Annals of Botany 104: 1353– 1361, 2009
doi:10.1093/aob/mcp245, available online at www.aob.oxfordjournals.org
Developmental morphology of strap-shaped gametophytes of Colysis decurrens: a
new look at meristem development and function in fern gametophytes
Naoko Takahashi, Mie Hashino†, Chieko Kami and Ryoko Imaichi*
Department of Chemical and Biological Sciences, Japan Women’s University, 2-8-1 Mejirodai, Tokyo 112-8681, Japan
Received: 12 May 2009 Returned for revision: 13 July 2009 Accepted: 26 August 2009 Published electronically: 6 October 2009
† Background and Aims The gametophytes of most homosporous ferns are cordate–thalloid in shape. Some are
strap- or ribbon-shaped and have been assumed to have evolved from terrestrial cordate shapes as an adaptation to
epiphytic habitats. The aim of the present study was to clarify the morphological evolution of the strap-shaped
gametophyte of microsoroids (Polypodiaceae) by precise analysis of their development.
† Methods Spores of Colysis decurrens collected in Kagoshima, Japan, were cultured and observed microscopically. Epi-illuminated micrographs of growing gametophytes were captured every 24 h, allowing analysis of the
cell lineage of meristems. Light microscopy of resin-sections and scanning electron microscopy were also used.
† Key Results Contrary to previous assumptions that strap-shaped Colysis gametophytes have no organized meristem, three different types of meristems are formed during development: (1) apical-cell based – responsible for
early growth; (2) marginal – further growth, including gametophyte branching; and (3) multicellular – formation
of cushions with archegonia. The cushion is two or three layers thick and intermittent. The apical-cell and multicellular meristems are similar to those of cordate gametophytes of other ferns, but the marginal meristem is
unique to the strap-shaped gametophyte of this fern.
† Conclusions The strap-shaped gametophytes of C. decurrens may have evolved from ancestors with a cordate
shape by insertion of the marginal meristem phase between the first apical-cell-based meristem and subsequent
multicellular meristem phases. Repeated retrieval of the marginal meristem at the multicellular meristem phase
would result in indefinite prolongation of gametophyte growth, an ecological adaptation to epiphytic habitats.
Key words: Apical cell, apical meristem, Colysis decurrens, cushion, development, evolution, ferns,
gametophytes, gametophyte adaptation, marginal meristem.
IN T RO DU C T IO N
Pteridophytes are characterized by having two independent
plant bodies, sporophytes and gametophytes. It is plausible
that both sporophytes and gametophytes have been subjected
to similar environmental conditions and then adapted to the
habitats where they grow (Watkins et al., 2007). As discussed
by Dassler and Farrar (1997), data on gametophytes are critical
in understanding pteridophyte reproduction, distribution,
ecology and evolution, but nevertheless adaptive morphology
in gametophytes has been largely ignored by researchers
until recently.
Although gametophytes of homosporous ferns are generally
cordate-thalloid with a midrib (cushion), some gametophytes
are strap- or ribbon-shaped or filamentous (Bower, 1923;
Orth, 1936; Nayar and Kaur, 1971). Strap- and ribbonshaped gametophytes are found in members of the
Hymenophyllaceae, vittarioids and Polypodiaceae, suggesting
parallel evolution in different clades of ferns as an adaptation
to epiphytic habitats (Farrar et al., 2008). Therefore, the study
of the evolution of gametophytes adapted to epiphytic habitats
may provide useful data for a better understanding of the adaptive evolution of fern sporophytes.
Nayar and Kaur (1969) claimed that strap- and ribbonshaped gametophytes have no organized meristems,
†
Present address: Midorimachi Junior High school in Chiba, 2-3-1
Midorimachi, Chiba 263-0023, Japan.
* For correspondence. E-mail [email protected]
unlike ordinary cordiform gametophytes, but their conclusion was based on a number of gametophytes fixed at
different developmental stages, not from long-term observations of the same gametophyte. Cell lineage analyses
focusing on meristem behaviour in growing gametophytes
are needed to clarify the development and evolution of
gametophytes. In the present study, a new sequential observation technique with epi-illuminated micrographs of
gametophytes growing in culture was used, permitting
precise cell lineage analysis.
