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The effects of light conditions from patchy natural canopies on the growth and morphology of white clover… Marcuvitz, Sheldon 1994

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THE EFFECTS OF LIGHT CONDITIONS FROM PATCHY NATURAL CANOPIESON THE GROWTH AND MORPHOLOGY OF WHITE CLOVER CLONESbySHELDON MARCUVITZB.A., Reed College, 1985A THESIS SUBMITTED IN PARTIAL FULFiLLMENT OF THE REQUIREMENTSFOR THE DEGREE OF DOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Botany)We accept this thesis as conforming to the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember 1994© Sheldon Marcuvitz, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree thatpermission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)____________________________Department of /?lzr(fThe University of British ColumbiaVancouver, CanadaDate ),1?’1’1DE.6 (2)88)11ABSTRACTIn a community such as a pasture, the success of an individual plant might beaffected by neighboring plants. One influence of neighbors on the environment of a plantis alteration of light conditions. Canopies in natural environments have only recently beenrecognized as heterogeneous in this regard, and responses of plants to alterations in lightconditions under heterogeneous canopies are poorly understood.In this investigation, heterogeneity in canopy conditions experienced by whiteclover (Trifolium repens L.) in a pasture was acknowledged. Three natural canopyconfigurations were used to examine whether light conditions established by neighborsalter the growth and morphology of white clover clones. Live grass neighbors were usedand at the same time, over one clover plant, patches of different light quality and/orquantity were provided.In the first set of experiments, light reflected from grass neighbors was providedsimultaneously with direct light. There were. no consistent effects on white clover growthand morphology, but there was evidence of phototropic movement of plant structures,which became located in positions that might minimize the effects of grasses on morepermanent features of the clones.In the second set of experiments, shade from three different species of grass waspresented to different clover clones for parts of each day, with full sun around noon. Thecanopies reduced overall growth and branching of clones, while increasing length of, andbiomass allocation to, petioles. Lolium perenne had different effects compared to Holcuslanatus or Dactylis glomerata, but between the latter two species, no differences weredetected.In the final set of experiments, two interconnected portions of white clover clones,the apical and basal regions of a primary stolon, were subjected to local canopies. Basalregion response was examined for independence from apical region conditions and vice111versa. Basal regions responded to apical conditions only when they were themselvesshaded, while apical regions responded to basal conditions regardless of their localillumination.The ways in which plants respond to neighboring vegetation are complex, anddifficulty exists in interpreting plant morphology in terms that are ecologically relevant.If we could identify advantages to particular strategies or responses, and then selectivelycontrol plant performance, we might be able to improve our use of plants through moreefficient production and management schemes.ivTABLE OF CONTENTSAbstract iiTable of Contents ivList of Tables viList of Figures viiList of Plates viiiAcknowledgement ixChapter One. GENERAL INTRODUCTION 1Chapter Two. GENERAL METHODS 282.1. Species used 292.2. Modular canopy design 372.3. Experimental chambers 442.4. Measurement of white clover 482.5. Data analysis 522.6. Microclimatic measurements 54Chapter Three. DOES LIGHT REFLECTED FROM NEIGHBORS (NORTHERNCANOPY) AFFECT THE GROWTH AND MORPHOLOGY OFWHITE CLOVER CLONES?3.1. Introduction 593.2. Methods 603.3. Results 653.4. Discussion 70VChapter Four. THE EFFECTS OF PARTIAL SHADE FROM THREE SPECIES OFGRASS NEIGHBORS ON THE GROWTH AND MORPHOLOGY OFWHITE CLOVER CLONES4.1. Introduction 904.2. Methods 924.3. Results 944.4. Discussion 103Chapter Five. IS THERE AN EFFECT OF REMOTE CANOPY CONDITIONS ONTHE GROWTH AND MORPHOLOGY OF LOCAL REGIONS OFWHITE CLOVER CLONES?5.1. Introduction 1285.2. Methods 1305.3. Results 1345.4. Microclimatic measurements 1375.5. Summary and discussion 139Chapter Six GENERAL DISCUSSION 155Literature Cited 164viLIST OF TABLESTable 3.1 773.2 813.3 813.4 823.5 823.6 83,843.7 864.1 1154.2 1154.3 1164.4 1164.5 1204.6 1214.7 1224.8 1244.9 1254.10 1264.11 1275.1 1465.2 1495.3 1505.4 1515.5 1525.6 154viiLIST OF FIGURESFigure 1.1 272.1 562.2 562.3 57,583.1 803.2 853.3 873.4 874.1 1114.2 112, 1134.3 1144.4 117, 1184.5 1194.6 1235.1 153LIST OF PLATESPlate 1.782 883 1094 1445 147viiiixACKNOWLEDGEMENTSI’d like to thank my supervisor, Roy Turkington, for the help on many aspects ofthis thesis. Thanks need to be given to the many friends who offered help at the criticalfinal harvests of the experiments. Pat Harrison deserves thanks, also, for the many callsto Plant Operations, trying to get help in repairing aspects of the greenhouse. Carolegreatly helped me wrap this up. I’d also like to thank all of the people who helped memaintain a perspective on this thesis, whether through agricultural and botanicaldiscussions, or through discussions over beer at the Grad Center.1Chapter OneGENERAL INTRODUCTION1.1. BACKGROUND ON Trifolium repens L.1.1.1. IMPORTANCE OF TifiS PASTURE LEGUMEPlants experience different problems capturing resources than do animals. Plantsare stationary and can not move around on the scale that animals do. However, plantshave remarkable abilities to modify their growth and morphology in order to captureresources. Plants with creeping growth habits, i.e., clonal plants with plagiotropic stemgrowth and rhizomatous and stoloniferous plants, have been called “foragers” as theywander through heterogeneous resource patches (Bell, 1984). One of the mostagronomically important of these foraging plants is white clover (Trifolium repens L.), apasture legume of mild temperate regions (Fig. 1.1). As a nitrogen fixing perennial,clover can continuously supply a grass sward with up to 60kg nitrogen/ha/yr and therebyindirectly provide protein grazing animals.White clover is primarily grown in mild temperate regions, where it canoverwinter with enough viable stolon material to continue growth in the next season(Harris et al., 1983). Even in these areas, its sensitivity to winter conditions makes itsperformance unpredictable from a farmer’s perspective, and its persistence requirespasture management in the form of controlled stocking or mowing (Frame andNewbould, 1986). With increasingly difficult economic times, unpredictability makes itsuse somewhat problematic, although at the same time the increased use of legumes iswarranted to reduce the use of artificial nitrogen fertilizer on farmland. It seems logical,then, that a better physiological understanding of the interactions between white cloverand other plants growing in a mixed sward should increase our ability to use white cloverefficiently in pastures. The biology, ecology and agronomy of the species have been2reviewed by Erith (1924); Ahlgren and Fuelleman (1950); Chestnutt and Lowe (1970);Burdon (1983); Turkington and Burdon (1983); and Frame and Newbould (1986).1.1.2. MANAGEMENT AND INTERACTIONS WITH NEIGHBORSIn most plant communities it is the proximity of neighboring plants that largelydetermine an individual’s success. Resources acquired by one plant are no longeravailable to others. Because different species have slightly different patterns of resourceuse, and because resources are patchily distributed, multispecies communities can persist(Grime, 1979; Tilman, 1988), and the composition of multispecies mixtures changesunder different environmental conditions (e.g. Pickett and Bazzaz, 1976). In pastures, themanagement regime often precisely determines the composition of species mixtures(Jones, 1933). For example, white clover persists because grazing or mowing maintainsthe sward at a height which prevents its elimination (Frame and Newbould, 1986). Intaller grass, white clover growth is greatly inhibited, and it contributes little to the totalyield of the pasture. The importance of white clover as a forage crop has led tomanagement regimes that strive to maintain a particular proportion of it in a pasture.As white clover grows through a sward, it encounters heterogeneity from twosources, and it responds phenotypically to this heterogeneity. First, it encounters variousspecies of plants, particularly grasses. The ability to respond in a specific manner todifferent species of grass (Chestnutt and Lowe, 1970) is a maj or factor contributing towhite clover persistence in pastures (Bulow-Olsen et al., 1984). Secondly, it encountersenvironmental variability caused by local soil conditions, dung and urine patches, molehills, and death of grass clumps (Parish, 1987). Turkington and Harper (1979), Burdon(1980), and Aarssen and Turkington (1985) have argued that the first component (theneighbors) has a much greater influence on success than the second component (abioticfactors). To continue growth and gathering of resources, the plant may need to modify itsphenotype as it continually encounters the patchiness in its environment.3White clover shows rather consistent patterns of growth when associated withparticular pasture conditions. When growing in a patch in the absence of neighbors,white clover will grow large, have more branches and have short petioles. These andvarious other characteristics of white clover such as the percentage of nodes withbranches, internode length, stolon growth rate, and leaf size show consistent patterns ofresponse when growing with different grass species (Chestnutt and Lowe, 1970;Turkington, 1979; Solangaarachchi, 1985). Turkington et al. (1991) compared twohalves (branches on either side of a primary stolon) of the same white clover plant whenthe two halves were growing with monocultures of different grass species. They showedthat white clover had many small modules when growing with Agrostis capillaris L.