reeTl CERVANTESET AL.: EMBRYOLOGYOF CHAMAEDOREAELEGANS I3l

Principes, 4l(3), 1997, pp. 13I-137 Embryologyof Chamaedoreaelegans ():Microsporangium, Microsporogenesis,' and Microgametogenesis

ErvntgunreGoi,+zAraz-CtRVANTES,T AruJel,ttRo Menrit'lnz MENA,T HrRuIro J. Qunnor2 ANDJuDrrH MAnqunz-GuzmArul rlaboratorio de Citologia, Facultad, il'e Ciencias, Uniaersidad.Nacional Autdnoma de Mdxico .Jard,fuBotdnico del Instituto dn Biolog{a, Uniuersid'adNacional Autdnoma de Mdxico

AnsrRecr moni 1970, Takhtajan 1980). Male flowersare yel- low and 2 mm long; they have six stamenswith elegans is a dioecious palm of great economic importance and is an endangered . Annually, the male short filaments and antherswhich are scarcelyvis- palm produces 2-3 inflorescences, each with a great number ible under the pistillode (Hodel 1992). The only of tiny yellow flowers. During anthesis, flowers produce a drop previouswork about embryologyof the genusCha- of nectar that is obserued over the apex of the pistillode that m,aedoreais from Mahabal6 and Biradar (1968), stands out from the triangular opening formed by the petals. The male flower has six . The anther wall is formed by where the investigationsof Sussenguth(192f) and six cellular strata: an epidermis, a monosiratified endothecium, Schnarf (1931) are cited. The type of division of a middle layer formed by three cellular strata, and the glan- the microsporemother cells in different speciesis dular . The mother cells begin and described. fom tetrads of tetrahedral and tetragonal . The mature anther wall consists of an epidermis and an endothe- When the need for efficient reproduction of this cium. Mature grains are two-celled and monosulcate, species was brought to our attention, we decided semitectate-reticulate. 1n uilro germination of pollen grains to study the basic reproductive biology of Cha- of the shows iheir high viability. The form, color, and structure nTaed,orea.The present study describesthe devel- pollen grains and nectar drop of the flower during anthesis hint at an entomophilous pollination. opment of the male flowers: anther development, microsporogenesis,microgametogenesis, and pol- Ien grains. Someof the data have taxonomicvalue. Chamaedoreais a neotropical dioecious genus, which ranges from to Brazil and Bolivia. The genuscomprises 133 speciesofwhich 54 can Materialsand Methods be found in Mexico (Quero 1987, Hodel 1990a-d, Flower and flowers at anthesis,from male 1991, Hodel and Castillo 1991, Barba and Rom- inflorescences of Chamaedorea elegatu, main- ero 1993). Charnaedoreaelegatus is an element of laboratory conditions at an average vegetationof medium and high evergreenrainfor- tained under of 25'C with a photoperiod of 16 est (Rzedowski 1978, Saldivia and Cherbonier temperature peri- 1982). At present ofthis palm specieshave hours of light and 8 hours of darkness,were great economicimportance because oftheir use in odically collected at different stages of develop- interior decoration. Their leaves are widely used ment. The material was fixed in FAA (formalde- in floral €urangements.Because of overexploitation hyde, l0 ml;96Vo ethanol 50 ml; glacial acetic this palm is on the list of endangered species acid, 5 ml; distilled water, 35 ml). From paraffin- (Vosters 1975, Vovides l98l, INIREB 1986, embeddedmaterial, sections (B-10 pm) were cut IUCN 19BB).Under natural conditions the repro- on a rotary microtome and stained with safranin- duction of Chamaedoreaelegans takes place only fast green (Johansen1940). Sections(0.8-2.0 pm) through seeding (Barba and Romero f993). obtained from JB4-embeddedmaterial cut on an Regarding its reproductive biology some studies ultramicrotome were stained with toluidine blue have been made on the pollen biology (Thanikai- (Valley 1976). The photomicrographs,which show PRINCIPES [Vor-.4l