Microsoroids, which are one of four clades of Polypodiaceae
(Schneider et al., 2004), produce various shaped gametophytes
depending on genera or species: strap-shaped (Colysis, Nayar,
1962), ribbon-shaped (Leptochilus and Paraleptochilus, Nayar
1963b), and cordiform or elongate cordiform gametophytes
(Microsorium, Nayar, 1963a; Microsorum (Microsorium), Pal
and Pal, 1962). Microsoroids grow in epilithic (rock surfaces),
epiphytic and also terrestrial habitats (Hennipman et al.,
2000). Thus, microsoroids have a key role to play in clarifying
the evolution of adaptive morphologies of gametophytes in
relation to habitat preferences. The aim of the present study
was to reveal the evolutionary course of strap-shaped gametophytes in microsoroids. Colysis decurrens grows in both epilithic and terrestrial habitats, and has strap-shaped
gametophytes (Momose, 1967). The development of
C. decurrens gametophytes was examined in comparison
with cordiform gametophytes of other ferns.
# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: [email protected]
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Takahashi et al. —Development of Colysis strap-shaped gametophytes
M AT E R IA L S A ND M E T HO DS
Spores of Colysis decurrens (Wall. ex Hook. & Grev.) Nakaike
(syn. Colysis pothifolia (Hamilt. Ex D. Don) Presl) were collected in June, 2003 in Sumiyo-cho, Amamishi City,
Kagoshima, Japan (RI-506), and in September, 2008 in
Kinkocho, Minami-osumi, Kagoshima (KF-19). Voucher
specimens are deposited in the herbarium of the University
of Tokyo (TI).
For sterilization, spores were immersed in 0.5 % sodium
hypochloride for 5 min and rinsed with sterilized water. The
sterilized spores were sown on 1 % solidified agar medium
with Parker and Thompson’s micronutrients (Klekowski,
1969). Cultures were grown under continuous, white, fluorescent illumination of 20 mmol m22 s21 at 24 + 1 8C.
The morphology of growing gametophytes was recorded by
sequential observation in which microscopic images of
growing gametophytes were captured using a metallurgical
microscope (Nikon-OPTIPHOT-2, Nikon Co., Tokyo, Japan)
with epi-illumination roughly every 24 h. Images at several
focal points of a gametophyte were synthesized into a clear
image using All-in-Focus DM-99i II (Synoptics Ltd,
Cambridge, UK) and Photoshop CS (Adobe Systems Inc.,
San Jose, CA, USA). Comparison of one image with the preceding image allowed tracing of cell lineages. Thirty-seven
individuals at early developmental stages, i.e. vegetative
stage, and around 20 individuals at later developmental
stages were observed in detail. There was no difference in
the development of gametophytes grown from spores collected
from the two localities mentioned above.
For scanning electron microscopy (SEM) observation,
gametophytes were fixed overnight with FAA (formalin –
acetic acid– 50 % ethanol, 5 : 5 : 90, v/v). The fixed materials
were dehydrated through a graded ethanol series, critical-point
dried and coated with platinum – palladium. Observations were
made using a Keyence VE-8800 (Keyence Corp., Osaka,
Japan) at 5 kV.
For anatomical observations, materials fixed with FAA were
dehydrated through a graded ethanol series, embedded in
Historesin (glycol methacrylate, Leica, Heidelberg,
Germany), cut into 2-mm-thick sections, and stained with
modified Sharman’s solution (Jernstedt et al., 1992). To
compare cell size in or between meristems of gametophytes
at different developmental stages, the length and thickness of
six cells (outermost and five inner cells behind) were measured
in three serial longitudinal sections (100 mm apart) of a gametophyte. Five and ten individuals, respectively, were used for
measurement of cell size at the marginal and cushion meristem
phases. Another individual with a newly initiating cushion
meristem was used for cell size measurement.
similarly by the secondary wall (wall II) at right angles to
wall I, resulting in an anterior half composed of four cells,
each of which is a quadrant (data not shown). In contrast,
the posterior half of the filament does not divide further, but
forms rhizoids (Fig. 1C, D). The terminal two quadrants,
which are side-by-side, each undergo periclinal (horizontal)
and anticlinal (vertical) divisions by wall III and IV, producing
triangular cells in both lateral sides of the anterior half
(Fig. 1C – F).
