Embryo sac development in some representatives of the tribe Cynodonteae ( Poaceae )

Chloris virgata Sw., Cynodon dactylon (L.) Pers., Harpochloa falx (L. f.) Kuntze, and Tragus berteronianus Schult. have a Polygonum type of embryo sac development. Unreduced embryo sacs were found in Eustachys paspaloides (Vahl) Lanza & Mattei, Harpochloa falx, and Rendlia altera (Rendle) Chiov. Both facultative and obligate apomixis were observed. The Hieracium type of embryo sac development was observed in the aposporic specimens.


INTRODUCTION
The subfamily Chloridoideae Rouy is divided into sev eral tribes.All the species included in this study belong to the tribe Cynodonteae Dumort.The chloridoid grasses that originated in Gondwanaland (Jacobs 1987), are pre dominantly species of arid and semi-arid areas, where their photosynthetic systems and associated anatomical and morphological adaptations appear to give them com petitive advantages (Jacobs 1987).The Cynodonteae com prises annual and perennial grasses that provide good grazing for domestic and wild animals.In addition, they play an important role in erosion control, and their pres ence gives an indication of the kind of management main tained (Van Oudtshoom 1991).
Cultivation and selection of the better adapted cultivars of these grasses may increase their grazing potential.Stud ies of their mode of reproduction can increase our knowl edge and enhance their improvement.The type of repro duction, asexual or sexual, can be established through a study of embryo sac development.Different types of em bryo sac development occur in different species.Diploid to tetraploid species usually reproduce sexually, whereas apomixis occurs at tetraploid and higher polyploid levels (Stebbins 1972).Facultative apomixis, where apomixis and sexual reproduction co-exist, occurs in most apomictic grasses.Species that reproduce sexually have theoretically a wider variability and a better chance of survival.In con trast, obligate apomicts have a limited variability which can only be improved by mutation.
The aim of the study was to determine how these grass es reproduce, whether sexually or apomictically.by estab lishing the types of embryo sacs that occur in the Cyno donteae.
Unlike Mogie (1992) we used the following definitions for the purpose of this study: reduced embryo sacs (em bryo sacs in which meiosis occurs) imply sexual repro duction, whereas unreduced embryo sacs imply asexual (or apomictic) reproduction.Sexual reproduction is the result of double fertilization, where the offspring contain genetic material from both parents.Asexual reproduction may include single fertilization (pseudogamous fertiliza tion), where the pollen donor does not contribute to the genetic composition of the offspring.With these stricter definitions of sexual and asexual reproduction, the double fertilization of unreduced embryo sacs, resulting in poly ploid lineages (Harlan & De Wet 1975;Lewis 1980), is excluded.We believe that it is justifiable to exclude these specific cases since no such lineages have been observed in any of the species studied (Strydom & Spies 1994).

MATERIAL AND METHODS
Voucher specimens are housed in the Geo Potts Her barium.Department of Botany and Genetics, University of the Orange Free State.Bloemfontein (BLFU) or the National Herbarium.Pretoria (PRE).
Inflorescences at different stages of development were collected and fixed in the field in C am oy's fixative.Ov ules were dissected from the inflorescences in 109c ethanol, serially dehydrated in ethanol and tertiary buthanol and embedded in a synthetic wax.Serial longitu dinal sections (7 (im) of the ovules of all the species, except Harpochloa falx and Retuilia altera were obtained by using conventional methods.The inflorescences of the last-mentioned species were sectioned transversely.Sec tions were stained in safranin and fast green according to the technique used by Spies & Du Plessis (1986) and mounted in Eukitt.At least 20 embryo sacs at various developmental stages were studied per specimen.