(bent grass) fewer, larger modules with longer internodes when growing with Holcuslanatus L. (velvet grass), and large modules on short internodes when growing withLolium perenne L. (ryegrass). In general, white clover is found to be more productivewhen growing in association with ryegrass than with any other companion grass(Turkington and Burdon, 1983). While the different responses of white clover to grasseswere quite marked, it is not clear what component of the physical environment thegrasses alter, although it has been variously attributed to physical prevention of rooting(Solangaarachchi, 1985), soil microbial populations (Turkington et al., 1988), or lightquantity and quality (Solangaarachchi, 1985; Solangaarachchi and Harper, 1987;Thompson and Harper, 1988; Thompson, 1993a).Besides differences induced by different species of grasses, different genotypes ofa single species, ryegrass, induced different morphologies in a particular white cloverclone (Hill, 1977; Aarssen and Turkington, 1985). Mixtures of white clover and ryegrasstaken from the same site of origin can result in higher yields than mixtures of plantsderived from different sites of origin (Turkington and Harper, 1979; Frame and4Newbould, 1986). This can result in widely different yields of white clover in apparentlysimilar white clover/ryegrass mixtures.Evans and Turkington (1988) showed that the morphological effects induced inwhite clover by different grasses can persist in a common garden, in the absence ofneighbors, for at least four months, but disappeared within two years. This carry-overeffect indicates that a particular morphology may be “programmed” into white cloverplants by their biotic environment, and internal controls might restrain phenoptypicchanges during short-term changes in conditions. It also demonstrates that the conditionsunder which a white clover plant previously grew are important in determining thatplant’s response to a newly encountered stimulus.1.1.3. CLONAL GROWTH1.1.3.1. PHENOTYPIC PLASTICITYAs a clonal plant, white clover has a modular design whereby each module, orramet, has the potential for independent existence and proliferation. This clonal growthhabit enables lateral spread, placing daughter ramets at a distance from the parent (Cook,1983). The ramets are constructed of resource-acquiring structures (leaves, roots) andspacing-associated structures (internodes). The selective placement of resource-acquiringstructures through differential elongation and branching of stolons in response toenvironmental heterogeneity is interpreted as “foraging behavior” in plants, and this termhas gained wide acceptance in recent years (e.g. Sutherland and Stillman, 1988;Silvertown and Gordon, 1989; Hutchings and Mogie, 1990; Grime, 1994; Hutchings andde Kroon, 1994).The dynamics of white clover in a sward are determined largely by vegetativegrowth, rather than recruitment from seeds (Turkington et al., 1979; Chapman, 1983).5The ability of white clover to persist in pastures shows that it is capable of gatheringsufficient resources even in these heterogeneous and competitive environments.Arguably, one of the main characteristics conferring this success is its phenotypicplasticity, the ability to make morphological and physiological adjustments. (Bradshaw,1965; Schlichting, 1986; Sultan, 1987; Grime, 1994; Hutchings and de Kroon, 1994).The “form” that a plant takes under particular conditions might allow for its most efficientuse of resources (Lovell and Lovell, 1985). Indeed, flexibility in morphology andphysiology is a character under genetic control, and thus is expected to be affected byselection and evolutionary change (Schlichting 1986; Thompson, 1991; Bell andLechowicz, 1994). In white clover, plasticity in response to phosphorus supply wasrecently observed to vary among genotypes, indicating that plasticity is a trait that can bebred (Caradus et al., 1993).White clover provides a good system in which to study the inheritance ofplasticity. In a well-studied permanent pasture in North Wales (around 100 years sinceestablishment), an individual white clover plant may co-exist with a single neighboringgrass species for several generations because grass patches may be up to lOOm2(Turkington and Harper, 1979; Thorhallsdotir, 1983; Turkington and Mehrhoff, 1986). Ina younger permanent pasture (50 years since establishment) in British Columbia, a morecomplex mosaic of neighboring grass species, with patches up to 1 m2, means that agrowing white clover may experience many different grass neighborhoods in a season(Aarssen and Turkington, 1985; Evans, 1986; Parish, 1987). It has been suggested thatthe genotypes of white clover persisting in the permanent pasture in N. Wales have beenscreened (presumably through initial mortality of genotypes arriving as seeds, or, sincethere is occasional seed-set, through generational-selection) for their ability to adopt aform allowing for continued existence with one particular species of grass (Turkingtonand Harper, 1979; Gliddon and Trathan, 1985). In the B.C. pasture with a more finegrained mosaic of neighborhoods, the same type of screening could be expected to occur,6except that it would be for the ability to be flexible in a heterogeneous environment,because particular morphologies are more productive with particular grass species(Turkington and Mehrhoff, 1990). PHYSIOLOGICAL INTEGRATIONThe extent to which connected ramets of a plant respond as an integrated unit, asopposed to independent units, has been debated for some time (reviewed by Pitelka andAshmun, 1985; Marshall, 1990; Hutchings and de Kroon, 1994). In some clonal species,the connections between ramets decay soon after establishment, while in others, theconnections may persist throughout a growing season or for many years, with the rametsremaining attached and perhaps more or less physiologically integrated, either as a wholeplant, or in groups of ramets defined as “integrated physiological units” (IPU’s sensuWatson, 1986). There seems to be variation in the extent of integration depending on thespecies examined, the architecture of the plant, and the conditions to which plants aresubject. It appears that physiological integration can occur through carbohydrate (Priceand Hutchings, 1992; Chapman et al., 1992a; Landa et al., 1992), water (Alpert, 1990;Evans and Whitney, 1992; Price et al., 1992) or mineral (Alpert, 1991; Marshall andAnderson-Taylor, 1992; Caradus et al., 1993) transfer, and possibly through signals,hormonal (Dong, 1993), or physiological (Hartnett and Bazzaz, 1983), transmittedbetween interconnected regions, although still no clear picture exists (Hutchings and deKroon, 1994). It seems that many plants have the ability to regulate their sharing ofresources between ramets, rather than simply sharing through diffusion from higher tolower concentrations (Caraco and Kelly, 1991), but the effects of physiologicalintegration on an ecological and evolutionary scale remain largely unknown.There have been many arguments put forth for the advantages of each plant’sparticular strategy (Pitelka and Ashmun, 1985; Caraco and Kelly, 1991; Hutchings and7Price, 1993). 1 Most of the attention in this area has focused on clonal, rhizomatous orstoloniferous plants and their ability to alleviate the effects of locally poor resourceconditions through physiological integration. In many. of the species tested, rametsgrowing in locally poor conditions were supported by interconnected ramets growing inmore favorable conditions (Hartnett and Bazzaz, 1983, in Solidago canadensis L.; Sladeand Hutchings, 1987a, in Glechoma hederacea L.; Lau and Young, 1988, in Lycopodiumflabelliforme (Fernald); Evans, 1991, in Hydrocotyle banariensis (Lam.); Alpert, 1991 inFragaria chiloensis (L.) Duchesne; Evans and Whitney, 1992 in Hydrocotylebanariensis). It appears that temporary growth in poor conditions can be sustainedthrough support from ramets growing in better conditions, and in some plants this may bea sound strategy, since by this time, an investment in stolon growth and ramet productionhas already been made by the plant. It would be advantageous to support the poorerramets for a while, either to allow their escape from the poor conditions or to allow bettermobilization of carbohydrate and nutrients back to the richer portion of the plant.Another possible effect of physiological integration between ramets is to intensify thelocal responses of ramets, i.e. ramets under poorer conditions would perhaps do worse,and ramets under richer conditions would do better than they would have done otherwisewithout this integration (Abrahamson et al., 1991; Hutchings and Price, 1993). Thiseffect of physiological integration was observed in Lolium perenne in response to locallysevere shading (Ong and Marshall, 1979), to some extent in nitrogen-limited ramets ofFragaria chiloensis to shared (translocated) nitrogen (Alpert, 1991), nutrient-starved1 In this context, ‘advantages’ of one growth strategy over another implies a comparison, and thecomparisons being made are not always clear. Many authors seem to be trying to synthesize a summaryabout the global strategies of plants regardless of the community structure. With respect to physiologicalintegration, there is often no distinction made between comparisons at this level and where individuals inone population vary in the degree to which they integrate or fail to integrate local conditions. Cautionneeds to be exercised, since comparisons made between different species (unless having very similargrowth form and found in very similar environments) have little relevance to their relative success orstrategies, even in the same community. Comparisons of this type should be restricted to differentindividuals in a population or to comparisons between ancestral and modem lineages where relative successcan be judged.8Solidago canadensis (Abrahamson et al., 1991), and in Carex bigelowii (Torr. ExSchweinitz) to chronically carbon-poor tillers (Jonsdottir and Callaghan, 1989).Physiological integration between ramets, however, is not always detected. There areexamples of a lack of integration in response to locally depleted light conditions, inLamiastrum galeobdolon (L.) Ehrend. and Polatschek. (Dong, 1993) and in response tolocal competition from grass in Glechoma hederacea (Hutchings and Price, 1993). Thismay depend largely on the characteristics measured, since in Kemball et al. (1992)physiological integration in white clover due to local shading was not detected inmorphological measurements, but radiolabelled 14C export from the shaded branch wasreduced.The designs of many of these experiments examining physiological integrationmake use of the concept that the phenotype (e.g., petiole and internode lengths, stolonbranching, physiological functioning) of localized portions of several clones growingunder identical conditions, would be indistinguishable if there were no dependence onconnected ramets living in remote conditions. Phenotypic variation detectable betweencorresponding portions growing under identical conditions is interpreted as evidence forthe existence of physiological