The right and left halves separated by wall II in the anterior
semicircular plate (Fig. 1E, F) soon become different in shape
and overall size. In Fig. 1E and F, the half demarcated by walls
I and II has slightly more cells than the other half, involving
the triangular cell with its own derivative cell. The triangular
cell (a) in one half continues to function as an apical cell,
dividing alternately at two lateral walls to produce derivative
cells that undergo periclinal and anticlinal divisions to form
rectangular cell packets (merophytes) (Fig. 1G – J). In contrast,
the other triangular cell in the other half soon ceases division
and its surrounding cells divide much less frequently
(Fig. 1G – J). After this stage, gametophyte growth is mainly
from the single apical cell and to merophytes derived from
it, and the gametophyte becomes ovobate in form (Fig. 1K, L).
Either one of the two triangular cells can become the apical
cell. However, sometimes (six of 37 individuals investigated
here) neither triangular cell differentiates into the apical cell,
producing a few derivative cells (Fig. 1O –R). Instead, one
of the rectangular surface cells between the triangular cells
becomes the apical cell through two continuous oblique divisions (Fig. 1Q– T). In this case, cells demarcated by walls II
and IV contribute to most of the gametophyte.
Growing gametophytes are inclined at around 458 from the
original long axis of the filamentous gametophyte due to
activity of the apical cell formed at the lateral side of the
anterior part of the young gametophyte (Fig. 1I, J). This
type of development occurred in 15 of 37 individuals but
not in 16 others where wall I in the anterior half is oblique
and the just-formed apical cell is at the apex of the growing
gametophyte rather than the lateral side (Fig. 1U, V). The triangular cell, which fails to become the apical cell, produces no
derivative cells (Fig. 1X). In this case, the gametophyte grows
vertically along the original long axis of the filamentous gametophyte (Fig. 1W, X).
Notably and unlike other homosporous-fern gametophytes, young gametophytes of Colysis do not become
cordate but are instead ovobate with no apparent notch, i.e.
a depressed portion. The ovobate shape may be due to merophytes surrounding the apical cell. In Colysis gametophytes, anticlinal and periclinal divisions occur regularly
so each merophyte keeps pace to maintain a rectangular
outline (Fig. 1K – N).
RES ULT S
Early development of gametophytes with apical-cell-based
meristems
About 10 d after spore sowing (DAS), a filament about four
cells long forms as a result of cell division by primary walls
(wall I), which are perpendicular to the long axis of the filament (Fig. 1A, B). The terminal and sub-terminal cells of
these four cells of the filamentous gametophyte divide
Loss of apical cells and further growth by marginal meristems
At around 40 DAS, the apical cell that produced five or six
(nine in one extreme case) derivative cells stops dividing at
two lateral walls, and instead undergoes periclinal division
to create an outer (surface) rectangular and an inner equilateral
triangular cell (Fig. 1M, N). By this stage, the apical cell
seems to have ceased functioning as the initial cell. Both the
Takahashi et al. —Development of Colysis strap-shaped gametophytes
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F I G . 1 Early stages of gametophyte development of Colysis decurrens. Epi-illuminated micrographs (A, C, E, G, I, K, M, O, Q, S, U, W) and their line drawings
(B, D, F, H, J, L, N, P, R, T, V, X). Numerals at the upper left corners indicate days after spore sowing (DAS). In the line drawings, triangular cells in red
are apical cells, those in grey are non-apical cells, and areas with different colours each show merophytes derived from the apical cell. I–IV show walls
formed by first to fourth cell divisions. (A– N) Different developmental stages of the same gametophyte. Merophytes are numbered from the oldest to the youngest (1, 2, . . . ). (O– T) Different stages of the same gametophyte with unusual apical cell formation. (U–X). Different stages of the same gametophyte with apical
cell at the summit, due to strong tilting of one of the primary walls (I). a, apical cell. Scale bars ¼ 50 mm.
outer rectangular and the inner triangular cells continue dividing in the periclinal and anticlinal planes to form a large merophyte maintaining an overall triangular shape (Fig. 2A – D).
