Annals of Botany 112: 1031–1043, 2013
doi:10.1093/aob/mct169, available online at www.aob.oxfordjournals.org
Ultrastructure of stomatal development in early-divergent angiosperms
reveals contrasting patterning and pre-patterning
Paula J. Rudall and Emma V. W. Knowles
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK
* For correspondence. E-mail [email protected]
Received: 18 February 2013 Returned for revision: 3 April 2013 Accepted: 12 June 2013 Published electronically: 21 August 2013
† Background and Aims Angiosperm stomata consistently possess a pair of guard cells, but differ between taxa in the
patterning and developmental origin of neighbour cells. Developmental studies of phylogenetically pivotal taxa are
essential as comparative yardsticks for understanding the evolution of stomatal development.
† Methods We present a novel ultrastructural study of developing stomata in leaves of Amborella (Amborellales),
Nymphaea and Cabomba (Nymphaeales), and Austrobaileya and Schisandra (Austrobaileyales), representing the
three earliest-divergent lineages of extant angiosperms (the ANITA-grade).
† Key Results Alternative developmental pathways occur in early-divergent angiosperms, resulting partly from differences in pre-patterning and partly from the presence or absence of highly polarized (asymmetric) mitoses in the
stomatal cell lineage. Amplifying divisions are absent from ANITA-grade taxa, indicating that ostensible similarities
with the stomatal patterning of Arabidopsis are superficial. In Amborella, ‘squared’ pre-patterning occurs in intercostal regions, with groups of four protodermal cells typically arranged in a rectangle; most guard-mother cells are
formed by asymmetric division of a precursor cell (the mesoperigenous condition) and are typically triangular or trapezoidal. In contrast, water-lily stomata are always perigenous (lacking asymmetric divisions). Austrobaileya has
occasional ‘giant’ stomata.
† Conclusions Similar mature stomatal phenotypes can result from contrasting morphogenetic factors, although the
results suggest that paracytic stomata are invariably the product of at least one asymmetric division. Loss of asymmetric divisions in stomatal development could be a significant factor in land plant evolution, with implications
for the diversity of key structural and physiological pathways.
Key words: Epidermal pre-patterning, meristemoids, mesogenous stomata, perigenous stomata, stomatal
development, ANITA, early-divergent angiosperms.
IN T RO DU C T IO N
Stomatal structure is highly conserved across land plants, in
which a symmetric pair of specialized guard cells delimits a
central pore (Sack, 1987; Ziegler, 1987). However, when
viewed from a developmental perspective, the patterning of the
stomatal complex (i.e. the stoma and neighbouring cells)
differs among taxa, depending on asymmetric divisions of one
or more specialized precursor cells and lateral divisions of neighbouring cells. Most hypotheses of stomatal evolution in angiosperms are based on comparative studies of mature stomata of
both extant and fossil taxa, with a primary focus on three
widely recognized stomatal types – anomocytic, paracytic and
stephanocytic – which differ in the patterning of their neighbour
cells (e.g. Wilkinson, 1979; Doyle and Endress, 2000; Carpenter,
2005; Rudall et al., 2012; for definitions see Table 1). In anomocytic stomata, the neighbour cells (located immediately adjacent to the guard cells) resemble other pavement epidermal
cells. Paracytic stomata possess one or more pairs of modified
lateral neighbour cells (termed subsidiary cells), whereas stephanocytic stomata possess a rosette of several distinct subsidiary
cells surrounding the guard cells. However, although such descriptive terms are useful for classification, they frequently
combine non-homologous patterns that have been achieved via
contrasting developmental pathways (e.g. Fryns-Claessens and
Van Cotthem, 1973; Payne, 1979).
In all angiosperm species that have been studied in detail to date,
stomatal development typically commences with the asymmetric
division of a meristemoid mother cell (MMC) to form a smaller
meristemoid and a larger stomatal-lineage ground cell (SLGC)
(Payne, 1979; Nadeau and Sack, 2003; Robinson et al., 2011;
Pillitteri and Torii, 2012). In many species, only one asymmetric
division occurs, and the resulting meristemoid forms a guardmother cell (GMC), which divides symmetrically to form a pair
of guard cells (e.g. in many monocots, including rice and
Tradescantia: Croxdale, 1998). This type of development, resulting in a single SLGC, is termed mesoperigenous (e.g. Payne,
1979). By contrast, in some eudicots (e.g. Arabidopsis), two or
more successive asymmetric divisions (termed amplifying divisions) can occur in the same cell lineage. In Arabidopsis, a
series of asymmetric amplifying divisions occurs, each producing
an SLGC and a meristemoid (Zhao and Sack, 1999; Robinson
et al., 2011; Pillitteri and Torii, 2012). After two or three amplifying divisions, the final meristemoid becomes a GMC and divides
symmetrically to form a pair of guard cells. The asymmetric divisions are orientated away from each other, so that the SLGCs form
a spiral surrounding the guard cells. This type of development,
resulting in an encircling ring of SLGCs, is termed mesogenous
(e.g. Payne, 1979). Molecular developmental studies, especially
on Arabidopsis, have identified numerous genes that together contribute to the regulation of stomatal development (e.g. Peterson
et al., 2010; Serna, 2011).