integration. One requirement of this type of analysis, isthat the portions of the plant are otherwise independent, i.e. the conditions experienced byone portion do not directly affect the remote portion, and it is unclear whether in many ofthe previous experiments this requirement was met. Experiments involving localdifferences in light conditions may need to pay special attention to this (see below).In many of the experiments described above, the locally-poor conditions wereimposed artificially and abruptly by the researchers. Under natural conditions resourcesare likely to vary in a much more continuous way with different intensities of gradients indifferent places. So it might be expected that the response of the portion of a plantgrowing in rich conditions to connected ramets would differ depending on the degree to9which the locally-poor ramets are depleted. If this were so, the use of artificially depletedconditions might evoke responses quite different from those in the field. The use of morenaturally-depleted conditions would greatly contribute to our understanding of interramet integration in the field. In a field situation, also, locally-poor conditions are morelikely to develop gradually, with the poorer ramets remaining under nearly idealconditions, at least for a while. For example, as a clonal plant approaches lower resourcepatches, e.g. lower light patches, a gradient might become established within the plant,where the ramets nearer the patch of low resource are under slightly poorer conditionsthan the rest of the plant. Under these conditions it might be advantageous for the richerramets with a more rapid growth rate to limit their support of these poorer ramets with aslower growth rate. There has been little experimental work enabling an evaluation of theplausibility or generality of this scenario under natural conditions, although suggestionsby de Kroon and Schieving (1990), Abrahamson et al. (1991), and Hutchings and Price(1993) indicate that in these situations, a lack of integration could lead to greater foragingefficiency. In a heterogeneous environment, it would also be advantageous to avoid theselower resource patches in the first place, and it remains unknown how the ability tointegrate conditions and respond accordingly might be involved in an early avoidanceresponse which may prevent resources from actually being limited.One of the potential benefits of physiological integration in white clover is that itpermits new daughter ramets to be supported through their establishment phase byreceiving photoassimilate from the parent or from older established ramets (Harvey,1970; Ryle et al., 1981). In white clover, there are usually several branches growing asone physically interconnected unit (IPU). Characteristic sinks for carbohydrate producedin photosynthesis are stolon apices, developing buds, expanding leaves, root apices, rootnodules, and flowers (Harvey, 1970, 1979; Chapman et al., 1992a,b). On a single stolon,it was suggested that all leaves acted as sources for all of the sinks, indicating thatbidirectional transport was possible in these plants. As plants aged, however, there was10reduced transport into older portions from the newer growth (Harvey, 1970). Developingsecondary stolons continued to import carbohydrate even after producing several of theirown leaves, indicating a close connection between the main stolon and daughter rametsproduced on branches. The same pattern was observed by Kemball et al. (1992)regardless of the illumination status of the branches themselves. They did notice,however, a reduction in the export of assimilate from a labelled branch if it was shaded.Chapman et al. (1991a,b) showed that carbohydrate exported from active leaves wentprimarily to nearby sinks, but labelled 14C was found in distant sinks, in both basipetaland acropetal directions from the leaf. Their observations led them to conclude thatrelative sink strength as well as the distance to the sinks determine the patterns ofcarbohydrate movement in white clover. They also observed that reserves ofcarbohydrate could be mobilized in response to stress such as defoliation, whicheliminates much of the active carbohydrate production. Phosphorus translocation inwhite clover has been recently studied (Chapman and Hay, 1993), and these authorsconcluded that the level of phosphorus transported from sources varied depending on theoverall phosphorus supply. The strongest sink for phosphorus appeared to be the brancharising at the node of an actively fed root. Continued translocation from main stolonroots to branch ramets allows the branches to enhance their development of leaves whilerelying on the parent stolon for mineral nutrition before the ramets on the branch stolonwere able to initiate their own roots. Chapman and Hay (1993) concluded thatphosphorus translocation patterns were not restrained to acropetal movement, as waspreviously suggested, but rather, were determined by the structure of the plants and thelocation with respect to principal sinks.In an examination of intra-plant integration under slightly more naturalconditions, Turkington et al. (1991) grew clover plants with ramets arising on oppositesides of the primary stolon directed to grow in different neighborhoods, formed bydifferent species of grass. The morphology of individual ramets responded to their11immediately neighboring grass independently, while the rate of new ramet productionwas constant over the entire plant, even though this differed under different neighbor-pairs. This suggests that the responses of some phenotypic characters vary locally,whereas others are integrated within the whole plant. In this case, the selective plasticityof some characteristics enables white clover to explore preferentially, favoring resource-rich patches but continuing to produce new ramets, regardless of local conditions. It wasalso suggested that since the development of individual ramets (leaves, internodes)depended on the acquisitions of resources locally, and that the development of an apexrequires resources from several interconnected ramets (Harvey, 1979; Newton, 1986),their data agree with what is known about the nutrition of ramets and apices in whiteclover.In summary, patterns of translocation within the plant provide detailed descriptionof short-term transport in plants, but information on the long-term effects on the fitnessand survival of ramets is needed when speculating about the ecological importance ofphysiological integration. In addition, experimental conditions unlike those experiencedin the field might yield information on mechanisms involved in establishment andmaintenance of integration within a plant, but might do little to increase understanding ofits ecological importance.1.2. MECHANISMS INVOLVED IN PLANT/PLANT INTERACTIONS1.2.1. IRRADIATION AND PHOTOMORPHOGENESISThe extent of spatial heterogeneity in pastures, shown to exist for both biotic andabiotic factors, is reflected in the heterogeneous structure of the pasture canopy.Arguably, some of the most important changes associated with different canopies are inthe light conditions. A clonal plant’s ability to respond to changes in surroundings maybe essential to its growth and survival within a heterogeneous canopy. The effects of12canopies found in the field on light conditions are very complex, and until recentlyspeculation about the eco-physiological effects of light on plants was difficult, since themajority of studies were conducted under relatively uniform and artificial light conditions(e.g. using cheese-cloth, colored filters, or brief pulses of monochromatic light).Following is (i) a description of what is known about the effects of light beneathrelatively uniform canopies on the growth of plants, and (ii) the consequences ofheterogeneity, or patchiness, in a canopy such as a pasture, on the light conditions at (a)one location on a plant, (b) over interconnected portions of a clonal plant, and (c)throughout the life of a plant. HOMOGENEOUS CANOPIESAbove the canopy surface in pastures and other plant communities, irradiancecomes from the sun, either directly in the sun’s beam, or indirectly from the clear sky.Diffuse radiation from the sky has a much lower photon flux density (PFD) than directradiation from the sun, and it has a higher proportion of blue wavelengths (Smith, 1982).The spectral energy distribution (SED), or quality, of the light from above a plant in anopen situation depends on the proportion of diffuse to direct light. This changes withsolar angle and aspect as well as proximity to opaque objects such as rocks or tree trunks(Stoutjesdijk, 1974; Smith, 1982). Under cloudy conditions, diffuse light from cloudsmay be homogeneous or mixed with direct light from the sun’s beam and diffuse lightfrom clear sky.Below the cover of a plant canopy, much larger changes in the SED occur thanabove a canopy, even under varying cloud conditions (Holmes, 1981; Smith, 1982).Extrapolation from absorption characteristics of chlorophyll largely describe the spectralchanges that occur beneath canopies of plants. There is great attenuation of blue and redwavelengths, as well as most of the energy in other visible wavelengths, but there is far13less attenuation of wavelengths longer than 700 nm (Holmes and Smith, 1977a;MacClellan and Frankland, 1985).Light quality in natural systems has most often been characterized by the ratio ofphotons in the red (R: 655-665 nm) region (greatly attenuated by vegetation) to the far-red (FR: 725-735 nm) region (attenuated little by vegetation). Phytochrome, aphotoreversible pigment having absorption maxima at 660 nm and at 730 nm, is thoughtto be the pigment responsible for plant response to variations in light quality formed bynatural canopies (Smith, 1982; Kasperbauer, 1987; Casal and Smith, 1989a; Smith andWhitelam, 1990; Ballare, 1994). For reviews of the photochemical properties ofphytochrome, see Kendrick and Kronenberg (1986), Furuya (1987), and Attridge (1990).Above a canopy the R:FR is usually between 1.1 and 1.3, depending on solar elevation,cloud conditions, and water vapor content (Smith, 1982; MacLellan and Frankland, 1985;Lee and Downum, 1991), and this is beyond the range of values that establish differencesin the phytochrome photoequilibrium (Smith, 1982). Measurement of light qualitybeneath natural plant canopies often shows the R:FR ranging from 0.1 to 0.5, and this iswithin the range where slight differences will cause large changes in the photoequilibriumbetween the two forms of phytochrome, and hence, alter its activity (Smith and Holmes,1977). Short-term monitoring of the R:FR beneath a Corylus avellana (hazelnut)canopy, however, showed that the R:FR fluctuated between 0.5 and 1.1 many timesduring a 20-second period, though this was bimodal and most often close to 0.5(Woodward, 1983). The bimodal distribution suggests that sunlight penetrates thecanopy intermittently as sunflecks, and brief periods of direct illumination can haveprofound effects on growth (Morgan and Smith, 1978; Baldocchi and Collineau, 1994;Pearcy et al., 1994).The patterns of growth observed under low R:FR conditions, such as beneath arelatively uniform closed canopy (e.g., beneath a forest canopy or beneath stands of many14cultivated crops) are consistent with postulated shade-avoidance responses (Grime,1981). Etiolation of seedlings emerging from the soil is accentuated by a low R:FR. Thispromotes elongation towards the surface of the canopy in plants with orthotropic shootorientation. Internode and petiole extension are also promoted by low R:FR light,branching is inhibited, and biomass partitioning to leaves, stems and roots is altered(Kasperbauer, 1971, 1987; Tucker and Mansfield, 1972; Field and Jackson, 1975;Holmes and Smith, 1977; Morgan, 1981; Casal and Kendrick, 1993). These responsesare generally weaker in plants from closed-canopy habitats than in plants from more openhabitats (Morgan and Smith, 1979). In plants with plagiotropic shoot orientation,responses to uniform low R:FR conditions are similar and include increased petioleelongation and decreased branching in white clover (Solangaarachchi, 1987; Thompsonand Harper, 1988; Thompson, 1 993a), increased internode and petiole length, increasedleaf area and decreased branching in Lamiastrum galeobdolon (Dong, 1993), and reducedleaf weight ratio (leaf weight to overall plant weight) and increased stem and petioleweight ratios in Veronica sp. (Dale and Causton, 1992). In white clover, conflictingresults have been obtained on the effects of low R:FR on internode length, and this isapparently due to the differences in PFD from 400-700 nm (photosynthetic photon fluxdensity, PPFD) in these experiments (Thompson, 1993a). All of the above responsespresumably serve to increase the chances that the plant will continue to intercept lightefficiently (Ballare, 1994). In several of the more recent experiments, fresh or livingplant material was used to filter the light and create low R:FR conditions. Use of thesenatural filters precludes the need for assumptions about responsible wavelengths. Otherwavelengths have been shown to elicit similar responses in plants. There have beensuggestions that decreases in UV-B can lead to increased internode elongation (Barnes etal., 1990; Ballare et al., 1991). There is also a specific bluefUV-A photoreceptor whichcan detect decreases in these wavelengths and promote internode elongation in seedlings(Gaba and Black, 1979) and in fully de-etiolated soybean plants (Britz, 1990). Distinct15groups of wavelengths may interact, e.g., blue and FR in photomorphogenic andphototropic responses (Drumm-Herrel and Mohr, 1984; Ballare et al., 1992; Janoudi andPoff, 1992) and green and FR on night-closure of Albizzia julibrissin leaflets (Tanada,1982). Work on this topic is scarce, so it remains largely unknown how groups ofdifferent wavelengths might interact in promoting plant responses to vegetation shade.However, correlations between growth responses under natural closed canopies and thoseunder controlled conditions known to elicit phytochrome responses, such as a brief pulseof red followed by far-red, have led to reasonable confidence implicating phytochrome inshade-avoidance responses under uniform canopy situations. HETEROGENEOUS CANOPIESMost plant communities present a very complex canopy structure that is far fromuniform. Besides intermittent penetration of sunlight through small spaces betweenleaves, as detected by Woodward (1983) beneath a complete canopy, there may be largergaps that allow penetration of sunlight for longer durations. These larger sunflecks cansupply a substantial portion of the energy for a planCs photosynthesis (Morgan and Smith,1978; Baldocchi and Collineau, 1994; Pearcy et al., 1994). A plant within a sunfleck willalso receive light from the clear sky that has been transmitted through leaves and in manycases, light that has been reflected off plants forming the boundary of the sunfleck(Anderson and Miller, 1974; Bazzaz and Wayne, 1994). In general, plants beneath acanopy that is not an entirely uniform surface of leaves will receive direct light from thesky or the sun and light that has been transmitted through and reflected off leaves.In recent years several studies (e.g. Casal and Smith, 1988; Woitzik and Mohr,1988; Rice and Bazzaz, 1989; Ballare et al., 1989, 1990, 1992a; Novoplansky et al.,1990; Casal and Kendrick, 1993; Dong, 1993) have reported progress toward unravelingmore complicated questions about how light affects plants growing in the field. Thesereports indicate how recent approaches have taken a more sophisticated and more16accurate view of a plant’s light environment, acknowledging, for example, the existenceof heterogeneity, which has several consequences for the way a plant responds to itscanopy (Ballare, 1994). In particular, this type of heterogeneity causes different inputsforming the global light environment to be present at one location on a plant, and causesconditions at a given time to be different at two interconnected portions of the plant.Each of these will change throughout a day and can also change in a predictable way overtime through the length of a growing season. This provides some theoretical backgroundfor hypothesizing how the particular light conditions created by grass neighbors mightinfluence the morphology of white clover.Consequences at one location within the plantIn a community such as a pasture, light received at one location will contain avariable proportion of transmitted, reflected and unfiltered light over time due to biotic(location, identity, and conditions of neighboring vegetation) and abiotic (diurnal andseasonal solar movement, cloud cover) factors. In addition, points in close proximity canhave widely different proportions of light from each source at any given time. Bothspatial and temporal heterogeneity mean that even though plants might receive light thathas a low R:FR, they are likely to receive some unfiltered light as well. Within a gap, aplant may experience these conditions even in direct sunlight. In this case, the source oflow R:FR light would be from reflection off upright leaves of neighboring plants (Sternand Donald, 1962; Wooley, 1971; Anderson and Miller, 1974; Bazzaz and Wayne, 1994).Reflection can create strong additions of FR wavelengths to a plant’s overall light regimeif the sun’s direct beam is the source of the reflected light (Ballare et al., 1987;Kasperbauer, 1987; Ballare et al., 1989; 1992a; Smith et al., 1990; Casal and Kendrick,1993). Kasperbauer (1987) suggests that “... light reflected from the leaves may be evenmore important [than light transmitted through leaves] in adaptation of a plant tocompetition from other plants.” The increase in FR that is associated with reflection, it is17now argued widely, provides an indication of the proximity to neighbors and, hence, anindication of the potential for competition, before competition for the light resource isexperienced. The responses to this increase in FR are consistent with shade-avoidancestrategies in orthotropic plants, i.e. increased internode elongation and decreasedbranching. The designs of many recent experiments have achieved a goal stated by Smith(1982) as necessary to further our Understanding of the function of phytochrome in thefield. Namely, they have provided differences in the R:FR at irradiation levels that arecommonly experienced by many plants growing in the field. There have as yet been fewstudies satisfying this criterion in plagiotropic plants, and therefore, the effects ofreflection from neighbors on plagiotropic plants remain largely unknown. An exceptionto this is the work by Novoplansky et al. (1990). They found that seedlings of Portulacaoleracea L. preferentially developed branches away from other seedlings, even thoughthey were widely enough spaced to receive nearly full illumination. Plants alsoresponded similarly to reflecting barriers that simulated neighbors. These results suggestthat plagiotropic plants can respond to increased FR in the proximity of neighboringplants and can selectively control placement of branches to avoid growing towardsneighbors.Consequences at more than one location within the plantThe horizontal growth form of white clover presents a situation whereheterogeneity in the light environment may occur throughout interconnected parts of asingle plant. The extent to which ramets of clonal plants respond independently to locallight conditions, as opposed to coordinating their response to whole-plant conditions, hasonly recently begun to be studied and is considered here (see also above section of the following studies is based on the idea that corresponding parts of separateclones can be compared when the interconnected portions of each clone are experiencingdifferent environments. Hartnett and Bazzaz (1983) examined the rates of18photosynthesis, growth and survival of ramets of Solidago canadensis connected througha common parental node to sibling ramets experiencing different conditions. If connectedto a ramet that received shade, the rate of photosynthesis in the target ramet was higherthan if it was connected to another fully-illuminated ramet. They suggested that a greaterassimilate demand on the fully-illuminated ramet was incurred by the shaded ramet, andthese ramets responded with a greater maximum rate of photosynthesis.2Growth andsurvival measurements also indicated that shaded ramets were supported with assimilatesfrom the other, fully-illuminated, ramet. These results suggest that translocation couldoccur in both basipetal and acropetal directions between ramets sharing a commonparental node. They hypothesized that in Solidago canadensis physiological integrationcould alleviate the effects of patch-specific limitations of light, and therefore increase thegenet’s survival in a heterogeneous environment. Slade and Hutchings (1987b) showedthat unshaded ramets of Glechoma hederacea formed internodes the length of which werenot dependent on the light conditions experienced by other parts of the stolon. However,shaded ramets formed shorter internodes if they were connected to basal ramets that wereunshaded. This only occurred if the unshaded basal ramets were within two or threenodes of the shaded ramets in question. This suggests that there is some translocation