Embed Size (px)
Citation preview
Leaf development in the anisophyllous shoots of Pellionia daveauana (Urticaceae)
Received April 28, 1983
MUELLER, P. A , , and N. G. DENGLER. 1984. Leaf development in the anisophyllous shoots of Pelliotlia doveauntla (Urticaceae). Can. J . Bot. 62: 1158-1 170.
'The dorsiventral shoot system of Pellionia daveauantl (Godefr.) N.E. Br. is characterized by opposite pairs of dimorphic leaves. The small dorsal leaves differ from the large ventral leaves by having a reduced leaf blade, fewer tissue layers in the epidermis and mesophyll, a reduced vascular system and significantly smaller cell size in all tissue layers. The observations reported here document the developmental basis of these morphological and histological differences. Although both dorsal and ventral leaves appear to be initiated simultaneously, the volume of the ventral leaf primordium is greater than that of the dorsal leaf primordium and growth in length occurs over a longer time period. Early plate meristem activity results in the elaboration of the ventral leaf blade, while plate meristem activity is lacking in dorsal leaves. During ventral leaf expansion periclinal divisions in adaxial and abaxial protoderm and ground meristem give rise to multiple epidermis and new mcsophyll layers, respectively. Similar periclinal divisions in dorsal leaves occur at an earlier developmental stage and are restricted in extent. Measurements of cell dimensions show that ccll enlargement also ceases at an carly developmental stage in dorsal leaves. Development of the ventral leaf is characterized by a relatively long period of ccll division and enlargement. In contrast, carly cessation of cell division and precocious cell maturation result in the distinctive structural features of the dorsal leaf blade.
MUELLER, P. A,, ct N. G. DENGLER. 1984. Leaf development in the anisophyllous shoots of Pelliotlia dnvenritrtzn (Urticaceae). Can. J . Bot. 62: 1158- 1170.
Le systkme dorsiventral des rameaux du Pelliot~ia clavenuntla (Godefr.) N.E. Br. est caractCrisC par des paires opposCes de feuilles dimorphes. Les petites feuilles dorsales diffkrent des grandes feuilles ventralcs par un limbc foliaire rkduit, un plus faible nombre de couches dc tissus dans I'Cpiderme et lc mCsophylle, un systkme vasculairc rCduit et unc taillc plus faible des ccllules dans tous les tissus. Les observations que nous prCsentons documentent le fondement ontogCnique dc ces differences morphologiques et histologiqucs. Bien que les feuilles dorsales et ventrales soient initiCes simultanCmcnt, le primordium de la feuille ventrale est plus gros que celui de la feuille dorsale et sa croissance en longueur dure plus longtcmps. Le limbe de la feuille ventrale s'Clabore t6t par I'activitC d'un mCristkme en plaque, mais une tclle activitC est abscntc chez les feuilles dorsales. Pendant I'expansion dc la feuille ventrale, des divisions periclines dans le protoderme adaxial ct abaxial et dans le mCrist6me fondamental donnent naissance respectivcment ii un Cpiderme multiple et 21 de nouvelles couches de mCsophylle. Dans les feuilles dorsales, de telles divisions pkriclines se produisent plus t6t au cours de I'ontogCnie et sont moins abondantes. La mesure de la dimension dcs ccllules montre que I'accroissement cellulaire cesse Cgalement plus t6t chez les feuilles dorsales. Le dCveloppement dc la feuille ventrale cst caractCrisC par une pCriode relativemcnt longue dc division et d'accroissemcnt cellulaires. Au contraire, I'arrCt hdtif dc la division cellulaire et la maturation prCcocc dcs ccllules produiscnt les caractkres distinctifs de la feuille dorsale.
[Traduit par le journal]
Introduction Interest in the ontogenetic basis of divergent form has com- Anisophylly refers to differences in leaf size and form which
are related to the transectional symmetry of the shoot. In some plant species anisophylly is facultative and occurs as a plastic response of the phenotype in which the amount of mutual shading by adjacent leaves is reduced by size differences between leaves of an opposite pair; under higher light intensi- ties facultatively anisophyllous shoots may be isophyllous (Wiehler 1978). In contrast, some plant species are character- ized by lateral anisophylly in which main shoots are isophyl- lous and lateral shoots anisophyllous (e.g., Sinnott and Durham 1923) or by habitual anisophylly in which all adult shoots are anisoph~llous. In both conditions, although the degree of anisophylly may be influenced by environmental conditions, the associated differences in leaf size and shape are expressed throughout the appropriate adult vegetative shoots. Habitual anisophylly occurs in a range of plant families, in- cluding the Urticaceae, Gesneriaceae, and Melastomaceae (Engler and Prantl 1889; Troll 1937). Within the subfamily Procrideae of the Urticaceae, variation in leaf form ranges from those species with opposite pairs of leaves of equal size through those in which one leaf of a pair is three to five times the size of the other to species such as Pellionia daveauana in which anisophylly is expressed to such an extreme degree that leaf arrangement is often described as alternate rather than opposite.
monly focused on expression of heteroblastic development in which the size and shape of early-formed organs differs from those produced later on the same shoot system (Foster 1935; Crotty 1955; Kaplan 1973; Franck 1976; Bruck and Kaplan 1980; Kaplan 1980). Often a correlation has been found to occur between the size and complexity of the mature leaf and the volume of the shoot apex at the time of initiation (Crotty 1955; Kaplan 1973; Franck 1976; Bruck and Kaplan 1980). Therefore, the shoot apex has generally been regarded as the determinant of this heteroblastic variation in size and complex- ity (Allsopp 1965, 1967; Cutter 1965). Since, in anisophyllous systems, the two leaf forms are initiated in close spatial and temporal proximity on the shoot apex, an investigation of leaf development should provide an opportunity to focus on the divergent ontogenetic pathways without the complication of concomitant structural and physiological changes in the shoot apical meristem.
