The evolution, morphology, and development of fern leaves

Abstract

Leaves are lateral determinate structures formed in a predictable sequence (phyllotaxy) on the flanks of an indeterminate shoot apical meristem. The origin and evolution of leaves in vascular plants has been widely debated. Being the main conspicuous organ of nearly all vascular plants and often easy to recognize as such, it seems surprising that leaves have had multiple origins. For decades, morphologists, anatomists, paleobotanists, and systematists have contributed data to this debate. More recently, molecular genetic studies have provided insight into leaf evolution and development mainly within angiosperms and, to a lesser extent, lycophytes. There has been recent interest in extending leaf evolutionary developmental studies to other species and lineages, particularly in lycophytes and ferns. Therefore, a review of fern leaf morphology, evolution and development is timely. Here we discuss the theories of leaf evolution in ferns, morphology, and diversity of fern leaves, and experimental results of fern leaf development. We summarize what is known about the molecular genetics of fern leaf development and what future studies might tell us about the evolution of fern leaf development.

Full text

REVIEW ARTICLE
published: 04 September 2013
doi: 10.3389/fpls.2013.00345
The evolution, morphology, and development of fern
leaves
Alejandra Vasco , Robbin C. Moran and Barbara A. Ambrose*
The New York Botanical Garden, Bronx, NY, USA
Edited by:
Madelaine E. Bartlett, Brigham
Young University, USA
Reviewed by:
Dan Chitwood, University of
California, Davis, USA
Hirokazu Tsukaya, The University of
Tokyo, Japan
Julia Nowak, University of British
Columbia, Canada
*Correspondence:
Barbara A. Ambrose, The New York
Botanical Garden, 2900 Southern
Blvd., Bronx, NY 10458-5126, USA
e-mail: bambrose@nybg.org
Leaves are lateral determinate structures formed in a predictable sequence (phyllotaxy)
on the flanks of an indeterminate shoot apical meristem. The origin and evolution
of leaves in vascular plants has been widely debated. Being the main conspicuous
organ of nearly all vascular plants and often easy to recognize as such, it seems
surprising that leaves have had multiple origins. For decades, morphologists, anatomists,
paleobotanists, and systematists have contributed data to this debate. More recently,
molecular genetic studies have provided insight into leaf evolution and development
mainly within angiosperms and, to a lesser extent, lycophytes. There has been recent
interest in extending leaf evolutionary developmental studies to other species and
lineages, particularly in lycophytes and ferns. Therefore, a review of fern leaf morphology,
evolution and development is timely. Here we discuss the theories of leaf evolution
in ferns, morphology, and diversity of fern leaves, and experimental results of fern
leaf development. We summarize what is known about the molecular genetics of fern
leaf development and what future studies might tell us about the evolution of fern leaf
development.
Keywords: leaf evolution, megaphyll, plant evo-devo, fronds, pteridophytes, Class I KNOX, Class III HD Zip
INTRODUCTION
“Nature made ferns for pure leaves to show what she could do in
that line”
-Henry David Thoreau
Ferns are the most diverse group of vascular plants after seed
plants. Recent morphological and molecular phylogenetic anal-
yses indicate that ferns are the sister group of seed plants
(Raubeson and Jansen, 1992; Stevenson and Loconte, 1996;
Kenrick and Crane, 1997; Pryer et al., 2001), and include the fam-
ilies Psilotaceae, and Equisetaceae, which have not always been
considered as ferns (Figure 1;Pryer et al., 2001). This phyloge-
netic circumscription and position of ferns has not been accepted
by all, especially paleobotanists who have argued that including
fossil taxa in the phylogenies resolves ferns as paraphyletic (e.g.,
Rothwell and Nixon, 2006; Tomescu, 2011). These controversies
along with the phylogenetic position of ferns as sister to seed
plants, and the fact that fern leaves display a great morphological
diversity, make ferns a key plant lineage for comparative stud-
ies on how leaves and vascular plants evolved. Studies in ferns
are important for resolving morphological interpretations within
particular fern groups and are crucial to our understanding of leaf
evolution and development in vascular plants.
Leaves have been the center of many evolutionary and develop-
mental studies, because they are the dominant, most conspicuous
organs of most plants, including ferns. Although typically envi-
sioned as compound, the leaves of ferns actually display great
morphological diversity (Figures 2,3). Historically, the leaves
of the eusporangiate Marattiaceae and of leptosporangiate ferns
(Figure 1) have been considered megaphylls, but there has been
great controversy on the definition of leaves in the Psilotaceae,
Ophioglossaceae, and Equisetaceae (e.g., Chrysler, 1910; Sen,
1968; Bierhorst, 1971; Kato, 1988; Kenrick and Crane, 1997).
These families have extremely modified leaves, and this has made
their interpretation difficult. If ferns are considered a mono-
phyletic group (Figure 1), then all fern leaves are considered to
be megaphylls or at least derived from megaphyllous ancestors.
Megaphylls then are present in seed plants and ferns and there
are several competing theories regarding their evolution and ori-
gin. It is still an open question whether the leaves of all fern are
homologous, let alone whether fern leaves are homologous with
seed plant leaves.
Here we review the following about fern leaves: their evo-
lution, their general morphology and diversity, unusual adap-
tations, the experimental biology, and the molecular genetic
studies to date. Integrating these different fields will not only
shed light on fern leaf evolution and development and help refine
hypotheses of fern leaf evolution, but also further our under-
standing of leaf evolution and development across the vascular
plants.
THE EVOLUTION OF LEAVES IN FERNS
In general, leaves are the main conspicuous organs of vascular
plants and often easy to recognize as such. It may therefore seem
surprising that they have had multiple origins. It has been hypoth-
esized that leaves evolved once in the ancestor of all vascular
plants (Kaplan, 2001; Schneider et al., 2002), twice (microphylls
in lycophytes and megaphylls in the remaining vascular plants;
Bower, 1935), three times (separately in lycophytes, ferns, and
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Vasco et al. Fern leaf evo-devo
FIGURE 1 | Relationships between vascular plants with an emphasis
on the ferns as summarized from morphological and molecular
phylogenetic analyses. Topology summarizes the results of previously
published studies (Kenrick and Crane, 1997; Doyle, 1998; Pryer et al., 2001;
Schuettpelz et al., 2007). Branches in gray correspond to exclusively fossil
lineages. Names in quotation marks indicate presumed paraphyletic lineages.
Only the fern families mentioned in the text are included here, for a complete
fern family phylogeny see Smithetal.(2006).
seed plants; Kenrick and Crane, 1997; Friedman et al., 2004;
Galtier, 2010; Corvez et al., 2012), four times (Boyce and Knoll,
2002), or six or more times (Tomescu, 2009). The number of
times leaves are thought to have evolved separately in vascu-
lar plants depends on the phylogenetic hypothesis used and the
inclusion and morphological interpretations of fossil taxa (Boyce,
2010).
Currently there is a general consensus that the leaves of lyco-
phytes and euphyllophytes are not homologous and have evolved
independently (Kenrick and Crane, 1997). Most morphological
and molecular phylogenetic analyses including either living or
fossil taxa or both, indicate that lycophytes are the sister group
of all other extant vascular plants, which are also called euphyl-
lophytes (Raubeson and Jansen, 1992; Stevenson and Loconte,
1996; Kenrick and Crane, 1997; Pryer et al., 2001; Schneider et al.,
2009)(Figure 1). Several studies have pointed out that the ear-
liest fossil relatives of both lycophytes and euphyllophytes were
leafless. If this is the case, then these two groups of plants must
have evolved leaves independently (Kenrick and Crane, 1997;
Friedman et al., 2004; Boyce, 2010).
The leaves of euphyllophytes have been called megaphylls
(Gifford and Foster, 1988), and there are several competing
theories regarding their evolution. The most widely accepted
is the one proposed by Zimmermann (1930).Concerningthe
evolution of leaves, his telome theory proposed that megaphyll
evolution involves three elementary processes that could occur
independently of each other (Figure 4). These three processes
transformed the dichotomous, leafless, photosynthetic axes of the
sporophyte of early Devonian plants into leaves (Zimmermann,
1930, 1952; Wilson, 1953; Stewart, 1964). One process involved
the elongation of some of the branches more than others, pro-
ducing a main central branch with subordinate lateral ones
(overtopping). A second process involved flattening the branch-
ing system into one plane (planation). A third process involved
the development of laminar tissue between the axes (syngenesis or
webbing). Paleobotanical evidence supports the existence of these
different processes and also suggests that there were anatomical
changes that correlated with the origin of megaphylls (Galtier,
2010). A fourth process, reduction of the branch system to sin-
gle scale-like leaf, was proposed to explain how leaves in Psilotum
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Vasco et al. Fern leaf evo-devo
FIGURE 2 | Examples of the diversity of size and shape in fern leaves.
