Botanical Journal of the Linnean Society, 72: 299-309. With 4 plates and 3 figures
J u n e 1976
Subsidiary-cell development in the Catasetinae
(Orchidaceae) and related groups
N. H. WILLIAMS, F.L.S.
Department of Biological Science, Florida State University,
Tallahassee, Florida 32306, U.S.A.
Accepted for publication December 197.5
Developmental studies of stomata in the tribes Cymbidieae, Maxillarieae and Gongoreae
(Orchidaceae) demonstrate the presence of definite subsidiary cells in these groups of the
Orchidaceae, contrary to recent reports that the Orchidaceae lack subsidiary cells. Fifteen
species in these groups were studied developmentally and an additional 36 species were studied
from mature leaves. In the Cymbidieae the subsidiary cells may be the lateral trapezoid cells
derived from the lateral contact cells, or the subsidiary cells may be derivatives of the trapezoid
cells. In the species of Maxillarieae and Gongoreae studied the subsidiary cells were derivatives
of the trapezoid cells. The type of subsidiary cell development in these groups is essentially the
same as has been found in other advanced groups of epidendroid orchids that have subsidiary
cells.
CONTENTS
Introduction
. . .
Materials and methods
Results and observations
Discussion
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Acknowledgements
References
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INTRODUCTION
Subsidiary cells have been known to be rather common in the Orchidaceae
for over 40 years (Solereder & Meyer, 1930), contrary to recent statements
that the Orchidales lack subsidiary cells (Stebbins & Khush, 1961; Cronquist,
1968, 1969; Paliwal, 1969; Garay, 1972; Withner, Nelson & Wejksnora, 1974).
Rosso (1966) illustrated subsidiary cells in several species of the
Cypripedioideae. Metcalfe (1961) has suggested that mature subsidiary cells are
of some importance in plant anatomy and taxonomy. Tomlinson (1969, 1974)
has stated that information concerning subsidiary-cell development is of more
importance than information derived from mature subsidiary cells. Tomlinson
(1974) and Williams (1975) have both mentioned that studies of mature
21
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N. €1. WILLIAMS
subsidiary cells without a developmental basis have led some workers to
erroneous conclusions about the nature of mature subsidiary cells. Such
erroneous conclusions are of little use in taxonomic or phylogenetic studies.
This paper reports on developmental studies of stomata and subsidiary cells
in the subtribes Catasetinae, Cyrtopodiinae and Cymbidiinae of the tribe
Cymbidieae and in groups in the tribes Maxillarieae (Zygopetalinae) and
Gongoreae. Studies of mature stomata of all groups are also included in this
paper. Several different viewpoints on the classification of these groups are
available. Dunsterville & Garay (1972) include the Catasetinae with the
Maxillaria alliance in one large subtribe, the Zygopetaliinae* (sic), whilst
Dressler (1974) divides these groups into separate tribes. I am following here
the recent system proposed by Dressler, which includes the Catasetinae,
Cyrtopodiinae and Cymbidiinae in one tribe (Cymbidieae Pfitzer) and divides
the other genera that Dunsterville & Garay (1972) include in the Zygopetalinae
among several subtribes of the Maxillarieae Pfitzer and the Gongoreae Pfitzer.
MATERIALS AND METHODS
Material of young plants was obtained from the cultivated collection at
Fairchild Tropical Garden, Miami, Florida; the collection of Dr Calaway H.
Dodson, Department of Biology, University of Miami, Coral Gables, Florida;
and the Marie Selby Botanical Gardens, Sarasota, Florida.
Young shoots were collected during the morning hours between 9 a.m. and
noon. The young shoots (1-10 cm long) were dissected and fixed in 3 : 1
100%ethanol : glacial acetic acid for 2 4 hours. Material was removed from the
fixative, cut into small strips about 1 cm long (from the base) and 2-5 mm
wide, and stained in aceto-carmine with gentle heating over an alcohol lamp.