# The Author 2013. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Rudall & Knowles — Stomata of early-divergent angiosperms
TA B L E 1. Glossary of stomatal terms (see also Bünning, 1952; Payne, 1979; Carpenter, 2005; Rudall et al., 2013)
Term
Definition
Guard cell
Guard-mother cell (GMC)
Meristemoid
Meristemoid-mother cell (MMC)
Stoma (plural stomata)
Stomatal complex
Stomatal-lineage ground-cell (SLGC), also termed a
mesogenous subsidiary cell
Mature stomata
Anomocytic
Paracytic
Stephanocytic
Developing stomata
Mesogenous
Mesoperigenous
Perigenous
One of a pair of specialized epidermal cells that together delimit the stomatal pore
Final stomatal precursor cell that divides symmetrically to form a pair of guard cells
Specialized precursor cell
Initial precursor cell that divides asymmetrically
Pair of guard cells plus the central pore that they delimit
Stoma plus all adjacent modified epidermal cells (subsidiary cells)
Larger daughter cell resulting from an asymmetric division of a meristemoid; can differentiate into
a pavement cell or can divide again asymmetrically
Lacking modified subsidiary cells (term applied to mature stomata)
Possessing one or more pairs of lateral subsidiary cells (applied to mature stomata; includes
laterocytic types)
Possessing a distinct rosette of subsidiary cells (applied to mature stomata; includes actinocytic
and tetracytic stomata)
Stomata with subsidiary cells formed from the same cell lineage as the guard cells, following one
or more asymmetric divisions
Stomata with a combination of both mesogenous and perigenous subsidiary cells
Stomata formed entirely by symmetric divisions; subsidiary cells not resulting from the same
immediate cell lineage as the guard cells
Understanding evolutionary pathways requires a more explicit
phylogenetic context than over-simplistic comparisons between
dicotyledons (a non-monophyletic group) and monocotyledons.
Such comparisons are most commonly exemplified by the model
organisms Arabidopsis and rice, respectively (e.g. Facette and
Smith, 2012). Modern classifications of extant angiosperms,
based primarily on molecular phylogenetic data (e.g. APG III,
2009), recognize two major species-rich clades – monocots
and eudicots – plus about five relatively species-poor
relictual lineages (Fig. 1). Three of these relictual lineages
(Amborellales, Nymphaeales, Austrobaileyales) form a stepwise
series of early-divergent angiosperms (sometimes termed the
ANITA-grade or ANA-grade) that is placed immediately above
the root node of the angiosperms in most analyses (e.g. Graham
and Iles, 2009; summarized by Rudall et al., 2013).
ANITA-grade angiosperms are disproportionately significant
as potential yardsticks for morphological evolution in more
derived angiosperms. Yet, although a few studies have examined
mature stomatal patterns in these taxa, remarkably little is known
about their development. Carpenter (2005) reported that
Amborella, Austrobaileya and Schisandra possess both paracytic
and stephanocytic stomata, but anomocytic stomata are rare. In
contrast, Nymphaeales possess anomocytic and more-or-less stephanocytic stomata but entirely lack paracytic stomata. However,
a comparative study of mature stomata cannot readily determine
whether a neighbour cell is mesogenous or perigenous (Table 1).
Developmental studies of these phylogenetically pivotal taxa
are essential to understand both the homologies of stomatal types
and the evolution of stomatal development in angiosperms. Here,
we present an ultrastructural study of developing stomata in
leaves of Amborella, Nymphaea, Cabomba, Austrobaileya and
Schisandra, together representing the three ANITA-grade
lineages (Fig. 1).
MAT E RI ALS A ND METH O DS
Leaves of five species representative of early-divergent angiosperms were examined for this study, all sampled from specimens
growing at RBG Kew (listed here as HK followed by the Kew accession number or sine numero). For each species, a selection of
foliage leaves at different stages of development was removed
from each plant. No cotyledons were examined in this study.
(1) Amborella trichopoda Baill., a shrub endemic to New
Caledonia, is the sole extant species of the family
Amborellaceae and order Amborellales; this species is
usually placed molecularly as sister to all other extant
angiosperms, or sometimes as sister to Nymphaeales.
Leaves were collected from a specimen that was kindly supplied by the Bonn Botanic Garden.
(2) Austrobaileyales includes three families of woody plants:
Austrobaileyaceae, Schisandraceae and Trimeniaceae, together encompassing approx. 70 species in five genera.
Material examined consisted of specimens grown at
RBG Kew (HK): Austrobaileya scandens C.T.White (HK
2012 – 64) and Schisandra rubriflora Rehder and
E.H.Wilson (HK 1969 – 19803).
(3) The order Nymphaeales consists of approx. 90 aquatic or
semi-aquatic species assigned to eight or nine genera. The
order includes the water-lily families Nymphaeaceae and
Cabombaceae. Two specimens grown at RBG Kew were
examined: Nymphaea violacea Lehm. (HK 2008– 566)
and Cabomba aquatica Aubl. (HK s.n.). Leaves of
N. violacea examined ranged from a full-sized leaf with
mature stomata to submerged leaves at different sizes.
C. aquatica has two leaf types: finely divided submerged
leaves that lack stomata, and floating peltate leaves that
have stomata on the upper surface only. A range of developmental stages of floating leaves were examined.
