Development of the axillary bud complex in Echinocystis lobata (Cucurbitaceae): interpreting the cucurbitaceous tendril†
The authors thank L. Hilton and J. Cunningham for field assistance and P. B. Tomlinson for the loan of the epi-illumination light microscope. Funding was provided by the College of Natural Sciences, University of Northern Iowa. This article represents a portion of the requirements for fulfillment of the M.S. degree of T. Guthrie.
In the Cucurbitaceae, the tendrils, coiling organs used for climbing and mechanical support, are part of an axillary bud complex (ABC). Although the morphological nature of tendrils and the branching pattern of the ABC in the Cucurbitaceae have been much studied, their homology remains unresolved, with hypothesized candidates being the leaf, flower, stem, or stem–leaf combination. We used Echinocystis lobata as a model to study the early ontogeny of the ABC with epi-illumination microscopy and serial resin sections. The ABC produces four structures (proximal to distal, relative to the subtending leaf) as the result of two successive subdivisions: an inflorescence of staminate flowers, a solitary pistillate flower, a lateral bud, and a tendril. The first separates the tendril primordium from the continuation of the ABC, and the second separates the staminate inflorescence and the ABC. The pistillate flower apparently forms between the staminate inflorescence and the lateral bud. Because there is no subtending leaf during these subdivisions and the first lateral appendages in the resulting primordia arise in the same plane, we conclude that the tendril and other organs formed by the ABC are lateral branches of equal morphological value. This study is the basis for continuing comparative and functional morphological studies.
Tendrils are thigmotropic coiling organs that aid in mechanical support of vines (8; 30). They are present in a number of widely divergent angiosperm families and represent modifications of all the various shoot components (17). Thus, they are most likely to have arisen many times and are good examples of convergent evolution. In some instances, it is relatively easy to determine the organ from which the tendril originated. For example, pea tendrils and leaflets can be considered as homologous structures (16), and tendrils in the Vitaceae have been shown to develop from an uncommitted primordium that differentiates into either an inflorescence or a tendril (27; 37; 12). However, in other taxa, tendril origin is less easily determined and remains controversial, as in the Cucurbitaceae, in which the tendril is part of a complicated nodal complex of organs. Because of this complexity, a simple examination of these nodal components at maturity is not sufficient to ascertain relationships. Likewise, a molecular approach to this question remains problematic until the range of possible origins for the cucurbitaceous tendril has been narrowed. Because an ontogenetic approach has proven effective in interpreting tendril homology in other taxa (e.g., Vitaceae, 12; 11), we have used a similar approach in this instance.
The Cucurbitaceae is a family of medium size, consisting of approximately 120 genera and about 850 species distributed predominantly in the tropical and subtropical regions of the New and Old World. Current classifications place it in the Order Cucurbitales, as part of the Eurosid I group (35 and onward). There are two subfamilies; the smaller Nhandiroboideae and the Cucurbitoideae, which contains the bulk of the taxa in the family (22). Over 90% of the species are found in three main areas: Africa and Madagascar, Central and South America, and Southeast Asia and Malaysia (21). In contrast to Old World representatives, which are mainly found in drier regions, New World taxa are most diverse in rain forest areas. There are 13 North American species (21), three of which are native to Iowa (10).
Most Cucurbitaceae are perennial, herbaceous vines (rarely shrubs or trees) that usually climb by means of branched or unbranched tendrils. Shoot growth is monopodial (5; 25; 26). Most taxa are monoecious, although there are some dioecious species (39; 38). The green to greenish-white flowers are typically small (20). The staminate flowers are borne on an inflorescence that may be racemose to cymose, and the pistillate flowers are generally solitary.
Tendrils in the Cucurbitaceae are generally considered to be part of an axillary complex (26), but their morphology differs between the two subfamilies. In subfamily Nhandiroboideae, a bifid tendril forms as part of the lateral shoot, which arises in the leaf axil (26). In contrast, in Cucurbitoideae, the “axillary” complex arises in an extraaxillary position and can consist of up to four components in the following order from distal to proximal to the leaf base: tendril, lateral shoot, solitary pistillate flower, and inflorescence of staminate flowers. This unusual morphology has meant that there is no strict consensus as to the morphological interpretation of the tendril in Cucurbitoideae. Some authors have postulated that the tendril represents a modified flower (8; 26; 2), but most authors have favored the interpretation that the tendrils are either modified stems (32; 5) or a stem–leaf combination (15; 28; 25; 3).
