Rev. Biol. Trop. 46(4): 1059-1067, 1998 www.ucr.ac.cr wWW.ots.ac.cr www.ots.duke.edu

Compared gametophytic development of threespecies of Phlebodium (Polypodiaceae, s.str.)

Blanca Pérez-Garéía1, Ramón Riba1,Aniceto Mendoza1 and Irma Reyes J.1

DepartaIÍlento de Biología, C. B. S., Universidad Autónoma Metropolitana. Ap. Postal 55-535. México, D. F. 09340. Fax: 7-24-46-88, e-mail: [email protected]

Received 22-1V-1998. Corrected 16-IX-1998. Accepted 3-X-1998.

Abstract: A comparison was made of the gametophytic deve10pment of Phlebodium araneosum, P. pseudoau­ reum and P. decumanum. Spores were collected from several Mexican localities ranging in altitude from 30 to 2300 m and sown on agar with mineral salts. Similarities in germination type and prothal1ial deve10pment were observed. Adu1t gametophytes are cordate-spatu1ate to cordate-reniforrn; garnetangia are of the typical type of the cornmon1ep tosporangiate fems. AlI three species share similar morphogenetic features of the gametophytes, and these do not 1end additional characteristics to support the distinction of Phlebodium from Polypodium, Microgramma and Niphidium, as regard the sexual phase.

Key words: Gametophyte, México, Micrograrnma, morphogenesis, Niphidium, Phlebodium, Po1ypodiaceae, Pteridophyta.

Phlebodium differsfrom areolated species cal epiphytic plant on palms, at altitudes from of Po(ypodium by the sori occurring on the . 200 to 500 m (Mickel andBeitel '1988).The " junction of double included veins in the are-,'. , morphologiciUcharacteristics distinguishing the , oles, whereas the sori in Polypodium are ter- three species arethe sori present in either one or minal on single included vein. several rows and the absence or presence of The genus Phlebodium comprises epi- scales and trichomes in the leaf blade. P. phytic to terrestrial plants (Mickel andBeitel decumanum has 3-7 series of sori between the 1988), . with three. species in the tropical and costa and the margin,while P. araneosum and P. subtropical areas ofMexico: pseudoaureum (P. areolatum fide Mickel and Phlebodium araneosum (M. Martens and Beite11988) have one series. P. araneosum has Galeotti) Mickel and Beitel, P. pseudoaureum scales in the abaxial surface of the lamina, and (Cav.) Lellinger and P. decumanum (Willd,) J. the lamina of P. decumanum and P. pseudoau- Sm. The three species are widely distributed reum is glabrous. and grow in Pinus-Liquidambar-Quercus The morphological features of develop- forests; Pinus-Quercus forests, tropical ever- ment from the spore to the young sporophyte green forests and mountain rain forests. in the three species are presented, in order to Phlebodium araneosum and P. pseudoaureum assess whether there are interspecific differ- are epiphytic or terrestrial,in altitudes ranging \:mces in the sexual phases of the studied from 600 to 2,500 m; P. decumanum is a typi- species. 1060 REVISTA DE BIOLOGIA TROPICAL

Previous studies have been conducted on Spores were obtained by sifting the envelopes the sexual phase of Phlebodium aureum contents through a sieve (# 2(0) with a 0.074 (Nayar and Kaur 1971). There are also studies mm mesh (Reyes and Pérez-García 1994, on fertilization and embryo development of Mendoza et al. 1996-1997). that species (Ward 1954a, 1954b, Ward and Spores for each species were sown with­ . Wetmore 1954). Other studies on lndian taxa out previous sterilization in 35 petri dishes (5 allied to Phlebodium are those ofNayar(1954, cm across) in Thompson medium (Klekowski 1962), Stokey (1959), Atkinson and Stokey 1969, Pérez-García 1989) to a density of 80- (1970). Studies on development of antheridia 150 spores/cm2. Cultures were grown at room and antheridial dehiscence have been conduct­ temperature (25-30 C); and 75 watt (Luz de ed by Davie (1951) and Hartman (1931). Día Solar brand t 38/ A ID, Slimline 75 v) lamps were used to provide Iight with a 12 hl12 h photoperiod. Photomicrographs were MATERIALSAND METHODS taken from live specimens.