For light microscopy (LM) and transmission electron microscopy (TEM) of all species except C. aquatica, samples from
the mid-regions of leaves were cut into small squares and fixed
in 3 % phosphate-buffered glutaraldehyde followed by 1 %
osmium tetroxide. Samples were taken through a graded
Rudall & Knowles — Stomata of early-divergent angiosperms
Major angiosperm clades
Austrobaileyales
Nymphaeales
Early-divergent angiosperms
Amborella
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magnoliids
monocots
eudicots
F I G . 1. Relationships of angiosperms, based on the Angiosperm Phylogeny Group classification (APG III, 2009).
ethanol and ethanol/LR-White resin series before embedding
and sectioning. Ultrathin sections (50 – 100 nm thick) were collected on Formvar-coated copper slot grids and imaged in a
Hitachi H-7650 TEM with integral AMT XR41 digital camera.
Semithin sections (0.5 mm thick) were mounted onto microscope slides, stained with toluidine blue, mounted using DPX
mountant (a mixture of distyrene, a plasticizer and xylene),
and examined using a Leitz Diaplan photomicroscope.
Composite images were merged using Adobe Photoshop.
Measurements were taken from scanning electron microscopy
(SEM) and LM images using the image processing program
ImageJ 1.46r (http://imagej.nih.gov/ij/docs/guide).
For SEM, leaves were fixed in 70 % ethanol. Dissected leaves
were critical-point dried using an Autosamdri 815B CPD,
mounted onto SEM stubs, coated with platinum using an
Emitech K-550 sputter coater, and examined at 2 kV using a
Hitachi cold-field emission SEM S-4700.
In C. aquatica, leaves at a range of developmental stages were
cleared using a modified version of Herr’s clearing fluid (lactic
acid/chloral hydrate/phenol/clove oil/Histoclear, in proportions
2 : 2 : 2 : 2 : 1, by weight) and examined using differential interference contrast optics on a Leitz Diaplan photomicroscope fitted
with a Leica DC500 digital camera.
R E S U LT S
Stomatal distribution
In all four species examined, most stomata are placed at least one
cell apart, a pattern that is often said to follow the
one-cell-spacing rule (e.g. Hara et al., 2007). However, occasional immediately contiguous stomata are not uncommon in
Amborella (Fig. 2I), Austrobaileya and Schisandra, although
they were not observed here in Nymphaea.
Amborella trichopoda (Figs 2– 5)
Mature stomata. The leaf is relatively thick (Fig. 2A) and bears
stomata only on the abaxial surface. Mature stomata are fairly
regular in both shape and size, ranging from approx. 30 mm
long in intercostal areas to approx. 40– 45 mm long in costal
areas; they are orientated apparently randomly with respect to
each other (Fig. 2B, C). The guard cells have dense, prominent
thickenings of the inner and outer periclinal walls that extend
across the top and bottom of the cell in transverse section
(Fig. 2E, F) and across the cell in paradermal section
(Fig. 2H – K). A thick cuticle is present, exhibiting prominent
outer cuticular ridges around the pore opening (Fig. 2E, F).
Inside the substomatal cavity, very small ridges are present on
the neighbouring pavement cells that partially underlie the
guard cells (Fig. 2E, F). Starch granules are present in the
guard cells (Fig. 2D), but are relatively infrequent in neighbouring epidermal cells. Some stomata have an irregular pattern of
neighbouring cells, but the majority have a narrow subsidiary
cell (or sometimes two smaller subsidiary cells) on each side.
Leaf abaxial epidermal development. In very young leaves, the
protodermal cells on the midrib and leaf margins are arranged
in linear cell files, so that each cell division is parallel with the
previous division (Fig. 3A). Stomata located on the midrib are
initiated before the intercostal stomata. In intercostal regions,
prior to GMC formation, protodermal cells show a ‘squared’ arrangement, consisting of groups of four cells roughly arranged in
a rectangle or less often a T-shape (Fig. 3B). (A squared pattern is
also present on the adaxial surface, where stomata are absent.)
To form this squared pattern during early leaf development,
each (approximately rectangular) epidermal cell divides symmetrically across its narrowest width, usually perpendicular to the
previous division (see Fig. 5A). Occasional groups of only
three cells resulted from failure of one of the cell divisions. The
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Rudall & Knowles — Stomata of early-divergent angiosperms
F I G . 2. Amborella trichopoda, abaxial surface of mature leaf. (A) SEM showing stomatal distribution, primarily intercostal, but with some stomata present over veins.
(B, C) Details of intercostal stomata (SEM). (D) Paradermal view of a single stoma (TEM). (E) Transverse section of a single stoma showing differential wall thickenings, prominent outer cuticular ridges and less conspicuous ridging inside stomatal chamber (TEM). (F) Transverse section (LM) of a single stoma showing differential
wall thickenings, prominent outer cuticular ridges and inconspicuous ridging inside stomatal chamber. (G) Transverse section of leaf (LM). (H) Paradermal section of
stoma (LM). Abbreviations: ir ¼ inner ridging, n ¼ nucleus, ocr ¼ outer cuticular ridge, s ¼ stoma, st ¼ starch, vb ¼ vascular bundle, wt ¼ wall thickening. Scale
bars: (A) ¼ 1 mm, (B) ¼ 100 mm, (C, G) ¼ 50 mm, (D) ¼ 2 mm, (E, F, H) ¼ 10 mm.
daughter cells then each iteratively expand and divide again symmetrically across their narrowest width, resulting in a squared
pattern that is only slightly disordered by different rates of expansion and division. The pavement cells expand as the leaf matures.