(A) Doryopteris nobilis, pedate laminae. (B) Deparia acrostichoides,lamina
1-pinnate-pinnatifid. (C) Pteris semipinnata, dimidiate pinnae.(D) Pilularia
globulifera, filiform, terete leaves attached to rhizome; globular structures
are sporocarps. (E) Microgramma megalophylla, simple and entire lamina.
(F) Dipteris conjugata, lamina divided into two halves from top of petiole.
(G) Hemionitis palmata, palmate lamina. (H) Lygodium flexuosum, rachis (at
right) with lamina of pinnule (other half of pinna not shown). (I)
Megalastrum subincisum (right side of the leaf partially cuted off). (J)
Ophioglossum vulgatum, ovate blade represents a phyllode (expanded
rachis). (K) Marsilea drummondii, lamina consists of two pairs of opposite
pinnae, these resembling a four-leaved clover. (L) Matonia pectinata,
lamina. (M) Matteuccia struthiopteris, sterile-fertile leaf dimorphism (fertile
leaf at right). (N) Thelypteris reptans, flagellate apex proliferous at tip.
(O) Bolbitis heteroclita, 1-pinnate lamina with elongate apical segment
proliferous at tip. (P) Diplazium tomit aroanum, pinnatifid leaf. (Q) Gleichenia
microphylla, pair of opposite pinnae.
(Psilotaceae) and lycophytes evolved (Zimmermann, 1930, 1952;
Wilson, 1953).
Determining homology of megaphylls among euphyllo-
phytes is challenging because the three processes of mega-
phyll evolution—overtopping, planation, and webbing—could
have developed independently at different times and in differ-
ent orders. Hypotheses of megaphyll homology depend on the
morphological and anatomical interpretations of extinct leafless
Devonian and Carboniferous plants. One hypothesis suggests that
megaphylls are not homologous within the euphyllophytes. This
is based on the phylogenetic placement of leafless fossils, such
as Psilophyton and Pertica, within the euphyllophytes, and the
placement of the Aneurophytales among lignophytes (Figure 1).
Such topology indicates that the common ancestor of lignophytes
(seed plants+fossil Aneurophytales) and that of ferns were leaf-
less (Kenrick and Crane, 1997; Friedman et al., 2004; Boyce,
FIGURE 3 | Examples of the diversity of size and shape in fern leaves.
(A) Pteris aspercaulis, enlarged basal pinnules on basiscopic side of basal
pinnae. (B) Adiantum lunatum, 1-pinnate. (C) Adiantum pedatum, pedate.
(D) Adiantopsis radiata, digitate pinnae. (E) Pyrrosia polydactyla, palmately
lobed. (F) Actiniopteris semiflabellata, incised leaf. (G) Trachypteris pinnata,
holodimorphic, with rosette of sterile leaves and erect fertile ones. (H)
Salvinia molesta, root-like (lower) leaf is submerged and bears sori, the two
round ones are floating. (I) Pteris ensiformis, holodimorphic, fertile leaf at
left. (J) Lemmaphyllum microphyllum, holodimorphic, longer leaf is fertile.
(K) Davallia heterophylla, holodimorphic, fertile leaf at left. (L) Olfersia
cervina, holodimorphic, fertile leaf at right. (M) Belvisia mucronata,
hemidimorphic, with caudate fertile apex. (N) Anemia adiantifolia,
hemidimorphic, with two basal pinnae fertile and long-stalked. (O)
Osmunda regalis, hemidimorphic, with fertile pinnae at apex. (P) Pellaea
cordifolia, decompound. (Q) Gymnocarpium robertianum, ternate.
(R) Adiantum raddianum, decompound. (S) Astrolepis sinuata,
1-pinnate-pinnatifid. (T) Polystichum tripteron, enlarged basal pinnae. (U)
Hemionitis ariifolia, hastate at left, deltate at right (from same plant). (V)
Vittaria lineata, linear leaves (shoe-string fern). (W) Cystopteris bulbifera,
long-attenuate apex. (X) Drynaria quercifolia, debris-collecting leaf at right.
(Y) Aglaomorpha meyeniana, hemidimorphic, with narrow distal pinnae
fertile and base expanded for collecting fallen organic debris. (Z) Cyrtomium
macrophyllum, 1-pinnate. (ZZ) Elaphoglossum crinitum, simple, entire.
2010; Corvez et al., 2012). Another hypothesis proposes that
megaphylls in the euphyllophytes may be homologous at the
level of lateral branches (megaphyll precursors), and that the
processes of flattening into one plane (planation, which some-
times also has been suggested to imply abaxial/adaxial anatomical
organization of leaves) and the formation of a lamina (web-
bing) developed independently in ferns and seed plants (Kenr ick
and Crane, 1997; Galtier, 2010). This hypothesis is based on
the statement that the earliest known megaphyll-like structures
are highly dissected and composed of segments that were short,
narrow, and single-veined, but lacked an expanded lamina and
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Vasco et al. Fern leaf evo-devo
FIGURE 4 | Three processes from the telome theory proposed to be
involved in megaphyll evolution (redrawn from Zimmermann, 1952).
These processes may have occurred in various sequences in different
groups of vascular plants.
the abaxial/adaxial anatomical organization of leaves (Rothwell,
1999; Boyce and Knoll, 2002). Fossil evidence has also allowed
some authors to hypothesize that the full set of megaphyllous
traits were acquired later, either at the same (Boyce and Knoll,
2002) or in a different (Sanders et al., 2009) sequence of events in
ferns compared to seed plants.
Even within ferns the homology of leaves is unclear.
There is currently no consensus whether the leaves of major
fern clades such as Equisetaceae (horsetails), Psilotaceae,
Ophioglossaceae, Marattiaceae, and leptosporangiate ferns are
homologous (Figure 1). There are three main causes for
this uncertainty. One is conflicting phylogenetic hypotheses.
Molecular phylogenetic hypotheses of extant taxa consider
ferns as a monophyletic group that includes the Equisetaceae,
Psilotaceae, and Ophioglossaceae (Figure 1;Pryer et al., 2001;
Qiu et al., 2006; Grewe et al., 2013). In contrast, morphologi-
cal phylogenetic hypotheses that incorporate fossils and extant
taxa place Equisetaceae, Psilotaceae, and Ophioglossaceae in dif-
ferent positions within the vascular plants, related to but not as
part of the ferns (Rothwell, 1996, 1999; Stevenson and Loconte,
1996; Rothwell and Nixon, 2006). If the molecular phylogenetic
hypothesis is accepted, then all ferns leaves should be homolo-
gous at some level. In contrast, if the morphological phylogenetic
hypotheses are accepted, then the leaves of ferns, Equisetaceae,
Psilotaceae, and Ophioglossaceae are not necessarily homologous.
The second reason for the uncertainty about megaphyll homol-
ogy in ferns is that there are conflicts about the interpretation and
codification of characters of extinct Devonian and Carboniferous
fernlike plants without laminate leaves. These conflicts make
the phylogenetic placement of the fossils equivocal, and there-
fore statements of leaf homology within ferns ambiguous. For
instance, although they did not analyze ferns in detail, Kenrick
and Crane (1997) considered modern ferns, sphenopsids, cla-
doxylopsids, and the Devonian genus Rhacophyton as part of the
same clade (Figure 1). However, Rothwell (1996, 1999) separated
living ferns from Rhacophyton and zygopterids, which he placed
with lignophytes based on secondary growth. This topology (not
shown in Figure 1) would mean that no Devonian members of
the line leading to modern ferns and no steps in the origin of the
fern leaf are known. The third reason for the difficulties is that no
fossils have been associated with ancestors of key lineages within
ferns such as Ophioglossales and Psilotales, whose extremely
modified leaves could suggest that they are not homologous to
those of other ferns. The lack of these fossils means that there
is no evidence concerning the mode of origin of their leaves.
For the Marattiales, which has an extensive fossil record from
the Carboniferous, all the fossils have planated leaves, thus giv-
ing no evidence as to the sequence of steps in how their leaves
evolved (Rothwell and Stockey, 1989; Rothwell, 1996). Fossil evi-
dence supports the view that Equisetum (sphenopsids) leaves
were derived from a single dichotomous branch (Lignier, 1908 in
Kenrick and Crane, 1997), and that Marattiales and leptosporan-
giate ferns have basically compound leaves more probably derived
from whole branch systems bearing dichotomous appendages
(Doyle, 2013).