Other merophytes surrounding the triangular merophyte also
expand in area due to an increase in cell number and cell
expansion, contributing to gametophyte growth (Fig. 2D).
Growing gametophytes appear to have no growth centre after
losing their apical cell.
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Takahashi et al. —Development of Colysis strap-shaped gametophytes
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F I G . 2 Gametophyte development after loss of the apical-cell-based meristem of Colysis decurrens. Epi-illuminated micrographs (A, C, F, H), micrograph taken
under transmitted light (E) and line drawings (B, D, G, I). Numerals at the upper left corners indicate DAS. Areas with different colours in line drawings show
merophytes derived from cells constituting the anterior marginal area at a given time. (A–D) Images of the same gametophyte at two stages: one just after apical
cell disappearance (A, B), and the next with initiating marginal meristem (arrowhead, C, D). Triangular cell packets in red indicate apical cell-derived merophyte.
(E –I) Different stages of the same gametophyte with a marginal meristem. (F) is close-up of the anterior end in (E). Asterisks in some basal cells in I indicate
fully differentiated cells that have ceased division. Scale bars: (A, C, F, H ) ¼ 100 mm; (E) ¼ 500 mm.
Takahashi et al. —Development of Colysis strap-shaped gametophytes
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F I G . 3 Gametophytes with marginal meristems of Colysis decurrens. Numerals at the upper left corners indicate DAS. (A– C) Micrographs of the same gametophyte undergoing branching by cessation of marginal meristem growth in the centre (arrowhead). (D, E) Micrograph of a gametophyte (D) and its median
longitudinal section (E). Both arrows indicate length measured for each cell. (F, G) Gametophyte with newly initiated antheridia. (G) is enlargement of (F)
at arrowhead. an, antheridium; rh, rhizoid. Scale bars: (A– C, F) ¼ 500 mm; (D, E, G) ¼ 100 mm.
After a short period of growth with no apical-cell-based
meristem, a new meristematic area is distinguishable in
or next to the apical-cell-derived triangular merophyte
(Fig. 2C, D). This marginal meristem enlarges to cover the
anterior margin of the gametophyte (Fig. 2E, F). It is composed
of rectangular cells with outermost (marginal) longest cells
(31.0 + 5.1 mm) and inner shorter cells behind them (25.3 +
5.3 mm; Figs 2F, H and 3E). Cell lineage analyses of growing
gametophytes shows that both the outermost and the inner
cells in the meristem undergo cell division in the periclinal
and anticlinal planes to form rectangular cell packets (merophytes) with larger merophytes near the margin (Fig. 2G, I).
However, even at the margin, no merophytes are substantially
larger than others (Fig. 2I), indicating that there is no growth
centre with a much higher division rate in the meristem. This behaviour is similar to that in young leaf laminae, and such the
structure is described here as a marginal meristem.
The marginal meristem expands further in area through
growth, but it does not cover the gametophyte margin entirely.
Cells in the proximal periphery of the marginal meristem stop
dividing and become fully differentiated and remain as an
inactive posterior portion of the thallus (Fig. 2H, I).
Therefore, young gametophytes with marginal meristems
first become ovobate (Fig. 3A), elongate ovobate (Fig. 2E)
and then strap-shaped (Fig. 4A). At the marginal meristem
stage, rhizoids are not restricted to the posterior region, but
arise from cells at the inner parts of the gametophytes
(Fig. 3B, F).
Gametophytes often branch terminally by division of the
marginal meristem. Occasionally when the marginal meristem
extends in area, meristematic activity stops in the middle of the
meristem (Fig. 3B). In these cases, both meristematic areas
separated by intervening differentiated cells maintain meristematic activity and grow further to give rise to independent
lobes (Fig. 3C). Finally, the gametophyte branches in two.
Branched lobes grow equally or unequally depending on the
individual.
Mature gametophytes
At around 50 DAS, antheridia begin forming from the relatively basal inner portion of the gametophytes, which are one
cell thick and still at the marginal meristem stage (Fig. 3F, G).
