Structure and development of fruits and seeds of weed species of Euphorbiaceae

(Structure and development of fruits and seeds of weed species of Euphorbiaceae). Euphorbiaceae s.l. presents several morphological types of fruits, of which the schizocarp is the most common. Th e fruits of weed species of Euphorbia graminea Jacq., Euphorbia heterophylla L., Euphorbia hirta L. (= Chamaesyce hirta (L.) Millsp.) and Euphorbia prostrata Aiton (= Chamaesyce prostrata (Aiton) Small) are objects of this study. Infl orescences and fruits in development were collected on the campus of the State University of Maringa (UEM), Brazil, fi xed in FAA 50 and glutaraldehyde, dehydrated and sectioned with a rotary microtome according to standard morphology and anatomy techniques. Th e species present several anatomical similarities, though there are some diff erences related to the outer epidermis of the ovaries, which is glabrous in E. graminea and hairy in the other species. Th e ripe fruits present a papilose exocarp and lack trichomes, except for E. hirta. Th e ovules are anatropous, bitegmic and crassinucellate; the seeds are exotegmic and the embryos are straight.


Introduction
Euphorbiaceae s.l.presents several morphological types of fruits, such as a bacca, coccarium and polospermatium (Spjut 1994) or schizocarp, capsule, drupe or a berry (Barroso et al. 1999).Judd et al. (2002) consider that Euphorbiaceae s.s.usually present a schizocarp with elastically dehiscent segments from a persistent central column.On the other hand, from a structural aspect, the fruit (pericarp) of studied Euphorbiaceae species are similar (Toledo 1963;Berg 1975;Oliveira & Oliveira 2009;Silva & Souza 2009), in which the formation of sclerenchymatous tissue may be considered one of the most important events in the ontogeny of the fruit (Silva & Souza 2009).
Th e ovules and seeds of Euphorbiaceae were widely studied by Landes (1946), Singh (1954), Toledo (1963), Singh & Chopra (1970), Berg (1975), Corner (1976), Paoli et al. (1995), Tokuoka & Tobe (1998;2002;2003), Añes et al. (2005) and Silva & Souza (2009).Th e studies of Tokuoka & Tobe (1998;2002;2003) have systematic implications and they showed that the fi ve following characters are likely to be useful for comparison between and within subfamilies: a) whether the inner integument is thick or thin; b) the presence or absence of vascular bundles in the inner integument; c) whether ovules or seeds are pachychalazal or not; d) whether seeds are arillate or not; and e) whether the exotegmen is fi brous or not.
Th e fruits of weed species of the Euphorbia L. are objects of the present study.Th e selected species were Euphorbia graminea Jacq., Euphorbia heterophylla L., Euphorbia hirta L. (= Chamaesyce hirta (L.) Millsp.) and Euphorbia prostrata Aiton (= Chamaesyce prostrata (Aiton) Small).Th e genus belongs to the large tribe Euphorbieae (Euphorbioideae), which is considered monophyletic on the basis of the infl orescence (cyathium) (Judd et al. 2002).Bruyns et al. (2006) argues that each cyathium consists of a cupular involucre (made up of four or fi ve fused bracts), and male fl owers inside this involucre surrounding a single female fl ower.Th e tribe Euphorbieae was divided into three subtribes, of which Euphorbiinae is the largest and nearly cosmopolitan.In this subtribe the involucre is made up of fi ve bracts, the male fl owers lack a perianth and the female fl owers are also mostly devoid of a perianth (Webster 1975).According to Webster (1994) the genus Euphorbia belongs to this subtribe.Numerous Euphorbieae specialists retain Chamaesyce Raf. as a subgenus of Euphorbia (Steimann & Porter 2002).With about 300 species, Chamaesyce is the largest segregate genus of Euphorbia and is widespread, although most species are confi ned to the New World (Steimann & Porter 2002).Th e present research deals with the structure and development of fruits (pericarp) and seeds of selected weed species, with emphasis on: a) the defi nition of the fruit type of the studied species; b) the discussion about the origin and diff erentiation of the sclerenchymatous tissue which occurs in the species; c) the evaluation of whether the ovules and seeds of the species present the basic characters of Euphorbioideae and Euphorbieae that were formulated by Tokuoka & Tobe (2002); and d) an analysis to see if there are signifi cant structural diff erences of the fruits and seeds among the species of Euphorbia and Chamaesyce.
Specifi c microchemical tests were carried out for lignin phloroglucinol with hydrochloric acid and for lipid substances with Sudan III (Johansen 1940).
Photomicrographs were prepared using a Leica EZ4D stereoscope camera and an Olympus BX50 optical microscope, which was fi tted with a digital Canon Power Shot A95 camera, and subsequently processed using the soft ware Leica Application Suite version 1.8.1 and Zoom Browser EX 4.6, respectively.
Micromorphological analyses (Bozzola & Russel 1992) were done with material fi xed in glutaraldehyde.Aft er washing in a 0.1M sodium cacodylate buff er, the samples were dehydrated in a graded acetone series, critical point dried with CO 2 , mounted on aluminum stubs, coated with gold, and subsequently examined using Scanning Electronic Microscopy (SEM) (Shimadzu SS-550 Superscan) to obtain digital images.