We observed GMCs, GCs and meristemoids in the same small
intercostal region, demonstrating that stomata develop sequentially. The stomata are all either perigenous or mesoperigenous;
none is entirely mesogenous (Glossary: Table 1). The perigenous
stomata develop by symmetric division of cells that have already
divided symmetrically (Figs 3G, H, 4D and 5B); no asymmetric
divisions have occurred in their developmental pathway.
Perigenous stomata are apparently among the earliest to
develop and are possibly relatively few in number, although
more data are required to confirm this hypothesis. In mesoperigenous stomata, one of the protodermal cells divides asymmetrically to form a smaller GMC and a larger SLGC;
subsequently, the GMC divides symmetrically to form a pair
of guard cells (Figs 3I– K, 4B, C, F and 5C). GMCs are identifiable by their relatively small size, small or absent vacuole, and
darker appearance, but especially by their angular shape,
which is triangular or trapezoidal, the longest wall being adjacent
to its sister cell (the SLGC). Intercostal GMCs appear randomly
distributed; distances between them differ, at least partly due to
the variable orientation of cell division. Asymmetric divisions of
cells adjacent to stomata are uncommon, and are typically orientated with the smaller cell (the GMC) furthest from the older
stoma, thus maintaining one-cell spacing (e.g. Fig. 3I).
Austrobaileya scandens (Figs 6 and 7)
Mature stomata. Mature intercostal leaf stomata are approx.
45 mm long, with occasional ‘giant’ stomata up to 60 mm long
(Fig. 6B). Each stoma is surrounded by a ring of 5 – 7 neighbour
cells, partly resulting from lateral divisions of neighbour cells
(Fig. 7A). Most stomata are bordered by a ring of concentric cuticular striations, although giant stomata reliably have radiating
striations (Fig. 6A – D). The cuticular ridges that extend over
the pore are not striated. The guard cells have thickened anticlinal
walls opposite the pore, and relatively thin walls bordering the
pore. Two prominent cuticular ridges (inner and outer) are
present around the pore opening. Stomata in older leaves have
prominent wall thickenings in the guard cells (Fig. 7A). In
younger stomata, before the walls become thickened, numerous
chloroplasts are present in the guard cells (Fig. 7B, C, E, F, I).
F I G . 3. Amborella trichopoda: light micrographs and diagrams illustrating patterns of cell divisions during early development of the abaxial leaf epidermis.
(A) Young developing leaf, with red lines outlining the likely boundaries of the original longitudinal cell files that existed prior to squared divisions. (B) Region
with squared groups of cells. (C, D) Two versions of the same micrograph, with a group of cells outlined in red in (D) to illustrate division pattern. More recent
walls are drawn as not interrupting older ones, to indicate their sequence of formation. (E) Group of cells highlighted in dark green box in (C), showing the
squared pattern of division (diagram, Fig. 7A), and illustrating how sister cells divide at different rates. Cell A was sister to cell B; they each divided into A1 and
A2 and B1 and B2, respectively. B2 has already divided again, forming B2a and B2b; A2 is in the process of dividing. (F) Diagram showing the series of divisions
highlighted in (D). The cells would initially have been smaller and more regularly shaped than they appear at this stage. (G, H) stomata formed directly by symmetric
division of protodermal cells, without asymmetric division (diagram, Fig. 5B). (I) Stoma and meristemoid both formed by asymmetric divisions (diagram, Fig. 5C).
(J) Meristemoid formed by asymmetric division. (L) Stoma formed by asymmetric division. (L) Older stoma; developmental origin not clear. Abbreviations: g ¼ guard
cell, gmc ¼ guard mother cell (GMC), m ¼ meristemoid, sd ¼ symmetric division, slgc ¼ stomatal lineage ground cell (SLGC). Scale bars: (A, B) ¼ 20 mm;
(C–E, G –K) ¼ 10 mm.
Rudall & Knowles — Stomata of early-divergent angiosperms
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Rudall & Knowles — Stomata of early-divergent angiosperms
Leaf epidermal development. Formation of stomata starts earliest
on the midrib, then begins over veins and near the midrib, and
finally in intercostal regions. Protodermal cells occur in linear
files over the midrib, the leaf margin and some veins (Figs 6E
and 7D). Orientation of older stomata suggests that midrib
GMCs are formed by a division parallel with the other divisions
within the cell file. Developing stomata over veins are slightly
longer than intercostal stomata on the same leaf. In intercostal
regions, protodermal cell division follows the ‘squared’ pattern
already documented in Amborella (Fig. 6F). As in Amborella,
cellular arrangement also indicates an underlying intercostal prepatterning that is linear (Fig. 6E). On both the midrib and the
intercostal regions, GMCs, GCs and meristemoids are all
present in the same small sample.
In intercostal regions, GMC orientation is often difficult to determine because of the circular cell shape (Fig. 7E, F). Intercostal
GMCs and new guard cell pairs appear circular or pentagonal in
paradermal section, probably depending on the angle and/or
level of sectioning (Fig. 7E – I). Guard cells are initially thin
walled and circular (Fig. 7B, C), but become more elongated
and their walls thicken (Fig. 7A).
Schisandra rubriflora (Fig. 8)
Mature stomata. As in Austrobaileya, the mature guard cells are
very thick-walled, leaving relatively little space for cytoplasm
(Fig. 8D). Two small cuticular ridges (inner and outer) are
present around the pore opening. Mature intercostal leaf
stomata are approx. 50 mm long, with occasional ‘giant’
stomata up to 75 mm long; they appear randomly orientated in
intercostal regions (Fig. 8C).