These interpretations relied mainly on mature structures and were not based on a complete ontogenetic examination of axillary bud complex development. In this study we chose to examine a readily available species native to both Ontario and Iowa, Echinocystis lobata Michaux (Torrey and Gray), to establish tendril ontogeny in a member of the Cucurbitaceae. Our objectives were twofold: to document the early ontogeny of the tendril, leaf, lateral shoot, and flower primordia in Echinocystis lobata and to use this information to interpret the homology of the cucurbit tendril.
MATERIALS AND METHODS
Echinocystis lobata (Michaux) Torrey & Gray is an herbaceous, monoecious annual vine that can reach 6–8 m high (14). It extends throughout the temperate regions of eastern North America, mainly in riverine habitats and rich, moist soils of deciduous forests, although it can also be found in waste areas and on drier sites. It is considered to be a weedy species in southern Canada (1).
In Iowa, 10 shoot tips of Echinocystis lobata were collected from plants at two sites in Black Hawk and Butler counties and stored in formalin-acetic acid-alcohol (FAA) (31). Seeds were harvested from these plants in October 1998, stratified at 4°C in University of Northern Iowa (UNI) potting soil mix for 26 wk (7), and germinated under natural greenhouse conditions. The resulting seven seedlings were transplanted to pots containing the standard UNI soil and grown outdoors during the summer. Plants were fertilized every two to three weeks with Peters 20–20–20 General Purpose liquid fertilizer (Scotts Co., Marysville, Ohio, USA). Observations were made daily, and a minimum of 50 shoot tips were harvested over the growing season and stored in FAA.
In Ontario, 10 seedlings from a forested site on the University of Guelph campus were transplanted at the first-true-leaf stage to pots containing Pro-Mix BX potting soil (Premier Horticulture, Rivière du Loup, Quebec, Canada) and grown to the flowering stage in the greenhouse. Plants were fertilized weekly with 20–20–20 fertilizer. A minimum of 50 shoot tips were harvested over the growing season and stored in FAA.
For resin sectioning, FAA-preserved material was dehydrated through a graded ethanol series, then infiltrated with LR White resin (London Resin, Theale, Berkshire, UK), in which it was embedded. The embedded material was serially sectioned at a thickness of 6 μm, using glass knives and a Sorvall Porter-Blum MT-2 ultra microtome (DuPont Co., Newton, Connecticut, USA). The sections were mounted on gelatin-covered slides and stained with toluidine blue O (31). Sections were viewed and photographed with an Olympus BX-40 photomicroscope (Olympus, Tokyo, Japan) equipped with an Olympus PM-20 exposure control unit with Kodak T-Max 100 and Technical Pan films (Eastman Kodak, Rochester, New York, USA).
Epi-illumination light microscopy
Preserved shoot tips were carried through a graded ethanol series and stained with either 0.1% chlorozole black in 95% ethanol, 0.1% nigrosin in 95% ethanol, or 0.5% alcoholic acid fuchsin at least overnight to improve contrast, then washed and stored in absolute ethanol. Dissected specimens immersed in absolute alcohol were examined and photographed with a Leitz photomicroscope fitted with Leitz Ultrapak (UO) dipping cones (Leitz Group, Oberkochen/Württemberg, Germany), a green wide-band interference filter, an Olympus PM-20 exposure control unit, and Kodak Technical Pan film (29; 6).
Echinocystis lobata climbs by means of a compound tendril that typically has a pair of first-order arms. The main tendril axis can extend to 20 cm or more. Leaves are palmately five-lobed, estipulate, and spirally arranged (Fig. 1A). Flowering usually occurs from middle June through August. Staminate and pistillate flowers are green to greenish-white with corollas measuring 3–10 mm in diameter. Staminate flowers occur on a racemose inflorescence with up to 15 individual flowers each (Fig. 1B). Pistillate flowers are generally solitary (rarely two per node). The maturing fruit is a greenish, ovoid to cylindrical capsule up to six cm long, covered with glabrous spines (Fig. 1C, 1D). At maturity, the capsule dehisces along two pores at its distal end, releasing two seeds per pore (Fig. 1C). Seeds are narrowly ovoid, 1–2 cm long, and brown to brown with black spots.
During the vegetative phase, each node consists of three elements: a leaf, a lateral bud, and a tendril (Fig. 2A). At maturity, leaves and tendrils are nearly 180° opposed at each node, with the lateral bud located more or less halfway between the petiole and the tendril. The nodal arrangement can be either right (clockwise) or left (counterclockwise) handed (Fig. 2B), with the handedness constant for each plant and the lateral organs displaced anodically (uphill) in the genetic spiral for that particular plant.
