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|Vol. 9(3), pp. 9-16||The McAllen International Orchid Society Journal||March 2008|
Past research by this worker concentrated on the members of the tropical genus Stanhopea, with only scattered samples from terrestrial orchids. For this line of research, it was fruitful to develop and employ a method of obtaining a casting of the leaf surface for certain epiphytes, but for terrestrial orchids in particular, there is really no substitute for working with live fresh leaf material. Specimens undergo deformation when dried, and although morphological studies may be productive to a certain extent when they are reconstituted, critical cell measurements remain compromised. The leaf casting system developed by this worker for his doctoral research is limited when studying decidedly pubescent to rugose surfaces, ones clad with a heavy waxy cuticle, or for leaves of very light texture as seen in some terrestrial orchid species. For these plants, with their thinner leaves, a small sample of fresh, living material is not only the best avenue, but--in fact--the only one that has provided good results. For practical purposes, the shipment of such material to a microscopic center is out of the question. Delays in transit are bound to degrade both the material and any results obtained. The microscopic and photographic equipment must be brought relatively close to the collection area to ensure the highest quality of data obtained photographically. This sounds complicated, but, in reality, is relatively simple. One simply packs equipment, travels to a location a short distance from the collection site, sets up equipment, and then collects, and photographs. The photographic record then becomes an enduring data sample that may not only be filed with a particular archival repository, but electronically shared worldwide, freeing archival centers with having to pack and mail material. While leaf material may be retained in a herbarium, it becomes relatively useless from a morphological standpoint.
Both anatomical and life cycle information need to be provided to field biologists nationally and internationally. To do this, plant specimens and seed must be allowed to be collected for investigations into the roles played by various soil fungi in the life cycles of different terrestrial orchid species. As a practical matter, this means collecting seed, flasking it using various solutions, and determining which soil fungus (or fungi) plays the optimum role in plantlet survival. In this field, seminal work has already begun (Zettler, 1997a, 1997b; Stewart & Zettler, 2002; Stewart, Zettler & Minso, 2003; Stewart & Kane, 2006, and others). However, if this work is to be expanded to the profit of field biologists globally, restrictions on collecting and propagating need to be removed for qualified workers to conduct serious studies. Such work also implies keeping a detailed photographic and text record of the life cycle of each species, for dissemination not only to domestic governmental field biologists, but particularly to those field biologists in countries where certain terrestrial orchids are at serious risk of being harvested (many illegally!) to extinction! None of these activities are suggested as commercial enterprises! They are suggested to learn more about these endangered plants, and provide field workers with detailed anatomical and life cycle information in order to aid in the recognition at all life cycle stages, and assist in the proliferation of these plants to the extent of their no longer being at risk of extinction. The ability to do the work exists. What is needed is massive cutting of the bureaucratic red tape that has resulted from an idealistic international treaty seriously flawed in the realities of the everyday world!
The plant leaf is a disposable factory. Most factories are a building with machinery installed, raw material supplied, work being done, and a finished product produced. Over time, machines are repaired or replaced, but the basic building endures. However, plant leaf factories are governed by the five basic phases of biology: beginning, growth, maturity, decline, and death! The leaf starts as a bud, then enlarges into being a fully grown leaf. At maturity, it does its work as a factory. The leaf utilizes water and minerals supplied by the roots and, through opened stomata, take in carbon dioxide from the air, while providing for the elimination of oxygen as waste material. With energy supplied by sunlight, the leaf produces the sugars which are transported by a vascular system which feeds all other plant parts. The leaf eventually ages, declines in ability to provide nutrition to the plant system, ultimately dies, is discarded by the plant system, and a whole new leaf factory is produced. Thus, while the leaf is a nutrition producing factory for the plant, it becomes not a disposable machine, but a disposable factory!