Leaf epidermal development. In the developing leaf, cells divide in
linear files along the midrib, but it was difficult to distinguish
cell-division patterning in intercostal regions, as the presence
of large oil cells disrupts the underlying pattern (Fig. 8A, B).
Nymphaea violacea (Figs 9 and 10)
Mature stomata. Stomata are present on the adaxial (upper)
surface of the floating leaf, which is very thick, including a palisade adaxial mesophyll and spongy abaxial mesophyll containing astrosclereids (Fig. 9B). Mature stomata are relatively small
(approx. 20 mm long) and are regular in size, shape and orientation (Fig. 9A, E). Each stoma is surrounded by 4 – 8 neighbour
cells, which have undulating anticlinal walls. Each neighbour
cell is often shared by two stomata. The guard-cell walls are generally thin, but slightly thickened bordering the pore. A single
small cuticular ridge surrounds the narrow pore. The pore
opens into a substomatal cavity (Fig. 9C, D, F). Ziegler (1987)
suggested that stomata in Nymphaea alba and Nuphar lutea are
non-functional because they lack substomatal cavities, but our
results clearly show substomatal cavities in the palisade mesophyll of Nymphaea violacea. In paradermal view, each guard
cell contains a row of starch plastids along the outer anticlinal
edge, and a large, elongated central nucleus (Fig. 10I).
The cuticle is apparently chambered and irregularly thickened
(Fig. 9C, D, F). A cuticular thickening above the wall that separates the guard cells eventually splits when the guard cells pull
apart to generate the stomatal pore, thus forming a cuticular
ridge. Although a narrow pore forms between the guard cells
when the leaf is still submerged, until it reaches the surface
the cuticular ridges keep the pore sealed to prevent water from
entering.
Leaf epidermal development. Prior to GMC formation, adaxial cell
division follows a squared pattern (Fig. 10A, B), except over the
main veins, where epidermal cells occur in approximately linear
files. We found no evidence of asymmetric divisions during stomatal development in Nymphaea violacea, indicating that protodermal cells give rise directly to GMCs. GMCs are formed
more-or-less simultaneously and are regularly spaced in a grid
pattern (Fig. 10C). Younger GMCs are usually pentagonal or
hexagonal, depending on the number of neighbour cells (typically 4 – 7). Their walls are straight or slightly convex in paradermal
section. GMCs each have a large, round central nucleus; they lack
the large vacuoles visible in developing pavement cells and
appear darker than pavement cells.
With a few exceptions, most neighbouring GMC divisions are
orientated in the same direction (Figs 9E and 10D, E). After
guard-cell formation, both the guard cells and the pavement
cells enlarge. The pavement cells probably do not divide
further, as the spacing between stomata appears to be the same
in developing and mature leaves. Their anticlinal walls become
sinuous. The guard cells elongate and differentiate; starch granules form soon after guard-cell formation, and the vacuole
enlarges as the leaf extends upwards towards the water surface.
Cabomba aquatica (Fig. 11)
Cabomba aquatica has two leaf types: finely divided submerged leaves that lack stomata, and floating peltate leaves that
have stomata on the upper surface. A range of developmental
stages of floating leaves were examined using LM of cleared
leaves. Very young leaves (Fig. 11A) show the squared patterning that is also typical of N. violacea. At this stage, it is impossible to determine which cells will form stomata. In slightly
older leaves (Fig. 11B, C), some of the protodermal cells have
become rounded and slightly domed; some of these cells have
already divided symmetrically to form a pair of guard cells.
Subsequently, all the cells enlarge, although pavement cells
enlarge more than the guard cells. Most stomata are orientated
on the same direction (Fig. 11D, E), with the division plane parallel to the veins that radiate from the centre of the peltate leaf.
However, occasional stomata are orientated differently, often
at right angles to the other stomata (Fig. 11F). The mature
surface is covered in tiny crystals (Fig. 11G, H). Each mature
stoma has a substomatal cavity (Fig. 11H, I).
D IS C US S IO N
Mature stomata: wall thickenings and ‘giant’ stomata
In fully expanded leaves, the guard cells of Amborella,
Austrobaileya and Schisandra possess similar strong thickenings
of their inner and outer periclinal walls. In paradermal view,
these characteristic wall thickenings traverse the cell entirely
(Figs 2H– K, 7A and 8D). The cell lumen is deeper at the poles
and hence almost dumbbell-shaped in profile, as Sack (1987)
noted for some other angiosperms with reniform (kidneyshaped) guard cells such as Quercus ilex. In contrast, such
Rudall & Knowles — Stomata of early-divergent angiosperms
1037
F I G . 4. Amborella trichopoda: TEM micrographs illustrating patterns of cell divisions during stomatal development on the abaxial leaf epidermis. (A) Group of protodermal cells showing squared arrangement. (B, C) Meristemoids formed by asymmetric division (diagram, Fig. 5C). (D) Pair of guard cells formed by symmetric
division of protodermal cells, without asymmetric division (diagram, Fig. 5B). (E) Pair of guard cells showing starch. (F) Pair of guard cells and SLGC.
Abbreviations: dp ¼ division plate, g ¼ guard cell, m ¼ meristemoid, sd ¼ symmetric division, slgc ¼ stomatal lineage ground cell (SLGC).