Spores were collected from different indi­ viduals at the localities shown in table 1. RESULTS Leaves with mature sporangia were placed in paper envelopesand sealed to prevent contam­ Spores: In all three species, spores are ination and to allow for the sporangia to open. monolete, ellipsoid, and golden in appearance.

TABLE I

Collectors, numbers, localities, and habitats of the material studied

Species ColJectorlNumber Locality HabitatlElevation Date

Phlebodium araneosum AMR-I07 Km 504 México-Oaxaca Hgy., BE, 201Om ll-IX-1995 (M. Martens & Galeotti) Oaxaca Mi:ckel &Beitel IRJ-IB Rincón de Ugarte BE. 1500 m 08-VIII-1993 Tejupilco de Hidalgo. México

BPG-1030, 1031 Tuxtepec, Oaxaca SAP, 930 m 09-IX-1995

Phlebodium pseudoaureum (Cav.) LeIJinger AMR-57 Cerro de Iztapalapa, Mpio. BPE, 2225 m 13-IX-1993 Tepoztlán, Morelos BPG-1012 Pedregal de de San angel, . SBC, 2300 m 14-IX-1993 Cd. Universitaria, Distrito Federal IRJ-68 Cañon de Iscahuasco, BE, 1580 m 1I-XI-1994 Tetela de Ocampo, Puebla IRJ-82 Behind Ometepetl, BPE, 1830 m 12-XI-1994 Tetela de Ocampo., Puebla.

Phlebodium decumanum BPG-1032, 1033 Tuxtepec, Oaxaca SAP, 100 m 09-IX-199 (Willd.) J. Smith BPG-1036 Tuxtepec,Oaxaca SAP,30 m 09-IX-1995 IRJ-IO1, 102 Tuxtepec, Oaxaca SAP, 85m 1O-IX-1995

" Abbreviations: AMR, Aniceto Mendoza Ruíz; BPG, Blanca Pérez-García; IRJ, Irma. Reyes Jaramillo; BE, oak forest; BPE, pine-oak forest; SAP, tropical evergreen forest; SBC, tropical deciduous fores!. PEREZ et al.: Compared gametophytic development of three species of Phlebodium 1061

The exospore is bilayered and the perispore is 12,13), which allows the growth and differen­ usually thin (Erdtman and Sorsa 1971, Tryon tiation of two wide wings and the beginning of and Lugardon 1991) and type 1 (Lloyd 1981). a central cushion impaiting to the prothalli a The spores of P. araneosum and P. pseudoau­ cordate shape mainly in P. araneosum and P. reum are 40-(45)-48 11m in diameter, with a pseudoaureum; in P. decumanum fue prothalli roughly papillated surface (Fig. 1). The papil­ are basically spatulate (Figs. 14,15). All three lae are blunt and irregular in size (2-5 11m) and species exhibit a Drynaria-type· prothallial the laesura ranges 30-35 11m; the spores of P. development (Nayar and Kaur 1969,1971). At decumanum are 36-(38)-40 11m in diameter, this stage, the prothalli of all three species .and tbe surface is verrucose; the verrucae are show uniceUular and capitate trichomes locat­ 2.5 11m high,and have an irregular distribution ed on the margins and surface (Figs. 16,17). pattem; laesura ranges from 25-30 11m in length (Fig. 2). Adult stage: Differentiation of gametangia and the central cushion takes Germination: Germination was detected place sometime between days 60-120. The with the growth of the fírst rhizoid cell typi­ adult prothalli are spatulate (Fig. 18) to cor­ cally within 7 to 15 days after sowing of the date-reniform (Figs. 19, 20), with wide spores (Figs. 3-5). The rhizoid is long and hya­ undulate wings and a central apical meris­ line in appearance with a large number of pro. tematic region, with unicellular and capitate plastids. A globose prothallial cell with abun­ secretory trichomes at the margins and on dant chloroplasts was formed subsequently. the surface of the prothalli. Allthree species exhibited a Vittaria-type ger­ mination pattern (Nayar and Kaur 19 71) with Gametangia: Full grown gametophytes refracting globules in the fírst prothallial cells with developed gametangia are already pre­ (Figs. 6,7). sent in 55 days old cultures. The archegonia of all three species show a uniform structure Filament stage: In all three species a (Figs. 21, 22). They grow from surface cells, uniseriate germination filament is apparent are bottle-shaped, with a globose venter after 12-30 days, consisting of 2-7 cells as. a embedded in the gametophyte containing the result of parallel divisions of the original pro­ egg. The neck is straight and slightly bent thallial cell (Figs. 7-9). The filament cells toward the apical notch, it has four rows of show abundant chloroplasts and refracting 5-7 cells each,with the ventral canal cell and globules scattered in the cytoplasm. The spore the neck canal cells inside. There are no wall was still attached to the basal cell and morphological differences in the archegonia there were one to three rhizoids present (Figs. of the three species. 7-9). The terminal cell was divided by a wall Differentiation of the antheridia in the parallel to the axis of the filament; further divi­ three species takes place on the surface of a sion follows an oblique direction, giving rise protruding cell already described for other to an obconic or meristematic apical cell. polypodiaéeous fems (Nayar and Kaur 1971) which is responsible for their pendulous shape. Laminar stage: The laminar confígura­ The antheridia in all three species are small tion of the prothalli is established approxi­ (Figs. 23, 24), spherical or subglobose to mately 30 days following germination through somewhat elongate in shape and are found oblique divisions of the apical meristematic along the margins and on the ventral surface of cell resulting in a small spatulate plate of 8-20 the gametophytes. The basic structure of the cells (Figs. 10, 11). Within 40-60 days, the antheridia consists of a basal cell with a flat to meristematic cell develops into an apical pluri­ concave shape, a ring cell lining the antheridi­ cellular meristem centrally located (Figs. al cavity, and an opercular cell. 1062 REVISTA DE BIOLOGrA TROPICAL