Currently the fossil record is insufficient to infer what interme-
diate characteristics of megaphylls may be homologous between
different fern lineages. Insights about this topic will probably
come from studies of morphology, developmental pathways, and
genetic networks of living taxa with different leaf morphologies
within a phylogenetic context (e.g., Stevenson and Loconte, 1996;
Harrison et al., 2005; Rothwell and Nixon, 2006). Even though
there is no complete sequenced genome or functional model
system for ferns, there is now a considerable amount of transcrip-
tome data for ferns available through the 1 KP project (http://
www.onekp.com/). This evidence, along with studies designed
to understand the molecular genetic basis of fern leaf diver-
sity will provide crucial data on leaf developmental pathways
in ferns and allow us to refine existing hypotheses on fern leaf
evolution.
THE GENERAL MORPHOLOGY OF FERN LEAVES
Although primarily for photosynthesis, fern leaves may also
assume other tasks such as propagating the plant vegetatively by
bulblets, harboring nitrogen-fixing cyanobacteria, forming nests
that collect humus falling from above, or efficiently dispersing
spores. Furthermore, ferns grow in many habitats—from man-
groves at sea level to alpine vegetation above tree line, temperate
forests to arctic tundras, and desserts to wetlands. Given this great
diversity in functions and habitats it is not surprising that fern
leaves exhibit a great diversity in size and shape (Figures 2,3).
This morphological diversity has helped taxonomists and mor-
phologists understand the evolution of ferns, but it can also be
used as a tool to unravel the developmental pathways underlying
fern leaf evolution.
In nearly all extant ferns, leaves constitute the dominant
organs of the plant. Fern leaves are typically envisioned as com-
pound (also termed dissected or divided) with pinnae or pinnules
arranged along a central axis (the rachis or costa) (Figure 5). Most
people probably envision ferns this way because, in fact, most
fern leaves are highly divided. Yet fern leaves exhibit enormous
diversity, especially in size, shape, and cutting (Figures 2,3).
Even though there is a great diversity of fern leaves, all share
common characteristics and consist basically of a stalk and a lam-
ina (see unusual fern leaves section for exceptions). The terms
applied to parts of a typical fern leaf differ sometimes from the
ones applied to seed plant leaves; thus, it is helpful to review the
terminology of a typical fern leaf (Figure 5). Fern leaves are often
called fronds, the stalk of the leaf is called the stipe or petiole.
Distal to this, the laterally expanded portion of the leaf is termed
the blade or lamina, whose central midrib is referred to as the
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Vasco et al. Fern leaf evo-devo
FIGURE 5 | Terminology of a typical fern leaf or frond.
rachis. The primary divisions of the blade are called pinnae, and
the secondary segments of these (if present) are called pinnules.
The stalk of a pinna could be called a petiolule, but this term is
seldom used in fern taxonomy. If the pinnules are further divided,
the divisions are termed segments, and sometimes their order is
specified, such as “tertiary segment” or in a more highly divided
leaf, “quaternary segment.” The midrib of the pinna is termed a
costa, and the midrib of a pinnule is called the costule. That half of
a pinna or pinnule that occurs on the side toward the distal apex
of the axis that bears it is called the acroscopic side, and accord-
ingly, the side that occurs toward the base is the basiscopic (for
more details of fern leaf terminology, see Tryon, 1960).
Fern leaves and megaphylls of other groups are defined by
a combination of characters that are a result of specific devel-
opmental processes. They are arranged in a fixed and predic-
tive phyllotaxy around the shoot apex (Schoute, 1938; Gifford
and Foster, 1988), they have adaxial/abaxial identities and with
laminar tissue supplied by numerous vascular traces, and most
grow for a finite amount of time to a finite size (Bower, 1923;
Hagemann, 1989; Kaplan and Groff, 1995; Kaplan, 2001).
Most seed plants (cycads excepted; Stevenson, 1990)havebuds
that form in the axils of the leaves as they are specified from the
shoot apical meristem and can subsequently grow out as shoots
(branches). However, nearly all ferns have extra-axillary branch-
ing, meaning that buds that will grow out as shoots may take
various positional relationships with respect to the point of leaf
insertion on the stem (Hagemann, 1989). Axillary branching is
extremely rare in ferns and, where investigated anatomically, the
vasculature of the leaves differs from that found in seed plants
because the steles do not come directly from the axil or are closely
associated with the leaf gap (Hébant-Mauri, 1984; Hagemann,
1989).
Nearly all mature fern leaves are bifacial, with well-defined
adaxial/abaxial identities. The condition is also present
anatomically two ways: first, with the mesophyll differentiated
adaxially as palisade tissue and abaxially as spongy tissue, and
second, by greater elongation of cells on the abaxial side vs.
adaxial. A few ferns, such as Enterosora (Polypodiaceae) have
a spongy mesophyll throughout, but this is rare among ferns
(Moran, pers. obs.). One exception to a mature bifacial leaf is
Pilularia (Marsileaceae, water ferns), whose leaves are terete and
unifacial. Its blade-less leaf is interpreted as a petiole that has lost
its apical pinnae (Eames, 1936). At least in fossil ferns, the defined
adaxial/abaxial identities are also present in the vascular bundles
of the petiole where the protoxylem is adaxial (in contrast to seed
plants that have abaxial protoxylem; Galtier, 2010).
Besides the typical characteristics of all megaphylls, fern leaves
have some additional characteristics that make them distinctive.
Their leaf primordia are often covered by hairs and/or scales
(Figure 6). Hairs are uniseriate and either one-celled or multi-
celled. Hairs develop from cell divisions of a single epidermal cell
(Bower, 1923). In contrast, scales are multicellular with the cells
arranged side-by-side in two or more rows. They develop from
cell divisions of several epidermal cells. Scales can be persistent in
mature leaves and may become smaller and reduced to uniseri-
ate proscales toward the margins of the laminae (Moran, 1986).
Such highly reduced, uniseriate scales are sometimes also called
microscales (Daigobo, 1972).
Another characteristic of fern leaves is that all cells of the
lower and upper epidermis contain chloroplasts (Copeland, 1907;
Wylie, 1948a,b, 1949). This condition is shared with the lyco-
phytes (Moran, pers. obs.). In contrast, seed plants (with few
exceptions) have chloroplasts only in the guard cells of the epi-
dermis, not in other epidermal cells (Cutter, 1978; Gifford and
Foster, 1988; Fahn, 1990). Among ferns, the few exceptions that
lack chloroplastsin all epidermal cells are species that grow in full
sun, such as high-canopy epiphytes (e.g., Elaphoglossum lingua,
Dryopteridaceae) or on sunny rock faces (e.g., Notholaena affinis,
Pteridaceae; Moran, pers. obs.). The absence of chloroplasts from
the epidermis of seed plants (except the guard cells) is best inter-
preted on the basis of outgroup comparison as a loss, and this loss
represents a synapomorphy for seed plants.
There are two characteristics typical of most fern leaves: a fid-
dlehead and aerophore lines (Figure 6). Fiddleheads, also called
croziers are circinately coiled leaf buds. Just as the whole leaf is
coiled in bud, so too are its subdivisions, the pinnae and pin-
nules. Presumably, the function of coiling is to protect the soft
meristematic parts concealed within the fiddlehead. Fiddleheads
are highly distinctive of ferns because they are absent from lyco-
phytes and nearly all seed plants (some exceptions in seed plants
are the cycads Cycas and Stangeria, and the insectivorous plant
Drosophyllum lusitanicum, although in the latter the coiling is
abaxial, not adaxial as in ferns). However, some ferns such as
Psilotum, Equisetum,Ophioglossum,Salvinia,andAzolla,lackfid-
dleheads. Aerophores or pneumatophores are also characteristic
of nearly all leptosporangiate fern leaves. They are apparent as
two light-colored lines on either side of the petiole (Davies, 1991).
These lines aerate the leaf. Their surfaces bear stomata that allow
air to diffuse into the loosely packed parenchyma beneath the
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Vasco et al. Fern leaf evo-devo
FIGURE 6 | Fiddlehead and aerophore (or pneumatophore) of Pteris
livida (Pteridaceae).
line. In some ferns the aerophores extend distally into the rachis,
or proximally onto the rhizomes (e.g., Mickelia and Polybotrya,
both Dryopteridaceae). In those few fern species that invest their
fiddleheads in a thick covering of mucilage, the lines are mod-
ified at the pinna bases into elongate peg-like structures that
protrude through the mucilage, thus aerating the developing leaf
(Hennipman, 1968). Even when aerophore lines are seemingly
absent, such as in the darkly sclerotized petioles of Adiantum
pedatum (Pteridaceae, Figure 3C), a line of stomata are present
in its place—a vestige of the ancestral aerophore.