ofthe effect of unshading in the acropetal direction for at least two nodes in this species.This is consistent with the patterns of labelled assimilate transport observed in thisspecies (Price et aL, 1992). Dong (1993) did not observe the same effect in Lamiastrumgaleobdolon, where shaded ramets apical of unshaded basal ramets showed nodependence on the conditions experienced by basal ramets. Instead, basal rametsproduced shorter internodes if the apical region was under high light conditions. No2However, in this experiment, the conditions used to impose shade on one sibling ramet (a shade tent)could have directly interfered with the light environment under which the other sibling ramet was grown,even though it did not receive its own shade ‘tent’. The details of the experiment are not clear, but it waslikely that these “fully-illuminated” ramets (the ones which were connected to shaded sibling raniets) grewunder lower illumination than the controls, which were connected to sibling ramets which received noshade “tent”, and this could have altered the way these ramets responded to the photosynthesismeasurements.19basipetally-transported effect on internode length was seen if the basal ramets werehighly illuminated and the apex was shaded. Dong (1993) suggests that this was an effectof hormones, rather than an effect of assimilate transport, because the apex is traditionallyconsidered to be a strong carbohydrate sink (Pitelka and Ashmun, 1985; Marshall, 1990).In white clover, Harvey (1970) found that localized shading of mature leaves reduced tonil the small amount of imported assimilate they would otherwise have received. Young,expanding leaves also imported less when locally shaded, but shading of stolon apiceshad no effect on their import of assimilate. Solangaarachchi (1985) grew apices of whiteclover into different neighborhoods of grasses while basal portions remained in the firstneighborhood. She showed that growth in the basal portion was inhibited by aneighborhood of grass around the stolon apex. She suggested that light conditionsexperienced by the apical region were responsible for the response of basal branches.Using the same technique on a smaller scale (with three connected ramets) Newton(1986) observed and concluded the same, suggesting that there may be basipetaltranslocation of the effects of shading which occurs only around the apical portion of astolon. Thompson (1993b) observed that supplemental Red light (without increasingPPFD) on one node of white clover plants, using light emitting diodes (LED), affectedthe branching and petiole length of ramets produced acropetally, and this effect wasreduced as the distance from the treated node increased. This suggests that the responseto R:FR can be communicated acropetally along a stolon in white clover. However, thediodes were illuminated 24 hours per day, and the effect of nighttime supplementation ofPPFD could not be determined, nor could the direct effects of scattering of the Red lightwithin and around the plants, especially at night. Kemball et al. (1992) shaded only onebasal branch on white clover clones. They found that there was no detectable growthresponse in other portions of the plant. Shading of the basal branch also did not changeits import of 14C labelled assimilate from the plant’s main stolon. However, labelling ofa basal leaf on shaded basal branches indicated an increase in its acropetal transport of20assimilate towards its stolon apex and a decrease of assimilate in basal portions of thatstolon and in the main stolon of the plant. This indibates that under unshaded conditions,there was both acropetal and basipetal transport of assimilate throughout the plant, andthat export of assimilate from a single branch was reduced by localized shading. Insummary, patterns of photoassimilate movement within several clonal plants have beenobserved to change due to local light conditions. However, the directions in whichfactors allowing for physiological integration (e.g. photoassimilate, hormones, etc.) movedo not seem to be consistent, depending on the particular plant (perhaps due to structurallimitations (Price et al., 1992)) and on the particular localized conditions. In severalcases, ramets under poorer light conditions received support from better-illuminatedramets. In these experiments low fluence rates were often established with artificialfilters, so these were examinations of the effects of locally low fluence rates. The effectsof locally altered R:FR conditions remains to be examined. The local differencesassociated with neighboring plants may involve changes to R:FR with little change toPPFD. It could be beneficial to the plant to increase growth in the region with a highR:FR and limit growth in the low R:FR region, tperhaps hrough carbohydrate supportfrom the low R:FR region.A requirement in these types of studies is that the separate environments createdover two (or more) portions of one plant need to be completely isolated to enable theconclusion that intra-plant integration had an effect on the growth of ramets. In none ofthe published accounts of the experiments described above is the reader assured that theconditions imposed around one portion of the clonal plant in question do not interferedirectly with the conditions experienced by the interconnected portion. The physicalarrangement of “shade tents”, and the necessary proximity to the interconnected ramets,makes this requirement logistically difficult to satisfy, yet it is necessary for inferring theexistence of intra-plant integration. Without this it remains uncertain how much of theresponse in proximal portions was due to conditions around the apex, and how much was21due to each ramet’s own local environment, which was itself altered by treatments onstolon apices. This requirement might be particularly stringent in experiments involvingmanipulations of light conditions, however, it can not be ignored in any experimentsrequiring independent manipulations of two portions of one plant.Temporal variation in light conditionsGreat complexity is added to understanding photomorphogenesis in the fieldwhen considering the temporal variation in the light environment. Plant response in thefield may be closely linked to changes in the light conditions occurring over manydifferent time scales from seconds to the life of the plant, a topic recently reviewed byBaldocchi and Collineau (1994), Pearcy et al. (1994), and Pearcy and Sims (1994).McLaren and Smith (1978) showed that petioles of Rumex obtusifolius (L.) elongatedmore if the R:FR decreased over time than if the R:FR remained unchanged at the lowestvalue throughout. A decrease in R:FR (without much decrease in PPFD) under naturalconditions would describe a canopy which was once open, but is now beginning to close,and elongation of partially-shaded plants could result in them overtopping the canopy.On the other hand, in a canopy that already presents a low R:FR from early in growth,there may be little chance for a plant beneath it to overtop the canopy. This suggests thatan increase in reflection off vegetation over time could be involved in white clover shadeavoidance, and there might be an ecological advantage, since it would indicate decreasingdistance from neighbors or increasing height of neighbors, potential sources of PPFDlimitation. Pearcy and Sims (1994) provide several examples of plant responses tochanges in PPFD over time, changes largely between quite low levels (as in shade) andhigh levels (as in the open). There are consistently observed effects on leafcharacteristics, and they suggest that many of these changes might be associated withacclimation to other environmental stresses that are concomitant with the newenvironment.22In summary, the photomorphogenic responses of plants in the field are onlyrelatively well studied with respect to complete shading by other plants, a condition thatmight not be very common, especially in communities with heterogeneous canopystructures such as pastures. In clonal plants under highly variable canopy conditions, onecould expect photomorphogenic responses to be expressed as horizontal movement ofstolons through a sward or as lengthening of petioles to ensure placement of leaves inhigh light positions (Boiler and Nosberger, 1985). Recent recognition of theheterogeneity in many communities has led to several investigations of shade-avoidanceresponses in many orthotropic plants with, for example, investigations of the role ofreflection off neighboring plants, although this has not been attempted on plagiotropicplants such as white clover. There have also been many recent investigations of theeffects of gradients in light conditions over interconnected portions of one plant, mainlyin cional, plagiotropic plants. There seems to be consistent evidence that there is sometype of intra-plant integration of the response to spatial heterogeneity, although theresponses are widely varied. Many of the experiments fail to meet the requirement ofeffectively isolating the plant neighborhoods. In addition, many of the conditions used toinvestigate photomorphogenesis in plants have been quite artificial, and extrapolation toplant responses in the field remains difficult. The response of plants to temporalheterogeneity is even less well understood.1.2.2. OTHER MECHANISMS INVOLVED IN PLANT/PLANTINTERACTIONSIn a plant community such as a pasture, neighbors have other less obviousinfluences on the nature of a white clover plant’s environment. Since the objective of thisthesis is to examine the influences of natural light conditions on the growth and form ofwhite clover, these are only briefly discussed here and reference is made to recentreviews addressing this topic. The various conditions associated with gaps of differentsize in vegetation and at different locations within a gap are reviewed by Bazzaz and23Wayne (1994). Above ground, air circulation patterns change with the proximity ofneighbors, perhaps increasing 02, water vapor, and ethylene, and depleting CO2. Theeffects of many of these influences on plant growth have been reviewed recently in aspecial issue of Plant, Cell, and Environment (13:7, 1990) entitled “Sensing theEnvironment”. Below ground, many precise microenvironmental conditions may beassociated with particular grass neighbors including depth of roots, affinity of roots forwater and nutrients, and soil microbe populations, all of which may influence the growthof associated white clover plants (Turkington et al., 1988; Stark, 1994; Caldwell, 1994).Accumulation of dead material within a Holcus lanatus or Agrostis capillaris sward mayactually prevent white clover stolon contact