l. Male flower at anthesis with ribs caused by fiber packagesananged longitudinally in the petals. SEM 30X. 2. LS floral at early stage of development.Pistillode (Pi), anther (An), fiiaments (Fi), vascular bundle (Vb). Scale : 42.2 pm. 3. TS anther. Epidemis (EP), outer secondary parietal layer (Opl), inner secondaryparietal layer (lpl), sporogenoustissue (St). Scale - 26.6 Fm. 4. TS anther. Anows show a periclinal division of the inner secondary parietal layer. Epidermis (Ep), inner secondary parietal layer (lpl), sporogenoustissue (St). Scale - 8.1 p,m. 5. TS anther. Epidermis (Ep), endothecium (En), sratified middle layer (Ml), tapetum (Ta). Sporogenoustissue (St). Scale : II.2 p,m. 6. TS anther with complete number of layers. Epidermis (Ep), endothecium (En), middle layer with three strata (M1),tapetum (Ta), sporogenoustissue (St). Scale - 10.9 pm. rw7) CERVANTESET AL.: EMBRYOLOGYOF CHAMAEDOREAELEGANS r33

7. TS anther. Mitotic divisions of the sporogenoustissue. Scale : 10.5 Fr,m.B. TS anther wall with degenerationof the middle layer. Epidermis (Ep), endothecium (En), middle layer (Ml). Scale : 12.4 1t"m.9. TS anther showing two-nucleate cells of the tapetum. Epidemis (Ep), middle layer (Ml), tapetum (Ta). Scale : 10.5 pm. 10. First meiotic division of microspore mother ""lls. S"u]" - I0.7 p,m. 11. Second meiotic division of microspore mother cells, which gives rise to tetrads with an isobilateral PRINCIPES [Vor-.4I

different stagesof development,were taken on a close to the sporogenoustissue is the secondary Zeiss photomicroscope. inner parietal layer (Fig. 3). The secondaryinner Some flowers at anthesis and mature pollen parietal layer divides periclinally and forms sub- grains were fixed in FAA, dehydrated through an sequently the tapetum, which is adjacent to the ethanol series (30-100Vo), and finally in absolute sporogenoustissue (Fig. 4). This middle layer acetone. The material was subjected to critical divides again periclinically to form another layer point drying with CO, in a pressurechamber (CPA (Fig. 5). The secondaryouter parietal layer divides II, Jeol JfC ll00) and coated with gold for obser- periclinally to form the endothecium,adjacent to vation under a scanning electron microscope the protodermis, and another layer of the middle (JEOL JMS-35). ceil;. The anther wall does not have any furthei periclinal divisions and remains as a single layer Results of protodermis, a single layer of endothecium, three cell layers of the middle region, and a sin- Under the laboratory conditions that were used gle-layer tapetum (Fig. 6). to maintain the Chamaed,oreaelegans plants, inflo- rescencesappeared throughout the year approxi- Microsporogenesis mately every three months. Flowers mature on the same inflorescence at the same time, until they As the anther wall develops, the sporogenous reach a size of 2.2-2.5 mm at anthesis. tissue divides frequently by mitosis (Fig. 7). The At the apex of the pistillode, which'emerges middle region begins to degenerate(Fig. B). The from the triangular opening formed by the petals, tapetum cells increase in volume, vacuolize, and a nectar drop is observed24 hours after anthesis, their nuclei divide (Fig. 9). The tapetum is of the which remains for 2-3 days before the abscission secretory type and has binucleate cells. The of the flower. When the anthers dehisce, pollen microsporemother cells, which occupy the central grains emergeand cover the internal walls of the region of each of the four microsporangiaof the petals and the floral cavity. No pollen grains were anther, are surrounded by a thick callose wall. observedoutside the flower, or on the nectar drop. Somemicrospores begin meiosiswith successive Under the scanning electron microscope,mul- cytokinesis(Figs. l0,ll) and others with simul- tiple ribs arranged longitudinally were observed taneous cytokinesis (Fig. 12) forming tetrads of on the flower (Fig. t). Transversesections showed tetrahedral(Fig. f3) and tetragonaltypes (Fig. l4). that these ribs are formed by fiber packageswith Endothecial cells have bar wall thickenings (Fig. thick walls. 15).