THE MORPHOLOGICAL DIVERSITY OF FERN LEAVES
There is a vast amount of leaf diversity in ferns, and here we give
only a few examples (Figures 2,3). Fern leaf morphological diver-
sity is exhibited in the lamina size, shape, dissection, reductions
in various parts, apical indeterminacy, petiole vasculature, and
petiole morphology.
LAMINA DISSECTION AND SHAPE
Although fern leaves are often stereotyped as being finely divided,
some are simple and entire, and others are merely lobed.
Simple leaves in ferns are found in Elaphoglossum (ca. 600 spp.;
Dryopteridaceae), Campyloneurum (ca. 50 spp.; Polypodiaceae),
Microgramma (35 spp.; Polypodiaceae), Grammitis (22 spp.;
Polypodiaceae), and Vittaria (6 spp.; Pteridaceae). On the basis
of outgroup comparison, the leaves of these ferns are believed
to be derived from ancestors with more divided leaves (e.g.,
Wagner, 1964; Moran et al., 2010,forElaphoglossum). One
example of simple leaves are the reniform ones that have
evolved independently in different fern families, resulting in
FIGURE 7 | Parallelism in four simple-leaved ferns belonging to
different families. (A) Lindsaea cyclophylla (Lindsaeaceae). (B) Adiantum
reniforme (Pteridaceae). (C) Trichomanes reniforme (Hymenophyllaceae).
(D) Schizaea elegans (Schizaeaceae).
some striking parallelisms (Figure 7). This ability to produce
similarly shaped laminae suggests similar developmental mech-
anisms may be at work. Lobed leaves are also found in ferns,
and the basic plan may be either pinnate, as in Aglaomorpha
meyeniana (Polypodiaceae, Figure 3Y), or palmately lobed as
in Hemionitis palmata (Pteridaceae, Figure 2G). Not surpris-
ingly divided fern leaves exhibit a great variety of archi-
tectures. The most common and widespread is the pinnate
plan. Here the petiole continues into the lamina as a single,
unbranched rachis that produces lateral pinnae. Examples are
Deparia acrostichodes (Thelypteridaceae, Figure 2B), Matteuccia
struthiopteris (Onocleaceae, Figure 2M), and Megalastrum subin-
cisum (Dryopteridaceae, Figure 2I). Sometimes the basal pinnae
are repeatedly branched on the basiscopic side—a condition
known as pedate. Examples are Adiantum pedatum (Pteridaceae,
Figure 3C)andDoryopteris nobilis (Pteridaceae, Figure 2A). In
highly divided leaves, branching patterns of the pinnae (pri-
mary divisions of the laminae) may be of taxonomic importance
(e.g., species of Megalast rum (Figure 2I)andLastreopsis, both
Dryopteridaceae).
LEAF INDETERMINACY
Leaves are generally considered to be determinate organs; that is,
they grow only to a certain length and no more. Fern leaves gener-
ally exhibit finite (determinate) growth but with longer mer istem-
atic activity of the apical portion and maturation toward the apex
(acroscopic growth). This prolonged meristematic activity is due
to an apical cell at the leaf tip and is a distinctive feature of ferns
compared to seed plants (Imaichi, 2008). A few ferns, however,
have indeterminate leaves. Generally such leaves are either pen-
dulous or scrambling over the surrounding vegetation. Alansmia
(Polypodiaceae) is a pendent epiphyte whose leaves exhibit small
continuously growing fiddleheads at their tips (Kessler et al.,
2011). Their laminae often exhibit constrictions where fiddlehead
activity diminishes during a less favorable part of the growing sea-
son. Many species of Nephrolepis (Nephrolepidaceae), particularly
N. exaltata (Boston fern) and N. pendula, exhibit pendulous inde-
terminate leaves. The latter species can have leaves up to three
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Vasco et al. Fern leaf evo-devo
meters long (Hovenkamp and Miyamoto, 2005; Rojas-Alvarado,
2008). Ferns with scrambling indeterminate leaves grow over
the surrounding vegetation and use it for support. Examples
include certain species of Gleicheniaceae (Figures 2Q,8;Moran,
2004), Jamesonia (Pteridaceae, Tryon, 1970), and Hypolepis
(Dennstaedtiaceae, Brownsey, 1987; Holtum, 1958). Scrambling
ferns often have leaf apices exhibiting intermittent growth
(Figure 8). The leaf apex (fiddlehead) rests while the subtend-
ing lateral pinnae develop. After extending laterally, the pinnae
come to rest on the surrounding vegetation, and the leaf apex
resumes extension growth. In this manner, and because of their
long-creeping rhizomes, the Gleicheniaceae often form dense
extensive thickets (Moran, 2004). Two genera of ferns that have
indeterminate leaves twining around a support are Salpichlaena
(Blechnaceae) and Lygodium (Lygodiaceae, Figure 2H;Mueller,
1983a,b). In Costa Rica, the leaves of Salpichlaena volubilis are
generally 10–12 m long, whereas those of Lygodium venustum are
usually 3–6 (Moran, pers. obs.). In these genera it is the rachis
that twines—a condition not found among the angiosperms (the
organ that twines in angiosperms such as Wisteria (Fabaceae) or
Convolvulus (Convolvulaceae) is the stem). The twining results
from the widely circumnutating leaf being interrupted by contact
with a support, and, when the rope hits a pole, the rope continues
its motion around the pole, wrapping upward and thus climb-
ing the pole (Darwin, 1876). This is not a thigmotropic grasping
response as exhibited, for instance, by the leaves of Clematis
(Ranunculaceae; Darwin, 1876).
PETIOLE VASCULATURE
Ferns exhibit a wide variety of petiole vasculatures (Figure 9).
One extreme is the polycyclic condition found in Acrostichum
(Pteridaceae) and the Marattiaceae. Here the petioles con-
tain several concentric circles (as seen in transverse sec-
tion), each circle composed of many individual leaf traces
(Figure 9A). At the other extreme is, for instance, Tri c h o manes
(Hymenophyllaceae, filmy ferns), whose petioles contain only
one vascular bundle. A single omega shaped vascular bundle,
with the open end of the omega oriented adaxially, is found in
Culcitaceae, Dennstaedtiaceae, Saccolomataceae (Figure 9C), and
many species of Pteris (Pteridaceae). The number and arrange-
ment of vascular bundles in the petiole is helpful in fern taxon-
omy. Of great importance in fern taxonomy is the distinction
between Eupolypods I and II (i.e., about 65% of extant ferns)
FIGURE 8 | Rhythmic leaf extension growth in the Gleicheniaceae
(from Moran, 2004).
based on petiole vasculature. With few exceptions, Eupolypods I
have several vascular bundles, all circular in cross section, with the
two adaxial ones enlarged (Figure 9F), whereas Eupolpods II have
only two vascular bundles elongated in cross section (Figure 9E;
Moran, pers. obs.).
PETIOLE MORPHOLOGY
In some ferns only the very base of the petiole develops and
the distal portion of the leaf does not. In these cases the dis-
tal part of the leaf is represented by either a vestigial fiddle-
head or necrotic tissue. In Matteuccia struthiopteris (Onocleaceae,
Figure 2M) and various species of Osmunda (Osmundaceae),
petiole bases are called cataphylls, and they protect the rhi-
zome apex (Goebel, 1905). In other ferns, the leaf bases store
abundant starch and are termed trophopods (Wag ner and
Johnson, 1983). These structures occur in Cystopteris protr usa
(Cystopteridaceae) and Onoclea sensibilis (Onocleaceae). In some
species of Diplazium (Athyriaceae) with erect subarborescent rhi-
zomes (e.g., Diplazium prominulum and D. striatastrum), the
rigid, starch-filled trophopods are tightly appressed to the trunk
and structurally support the rhizome (Moran, pers. obs.).
FERNS WITH UNUSUAL LEAF MORPHOLOGIES
Some fern leaves present unusual shapes and adaptations. The
subject is vast and only a small sample is covered here.
OPHIOGLOSSACEAE
Relative to other ferns, unusual features of the leaves of this
family include sheathing leaf bases, buds rarely circinate, and
adaxial position of the sporangia (Bower, 1926; Kato, 1988).
Furthermore, species of Ophioglossaceae usually produce only
one leaf at a time. The principal attribute distinguishing
Ophioglossaceae from other ferns is the division of the leaf into
separate vegetative (sterile segment) and sporangium-bearing
(fertile segment) portions (Figure 2J;Bower, 1926; Gifford and
Foster, 1988; Wagner, 1990). The fertile segment has been
FIGURE 9 | Examples of petiole vasculature in ferns, as seen in cross
section. (A) Eupodium laeve (Marattiaceae). (B) Dicksonia sellowiana
(Dicksoniaceae). (C) Saccoloma chartaceum (Saccolomataceae). (D)
Acrostichum danaeifolium (Pteridaceae). (E) Diplazium hians (Eupolypods II,
Athyriaceae). (F) Polystichum concinnum (Eupolypods I, Dryopteridaceae).