with the soil, and this is likely to reducerooting and branching from nodes (Chapman, 1983). These factors surely interact witheach other and with light conditions in the field, and the responses to neighbors in thefield are undoubtedly complex.1.3. OBJECTIVESWhen considering through what mechanisms neighbors affect white clovermorphology, it may at first be beneficial to simplify the environment and only allowpossible interactions through alterations in the light conditions. This study represents adeparture from traditional studies of the effects of light conditions on plants, whichgenerally have used artificial conditions. Providing a SED that is altered by livevegetation more accurately mimics light conditions in the field, and assumptions aboutresponsible wavelengths need not be made. This study investigates the importance ofalterations in the light conditions as a possible mechanism responsible for neighbordetection, rather than being a study of the mechanisms involved in detecting lightchanges (e.g., phytochrome or other receptors, signal transduction, molecular basis forresponse, etc.). Three different canopy arrangements are used, all with separation belowground, which recognize the existence of patchiness in the above-ground environment.Each of these puts target white clover clones in a different arrangement with respect to24the canopy of grass neighbors, mimicking a field situation where there is a gap in which awhite clover plant resides, either wholly or partially. These arrangements createcontrasting light conditions dictated by the arrangement of the canopy and the position ofthe white clover clones with respect to the canopy and the sun.1.3.1. NORTHERN CANOPY: REFLECTIONIt has been shown in several orthotropic plants that neighbors can be detectedthrough changes in the R:FR of light impinging in the horizontal direction, and thisallows them to detect a signal of impending competition before there is photosyntheticlimitation (Ballare et al., 1987, 1988, 1989, 1990; Smith et al., 1990). The responses ofplants in these experiments were similar to the responses under complete vegetationshading, e.g. longer internodes and reduced branching. In white clover, the responses toreflection are also expected to be qualitatively similar to responses under complete shade,e.g. increased petiole length and reduced branching (Solangaarachchi and Harper, 1987;Thompson and Harper, 1988; Thompson, 1993a). In plagiotropic plants, the possibilitythat the placement of ramets horizontally, in the plane of the growing surface, is alteredthrough reflection from neighbors, has been suggested for Portulaca oleracea seedlings(Novoplansky et al. 1990; Novoplansky, 1991), however the effects of reflection have notbeen examined in white clover. In five very similar experiments, the effects of lightreflected from grass neighbors on the growth and morphology of white clover clones wasinvestigated (Chapter 3). In each of these experiments, a canopy of grass, Dactylisglomerata (orchard grass), was placed to the north of target white clover clones. In thisarrangement, the target plant received direct light from the sun and reflected light fromthe grass barriers. In the first four experiments, control clones grew in front of a control“canopy” of pots with potting-medium only, and in the fifth experiment, control clonesgrew in front of a barrier of bleached grass. By describing clonal growth in response toreflected light, these experiments are intended to establish the sensitivity of white cloverto potential signals of impending competition from neighboring grasses.251.3.2. EASTERN AND WESTERN CANOPY: PARTIAL SHADEIn one experiment the morphologies produced by a single clover clone in responseto different species of grass forming the borders of a standard gap are described (Chapter4). In this experiment, target white clover clones grew inside a corridor bordered to theeast and west by Dactylis glomerata, Lolium perenne, Holcus lanatus, or control (empty)pots. In these arrangements, light was transmitted through and reflected off the grassescasting shade in a natural pattern. In the middle of the day, direct light was also receivedfrom the sun by the clover clones. This experiment provides a description ofphytochrome-mediated responses to light altered by leaf-filters, rather than cellophanefilters or monochromatic light, under conditions where plants also receive some directunfiltered light (unlike the homogeneous leaf filters used in Solangaarachchi and Harper,1987; Thompson and Harper, 1988; Thompson, 1993a). It is possible that thesephotomorphogenic responses are quite sensitive to natural changes in light brought aboutby different species of neighbor, and that gaps within different species of grass arerecognized as different from white clover’s perspective.1.3.3. SOUTHERN CANOPY OVER PART OF THE PLANT: LOCALIZEDSHADEA series of experiments was designed to investigate the extent of integrationbetween portions of a clover clone when subject to a natural canopy placed to the south oflocalized portions of target white clover clones (Chapter 5). The extent and direction ofintegration was determined by examining the effects of a localized apical or basal-regiongrass canopy on the growth and morphology of the remainder of the plant. Theseallowed the following questions to be investigated: Do the conditions experienced by theapical region affect the response of the basal region in these clones? Do the conditionsexperienced by the basal region influence the response of the apical region? Thesequestions were both investigated when the region under examination (not receiving the26variable local canopy) was behind both open and grass canopies. These experimentsallowed an assessment of the importance of physiological integration in determining thegrowth and morphology of white clover clones growing under different localized natural-canopy conditions.1.3.4. SUMMARY OF OBJECTIVES1. Northern canopy, reflected light: to determine if light reflected fromneighboring grasses influences the growth and morphology of clover clones otherwisegrowing under open conditions.2. Eastern and western canopy, partial shade: to determine if shade cast fromneighboring grasses influences clover growth and morphology when direct light is alsoreceived for part of the day, and to determine if the effects of shade cast in this fashionfrom different species of grass differ.3. Southern canopy over parts of a single plant, localized shade: todetermine if and under what canopy conditions a localized region of a clover cloneresponds to shade experienced by a remote region of the clone.Figure 1.1. Mature stolon of white clover (Trifolium repens L.). This stolon containsseveral ramets, each consisting of a leaf, the axil containing either a branch, flower, orbud, and the root.10 CM28Chapter TwoGENERAL METHODSThree sets of experiments were designed to assess the effects of reflected, partialshade, and localized shade conditions on the growth and form of white clover. Theseexperiments have many aspects in common, e.g., the species used, the modular canopy ofgrasses which formed the treatments, the experimental chambers, and the techniques formeasuring and evaluating the temperature, the spectral energy distribution (SED), and theclover clones. To avoid excessive overlap in Methods, the characteristics in common willbe described in this chapter. The particular characteristics which distinguish each set ofexperiments, e.g., the arrangement of the canopy, the details of the growth andmeasurement of clover, and the particular data analyses, are also described in this chapter,but specific methods used for the individual experiments within each set are described intheir respective chapters.The designs of the canopies used in these experiments allowed clover clones toreceive light conditions that mimicked different arrangements of neighbors growing infield while preventing below-ground interaction. Each of these canopy arrangementsemphasized a different influence of the canopy on light conditions. In the firstexperiment, canopy modules were arranged to the north of target clover clones (Fig. 2.1)to emphasize reflected light. In the second experiment canopy modules were arranged tothe east and west of clover clones (Fig. 2.2) creating a standard gap and allowing shadefrom different species to be received along with some direct light. In the third set ofexperiments, two portions of clover clones (apical and basal along a primary stolon) wereisolated but remained connected, and each received a canopy treatment independent ofthe interconnected portion (Fig. 2.3a,b,c,d). The canopy modules were arranged to thesouth and east (over apical regions) or south and west (over basal regions) of clover29clones creating local shade conditions similar to a uniform sward, with little or no directlight.2.1. SPECIES USED2.1.1. WHITE CLOVER2.1.1.1. COLLECTION AND PROPAGATION OF MATERIALAll of the white clover material used in these experiments was obtained from asingle clone collected approximately six months earlier from an old permanent pasture(sown in 1939) in Aldergrove, B.C. described in Aarssen and Turkington (1985). Thispasture is considerably well studied and the clover persists largely through vegetativepropagation (Parish, 1987; Evans and Turkington, 1988). Clover clones from this pasturemight possess a high degree of phenotypic plasticity because of their ability to persist inthe long term among a complex mosaic of grass neighbors, an argument detailed byTurkington and Mehrhoff (1990). These clones would therefore make good test subjectsfor examining responses to varying above-ground canopy conditions. The clone whichwas selected possessed three qualities that made it desirable over several other clonesderived from this pasture; i) it was vigorous and healthy growing in pots in thegreenhouse, ii) its phenotype was observed to vary quite markedly with illuminationconditions in the greenhouse, and iii) it was a relatively large-leaved clone, whosemeasurable morphological characters can vary to a greater degree than smaller-leavedclones (Caradus and Chapman, 1991). The use of a single clone makes these experimentsexamples of the use of a phytometer to measure the environment by mimicking theresponse of a plant growing within it. This type of experiment can then be conductedusing several genetically distinct clover plants to establish the level of variation inplasticity that is genetically-based (Bell and Lechowicz, 1994).30In September 1988 several cuttings of white clover from this stock material wererooted and transplanted into 25x50x8 cm flats containing Fison’s Sunshine Mix #1, anutrient-rich soilless potting mix. The flats of clover were irrigated as needed on aconstant liquid-feed program (i.e. fertilized during at least 3 out of every 4 irrigations) atlg/l of 20-20-20, N-P-K to at least 10% beyond their point of saturation. These weregrown in a 18/18°C day/night greenhouse at the University of British Columbia south-campus field station. The clover was kept free from pests with regular use of airborneinsecticides (Malathion, Ambush, and Sevin), an airborne fungicide (chlorothalonil), anda soil insecticide (Diazanon granular) at recommended rates.When the flats had a dense population of clover, cuttings were taken from theends of the longest stolons. Taking cuttings from the ends of stolons eliminated potentialage-dependent responses by the clover to the imposed treatments. Six of the cuttingswere transplanted into each of eight new flats containing fresh potting medium. Thus, thematerial was sub-cloned periodically, and when grown in this fashion, the morphology ofthis stock material, e.g., leaf size, internode length, and stolon diameter, remainedrelatively consistent. This method of subdividing and planting in fresh media ensured aconsistent supply of healthy, uniform stock material from which cuttings could be takenfor use in the experiments. PREPARATION FOR EXPERIMENTSIt was important to start the experiments with uniform cuttings, because earlyvariation in size could lead to large differences later, perhaps altering the effects of thegrass. There was always a phase after the stock material was subdivided and transplantedinto new containers, when each flat of clover had several rapidly-expanding stolonsadvancing along the potting-medium surface. This was the stage at which cuttings weretaken for use in the experiments; before the flats became overly crowded, and whenstolon apices from the same order of branching could be selected. At this stage there31were nearly always advancing apices from established stolons containing severalintemodes which made ideal material for propagating and for growing in the experiments.Stolon tips from the stock material were selected for uniformity and excisedbefore the newest node had rooted in the potting medium. These cuttings consisted of thenewest expanded leaf and the region apical to it, and a small root initial at the node. Thecuttings were placed horizontally on 3.0 cm x 3.0 cm x 1.5 cm rock-wool cubes, withplastic-coated wire hooks placed over the stolon at the point where the expanded leaforiginated. These were placed under shade on a heated mist-propagation bench for 5-7days and then transferred to the greenhouse where they were allowed to grow into therock-wool cubes. The rooted clones were placed in a flat with drainage holes inside a flatwithout holes, so that they could be bathed in a nutrient solution (20-20-20; NPK lgIl)which conveniently could be changed every second day. The cuttings were allowed toacclimate thus for 7 more days.An excess of visually uniform cuttings were then selected and transplanted intothe appropriate containers to be used for each experiment, either 30 x 15 x 8 cm deep, forthe partial shade experiment, or 50 cm x 25 cm x 8 for all others. One cutting wasplaced near the end of each flat, with the stolon pointing straight down the middle of theflat. All flats were filled with the same potting mix, Fison’s Sunshine Mix #1. Thetransplanted clones were allowed to root-in in a common area of the greenhouse (for 4-8days) with their position rerandomized every second day. The plants were continued onthe same constant liquid feed program described above for clover stock material, andwere always being irrigated whenever the driest flat required. This allowed plants to begrown more uniformly and reproducibly. The clones were also treated with pesticides(airborne insecticides as above, Diazanon granular watered-in to kill fungus-gnat larvae,and Captan fungicide sprayed on the potting-medium surface to prevent algal growth),which began the bi-weekly program which was to continue throughout the experiment.32After 4-8 days, the length of the stolon, the number of ramets, and the length of thenewest petiole were measured on each clone. These clones were then selected foruniformity, randomly assigned a treatment, and then placed into the experimentalcompartments. The clones were allowed to acclimate in the experimental compartmentsfor 2-4 days (reflection and partial shade experiments) or 18-40 days (localized shadeexperiments) before the treatments were imposed. TREATMENT DURING THE EXPERIMENTSDuring all experiments clover clones continued on the constant liquid feed and biweekly pesticide programs described above. During the reflection and partial shadeexperiments, the position of clover clones was rerandomized every four days. In thereflection experiments, to accomplish the rerandomization without compromising theexperimental conditions, clover clones were moved only after all of the canopy moduleshad been removed from the compartments. The clones were then moved to theappropriate new compartments, and the canopy modules were replaced. This way,control clones were prevented from being close to neighboring grasses while beingrerandomized, and therefore, they never received pulses of FR which could have had aneffect on their morphology. During the partial shade experiment, a canopy of each typewas prepared but remained vacant (without clover clones). After each rerandomizatioñ adifferent canopy became the spare. This system was used so that the grasses could beclipped (see below) when the clover was removed during rerandomization. First, eachspare aisle was clipped, after which clover clones from the same treatment in a differentblock were moved into this aisle, vacating the next aisle to be clipped. This procedurewas followed until the final aisle of each species was vacated and the grasses clipped.During the localized shade experiments, the clover clones were fixed into position andwere not rerandomized. In these experiments, the two regions of clover clones alwaysreceived irrigation and pesticide treatment at the same time.332.1.2. GRASSES2.1.2.1. COLLECTION AND PROPAGATION OF MATERIALThe grass species chosen to be neighbors of the clover are species that co-occurwith it in pastures of British Columbia. Dactylis glomerata (Dactylis), Holcus lanatus(Holcus), and Lolium perenne (Lolium) are found growing largely in monospecificclumps or in patches also containing clover (Aarssen and Turkington, 1985). Patches ofthese grass species offer varying degrees of competitiveness to white clover (Haynes,1980; Frame and Newbould, 1986; Evans and Turkington, 1988). Seed of the threespecies was obtained from Buckerfields Seed Co, Vancouver, and sown with 50-100seeds per pot in approximately one hundred 800 cm3 (10-cm) pots containing a mixtureof 1/3 peat, 1/3 sand, and 1/3 perlite. The seeds were covered lightly with sand andplaced in the heated greenhouse at the University of British Columbia south-campus fieldstation in September 1988.To produce material suitable for the experiments, it was important to grow thesegrasses under nearly-ideal conditions. It was necessary for the pots of grass to (i) alterlight conditions comparably to grasses in the field, (ii) be easily reproducible from thegrower’s standpoint, and (iii) form a canopy that was uniform and quantifiable. Underthese conditions, replicates of treatments in individual experiments would more closelyresemble each other, and an experiment could be repeated using almost identical canopyconditions. Plants growing in containers in a greenhouse generally find conditionsrelatively resource-poor compared to the locations in which they normally grow in thefield, where they often have greater access to light and a larger rooting-space than in agreenhouse. Examples of these three species of grass were available in fields outside thegreenhouse. It was only under extremely rich conditions in the greenhouse, i.e. growingwith heavy fertilization and in spaced rows allowing one side full exposure, that these34canopy modules closely mimicked grasses growing in the field and, hence, would alterthe light conditions similarly. The consistency to be gained by growing canopy modulesin this fashion came from each species’ ability to self-thin under rich conditions. Thiswas accomplished by growing the grasses in a relatively open situation with consistentfertilization allowing each species to reach its own natural density.The grasses were irrigated as needed on a constant liquid-feed program (i.e.fertilized during at least 3 out of every 4 irrigations to at least 10% beyond saturation) atlg/l of 20-20-20, N-P-K. These were placed on benches in the greenhouse in spacedrows grouped by species, in order to encourage uniformity within each species. The potswere kept free from pests with a regular program using airborne insecticides (Ambush,Malathion, and Sevin), airborne fungicides (chlorothalonil, Maneb, and benomyl), a soil-applied insecticide (Diazanon granular), and a fungicide soil-drench (chlorothalonil) atthe recommended rates. The soil was also limed (1 gm/pot) twice yearly.Twice per week beginning early in growth, the grasses were clipped to encouragesturdy growth and tillering and to fill the pots with lateral growth. Material overhangingthe bounds of each pot was also clipped, helping to maintain a tidy, self-supportingcanopy which had the same lateral dimensions as the pot. Progressively, the grasses wereallowed to grow taller before being clipped, until they formed a sturdy, square “hedge”,lOxlOx2O cm tall. They were then clipped weekly to this height. During this time thepots were allowed to fill and thin without interference, except for occasional removal ofthe thatch layer before it became a significant feature of the canopy.Eventually, the stock of grass material for the experiments, consisting of thesethree species of grass growing as uniform hedges above the pots, could be maintainedindefinitely. Each pot of grass formed a pillar-shaped canopy that could be arranged withothers like it in a standard, repeatable fashion. These pots became the canopy-forming35modules that were used to present the desired above-ground canopy conditions to cloverclones.Canopies that were used in control treatments were designed to present cloverclones with no influence from grass, though otherwise be the same. These “empty”canopies were formed by the same pots filled with the same potting medium butcontaining no grasses, and in one reflection experiment control pots contained bleachedgrass (see below). Control pots were treated in the same manner as the pots containinglive grass; i.e. they were irrigated and treated with pesticides as if grasses were growingthem. In the experiments, they were placed in the same arrangement as the grass-canopymodules, creating a “canopy” of empty pots (or bleached grass). PREPARATION FOR EXPERIMENTSAn excess of pots of all the types needed for each experiment was arranged withspecies in rows 10cm apart, on benches in the greenhouse. The orientation of the potswas kept consistent, so the sides next to neighboring pots in the rows would remain thesame. These also would be the sides that were next to neighboring pots of grass in theexperiments, on the inside of a row of canopy modules. Of the two remaining sides onthe grass pillars, one consistently received the irrigation, which came from a tight showerspray and matted down some lower leaves and thatch. The remaining side was directedtoward the south wherever possible. This south face of the pillars of grass most closelyresembled the appearance of a clump of grass growing in the field. This was the side thatfaced clover clones during the experiments. Throughout this time the average tillerdensity in the pots of grass remained between 40-150 per pot (4000-15,000 per m2), theleaf density remained between 80-300 per pot, and the estimated LAI remained betweenca. 