A
B
C
F I G . 5. Amborella trichopoda: diagrams to illustrate different orientations of
cell division. (A) Protodermal ‘squared’ division. Each cell divides symmetrically across its narrowest width, so that each division is usually perpendicular to the
previous one. (B, C) Two contrasting trajectories of stomatal formation from
squared pattern: (B) perigenous stoma formed by symmetric division of protodermal cells; (C) mesoperigenous stoma formed by asymmetric division of protodermal cells to form a guard-mother cell (GMC: red) and a stomatal-lineage ground
cell (SLGC: yellow). Stomata are coloured green, GMC red and SLGC yellow.
Other cells are not coloured.
pronounced differential wall thickenings are entirely lacking in
Nymphaea, in which the wall is only slightly thickened on the
side bordering the pore (Fig. 9E). It is tempting to speculate
that such contrasting wall thickenings reflect differences in stomatal mechanics in different taxa, although Sack (1987) was
sceptical of their significance in this respect.
So-called ‘giant’ stomata are unusually large stomata that are
interspersed among more normal-sized stomata on a leaf surface;
recorded examples are present on leaves of the eudicot genera
Mangifera and Limoma (Sitholey and Pandey, 1971). We
observed giant stomata dispersed among smaller stomata
across the leaf blade in Austrobaileya scandens (Fig. 6B);
indeed, the giant stomata are distinguishable by their radiating
cuticular striations (see also Wilkinson, 1979). The difference
in cuticular patterning between giant and regular stomata in
this species indicates a difference in developmental timing, as
they are initiated at different times as the leaf expands. We hypothesize that the giant stomata are formed before smaller
stomata. In Austrobaileya the midrib stomata are generally
larger than those on the lamina, and are probably formed
earlier. In some other angiosperms, specialized enlarged
stomata occur over hydathodes on marginal leaf teeth. Such specialized stomata usually develop relatively early (e.g. Payne,
1979) and represent water pores with a different function to
typical photosynthetic stomata, although studies of
Arabidopsis have shown that the same genes control their early
development (Pillitteri et al., 2008). Thus, it seems likely that
differences in stomatal size across a single leaf are influenced
by differences in developmental timing rather than by contrasting spatial constraints.
Stomatal development and epidermal pre-patterning
Stomatal diversity is governed by several primary morphogenetic factors, which are regulated by a complex signalling
cascade of genes from several families (e.g. Peterson et al.,
2010; Rychel et al., 2010; Serna, 2011; Facette and Smith,
2012). These morphogenetic factors include: (1) the presence
of an asymmetric division in the stomatal cell lineage preceding
GMC formation; (2) a subsequent series of asymmetric cell divisions (termed amplifying divisions: Nadeau, 2009) in the GMC
1038
Rudall & Knowles — Stomata of early-divergent angiosperms
F I G . 6. Austrobaileya scandens: SEM micrographs of abaxial leaf surfaces. (A– C) Mature leaves, showing giant stoma with radiating striations in (B) and broken
stoma with encircling striations in (C). (D –G) Developing leaves. (D) Stoma with encircling striations starting to develop. (E, F) Young surfaces with pre-patterning
still visible, linear patterning outlined in (E) and squared patterning outlined in (F). (G) Young surface with a single pair of guard cells. Scale bars: (A, B, E, F) ¼
100 mm, (C) ¼ 10 mm, (D, G) ¼ 50 mm.
lineage; and (3) the developmental origin of neighbouring cells
by lateral divisions in adjacent cell lineages. Stomata of
Arabidopsis are characterized by both asymmetric and amplifying divisions, whereas asymmetric and lateral divisions characterize many monocots, most notably maize.
Our investigation necessarily incorporates epidermal prepatterning, which occurs prior to GMC initiation and has rarely
been described in detail in studies of stomatal development. In
Amborella, abaxial epidermal cells divide in linear cell files
during the initial elongation phase of the leaf, prior to leaf expansion (Fig. 5A). This linear pattern resembles the linear cell files
found in most monocot leaves, which are typically relatively
narrow. Thus, we speculate that the persistence of linear cell
files in monocots represents a neotenous condition, resembling
early development of laminar leaves. This hypothesis will be
tested in a future investigation. Marx and Sachs (1977) also
noted that in the eudicot Anagallis arvensis, epidermal cells
are arranged in files prior to stomatal formation. We also
observed linear pre-patterning in Austrobaileya, although the
early initiation of numerous large oil cells distorts protodermal
patterning in Schisandra.
In Amborella, following the onset of lateral expansion of the
leaf blade, cell divisions remain in linear files on the midrib
and margins but adopt a squared (or sometimes irregular)
pattern in intercostal regions. The squared groups of cells are
formed by perpendicular (rather than parallel) protodermal divisions, illustrated diagrammatically in Fig. 5A. Each cell divides
symmetrically across its narrowest width, so that each division
usually occurs perpendicular to the previous division.
Sometimes a daughter cell is square or unusually wide or short,
so that its division is approximately parallel to the previous one.
A similar squared pre-patterning is also evident in Austrobaileya
(Fig. 6F), Nymphaea (Fig. 10A, B) and Cabomba (Fig. 11A),
thus characterizing all three ANITA-grade lineages and indicating
that the squared condition could be ancestral (plesiomorphic) in
angiosperm leaves.