In better developed gametophytes, antheridia are scattered in
the middle along the long axis of the gametophyte. When
the gametophyte starts forming archegonia, functional antheridia, when present, are restricted to the posterior portion of the
thallus. No antheridia were observed on any branched gametophytes during the course of the study.
The archegonia form first at 55– 68 DAS for non-branched
gametophytes (observations from seven individuals) and 99–
105 DAS for branched gametophytes (two individuals) from
a small cushion two layers thick behind the anterior margin
(Fig. 4A – E). As the gametophyte grows further, the small
cushion becomes extended and narrowly elongated, forming
more archegonia in vertical lines (Fig. 4J). Concomitantly,
the gametophyte anterior margin shape changes from round
(Fig. 4A) to flat (Fig. 4K), and finally becomes slightly
depressed, forming a very shallow notch (Fig. 4F, G, J). In
the present culture condition, archegonia arose at both upper
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Takahashi et al. —Development of Colysis strap-shaped gametophytes
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F I G . 4 Mature Colysis decurrens gametophytes with cushion meristems. All gametophytes (A, F, G, J, K, M) are well-grown branched lobes that have been
produced by terminal branching of the original gametophyte (not shown). Micrographs taken with transmitted light (A, F, G, K, M), epi-illumination (B, H,
L, N), SEM (J) and longitudinal sections (C– E, I). (A –E) Gametophyte with newly initiated cushion at almost 100 DAS. A part of the anterior end (arrowhead)
in (A) is enlarged in (B). Arrows (left, middle and right) in (B) indicate three sites of longitudinal sections shown in (C), (D) and (E), respectively. The right side
of (C– E) is the dorsal side of gametophyte. (F) Gametophyte with developed cushion. (G– I) Gametophyte with better-developed cushion. (H) shows an enlarged
image of cushion meristem of (G). (I) is a median longitudinal section of (G). Inset to (I) is an enlarged image to show paradermal cell division (arrows) parallel
to substratum. (J) SEM of gametophyte with shallow notch region, below which archegonia are arranged in line. (K, L) Gametophyte with intermittent cushion.
(L) is enlarged image of the cushion meristem in (K). Inset to (L) is close-up of the middle outer region (arrowhead). Right and left arrows in the inset indicate
anticlinal and unequally periclinal divisions, respectively. (M, N) Gametophyte with two new cushion meristems (arrowheads) after long cessation of cushion
formation. (N) is an enlarged image of the cushion meristem. ar, archegonium; ch, cushion. Scale bars: (A, F, G, K, M) ¼ 500 mm; (B–E, H–J, L, N) ¼ 100 mm;
insets to (I) and (L) ¼ 50 mm.
(dorsal) and lower (ventral) surfaces of the cushion two or
three cells thick (Fig. 4I).
Notably, no matter how large the gametophyte becomes, the
distance between the anterior margin and cushion starting
point, i.e. first paradermal division ( parallel to the substratum),
remains nearly the same, i.e. about ten cells behind the anterior
margin (Fig. 4D, I). This indicates that the cushion is formed
by the activity of a meristem that occupies the region anterior
to the cushion (Fig. 4H). In this context, this meristem is here
called the cushion meristem.
Takahashi et al. —Development of Colysis strap-shaped gametophytes
Young cushion meristems are constructed of vertical cell
files that are clearly recognizable probably due to recent frequent divisions in the periclinal plane (Fig. 4L). Close-up
views of the meristem show that somewhat longer outermost
cells divide both in an unequal periclinal plane to produce
small cells proximally and slightly longer cells distally, as
well as in an anticlinal plane to produce long outermost cells
horizontally (inset to Fig. 4L). In mature cushion meristems,
the marginal and submarginal cells undergo anticlinal cell divisions more frequently than inner cells, so the number of cell
files increases in the horizontal direction near the anterior
margin (Fig. 4H). As a result, the mature cushion meristem
often becomes triangular in outline (Fig. 4H). The cushion
meristem sometimes produces cells rapidly in the horizontal
(lateral) and vertical direction, so growth is out of line with
the neighbouring area. In this case, the cushion meristem
forms either a slightly protruding anterior margin (Fig. 4N)
or an enlarged part growing away from the substrate (agar)
surface (data not shown).