Anther Wall Development PollenGrains During the early developmentalstages, in lon- The callose wall loosensand dissolves,freeing gitudinal sections of floral buds (0.8-1.0 mm the young pollen grains, which emergewith their long), sepals already cover the petals, pistillode, walls already formed (Fig. 16). As a monad, the and primordia (Fig. 2). The latter are microspore divides, forming generativeand vege- formed by meristematiccells surroundedby a pro- tative cells (Fig. l7). Pollen grains contain starch todermis. The hypodermal cells divide periclin- in the cytoplasm of the vegetative cell. The tape- ally, forming two layers. The outer layer, adjacent tum cells break and their contents adhere to the to the protodermis, is the primary parietal layer, pollen grain wall. At this stage the anthers lose which differentiatesinto the anther wall. The inner the septum that separateseach sporangiumfrom layer becomes the sporogenous tissue. Subse- the theca and open by longitudinal dehiscence, quently, the primary parietal layer divides peri- allowing the release of a large amount of morpho- clinically forming two layers: adjacent to the pro- logically well-formed pollen grains (fig. 1B). todermis is the secondaryouter parietal layer and Our observationsindicate that pollen grains are

: anangement. Scale IO.2 1tm. 12. Second meiotic division of the microspore mother cells, which gives rise to tetrads with tetrahedral anangement. Meiotic division (Md). Scale : 13.3 p,m. 13. Microspore tetrads with tetrahedral anangement (Tt). Scale : 10.9 p,m. 14. Microspore tetrads with tetragonal (Ti) and tetrahedral (Tt) anangement. Scale : 10.9 p,m. 19971 CERVANTESET AL.: EMBRYOLOGYOF CHAMAEDOREAELEGANS t35