RESULTS AND DISCUSSION
The Cynodonteae ovule consists of the nucellus.inte guments (Figure 1A), chalaza, raphe, and funicle.The nu cellus is enveloped by two integuments.They grow beyond the nucellus and arch over its apex forming the micropyle.Both Harpochloa fa lx and Rendlia altera have ovules that are amphitropous, whereas the other species studied have anatropous ovules.
The female gametophyte develops from the archesporium formed in the nucellar tissue of the ovule (Figure 1A).The archesporium divides meiotically, to form a lin ear tetrad of macrospores, of which only the chalazal one is functional.This macrospore enlarges and after three mi totic divisions (Figure 1B-F) a small coenocyte differen tiates.After cytokinesis the macrogametophyte consists of an egg cell, two synergids, the central cell with two polar nuclei, and three antipodal cells (Figure 1G-J).Therefore, up to this stage, embryo sac development conforms to the Polygonum type.The egg cell and the two synergids show a triangular arrangement.The largest portion of the em bryo sac is occupied by the central cell with the two polar nuclei.Usually, the antipodal cells are the smallest cells of the embryo sac.However, the antipodal cells of the Cynodonteae often undergo numerous divisions, which re sult in the formation of a mass of antipodal cells or an antipodal complex (Figure IK.L) in the mature embryo sac.This phenomenon was also observed in other grasses (Willemse & Van Went 1984).
The specimens of Chloris virgata had reduced embryo sacs, and antipodal complexes were present (Table 1; Fig ure IB, D, K).The reduced embryo sacs of the specimens of Cynodon dactylon had no antipodal complexes (Table 1).Some specimens of Harpochloa falx had reduced em bryo sacs with the egg cell, the polar cells, and two syn ergids clearly visible in most of the embryo sacs (Figure 1A, C, E, G-J).Antipodal complexes were present in some of these embryo sacs (Table 1; Figure 1L).The em bryo sacs of Tragus berteronianus were reduced (Table 1).
The specimens of Eustachys paspaloides, Harpochloa falx and Rendlia altera had unreduced embryo sacs (Table 1; Figure 2A-E).Apomixis means asexual (agamic) re production by seed, that is, agamospermy (Nogler 1984).There are two kinds of apomicts.namely, obligate apomicts, which have a completely closed recombination sys tem and heterozygotic genotypes which are preserved at the cost of evolutionary flexibility, and facultative apo micts, where unreduced and reduced modes of reproduc tion coexist (Mogie 1992).Apomixis leads to maternal offspring which are normally genetically exact copies of the mother plant (Nogler 1984).
Eustachys paspaloides and Harpochloa falx were fac ultative apomicts with both reduced and unreduced em bryo sacs.In most cases the reduced embryo sacs degene rate eventually.Rendlia altera was obligate apomictic be cause all reduced embryo sacs degenerated at an early stage.The embryo sacs of these species were aposporous.There are two types of aposporous embryo sacs, the Hieracium type, a bipolar, eight-nucleate embryo sac.and the Panicum type, a monopolar, four-nucleate embryo sac (Nogler 1984).The specimens of Eustachys paspaloides, Harpochloa fa lx , and Rendlia altera had bipolar embryo sacs, therefore the Hieracium type.Antipodal complexes were absent in Eustachys paspaloides and Rendlia altera.
The ovules of Eustachys paspaloides contained reduced embryo sacs with the synergids or the egg cell visible, whereas other ovules had up to two or three different em bryo sacs each (Figure 2E).One specimen of Harpochloa fa lx had two different embryo sacs (Figure 2B).The ovules of Rendlia altera had up to five different embryo sacs per ovule (Figure 2A, C, D).
In conclusion, the grasses of Cynodonteae reproduce both sexually and apomictically.Reduced embryo sacs of the Polygonum type occur.The apomicts have unreduced bipolar Hieracium type embryo sacs.Sexual reproduction occurs at different polyploid levels as seen in Harpochloa fa lx and Rendlia altera.Some species have reduced em bryo sacs at the tetraploid and higher polyploid levels (Harpochloa falx), whereas others have unreduced em bryo sacs at these polyploid levels (Rendlia altera) (Stry dom & Spies 1994).Eustachys paspaloides and Har pochloa fa lx were both facultative apomicts and Rendlia altera was an obligate apomict.The types of ovules differ as seen in Harpochloa falx and Rendlia altera.Both have amphitropous ovules, whereas the other species have anatropous ovules.

TABLE 1 .
-Type of embryo sac development in some representatives of the tribe Cynodonteae (chromosome numbers from Strydom & Chloris virgata, Spies 4783: C, binucleate embryo sac in H. falx, Spies 5078; D, tetranucleate embryo sac in C. virgata with only three nuclei visible in section.Spies 4783; E. tetranucleate embryo sac in H. falx with only three nuclei visible.Spies 5125: F, tetranucleate embryo sac of Eustachys paspaloides.Spies /5 2 /, with only two nuclei visible; G, two synergids in H .falx, Spies 4827; H. I. egg cell and synergids of H .falx, Spies 5113; J, two polar nuclei in H .falx, Spies 4827\ K, antipodal complex with only three antipodal cells visible in this section of C. virgata.Spies 4799: L, antipodal complex with only three antipodal cells visible in this section of H.falx. Spies 5118.a, archesporium; ac, antipodal complex; e, egg cell; i, integument; k. nucleus; n. nucellus; p. polar nucleus; s, synergid.Scale bar; 80 nm.
FIGURE 2.-Photomicrographs of unreduced embryo sac development in Cynodonteae.A, reduced and unreduced embryo sacs in Rendlia altera, Spies 5129\ a degenerating macrospore can be seen next to a reduced one; B, two developing unreduced embryo sacs in Harpochloa falx.Spies 4695; C, three unreduced embryo sacs in R. altera, Spies 5072, two with one nucleus and one with two nuclei; D, five unreduced embryo sacs in R. altera, Spies 5129, each with one nucleus; E, two unreduced embryo sacs in Eustachys paspaloides, Spies 1521, each with one nucleus, a, unreduced embryo sac; d, degenerating embryo sac; s, reduced embryo sac.Scale bar: 80 nm.