INTRODUCTION

Archangiopteris contains about ten species and belongs

to an ancient family of ferns, the Marattiaceae (Ching,

1958; Tryon and Lugardon, 1991). The genus has been

traced to the Middle Jurassic period in the fossil record

(Hill and Camus, 1986) and is thought to be a lineage

derived from Angiopteris (Ching, 1958; Chang, 1973).

Extant species occur primarily in China, including Yunnan,

Kwangsi, Kwangtung and Hainan, Tonkin and Taiwan,

with high levels of endemism in local floras (Tryon

and Lugardon, 1991). Achangiopteris somai Hayata is

endemic to Taiwan (DeVol and Shieh, 1994) and is listed

as an endangered species (Kuo, 1997). There are two

populations of A. somai: one in Wulai (north Taiwan) and

one in Lienhwachi (central Taiwan). Altogether, less than

1000 individuals survive. To aid conservation of this rare

fern, we studied their reproductive biology, especially the

gametophyte generation.

In the Marattiaceae, gametophytes have been

Botanical Studies (2007) 48: 205-213.

*

Corresponding author: E-mail: chiou@tfri.gov.tw; Tel:

886-2-23039978; Fax: 886-2-23076220.

documented for a number of species, including Angiopteris

evecta (Farmer, 1892; Haupt, 1940), Danaea simplicifolia

(Brebner, 1896), Marattia sambucina (Stokey, 1942),

Marattia douglasii (Campbell, 1894), and Macroglossum

smithii (Stokey, 1942). The gametophytes of these spe-

cies are large, conspicuous

,

and perennial. They are dark

green, relatively thick, and look more like liverworts than

the gametophytes of leptosporangiate ferns. The antheridia

and archegonia are sunken (Nayar and Kaur, 1971).

Endophytic fungi are found in some species (Haupt, 1940).

The shapes of field-collected A. somai gametophytes

and young sporophytes were first described, though

not in great detail, by Sasaki (1928). Yang et al. (2004)

successfully induced spore germination of A. somai

cultured in soil collected from its natural habitat. However,

the spore germination type and detailed information on

gametophyte development and sporophyte formation await

further study.

Ploidy is the primary indicator of the reproductive

mode. Most diploid and tetraploid ferns undergo sexual

reproduction to form new sporophytes, but triploid ferns

usually produce sporophytes apomictically (Moore et

al., 2002; Chiou et al., 2006; Huang et al., 2006). In the

mORphOlOgy

Observations on gametophytes and juvenile sporophytes

of Archangiopteris somai hayata (marattiaceae), an

endangered fern in Taiwan

Hsueh-Mei CHOU

1

, Yao-Moan HUANG

2

, Shau-Lian WONG

3

, Tsung-Hsin HSIEH

4

, Sheng-Yuan

HSU

2

, and Wen-Liang CHIOU

2,

*

1

Department of Biotechnology, Yuanpei University, Hsinchu 300, Taiwan

2

Division of Forest Biology, Taiwan Forestry Research Institute, Taipei 100, Taiwan

3

Division of Interpretation and Education, Endemic Species Research Institute, Nantou 552, Taiwan

4

Department of Biological Science and Technology, National University of Tainan, Tainan 700, Taiwan

(Received February 15, 2006; Accepted August 30, 2006)

ABSTRACT

. Archangiopteris somai is an endemic eusporangiate fern in Taiwan. It is classified as an

endangered species because there are only a few small populations. In culture, spores germinated two weeks

after sowing. Gametophyte development is of the "Marattia" type. Gametophyte shape changed from circular,

to cordate, to branched. Antheridia and archegonia appeared 10 and 12 months, respectively, after spores were

sown. Antheridia occurred in the cushion on the dorsal and ventral surfaces of gametophytes while archego-

nia were observed on the ventral surface only. On hermaphroditic gametophytes, antheridia and archegonia

were usually intermingled. Both types of gametangia were eusporangiate. After 13 months culture, sporo-

phytes began to form. Each gametophyte had several archegonia, and only one sporophyte was produced per

gametophyte. The first sporophyte frond was simple and had pinnate venation. After 3 years of culture, only

3% of the gametophytes produced a sporophyte. It took a minimum of 13 months after a spore sowing for a

sporophyte to be produced by a gametophyte. Increasing the wild population will likely be slow and difficult.

Keywords: Archangiopteris somai; Chromosome; Eusporangiate fern; Gametophyte; Juvenile sporophyte;

Marattiaceae; Spore.