The extreme reduction of the dorsal leaf in Pellionia daveauana also raises the question of homology between the two leaf types. According to the concept of Eichler (1861), a leaf primordium consists of an upper leaf zone and a lower leaf zone; the leaf base region, including stipules when present, is derived from the lower leaf zone, and the leaf blade and petiole are derived from the upper leaf zone. A developmental analysis should also determine whether the dorsal leaf is apparently
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
MUELLER AND DENGLER 1159
devoid of a lamina because (i) the upper leaf zone is lost distally or ( i i ) the upper leaf zone is present but is not differentiated into a blade and petiole. The objectives of this investigation are to distinguish between these alternatives and to document the morphogenetic and histogenetic processes which result in these strongly dimorphic leaves.
Materials and methods Material of Pellionit? davetzuana (Godefr.) N.E. Br. was obtained
from the greenhouse collection of the University of Toronto. Cuttings were rooted in individual pots and were allowed to become established under uniform growth-chamber conditions. Terminal shoot tips were then removed and the expansion of the most distal lateral bud provided material for these observations of leaf development. Since growth of the new lateral shoots was variable, material from the 64 established cuttings was separated into three treatmcnt groups using a random- numbers table. The 22 plants in the treatment-1 group were maintained in the growth chamber and measurements of the length of expanding leaves were made every 2 days over a period of 82 days. At the end of 82 days the tips of the lateral shoots were removed and fixed in formalin - acetic acid - alcohol (FAA). Lengths of ventral and dorsal
For characterization of the anatomy of mature leaf blades and of histogenesis in expanding leaves, nine parameters were measured from leaf cross sections taken at the midpoint between blade apex and base: leaf thickness, cell height in both layers of the adaxial epidermis (layers 1, 2) and of the uppermost and lowermost layers of mesophyll tissue (layers 3, 4). The locations of these tissue layers are indi- cated in Figs. 10 and 13. Average mature cell dimensions were com- pared by a t-test and by calculation of the coefficient of variation (Snedecor and Cochran 1980). For the developmental study, ventral and dorsal leaves from the cross sectional series of treatment-2 apices were placed into the following size categories: 0-99, 100-199, 200-399,400-599,600-799,800-999, 1000- 1199, 1200- 1399, 1400- 1599, and 1600- 1799 km. Ventral leaves were also placed into the following additional size categories: 1800- 1999 k m , and 2-4 ,4-6 ,6-8 , 8- 10, 10- 15, and 15-20 mm. A two-way analysis of variance was performed on the quantitative anatomical data for ventral and dorsal leaves of similar size classes and a one-way analysis of variance on the remaining size classes of ventral leaves. To deter- mine the size classes between which differences occurred, Duncan multiple-range tests were pcrformed (Sokal and Rohlf 1969).
Results leaves not.yet expanded from the bud were calculated from serial Morpho~ogy of the shoot system cross sections of these shoot tips. Treatment-2 plants were allowed Pellionia daveauatza is a creeping fleshy herbaceous plant to grow undisturbed for the same time period; then shoot tips and whole leaves or portions from the midregion of ventral leaves were with plagiotropic dorsiventral shoots which are characterized
removed and fixed in 2% glutaraldehyde. Also, after 82 days, shoot by leaf dimorphism. The larger laminar leaves are born tips from treatment 3 were fixed in FAA. In addition, terminal in two ranks on the stem (Figs. 1, 3-5). Although the dorsal shoot tips from the original 64 established cuttings were fixed in leaves are ephemeral and often have fallen from older portions FAA and some werc used for an analysis of leaf arrangement. Shoot of a stem, close examination of vigorously growing shoots tips used in scanning electron microscopy and mature ventral and reveals two ranks of small peglike leaves borne on the dorsal dorsal leaves used for a description of mature anatomy were col- surface of the stem (Figs. 1, 3). Although ventral and dorsal lected from the original greenhouse-grown plants and were fixed in leaves are borne in opposite to s u ~ o p p o s i ~ e pairs and alternation 2% glutaraldehyde. in orientation of successive pairs occurs, leaf arrangement is
Shoot tips and leaves fixed in 2% glutaraldehyde in 0.05 M phos- not strictly decussate (Fig, 2). Measurements made from phate buffer (pH 6.8) for 6 h were subsequently rinsed and stored overnight in 0.1 M phosphate buffer, postfixed with 1% osmium shoot tip cross sections indicate that the mean divergence
tetroxide in 0.05 M phosphate buffer, dchydratcd through an acetonc between the and leaf of a pair (e.g., V2 series, and embedded in Spurr resin. Embedded tissue was sectioned and D2, Fig. 2) is g4.0 * 2.403 between successive ven- at 4 Ltm on a Porter Blum MT-2 ultramicrotomc and stained with tral leaves (e.g., V3 and V4, Fig. 2) is 124.9 * 3.6", and toluidine blue - basic fuchsin. Other shoot tips. fixed for 24 h in FAA, were dehydrated through a tertiary butyl alcohol series and embedded in Paraplast. Serial cross and longitudinal sections, I0 k m in thickness, were made on a rotary microtome and stained in safranin - orange G. All light microscope observations were made on a Zeiss Jena Ergaval microscope and recorded on Kodak Panatomic X film. For scanning electron microscopy, shoot tips fixed in 2% glu- taraldehyde in 0.05 M phosphate buffer (pH 6.8) were rinsed and left overnight in the same buffer. Tissue was then dehydrated through an acetone series and critical point dried with liquid COz. Shoot tips were mounted on stubs with Scotch brand double-sided stick tape and coated with gold in a Sputter coater PE-5000. Observations were made on a Cambridge S-180 scanning electron microscope and recorded on Polaroid P55 film.
Observations of leaf arrangement werc made from tracings of cross sections of 10 shoot tips on which lines werc drawn from the center of the shoot apex to the midvein procambium of each leaf of the youngest three or four pairs. Angles between lines drawn for corre- sponding ventral and dorsal leaves and between successive ventral and dorsal leaves were measured with a protractor (Richards 1951). The allometric relationship between length and width of ventral and dorsal leaves was obtained by performing a regression analysis of data obtained from the leaves of treatment-l and -2 plants. The data werc transformed to natural logs to make the relationship between the two variables more linear and to stabilize residual variance. Since com- parison of slopes of the regression lines for ventral and dorsal leaves of F ratio showed that the assumption of homogeneity of variance did not hold, a conservative t-test was used to compare the slopes (Snedecor and Cochran 1980).
between successive dorsal leaves (e.g., D3 and D4, Fig. 2) is 55.6 1+- 2.5".