Material prepared in this manner usually had all stages of developing stomata
on any given strip. Semi-permanent mounts were made in 3 : 1 glycerin :
ethanol, although fresh mounts in aceto-carmine were quite satisfactory for
examination. N o attempt was made to make permanent mounts, but after
examination the material was returned to the fixative. Only the development
of abaxial stomata was studied, since all leaves of these groups possess
abaxial stomata, while adaxial stomata are rather limited in distribution in the
Orchidaceae. Material prepared as outlined above is rarely suitable for
photographic purposes, although it is excellent for examination; therefore,
most of the documentation is by camera lucida drawings. The terminology of
the stomata1 complex follows that of Tomlinson (1974).
Mature leaves were prepared for study following the scrape technique of
Metcalfe (1960), in which the leaf is scraped with a razor blade until only the
epidermis remains. The epidermis is washed with water or 70% ethanol and
mounted unstained in 3 : 1 glycerin : ethanol.
The species studied developmentally and as mature leaves are listed in
Table 1. All species studied developmentally were also studied when mature.
* The correct subtrihal name for the Zygopetalum and Maxillaria alliance, if they are put together, is
Maxillariinae Bentham, not Zygopetalinae Schlechter. See also Butzin (1971), who makes a case for
using Maxillariinae Lindley, which would also have priority over Zygopetalinae Schlechter.
SUBSIDIARY CELLS IN T H E CATASETINAE
301
Table 1. Species examined in which subsidiary cells have been foundt
Tribe Cymhidieae Pfi tzer
Subtribe Catasetinae Schltr.
* Catasetum bicolor Klotzsch
* Catasetum e x p a n s u m Reichenb. f.
* Catasetum luridum (Link) Lindl.
* Catasettrm macrocarpum Rich.
* Catasetum pileatum Reichenb. f.
* Catasetum randii Rolfe
* Catasetum saccatum Lindl.
* Catasetum tabtrlare Lindl.
* Catuseturn tenebrosum Kranzl.
* Catasetum warczewitzii Lindl. & Paxton
Cycnoches loddigesii Lindl.
Subtribe Cyrtopodiinae Bentham
Cyrtopodium andersonii (Lamhert) R. Br.
* Cyrtopodium paranense Schlechter
Cyrtopodium p u n c t a t u m (L.) Lindl.
Eriopsis helenae Kranzl.
Eulophia alta (L.) Fawcett & Kendle
Eulophia sp. cultivated from Africa
Tribe Gongoreae Pfitzer
*
Acineta chrysantha (Morr.) Lindl. & Paxton
Coeliopsis h yacin thosma Keichen b. f.
Gongora galeata (Lindl.) Keichenh. f.
Gongora quinquenervis Kuiz & Pavon
Kegeliella kupperi Mansf.
L y c o m o r m i u m squnlidum Reichenb. f.
Stanhopea in termedia Klotzsc h
Stanhopea jenishiana Kramer ex Reichenb. f
Stanhopea wardii Lodd. ex Lindl.
Subtribe Cymbidiinae Bentham
Ansellia gigantea Reichenb. f.
Cymbidiella flabellata (Thou.) Kolfe
* C y m b i d i u m aloifolium (L.) Swartz
C y m b i d i u m canaliculatum R. Br.
C y m b i d i u m m a d i d u m Lindl.
Grammatophyllum papuanum J. J . Sm.
Grammatophyllum scriptum (L.) Blume
Tribe Maxillarieae Pfitzer
Subtribe Zygopetalinae Schlechter
Anguloa cliftoni Rolfe
Bollea coelestis (Reichenb. f.) Keichenb. f.
Cochleanthes discolor (Lindl.)
Schultes & Garay
Cochleanthes marginatn (Reichenb. f.)
Schultes & Garay
Huntleya sp.
Kefersteinia lactea Reichenb. f.
Lycaste brevispatha Klotzsch ex Lindl.
I.ycaste cruenta Lindl.
Lycaste lanipes (Ruiz & Pavon) Lindl.
Lycaste macrophylla Lindl.
* Lycaste schillerana Reichenb. f.
* Lycaste tricolor (Klotzsch) Reichenb. f.
Neomoorea irrorata Rolfe
Promenaea stapelioides (Lindl.) L i d .
Stenia pallida Lindl.
X y l o b i u m elongatum (Lindl. & Paxton)
Hemsley
X y l o b i u m squnlens Lindl.
Zygopetalum mackayi Hook.
t N o species examined in these groups has lacked subsidiary cells.