No obvious pre-patterning has been reported in Arabidopsis,
except in seedling hypocotyls, where stomata are formed in
files of cells overlying the junctions between the mesophyll
cells (Berger et al., 1998). The apparent lack of an obvious
squared pattern in Arabidopsis could be related to the presence
of amplifying divisions during stomatal development (see
below), perhaps due to differences in timing of leaf expansion.
Studies of pre-patterning in a taxonomically broad range of
angiosperms will help to determine the evolutionary significance
of this feature, which is clearly related to broader – and inevitably complex – issues of leaf development.
Lateral divisions in neighbouring cells
The presence of lateral divisions in the cells neighbouring the
stomata can increase the complexity of the epidermis and often
makes interpretation of stomatal patterns difficult. For example,
lateral divisions of neighbour cells are frequent, although not
Rudall & Knowles — Stomata of early-divergent angiosperms
1039
F I G . 7. Austrobaileya scandens: abaxial leaf surfaces, all LM, except (C), TEM. (A) Mature stomata with wall thickenings. (B, C) Stomata before wall thickenings
developed, with starch plastids. (D–I) Developing surfaces with a range of stomatal stages. Scale bars: (A– C, E –I) ¼ 10 mm, (D) ¼ 20 mm.
ubiquitous, during stomatal development in leaves of the ‘living
fossil’ gymnosperm species Ginkgo biloba (Rudall et al., 2012).
Highly consistent lateral divisions of neighbour cells occur in
commelinid monocots such as maize and Tradescantia (e.g.
Tomlinson, 1974; Croxdale, 1998; Cartwright et al., 2009).
At least in maize, asymmetric division of neighbouring cells is
promoted by the PANGLOSS1 (PAN1) gene (e.g. Facette and
Smith, 2012). The function of narrow lateral subsidiary cells is
not clear; they could have a physiological role, or (perhaps more
likely) they could help to compensate for the contrasts in growth
rate between stomata and their neighbours (see also Payne, 1979).
In the present study, Amborella proved an unexpectedly useful
subject for studying stomatal development because it apparently
lacks lateral divisions of neighbour cells. We also found no
evidence for lateral divisions of neighbour cells during stomatal
development in Nymphaea violacea. In contrast, lateral divisions
are common in Austrobaileya and Schisandra, in which the
lateral neighbour cells divide parallel to the guard cells and often
asymmetrically, thereby generating narrow neighbour cells. This
finding supports Carpenter’s (2005) observation that stomata
encircled by two concentric rings of neighbours are common in
Austrobaileyales but not in Amborella or water-lilies. Studies of
Austrobaileya and Schisandra are also complicated by the
presence of oil cells in the epidermis that are relatively large at
early developmental stages, although less obvious in the
mature leaf.
Presence of an asymmetric division preceding GMC formation
Our study shows two clear pathways to stomatal formation in
Amborella. Either a protodermal cell directly forms a GMC and
divides symmetrically (Fig. 5B), or a protodermal cell divides
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Rudall & Knowles — Stomata of early-divergent angiosperms
F I G . 8. Schisandra rubriflora: abaxial leaf surfaces. (A, B) Developing leaves
with patterning distrupted by the presence of large oil cells (LM). (D) Mature
surface with stomata randomly orientated (SEM). Abbrevaitions: g ¼ guard
cell, oc ¼ oil cell, wt ¼ wall thickening of mature guard cell. Scale bars:
(A, B, D) ¼ 10 mm, (C) ¼ 100 mm.
asymmetrically to give rise to a GMC and an SLGC (Fig. 5C).
Similar pathways probably exist in both Austrobaileya and
Schisandra, although the presence of lateral divisions and oil
cells both obscure the fundamental stomatal patterning in these
taxa. Carpenter (2005) also noted some evidence for asymmetric
divisions in Illicium, another genus of the order Austrobaileyales.
In contrast, we found no evidence of asymmetric divisions
during stomatal development in either species of Nymphaeales
examined (Cabomba aquatica and Nymphaea violacea), indicating that protodermal cells always give rise directly to GMCs in
this species. In these water-lily species, following squared prepatterning of protodermal cells, there emerges a highly regular
arrangement of pavement cells in mature leaves, with stomata
of a relatively consistent size, orientation and spacing. The relatively regular spacing of stomata could be due to lateral inhibition
of meristemoids or GMCs, and is a common feature of angiosperm leaves. However, consistent alignment of stomata is
highly unusual in a species with non-linear leaves. In species
with protodermal cells in linear files (such as monocots),
stomata are regularly aligned simply by orientating the division
of the GMC perpendicular to previous divisions. Similarly, in
Amborella, persistent linear patterning over the midrib results
in regular stomatal orientation in this region, as GMC divisions
are orientated perpendicular to the GMC-forming division.
However, this linear patterning does not explain stomatal
alignment in Nymphaea and Cabomba, in which protodermal
F I G . 9. Nymphaea violacea. (A) Adaxial surface of floating leaf (SEM). (B)
Transverse section of leaf. (C, D, F) Transverse sections of stomata. (E)
Paradermal section of adaxial epidermis with mature stomata. Abbreviations:
ch ¼ cuticular chamber, g ¼ guard cell, sc ¼ sclereid, ss ¼ substomatal
chamber. Scale bars: (A, C, E, F) ¼ 10 mm, (B) ¼ 100 mm, (D) ¼ 5 mm.
pattering is squared rather than linear. In Amborella, asymmetric
divisions that follow squared pre-patterning result in apparently
random orientation of stomata in intercostal regions. The reason
that stomatal orientation is so regular in water-lilies is that they
apparently lack asymmetric divisions in the stomatal cell
lineage, so divisions are always aligned with other cells.