The blade of the ventral leaf is asymmetric and roughly ovate with an acute apex, lobed margins, and an asymmetrically lobed base (Fig. 4). Anthocyanin pigments in the adaxial epi- dermal layer impart a deep green colour to the leaf margins and distal portion of major veins; in contrast, the leaf center remains light green. Stomata and hydathodes are restricted to the abaxial lamina surface. The lamina is attached to a short petiolar region and the leaf base bears a single lanceolate, membranaceous stipule which partially enfolds the stem (Figs. 1, 3 , 5).
The oblong lamina of the dorsal leaf is only slightly dorsi- ventrally flattened and is weakly differentiated into blade and petiolar regions (Figs. 6, 7). The leaf blade is light green in colour, and stomata and a single apical hydathode occur on the abaxial surface. The stipular appendage of the dorsal leaf is similar in appearance to that of the ventral leaf (Figs. 3, 6). In portions of the shoot in which the rest of the dorsal leaf is abscised, the dorsal stipule remains attached to the stem.
Anatomy of the mature leaf blade Ventral leaf blade histology is characterized by a multiple
epidermis consisting of two cell layers of approximately iso- diametric cells on the adaxial side of the leaf and three (range 2- 10) cell layers on the abaxial side of the leaf (Fig. 10). Darkly stained mucilage cells occur in the innermost layer of
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
CAN. J. BOT. V 'OL. 62. 1984
Frc. 1 . Diagram of the shoot system of Pellioi~in dnvenuclnn as viewed from the dorsal side. The labelled dorsal leaf and ventral leaf form an opposite pair.
FIG. 2. Tracing of a cross section of a shoot tip at the level of the apical meristem (A), showing the arrangement of ventral leaves (V I -V4), ventral stipules (VS2-VS4), dorsal leaves (Dl -D4), and dorsal stipules (DS2-DS4).
the adaxial epidermis as idioblasts (Figs. 10, 11). Large styloid-containing lithocysts are also found in the adaxial epidermis (Figs. 1 1, 12). During tissue differentiation, the protodermal cells giving rise to the lithocysts do not undergo periclinal division but rather enlarge in the paradermal plane at the level of the second adaxial epidermal layer. The crystals are attached by a thin stalk to the outer tangential wall of the epidermal cell. The abaxial epidermis lacks these lithocysts and is characterized by a lower density of mucilage cells. The abaxial surface layer has slightly raised stomata, each con- sisting of a pair of guard cells surrounded by three subsidiary cells (Fig. 8). Large substomatal cavities traverse the abaxial epidermal layer, connecting mesophyll with external air space (Figs. 10, 12). The mesophyll tissue is composed of one to three layers of elongate to conical palisade parenchyma cells with densely stained chloroplasts aggregated toward the abaxial end of the cell and two or three layers of elliptical spongy parenchyma cells with few and (or) smaller plastids scattered throughout the cell (Figs. 10- 12). The pattern of major vena- tion is eucamptodromous, and the reticulate minor venation can be resolved into tertiary, quaternary, and quinternary size cate- gories. Observations of cleared leaves show that hydathodes located at the leaf margin are associated with a single quin-
ternary vein, while those located in a nonrnarginal position are supplied by the confluence of three quinternary bundles. The epidermis of a hydathode region is one cell layer thick and contains small reduced stomata (Figs. 9, 12). The associated epithem consists of small, closely packed ground parenchyma cells.
The epidermis of the dorsal leaf blade is one or two cell layers in thickness; the region with a single epidermal layer is usually restricted to the margins of the leaf (Fig. 13). In the midrib region mucilage cells and lithocysts containing styloid crystals are found in the multiple epidermis. Stomata are re- stricted to the abaxial epidermis and small substomatal cavities traverse the multiple portion of the epidermis. Mucilage- containing cells may also occur in the abaxial epidermis. The mesophyll tissue of the dorsal leaves is best developed adjacent to the midvein where there may be three or four layers of tissue; this is reduced to one or two in the marginal portion of the leaf. Usually only the uppermost mesophyll layer (layer 3) is com- posed of palisadelike spherical to conical cells with darkly stained plastids. Cells of the other mesophyll layers are ellip- tical in shape with fewer plastids and are associated with more extensive intercellular space. The vascular system of the dorsaI leaf consists of a single midvein which is comparable in size and in number of conducting cells with the quaternary veins of the ventral leaves. A single hydathode is associated with the distal end of the midvein and is similar in structure to that described for ventral leaves.
Although the component cell types and their organization into tissues are similar in the blades of both leaf types, there are striking differences in cell size, cell number, and the number of tissue layers between ventral and dorsal leaves. The observed differences in cell size were confirmed by measurements of the cell dimensions which are listed in Table 2 and which show significant differences between ventral and dorsal leaves for all characters except palisade parenchyma cell height. 'The average thickness of the ventral leaf is also significantly greater than that of the dorsal leaf (Table 1) and is associated with both a greater number of tissue layers and larger cell sizes. Calcula- tion of the coefficient of variation for the data presented in Table 2 indicates that, except for height of cells in the most abaxial spongy mesophyll layer (layer 4), there is greater inher- ent variability in cell size in ventral leaves as compared with dorsal leaves.
Leaf morphogenesis The ventral and dorsal leaves of Pelliorlia daveauarza are
formed on a flat to slightly dome-shaped apex (Figs. 14, 15). Because of skewed orientation of the shoot apex in relation to shoot symmetry, median longitudinal sections were difficult to obtain and the earliest stages of leaf initiation were not directly observed. However, since all sectioned material and scanning electron micrographs show both the dorsal and ventral pri- mordia of a pair are present, initiation of the two leaf types is most likely stimultaneous. The ventral leaf primordium has a greater tangential extent and a larger volume from the earliest stages observed and growth of the leaf blade is initiated when the leaf is about 0.2 mm in length (Figs. 14, 15). The lobing which characterizes the blade margin appears when the leaf is about 0.3 mm in length (Figs. 16- 19). In contrast, the dorsal leaf primordium has a much smaller volume from the outset and lacks the differentiation of the leaf blade zone which char- acterizes the ventral leaf at comparable stages (Figs. 14- 19).