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Vasco et al. Fern leaf evo-devo
variously interpreted. One interpretation is that it develops from
a single pinna or two fused pinnae so that the plant is composed
of a leaf with a sterile portion that is proximal and a fertile por-
tion that is distal (Chrysler, 1910, 1911). Another interpretation
is that the fertile spike is derived from a shoot or a modified
branch system so that the plant consists of an organ that is inter-
mediate between a true leaf and a branch (Zimmermann, 1952;
Wilson, 1953; Sen, 1968). The sterile segment of Ophioglossum
(Figure 2J) lacks a midrib, and this has been proposed to be
theresultofthelossofthelaminatissueandretentionof
the rachis. The latter expanded laterally by intercalary divisions
and, at the same time, elaborated a system of reticulate veins
(Wagner, 1990). In other words, the lamina of Ophioglossum is a
phyllode.
PSILOTUM
The sterile leaves of Psilotum (Psilotaceae) are unusual among
ferns by being scale-like and generally lacking a vascular sup-
ply. The “simple” structure of the leaves and the dichotomizing
axes that constitute the entire plant, led several authors to relate
Psilotum to the earliest vascular plants (Bower, 1935; Eames, 1936;
Wilson, 1953; Rothwell, 1999). Molecular phylogenetic analyses
of chloroplast and nuclear markers (Pryer et al., 2001)andfrom
complete plastid genomes (Grewe et al., 2013), however, have
confirmed that Psilotaceae are ferns (Figure 1). The inclusion
of Psilotaceae within the ferns suggests that the small scale-like
appendages in Psilotum are probably best interpreted as highly
reduced leaves.
EQUISETUM
The leaves of Equisetum (Equisetaceae) are unique among ferns
in form and function. Unlike most ferns that have leaves as
the dominant organs of the plant, in Equisetum the stem is the
dominant organ and performs most of the plant’s photosynthe-
sis. The leaves are highly reduced and connate laterally to form
a sheath around the base of each segment of the aerial stem.
The teeth along the rim of the sheaths represent free leaf tips
(in some species, such as E. hyemale, the teeth are deciduous).
At the base of each leaf sheath is a node at which there is an
intercalary meristem. New stem tissue produced by these meris-
tems is responsible for nearly all of the elongation of the aerial
stem. In temperate species, the apical meristem differentiates as
parenchyma by the end of the summer and therefore is no longer
active. In the following spring, nearly all the elongation of the
aerial shoots comes from the activity of the intercalary meris-
tems (Hauke, 1985). At each node, the youngest—and therefore
softest and structurally weakest—stem tissue is located just above
the intercalary meristem and surrounded by the leaf sheath. The
function of the sheath is to structurally support this weaker stem
tissue, just as leaf sheaths do in other plants with intercalary
meristems at the nodes, such as grasses, sedges, and commelinid
monocots (Fisher and French, 1976). In Equisetum,therela-
tionship of the vegetative and fertile portions has been debated:
the sporangiophore (the fertile portion) has been interpreted as
being a novel organ “(organ sui generis)” or homologous to the
leaves (Goebel, 1905; Bower, 1935; Zimmermann, 1952; Page,
1972). Complete serial transitions between the leaf sheaths and
the sporangium-bearing structures have been found, suggesting
that the latter are sporophylls (Page, 1972).
FILMY FERNS
In the filmy ferns (600 spp., Hymenophyllaceae), the laminae are
one cell layer thick between the veins. A few species are known
with 2–4 cell layers, but these species lack intercellular spaces and
stomata (Copeland, 1938). False veins occur in some filmy fern
genera, such as Didymoglossum (Wessels Boer, 1962). These veins
are one cell wide and appear as faint streaks that do not connect to
the true veins. The thin laminae of filmy ferns dry out readily and
then, upon rehydration, rapidly expand and resume photosynthe-
sis (Proctor, 2003, 2012). They are capable of directly absorbing
water and nutrients.
HETEROSPOROUS WATER FERNS
The leaves of all heterosporous water ferns are unusual, having
been highly modified for their aquatic or semi-aquatic habi-
tats. Salvinia (Salviniaceae) has two kinds of leaves produced in
afalsewhorlofthree(Croxdale, 1978, 1979, 1981). The first
kind of leaf, of which there are two in the false whorl, is float-
ing, green, entire, and conduplicate in bud (i.e., not circinate).
The other kind of leaf is whitish, highly divided, and hanging
down in the water. Only this submerged leaf bears sori. The dor-
sal surfaces of the floating leaves are covered by erect papillae.
In some species (e.g., S. auriculata and S. molesta, Figure 3H)
these papillae have four hairs at their apices, the complete struc-
ture (papillae and hairs) resembling an egg-beater. These peculiar
structures help water bead-up and roll off the leaf. The sur-
face of the leaf, except for those apical cells of hairs united
by their tips, bear a super-hydrophobic wax that prevents wet-
ting the surface, thus helping to keep the plant afloat (Barthlott
et al., 2010). Azolla has bilobed leaves up to 1 mm long—the
smallest leaves of any fern. A colorless ventral lobe rests on the
water and a thicker green dorsal lobe arches upward. This thicker
lobe contains a cavity that harbors nitrogen-fixing cyanobac-
teria (Anabaena azollae). Marsilea (Marsileaceae, Figure 2K)is
unusual because its leaves resemble a four-leaved clover.Its leaflets
(pinnae) consist of two pairs of opposite pinnae, each pinna pro-
vided with a pulvinus at its base. Through the action of the
pulvini, the pinnae fold forward and upward at night, form-
ing a vertical packet with the basal pair enclosed within the
distal one. At dawn the packet unfolds to present the pinna per-
pendicularly to the sun (Darwin, 1896). Marsilea is the only
nyctinastic fern. Also unusual about Marsilea (and the other
two genera of Marsileaceae, Pilularia, and Regnellidium)areits
sporocarps, hardened bean-like structures that represent folded
and marginally sealed pinnae containing the sori (Eames, 1936;
Nagalingum et al., 2006).
TREE FERNS
Among the tree-fern family Cyatheaceae, several species of
Alsophila produce at the base of their petioles skeletonized pin-
nae with linear or filiform segments. They bear stomata on their
abaxial surfaces and have compact mesophyll with little intercel-
lular spaces (Goebel, 1905). These pinnae, called aphlebiae, are of
unknown function.
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Vasco et al. Fern leaf evo-devo
DRYNARIOID FERNS
As an adaptation to their epiphytic life style, the drynarioid gen-
era of Polypodiaceae have leaves modified for collecting organic
debris that falls from above, mostly bits of bark and leaves
(Hennipman and Roos, 1982; Janssen and Schneider, 2005). As
the debris decomposes, it forms humus into which the plant
grows roots to absorb water and nutrients. This is important
nutritionally because these ferns are epiphytic, not in contact with
water and minerals in the soil. One of these genera of humus col-
lectors, Aglaomorpha (Polypodiaceae, Figure 3Y), has sterile and
fertile leaves of one type (monomorphic) but modified within
the same leaf to accumulate organic debris. The leaves are ses-
sile with deeply pinnatifid laminae and expanded bases that turn
brown and papery with age. The bases resist decay and persist on
the plant long after the green laminar tissue has decayed away
(the rachises are persistent, though). Thus, the old leaf bases
retain humus for a long time. In the related genus Drynaria
(Polypodiaceae; Figure 3X), humus-collecting leaves are com-
pletely differentiated from the green foliage leaves that produce
spores—an example of holodimorphism (see section on sterile-
fertile leaf dimorphy). The humus-collecting leaves are brown,
stiff, papery, and dead at functional maturity. They grow over
the fern’s creeping rhizome, covering it completely. The leaf bases
are sessile and wide so that the accumulated humus does not fall
through. The humus-collecting leaves are much smaller than the
green fertile ones, which also differ by being long-petiolate to ele-
vate the sori away from the substrate where they are more likely to
encounter air currents for spore dispersal. The related and widely
cultivated staghorn ferns, Platycerium (Polypodiaceae), also have
holodimorphic humus-collecting and fertile leaves. The humus-
collecting leaves are also brown, stiff, papery, and dead at func-
tional maturity. They grow tightly overlapped and appressed or
ascending over rhizome, with sessile and broad bases—all modi-
fications so that trapped organic matter does not fall through. The
mesophyll is thick and, when dead at functional maturity, absorbs
water like a sponge. In contrast, the fertile leaves are green, much
longer, and arching away from the substrate. They are of relatively
short duration on the plant, compared to the humus-collection
leaves that persist for a long time. These drynarioid ferns provide
an outstanding example of leaf modification as adaptations for
life in the tree tops.