A gametophyte with a newly initiating cushion meristem
has the round anterior margin typical of the marginal meristem
stage as mentioned above (Fig. 4A), and there are no gametophytes with both round marginal and cushion meristems near
each other. These facts suggest that the cushion meristem
may be initiated through changes in the marginal meristem
that modify its shape and organization. To clarify the initiation
of the cushion meristem, the typical cushion and marginal
meristems were first compared morphometrically (for ten
and five individuals, respectively) using median longitudinal
sections: (1) the thickness of the inner cells is almost identical
at 45.2 + 3.5 vs. 42.6 + 3.0 mm for the cushion vs. marginal
meristems, respectively; (2) the outermost cells are similarly
longer than inner cells (compare Fig. 2H with Fig. 4L);
(3) the inner cells of the cushion meristem are significantly
shorter than those of the marginal meristem (19.0 + 3.7 vs.
25.3 + 5.3 mm, respectively, Student’s t-test P , 0.05), while
outermost cells are not (28.0 + 3.0 vs. 31.0 + 5.1 mm); and
(4) the length ratio of the outermost to inner cells is larger
in the cushion (28.0/19.0 mm ¼ 1.47) than in the marginal
meristem (31.0/25.3 mm ¼ 1.23). In summary, the cushion
meristem can be distinguished from the marginal meristem
by larger length ratios of the outermost to inner cells.
Next, we examined a cushion meristem that was just initiating and was not yet arranged in clear cell files for one individual (Fig. 4A – E). Two serial longitudinal sections of the
meristem show that the inner cells (15.5 + 3.1 mm long,
Fig. 4D, and 12.5 + 5.4 mm long, Fig. 4E) anterior to the
cushion are more similar in size to those of the typical
cushion meristem (19.0 + 3.7 mm long, n ¼ 10) than to the
marginal meristem (25.3 + 5.3 mm long, n ¼ 5). In contrast,
inner cells in both lateral sides of the initiating cushion meristem are longer (21.8 + 5.2 mm long, Fig. 4C) and comparable
with cells of the marginal meristems (25.3 + 5.3 mm long, n ¼
5). The length ratio of the outermost to inner cells is 1.01
(22.0/21.8 mm), 1.08 (16.9/15.7 mm) and 1.53 (21.4/
14.0 mm) in Fig. 4C, D and E, respectively. This ratio 1.01
(Fig. 4C) is similar to the length ratio of the outermost to
inner cells of the marginal meristem (31.0/25.3 mm ¼ 1.23),
and the ratio 1.53 (Fig. 4E) is similar to that of the cushion
meristem (28.0/19.0 mm ¼ 1.47), supporting the proposal
1359
that the cushion meristem may have been established within
the marginal meristem by changes in frequency and orientation
of cell divisions.
In Colysis gametophytes, the cushion is usually formed in
line but intermittently with intervening one-cell-thick regions
(Fig. 4K, M). A gametophyte in Fig. 4K has just begun to
initiate the archegonia after a long interruption of cushion formation. Another gametophyte (Fig. 4M) bears two cushion
meristems with newly initiated archegonia of their own
(Fig. 4N), with the single cushion on the basal common part
of the gametophyte. There are no gametophytes with cushion
meristems not accompanied by cushions. As shown in
Fig. 3A– C, the gametophyte branches only when elongating
behind a typical, rounded marginal meristem, and hence the
gametophyte in Fig. 4M with a long extension and no
cushion is assumed to have gained its marginal meristem
after ceasing cushion formation. It seems likely that marginal
and cushion meristems appear repeatedly alternately in a
growing strap-shaped gametophyte of C. decurrens.
DISCUSSION
Three distinct meristems in Colysis gametophytes
Strap-shaped gametophytes of Colysis, Leptochilus and
Paraleptochilus have been described as exhibiting the
Kaulina type of development (Nayar, 1962, 1963b; Pal and
Pal, 1962; Nayar and Kaur, 1971), in which neither apical
cell nor organized meristems ( pluricellular meristem) are
ever established (Nayar and Kaur, 1969). Contrary to this
idea, our cell-lineage analysis of C. decurrens clearly shows
that both apical-cell-based meristems and organized meristems
are produced in gametophytes.