Ovary structure and fruit development
Th e 3-carpellate ovary (Fig. 1-2) is connate, 3-lobed, has one ovule in each locule and axile placentation.Th e outer epidermis is uniseriate and papillose, and is glabrous in E. graminea (Fig. 1) and hairy in the other species (Fig. 2, 3) (unicellular nonglandular trichomes in E. heterophylla and long multicellular nonglandular trichomes in E. hirta and E. prostrata).Th e mesophyll (Fig. 3) is a little narrower in E. hirta and E. prostrata than in the other species and presents three diff erent regions: a) an outer parenchymatous region that consists of thin-walled and isodiametric cells; b) a middle region with two to four layers of slightly tangentially elongated cells; and c) an inner uniseriate tissue with short palisade-like cells.Th e inner epidermis consists of two or three cellular layers with pluricellular trichomes (Fig. 4).Th e pluriseriate middle mesophyll and the inner epidermis result from the meristems, which develop on the mesophyll and adaxial epidermis of the bud ovary wall.
Th e pericarp (Fig. 5) derived from the ovary wall is composed of a uniseriate exocarp, mesocarp with three regions of tissues and an endocarp with two or three layers of cells.An enlargement of the cells contributes in the growth   of the fruit wall.Th e middle mesocarp and endocarp cells undergo tangential elongation (Fig. 5).Th e cells of the inner mesocarp become conspicuous by their radial elongation (Fig. 5).Th e walls of middle mesocarp, inner mesocarp and endocarp cells gradually become thicker.Th e septum originates from cohesion of the carpel walls.Most of the septum has a structure similar to the remaining part of the carpel.
In ripe fruits (Fig. 6, 7, 8, 9), the exocarp cells resemble the ones in the single-layered outer epidermis of the ovary.Th e exocarp is papillose and lacks trichomes, except for E. hirta that has pluricellular, nonglandular trichomes.Th e outer mesocarp is parenchymatous, although much larger in E. heterophylla.In this layer there are collateral vascular bundles.Th e middle mesocarp is composed of lignifi ed or non-lignifi ed fi bers.Th e inner mesocarp is formed by lignifi ed, thick-walled macrosclereids that are more or less perpendicularly arranged to the middle mesocarp.Th e biseriate or triseriate endocarp is composed of non-lignifi ed fi ber-like cells and an epidermis with trichomes.
Th e fruit presents two dehiscent regions: a dorsal region and another region along the carpel sutures.In the dorsal dehiscence of each carpel the exocarp and outer mesocarp undergo a few structural alterations, but the middle mesocarp fi bers, and inner mesocarp macrosclereids, are substituted gradually by isodiametric cells; the endocarp cells become thick-walled (Fig. 7).In the ventral suture there is dehiscence abscission tissue among the carpel walls; this consists of spongy parenchyma with cells that develops arms.Th e carpophore is separate from the septum for the abscission tissue (Fig. 9).

Ovule structure and seed development
Th e ovule is anatropous, bitegmic, crassinucellate with the micropyle formed by both inner and outer integuments (Fig. 10).Th e exostome and endostome are both in line in E. hirta and E. prostrata and the micropylar canal has a kind of zigzag outline in E. graminea (Fig. 10) and E. heterophylla.Th rough division of the cells in the micropylar end of the nucellus, a cell proliferation is developed and it extends beyond the micropyle.Th e outer integument consists of three or four layers of cells (Fig. 12).Th e inner integument of E. graminea and E. heterophylla (7-10 cell-layers) is much thicker than in E. hirta and E. prostrata.
In the developing seed, the number of cell-layers of the inner integument multiplies slightly in E. graminea and E. heterophylla, whereas the number of cell-layers in the testa remains the same (Fig. 12).Th e cells of the testa maintain thin walls.Especially, the cells of the outer tegmen epidermis become conspicuous because of their radial elongation and deep staining capacity.Th e aril (Fig. 11) is formed in the exostome of the young seeds of the Euphorbia species in which cells of the mesophyll of the outer integuments undergo division and radial elongation.Th e endosperm is nuclear.
At maturity the seed coat consists of both testa and tegmen, but the former is unspecialized (Fig. 13).Th e ex-otesta of E. hirta, E. prostrata and E. heterophylla consists of shortly projecting cells which extend towards the endocarp (Fig. 6,14,15).Th e mesotesta is crushed and the endotesta cells become slightly thick-walled.Th e exotegmen cells of E. graminea and E. heterophylla undergo elongation and a secondary thickening of the cell wall becomes lignifi ed thick-walled macrosclereids arranged in palisade layer in the mature seed; E. hirta and E. prostrata have an exotegmen with a short palisade layer (Fig. 13).Th e mesotegmen is parenchymatous and the endotegmen consists of thin-walled and irregular cells (Fig. 13); both may be crushed in the ripe seed.Th e aril was not observed in the ripe seeds.Th e embryo is relatively large and straight (Fig. 16, 17).