STERILE-FERTILE LEAF DIMORPHY
A special case of fern leaf morphological diversity is sterile-fertile
leaf dimorphy. This phenomenon is generally thought to consist
of narrower and taller fertile leaves compared to the sterile ones,
but it is much more than that. Dimorphism is a syndrome of
many characters, and these may be anatomical or morphological.
The character differences maximize spore dispersal and mini-
mize the metabolic cost of construction of fertile leaves (Moran,
1987). Narrower laminae or pinnae have a thinner boundary
layer of air (Salisbury and Ross, 1992). This thinner layer of
air would promote drying of sporangia and increase the chance
that spores, after being catapulted from sporangia, would soon
encounter moving air currents. Also maximizing spore dispersal
are the longer and more erect petioles that elevate the fertile lam-
ina away from the substrate where the spores are more likely to
be picked up by air currents. After shedding their spores, fertile
leaves have completed their function and soon wilt. Because fer-
tile leaves are ephemeral relative to the sterile ones, they tend to
be of “cheaper” construction, built with energetically less expen-
sive tissues such as parenchyma and collenchyma, not harder and
denser sclerenchyma.
Sterile-fertile leaf dimorphy is common in ferns. If sterile
and fertile leaves are the same size and shape, they are said to be
monomorphic. If differentiated, they are said to be dimorphic.
The dimorphy may be of two types. In the first, only part of
the leaf is fertile—the hemidimorphic condition—as exempli-
fied by Aglaomorpha meyeniana (Polypodiaceae, Figure 3Y),
Belvisia (Polypodiaceae, Figure 3M), Osmunda (Osumundaceae,
Figure 3O), and Anemiaceae (Figure 3N). In the second, the
complete fertile leaf is differentiated from the sterile—the
holodimorphic condition—as seen in Davallia heterophylla
(Davalliaceae, Figure 3K), Matteuccia (Onocleaceae, Figure 2M),
and Olfersia (Dryopteridaceae, Figure 3L), Osmundastrum
(Osmundaceae), and Trachy p t e r is (Pteridaceae, Figure 3G).
These two forms of dimorphy have evolved many times among
ferns (Wagner and Wagner, 1977).
The fertile leaves of the sister families Psilotaceae and
Ophioglossaceae are unusual in that the synangia in the former
and the sporangia-bearing portion in the latter arise adaxially
(Imaichi and Nishida, 1986; Wagner, 1990; Schneider, 2013). The
fertile leaves of all other ferns bear sporangia abaxially (marginally
in some groups such as the Hymenophyllaceae and Culcitaceae).
Both families are further atypical in that a single vein runs to the
base of the sporangium or synangium (Eames, 1936). In no other
ferns are the sporangia similarly supplied.
Another aspect of dimorphy is the phenology of sterile and
fertile leaf production (Sharpe and Mehltreter, 2010). This is
especially unknown for tropical ferns not limited by an unfavor-
able winter growing season and thus capable of producing leaves
throughout the year. In these cases, fertile leaves may be produced
at certain times of the year, such as during the wet season or dry
season. Also, in almost all tropical ferns with strong sterile-fertile
leaf dimorphy, the fertile leaves tend to be shorter-lived than the
sterile. Yet the timing of production and duration on the rhizome
are unknown for all but a handful of ferns. Also, it is rarely known
how many fertile leaves are produced annually in proportion to
sterile ones. Phenology is one area of fern leaf biology where field
studies are greatly needed.
FOLIAR BUDS
About 5% of fern species worldwide have foliar-borne buds
(Moran, 2004). Buds may form anywhere on the leaf: along
the petiole, in the angle between the rachis and the pinnae,
at the apices of the lamina or the pinnae, or above the point
that sori would normally form. Buds contact the soil by var-
ious means. Most commonly, as the leaf senesces, the petiole
weakens at the base, and the leaf gradually reclines toward the
ground. Eventually the leaf comes to rest on the soil and the bud,
still attached to the leaf, is “planted” and takes root. In other
ferns, such as Bolbitis heteroclita (Dryopteridaceae, Figure 2O),
Thelypteris reptans (Thelypteridaceae, Figure 2N)orAsplenium
rhizophyllum (Aspleniaceae), the lamina apices are long-attenuate
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Vasco et al. Fern leaf evo-devo
or flageliform (whip-like) with buds along their length or at their
tips. These leaves arch over and touch the ground, placing the bud
in contact with the soil. Rarely, foliar-borne buds abscise from
thematureleafanddroptotheground,asinCystopteris bu lbifera
(Cystopteridaceae, Figure 3W).
RHEOPHYTIC FERNS
The leaves of rheophytic ferns exhibit several morphological
and anatomical adaptations to their unusual habitat. Rheophytic
species are those confined to the beds of swift-running streams
and grow submerged during regularly occurring flash floods (Van
Steenis, 1981; Kato and Iwatsuki, 1991). Some examples of rheo-
phytic ferns are Asplenium obtusifolium (Aspleniaceae), Osmunda
lancea (Osmundaceae), and Tectaria lobbii (Tectariaceae). These
ferns are characterized by flood-resistant morphological fea-
tures such as narrow leaf blades or narrow leaflets with cuneate
bases (a phenomenon called stenophylly) and petioles that are
flexible—both characters that reduce drag from rushing water.
The root system is matted and tightly anchored to the substrate.
Anatomically, fern rheophytes have a smaller mesophyll with
more cells in the spongy layer and with fewer intercellular spaces
compared to related upland species. The cuticle is thicker and the
epicuticular wax deposits on the epidermis are denser. Finally, the
frequencyofoccurrenceofstomataperunitareaofleafishigher
in rheophytes than in related upland species (Kato and Imaichi,
1992).
EXPERIMENTAL ANALYSES OF FERN LEAF DEVELOPMENT
There is a rich history of experimental studies in ferns (reviewed
in White, 1971; White and Turner, 1995). These studies were
designed to understand how fern leaves, and ferns in general,
develop. Experimental studies of fern leaves have investigated
the growth of heteroblastic leaf sequences, cataphylls, and sim-
ple and compound leaves (Wardlaw, 1963; White, 1971). Here
we will focus on leaf-development studies designed to under-
stand phyllotaxy, adaxial/abaxial identity, and when and how a
leaf is determined. These studies used microsurgery to isolate
primordia from the shoot and/or adjacent leaves and also ster-
ile techniques to grow leaves or shoot apices in isolation. Most
studies use the terminology “P” and “I” where P1 is the youngest
leaf primordium and P10 is the 10th oldest primordium. The
“I” indicates the incipient leaf primordia where I1 would be
the first primordia to develop after P1, and I3 would be the
third primordium to develop after P1. The positions of incip-
ient primordia are estimated by the phyllotaxy of the species.
Many of the experimental studies on fern leaf development
that we will discuss were performed on two different leptospo-
rangiate fern species Dryopteris aristata (Dryopteridaceae) and
Osmunda cinnamomea (Osmundaceae). Both species have com-
pound leaves. Although the former species is now classified in
Arachniodes (Dryopteridaceae) and the latter in Osmundastrum
(Osmundaceae), we will use here the older names employed by
the researchers in the original developmental studies cited.
Fern leaves arise from the periphery of a shoot apical meris-
tem (SAM) in a distinct phyllotaxy as do seed plant leaves
(Wardlaw, 1963; Gifford and Foster, 1988). The SAM in ferns
typically has 1 or 2 large apical cells surrounded by small
cytoplasmically dense cells that divide frequently (Bower, 1884;
Wardlaw, 1963; Bierhorst, 1971; White, 1971; McAlpin and
White, 1974; Stevenson, 1976; White and Turner, 1995). Although
the SAM of the ferns and seed plants differ anatomically (because
of the presence of apical initial(s) in ferns), they are similar by
having distinct zones of cells in the meristem (Wardlaw, 1963;
White, 1971; McAlpin and White, 1974; Stevenson, 1976; Steeves
and Sussex, 1989). Fern leaf primordia arise from one or a group
of cells on the flank of the SAM (Bower, 1923; Wardlaw, 1949b;
Steeves and Briggs, 1958; Bierhorst, 1977; Imaichi, 1980, 1982). A
single apical cell forms at the tip of the leaf and has 2 cutting faces
(Wardlaw, 1963; White and Turner, 1995). The leaf is formed by
divisions of the apical initial as well as by divisions in the marginal
meristem. Divisions in the marginal meristem and sub-marginal
cells, and cessation of division in groups of cells regularly spaced
alongthemarginalmeristem,giverisetopinnae(Wardlaw, 1963).