A single stipule arises from the adaxial base of the ventral
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
MUELLER AN11 DENC;LEI<
FIG. 3. Portion of a shoot showing the position of a ventral leaf (V. lamina renioved). vcntral stipule (VS). dorsal leaf (D). and dorsal stipule (DS). X6.5. FIG. 4. Adaxial surhce of mature ventral Ieaf. x 3 . FIG. 5 . Stipulc of Ieaf shown in Fig. 4. X3. FIG. 6. Abaxial surfacc of a dorsal leaf (D). X 10. FIG. 7. Scanning electron micrograph of the adaxial surface of a mature dorsal leaf showing stalked glandular. trichonies (T). X 370. FIG. 8. Scanning electron micrograph of the abaxial epidermis of a mature ventral leaf showing stomates (S) and a hydathode (H). X 110. FIG. 9. Scanning electron micrograph of the abaxial surface of a mature ventral leaf cornparing normal stoniates (S) with reduced hydathode stomates (HS). X 370.
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
CAN. I . BOT. VOL. 62. 1984
FIG. 10. Cross section of a mature ventral leaf showing tissue layers (1 -6) as described in text. Note mucilage cells (M) in adaxial epidermis and substomatal cavity (SC), X 135. FIG. 11. Cross section of a mature ventral leaf illustrating a styloid-containing lithocyst (C) and quaternary minor vein (B). X 135. FIG. 12. Cross section through a hydathode of a mature ventral leaf. Note compact epithem tissue (E) and reduced stomate (HS). X 135. FIG. 13. Cross section of a mature dorsal leaf showing tissue layers ( 1 -6) as described in text. Note mucilage cells (M), stomatc (S), and midvein (B). X 170.
leaf primordium. The zone of stipule initiation appears to occur first on the ventral side of the leaf (Fig. 14) and then to extend across the leaf base (Figs. 16- 19). In leaves at the L1 position, the ventral stipule appears as a thin, awl-shaped flap of tissue with an asymmetrically placed apex (Figs. 16- 19). Extension of the margins of the corresponding ventral leaf blade occurs in a dorsal direction, away from the stipule apex, resulting in the placement of the leaf blade and stipule diagrammed in Fig. 2. Initiation of the dorsal stipule occurs on the dorsal side of the adaxial base of the dorsal leaf primoridum (Figs. 16, 18). At early stages, the stipule appears as a buttresslike mound of cells, and its growth lags behind that of the corresponding ventral stipule (Fig. 19). Based on macroscopic appearance in dissected shoot tips, the dorsal stipule acquires a flattened shape, similar to that of the ventral stipule, by the L2 position. The margins of the dorsal stipule extend more or less sym- metrically from the site of origin, resulting in a position which is placed to the inside, and dorsally, from the dorsal leaf lamina (Fig. 2).
Expansion of the ventral leaves from the apical bud was documented by measurements of treatment-1 plants over an
TABLE 1. Dimensions of mature ventral and dorsal leaves of Prllionia daveauantr (means ? standard error)
Significance Dimension Ventral leaf Dorsal leaf level, P
Length, mm 24.4k0.71 1.28?0.02 50.05 Width, mm 18.5?0.5 0.37?0.01 50.05 Thickness, p n 444.5k 12.3 131.6k21.6 50.05
82-day period (Fig. 20). Although considerable variability among individual shoots was observed, the first-formed ventral leaves tend to have a slower rate of expansion, are shorter in final length than subsequent leaves, and usually abscise 8- 13 days after emergence from the terminal bud under the growing conditions used. Later formed ventral leaves are char- acterized by a rapid initial rate of expansion and a longer period of expansion, and they are usually retained on the shoot.
The pattern of expansion of ventral and dorsal leaves was compared by averaging leaf lengths at each nodal position (Fig. 21). The mean length of ventral leaf blades increases
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
MUELLER AND DENGLER
TABLE 2. Comparison of cell dimensions in mature ventral and dorsal leaves of Pelliorzin dnven~rnna (means 2 standard error)
Cell dimension Ventral leaf Dorsal leaf Significance level, P
Adaxial epidermis cell height, p,m (layer 1 ) 24.32 1.6
cv, 6 .5 Adaxial epidermis cell
height, p,m (layer 2) 75.02 19.3 cv, 25.8
19.824.1 cv, 20.7
Abaxial epidermis cell height, p,m (layer 6) 33.324.2
cv, 12.6 24.121.7
cv, 6.8 Abaxial epidermis cell
height, p,m (layer 5) 105.2525.3 cv, 24.1
17.722.8 cv. 15.8
Palisade parenchyma cell height, p,m (layer 3) 15.62 1.8
cv, 9.1 27.753.3
cv, 11.8 Palisade parenchyma cell
width, p,m (layer 3) 13.75 1.4 cv, 13.1
16.62 1.3 cv, 7.6
Spongy parenchyma cell height, p,m (layer 4) 22.623.0
cv. 13.5 15.72 1.3
cv, 25.7 Spongy parenchyma cell
width, p,m (layer 4) 33.624.0 cv, 3.9
20.222.2 cv, 1.06
NOTE: cv, coefficient of variation
from position L1 to L7 or L8 (Fig. 21A); apparent decrease at older nodes reflects heteroblastic variation within each stem. Mean length of the dorsal leaf blade increases between L1 and L3 or L4 (Fig. 21B); no heteroblastic variation in size was
and ground meristem are clearly distinguished (Fig. 24). After this stage periclinal divisions occur in-the middle layer of the ground meristem, increasing the thickness of the lamina to six cell layers. The three most abaxial layers of the ground meri- stem begin to enlarge more rapidly than the adaxial layer; these recognized. Expansion of both the ventral and dorsal stipules
appears to be complete by position L5 or L6 (Figs. 21A, 21B). Mean length of the dorsal stipule exceeds that of the corre- sponding leaf blade, while mean length of the ventral leaf blade
future spongy mesophyll cells become vacuolated, and associ- ated small intercellular spaces begin to develop. When ventral leaves are 4-5 mm in length (L3-L4), cells of the adaxial
is greater than that of the corresponding stipule at all stages observed.
