Cytotaxonomic studies in Themeda triandra Forssk.: Part III: Sexual and apomictic embryo sac development in 53 collections

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1 S.-Afr.Tydskr.Plantk., 19,56(5); Cyttaxnmic studies in Themeda triandra Frssk.: Part III: Sexual and apmictic embry sac develpment in 53 cllectins H. Liebenberg Departmentf Genetics, University f Pretria, Pretria, 0002 Republic f Suth Africa Accepted 8 June 19 The embry sac develpment f 53 cllectins f Themeda triandra Frssk. was studied. All the diplids and tw f the tetraplids had f the Plygnunrtype, whereas the remaining plyplids pssess 4-nucleate apspric. Sme f the cllectins are facultative apmicts, while thers (in particular the penta- and hexaplids) are essentially bligate apmicts. Interesting variatins in the type f apspry was encuntered in the different apmicts. This variatin might be due t envirnmental influences. On the whle it wuld appear frm the results that Themeda triandra is a yung and therefre still actively develping agamic cmplex in suthern Africa. Versamelings (53) van Themeda triandra Frssk. is ndersek ten psigte van hul kiemsak-ntwikkeling. Die diplo"iede swel as twee van die tetrapi"iede het geslagtelike Plygnunrtipe kiemsak-ntwikkeling vertn. Die rblywende plipi"iede het 'n 4-kernige apspriese kiemsak-ntwikkeling. Beide fakultatiewe swel as rwegend verpligte apmikte km vr nder die verskillende versamelings. Verpligte apmiksie is veral vlp nder die penta- en heksapl iede. Interessante variasie ten psigte van die tipe apsrie wat vrkm, is verkry. Hierdie variasie mag egter slegs as gevlg van mgewings-effekte wees. Or die algemeen dui die resultate daarp dat T. triandra ng 'n jng en gevlglik aktief-ntwikkellende agamiese kmpleks in SuiderAfrika is. Keywrds: Agamic cmplex, apmixis, embry sac, plyplidy, Themeda triandra Intrductin In a previus article (Liebenberg 1986) the present authr reprted n the chrmsme numbers and micrsprgenesis f 53 Themeda triandra Frssk. cllectins. The embry sac develpment f T. triandra has been studied by Brwn & Emery (1957, 1958), wh fund that a tetraplid frm Suth Africa and an ctplid frm India were 4-nucleate apspric apmicts, and by Liebenberg (Liebenberg 1961, Liebenberg & Pienaar 1962), wh studied a tetraplid and a diplid (included in this reprt as Williams 37 & 65). Whereas the embry sac develpment f the tetraplid agreed with that fund by Brwn & Emery (p cit), the diplid prved t have a embry sac develpment f the nrmal 8-nucleate, Plygnum-type accrding t the terminlgy f Maheshwari (19 & 1963). These studies shw that Themeda triandra frms an agamic cmplex. It was therefre decided t study the embry sac develpment f all 53 cllectins previusly studied, in rder t gain mre infrmatin n the extent f apmixis in this species in suthern Africa. Materials and Methds The 53 plants reprted n by Liebenberg (1986) were selected fr this study. Inflrescences were cllected frm plants grwn in a unifrm nursery at Pretria. The riginal lcalities f each cllectin as well as their chrmsme numbers, are listed in Tables 1 and 2. Bi flwers frm these inflrescences were fixed in Navashin (Stckhlm mdificatin) (Maheshwari 1939) and embedded in paraffin wax, using a tertiary buthyl alchl schedule (Jhansen 19). Sectined slides were stained in Heidenhains haematxyline and cunterstained with range G (Jhansen 19). The number f scred fr each cllectin are given in Tables 1 and 2. Due t the scarcity f material in sme cllectins (nly ne ut f seven flwers are bi) as well as the fact that many sectins were useless as a result f the hardness f the bi flwers, nly a few embry culd be scred in sme f the cllectins. Results The results f the embry sac evaluatins are given in Tables 1 and 2. In general the results agree with previus findings. The diplids (2n = = 2x), as well as the aneuplids at diplid level, all have, 8-nucleate, Plygnum-type embry sac develpments with the custmary increase in antipdal cells cmmn t the Paceae (Figure la-f). An interesting deviatin frm the nrmal situatin encuntered was the ccurrence f multiple in the same vule. These multiple apparently develp frm separate macrspre mther cells rather than frm mre than ne macrspre f the same tetrad (Figure 2f). Since in sme cllectins nly a few vules culd be evaluated, the variatin in this respect, between the different diplids studied, was difficult t quantify. Hwever, the values btained fr the different diplid cllectins (Table 1) differ s much in sme cases that there can be n dubt that actual differences d ccur between the different diplid cllectins. Of the 19 diplids (and aneuplids at diplid level) studied, eight (Williams, A, 31,, 65, 76A, 91 & 6) cntained nly ne embry sac in all vules scred (Williams 19 is excluded since nly tw vules culd be evaluated). Williams 4 and 7A may be included in this grup althugh bth als had vules that cntained degenerated remnants f (Table 1). Fur diplids (Williams 2,,11 &