The apical cell cuts off several derivatives as the single
initial cell and then disappears by periclinal division. The
number of derivatives formed by the apical cell before its disappearance (5 – 9 in C. decurrens) is similar to that of other
cordiform gametophytes (6– 8 in Lygodium japonicum by
N. Takahashi, five and six in Osmunda japonica by
A. Inoue, unpubl. data). Our preliminary studies show that
strap- or ribbon-shaped gametophytes in vittarioids and some
genera of Polypodiaceae similarly have single apical cells
when young, suggesting that the apical cell is necessary for
early growth of fern gametophytes regardless of gametophyte
morphology.
In Colysis, an organized multicellular meristem, described
here as a cushion meristem, arises later at the reproductive
stage. This meristem shows an organization similar to a multicellular meristem ( pluricellular meristem, sensu Nayar and
Kaur, 1971) located in the notch of cordiform gametophytes:
a row of several narrow initial cells at the anterior end
elongates parallel to the long axis of the gametophyte and
actively divides at walls parallel to either lateral or basal
walls. Furthermore, the Colysis cushion meristem shares
another unique character with the multicellular meristem of
cordiform gametophyte: cells proximally cut off from initial
cells undergo divisions in a plane parallel to the substratum
to form the multi-layered cushion. These facts suggest that
the Colysis cushion meristem is similar to the multicellular
meristem typical of cordiform gametophytes. In this context,
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Takahashi et al. —Development of Colysis strap-shaped gametophytes
the strap-shaped Colysis gametophytes may have evolved from
ancestral cordiform gametophytes by insertion of the marginal
meristem phase between the apical-cell-based and the multicellular meristem phases.
However, it might be possible that the Colysis cushion meristem is an altered or modified marginal meristem, because it is
developed from the marginal meristem, and when cushion production ceases, the apex regains the rounded aspect of a
typical, non-cushion-producing marginal meristem. Wagner
and Farrar (1976) also reported that production of an intermittent cushion in non-cordiform gametophytes of Hyalotricha
(Polypodiaceae) seems to be correlated with width (robustness)
of its gametophyte apex; when growing conditions are poor,
the gametophyte apex narrows and ceases production of an
archegonial cushion. It could be interpreted that the Colysis
marginal meristem has gained the ability to form cushions
but it is more parsimonious to suggest that the ability to
form the cushion has been retained in Colysis gametophytes
during evolution from ancestral cordiform gametophytes.
The presence of both the apical-cell-based meristem and the
multicellular meristem may have been overlooked in earlier
Colysis research because growing gametophytes have an
anterior end that is round, flat or shallowly notched
accompanied by much less developed wing portions. The difference in wing shapes and resultant difference in depth of the
notch between strap-shaped and cordiform gametophytes is
due to the different shapes of merophytes as a whole constructing the wings; Colysis merophytes are rectangular, but those of
cordiform gametophytes are fan-shaped with the widest part at
the outermost part in cordiform gametophytes (Döpp, 1927 for
Onoclea, Cystopteris, Aspidium and others). In addition, at the
multicellular meristem stage, cells behind the outermost
initial cells divide in the periclinal plane more frequently in
Colysis gametophytes than in the cordiform gametophytes of
other ferns, resulting in cushion formation further from the
anterior end than in cordiform gametophytes: nearly ten cells
(C. decurrens in the present study; Stokey and Atkinson,
1958a for Elaphoglossum angustissimum) vs. one or two cells
(cordiform gametophytes, e.g. Döpp, 1927 for Onoclea
struthiopteris; Stokey, 1945 for Dipteris conjugata).
The marginal meristem is the most important feature distinguishing the strap-shaped gametophyte of C. decurrens
from the cordiform gametophytes of other ferns. The occurrence of the so-called marginal meristem has been noted previously for ribbon-shaped gametophytes, but that meristem
was an apical meristem that resided in the anterior margin
(Farrar et al., 2008). However, the marginal meristem of
Colysis is easily distinguished from the apical meristem (the
apical-cell-based and the multicellular meristems) in having
no growth centre and in not producing the cushion. This meristem is comparable in its organization to the marginal meristem of fern leaves where the marginal meristem consists of
conspicuous marginal initials and submarginal cells, and
undergoes fractionation to form pinnae (Hagemann, 1965,
1984; Mueller, 1982). Fractionation of the meristem is similarly involved in gametophyte branching in Colysis due to cessation of meristematic activity in the centre of the
gametophyte.