An asterisk denotes species studied developmentally.
RESULTS AND OBSERVATIONS
The early stages of stomata1 ontogeny in the Catasetinae are quite similar to
the stages found in the Oncidieae (Williams, in prep.), Zygopetalinae and
Gongoreae. In very young material the cells are not differentiated and the
guard cell mother cell (GMC) is not distinguishable from the other epidermal
cells. Soon, however, the GMC becomes recognizable from the other epidermal
cells. The GMC is usually shorter and more barrel-shaped than the other
epidermal cells, and its cell walls are more refractive than those of the other
cells (Fig. 1A-D; Plate 2A). The nucleus of the GMC is usually denser and stains
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N. H. WILLIAMS
SUBSIDIARY CELLS IN THE CATASETINAE
303
more deeply than do the nuclei of the remaining epidermal cells. The first
divisions in the formation of the stomatal complex occur in the files of cells on
either side of the GMC in the cells next to the GMC (Fig. 1B; Plate 2A). These
divisions produce cell walls that are oblique t o the long axis of the leaf. Usually
there is an oblique division at each end of each of the lateral neighbouring cells
(Fig. 1 D ; Plate 2A). The result of these oblique divisions is a trapezoid cell on
each side of the GMC.
In the Catasetinae the divisions in the neighbouring cells usually stop with
the production of the trapezoid cells (Fig. lE), although occasionally further
divisions occur in the trapezoid cells (Fig. 1F). I t is very rare in the Catasetinae
for both of the trapezoid cells to undergo later divisions, although they do so at
times (Fig. 1F). The last division in the stomatal complex is in the GMC, which
divides to produce the two guard cells (Fig. lE,F). The three types of stomatal
complex found in the Catasetinae are illustrated in Fig. 1E,F. In those cases
where the trapezoid cell fails to undergo further divisions, the trapezoid cell
itself is considered to be a subsidiary cell.
The early stages of stomatal development in the Cymbidiinae are quite
similar to those described above. The guard cell mother cell becomes distinct
very early in ontogeny. Oblique divisions occur in the lateral neighbouring cells
prior to division of the GMC t o produce trapezoid cells (Plate 1A). Divisions in
the trapezoid cell occur just prior to division of the GMC (Plate 1B). If polar
subsidiary cells are formed, they are usually produced prior t o division of the
GMC (Plate 1B). In the Cymbidiinae both trapezoid cells and derivatives of
trapezoid cells are found, their proportions varying from species to species. In
Cymbidium aloifolium (the one species studied developmentally; Fig. 2A) both
trapezoid cells and derivatives of trapezoid cells are found, but trapezoid cells
are the main type of subsidiary cell found. In Cymbidium madidum, C.
canaliculatum and Cymbidiellu flabellata (Fig. 2B) both types are found, but in
these species the derivatives of the tfapezoid cells (or distinct subsidiary cells)
far outnumber these cells themselves. In Gramrnatophyllaimpapuanum the two
types are about equally distributed, but in G. scriptum (Fig. 2D) the subsidiary
cells are much commoner than the trapezoid cells. In Anselliu giguntea
(Fig. 2C) trapezoid-cell-derivative subsidiary cells are found but not trapezoid
cells. Thus in this subtribe varying combinations of the two types of subsidiary
cells occur: in some species trapezoid cells with few divisions in the trapezoid
Figure 1. Stages of stomatal development and mature stomata of Cataserum. A-D, Progressive
stages in stomatal development: A, C. warczewitzii, very early stage in which the guard cell
mother cell (CMC) is already distinct (arrows)-note the short barrel shape of the guard cell
mother cells; B, C. macrocarpurn, GMC with thick walls, one neighbouring cell undergoing the
first oblique division next to GMC (arrow); C , C. warczewitzii, stage in which most
neighbouring cells have undergone the first oblique division t o produce oblique cross walls
(arrows); D, C. macrocarpum, GMC prior t o division, both lateral neighbouring cells have
finished both oblique divisions and are now trapezoid cells (stippled). E, F, Mature stomata: E ,
C. expansum, mature stomata-note that the trapezoid cells have n o t undergone further division
and are the actual subsidiary cells; F, C. expansum, mature stomata-the trapezoid cells of the
upper stoma have divided to produce derivatives of the trapezoid cells in which the cell walls
run from one original oblique wall t o the wall of a polar contact cell-the lower stoma has one
complete trapezoid cell and one trapezoid cell t h at has divided with the cell wall going from
one original oblique wall of the trapezoid cell to the wall of a polar contact cell. A-D to same
scale, E, F t o same scale. Scale in D equals 0.01 mm, scale in E equals 0.05 mm.