Some plants have relatively large leaf cells while others have small-to-relatively-minute ones. To understand why this is so, it may be useful to consider an evolutionary comparison. Assume a large pool filled with water in which are balloons filled, for the most part, with water, but each one with a small amount of oil or air. Some balloons are filled to their capacity, but others less so. In the pool, all float, and are relatively weightless, much like fishes or whales in an ocean. However, when a plug is pulled and the water is drained from the pool all these "cells" are stranded on the land surface. Now the walls of the larger balloons are subjected to much more stress than those of the smaller ones, so the walls of the larger ones tend to break. The same may be noted with plant cells. As the genus Stahopea progressed from tropical to temperate zone environments the climatic selection pressures become progressively greater. Likewise, the same may be said for plants spreading from wet to dry environments, shady to sunlit environments, and so forth. This was graphically illustrated to this worker while investigating the leaf cells of members of the genus Stanhopea, an orchid genus that originated in the New World tropics and--over time--radiated into near-temperate zones. Tropical members of Stanhopea have relatively large leaf cells, but as the genus radiated, the leaf cells become smaller as speciation progresses. More might be explored concerning this topic, but for this work it is sufficient to note that as plant cells have relatively rigid walls, any expansion and contraction activity will be correspondingly greater in larger celled leaves, and will necessitate greater cushioning between any expanding members and the rigid cell walls surrounding them, with the reverse being true of smaller-celled leaves.
Fig. 1. Adaxial epidermal leaf cells, x80, Stanhopea oculata. Plant Pl#290496-5 C6. Xylol strip Adα-4, 26 May, 1996. 35mm Photomicrograph transparency, 19 July, 1996.
Fig. 2. Abaxial epidermal leaf cells, x80, Stanhopea oculata. Pl#290496-5 C6. Xylol strip Abα-2. 26 May, 1996. 35mm Photomicrograph transparency 19 July, 1996.
Fig. 3. Stomata guard cells. Drawing, 01 February, 2008.
In many mature leaves, the upper (adaxial) surface (the one exposed to the sunlight) is covered with a waxy coating. This acts as not only a form of sunscreen, but aids in shedding rain so the leaf cells don't become overly saturated with water and burst. This upper surface may look like just so many bricks with an occasional wax-secreting gland present (Fig. 1). The adaxial surface may even sprout prickles, thorns or hairs which may aid in deterring large or even tiny predators. The underside of the leaf (the abaxial side) differs. Many of these surfaces look like just so many cells rather irregularly arranged; punctuated with small openings looking much like a pair of bananas joined at their ends (Fig. 2). As these cells shrink, an opening into the leaf's interior is provided. When they swell, the opening into the interior of the leaf is closed. These specialized cells are known as guard or stomata cells and may be assisted by subsidiary cells (also seen in Fig. 2). When the stomata cells shrink, the opening looks something like a "stoma" (mouth) into the plant interior (Fig. 3), and they act to "guard" the "mouth" entrance into the plant's interior.
Land plants may have stomata on one or both leaf surfaces. Stomata are common on the abaxial surface; but are usually sparse or absent on the adaxial surface. The guard cells are very different from other epidermal cells, and subsidiary cells are also structurally unlike the surrounding epidermal cells, and may be quite distinctive. Certain plant groups have additional cells arranged around their stomatal cells, but in some genera no additional specialized cells are present. These additional specialized cells are referred to as subsidiary or accessory cells. They may act to assist, reinforce, or protect the stomatal cells. Given that plant cells have relatively rigid cellulose cell walls, and that the stomata must expand and contract, subsidiary cells afford a cushioning effect for the adjoining (more rigid) cells from the stomatal expansions and contractions. Subsidiary cells have been noted by this worker as more prevalent in plants with larger leaf cells, and less so in ones with small-to-tiny leaf cells. This evolutionary adaption may have evolved, in part, because the expansion and contraction of the stomata in the smaller celled plants does not generate comparable stresses on the adjoining leaf cells.
N. H. Williams (1979) suggests the presence of four subsidiary cells with each stomata may be a derived condition, but outgroup comparison suggests the presence of subsidiary cells as an ancestral condition for the Orchidaceae. The subsidiary cell pattern in the Cranichideae is distinctive, and may be characterized by the mesoperigenous development of the subsidiary cells (Williams, 1975). Dressler (1993) states the ontogeny of subsidiary cells is considered important in plant classification, but he instantly qualifies this statement by noting that there are many systems of classifying stomatal types, each with its own abstruse terminology.
Clinical research by this worker (1999), utilizing extensive measuring of guard and subsidiary cells, found no statistical support for these cells having taxonomic significance as regards species identification in the genus Stanhopea. Likewise, no similar taxonomic significance was found in other orchids studied, albeit research was less exhaustive with members of Govenia, Laelia, Malaxis, Phalaenopsis, and other genera. Dressler's offering on stomata and subsidiary cells is limited to less than a page (three paragraphs), in which he notes the following, "As is too often the case, we have good information on the pattern and ontogeny of the epidermis in the advanced Epidendroideae, but more study of the primitive groups is needed." (page 23)
Fig. 4. Anisocytic Subsidiary cells. Drawing, 01 February, 2008.