Similar to the fern SAM, the meristematic regions of the leaf also
have distinct zones of cells, however, unlike the fern SAM the leaf
meristems have a bilateral symmetry. Maturation of the develop-
ing fern leaf is acroscopic, that is, toward the apex (Figure 5).
Its proximal portions mature first, with a wave of maturation
proceeding distally (Briggs and Steeves, 1958; Voeller, 1960).
As in other vascular plants, the leaves of ferns are arranged in a
fixed and predictive phyllotactic sequence around the shoot apex
(Schoute, 1938; Gifford and Foster, 1988). Early experimental
studies in leaf development were performed to understand how
and what determined where a leaf developed on the flank of the
SAM (White, 1971; Steeves and Sussex, 1989). In Dryopteris aris-
tata, incisions were made to isolate incipient leaf primordia from
the SAM and/or older primordia. The plants were allowed to grow
and the resulting phyllotaxy was examined (Wardlaw, 1949a,b,c).
This series of experiments showed that incisions around I1,
disrupted the phyllotaxy, and the primordia that subsequently
formed were misplaced. These studies supported earlier ones in
angiosperms concluding that adjacent primordia influence the
position of the incipient leaf primordia (Snow and Snow, 1932;
Wardlaw, 1949a,b). The angiosperm data was interpreted as leaf
primordia develop in the first available space on the meristem
(Snow and Snow, 1932; Wardlaw, 1949a,b; Steeves and Sussex,
1989).
However, space is not limited in the Dryopteris aristata shoot
tip compared to the angiosperm shoot tip, and therefore space
constraints did not provide the best explanation for the control of
phyllotaxy in ferns (Wardlaw, 1949a,b; Steeves and Sussex, 1989).
In addition, in the Dryopteris aristata phyllotaxy experiments, the
“misplaced” primordia grew faster than the adjacent older leaves,
which suggested that nearby older leaves inhibit the growth of
younger leaves (Wardlaw, 1949a,b,c). Therefore, the phyllotaxy
experiments in ferns were better interpreted using the field the-
ory of phyllotaxy as opposed to being directed by available space
(Wardlaw, 1949a; White, 1971; Steeves and Sussex, 1989). This
theory states that there are regions of inhibition around the
SAM and around the leaves and those regions are the ones that
control when and where a leaf primordium develops (Ward law ,
1949a,b,d; White, 1971; Steeves and Sussex, 1989). Experimental
studies have shown that older leaves not only inhibit the growth
and placement of nearby incipient leaf primordia but also have a
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Vasco et al. Fern leaf evo-devo
role in determining whether a leaf primordium develops as a leaf
or a shoot (reviewed in White, 1971).
Experiments were performed to understand the relationship
between the SAM and leaves in ferns. In Dryopteris aristata, if
all of the surrounding primordia are removed from a growing
shoot tip, then the apical meristem continues to grow and pro-
duce leaves (Wardlaw, 1947, 1949a,c; White, 1971). In addition,
isolated SAMs grown in sterile culture on media supplemented
with sucrose and auxin formed adult plants (White, 1971). The
results of these experiments indicate that the SAM is capable
of autonomous development and there are no signals coming
from leaves that direct shoot development. Experiments were
also developed to understand how a determinate dorsiventral
leaf arises from an indeterminate radial SAM. Sterile culture and
microsurgery experiments were designed to understand whether
the signals for a leaf to develop as a leaf come from the SAM, the
developing leaf itself, or the surrounding leaves.
Microsurgery experiments have provided further insight into
the relationship between the shoot apical meristem and leaf devel-
opment. If a shallow incision is made between an incipient leaf
primordium and the apex of the SAM, then the primordium still
develops as a leaf (Wardlaw, 1956). However, if a deep incision
separates an incipient leaf primordium and the SAM, then the
incipient leaf primordium develops as a shoot (Wardlaw, 1949b).
These results suggest that a leaf determining signal comes from
the SAM and that fern leaves are not determined as leaves imme-
diately upon arising from the SAM. To better understand when a
leaf is determined as a leaf, deep incisions were made between leaf
primordia in Dryopteris aristata at various developmental stages
(Cutter, 1954, 1956; White, 1971). If incisions were made between
the SAM and the leaf primordia at developmental stage P1, then
these isolated primordia were more likely to grow out as buds
(Cutter, 1954, 1956). However, if incisions were made between
the SAM and leaf primordia at developmental stage P4, then these
isolated primordia were more likely to grow out as leaves (Cutter,
1954, 1956). Leaf primordia P1, P2, and P3 are more plastic in
their development and after incisions, may grow out as buds,
although a few still develop as leaves. These experiments indicate
that a Dryopteris aristata leaf is determined as a leaf sometime
after stage P1. Although some results showed that if I1 is isolated
then it more likely develops as a bud, but in rare cases it devel-
oped as a leaf (Amer and Williams, 1957). Similar results were
found in Osmunda cinnamomea leaf primordia at developmen-
tal stages P1-P10 that were isolated and grown in sterile culture
(Steeves, 1961, 1963; White, 1971). These excised leaves varied in
their development, but generally the isolated younger primordia
developed as shoots and the older ones as leaves.
Sterile culture experiments were also used to investigate if a
leaf determining signal also came from leaves. In these experi-
ments, Osmunda cinnamomea leaf primordia were excised and
grown with other leaves. If isolated P3 leaves, which are still
plastic in their developmental potential, were grown with older
leaves (isolated P10 or P12 leaves), then the P3 leaves were more
likely to grow out as leaves instead of buds. (Kuehnert, 1967,
1969a,b,c; Haight and Kuehnert, 1969; White, 1971). However,
if isolated P3 leaves are grown with mature P14 leaves, then the
P3 leaves mostly developed as buds (Kuehnert, 1969a,b; White,
1971). These experiments indicate that a leaf determining signal
is coming from older yet still developing leaves. Further sterile
culture experiments were performed to determine if the signal
coming from leaves involved a diffusible substance. Isolated P3
primordia were grown on media containing homogenized leaves
(P10 or P12), which resulted in most of P3 primordia developing
as leaves (Kuehnert, 1969b; White, 1971). These results are similar
to those found with isolated P3 primordia grown with P10 or P12
leaves. Isolated P3 leaves were also grown with older leaves that
were not in physical contact with each other. In these experiments
the leaf pairs were separated by either an impermeable barrier or
a permeable barrier. The results of these experiments were similar
to the previous experiments that show that the older primordia
have a leaf determination effect on P3 and that the determination
effect appears to be mediated by a diffusible substance.
In the experiments with Osmunda cinnamomea,mostofthe
excised P3 leaves develop as buds; however, some still develop as
leaves. This suggests that another part of the plant has a deter-
mining influence on leaf development besides older leaves. If all
of the leaf primordia are removed from the SAM, then the incip-
ient leaf primordium (I1) develops as a leaf (Hicks and Steeves,
1969; White, 1971). In addition, if I1 is isolated from the apex then
the incipient primordium develops as a shoot. These experiments
indicate that the SAM is not only capable of autonomous devel-
opment but also has a leaf-determining influence on incipient
primordia.
A defining characteristic of nearly all vascular plant leaves is
that they have adaxial/abaxial identities. This distinguishes leaves
from shoots, which are radially symmetric. However, Ward l aw
(1949b,d) concluded that initially leaf primordia and shoot buds
of Dryopteris aristata are histologically indistinguishable. In some
fern microsurgery experiments where leaf primordia (P4-P9)
were isolated from the SAM by incisions, some determinate leaves
grew out that had near radial symmetry and buds in their axils
(Wardlaw, 1945, 1947, 1949c; Cutter, 1954). Similar results were
found in angiosperms where incisions separating I1 from the
shoot apex resulted in the development of radially symmetric
organs (Snow and Snow, 1935; Sussex, 1951). The depth of the
incisions and the location of the cuts had an impact on the fate
of I1 (Sussex, 1951; Cutter, 1954). If incisions were made close
to I1 then the primordia that grew out were radially symmet-
ric. These microsurgery experiments in ferns and angiosperms
indicated that the shoot apical meristem had an influence on the
adaxial/abaxial identity of leaves (Sussex, 1951; Cutter, 1954).
In summary, the microsurgery experiments in ferns supported
the field theory of phyllotaxy; that is, there is a field of inhibition
around the SAM and existing primordia that prevents incipient
leaf primordia developing too closely to these fields. Microsurgery
and sterile culture experiments demonstrated that the SAM does
not require attached leaf primorida or leaves to continue the
development of the adult shoot and therefore the SAM is capa-
ble of autonomous development. Microsurgery and sterile culture
experiments also revealed that the SAM and developing leaves
influence the developmental fate of leaf primordia. In addition,
the signal from developing leaves involves a diffusible substance.