304
N. H. WILLIAMS
SUBSIDIARY CELLS IN THE CATASETINAE
305
cells are mainly produced, in others divisions in the trapezoid cells occur to
varying degrees. Of those species in which divisions in the trapezoid cells occur,
some show these divisions infrequently while in others all (or almost all) of the
trapezoid cells undergo a further division to produce distinct lateral subsidiary
cells.
The developmental sequences of the subsidiary cells in the Cyrtopodiinae are
the same as those described in the other two subtribes mentioned above. In
some cases the trapezoid cell does not divide, but in others it does. Both
divided and undivided trapezoid cells can occur in the same stomatal complex
(Plate 2B). The mature trapezoid cells are about equal in distribution to the
trapezoid-cell-derivative subsidiary cells in Cyrtopodium paranense and C.
andersonii (Plate 3A). In Briopsis helenae there are mainly trapezoid-cellderivative subsidiary cells, with a few trapezoid cells. In Eulophia alta and
Euloplzia sp. there are mainly trapezoid-cell-derivative subsidiary cells with very
few trapezoid cells.
The early stages of stomatal development in the Zygopetalinae are quite
similar to those of the other groups. At first the GMC becomes recognizable by
its shape and staining characteristics. The lateral neighbouring cells undergo
oblique divisions at each end of the cell to produce trapezoid cells. In the
Zygopetalinae the rule is for each trapezoid cell to undergo one additional
division to produce a lateral subsidiary cell. These divisions are usually oblique,
with the last cell wall running from a polar cell to an original oblique wall of
the trapezoid cell. At times this division is not oblique and the cell wall runs
from one oblique wall of the trapezoid cell to the other oblique wall. There are
occasionally divisions in the polar neighbouring cells which produce terminal
subsidiary cells (Plate 3B). The final division is in the GMC to produce the two
guard cells. The mature subsidiary cells in this group are derivatives of the
trapezoid cell. Some variation exists in the shape of the mature subsidiary cells
in this group, both interspecifically (Plates 3B and 4: Fig. 3C-H) and
intraspecifically (Plate 4A,B). This type of development is also found in the
species of the Gongoreae studied developmentally.
DISCUSSION
Subsidiary cells have been defined in the past as cells next to or closely
associated with the guard cells which are different in morphology from the
remaining epidermal cells (Esau, 1965). Pant (1965), Paliwal (1969), and
Fryns-Claessens & Van Cotthem (1973) have stressed a definition of subsidiary
Figure 2. Abaxial surfaces showing stomata and subsidiary cells of some species of the
Cymbidiinae (A-D) and Gongoreae (E-H). A, Cymbidium aloifolium-note the subsidiary cell
(arrow) which is the mature trapezoid cell-the outline of the trapezoid cell is apparent; B ,
Cymbidiella flabellata-in some cases the trapezoid cell did n o t divide (arrows) and is the
subsidiary cell, in other cases the trapezoid cell divided and produced smaller subsidiary cells; C ,
Ansellia gigantea-in all cases shown the trapezoid cell has divided; D, Grammatophyllum
scn'ptum-the division of the trapezoid cell itself is horizontal rather than oblique; E, Acineta
chrysantlra; F, Coeliopsis hyacinthosma; G , Gongora quinquenervis-note that the derivatives of
the trapezoid cells in G. quinquenervis in some instances have divided to produce four
subsidiary cells around each pair of guard cells; H, Stanhopea jenishiana. All t o same scale, scale
equals 0.1 mm.
306
N. H. WILLIAMS
SUBSIDIARY CELLS IN THE CATASETINAE
307
cells in which the subsidiary cells have a developmental relationship to the
guard cells. Tomlinson (1974) has proposed a more simplified, usable
classification of stomatal types, based on developmental sequences, which is
followed here.