Measurement of leaf width over a range of leaf lengths indicates that the ratio of length to width does not change substantially during the developmental stages of ventral and dorsal leaves examined. This was confirmed by regression analysis, which showed a linear relationship between the vari-
protoderm are considerably vacuolated and enlarged in com- parison with abaxial protoderm (Fig. 25). Guard cells and mucilaginous cells begin to differentiate in dermal tissue at the leaf margin. Periclinal divisions, giving rise to the multiple epidermis, occur near the leaf margin and are present through- out the leaf lamina by the time the leaf is 6-7 mm in length (L4) (Fig. 26). In most ventral leaves periclinal divisions also occur in the adaxial ground meristem layer, giving rise to two ables. The slopes of the regression lines (or growth constants)
for ventral and dorsal leaves were found to be significantly different (Table 3).
(or more) layers of palisade parenchyma ( ~ i ~ l 26). Lithocysts, mucilage cells, guard cells, and substomatal cavities are recog- nizable throughout the leaf in leaves 8- 10 mm in length (Fig. 27). Cells of the palisade layers enlarge later than those Leaf blade histogenesis
Observations of cross sections of primordia and young ventral leaves indicate that the leaf blade becomes dorsiventral at an early stage (Figs. 22, 23). The early development of a lamina is primarily associated with plate meristem activity in which five tissue layers are extended through anticlinal di- visions (Figs. 23, 24). Although mitotic figures were rarely seen, the presence of thin walls between adjacent sister cells indicates that plate meristem activity continues until the leaves are 3-4 mm in length (L3). While cell vacuolation of proto- derm and ground meristem tissue and blocking out of midvein procambium occur early in the midrib region, the developing lamina is characterized by five layers of cells of similar staining density. By the time ventral leaves are 1.0 mm in length, cells in all layers in the lamina have increased in size; however, cell enlargement is greater in the surface layers so that protoderm
of the spongy mesophyll, and associated intercellular space does not develop until ventral leaves are 16-20 mm in length (L5). At these same stages rapid enlargement and vacuolation of cells in the multiple epidermis differentiate this tissue from the mesophyll (Fig. 27). This description of the timing of his- tological events is based on observations from the midregion of the leaf blade; however, observations throughout the length of the blade indicate that a basipetal maturation gradient charac- terizes development of the ventral leaf. Maturation also pro- ceeds from the margin to the midrib region.
Histogenesis of the dorsal leaf blade is distinguished by the lack of plate meristem activity. The transition from the primor- dial stage to a slightly dorsiventral organ is associated primarily with increase in cell size with few oriented divisions (Figs. 28, 29). Cell enlargement and vacuolation of dermal and ground
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
CAN. J . BOT. VOL. 62, 1984
M e o - u z z c 2 n ' s z B 6 ,$
a 04,o;; 2 5 G > = ? 3 E Z Z r , , Z 0 = m - C " t.3; 22 > 2 5 . E . S g m.iu.- . - 5 E Z e a~ - E g z2g z- .o .- - i u & 0 5 a y a o m " & c c d z $ ,2.- .- a a .- - - E E > >,z n 5 . z 3 & E -.- 0 a 7 - 3 5 t z o
5 $ : J z % m a 2 % .%:z
-a - z 2 m 2 5 8eZu 2; - & Z s z L ? - - 2 .23 gu 5 - . c c O K J L 005 o i f ; o > " > c b . 5 c a 56 ,s O 5 - .- - 3 * ." E .s x X . Z ? a O . .- 2 arJ .o m ;j 2 .. ,522 0 0 3 g,s 0 2 - % v,&5 E 2 O - w ' g ~ " S . E : ~ - 2 - 3,5
MI- o m > ~ p , $ 5 " ~ ~ 2 2 5 3 ' 5 0 -z 0 a - $ E d g . 2 a m ' = - >, - a 8 u o % Z & C c z
;J mv,.- . - i - 2 . Z " Z L 3 " 5 - - - 2 2 3 . o " G ~ a . m5;'c'e," : > c l r ; g . A 5 -PI V] - U " V ] X ::L&.: x a "3 a a? ,o a 3 3 - .Z.E if; ,$ , , o m - - x s - m " m s(ngss5; * a l r ; 5 ? X Z 5 - > 0 . -
. n 5 c z c : oric UY oif; m%,,"m
z o a x a 0 4 !5 E P d - - a0 d A k i J * D zg5;e g x . 3 E k > m .- c - - a E , Z b 2 2 - 5 0 ? 3 2 c d u.cLz 2 3 0.; z . 5 2 0 5 ' - 2 " % % . s o n 02 -2 2 2 g.a;3;;z . A .- ;; 9 m - c V] =-s S E- 'n_ 5 E z u 3 " $ Z c a S E 2 2
a > , , b o g , ~ 5 $ - m o , s , i . z
0 E - i 1 " & ~ a z z 2.5 0 ",%S 2
0 , G Y " m 0
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
MUELLER A N D IIENGLER
TABLE 3. Regression equations for log transformatio~is of length and width data for developing vcntral ancl dorsal Icavcs of Pelliortirr rlrt~~eaurrrrrr
Correlation Dcgrccs of Slopc Lcaf ty pc Rcgrcssion equation cocfficicnt frccdom cornpa~.ison
Ventral In y = - 1.09 + 1.08 In x 0.97 152 Significantly different
Dorsal In y = 2.42 + 0.46 In .I 0.7 1 86 P 5 0.05
6.0 - followed by rapid cell enlargement, resulting in cell dimen- 5.6- sions larger than those of layer 1 at maturity. The formation of 5.2- multiple epidermis in the dorsal leaf was not expressed in these 4.8- 1: measurements of dimensions of layer-1 cells, presumably as a
- 4.4- result of the restricted distribution of these periclinal divisions. 4.0- - 'O The pattern of cell enlargement in the abaxial epidermis is 3.6- f 14 similar to that of the adaxial epidermis in both ventral and 3.2-
- dorsal leaves (Fig. 32B). However, the periclinal divisions - 2.8- giving rise to multiple epidermis did not result in a net decrease P 2.4- 2
2.0- in cell size. Cell height of layer-5 cells is considerably greater than that of layer-6 cells at the earliest stage in which this dimension could consistently be measured, indicating that the periclinal divisions giving rise to this layer are associated with
0.8
0.4 a period of rapid cell enlargement. Increase in both height
I t I , , I , 1 1 1 1 1 1 1 1 1 1 1 1 , 1 , 1 ~ 1 1 1 1 1 1 1 1 1 1 1 1 1 , 1 and width of the most adaxial mesophyll cells, layer 3, occurs 16 22 28 1 7 13 19 25 312 8 14 20 26 2 8 most rapidly in the dorsal leaves (Fig. 32C). Enlargement of I980 Dec. 1981 Jon. Fe b. M a r .