2 S.Afr.1. Bt., 19,56(5) Table Embry sac types fund in the diplid cllectins f Themeda triandra Percentage f vules cntaining: Cllectin nly with 1-2 Number f n. embry embry embry apmictic 2n embry sac evaluated Williams Lcality** 2 Wrcester 4 Rbertsn 7A Malmesbury Malmesbury * * CapeTwn Smerset West Mssel Bay 19 Gerge Bedfrd A 29 6B 2 Alexandria Bathurst Peddie Bergville 65 Middelburg c.p. 4B 12 4B Cradck Stellenbsch 91 Christiana 16 6 Richards Bay 6367 Nabmspruit A *The remaining vules f these tw cllectins cntained nly degenerated remnents f the **The exact lcalities fr these cllectins can be fund in Liebenberg (1986) 63167) exhibited a high prprtin f vules with ne embry sac and a lw prprtin with tw. In ne diplid (Williams 18) all vules scred had tw per vule. Tw cllectins (Williams 29 & ) exhibited three t fur per vule in 85% and % f the vules studied, respectively. In nly ne diplid (Williams 13 frm Smerset West) was any trace f 4-nucleate apspric fund. This was encuntered in nly ne f the vules studied. Neither aneuplidy (Williams 4,, & 63167) nr B-chrmsmes (Williams 18. & ) appear t have any significant effect n the ccurrence f multiple r apspric embry. The relatively lw numbers f vules evaluated per cllectin in this study, wuld hwever make it unlikely that such effects wuld have been nticed. Except fr tetraplids (2n = = 4x; Williams 32 & 34), in which nly 8-nucleate were encuntered, all ther plyplids were apspric apmicts. The vules which will eventually cntain apspric embry, initially develp a, Plygnum-type embry sac. This embry sac may develp t maturity and persist until fertilizatin ccurs. Mre ften, hwever, it degenerates and all traces f it has disappeared in the mature vule (Figure 2a). During the early stages f vule develpment ne t seven (Figure 2e) apspre mther cells differentiate frm nucellar cells (Figure 2a-e). These apspre mther cells underg tw mittic divisins resulting in a 4-nucleate, mnplar apspric embry sac (Figure 2c-<1). These cntain ne egg cell, tw synergids and ne plar nucleus. N deviatin frm this was encuntered. Due t the cmpetitin between the different apspric and at times,, the frm f the 4-nucleate apspric may vary cnsiderably between different vules. The are easy t distinguish frm the apspric. They are biplar, 8-nucleate with a later increase in the number f antipdal cells (Figure If) which d nt ccur in the apspric embry at all. Table 2 shws in what prprtin f the vules ccurred with r withut apspric. Sme interesting variatins f this general pattern were encuntered amngst the apspric plyplids. In the tetraplid, Williams 75, all the vules cntained nrmal. In additin.7% f the vules als cntained ne r mre apspric. Anther tetraplid (Williams 38) pssessed vules cntaining nly in 61% f the vules studied. The remaining vules cntained n but three r mre apspric. Only a few ther plyplid cllectins had high frequencies f vules with nly (fr example the tetraplids Williams and t a lesser extent Williams A131 & 6519 and the hexaplids Williams 45 & 85). In all the remaining plyplids, less than % f the vules studied cntained nly. In tw tetraplids (Williams 99Gr & 3), tw pentaplids (Williams 84 & 99B1) and fur f the hexaplids Williams & 63171) n ccurred in any f the vules evaluated. In sme f the vules the apmictic ccurred clustered tgether, whereas in thers they tend t be mre