During the reproductive phase, each node possesses five elements in the following order: a leaf, a raceme-like inflorescence of staminate flowers, a solitary pistillate flower (rarely 2), a lateral bud (which may develop into a lateral shoot), and a tendril (Fig. 2A). As with the vegetative shoots, these structures are offset laterally from the leaf axil, with the tendril the most distal and approximately 180° opposite the leaf (Fig. 2C).
Leaves are initiated spirally on the flank of the shoot apical meristem, with each leaf primordium (L) arising as a slight swelling (Fig. 3A). By the second plastochron, the leaf is more obviously dorsiventral and has begun to curve over the shoot apical meristem (Fig. 3A, L2). By the third plastochron, the axillary bud complex (ABC) arises, offset anodically (uphill and to the side of leaf L3), never appearing physically continuous with its associated leaf (Fig. 3A, ABC3). The timing and position of the developmental events for lateral shoot and tendril primordium production from the ABC are similar for both vegetative and reproductive phases and will be described together. Differences in timing and position occur during flower formation and will be discussed separately.
The axillary bud complex subdivides unequally to form a smaller tendril primordium distal to its associated leaf and a larger continuation of the axillary bud complex (Fig. 3B, 3C, T, cABC). This subdivision may occur as early as the third plastochron, but more typically it occurs during the fourth. In transverse section, the two regions, although clearly part of the same complex, are separated from one another by a layer of less densely stained cells (Fig. 3C, arrowhead). By the sixth plastochron, the two organ primordia, although still spatially close, are easily distinguished (Fig. 3B, T, cABC). In vegetative material, the continuation of the axillary bud complex will form the lateral (axillary) shoot (Fig. 3B, Ax9). In longitudinal section, the lateral shoot apex has a clearly defined two-layer tunica-like organization (Fig. 3D, arrowhead). The first leaf to be initiated on the flank of the lateral shoot is in an adaxial position (Fig. 4D, L1), and as successive leaves are initiated, a typical lateral shoot forms (Fig. 5D, Ax12).
As stated, the tendril primordium forms from the smaller, distal portion of the subdivision of the axillary bud complex, usually at the third or fourth plastochron (Fig. 3B). In section, the tendril can be distinguished from the continuation of the axillary bud complex, although at this stage, both are clearly components of the same structure (Fig. 3C). By node 5, the tendril and the continuation of the axillary bud complex are roughly equal in size (Fig. 4A), and a leaflike structure has been initiated on the adaxial flank of the tendril primordium (Fig. 4B, arrowhead). It is this leaflike structure that will form the central, primary growth axis of the tendril (Figs. 4C, black arrowhead; 4E, TA1). Subsequently, two arms originate in what appears to be an axillary position of the primary growth axis (Fig. 4C, white arrowhead). By node 8, the tendril primary axis has elongated, taking on a cylindrical appearance (Fig. 4D, T8). As growth of the primary axis continues, it continues to curve over the developing first order arms, which also become cylindrical and curve inward by node 11 (Fig. 4E, TA).
Flower initiation and development
The first indication of the development of the floral primordium occurs at node 5, when the continuation of the axillary bud complex subdivides unequally to form the floral and lateral bud primordia (Figs. 4D, 5A, F, Ax). As with the tendril primordium, it is the floral primordium that is the smaller. By node 6, the floral primordium has enlarged and is roughly dome-shaped (Fig. 5B, F6). Subsequently, the staminate inflorescence is established as individual staminate flowers are initiated on the floral dome (Fig. 5C, MI10). At the same time, the pistillate floral primordium appears, located between the staminate inflorescence axis and the axillary bud (Fig. 5C, FF10). By node 12, the pistillate flower has enlarged, and both sepal and petal primordia are evident (Fig. 5D, FF12).
Workers who have described the positions and morphological relationships of the nodal organs in Cucurbitaceae have, for the most part, based their observations on mature structures and have not examined the initiation of the organs (8; 15; 19; 32; 5; 25; 26). Similarly, studies of Echinocystis lobata (4; 7; 36; 28; 33) were not focused on development, nor with the morphological interpretation of the tendril. The present report is the first complete ontogenetic study of the initiation and early development of the axillary organs in a member of the Cucurbitaceae. Our data suggest that all the nodal organs arise as a result of successive subdivisions of the original axillary bud complex or its continuation and are not initiated as successive branch orders. Thus, if the main shoot is assigned a morphological value N, the axillary bud complex is a first order branch of morphological value N + 1, and all the nodal organs have this same morphological value. Successive branches of the axillary bud would have the value N + 2.