Time ( d o ~ s ) these palisade cells is considerably delayed in the expanding
FIG. 20. Elongation of thc ventral lcavcs of a rcprcscntativc treatment-l plant. Lcavcs arc numbered in order of expansion from the bud.
tissue and formation of intercellular space in the ground tissue occur before young dorsal leaves a r e 0.2 nlm in length (L1 -L2, Fig. 29). Formation of guard cells and substoniatal cavities as well as continued enlargement of epidermal cells are observed in leaves 0.3-0.4 mm in length (Fig. 30). The results of periclinal divisions in both the adaxial and abaxial epidermis are observed in leaves 0.4-0.6 mm in length (L2-L3, Fig. 31). At this stage developing lithocysts and mucilage cells occur in the dermal tissues and intercellular space is conspicuous in the ground tissue. Although there appears to be a slight increase in number of cells in the nieso- phyll, the time and location of divisions were too irregular to be detected.
To put the description of cell enlargement on a more quan- titative basis. measurements of the cell dimensions were made from cross sections of the midregion of leaves over a range of developniental stages. Because of the wide variation in- leaf length and stage of tissue maturation at each leaf position, developing ventral and dorsal leaves were placed into size categories that allowed comparison of ventral and dorsal leaves of overlapping length during the growth period; the cate- gories corresponded roughly to the size increments associated with leaf position in older ventral leaves. Figures 32A-32D illustrate the trends observed in this analysis of histogenesis. Enlargement of the adaxial epidermis (layer 1 ) occurs rapidly in the dorsal leaves and approximates mature dimensions when leaves are 0.8 min in length (Fig. 32A). Enlargement of the adaxial epidermis proceeds more slowly in ventral leaves but occurs over a longer time period. The periclinal divisions giving rise to the multiple epidermis are reflected in a decrease in cell height of layer 1 when the ventral leaves are 6-8 mm in length. Formation of layer 2 of the adaxial epidermis is
ventral leaves measured. As for other cell types, cell enlarge- ment in the spongy parenchyma, layer 4, occurs most rapidly in dorsal leaves. while expansion occurs later in ventral leaves (Fig. 32D). In both leaf types, there is greater increase in cell width as compared with cell height, contributing to the mature elliptical shape of these cells.
Discussion The observations reported here provide evidence that the
ventral and the dorsal leaves of Pelliorzia davea~latza may be regarded as homologous structures. In both leaf types, the lower leaf zone gives rise to a single membranous stipule on the adaxial surface of the leaf primordiuni. While pattern of stipule expansion and form and anatomy of mature stipules are similar for both leaf types, the growth of the upper leaf zone differs strikingly between ventral and dorsal leaves. In ventral leaves, this zone beconies differentiated into a large flattened leaf blade and a short cylindrical petiole. In dorsal leaves the upper leaf zone is only weakly differentiated into blade and petiole. Despite the differences in size and form of the leaf blade region, a histological analysis shows that the blades of both leaf types have basically the same arrangement of tissues and are characterized by the same specialized cell types and structures (e.g., styloid crystals. mucilage cells, hydathodes). The small size of the dorsal leaf lamina is acconipanied by a reduction in number of tissue layers, significantly smaller cell size as ob- served in leaf cross section, and a simplified vascular system. Since ventral and dorsal leaf blades meet the criteria of equiv- alent position and of equivalent special qualities (Remane 1952), they may be regarded as hon~ologous structures which can be directly compared to document the developmental basis of habitual anisophylly.
Scanning electron micrographs and cross sections of shoot tips show that from the earliest stages observed, the volume of the primordiuni of the dorsal leaf is less than that of the ventral leaf. The plate meristem which gives rise to the ventral
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
CAN. J . DOT. VOL. 62. 1984
T
401 ( A I V e n t r a l l e a f
5
I LIO L I I L IZ L13
L e a f ~ o s i t i o n
FIG. 21. Mean lengths + standard error of ventral and dorsal leaves of treatment-l plants based on analysis of serial cross sections of shoot tips and n~acroscopic measurements. Solid bar represents lamina length; open bar represents stipule length. (A) Ventral leaf: (B) dorsal leaf.
leaf blade becomes active in leaves about 0.2 mm in length. There is no indication of organized plate meristem activity in dorsal leaves; rather cells already present in the primor- dium may divide in a less conspicuously oriented manner and subsequently differentiate. This absence of laminar growth is reflected in the greater length to width ratio of dorsal leaves, an allometric relationship which is maintained over all stages of development. Measurements of leaf length at nodes succes- sively farther from the shoot apex indicate that, although the pattern of expansion of ventral and dorsal stipules is similar, rate of expansion of ventral leaf lamina is more rapid than that of dorsal leaf lamina and also occurs over a longer time period. During the period of expansion of ventral leaves, and appar- ently after the period of plate meristem activity, periclinal divisions in the enlarging protoderm give rise to multiple epi- dermis, and periclinal divisions in the abaxial portion of the ground meristem increase the number of cell layers that differ- entiate as spongy parenchyma. At a slightly later stage of development, periclinal divisions in the adaxial layer of ground meristem determine the number of cell layers that will differ- entiate as palisade parenchyma. These divisions result in a greater number of tissue layers in the ventral leaves as com- pared with the dorsal leaves. Both the number of tissue layers and cell size in all measured tissue layers contribute to the greater thickness of the ventral leaves. Although differences in cell dimensions have been documented from cross sections of dorsal and ventral leaves and also occur in cell area as seen in surface view, these differences are in the order of 1.5- to 5-fold
and do not alone account for the dimorphism in leaf area. Probably the most important mechanisnl in the elaboration of the lamina of the ventral leaf is cell division associated with the activity of the plate meristem.