: 15. TS anrher wall. Endothecium (En) with thickened walls. Middle layer remains (M1). Scale 10.7 p,m. 16. Young pollen grains (Ypg). Exine (ex). Tapetum remains (ta). Scaie : 10.0 !r,m. 17. Two-celled pollen grain (Pg),generative cell (Gc), spermatic : : pm. 19. Mature polien grain, monosulcate, I"ff 1S"1.'5cal" 11.4 pm. 18. TS mature dehiscentanther. Scale 126.O semitectate-reticulate.SEM 2000X. PRINCIPES lVoL.41 morphologicallywell formed and apparently capa- type of tetrad in a single sporangiumof Hyphaene ble of reproduction. Preliminary studies on the indica (:H. dichotoma),Areca catechu, and.Chry- germination in vitro show a high viability of the salidocarpus lutescens,In Cham,aedoreaelegans pollen grains. the tetrads are of both tetragonal and tetrahedral rypes. PollenMorphology The pollen grain types found in the palms are highly variable (Mahabal6 1967, Parthasarathy elegans the pollen is monosul- In Chamaedorea 1970, Uhl and Moore1973, Takhtajan1980, Fer- The exine is semitec- cate, heteropolar-bilateral. gusonet al. 1983, Dahlgrenet al. 1985, Harley et the reticulum presents muri of I pm tate, and al. l99f). Thanikaimoni(1970) described a mono- and lumen ranging from I to 3 pm. The lon- wide sulcatepollen type, reticulate-semitectatefor the is 36.3 pm (34-38) and the shorrest 27 .2 gest axis genlrs Chamaedorea;these characteristics agree pm (18-26). with the resultsobtained. Discussion Acknowledgments Male flowers of Charnaedoreo, elegans possess a Ana Rivas and Verena Rubio for the pistillode, which is only present in some genera We thank translationand Biol. Yolanda Horne- of the family Arecaceae (Essig 1973; Uhl I976a,b, manuscript photographsand Dr. SocorroLoz- 1977,l97&a,b, 1980; Uhl and Dransfield 1987). las for the SEM Dr. BeatrizLudlow for pollen description. The presence ofthe pistillode that produces nectar ano and drops at anthesis suggests zoophilous pollination for this sDecies. LItnRarunp Cnel The characters found during anther develop- BARBA,A. A. ANDA. J. RoMERo.1993. La palma Cametlora: ment in the microsporogenesis and microgameto- Historia Evolutivay Cultivo.En prensa.ENEP Zaragoza. geneis of Chamaedorea elegans agree with Davis 163 pp. (1966) who established these characters for the BIRADAR,N. V. 1968. Studieson palms:embryology of Phoe- family. nix pusilla, P, acaulis and.P. reclinata. Proc. Ind. Sci. 67(2)8: 165-173. Anther wall formation in Chamaed,orea elegans - ANr) T. S. M,qH,qe,lLE.1968. Studies on palms: is of a basic type, although for , in embryology ol Phoenix robusta Hook. Proc. Indian Acad. general, the formation of the walls almost always Sci.Sect.B.68: I B. proceeds according to the monocotyledonous type DeHLcRET\r"R. M. T., H. Crrrronn, ANDP. Y[o. 1985. The of the monocotyledons.Springer Verlag, Berlin. (Davis 1966, Dahlgren et al. 1985). The type of families Germany. pre- anther wall development is not established in D,tvIs, G. L. 7966. Systematicembryology of the angiosperm". vious works on palm embryology (Biradar 1968, John Wiley & Sons,New York, New York, USA. Biradar and Mahabal6 L968, Mahabal6 and Bira- Esstc, B. F. 1973. Pollination in some New Guinea palms. dar 1968, Kulkami and Mahabell1974), only the Principes17: 75-€3. F|RGUSON.J. K.. J. DnersnELD, F. C. P,lcn, ,lun C. TH,twt- number of cell layers were determined. KAIMONI.1983. Noteson the pollen morphologyof Pjn- Dahlgren et al. (1985) reported the presence of azga with special reference ro P. aristata and P' pilosa- a unique cellular stratum forming the middle layer (Palmae: Arecoideae). Crana 22: 65-72. in monocotyledons. Nevertheless, the number of HARLEy,M. M., M. H. KunltlruN,AND I. K. FsRGusoN.1991. Pollen and spores: patterns of diversification.-1n: S. strata is variable in different members of Areca- Blackmoreand S. H. Barnes(eds.). Systematics Associ- ceaei Phoenix syluestris has two, P. pusilla and P. ation Special 44: 225-238. acaulis one or two (Biradar 1968, Kulkami and HooEr-.D. R. I990a. Chomaedoreaamabilis: an ornamenlal Mahabal6 I97 4i).ln C. elegans we found that three speciesfrom central America.Principes 34(f): 4-10. in strata form the middle layer. 1990b. New species and notes on related taxa Chamaedoreasubgenus Stephanostachys. Principes 34(4): Regarding the tapetum, although the amoeboid I60-l 76. or plasmodial type is described as the most com- I990c. Three new species ol Chamaedorea.Areca- mon for monocotyledons (Dahlgren et al. 1985), in ceae from ,M6xico. Phytopatologia68(5): 401- the family Arecaceae the secretory type is more 409. Chamue,d.oreaCastillo-mozrii. New speciesof frequent. The arrangement of microspore tetrads l99}d. Arecaceae from Cuatemala. Phytopatologia 68(5): 397- in palms is very variable (Davis 1966, Mahabal6 400. and Biradar 1968, Dahlgren et al. 1985). Rao 1991. New species of Chamaedorea{iom Central (1959) described the presence of more than one America.Principes 35(2): 7 2-42. re97l CERVANTESET AL.: EMBRYOLOGYOF CHAMAEDOREAELEGANS