Figs. 1-9. Germinatíon pattern. 1-2. Spores of P. araneOSllm and P. decurnanum respectively. 3. Germinatioll initi­ ation oI P. decumanum. 4. 5. GelTI1ioatíon of P decumanul/l. 6. Germination of P. araneosum. 7-9. GerminalÍon fil­

amem of P. araneosum. P. decllmanwn and P. pseudoaureulI! respectively. ce = spore coat; cp = prothalliaJ cell; ¡ =

laesura; p = papilla; r = rhizoid. Bar = 50 pm. PEREZ el al.: Compa.red gametophytic development of three species of Phlebodium 1063

Figs. 10-18. Prothallial plate and trichomes. lO-l!. Prothallial plate oí' P. decumanwn and P. pseudoaureum. 12-13. Spatulate and cOI'date gametophytes of P pseudoaurewn. 14. Spatulare gametophyte of P. decumanum. 15. Spatulate gameophyte of P araueosltln. 16-17. Marginal trichomes of P araneosum alid P. pseudoQurewn respectively. 18. Adult pro­ thallus of P decwna/1.um. cm '" meristematic cell; m = apical pluri.cellular meristem; t = trichomes. Bar = 45 11m in Fígs.

10,11,16, 17. Bar = 0.2 mm in Figs. 12, 13, 14,15.18. 1064 REVISTA DE BIOLOGrATROPICAL

Figs. 19-26. Adult stage, gametangia and young sporophyte. 19-20. Adult prothalli oLP. araneoswn and P. pseudoaureum respectively. 21-22. Neck and mouth of archegonia in P. pseudoaureul11. 23-24. Antheridia of P. pseudoaureul11. 25-26. First

leaves of P. pseudoaureum. a = antherozoids, ca = annular cel!, eb = basal cell. co = opercular cell. Bar = 0,8 mm in Fig.

19. Bar = 0.30 mm in Figs. 20,25, 26. Bar = 50 11m in Figs. 21,22, 23, 24. PEREZ et al.: Compared gametophytic development of three species of Phlebodium 1065

Fígs. 27-29. Sporophyte trichomes and stomata. 27. Two-celled trichome of petiole in P pseudoaureum. 28. Two-celled tri­

29. = 50 chome on. the leaf margin of P pseudoaureum. Anomocytic stomata of P pseudoaureum. Bar �m.