There are some developmental data for strap- and ribbonshaped gametophytes in Hymenophyllaceae (Stokey, 1948),
Vittariaceae (Farrar, 1974), grammitids (Stokey and
Atkinson, 1958b; Dassler and Farrar, 1997) and other
Polypodiaceae (Masuyama, 1975). Some figures presented by
these authors suggest the presence of the apical-cell-based,
marginal and multicellular meristems, but the data are very
fragmentary. Extensive studies of strap- and ribbon-shaped
fern gametophytes focusing on meristem behaviour would
provide a better understanding of the morphological evolution
of such unusually shaped gametophytes.
Evolution and ecology of strap-shaped gametophytes
Nayar (1962) reported that Colysis gametophytes take more
than 1 year to form antheridia and more than 2 years to form
archegonia (on Knop’s agar medium). The present study
showed archegonia forming at almost 4 months (using Parker
and Thompson’s agar medium). The time until archegonia
are formed may depend on culture medium and/or conditions,
but 4 months is longer than the time taken for archegonia to
form in culture on the cordiform gametophytes of other
ferns (30 DAS in Lygodium japonicum cultured using the
same medium and photoperiod as C. decurrens,
N. Takahashi, unpubl. data). Perhaps the longer period of
vegetative growth of C. decurrens is partly caused by the
insertion of the marginal-meristem phase between the
apical-cell-based and pluricellular meristem phases.
A gemmiferous strap gametophyte is better adapted to epiphytic habitats, because the continuous production and dispersal of gemmae may support metapopulation dynamics whereby
new habitats can be colonized as older sites become unsuitable, thus accommodating individual population extinction
(Farrar et al., 2008). Our studies of C. decurrens to date
have revealed no gemmae, although Nayar and Kaur (1971)
reported gemmae in all genera of ribbon-like gametophytes
of Polypodiaceae, so a longer term study (and observations
of gametophytes from natural substrata) would be interesting.
However, regardless of whether gemmae occur or not, the
indefinite longevity of all perennial gametophytes must be
especially significant in colonization of new habitats that are
kilometres or thousands of kilometres from the spore source
(Rumsey et al., 1998; Dassler and Farrar, 2001). The Colysis
gametophyte often reverts to the marginal meristem from the
cushion meristem phase. As gametophytes do not form archegonia in the marginal meristem phase, retrieval of the marginal
meristem allows the gametophyte to live indefinitely and
survive adverse environmental conditions.
In conclusion, strap-shaped gametophytes of C. decurrens
are formed by three different developmental phases, each
characterized by its own meristem: (1) the apical-cell-based
meristem responsible for early growth, (2) the marginal meristem responsible for further growth involving terminal branching of gametophytes and (3) the multicellular cushion
meristem for reproductive growth forming the cushion with
archegonia. The strap-shaped gametophyte may have evolved
from insertion of the marginal meristem phase between the
apical-cell-based and multicellular meristem phases, the
latter of which is probably common to cordiform gametophytes of other ferns. The marginal meristem repeatedly alternates with the cushion meristem phase, resulting in indefinite
gametophyte growth with intermittent cushion formation.
Takahashi et al. —Development of Colysis strap-shaped gametophytes
ACK N OW L E DG E M E N T S
We thank Dr Atsushi Ebihara of the Department of Botany of
the National Museum of Nature and Science, Tokyo, and
Professor Hirokazu Tsukaya of Gradate School of Science,
University of Tokyo, for suggesting the scientific name of
Colysis decurrens, and helping in spore collection and fieldwork, respectively. We also thank Professor Donald
R. Farrar and an anonymous referee for detailed and helpful
suggestions and comments. This study was supported by
Grants-in-Aid for Scientific Research, Japan (grant number
16370046).
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