Fig. 5. Paracytic Subsidiary cells. Drawing, 01 February, 2008.
Fig. 6. Diacytic Subsidiary cells. Drawing, 01 February, 2008.
In the course of researching guard and subsidiary cells, and reviewing the classification systems of other workers, this worker found it useful to simplify the classification of subsidiary cell formations into three groups: anisocytic, paracytic, or diacytic. Each of these three groups does essentially the same job and is composed of the same cellular material, but these cells differ from the composition of the guard cells and the other normal epidermal leaf cells. Anisocytic cells (aniso: "unequal) are unequal in appearance to each other (Fig. 4). An anisocytic cell group may be composed of three or more cells which surround the guard cells, buffering it from the other epidermal leaf cells. Paracytic subsidiary cells (para, around, and cytos: cells, not "parasitic," which has to do with being a parasite!) are arranged about the long axes of the stomata cells (Fig. 5). The third group of subsidiary cells are called Diacytic (dia, across) because they are arranged at right angles to the stomata cells (Fig. 6).
Fig. 7. Abaxial epidermal leaf cells, x80, Govenia utriculata. Pl#230796-2 C35. Xylol strip Abα-1: 23 July, 1996. 35mm Photomicrograph transparency digitalized: 31 January, 2008.
The bewhiskered subsidiary cells shown in Fig. 2 are clearly paracytic ones, and although they vary in size and frequency from one species to another, this type of subsidiary cell formation is a general characteristic of members of the genus Stanhopea; a decidedly tropical to an almost-temperate genus. Compare these cells with the ones from the abaxial surface of Govenia utriculata, a terrestrial orchid species (Fig. 7). The subsidiary cell areas of Stanhopea oculata specimen in Fig. 2 averaged 1632.02µ2, while those of the terrestrial Govenia superba averaged a mere 865.51µ2. The guard cells of plants with relatively large epidermal leaf cells obviously require much more "cushioning" than do the guard cells of ones of smaller sized epidermal leaf cells.
Less obvious, albeit not surprisingly, as epidermal leaf cells are smaller, the need for "cushioning" of subsidiary cells becomes minimal or may vanish altogether. In fact, in the Orchidoideae, Neottieae, and Pogoniinae, recognizable subsidiary cells are consistently lacking (Dressler, 1993, and H. Rasmussen, 1981, 1987) indicates that some cells in the Orchioideae may be mesoperigenous in origin, but not recognizable as subsidiary cells at maturity.
Fig. 8. Abaxial epidermal leaf cells, S. stellata. Dried leaf, X80 mag, 35mm transp#5, 19Jan08.
Fig. 9. Abaxial epidermal leaf cells, S. stellata. Dried leaf, X320 mag, 35mm transp#7, 19Jan08.
Another worker had suggested that the subsidiary cells in Spiranthes might be taxonomically significant. Accordingly, dried-leaf and live-leaf samples were examined using a Microscopics trinocular compound microscope fitted with the Nikon HFM photographic computerized system. Examinations were done, and 35mm Kodachrom color photomicrographs were made of dried leaf material of Spiranthes stellata and S. romanzoffiana was examined. Material of S. stellata was examined at magnifications of X80 (Fig. 8) and X320 (Fig. 9).
Fig. 10. Abaxial epidermal cells, S. romanzoffiana. Dried leaf, X80 mag, 35mm transp#16. 19Jan08.
Dried leaf material of S. romanzoffiana was also examined at X80 magnification, and the guard cells examined and photographed. A small area of that 35mm transparency was digitally scanned and the digital scan greatly enlarged (Fig. 10). As in examinations of the S. stellata material, no subsidiary cells were observed at magnifications of X80 or X320. In all examinations, of dried leaf material, guard cells could be observed at both X80 and X320, although they could be more critically observed at the higher magnification. However, even at the higher magnification (X320), and intently visually scanning each of the specimens, no subsidiary cells were ever detected.
Fig. 11. Abaxial epidermal leaf cells, Spiranthes cernua. Live leaf, X80 mag, 35mm transparency #23. 19Jan08.