-AND N. J. M. Cesrrr-r-o.1991. Additional of Cha- SussENGUTH,K. 192I. Betrage zur frage der systematischen maedoreafrom M6xico. Principes 34(2): 5843. ausschlussesder monokotyl.Beih bot. ZbI.38: l-70. INIREB. 1986. Plantas Mexicanas en peligro. INIREB' Fol- TAKHTAJAN,A. L. 1980. Outline of the classification of flow- leto. ering plants. Bot. Rev. 16:22*359. IUCN. I9BB. Botanic garden and germoplasm conseroation. THANIKAIMoNI,G. 1970. Les palmiers: palynologie et syste- Botanic Gardens ConseroationsNews 1(3): 56-59. matique.Trav. Sect.Sci. Tech. Inst. Franc.Pondich6 ll: JoseNsolu, D. A. 1940. microtechnique. McGraw Hill, L_286, Uur-, N. W. 1976a. Developmental studies in Ptychosperma New York, New York, USA. ' (Palmae) KULKARNT,K. M. er,lo T. S. Meuaser{i. 1974. Studies on I. The inflorescence and the flower cluster. palms: embryology o{ Liaistona chinemis. R. Br. Proc. Amer. J. Bot. 63(1): 82-96. in Ptychosperma(PaI- Indian Acad. Sci. Sect.B. B0: 1-17. 1976b. Developmentalstudies and pistillate flowers. Amer. B*t. MAHABALf, T. S. 1967. Pollen grains in the Palmae. Rev. mae) II. The staminate J. Paleobot. Palynol. 4: 299-304. 63:97-1O9. 1977. Centrifugal stamen initiation in Phytelephan- -AND N. V. Brnelen. 1968. Studieson palms:embry- toid palms. Amer. J. Bot.64(9): lt52-1161. ology of Phoenix sylaestis Roxb. Proc. Indian Acad. Scr. l978a. Floral anatomy of the five species of Sect.B. 67:77-96. Hyophorbe(Palmae). Gentes Herbarium. II: 246-267. PARTHASARATHv,M. V. 1970. Fine structure of pollen sur- 1978b. Floral anatomy of Maxburretia (Palmae). face in palms. Principes4;52-62. GentesHerbarium 1l: 200-211. H. f987. Notes sobre el g6nero Chamaedoreaen QuERo, J. 1980. The structure of the acervulus-the flower M6xico. 1n: Memorias del X congresoMexicano de Bot6n- cluster of Chamaedoreoid palms. Amer. J. Bot. 65(2): Iztacala. M6xico. ica. Contribuci6n 541. ENEP 197-204. Reo, C. V. 1959. Contribution to the embryology of Palmae. -AND H. E. MooRE. 1973. The protectionofpollen and Partll. Ceroxylineae.Iotr. Indian Bot. Soc. 38: 46-75. ovules in palms. Principes 17: llI-I49. Rztlowsrt, J. f978. VegetaciSnde M6xico. Limusa, M6xico. -AND J. DR.qnsrrrr_o.1987. GeneraPalmarum. Allen SeLltvre, G. T. el[o V. CHERBoNIER.1982. De la recoleccirin Press, Lawrence, Kansas, USA. silvestre al cultivo de la palma camedor. Perspectivasde VALLEv, P. 1976. Industrial Park JB4 embedding kit. Poly- aprovechamiento.1n.'Altemativas para el uso del suelo sciences, Warrington, PA, USA. en 6reasforestales del tr6pico hrimedo. Publicaci6n espe- VosrERS,J. 1975. Commercial tse of Chamaed'oreaelegam. cial. Nrimero 26 INIF, pp. 49-73. Princines 19: 149-150. ScHNARF,K. 1931. Vergleichende Embryologie der Angto- VovIDES, A. P. 1981. Lista preliminar de plantas mexicanas spermen. Berlin, Germany. raras o en peligro de extinci6n. Biotica 6: 2.

CHAPTER NEWS AND EVENTS (Continued.fromp. 130)

Mooreanals No More significant rise in membership to The Friends of The Palmetum,it was perceived that there existed The journal Mooreana, named in honor of Dr. the need for a journal of improved quality and Harold E. MooreoJr. and which has been pub- substance.Mooreana first appearedin June l99I lished by the Townsville City Council in North under the editorship of Robert Tucker. Following Queensland,has printed its last issue. Its history Robert's untimely death in early 1992, I fJohn was well summaxizedin John Dowe's editorial in Dowe] assumed editorship of Mooreana in April the last issue: of that year as part of my duties as the incumbent Botanic Collections Officer." "The journal had its beginnings in 1988 when Robert Tucker, then Botanic Collections Officer, Palm enthusiasts around the world will miss began publishing News from The Palmetum this quality publication. The Intemational Palm shortly after the official opening of The Palmetum Societywould like to thank John Dowefor his ded- in Septemberof that year. . . . With an increased icated and quality work in the past on Mooreana. interest in The Palmetum both locally and inter- nationally in the early 1990s, accompaniedby a (Continued,on p. 162)