206

Botanical Studies, Vol. 48, 2007

Marattiaceae, sporophytes are either diploid (2n = 78,

80, Li, 1989; Nakato, 1988, 1996) or tetraploid Marattia

(2n = 156 or 160, Manton and Sledge, 1954; Ninan,

1956; Brownlie, 1961; Ghatak, 1962; Walker, 1966). The

chromosome number of Archangiopteris has not been

published previously.

In this study, the sporophyte chromosome number of

A. somai was determined, and the spores, gametophytes,

and young sporophytes were described and imaged. Based

on these findings, recommendations for conserving this

endangered species ex situ are provided.

mATERIAlS AND mEThODS

plant materials

Three mature plants were randomly sampled from

Lienhwachi, Nantou County, Central Taiwan (23¢X54¡¦N,

120¢X54¡¦E, elevation 650-660 m). The root tips of the plants

were collected to observe the number of chromosomes.

Spores were collected from one, mature, spore-bearing

frond from each of these three plants using the method de-

scribed by Huang et al. (2003) and Yang et al. (2004) for

culture.

Culture

To observe the early stages of the gametophyte, spores

were sown directly on a membrane filter (pore size 0.45

£gm, 47 mm in diameter, Gelman Laboratory) lying on

soil collected from the ferns¡¦ native habitat. The num-

ber of gametophytes cultured from the spores of each

plant was counted once a month. After 6 months culture,

gametophytes were transferred from the filter to a new

medium containing soil from the native habitat. Ten

gametophytes cultured for 12 months were collected to

observe the development of gametangia. All cultures were

maintained under white fluorescent illumination at about

24 £gmole m

-2

s

-1

, 12 h/d. Temperature ranged from 20 to 28

¢XC.

light microscopy

The root tips of three plants were pretreated for 3-4 h

with a mixture of 70 ppm cycloheximide and 250 ppm

8-hydroxyquinoline (1:1) at about 18-20¢XC. These root tips

were fixed for 1-3 h in a mixture of 45% acetic acid and

absolute ethanol (1:3) at about 20¢XC, and preserved in 70%

ethanol at 4¢XC. Then they were macerated for 1-3 min in 1

N HCl at 60¢XC, washed for 10 s and digested for 1-2 h in

4% pectinase. Finally, the root tips were squashed in modi-

fied Sharma¡¦s solution (Sharma, 1982). The chromosomes

were counted using Leica Wild M8 and Leitz Dialux 20

light microscopes (Wetzlar, Germany), and they were

photographed with a Nikon Coolpix 995 digital camera

(Tokyo, Japan). Moreover, gametophytes were processed

through fixation (in FAA, 37% formalin: 99% propionic

acid : glycerin : 95% ethanol : ddH

2

O v/v = 5 : 5 : 15 : 35 :

40, 2 days), dehydration (in an ethanol-acetone series), and

were then embedded in Spurr¡¦s resin (Spurr, 1969). Semi-

thin sections (1 £gm) were made using an Leica Ultracut

R ultramicrotome (Wien, Austria) and were stained with

0.5% toluidine blue O in Ca

2

CO

3

buffer, pH 11.1. Sections

were imaged with a Leitz Dialux 20 light microscopes

(Wetzlar, Germany). The outline of each new frond and

venation was observed and imaged. The cumulative rate

of sporophyte production during the three years of culture

was calculated.

Scanning electron microscopy

To observe the fine external feature of spores, air-

dried spores were mounted on double-stick mending tape,

coated with gold, and imaged with a Hitachi Scanning

Electron Microscope S2400 (Tokyo, Japan). Further, the

gametangia of gametophytes were processed through

fixation in FAA for 2 days, dehydration (in an ethanol-

acetone series), critical point drying, being coated with

gold and imaged with the same Scanning Electron Micro-

scope described above.

RESUlTS

Chromosome number of sporophytes

Assuming the basic chromosome number is 40, all A.

somai were tetraploid (2n = 4x = 160) (Figure 1).

External feature of spore

The spores were globose in shape, pale to yellowish-tan

in color, and had dense unbranched spines. The proximal

surface was concave and lacked an arm (Figure 2A-B).