19 erroneously considered the Cucurbitaceae branching pattern to be sympodial; the tendril represents the shoot apical meristem, which has been pushed to the side almost directly opposite the leaf, and shoot growth is continued by the lateral bud. Later workers disagreed (5; 25; 26) and interpreted the shoot branching pattern to be monopodial. We agree with these authors and found that for E. lobata branching is monopodial, given that the main axis is indeterminate and produces leaves on its flank, with all nodal elements arising from an axillary bud complex (Fig. 3A).
Axillary bud complex
The bud complex in the Cucurbitoideae is extraaxillary from inception (Fig. 3A). 34 explained this extraaxillary position using his space-filling theory of phyllotaxis. In this theory, leaf primordia (and their associated axillary buds) are displaced in the anodic (uphill) direction of the genetic spiral by the influence of the axillary bud three nodes below. We agree, given the large number of organs that are present at each node and derived from this structure (Fig. 5C, 5D).
Because we can assume that the cucurbitaceous tendril is modified from an existing organ such as a stem, leaf, or flower, the question is, “Which organ was modified” As we stated in our introduction, previous workers’ interpretations can be categorized as (1) modified flower (8; 26; 2), (2) modified stem (5; 32), or (3) a stem–leaf combination (15; 28; 25; 3).
The hypothesis that the tendril is a flower homologue in which the tendril stalk is a modified flower peduncle and the tendril arms are prophylls of the flower is based in part on the branching orders of the nodal organs. 18 viewed the single female flower in the Cucurbitaceae as the true axillary shoot (N +1) and the other nodal elements branches (N +2) from its axis. This interpretation was refuted by 5, 25, and 26, and they regarded the axillary bud (or lateral shoot) as the primary axillary element (N +1) and the floral shoot as a branch of the axillary shoot (N +2).
Although 26 concluded that the tendril is shootlike, he interpreted the tendril stalk to be a modified flower peduncle with the tendril arms representing modified prophylls of the flower. As evidence, he cited instances in which tendrils are derived from solitary flowers or combinational forms of tendril and flower. The evidence that tendrils are sometimes transformed into flowers does not necessarily mean that tendrils are modified flower shoots, but only that the tendril retains the capacity for floral initiation and development, which would seem to imply that the tendril has stem (axillary) characters. Also, there is no documentation of flowers in the Cucurbitaceae transforming into tendrils, which would be expected if the two organs are truly homologous structures.
In E. lobata, floral and tendril initiation are separated by both time and space. The tendril develops early and is spatially separated from the floral region of the ABC by the lateral shoot. Thus, after the inception of the ABC, floral and tendril primordia do not share any common area of the ABC, nor does any portion of the tendril primordium appear clearly prophyll-like in position, timing, or structure relative to the axillary bud complex.
Mature angiosperm leaves share a number of attributes, including a lateral position on the stem; a determinate growth pattern; an association with an axillary bud on the adaxial side of the leaf base; subregions of blade, petiole, and leaf base; stipules (if present) along the longitudinal axis; and typically a dorsiventral symmetry with a flattening in the transverse plane (9). Therefore, if the tendril in E. lobata is homologous to a leaf, it would be expected to share a majority of these characteristics with a leaf.
Both leaves and tendrils have stomata and epidermal, glandular hairs. The arrangement and anatomy of the vascular cylinder is similar for the leaf petiole and the tendril arms (28; T. Guthrie, personal observations). The growth pattern of both is determinate and occurs laterally. However, the tendril is not dorsiventral in the sense that it is flattened in a transverse plane. It is more radially symmetrical in appearance, although there is an adaxial/abaxial plane in the sense that the tendril arms are not medially positioned on the tendril main axis. The tendril does not have comparable subregions to that of the leaf (with the exception of proximal/distal domains).
However, the ontogeny of the leaf and tendril differ markedly. The unequal subdivision of the ABC into the tendril and the continuation of the axillary bud complex do not resemble the initiation of the leaf primordium at the shoot apical meristem (Fig. 3B). The tendril is initiated at the flank of the ABC, but it is as a subdivision into two separate primordia. According to 23, all angiosperm leaves are dorsiventral from the onset of their initiation, and their subsequent growth further elaborates this theme. However, in E. lobata the tendril primordium develops from a more or less radially symmetrical rounded mass of tissue from a subdivision event (Fig. 3B). While the leaf develops lamina lobes and trichomes very early (by node 4), in contrast, the tendril does not develop something resembling a dorsiventral plane until much later, when the main growth axis is established and the first order branches (arms) are initiated at node 7.