The subsidiary cells of the Catasetinae are essentially the trapezoid cells,
which have a definite developmental relationship to the guard cells. This
relationship is especially noticeable if one studies the complex developmentally. In addition, subsidiary cells which are the result of further divisions
in the trapezoid cells are also found in the Catasetinae; although not
frequently. In the Catasetinae the subsidiary cells are not always easily
recognized in mature tissue, and it is for this reason that developmental studies
are so necessary.
In the tribe Cymbidieae the Catasetinae are somewhat distinct from the
other two subtribes studied in that the development of the subsidiary-cell
complex in the Catasetinae does not proceed as far as it does in the
Cyrtopodiinae or Cymbidiinae, although definite subsidiary cells are produced.
The Catasetinae have the ability to produce the early stages of the complex
(the trapezoid cells), but the later divisions are not regularly produced. The
Cymbidiinae and Cyrtopodiinae have the ability to produce the early stages as
well 'as the late stages in the sequence of development of the subsidiary-cell
complex. In view of the variation that seems to exist in the tribe Cymbidieae, I
believe that this situation should be noted but not unduly stressed in the
taxonomy of the tribe.
The trapezoid cells of the Zygopetalinae and Gongoreae regularly divide to
produce morphologically distinct subsidiary cells. In material examined to date
n o species from either of these groups has been found to lack distinct
subsidiary cells.
The regular production of subsidiary cells by the division of the trapezoid
cells is interpreted as an advanced condition in the Orchidaceae (Williams, in
prep.), and the Zygopetalinae and Gongoreae are interpreted as being more
advanced than the Cymbidieae, especially the Catasetinae, in the evolution of
the stomatal complex.
ACKNOWLEDGEMENTS
I thank Dr C. H. Dodson for reading an early version of this paper. This
research was supported by grants from the National Science Foundation.
Washington D.C. U.S.A. (GB-35942 and GB-40880).
Figure 3. Representative adaxial (A, B ) and abaxial surfaces (C-H) of species of the
Zygopetalinae. A, Zygopetalum mackayi, adaxial surface lacking stomata; R, Promenaea
stapelioides, adaxial surface lacking stomata; C , Stenia pallida, abaxial surface, note lateral
subsidiary cells-the outline of the original trapezoid cell is no longer apparent; D , Kefersteinia
lactea, abaxial surface with elongate subsidiary cells; E, Cochleanthes discolor, abaxial surface
with stomata and subsidiary cells; F, BoNea coelestis, abaxial surface-note slight variation in
size and shape of subsidiary cells; G, Zygopetalum mackayi. abaxial surface with distinct
subsidiary cells; H, Cochfeanthes marginata, abaxial surface with subsidiary cells. A-M t o same
scale, scale equals 0.1 mm.
308
N. H. WILLIAMS
REFERENCES
BUTZIN, F., 1971. Die Namen der supragenerischen Einheiten der Orchidaceac. Willdenowia, 6: 301-40.
CONQUIST, A, , 1968. The evolution and classification offlowering plants. Boston: Houghton Mifflin Co.
CKONQUIST, A.. 1969. Broad features of the system of Angiosperms. Taxon, 18: 188-93.
DRESSLEK, R. L., 1974. Classification of the orchid family. Proceedings of the Seventh World Orchid
Conference: 259-79.
DUNSTERVILLE, G. C . K. & GAKAY, L. A,, 1972. Venezuelan orchids illustrated, 5: 16-18. London:
Andre Deutsch, Ltd.
W A U , K., 1965. Plant anutomy, 2nd ed. New York: J o h n Wiley and Sons, Inc.
FRYNS-CLAESSENS, E. & VAN COTTHEM, W., 1973. A new classification of the ontogenetic types of
stomata. Botanical Review, 39: 71-1 38.
GARAY, L. A., 1972. On the origin of the Orchidaceae. 11. Journal of the Arnold Arboretum, 53:
202-15.
METCALFE, C. R., 1960. Anatomy of the Monocotyledons. I . Gramineae. Oxford: Clarendon Press.
METCALFE, C. R., 1961. T h e anatomical approach to systematics. I n Recent advances in botuny:
146-50. Toronto: University of Toronto Press.