3 S.-Afr.Tydskr.Plantk., 19,56(5) 556 Table 2 Embry sac types fund in the plyplid cllectins f Themeda triandra Percentage f vules cntaining: Cllectin nly I n. embry apmict apmict embry sac Williams Lcality* 2n I aprni ct widespread cmpactly Number f apmict gruped embry sac evaluated + Stutterheim East Lndn 34 Barkley East 37 Lady Grey Pietennaritzburg Zululand? A Estcurt Bergville Middelburg c.p Graaff-Reinet Cles berg Wdhuse Blemfntein Fauresmith Barkley West Weenen Ween en Gr Magut Nelspruit A 131 Candver Piet Retief Sabie B 78 Ennel Zastrn Magut Pietennaritzburg Brits 17 Rustenburg BI Reddersburg Mafekeng Aliwal Nrth Pta Krnstad Nabmspruit Pretria *The exact lcalities fr these cllectins can be fund in Liebenberg (1986) spread thrugh the nucellus. Data in this cnnectin are listed in Table 2 as percentages. In tw f the tetraplids and ne pentaplid Williams 38,54 & 78) nly dispersed were fund. In three ther tetraplids nly a clustered gruping was encuntered (Williams 75, 3 & Al31). The prprtins fr the remaining apmicts varied cnsiderably between these tw extremes. Discussin and Cnclusins The results f this study further supprt the previus cnclusins that Themeda triandra frms an agamic cmplex in suthern Africa (Brwn & Emery 1957, 1958; Liebenberg 1961, 1986; Liebenberg & Pienaar 1962). This als agrees with similar findings f T. triandra and ther Themeda species frm the Ind-Australian regin (Wdland 1964; Faruqi et al. 1975; Birari 19). As is cmmn t all such cmplexes (Stebbins 19; Grant 1971; Lewis 19) the diplids are usually whereas the plyplids tend t be apmicts; in the case f Themeda apspric apmicts. The pattern f the apspric embry sac develpment fund, des nt deviate significantly frm that f previus reprts. An interesting finding nt previusly reprted in Themeda, is the ccurrence f multiple in the same nucellus. This phenmenn is hwever nt unknwn in ther Angisperm taxa (Maheshwari 19, 1963; Buman 1984). Since this study did nt include pst-fertilizatin flwers, it is nt knwn what effect these multiple have n pssible plyembryny nr whether the multiple remain separate r tend t fuse at a later stage. Cnsiderable variatin in the pattern and frequency f apspry was fund. It is pssible that this variatin, as well as the multiple, are determined by

4 S.Afr.J.Bt., 19,56(5) 557 b c d ',.:, ~;, ~.~ ~'::~, e s s Figure 1 Sexual embry sac develpment in a diplid (Williams 65). (a) Macrspre mther cell, (b) tetrad stage with 3 degenerating macrspres, (c) 2-nucleate stage - nte biplar nuclear arrangement, (d) 4-nuc1eate stage, (e) 8-nuc1eate stage, (f) mature Plygnum-typeembry sac - nte increase in number f antipdal cells. S = synergids,o = egg cell. envirnmental effects. The plants were cllected ver eclgically diverse habitats, but were grwn in a unifrm nursery in Pretria. A marked effect n the general phentype f the plants transplanted frm the suthern Cape Prvince was nticeable when plants in the nursery were cmpared with their herbarium specimens. Envirnmental effects n chrmsme behaviur and in particular n apmictic embry sac develpment has been reprted previusly (Heslp-Harrisn 1959; Grazi et ai. 1961; Knx & HeslpHarrisn 1963; Knx 1967; Evans & Knx 1969; Ngler 1984). Frm the results f the embry sac, cytgenetic and mrphlgical studies, Themeda triandra in suthern Africa, appears t be a cmparatively yung, actively evlving, agamic cmplex. Nt nly are there still many diplids present in the cmplex, but amngst the plyplids - in particular many f the tetraplids - a relatively high degree f ity ccurs. Accrding t the terminlgy f Grant (1971) the Themeda triandra agamic cmplex can be classified, alng with ther agamic cmplexes such as Crepis, Rubus, Taraxacum, and Hieracium, as 'early mature' in its develpment. Tw f the tetraplids were fund t be entirely. In yunger agamic cmplexes, such as, amngst thers, the Butelua curtipendula cmplex, the lwer plyplids tend t be fully (Grant 1971; Stebbins 19). These tw tetraplids may be remnants f such an earlier stage f develpment in Themeda triandra. It is hwever difficult t determine the exact percentage f seeds frmed in the facultative apmicts, because f the fact that in many the embry ccur tgether with ne r mre apmictic in the same vule. It is almst impssible t determine which

5 S.-Afr.Tydskr. Plantk., 19,56(5) 558 a b c d e f f' ' _ ' '.' Figure 2 Apspric embry sac develpment in a tetraplid (Williams 37) and multiple in a diplid. (a) Tw apspre mther cells and remnants f tetrad, (b) fur I-nucleate apspric in the same vule, (c) tw mature 4-nucleated apspric and ne 2-nucleate stage apspric embry sac in the same vule. (It is nt pssible t distinguish clearly between the synergids and egg cells in these due t the distrted character f the ), (d) five apspric in the same vule, at different stages f develpment, (e) seven apspric in the same vule, (f) at least three separate tetrads in the same diplid vule (Williams 29). wuld fnn the eventual effective embrys. In sme cllectins, n the ther hand, a fair amunt f vules ccurred cntaining nly a embry sac (Williams 38, 45,64,75,85, Al31, 261 & 6519, see Table 2). These findings are brn ut by the fact that T. triandra is very variable mrphlgically in sme lcalities where plyplidy ccurs, whereas at ther lcalities the mrphlgy f the lcal plants are very unifrm due t a high degree f apmixis. This is generally true f many f the higher plyplid ppulatins (penta- and hexaplids). Acknwledgments The authr wishes t thank the Department f Agriculture f the Republic f Suth Africa wh made this study pssible.

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