Thus, the hypothesis that the tendril is a leaf homologue, either modified from the first leaf of an axillary shoot or from a leaf originally opposed to a basal leaf displaced laterally by the growth of the axillary bud, is not well supported. The tendril and the continuation of the axillary bud complex can be thought of as independent of one another as if these nodal organs have the same morphological value and any displacement along the node that occurs is the result of their simultaneous and mutual growth.
The authors who consider the tendril to be a shoot axis regard the lateral bud (vegetative shoot) to be the principal axillary element, but differ on the morphological value of the floral and tendril shoots. 15, 18, 25, and 3 consider the tendril to be a compound structure consisting of an axillary shoot (N +2) fused with its subtending leaf. Thus, the subtending leaf, which forms the outer tendril arm, is the α-prophyll (the first prophyll produced) on the primary axillary shoot. Both 5 and 26 demonstrated that no subtending leaf is involved in the tendril architecture, but they differ in their interpretations of tendril homology. 5 considered that the tendril represents a shoot of equal value to the axillary shoot N +1 and that the floral shoot has the value N +2. 26 also considered the tendril to have the value N +1, but interpreted the tendril to be a modified floral shoot (see section Flower hypotheses).
For E. lobata, the tendril does appear to represent a shoot of the value N +1. There is no subtending leaf involved in its development. The axillary bud complex (ABC) at node 3 appears as one entire structure which then goes through two subdivision events. The first event occurs at node 4 in which the ABC divides unequally to form the tendril primordium and the continuation of the ABC (Figs. 3B, 6). This event does not involve a subtending leaf, and neither portion resembles a prophyll-like structure. The tendril primordium is initiated as a rounded mass of tissue and remains dome shaped until the main tendril arm is established at node 7, when it resembles a prophyll-like appendage (Fig. 5C, T8). The secondary axes (first order branches) are then initiated in an axillary-like position on the flank of the dome-shaped primordium at nodes 8–9. It is important to note that both the main tendril axis and the secondary axes (arms or branches) remain cylindrical throughout their development (Fig. 4E).
The second subdivision event involves the separation of the floral primordium from the lateral bud primordium at node 5 (Fig. 6). The primordium, which will differentiate into the staminate inflorescence, remains as a more or less rounded mass of tissue until a lateral bractlike structure forms on the flank at node 8 (Fig. 5B). At this time, the inflorescence axis is established, and the individual staminate flowers begin to develop. It seems likely that the staminate inflorescence is of the value N +1 because neither it nor the tendril appears to be axillary to the lateral bud, which separates the two nodal organs (Figs. 5C, 6). We hypothesize that they are similar to the supernumerary buds found in some species of the Vitaceae (13). It is unclear precisely when or how the solitary pistillate flower is initiated, because in our material it was first noticeable as a small, rounded mass situated between the staminate inflorescence and the axillary bud by node 10 (Figs. 5C, 6).
Those authors who consider the tendril in the Cucurbitaceae to be an axillary shoot (stem) of the value N +2 invoke the common ideal that an axillary shoot must be associated with a subtending leaf (15; 25; 3). Even though there is no evidence of a leaf subtending a tendril in any of the Cucurbitaceae, this interpretation relies upon the notion of a congenital loss of function and/or structure (the subtending leaf). This hypothesis is untestable by definition, in that there would be no morphological evidence of this loss. These same authors also consider the tendril arms to be foliar in nature and cite evidence in which tendril arms possess leaflike laminas (15) or an anatomical resemblance to leaves (25; 3). While the tendril arms and the leaf have anatomical similarities, 15, p. 425) himself states an “anatomical relationship alone can never solve a morphological problem.” When the ontogeny of the tendril is considered, the tendril arms do not seem to possess truly leaflike characters, either in form or position.
The tendril in E. lobata most likely represents an axillary shoot (stem) of the morphological value of N +1, and the tendril arms represent first-order branches. The tendril and the leaf do not share enough characteristics, either in ontogeny or form, to be considered homologous organs. Floral and tendril initiation and development are separated by both time and space in such a manner as to be considered independent events. We consider the tendril arms to be first-order branches because they develop in an axillary-like position, not a prophyll-like position. It is most reasonable to consider that the ABC itself is the true axillary structure and that all the structures produced by the ABC have the same morphological value N + 1.
We have focused on the initiation and early development of the axillary bud complex in one species and have used this information to create a developmental model that results in a more likely interpretation of the morphological nature of the tendril. Recent molecular phylogenetic hypotheses for the family (24) now provide a framework for choice of taxa and character mapping in further developmental and functional morphological studies as our model is further tested and refined.
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