PALIWAL, G. S . , 1969. Stomatal ontogeny and phylogeny. I . Monocotyledons. Acta botanica
neerlandicu, 18: 654-68.
PANT, D. I)., 1965. O n the ontogeny of stomata and other homologous structures. Plant Science Series,
Allahabud, 1: 1-24.
ROSSO, S. W., 1966. The vegetative anatomy of the Cypripedioideae (Orchidaceae). Journal of the
Linnean Society (Botany), 59: 328-3 1.
SOLEREDER, H. & MEYER, F. J., 1930. Systematische Anatomie der Monokotyledonen. VI.
Microspermae. Berlin: Verlag von Gehriider Borntrager. English translation 1969. Jerusalem: Israel
Program for Scientific Translations.
STEBBINS, G. L. & KHUSH, G. S . , 1961. Variation in the organization of the stomata1 complex in the
leaf epidermis of Monocotyledons and its bearing on their phylogeny. Americun Jottrnal of Botany,
48: 51-9.
TOMLINSON, P. B., 1969. .Anutomy of the Monocotyledons. III. Commelinales-Zingiberules. Oxford:
Clarendon Press.
TOMLINSON, P. B., 1974. Development of the stomata1 complex as a taxonomic character in the
Monocotyledons. Taxon, 23: 109-28.
WILLIAMS, N. H., 1975. Stomatal devclopment in Ludisia discolor (Orchidaceae): mesoperigenous
subsidiary cells in the Monocotyledons. Tuxon, 24: 281-8.
WITHNIIR, C. L., NELSON, P. K. & WEJKSNORA, P. J., 1774. T h e anatomy of orchids. I n C. L. Withner
(Ed.), The orchids-scientific studies: 267-347. New York: John Wiley and Sons.
EXPLANATION O F PLATES
PLATE 1
A. Guard cell mother cell between two trapezoid cells of Cymbidium aloifolium. Note the small
size of the guard cell mother cell in relation to the size of the other epidermal cells. Focal plane
below surface. B. Very young guard cells of C. aloifolium. The lower trapezoid cell and b o t h
terminal contact cells have divided to produce lateral and polar subsidiary cells. T h e upper
trapezoid cell has n o t divided yet. T h e polar subsidiary cells will probably be indistinct a t
maturity. Note that the t w o young guard cells occupy approximately the same space as the
guard cell mother cell in A above. Focal plane at surface.
PLATE 2
A. Young guard cell mother cell and trapezoid cell of Catasetum macrocarpum Note distinctive
shape of the guard cell mother cell. B. Young guard cells of Eulophiu sp. T h e trapezoid cell on
the right has divided t o produce a lateral subsidiary cell; the trapezoid cell o n the left did n o t
divide
Botanical Joumal of the Limzemz Society, 72 (1976)
N. H. WILL IAMS
Plate 1
(Facing p. 308)
Plate 2
Botanical Journal of the Limzean Society, 72 (1976)
•
I
I
B
N. H. vYILLIAl\lS
[J()fanical Journal of the Linn('all SociefJ', 72 ( 1976)
Plate 3
A
N. II. WILL1Al\1 S
Botanical Journal of the Linnecm Society, 72 (1976)
Plate 4
A
•
I
I
J
N. H. \YILL IA1\1S
B
SUBSIDIARY CELLS IN THE CATASETINAE
PLATE 3
A. Nearly mature stomata of Cyrtopodium andersonii. Note distinct lateral subsidiary cells. The
shape of the original trapezoid cell on the right is still obvious. B. Mature stoma of
Cochleanthes discolor. Both lateral and terminal subsidiary cells are distinct. The shapes of the
original trapezoid cells are no longer obvious.
PLATE 4
Variations in the mature stomatal patterns of Lycaste schil/erana. A. A stoma with lateral
subsidiary cells and one obvious polar subsidiary cell. B. A stoma with lateral subsidiary cells
but lacking obvious polar subsidiary cells. Note the striking size differences between the lateral
subsidiary cells and the other product of the division of the trapezoid cell.
gmc, Guard cell mother cell; tc, trapezoid cell; psc, polar subsidiary cell; scale bar equals
0.01 mm on all plates.
309