Gametophyte morphology and development of six Chinese species of Pteris (Pteridaceae).

ABSTRACT.--Spores of six Chinese species of Pteris (Pteridaceae) were sown on soil and subsequent gametophyte morphology and development were studied. Spores of all species are trilete, tetrahedral and with a distinct equatorial flange. Germination is Vittaria-type and the prothallial development is Ceratopteris-type in all of the species. Adult gametophytes are cordate and gametangia are of the common type for leptosporangiate ferns. Differences among species include spore size, germination time, formation time of the gametangia, gametophyte margin shape, number of archegonial neck cells and shapes of the antheridial dehiscence.

KEY WORDS.--Pteris. gametophyte, Vittaria-type, Ceratopteris-type, Pteris vittata, Pteris ensiformis, Pteris excelsa, Pteris fauriei, Pteris finotii, Pteris wallichiana

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The genus Pteris L. (Pteridaceae) is found in the tropics and is in need of redefinition (Smith et al., 2006), however it is estimated to comprise about 250 species (Tryon et al., 1000). Differences in characteristics of fern spore germination and gametophyte development can offer compelling criteria for taxonomic and phyletic studies (Holttum, 1040; Stokey, 1951, 1060; Atldnson and Stokey, 1964; Atkinson, 1073; Raine et al., 1906; Chiou and Farrar, 1997; Chiou et al., 1008; Chandra et al., 2003). Type of spore germination, development of the prothallial plate and the meristematic regions, form of the mature and old thallus, type, position, and time of appearance of hairs when present, and form of the sex organs (especially the antheridium) may prove of value to the taxonomists. Perez-Garcia and Mendoza-Ruiz (2004) indicated that gametophytes may be useful for taxonomic and phyletic studies at the family and generic levels, as well as among species within the same genus. The combination of characters of hair type and position, margin, antheridial structure, shapes of the antheridial dehiscence, antherozoid liberation, and number of archegonial neck cells were used by Atkinson (1973), Pryer et al, (1995), and Perez-Garcia and Mendoza-Ruiz (2004) to delimit subgenera, species, or groups of species within the Thelypteridaceae.

Though the Pteridaceae is large, studies on its gametophyte morphology are limited. However, based on the limited information, the gametophytes of Pteris can be summarized as the following: germination is Vittaria-type and the prothallial development is Ceratopteris-type. The adult prothallus is cordatethalloid, with broad wings, growing very fast, with a distinct cushion. Rhizoids are nearly hyaline or pale brown, distributed in the lower surface of the cushion, with thin cell walls. The adult prothallus is naked. Gametangia are of the common leptosporangiate-type: antheridia are formed from early development stages of the prothallus. The cap cell becomes loose and is pushed off, releasing the spermatozoids. The neck of the archegonia is elongated, curving away from the apex of the prothallus (Nayar and Kaur 1971).

This study describes the gametophyte morphology and development of P. vittata L., P. ensiformis Burm, P. excelsa Gaud., P. fauriei Hieron., P. finotii Christ. and P. wallichiana Agardh.

MATERIALS AND METHODS

Spores were obtained from live plants collected from several sites in China (Table 1). Fertile pinnae were kept in clean paper bags under dry conditions until spores were shed. About one week later, the sporangia and indusia were separated from the spores by a mesh with pores 0.054 mm in diameter. Spores were cultured in plastic basins (measuring 25 cm x 20 cm x 5 cm) with a sieved mixture of black soil and sand (1:1). Thickness of the mixture was about 3 cm. The surface of the mixture was made smooth and substantial and the basins were then watered. Spores of each species were sown evenly at an average density of 250-300 spores per [cm.sup.2]. Basins were covered with transparent plastic film on which two to three small holes were made in order to avoid contamination and desiccation. They were placed in the dark at 25[degrees]C for 24 h then transferred to fluorescent light (10 000 [micro]mol x [m.sup.-2] x [sec.sup.-1]) at 25[degrees]C under a diurnal cycle of 12/12 hr. Cultures were moistened with tap water to prevent desiccation and, in the last stages, to help antheridial opening and movement of antherozoids.

Spore sizes were measured from material in water with a compound microscope (No. XTS 20130, Beijing Tech Instrument Co., LTD] equipped with an ocular micrometer. Measurements of the spore length and width were obtained from an average sample of fifty spores per species (Table 1). Spore morphology was observed under the compound microscope from material in water. All pictures of microscopic material were taken from living materials under lab conditions with a Nikon ECLIPSE E600 camera.