Water-lilies are also apparently unusual in that the stomata are
initiated almost simultaneously and mature synchronously.
Epidermal cell division ceases soon after stomatal formation;
thereafter, the leaf enlarges primarily by cell expansion.
Conclusions: amplifying divisions and asymmetric divisions
With respect to mature stomatal types, Carpenter (2005)
reported that Amborella, Austrobaileya and Schisandra
produce mostly paracytic and stephanocytic and rarely anomocytic stomata, whereas Nymphaeales have anomocytic and stephanocytic stomata but lack paracytic stomata entirely. We found
only anomocytic stomata in mature leaves of Nymphaeales,
Rudall & Knowles — Stomata of early-divergent angiosperms
1041
F I G . 10. Nymphaea violacea. Adaxial leaf surfaces: (A– E) LM, and (F–I) TEM. (A, B) Two versions of the same micrograph, with a group of cells outlined in (B) to
illustrate squared patterning, prior to GMC fomation. (C) Stage with GMCs. (D) Stage with GMCs just divided. (E) Stage with maturing stomata. (F– I) Successive
stages of developing stomata. Scale bars: (A) ¼ 5 mm, (C– E) ¼ 10 mm, (F– I) ¼ 2 mm.
and it appears that paracytic stomata are absent from water-lilies. Is
there a developmental basis for this difference? Our developmental study shows that stomata of water-lilies (at least in Nymphaea
violacea and Cabomba aquatica) entirely lack asymmetric divisions in their developmental pathway, and are therefore always
perigenous. In contrast, stomatal development in Amborella and
Austrobaileya (and probably Schisandra) is often mesoperigenous
(i.e. with a single asymmetric division in the stomatal cell lineage:
Table 1). Thus, in water-lilies there is a clear correlation between
anomocytic stomata and perigenous development. However, this
correlation fails for most other taxa. In linear-leaved species, such
as many monocots and conifers, anomocytic stomata invariably
result from asymmetric divisions (e.g. Tomlinson, 1974;
Johnson and Riding, 1981). These results demonstrate that anomocytic and stephanocytic stomata can result from contrasting morphogenetic factors, thus confirming earlier assumptions that
mature stomatal types rarely reflect developmental patterns (e.g.
Payne, 1979).
On the other hand, our results suggest that paracytic stomata
are invariably the product of at least one asymmetric division,
at least in early-divergent angiosperms. This finding is potentially significant in assessing stomatal evolution in taxa known only
from fossils (Rudall et al., 2013). Paracytic stomata are highly
characteristic of angiosperms (e.g. Doyle and Endress, 2000;
Carpenter, 2005). If the topology of relationships of extant
angiosperms shown in Fig. 1 is correct, then our observations
suggest that asymmetric divisions were lost in Nymphaeales,
in which the aquatic environment could promote a neotenous
habit. This hypothesis contrasts with Carpenter’s (2005) suggestion of recurrent formation of paracytic types in early angiosperms, but these alternative hypotheses both appear plausible,
and more data are needed to test them. Evolutionary loss of
highly polarized asymmetric divisions that form meristemoids
has also been reported in other angiosperms, albeit in nonstomatal cell lineages; this phenomenon is most obvious when
it results in a loss of epidermal long – short cell alternation, as
in the silica cells and root epidermis of rice and its close allies
(Kim and Dolan, 2011; Rudall et al., in press). It remains
unclear whether this evolutionary switch has adaptive significance, although clearly the diverse range of stomatal patterning
could imply functional and mechanical diversity (Franks and
Farquhar, 2007).
We found no evidence for amplifying divisions in either
Amborella or any of the other ANITA-grade angiosperms examined here, indicating that ostensible similarities with the stomatal
patterning of Arabidopsis are superficial. Among other angiosperms that are putatively early-divergent, Peterson et al. (2010)
hypothesized that the extended spiral of cells around each stoma
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Rudall & Knowles — Stomata of early-divergent angiosperms
F I G . 11. Cabomba aquatica: adaxial leaf surfaces (DIC). (A) Very young leaf (located above water surface, but with margins still tightly rolled) showing squared
protodermal patterning prior to GMC differentiation. (B, C) Slightly older (still rolled) leaf in which some prodermal cells have directly differentiated into GMCs;
some have already divided symmetrically to form a pair of GCs. (D–F) Older (unfurled, floating) leaf with stomata present, mostly orientated in the same direction,
with a few exceptions. (G– I) Fully enlarged floating leaf with surface crystals. Images (H, I) show optical sections of a single stoma (H) and substomatal cavity (I). Scale
bars are all 10 mm, except (D, G) ¼ 20 mm.
in Houttuynia, a magnoliid, develops by amplifying divisions, as
in Arabidopsis. To test more broadly the evolutionary origin and
phylogenetic distribution of both asymmetric and amplifying divisions in angiosperms, our future studies will examine stomatal development in magnoliids and net-veined monocots, as well as in
well-preserved fossils of putative angiosperm relatives.
AC KN OW LED GEMEN T S
We thank Carlos Magdalena and Sara Redstone for growing the
plants examined, and Richard Bateman for critically reading the
manuscript.
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