In a broad co~nparision of the n~orphological differences between mature leaves in heteroblastic series, Goebel (1900) proposed that all honlologous organs in such a series share the same early stages of development and differ in mature form through the process of arrest at a particular developmental stage and subsequent divergence of ontogenetic pathways. Despite the wide acceptance of this proposal in general reviews of work on leaf development (Jones 1956; Allsopp 1965, 1967), careful developn~ental analysis of heteroblastic leaf series has shown that differences in mature form are often expressed at, or shortly after, leaf initiation (Foster 1935; Kaplan 1973; Bruck and Kaplan 1980). The observations reported here for the anisophyllous shoots of Pelliorzia davefluflrz~ show that the ventral and dorsal leaves are readily distinguished at the earliest primordium stage and that the primary morphogenetic mecha- nism in the elaboration of the blade of the ventral leaf, activity of the plate meristem. is completely absent in the developnlent of the dorsal leaf. Similar observations of early divergence have been made for other examples of anisophylly. Croxdale (1978) showed that the primordia of the highly dissected sub- merged leaves of Snlvirlin can be distinguished from those of the floating leaves in the same whorl when 70-90 pm in length and that expansion of the submerged leaves occurs at a reduced rate over a longer time period. Dengler (1983a, 1983b)
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
MUELLER AND DENGLER
FIGS. 22-27. Cross scctions from midregions of dcveloping ventral Icavcs. Fig. 22. Leaf 164 p m in length. Adaxial protodcrn (AD), abaxial protoderm (AB), and procambium (PC) of the midvein are labelled. x850. Fig. 23. Leaf 320 p m in Icngth, showing lamina formation. X380. Fig. 24. Leaf 1304 p m in Icngth. Note devcloping trichome (T) and ccll enlargement in adaxial (AD) and abaxial (AB) dcrmal layers. x250. Fig. 25. Lcaf 4-5 mm in length. Notc devcloping lithocyst (C) in the adaxial epidermis and prcscnce of four layers of ground tissue. x325. Fig. 26. Leaf 6-7 mm in Icngth. Arrows indicate new pcriclinal walls in the most adaxial layer of ground tissue; arrowheads indicate cells in the adaxial epidermis with new periclinal walls. x200. Fig. 27. Lcaf 8- 10 mm in length. Note mucilage ccll (M) and dcvcloping substomatal cavity (SB). X200.
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
CAN. J . DOT. VOL.. 62. 1984
Frcs. 28-31. Cross sections of developing dorsal leaves taken from the niidregion of the leaf. Fig. 28. Leaf 56 Fm in length. Adaxial protoderm (AD) and abaxial protoderm (AB) are labelled. X850. Fig. 29. Leaf I92 p n in length. showing vacuolation in cells of the adaxial (AD) and abaxial (AB) protoderm and ground t~sslie (G). X575. Fig. 30. Leaf 348 p n in length. x500. Fig. 3 I . Leaf 528 Fm in length. showing develop~ng lithocyst (C) and periclinal wall which has resulted in localized multiple epidermis (arrow). x350.
also described differences in the volume of ventral and dorsal leaf primordia of Selaginella martensii, although subsequent differences in leaf expansion and histogenesis of the two leaf types were less marked than those described here. Kaplan (1980) has emphasized that the unique morphological and histological features of an organ can have their inception at any phase of development and do not have to be confined to the later stages of ontogeny. In Pelliot1ia clavealrann and in other examples in which there is extreme dimorphism between homologous organs, these differences are expressed at the earliest stages of development.
In addition to describing differences in cell enlargement between dorsal and ventral leaves, these data also document some of the patterns of cell expansion which results in the distinctive features of certain cell types. For instance, the somewhat elongated shape of the palisade cells of ventral leaves is the product of little change in width and a twofold increase in height during leaf expansion. In contrast. the spongy mesophyll cells grow less in cell height than they do in cell diameter, contributing to an elliptical shape. Similar 3bser- vations have been made of the differentiation of epidermal and
mesophyll cells in other species (Maksymowych 1963; Dale 1968; Dengler et al. 1975). The observations of epidermal cell dimensions in Pelliotlia cla\~ealrann underscore some of the problems in making deductions about the relative contributions of cell division and cell enlargement in developing leaves on the basis of structural data alone. When the periclinal divisions giving rise to a multiple epidermis are more or less synchronous and are associated with a slower rate of enlargement, as in the adaxial epidermis, a net reduction in cell size was observed. In contrast, similar periclinal divisions in the abaxial dermal layers appear to be associated with a greater amount of cell enlargement and data on cell size alone do not reflect the occurrence of these divisions.
These data on cell enlargement also document the precocious cell enlargement and tissue maturation which distinguish histo- genesis of the dorsal leaf blade from that of the ventral leaf blade. By the L3 stage, when dorsal leaves are 0.8 mm in length, individual cell dimensions and leaf thickness as measured in leaf cross sections have reached almost mature size. In contrast, in ventral leaves of comparable age, cell size has not increased significantly over that for the youngest
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
MUELLER AND DENGLER
4 4 r (A) Adaxial epidermis
40 1 layer
layer 1
(B) Abaxial epidermis
layer 6
layer 6
e * Ventra l l e a f 8-
4 t o Dorsal lea f
* Ventral lea f
- o Dorsal leof
(C) Palisade mesophyll (layer 3) (D) Spongy mesophyll (layer 4 )
r * ~ e n t r o l lea f re Ventra l l e a f
A O Dorsal leaf no Dorsal lea f
--, 20
16
height +
0 I I I I I I I I I
t . 0 2 1 6 1; 1; 114 1 6 1 8
Leaf length (rnrn) Leaf length (mm)
FIG. 32. Changes in cell dimensions in developing ventral and dorsal leaves. Tissue layers are indicated in Figs. 10 and 13. Bars equal 2 standard error of the mean. Asterisk indicates significant change from previous size class. P 5 0.05. (A) Height of adaxial epidermal cells (layers I , 2); (B) height of abaxial epidermal cells (layers 5, 6); (C) height and width of palisade parenchyn~a cells (layer 3): (D) height and width of spongy parenchyma cells (layer 4).