Fig. 12. Abaxial epidermal leaf cells, Spiranthes cernua. Live leaf, X320 mag, 35mm transparency #24. 19Jan08.
Continuing the investigation, a live leaf sample was taken of Spiranthes cernua, placed on a glass slide, and examined and photographed under the microscope. Guard cells were clearly visible at X80 magnification, but no subsidiary cells were evidenced (Fig. 11). Although the abaxial leaf cells could be clearly seen, no formations surrounding the various guard cells gave any hints of having any paracytic, anisocytic, or diacytic patterns that would indicate subsidiary cells. The abaxial leaf surface was then examined at X320 magnification (Fig. 12) and again, even more clearly, no subsidiary cells were visible.
No other species of Spiranthes were examined, but from examinations of adaxial and abaxial specimens of Spiranthes stellata, S. romanzoffiana, and S. cernua, it is inferred that at least at maturity, subsidiary cells are absent in members of this genus. In no case were stomata observed on the adaxial leaf surfaces of the Spiranthes specimens examined in this limited study. The examination of leaf samples at X32 magnification shows the generalized cell pattern for both the adaxial and abaxial surfaces. However, if critical measuring of epidermal leaf cells is to be done, as this worker did extensively with members of the genus Stanhopea, magnification at X80 is recommended.
Few specimens were examined in this study, and it may be that taxonomically significant patterns would emerge if a large number of species of several genera were surveyed and the data submitted to an analysis of variance treatment (ANOVA). However, in order to ensure statistical validity, this worker would insist on an absolute minimum of five samples of of each species, with ten randomly collected specimens of each species considered as a more robust sampling. If this work is done, it is suggested that data from the adaxial leaf cells probably will be found to be more significant from a taxonomic standpoint than the data collected from abaxial leaf surfaces.
For general use by field biologists, for example, in a national park environment, it would be useful to have access to certain readily confirmed general identification markers such as the presence or absence of subsidiary cells, or the general appearance of wax glands on the adaxial leaf surface or the appearance and general frequency of stomatal guard cells on adaxial leaf surfaces. If so, these could be additional tools for the recognition of certain species during non-flowering periods.
Dressler, R. L. 1993. Phylogeny and Classification of The Orchid Family. Portland Oregon: Dioscorides Press. 314pp.
Ferry T., R. J. 1999. Estudio Anatómico Epidermico en Las Hojas Del Genero Stanhopea (Orchidaceae) y Sus Implicaciones Taxonómicas. Monterrey, NL, México: Universidad Autónoma de Nuevo León. 49pp y Anexo I (68pp.), II (27pp.), III (44pp.), y Epilogo (1p.).
Rasmussen, H. 1981. The diversity of stomatal development in Orchidaceae subfamily Orchidoideae. Botanical Journal of the Linnean Society 82: 381-393.
_______ . 1987. Orchid stomata-structure, differentiation, function, and phylogeny. In Orchid Biology, Reviews and Perspectives, Vol. 4. Ed. J. Arditti. Ithaca: Cornell University Press. 349pp.
Stewart S. L., Zettler, L. W. 2002. Symbiotic germination of three semi-aquatic rein orchids (Habenaria repens, H. quinqueseta, H. macroceratitis) from Florida. Aquat. Bot.: 72: 25-35.
_______ ., Zettler, L.W., Minso, J., Brown, P.M. 2003. Symbiotic germination and reintroduction of Spiranthes brevilabris Lindley, an endangered orchid native to Florida. Selbyana: 24: 64-70.
_______ ., Kane, M. E. 2006. Symbiotic seed germination of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell Tissue Organ Cult. 86: 159-167.
Williams, N. H. 1975. Stomatal Development in Ludisia discolor (Orchidaceae): mesoperigenous subsidiary cells in the mocotyledons. Taxon 24: 291-288.
_______ . 1979. Subsidiary cells in the Orchidaceae: their general distribution with special reference to development in the Oncidieae. Botanical Journal of the Linnean Society 78: 41-66.
Zettler, L. W. 1997a. Orchid fungal symbiosis and its value in conservation. McIlvania. 13: 40-45.
_______ . 1997b. Terrestrial orchid conservation by symbiotic seed germination: techniques and perspectives. Selbyana 18: 188-194.