RESULTS

Spores.--Spores of all species are trilete, tetrahedral, brown and possess a distinct equatorial flange. Spores vary in size from (19) 27.4 (40) x (10) 10.3 (12) [micro]m (P.fauriei) to (92) 104 (110) x (90) 96 (100) [micro]m (P.finotii) (Figs. 1-3, see Table 2).

[FIGURES 1-9 OMITTED]

Germination.--Spores begin to germinate between day 2 and day 13 after they are sown (Table 2). Germination is Vittaria-type in all species (Figs. 4-5). Gametophytes of all the species first develop a rhizoid. Of all species, division begins in the first prothallial cell with a transverse wall and finally forms a short germ-filament, 2-25 cells long (Figs. 6-7).

Laminar phase.--The differentiation of this phase is asynchronous in all species and the development occurs between days 6 and 40 (Fig. 8; see Table 2). In P. vittata, as the prothallial plate grows, meristematic activity gradually becomes focused on a group of marginal cells on one side of the plate, away from the apical region. This lateral meristematic region soon locates at the bottom of a notch, which increasingly becomes more obvious as growth proceeds. The position of the meristem results in the asymmetrical young prothallus with one wing larger than the other. When the meristem is formed farther away from the apex, the thallus remains distinctly lopsided longer. The thallus becomes nearly symmetrical by growth of the sides of the wings, making the meristem nearly apical. At last, the prothallial plate becomes cordate after 5-30 days, so development of the prothallial plate is Ceratopteris-type (as defined by Nayar and Kaur, 1969). Then a cushion with the gametangia forms. The adult gametophyte is cordate. The time for the first adult cordiform gametophytes of all species to differentiate ranges between days 17 and 50 (Figs. 9-11, see Table 2). The prothallial development pattern of the other species is identical to P. vittata.

Gametangia.--Once the gametophytes have reached sexual maturity (20-90 days), the gametangia differentiation and development begins. The gametangia are all of leptosporangiate, homosporous ferns. Antheridia of all species are distributed on the lower surface of the gametophyte at the basal end of the cushion. Antheridia are globose and are composed of a basal cell, a ring cell and an opercular cell (Figs. 12-13). During antheridial dehiscence the opercular cell becomes loose and is pushed off, releasing the spermatozoids.

In all species, the archegonia differentiate at about the same time as antheridia. Archegonia are distributed on the lower surface of the gametophyte at the apical end of cushion and near the meristematic region. The necks are oriented toward the basal region of the gametophytes, with 4 rows of cells, 3-5 cells per row (Figs. 14-15).

Sporophytes.--The first sporophytes were observed by about 5-8 weeks after sowing. Fertilization occurred on almost all gametophytes to produce sporophytes.

DISCUSSION

The spores of all species share features such as trilete spores with a distinct equatorial flange, however the spore sizes of the six species are different.

For all species, the germination pattern is of the Vittaria-type. It is the most common type in ferns (Nayar and Kaur, 1971). In this type, the rhizoid develops first after a wall perpendicular to the polar axis of the spores is formed. The first rhizoid of P. fauriei is chlorophyllous, but according to Nayar and Kaur (1971), the rhizoids of Pteris are nonchlorophyllous. The first prothallial cell divides and then the apical cell continues to divide, producing a short filament 2-25 cells long. Germination time differs among all species; spores of P. vittata germinate faster than the other species.

[FIGURES 10-15 OMITTED]

Prothallial development in all species is of the Ceratopteris-type in which the prothallial plate is nonmeristic at the beginning. With its growth, a multicellular meristem emerges on one side of the plate. Cell divisions in the meristem make the young thallus asymmetrical. With the growth of the smaller wing of the thallus, it becomes symmetrical. The adult gametophyte develops faster in P. vittata than the other species.

Sex organs are of the common leptosporangiate type. The dehiscence type in antheridia is consistent with the description given by Nayar and Kaur (1971). The uniform development of the gametophyte in all species has been mentioned above. Distinguishing characteristics among the six species such as size of the spores, germination time, time of formation of the gametangia, thallus margin shape, number of archegonial neck cells was also observed.