Sporophyte: The embryo development is smaller (by about 10 f.lm), and show a less similar to that of Phlebodium aureum (Ward conspicuous omamentation, with underdeveI� 1954b). The first leaves, which appear within oped papillae as compared to the spores of P. 150-400 days after sowing, in all three species araneosum and P. pseudoaureum. (Figs. 25, 26), are simple, spatulate, and have a dichotomous vascular bundle, As in the gametophytes, the young sporophyte shows unicellular, short, capitate trichomes along the . margin and on the leaf surface, although two­ 8 celled trichomes can also be found on the peti­ 7

ole (Fig. 27) and on the margin of the lamina 6 (Fig. 28). Stomata are anomocytic (Van U'J 5 Cotthem 1973), with two occlusive cells sur­ 4 rounded by epidermal cells with wavy lateral � walls (Fig. 29), and are present on the abaxial 3 -�- P pseudoaureum surface of the lamina, but absent on the adaxi­ 2 -�- P decumanum al surface. -�- Paraneosum

DISCUSSION 50 100 150 200 CULTURE TIME (DAYS) Comparative analysis of the gameto­ phyte morphogenesis of. these three species Fíg. 30. Tímíng of events during development of game­ 1. of Phlebodium showed similarities (Fig. 30). tophytes. Events: Inítiation of gerrnínatioll. 2. Phase of the germfilament. 3. Illitiatioll of prothallial plateo 4. Prot­ Spores are aH monolete, ellipsoid, and gold­ hallial plate with differentiation of apícal cel!. 5. Adult en although spores from P. decumanum are prothallus. 6. Archegollia. 7. Antheridium. 8. Sporophyte. 1066 REVISTA DE BIOLOGIA TROPICAL

Differences in shape were evident in the RESUMEN gametophytic phase. In P. araneosum and P. pseudoaureum prothalliare mainly cordate, Una comparación del desarrollo del gametofito de y P. although sornetimes reniform and with wide Phlebodium araneosum, P. pseudoaureum decumanum fue hecha. Las esporas fueron sembradas en P. decumanum, wings are wings, whereasin agar con sales minerales. Similitudes en el tipo de germi­ shorter and spatulate. nación y desarrollo protálico fueron observadas. Los qametangia are of the leptosporangiate gametofitos adultos fueron cordiforme-espatulados a type in all three species, with no significant cordiforme-reniformes; losgametangios son del tipo morphological differences arnong them. común de los helechos Ieptosporangiados. Las tres especies muestran caracteristicas morfogenéticas del with Stolze (198 1), there are According gametofito similares y no añaden caractenstic<\s adi­ several ideas about the circumscription of the cionales que apoyen la distinción �ntre Phlebodium, genera segregated from Polypodium. Recent Polypodium, Microgramma y Niphidium, por lo .que se papers recognize the following genera: refiere a la fase sexual. Polypodium, Microgramma, Niphidium, Campyl()fleWUm, Phlebodium and Pleopeltis. The sporophytes at those genera are different REFERENCES between them, but the development and mor­ & phology of their gametophytes are very simi­ Atkinson, L.R. A.G. Stokey. 1970. Garnetophyte of Polypodium clmoodes.Phytomorphology 20: 364-367. lar, as shown by the present study and those of