Spore germination and gametophyte

development

After one week of spores sowed, the spore cell

synthesized numerous chloroplasts and became distinctly

green before the spore coat ruptured (Figure 2C). In two

weeks culture, the first unicellular rhizoid formed from the

basal cell (Figure 2D). The first gametophyte cell began to

form after three weeks culture, and by four weeks it had

grown into a round plate with four cells (Figure 2E). The

shape of the gametophyte changed gradually from round

to spatulate (Figure 2E-H). After five months culture, the

gametophyte was cordate.

mature gametophyte

Mature gametophytes of A. somai were dark-green,

naked, and more or less cordate (Figure 3A). The midrib

of the gametophyte was very thick and protruded strongly

from the ventral surface. Unicellular white rhizoids, less

than 5 mm long, appeared only on the ventral side of the

midrib. The margin of the wings was only one cell thick

while other parts of the wings were two or more cells

thick. Because both the midrib and the wings are several

cells thick where they join, the boundary between them is

vague. Gametophytes grew vigorously for 3 years. Game-

tophytes elongated with time (Figure 3B), and produced

CHOU et al. ¡V Gametophytes and sporophytes of

Archangiopteris somai

207

several branches, each with a meristem, gametangia, and

rhizoids. Branches varied in shape from cordate to oblong

strap-like (Figure 3C, D).

Gametophytes were potentially monoecious. An-

theridia formed two months earlier than archegonia.

Both gametangia were completely embedded in the

gametophyte tissue. Some of the gametophytes that grew

vigorously only produced archegonia while some that

grew slowly only produced antheridia. On hermaphroditic

gametophytes, antheridia and archegonia usually intermin-

gled with each other on the ventral surfaces (Figure 3E,

F). Antheridia and archegonia were distinguished by their

triangular or rectangular apertures, respectively (Figure

3G-H).

The first antheridia formed on 10-month-old

gametophytes, mainly on the midrib behind the apical

meristem with a smaller number forming on the wings.

The development of the antheridia resembled that of other

Marattiaceae (Nayar and Kaur, 1971). At first, a superficial,

initial cell underwent periclinal division to form an outer

primary wall cell and an inner primary spermatogeneous

cell. The primary wall cell then underwent three anticli-

nal divisions, which formed a triangular cover cell in the

central position. The inner primary spermatogeneous cell

underwent a series of mitotic divisions that eventually pro-

duced a large number of sperm (Figure 4A). Finally, sperm

were liberated from the antheridium when the cover cell

opened (Figure 4B).

In one-year of culture, the superficial cell divided into

an outer primary neck cell and an inner cell on the ventral

midrib (Figure 4C). The inner cell divided periclinally

to form a ventral cell and a neck canal cell. Soon, the

ventral cell formed the ventral canal cell and egg. This was

followed by division of the nucleus of the neck canal cell

to form two nuclei that were not separated by a wall. The

primary neck cell formed four neck cells by two succes-

sive anticlinal divisions at right angles to each other. The

neck cells increased in number, forming three tiers of four

cells each (Figure 4D). Fertilization was followed by the

breakdown of the ventral canal cell, neck canal cell, and

neck cells (Figure 4E, F). As soon as fertilization occurred,

an embryo began to develop. Although many archegonia

formed on each gametophyte, only one archegonium

produced the new sporophyte (Figure 5C).

Juvenile sporophyte

After 13 months from spore sown, the embryo

protruded from the dorsal surface of the gametophyte

(Figure 5A, B) and formed its first frond. At the same time,

the first root appeared on the ventral surface. The first

frond was simple and had a midrib and a serrated margin

(Figure 5C). The first four fronds were similar in shape

and venation. The fifth frond was pinnatifid. The sixth and

all later fronds were pinnate (Figure 5D-L). During this

three-year

-

study, female/hermaphroditic gametophytes

totaled 1419, but only 3% (42/1419) of the gametophytes

produced a sporophyte.

In summary, this observation of the culturing life cycle

of A. somai included the spore germination, antheridium

Figure 1. Chromosomes of a root tip cell of A. somai. Bar = 10 £gm.

208

Botanical Studies, Vol. 48, 2007

Figure 2. External feature of spores and young gametophytes of A. somai. A, Spore lateral view; B, Spore proximal view; C, Spore

germination; D, The first rhizoid; E-F, Circular gametophytes; G-H, Spatulate gametophytes. A-D, Bars = 10 £gm; E, Bar = 30 £gm; F-H,

Bars = 40 £gm.

CHOU et al. ¡V Gametophytes and sporophytes of

Archangiopteris somai

209

and archegonium formation, and young sporophyte

production. Those events occurred 2 weeks, 10 months, 12

months, and 13 months, respectively, after the spore had

been sown.

DISCUSSION

Cytogenetic studies have found that the sporophytes of

Marattiaceae, including Angiopteris, Marattia, Danaea,

are either diploid (2n = 78, 80, Li, 1989; Nakato, 1988,

1996) or tetraploid (2n = 156 or 160, Manton and Sledge,

1954; Ninan, 1956; Brownlie, 1961; Ghatak, 1962; Walker,

1966). The chromosome number of A. somai, 2n = 160, is

the first number reported for this genus.