ACKNOWLEDGMENTS

This work was supported by the Project of the Ministry of Science and Technology of the People's Republic of China (2005DKA21006, 2006GH559901) and the Project of Chinese Academy of Sciences (KSCX2-YW-N-52). We thank Aniceto Medoza-Ruiz and Dr. Blanca Perez-Garcia for valuable comments on the manuscript.

LITERATURE CITED

ATKINSON, L. R. 1973. The gametophyte and family relationships. J. Linn. Soc. Bot. 1:73-90.

ATKINSON, L. R. and A. G. STOKEY. 1964. Comparative morphology of the homasporous ferns. Phytomorphology 14:51-70.

CHANDRA, S., M. SRIVASTAVA and R. SRIVASTAVA. 2003. Contribution to the gametophyte morphology of the fern genus Lomagramma J. Sm. in India. Amer. Fern J. 93:25-31.

CHIOU, W. L. and D. R. FARRAR. 1997. Comparative gametophyte morphology of selected species of the family Polypodiaceae. Amer. Fern J. 87:77-86.

CHIOU, W. L., D. R. FARRAR, D. R. and T. A. RANKER. 1998. Gametophyte morphology and reproductive biology in Elaphoglossum Schott. Can. J. Bot. 76:1967-1977.

HOLTTUM, R. E. 1949. The classification of ferns. Biol. Rev. 24:267-296.

NAYAR, B. K. and S. KAUR. 1969. Type of prothallial development in homosporous ferns. Phytomorphology 19:179-188.

NAYAR, B. K. and S. KAUR. 1971. Gametophytes of homosporous ferns. Bot. Rev. 37:295-396.

PEREZ-GARCIA, B. and A. MENDOZA-RUIZ. 2004. A contribution to the gametophyte morphology and development in several species of Thelypteris, Thelypteridaceae. Amer. Fern J. 94:143-154.

PRYER, K. M., A. R. SMITH and J. K. SKOG. 1995. Phylogenetic relationships of extant ferns based on evidence from morphology and rbcL sequences. Amer. Fern J. 85:205-282.

RAINE, C. A., F. J. RUMSEY and E. SHEFFIELD. 1996. The use of gametophytic characters in systematics with specific reference to the Hymenophyllaceae. Pp. 343-345. In Pteridology in Perspective. Edited by J. M. Camus, M. Gibby and R. J. Johns. Royal Botanic Garden, Kew.

SMITH, A. R., K. M. PRYER, E. SCHUETTPELZ, P. KORALL, H. SCHNEIDER and P. G. WOLF. 2006. A classification for extant ferns. Taxon 55:705-731.

STOKEY, A. G. 1951. The contribution by the gametophyte to classification of the homosporous ferns. Phytomorphology 1:39-58.

STOKEY, A. G. 1960. Multicellular and branched hairs on the ferns gametophytes. Amer. Fern J. 50:78-87.

K. M. ZHANG

Institute of Botany, the Chinese Academy of Sciences, Beijing, 100093, China

Graduate University of Chinese Academy of Sciences, Beijing, 100040, China

L. SHI *, X. C. ZHANG, and C. D. JIANG

Institute of Botany, the Chinese Academy of Sciences, Beijing, 100093, China

W. L. TIM-CHUN

Kadoorie Farm & Botanic Garden Corporation, Hong Kong, China

* Corresponding author (telephone: [phone], fax number: 01082593616, e-mail: shilei67@ 263.net).

TABLE 1. Collection data for materials used in current study.

                                   Collection No.
Scientific name   Collector name      and date

P. vittata        W. L. Tim-Chun   141
                                   04/2004

P. finotii        W. L. Tim-Chun   1894
                                   04/2004

P. fauriei        W. L. Tim-Chun   1941
                                   04/2004

P. excelsa        B. D. Liu        151
                                   07/2004

P. wallichiana    B. D. Liu        192
                                   07/2004

P. ensiformis     X. C. Zhang      172
                                   06/2004

Scientific name     Site location           Deposit herbarium

P. vittata        Hongkong, terraces        Herbarium of KFBG
                    near herbarium of
                    Kadoorie Farm &
                    Botanic Garden (KFBG)

P. finotii        Fern Walk at KFBG         Herbarium of KFBG

P. fauriei        Orchid Fall at KFBG       Herbarium of KFBG

P. excelsa        Yunnan, Kunming           Herbarium of
                                              Harbin Normal
                                              University

P. wallichiana    Yunnan, Kunming           Herbarium of
                                              Harbin Normal
                                              University

P. ensiformis     Institute of Botany,      Herbarium of
                    the Chinese Academy       Chinese National
                    of Sciences (IBCAS)       Herbarium,
                                              IBCAS (PE)

                  Spore
Scientific name   numbers

P. vittata        50

P. finotii        51

P. fauriei        50

P. excelsa        50

P. wallichiana    49

P. ensiformis     50

TABLE 2. Developmental stages of the gametophytes of the six species
of Pteris.