Discussion
As a dorsiventral structure, the carpel can develop a so-called ventral meristem on its inner surface, either in subepidermal layers or in the inner epidermis itself (or both strata); in very peculiar cases, a dorsal meristem can develop (Roth 1977).Leguminosae and Rutaceae fruits have a ventral or adaxial meristem (Souza 1984;1993;Souza et al. 2003) and Bignoniaceae fruits present a middle meristem (Souza et al. 2005).Fruits of Euphorbia species develop two meristems, one in the middle region of the mesophyll and the other in the inner epidermis of the fl oral bud ovary.Euphorbia fruits are similar to those of Dalechampia stipulaceae Müll.Arg.(Silva & Souza 2009).Manihot fruits also present two meristems (subadaxial and adaxial) that originate in the inner mesocarp and endocarp, respectively (Oliveira & Oliveira 2009).
Th e fruit wall may be arbitrarily divided into layers that are referred to as the exocarp (outer layer), mesocarp (middle layer) and endocarp (inner layer) (Spjut 1994), or may involve ontogenetic study of the pericarp (Roth 1977;Souza 2006).Based on the ontogeny study, the Euphorbia fruits were found to be relatively uniform presenting an epidermal exocarp, parenchymatous and sclerenchymatous mesocarp, and endocarp which consists of an epidermis and fi brous cells.Dalechampia stipulaceae fruits (Silva & Souza 2009) match Euphorbia fruits in having the epidermal exocarp and mesocarp with parenchyma and sclerenchyma (fi bers and macrosclereids), although they diff er by the endocarp, which only has fi bers.On the other hand, Manihot fruits have no macrosclereids in the mesocarp, but they present gelatinous fi ber-sclereids in the middle mesocarp, stone cells in the inner mesocarp and a collenchymatous endocarp (Oliveira & Oliveira 2009).
Fibers and macrosclereids of Euphorbia fruits are arranged in the mesocarp so that the cells of one layer cross the cells of another layer.It is probable that this crossed arrangement of cells is related to the movement of water lost during the dehiscence mechanism called xerochasy (Roth 1977).In agreement with this author, during water loss stress is exercised on the pericarp, and the fruit consequently opens.In the dehiscence mechanism of the Euphorbia fruits the presence of the separation tissue placed among the carpels is essential, occurring in the median plane of the carpel and among the carpels and carpophore.
Based on the dehiscence type and persistent carpophore (Barroso et al. 1999), the Euphorbia fruits should be classifi ed as schizocarps.According to Spjut (1994), these schizocarpic fruits can be considered as the coccarium type, a fruit derived from a schizocarpous gynoecium characterized by fruitlets opening along their ventral sutures, and sometimes the dorsal sutures, as a result of their separation from one another or from the central axis.
The ovule and seed structure of the Euphorbia species was found to be relatively uniform.However, comparisons of the species show variations in the thickness of the inner integument, micropyle morphology (exostome and endostome) and size of the exotegmen cells.Both E. hirta (= C. hirta) and E. prostrata (= C. prostrata) are distinct from the other Euphorbia species in having a thin inner integument, exostome and endostome in line and short palisade cells.Other Euphorbiaceae species, such as Euphorbia esula L. (Selbo & Carmichael 1999), present the same characteristic as E. heterophylla and E. graminea, such as a micropyle with an exostome and endostome in a zig-zag.
Th e seeds of the studied species have the basic structural characters of Euphorbiaceae and Euphorbia (Corner 1976).According to this author, the seeds are exotegmic in which the mechanical layer consists of an outer epidermis with palisade and more or less prismatic cells.Based on the embryo classifi cation formulated by Martin (1946), all Euphorbia embryos have been placed within the major group termed axile division; the type is foliate subdivision, spatulate subtype.
Regardless of their few diff erences, the seeds of Euphorbia species present the general structure of the ovules and seeds reported by Tokuoka & Tobe (2002) for the subfamily Euphorbioideae: bitegmic ovules, inner integuments without vascular bundles, outer integument without postchalazal branching, ovules with an aril, and exotegmic seeds with radially elongated cells.Based on the diff erences in the thickness of the inner integument in the tribe Euphorbieae and Euphorbia species, Tokuoka & Tobe (2002) suggested a more extensive study to confi rm if this character is related to evolutionary relationships within the genus.
Although the sample of the species is not broad enough to make a defi nitive conclusion, this fruit/seed study supports the proposition of the Euphorbieae specialists, which is to retain Chamaesyce as a subgenus of Euphorbia.