Developmental morphology of strap-shaped gametophytes of Colysis decurrens: a new look at meristem development and function in fern gametophytes.
ABSTRACT 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.
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.
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.
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.
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ABSTRACT: Factors that influence the distribution of ferns are poorly understood and likely reflect the ecology of both the sporophyte and the gametophyte generation. Little study has been done on the ecology of the gametophyte generation, especially in regard to tropical species. The goal of this study was to examine demography and the influence of light and disturbance on the distribution of the gametophytes of several tropical epiphytic, hemiepiphytic, and terrestrial fern species. Through a series of observational and experimental studies, we found that increased terrestrial gametophyte density and richness were related to both increased light and disturbance. By contrast, increased light had no influence, and increased disturbance negatively affected epiphytic density. Over a 25-mo demographic study, epiphytic and hemiepiphytic species had significantly greater longevities and lower recruitment rates than terrestrial species. Such unique strategies may have evolved in response to different disturbance regimens between the two habitats. Terrestrial species encounter and are adapted to more frequent disturbance and have invested in rapid gametophyte growth and recruitment. Epiphytic species may be more influenced by bryophyte competition, and in habitats of relatively low disturbance, they have invested in greater size and longevities. In such systems, gametophytes are able to survive for years waiting for favorable recruitment conditions.American Journal of Botany 04/2007; 94(4):701-8. · 2.59 Impact Factor
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ABSTRACT: We explore the phylogeny of the polygrammoid ferns using nucleotide sequences derived from three plastid loci for each of 98 selected species. Our analyses recovered four major monophyletic lineages: the loxogrammoids, two clades consisting of taxa restricted to the Old World, and a largely neotropical clade that also includes the pantropical Grammitidaceae. The loxogrammoid lineage diverges first and is sister to a large clade comprising the three remaining species-rich lineages. One paleotropical clade includes the drynarioid and selligueoid ferns, whereas the second paleotropical clade includes the platycerioids, lepisoroids, microsoroids, and their relatives. The grammitids nest within the neotropical clade, although the sister taxon of this circum-tropic, epiphytic group remains ambiguous. Microsorum and Polypodium, as traditionally defined, were recovered as polyphyletic. The relatively short branch lengths of the deepest clades contrast with the long branch lengths leading to the terminal groups. This suggests that the polygrammoid ferns arose through an old, rapid radiation. Our analysis also reveals that the rate of substitution in the grammitids is remarkably higher relative to other polygrammoids. Disparities in substitution rate may be correlated with one or more features characterizing grammitids, including species richness, chlorophyllous spores, and an extended gametophytic phase.Molecular Phylogenetics and Evolution 07/2004; 31(3):1041-63. · 4.07 Impact Factor
- American Journal of Botany - AMER J BOT. 01/1963; 50(4).
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 2009Accepted: 26 August 2009Published 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 microscopi-
cally. 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 mer-
istem, 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 multi-
cellular 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.
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
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 ribbon-
shaped gametophytesare found
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 adap-
tive evolution of fern sporophytes.
Nayar and Kaur (1969) claimed that strap- and ribbon-
in membersof the
unlike ordinary cordiform gametophytes, but their con-
clusion was based on a number of gametophytes fixed at
different developmental stages, not from long-term obser-
vations 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 obser-
vation 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 gameto-
phytes in microsoroids. Colysis decurrens grows in both epi-
C. decurrens gametophytes was examined in comparison
with cordiform gametophytes of other ferns.
†Present address: Midorimachi Junior High school in Chiba, 2-3-1
Midorimachi, Chiba 263-0023, Japan.
* For correspondence. E-mail email@example.com
# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: firstname.lastname@example.org
Annals of Botany 104: 1353–1361, 2009
doi:10.1093/aob/mcp245, available online at www.aob.oxfordjournals.org
MATERIALS AND METHODS
Spores of Colysis decurrens (Wall. ex Hook. & Grev.) Nakaike
(syn. Colysis pothifolia (Hamilt. Ex D. Don) Presl) were col-
lected 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, fluor-
escent illumination of 20 mmol m22s21at 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
Cambridge, UK) and Photoshop CS (Adobe Systems Inc.,
San Jose, CA, USA). Comparison of one image with the pre-
ceding 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
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 game-
tophyte. 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.
Early development of gametophytes with apical-cell-based
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 fila-
ment (Fig. 1A, B). The terminal and sub-terminal cells of
these four cells of the filamentous gametophyte divide
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
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 div-
isions (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 tri-
angular 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 game-
tophyte (Fig. 1W, X).
Notably and unlike other homosporous-fern gameto-
phytes, 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 mer-
ophytes surrounding the apical cell. In Colysis gameto-
phytes, anticlinal and periclinal divisions occur regularly
so each merophyte keeps pace to maintain a rectangular
outline (Fig. 1K–N).
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 gametophytes1354
outer rectangular and the inner triangular cells continue divid-
ing in the periclinal and anticlinal planes to form a large mer-
ophyte 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.
FIG. 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 young-
est (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.
Takahashi et al. —Development of Colysis strap-shaped gametophytes1355
FIG. 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 gametophytes1356
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 (mero-
phytes) 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
haviour 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 meriste-
matic 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
At around 50 DAS, antheridia begin forming from the rela-
tively 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 anther-
idia, when present, are restricted to the posterior portion of the
thallus. No antheridia were observed on any branched gameto-
phytes 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
FIG. 3 Gametophytes with marginal meristems of Colysis decurrens. Numerals at the upper left corners indicate DAS. (A–C) Micrographs of the same game-
tophyte 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.
Takahashi et al. —Development of Colysis strap-shaped gametophytes1357