Previously, some taxonomists considered A. somai a

variety of A. henryi, which grows in Yunnan, southern

China (Wu and Ching, 1991). Other taxonomists treated

A. somai as a separate species (Chang, 1973; DeVol, 1975;

Figure 3. Mature gametophytes in different shapes. A, Cordate; B, Elongate cordate; C-D, Branched. Bars = 1 cm; E, Ventral view of

hermaphroditic gametophyte. Bar = 1 mm; F, Enlarged view of the midrib near the notch. An: antheridium, Ar: archegonium. Bars = 1

mm; G, Antheridium; H, Archegonium. Bars = 50 £gm.

210

Botanical Studies, Vol. 48, 2007

DeVol and Shieh, 1994). Based on characteristics of the

exine, Zhang et al. (1990) divided A. somai and A. henryi

into two separate species. The exine of A. henryi spores

bears branched spines (Hill and Camus, 1986). In this

study, we found that the exine of A. somai spores bore

unbranched spines only.

The shape of marattiaceous gametophytes is

quite uniform (Stokey, 1942). They differ from the

gametophytes of leptosporangiate ferns by having a

distinctly more massive body (Campbell, 1894). The

development of A. somai gametophytes (marattia-type)

and young sporophytes (midribbed-type) is almost

identical to that of other species of Marattiaceae (Wagner,

1952; Nayar and Kaur, 1971). Gametophytes are evidently

long-lived and can achieve considerable sizes by forming

multiple branches, a common phenomenon in the

Figure 4. Longitudinal sections of A. somai antheridia and archegonia. A, Sperm in antheridium; B, Sperm have been released. Bars

= 50 £gm; C-F, Developing archegonia; E, A mature archegonium with egg; F, A mature archegonium, neck cells open. NC = neck cell;

NCC= neck canal cell; PNC = primary neck cell; VC = ventral cell; VCC= ventral canal cell. Bars = 50 £gm.

CHOU et al. ¡V Gametophytes and sporophytes of

Archangiopteris somai

211

Figure 5. Developing A. somai sporophytes. A, An embryo (dashed loop) embedded in the gametophyte; B, An embryo (dashed loop)

protruding from the dorsal side of a gametophyte. Bars = 200 £gm; C, A new sporophyte arising from the dorsal side of gametophyte;

D, A young sporophyte with its first four fronds. Bars = 1 cm; E-F, First frond; G, Second frond; H, Third

frond; I-J, Fourth fronds; K,

Fifth frond; L, Sixth frond. Bar = 5 cm.

212

Botanical Studies, Vol. 48, 2007

Marattiaceae (Campbell, 1914). Endophytic fungi have

been found within gametophytes of some Marattiaceae,

such as Angiopteris evecta (Haupt, 1940), but none was

found in A. somai during this study.

Most ferns have young leaves that lack midribs. Howev-

er, ferns in the Ophioglossaceae and Gleicheniaceae, some

in the Marattiaceae, and ferns in the genus Nephrolepis

have midribs in juvenile leaf-blades (Wagner, 1952). A.

somai also has leaves with midribs.

Some species of Marattiaceae, such as Marattia

sambucina, are apomictic (Stokey, 1942). However, in

this study, all A. somai sporophytes resulted from sexual

reproduction. Only 3% of the gametophytes produced

sporophytes. It may be that A. somai has a high genetic

load that limits reproduction and contributes to its

endangered status (e. g., Peck et al., 1990). However, the

low reproduction observed in this study may also be due

to the culture condition being ill-suited to the sporophyte

formation of A. somai.

The best strategy for the long-term protection of a

rare and endangered species is the preservation of natural

communities and populations in the wilds, known as in

situ preservation (Primack, 2004). However, if the last

surviving populations are in continuous decline, and the

original habitats are not protected, as is the case with A.

somai, the only way to prevent extinction is to maintain

individuals in culture (Primack, 2004). Sowing spores and

growing gametophytes yielded few sporophytes (3%).

However, a single A. somai plant may produce billions of

viable spores every year, so it should be possible to obtain

numerous sporophytes. Sporophytes grown in culture

can be released periodically into the wild to augment the

original population and maintain the existence of this rare

species.

Acknowledgements. The authors thank Mr. Shih-Min

Yang for taking photographs (Figure 2), and Dr. Alan

Warneke for editing assistance. This research was partly

supported by the National Science Council of Taiwan

(NSC 91-2313-B-003-004) and Taiwan Forestry Research

Institute.

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