according to the Compton (1912) classification.The low and intermediate types of transition seem to be common of the families Amaranthaceae, Annonaceae, Bignoniaceae, Cactaceae, Fabaceae and Hypericaceae (SOUZA, 2009).
The hypocotyl epidermis (Figure 2D and E) is uniseriate and contains stomata; the nonglandular trichomes occur in C. prostrata and E. heterophylla.The hypocotyl cortex (Figure 2D and  E) shows parenchyma and eventually collenchyma; there is no endodermis with Casparian strips.The central cylinder consists of collateral vascular bundles (Figure 2D and E) which enclose the parenchymatous pith.The pith is partially destroyed in E. graminea (Figure 2D) and E. heterophylla, while in C. prostrata it is complete and reduced.Cotyledons of the three Euphorbiaceae species have three leaf traces (Figure 3A).The trilacunar condition seems to be uncommon among the studied seedlings (SOUZA, 2009).Bailey (1956) registered that of the 99 dicotyledon families studied, 77% of seedlings have an even number of traces at the cotyledonary node, and 60% of cotyledons have two independent traces related to a single gap.
The epicotyl of both species of Euphorbia has the stem structure (Figure 3B).The epidermis is uniseriate and the cortex presents parenchyma and collenchyma (Figure 3B).Numerous collateral vascular bundles occur in the central cylinder of Euphorbia (Figure 3B), approximately 12 in E. graminea and 16 in E. heterophylla.The parenchymatous pith of E. heterophylla may be partially destroyed.Seedling of C. prostrata are devoid of epicotyl, made up of stem (Figure 3C) with uniseriate epidermis presenting thick-wall cells and non-glandular trichomes, parenchymatous and collenchymatous cortex, central cylinder with six or seven collateral vascular bundles, and parenchymatous pith.
Cotyledons (Figure 4A and C) of the three species consist of uniseriate, glabrous and amphistomatic epidermis, and dorsiventral leaf with a layer of palisade parenchyma and bi-or multiseriate spongy parenchyma.Typical palisade parenchyma is wanting in C. prostrata in which cells are commonly elongated but much larger, except in the vein regions (Figure 4A).Small and medium-sized veins are immersed in the mesophyll and present endodermis (bundle sheath).
Cotyledons of C. prostrata present Kranz-type leaf anatomy with prominent bundle sheath cells and a layer of radial mesophyll cells that surrounds the vascular bundles (Figure 4A).Eophylls (Figure 4B) of both Euphorbia species consist of uniseriate and amphistomatic epidermis; multicellular non-glandular trichomes are present in E. graminea, never in E. heterophylla.Mesophyll is dorsiventral and made up of a palisade parenchyma layer in E. graminea, and parenchyma with funneled cells in E. heterophylla (Figure 4D).The structure of C. prostrata prophylls resembles the cotyledons, besides presenting Kranz structure (Figure 4B) The Kranz type of leaf anatomy, observed in cotyledons and prophylls of C. prostrata, has been registered in Euphorbiaceae (FAHN, 1990;LAETSCH, 1974).For Koutnik (1984) Chamaesyce is characterized by unusual synapomorphies, among them the C 4 photosynthesis (Kranz structure).
The current taxonomic trend is going towards the split of the genus Euphorbia (STEINMANN; PORTER, 2002).This has already been observed with the removal of Chamaesyce by Webster (1967).The present study points out few differences among the seedlings of both genera.Morphological variations occur in the cotyledon shape in the seedlings of Euphorbia, and epicotyl and eophylls are entirely lacking in Chamaesyce seedlings.Under the anatomical aspect it is especially remarkable the Kranz structure of the leaves that occurs only in Chamaesyce.Other potentially significant seedling anatomical features for species characterization are listed in Table 1.

Conclusion
The study showed morphological variations in the cotyledon shape in Euphorbia seedlings.Epicotyl and eophylls are lacking in Chamaesyce Kranz structure just occurs in Chamaesyce.
of the three species were collected at the campus of the State University of Maringá, Paraná State, Brazil.Voucher materials were deposited at the UEM Herbarium, collection numbers: C.

Table 1 .
Other seedling anatomical features potentially significant for the characterization of Chamaesyce prostrata (Aiton) Small, Euphorbia graminea Jacq.and E. heterophylla L.