Polypodium lepidotrichum (Reyes and Perez­ Davíe, J.H.1951. The development of the antheridium in García 1994), Niphidium crassifolium (Reyes the Po!ypodiaceae. Amer. 1. Bol. 38: 621-628. el al. 1996) and Microgrammanítida (Ramírez & and Pérez-García 1998). Erdtman, G. P. Sorsa. 1971. Pollen and spore morphol­ ogy / Plant taxonomy, Pteridophyla. 302 pp, The sexual phase of the species mentioned Almqvist and Wíksell, Stockholm. above and the species studied of Phlebodium share several characters as monolete ellipsoidal Hartman, N .E. 1931. Antherídíal dehiscence in the spores, with oil globules typical of Polypodiaceae. Bo!. Gaz. (Londres) 91: 252-276. Polypodíaceae s.str., germination and pattern Klekowskí, E. J., Jr. 1969. Reproductive bíology of Ihe of prothallial development, archegonia and Pteridophyta. III. A study of the Blechnaceae. J. antheridia typical of the leptosporangiate ferns Linn. Soc., Bot. 62: 361-377. and unicellular trichomes in margins and SUf­ faces of the adult gametophytes. Lloyd, R.M. 1981. The perispore in Polypodium and related 1. A1though there are differences in the genera (Polypodíaceae). Canad. Bol. 59: 175-189. time shape of the adult gametophytes and the Mendoza, A., B. Pérez-García, I. Reyes Jaramillo & M. apparitionof the sporophytes those are not sig­ Ricci. 1996-1997. Desarrollo del gametofito de nificative and do not lend additional support Pteris berteroana (Pteridaceae: Pterideae). Rev. for the circumscription of the genera as regard Biol. Trop.44/45: 51-57. their sexual phase. Mickel, J.T. & J.M. Beitel. 1988. Pteridophyte Flora Of Oaxaca, Mexíco. Mem. New York Bo!. Gard. 46: 1-568. ACKNOWLEDGMENTS Nayar, B.K. 1954. Studies in Polypodiaceae-II. Contribu­ lions to the morphology oE Pseudodrynaria coronans The authors thank Laura E. Riba for the (Wall.) C. Chr. Phytomorphology 4: 379c390. EngIish transIation and Robbin C. Moran for

extremely valuable comments to the manu­ Nayar, B.K. 1962. Morphology oE spores and prothalli of scrípt. sorne species of Polypodiaceae. Bol. Gaz. (Crawfordsville) 123: 223-232. PEREZ et al.: Compared garnetophytic development oC three species oC Phlebodium 1067

Nayar, B.K. & S. Kaur. 1969. Types oC prothallial deve- Stolze, R. G. 1981. Feros and Cero allies oC Guatemala. 10pment in homosporous Ceros. Phytomorphology Fieldiana. Bot., N. S., No. 6: 358-359. 19: 179-188. Stokey, A.G. 1959. Polypodium pectinatum and P. plumu­

Nayar, B.K., & S. Kaur. 1971. Garnetophytes oC homospo­ la - Polypodiaceae 'or Grarnmitidaceae? Amer. Fero rous Ceros. Bot. Rev. (Lancaster) 37: 350-351. J. 49: 142-146.

Pérez-García, B. 1989. Morfogénesis de garnetofitos de Tryon,A.F. & B. Lugardon. 1991. Spores oC the Pteridophy­ Cyatheaceae (Pterophyta: Filicales). Tesis Doctoral. ta: surface, wall structure,and diversity based on elec­ Fac. Ciencias, Universidad Nacional Autónoma de tron microscope studies. Springer,New York. 648 p. México, México, D. F. Van Cottbem, W.RJ. 1973. Stomatal types and systema­ Ramírez T.R. & B. Pérez-Garcia. 1998. Fase gametoCítica tics./n: A. C. Jermy, J. A. Crabbe and B. A. Thomas de Microgramma nitida (J.Sm.) A. R. Sm. (Polypo­ (Eds.). Tbe Pbylogeny and classification oC the Ceros.

diaceae s.str.). Rev. Bio!. Trop. 43: 000-000- Academic, New York. p. 59-71.

Reyes, l. & B. Pérez-García. 1994. Morfología y estrate­ Ward, M. 1954a. Fertilization in Phlebodium aureum J. gias reproductivas del gametofito de Polypodium le­ Sm. Pbytomorphology 4: 1-17. pidotrichum (Fée) Maxon (Polypodiaceae). Acta Bot. Mex. 28: 71-78. Ward, M. 1954b. The development oC the embryo oC Phle­ bodium aureum 1. Sm. Phytomorphology 4: 18-26. Reyes Jaramillo,l., B. Pérez-García & A. Mendoza. 1996. Desarrollo del garnetofito y del esporofito joven de Ward, M. & R.H. Wetmore. 1954. Experimental control oC Niphidium crassifolium (Filicales: Polypodiaceae development in the embryo oC the Cero Phlebodium s.str.). Rev. Bio!' Trop. 44: 485-490. aureum. Amer. J. Bot. 41: 428-434.