Scientific
name                 Spores                     Germination

P. vittata       Trilete, tetrahedral,      Vittaria-type, day
                   brown and possess a        2-3, a rhizoid first
                   distinct equatorial        develops and the
                   flange, (90) 95 (100)      first prothallial cell
                   X (80) 87.5 (90)           divides with a trans-
                   [micro]m                   verse wall

P. finotii       Trilete, tetrahedral,      Vittaria-type, day
                   brown and possess a        4-6, a rhizoid first
                   distinct equatorial        develops and the first
                   flange, (92) 104 (110)     prothallial cell di-
                   X (90) 96 (100)            vides with a trans-
                   [micro]m                   verse wall

P. fauriei       Trilete, tetrahedral,      Vittaria-type, about
                   brown and possess a        one week, a rhizoid
                   distinct equatorial        first develops and the
                   flange, (19) 27.4 (40)     first prothallial cell
                   X (10) 10.3 (12)           divides with a trans-
                   [micro]m                   verse wall

P. excelsa       Trilete, tetrahedral,      Vittaria-type, day
                   brown and possess a        7-11, a rhizoid first
                   distinct equatorial        develops and the first
                   flange, (29) 31 (34)       prothallial cell di-
                   X (20) 24 (29)             vides with a trans-
                   [micro]m                   verse wall

P. wallichiana   Trilete, tetrahedral,      Vittaria-type, about
                   brown and possess a        day 13, a rhizoid first
                   distinct equatorial        develops and the first
                   flange, (44) 50 (56)       prothallial cell di-
                   X (40) 46 (51)             vides with a trans-
                   [micro]m                   verse wall

P. ensiformis    Trilete, tetrahedral,      Vittaria-type, about
                   brown and possess a        one week, a rhizoid
                   distinct equatorial        first develops and the
                   flange, (27) 30 (32)       first prothallial cell
                   X (23) 28 (29)             divides with a trans-
                   [micro]m                   verse wall

Scientific
name                 Filamentous phase          Plate phase

P. vittata       Germinal filaments         Ceratopteris-type,
                   2-19 cells long,           about one week
                   spore coat remains
                   attached

P. finotii       Germinal filaments         Ceratopteris-type,
                   2-12 cells long, spore     10-40 days
                   coat remains attached

P. fauriei       Germinal filaments         Ceratopteris-type,
                   2-7 cells long, spore      7-14 days
                   coat remains attached

P. excelsa       Germinal filaments         Ceratopteris-type,
                   2-22 cells long, spore     about 20 days
                   coat remains attached

P. wallichiana   Germinal filaments         Ceratopteris-type,
                   2-7 cells long, spore      about 36 days
                   coat remains attached

P. ensiformis    Germinal filaments         Ceratopteris-type,
                   2-25 cells long, spore     about 25 days
                   coat remains attached

Scientific
name                 Adult phase          Gametangia

P. vittata       17-50 days, asym-      [female] [male]
                   metrical spatulate
                   to cordiform
                                        20-40 days

P. finotii       about 26 days,         [female] [male]
                   asymmetrical
                   spatulate to
                   cordiform
                                        26-60 days

P. fauriei       about 24 days,         [female] [male]
                   asymmetrical
                   spatulate to
                   cordiform
                                        28-80 days

P. excelsa       about 25 days,         [female] [male]
                   asymmetrical
                   spatulate to
                   cordiform
                                        about 25 days

P. wallichiana   about 43 days,         [female] [male]
                   asymmetrical
                   spatulate to
                   cordiform
                                        about 36 days

P. ensiformis    about 50 days,         [female] [male]
                   asymmetrical
                   spatulate to
                   cordiform
                                        about 90 days