Finally, the SAM was found to play a role in the specification
of adaxial/abaxial identity in leaves. Molecular genetic studies
www.frontiersin.org September 2013 | Volume 4 | Article 345 |11

Vasco et al. Fern leaf evo-devo
in angiosperm leaf development are beginning to provide clues
into the nature of the signaling that occurs between the shoot
apex and leaf primordia (Byrne, 2012). Auxin signaling has been
shown to play an integral role in angiosperm leaf development
(Byrne, 2012; Traas, 2013). In angiosperms, polar auxin trans-
port is important in specifying phyllotaxy, leaf patterning, and
is integral in the leaf development network involving 2 key tran-
scription factors, Class I KNOX and Class III HD-Zip (see below).
The experimental evidence indicates that similar signaling occurs
between the shoot apex and leaf primordia in ferns, however, lit-
tle is known about the molecular nature of these signals in ferns.
Auxin is a likely candidate for signaling in fern leaf development.
Auxin has been shown to effect the leaf complexity in the fern
Marsilea as well as crozier development (Allsopp, 1952; Steeves
and Briggs, 1960). However, much work remains to be done on
the molecular genetics of leaf development in ferns.
MOLECULAR GENETICS OF FERN LEAF DEVELOPMENT
Much is known about the molecular genetics of the leaf develop-
mental network in angiosperms (Byrne, 2012). However, outside
of the seed plants, comparative molecular studies so far have
focused on only two transcription factors: Class I KNOX and
Class III HD-Zip (Harrison et al., 2005; Prigge and Clark, 2006;
Floyd and Bowman, 2006). In all vascular plants, leaves arise from
the SAM. Two criteria can be used to differentiate the leaf from the
shoot in most vascular plants: determinacy and adaxial/abaxial
polarity (Arber, 1941). Interestingly, these are exactly the roles
played by Class I KNOX and Class III HD-Zip genes, respec-
tively. These two genes have been used in comparative studies
to better understand leaf evolution and development in vascular
plants. Class I KNOX genes, have important roles in maintain-
ing the indeterminacy of the SAM (reviewed in Hay and Tsiantis,
2010; Byrne, 2012; Townsley and Sinha, 2012). They are downreg-
ulated in determinate simple leaves but upregulated in compound
leaves to specify pinnae (Bharathan et al., 2002;reviewedinHay
and Tsiantis, 2009). Class III HD-Zip proteins have important
roles in the development of the SAM, vasculature, and the adaxial
region of the leaf in flowering plants (Prigge et al., 2005; Floyd and
Bowman, 2006, 2007). The specification of adaxial and abaxial
identities has been suggested to be important for lamina out-
growth (Waites and Hudson, 1995). Therefore, we know the genes
that are important for specifying determinacy and polarity—two
key characteristics of leaves, and can study the role of these two
genes in vascular plants to better understand the evolution and
development of leaves.
Class I KNOX and Class III HD Zips genes have been well-
studied across the flowering plants and to a lesser extent in
gymnosperms (reviewed in Floyd and Bowman, 2007; Efroni
et al., 2010; Floyd and Bowman, 2010; Hay and Tsiantis, 2010;
Byrne, 2012; Townsley and Sinha, 2012; Yamaguchi et al., 2012).
Studies of Class I KNOX and Class III HD Zip expression have
also been performed in lycophytes. These studies came to dia-
metrically opposite conclusions about the conservation of a leaf
developmental mechanism between microphylls and megaphylls
(Harrison et al., 2005; Floyd and Bowman, 2006; Prigge and
Clark, 2006). The expression of Class III HD Zip genes have not
been studied in ferns; however, there have been several studies of
Class I KNOX gene expression in ferns. Because ferns occupy a
key phylogenetic position as sister to the seed plants (Figure 1),
comparative studies in diverse fern species may help to elucidate
the evolution of these leaf developmental regulators and their role
in leaf development.
Comparative studies of the Class I KNOX genes have been
performed in the leptosporangiate ferns Anogramma chaerophylla
(Pteridaceae), Ceratopteris richardii (Pteridaceae), and Osmunda
regalis (Osmundaceae, Figure 3O), all of which have divided
leaves (Bharathan et al., 2002; Harrison et al., 2005; Sano et al.,
2005). These studies found that, as in seed plants with compound
leaves, Class I KNOX genes are expressed in the meristem and in
the developing leaves. Yet unlike seed plants, Class I KNOX genes
were not found to be down-regulated in leaf primordia. These
results might suggest either the independent origin of megaphylls
in ferns and seed plants, or may simply reflect the prolonged inde-
terminacy of fern leaves. In flowering plants, Class I KNOX genes
are down-regulated while ARP genes are up-regulated in leaf pri-
mordia (reviewed in Floyd and Bowman, 2007, 2010; Hay and
Tsiantis, 2010; Efroni et al., 2010). In addition to Class I KNOX
expression, ARP protein expression was also studied in Osmunda
(Harrison et al., 2005). This study found that the expression of
Class I KNOX and ARP genes overlapped in the meristem and
leaves unlike the complementary expression patterns in flowering
plants (Harrison et al., 2005). In support of their results in lyco-
phytes, Harrison et al. (2005) suggested that this may reflect the
ancestral role of the leaf developmental module of Class I KNOX
and ARP in shoot branching, and that this module was recruited
independently during leaf evolution in vascular plants.
FUTURE DIRECTIONS IN FERN LEAF EVO-DEVO
The results of molecular genetic studies of KNOX/ARP in
Osmunda and the KNOX regulatory network in compound-
leaved angiosperms have brought the partial shoot theory of
Agnes Arber back into discussions of leaf evolution and devel-
opment (Arber, 1941; Barkoulas et al., 2007; Koenig et al., 2009).
Arber considered the shoot to be the fundamental organ of the
plant, and that all leaves are “partial shoots, and only partial
shoots” because their indeterminate growth and radial symmetry
are repressed (Arber, 1941).
The morphology of fern leaves, in particular, has shoot-like
characteristics. Fern leaves have extended indeterminacy, and
some have indeterminate leaves (see Leaf indeterminacy section
above). Anatomically, Wardlaw (1949b,d) found the shoot and
leaf primordia indistinguishable from each other. In addition,
experimental studies found that fern leaves are not determined
as leaves until later in their development but when first specified
have more of a shoot identity (reviewed in White, 1971). Further
support for the shoot-like characteristics of fern leaves comes
from recent studies in Nephrolepis exaltata (Nephrolepidaceae)
that found that there is a reiteration from shoot apical meristem
to leaf to pinnae that suggests a reiteration of a shoot program
(Sanders et al., 2011).
The results from experimental leaf studies performed in ferns
parallel the results found in angiosperms. These results sug-
gest that similar developmental processes are at work in both
plant groups, furthermore paleobotany studies suggest that these
Frontiers in Plant Science | Plant Evolution and Development September 2013 | Volume 4 | Article 345 |12

Vasco et al. Fern leaf evo-devo
processes may not be occurring in the same sequence or have
similar timing (Boyce and Knoll, 2002; Sanders et al., 2009).
Molecular genetic studies of leaf evo-devo in ferns could not
only fill the gap that exists in our understanding of fern leaf
development, but also provide crucial data to the debate on the
evolution and origin of megaphylls. For example, developmental
genetic studies in ferns with diverse morphologies (e.g., simple
vs. compound leaves) could provide the molecular basis for their
morphological diversity. In addition, studies of leaf development
genes in Psilotum (Psilotaceae), Ophioglossum (Ophioglossaceae),
and Equisetum (Equisetaceae) could provide crucial molecular
data to better interpret the leaf morphology of these distinctive
genera. A vast amount of fern research has been performed in
the fields of paleobotany, morphology, development, and experi-
mental biology. The field of developmental genetics is the missing
piece and when integrated with data from other fields will help
us develop more robust hypotheses of fern leaf evolution and
development and more broadly hypotheses of leaf evolution and
development in vascular plants.
ACKNOWLEDGMENTS
We thank the issue editors for inviting us to write a review for
the special issue. Dennis Stevenson for his insightful ideas and
bibliography. This research was supported by a grant to Moran
and Ambrose from the United States National Science Foundation
(DEB-1020443).
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Conflict of Interest Statement: The
authors declare that the research
was conducted in the absence of any
commercial or financial relationships
that could be construed as a potential
conflict of interest.
Received: 28 May 2013; accepted: 15
August 2013; published online: 04
September 2013.
Citation: Vasco A, Moran RC and
Ambrose BA (2013) The evolution, mor-
phology, and development of fern leaves.
Front. Plant Sci. 4:345. doi: 10.3389/fpls.
2013.00345
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