primordial stages observed. Cell enlargement continues over a long developmental period in the ventral leaves and in fact probably was not complete in the treatment-2 develop- mental material observed here, since the final cell dimensions obtained were smaller than those observed in a population of mature leaves. The greater coefficient of variation observed for cell dimensions in ventral leaves may be a reflection of this longer developmental period and the greater plasticity that it allows. Precocious development of simple or reduced leaves also has been shown to occur in heteroblastic shoots in a num- ber of species (Crotty 1955; Kaplan 1973; Bruck and Kaplan 1980) and in the anisophyllous shoots of Selaginella nzarteizsii (Dengler 19830, 19836).
Acknowledgements We gratefully acknowledge the helpful suggestions of
Dr. R. E. Dengler, Dr. D. R. Kaplan, and Dr. U . Posluszny and financial support from the Natural Sciences and Engineering Research Council of Canada.
ALLSOPP, A. 1965. Heteroblastic development in cormophytes. Handb. Pflanzenphysiol. 15: 1 102- 122 1.
1967. Heteroblastic development in vascular plants. Adv. Morphog. 6: 127-171.
BRUCK, D. K., and D. R. KAPLAN. 1980. Heterophyllic development in Muehlenbeckin (Polygonaceae). Am. J. Bot. 67: 339-346.
CROTTY, W. J. 1955. Trends in the pattern of primordial develop- ment with age in the fern Acrosrich~rtn tlrt~nc.fbli~rtn. Am. J . Bot. 42: 627-636.
CROXDALE, J . G. 1978. Salvit~itr leaves. I. Origin and early differ-
entiation of floating and submerged leaves. Can. J. Bot. 56: 1982- 1994.
CUTTER, E. G. 1965. Recent experimental studies of the shoot apex and shoot morphogenesis. Bot. Rev. 31: 7- 1 13.
DALE, J . E. 1968. Cell growth in expanding primary leaves of Phnseolus. J . Exp. Bot. 19: 322-332.
DENGLER, N. G. 1983tr. The develop~nental basis of anisophylly in S~lnginelln vntzrr~t~sii. I . Initiation and morphology of growth. Am. J . Bot. 70: 181- 192.
1983b. The developmental basis of anisophylly in Selngit~elkz mtrrrensii. 11. Histogenesis. Am. J . Bot. 70: 193 -206.
DENGLER, N. G. , L. B. MACKAY, and L. M. GREGORY. 1975. Cell enlargement and tissue differentiation during leaf expansion in beech. Fng~ l s grcrndifolicz. Can. J . Bot. 53: 2848-2865.
EICHLER, A. W. 1861. Zur Entwicklunps,~eschichte des Blattes mit besonderen Beriicksichtigung der ~ebe~bla t tb i ldungen. Disserta- tion, University of Marburg. West Germany.
ENGLER, A., and K. PRANTL. 1889. Die natiirlichen Pflanzenfamilien. Ill. Teil 1. Verlag von Wilhelm Engel~nann. Leipzig.
FOSTER, A. S . 1935. A histogenetic study of foliar determination in Cnryn brtcklwi var. c~rkarlstrtln. Am. J . Bot. 22: 88- 147.
FRANCK, D. H. 1976. Comparative morphology and early leaf histo- genesis of adult and juvenile leaves of Dtrrlit~grot~icr califort~ictr and their bearing on the concept of heterophylly. Bot. Gaz. (Chicago), 137: 20-34.
GOEBEL, K. 1900. Organography of plants. Past I. (English trans- lation.) Clarendon, Oxford.
JONES, H. 1956. Morphological aspects o l leaf expansion in relation to changes in leaf form. It1 The growth of leaves. Etliretl I7.v F. L. Milthorpe. Butterworth Scientific Publ.. London. pp. 93- 105.
KAPLAN, D. R. 1973. Comparative developmental analysis of the heteroblastic leaf series of axillary shoots of Acorlts ctrlntn~rs L.
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
1170 CAN. J . BOT.
(Araceae). Cellule, 69: 253-290. 1980. Heteroblastic leaf development in Acacia. Morpho-
logical and morphogenetic implications. Cellule, 73: 137-203. MAKSYMOWYCH, R. 1963. Cell divisions and cell elongation in leaf
development of Xanthirtrn pennsylvanicurn. Am. J . Bot. 50: 891-901.
REMANE, A. 1952. Die Grundlagen des natiirlichen Systems, der vergleichende Anatomie und der Phylogenetik. Akademische Ver- lagsgesellschaft, Leipzig.
RICHARDS, F. J . 1951. Phyllotaxis: its quantitative expression and relation to growth in the apex. Philos. Trans. R. Soc. London
VOL. 62. 1984
Ser. B, 235: 509-564. SINNOTT, E. W., and G. B. DURHAM. 1923. A quantitative study of
anisophylly in Acer. Am. J . Bot. 10: 278-287. SNEDECOR, G. W., and W. G. COCHRAN. 1980. Statistical methods.
Iowa State University Press, Ames. SOKAL, R., and J . ROHLF. 1969. Biometry. W. H. Freeman and Co.,
San Francisco. TROLL, W. 1937. Vergleichende Morphologie der hoheren Pflanzen.
Bd. I . Vegetationsorgane G. Borntraeger, Berlin. WIEHLER, H. 1978. The genera Episcia, Alsobia, Naittilocaly,~ and
Paradrytnonia (Gesneriaceae). Selbyana, 5: 1 1-60.
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y Sa
nta
Cru
z (U
CSC
) on
12/
04/1
4Fo
r pe
rson
al u
se o
nly.