2 and 5. These pots of grass resembled clumps growing in the field, and altering theSED similarly, could present clover clones with light conditions found in a sward while36preventing interaction below-ground. The control pots were placed in the samearrangement and treated identically before the experiments. TREATMENT DURING THE EXPERIMENTSA two-fold excess of uniform canopy modules (either pots of grass or control-canopy pots) was prepared for the experiments to ensure uniformity in the canopiesthroughout the experiments. It was uncertain as to how consistent the grass canopymodules would remain when growing in the experimental treatments. With an excess,pots could be rotated in and out of the experimental chambers as needed, ensuring that ahealthy, field-like, grass-canopy could be presented to each clover clone receiving thattreatment. During the experiments all excess canopy modules remained in similar rowsoutside of the treatments, and these were irrigated and sprayed the same as modules in theexperiment.The canopy modules were fed throughout the experiment on the same constantliquid feed program described above, with irrigation occurring as soon as any pots beganto dry out. The same pesticides as above were applied to grasses and control pots, butsince there was an excess, this could always take place outside the experimentalcompartments. In the reflection experiments, the pots forming the canopies werereplaced with pots freshly clipped to 20 cm every 4 days, at the same time that theposition of clover clones was rerandomized. In the partial shade experiment, the pots ofgrass were clipped to 15 cm every 4 days in the rows when clover clones were removed.In localized shade experiments pots of grass were clipped every 4 days to 20 cm, outsideof the experimental compartments. The edges of each pot were clipped at the same timeensuring that leaves of grass canopies did not overhang the flat of clover, thus confiningthe canopies’ effects to the hedge of grass forming the standard canopy, and preventinggrass contact with clover leaves. Clipping of grasses always took place at a distance fromclover clones, preventing cut leaves, motion, etc. from affecting them.372.2. MODULAR CANOPY DESIGN--CONSTRUCTION OFNEIGHBORHOODSAn individual treatment in all experiments consisted of a single cutting of theclover clone rooted in its own container and a canopy formed by either a hedge of livegrasses growing in pots (treatments) or control pots. The different canopy conditionsused in each experiment can be described by the species, density, and orientation of thecanopy-forming modules with respect to the clover clones. For controls in theseexperiments, pots containing potting-medium only were placed in the same arrangementas pots with grass, creating an “empty canopy”. Thus, the differences between controlsand experimental treatments were confined to the zone where the grass plants werelocated, restricting to above-ground the possible mechanisms used for detection of thesecanopies by clover clones.It was important to design the neighborhoods so the grass hedges were preventedfrom interacting with the clover clones through leaf contact with clover foliage orpotting-medium, splashing from the potting medium, and wicking of drainage water.These and other potential modes of interaction had to be minimized to ensure that theeffect of the canopy was primarily due to alterations in the light conditions. Care wastaken to avoid these, by treating the different types of canopy used in each experimentidentically. For example, water, fertilizer, and pesticides were applied equally to theregions occupied by the canopy pots regardless of the canopy design or the species ofgrass used.All pots used for the canopies were positioned at or above the level of the flatcontaining clover. If the pots had been lower, grass leaves in experimental treatmentswould have covered the sides of clover flats, while in controls the sides of clover flatsremained exposed. This location assured that the canopy modules would insulate the soil38similarly regardless of its contents, since in all cases the sides of each flat of clover wereequally exposed. In addition, it was important for grasses to be high up towards thezenith in order for the canopy to have the desired effects for the experiments.As mentioned, the species chosen to form the canopy could be varied, and in thepartial shade experiment each species could produce its own distinct neighborhoodinfluences. Pots of different grass species consistently differed in many parametersincluding tiller and leaf density, leaf width, pubescence, and leaf angle, as they do in thefield (Burdon, 1982; Burdon and Harper, 1982). These canopy modules, however, wereconsidered to be equivalent, well-defined, and reproducible because of the similarconditions under which they were grown. Since each pot (within species as well asbetween) determined its own density (through self-thinning), the final outcome of all thespecies (and pots within species) would be determined by the amount of resourcesprovided, and this can be duplicated. A reliable way of providing equal resources todifferent plants is to saturate them. The constant liquid-feed program and spaced rows,both before and during the experiments, were designed to accomplish this.The density of modules used to form the canopy could be varied, and this wouldproduce a distinct neighborhood. The traditional definition of density as plants per m2does not adequately describe conditions in these experiments where below-groundinteraction is prevented. A one or two-dimensional measurement does not adequatelydescribe density, even with below-ground interaction, because it does not describe therestrictions to the space which provides resources (Ross and Harper, 1982). For theabove-ground applications in these experiments, density can be described better byquantifying the amount of sky obscured, or the amount of the global-radiation hemispherethat is occupied, using an angle, wedge, or percentage of a hemisphere. Using thisdefinition density is determined by both the distance and height of neighboring plants.39For example, tall grass at a greater distance would cause the same amount of skyobstruction as shorter grass that was closer.Although a short and very close canopy arrangement would obscure the sky thesame as a tall, distant one, the two canopies could differ in factors such as temperature,humidity, and other airborne factors. The further away the canopies are located, inaddition, the less these proximity factors are involved, while changes to the lightconditions will still occur. To focus the differences between canopies on alterations tothe light environment rather than to these other proximity factors, the distance to thecanopy was maximized and circulating fans were used to increase air flow. To maximizethe distance, the height of the canopy also had to be maximized to present the desiredamount of sky obstruction. The height of the canopies was constrained by the height thegrasses could grow and still be uniform in the pots and by the maximum depth of the sub-canopy of pots, which could be elevated slightly above the clover soil level before itinfluenced a large a proportion of the global radiation hemisphere itself. The specificdistance and height used in each type of experiment is described below.The orientation of canopy modules with respect to the sun was the most importantfactor determining the light conditions experienced by clover clones. The specificplacement of the canopies in each experiment (Figs. 2.1, 2.2, 2.3) created a particularlight environment. If the orientation with respect to the sun stayed the same, twocanopies of the same species at a constant height and distance would create the same lightconditions. This feature was utilized so that replicates of each treatment could be created.Following is a description of the orientations used in the three types of experiments.2.2.1. REFLECTION (NORTHERN CANOPY)Grass canopies used in these experiments (Chapter 3) were designed to provideclover clones with a signal of impending interspecific interaction largely through40reflection from neighboring grasses, without limiting the available PPFD. A solid barrierof live grasses was used to reflect light with an SED, and in a pattern, that far moreaccurately simulates field conditions than artificial filters. Experimental clover cloneswere divided into two groups, both to be grown under high levels of PPFD, with onegroup receiving additional light reflected from live grass neighbors. Experimentaltreatments consisted of a row of five Daclylis canopy modules (described above) placedin an east-west orientation 15cm to the north of, and totally separated from, target cloverplants, which were arranged with the primary shoot growing parallel to the row of canopymodules (right side of Fig. 2.1). Controls were arranged with clover clones growing infront of pots with no grasses and containing only the growing medium, a soilless potting-mix (left side of Fig. 2.1). In this arrangement the two sets of clover clones both receiveddirect light from the south, while experimental clones received, in addition, reflected lightfrom the row of grasses to the north. This design simulates a field situation where aclover stolon is growing within a large gap but is near grass neighbors at the northernedge.In two of the experiments in this set (Exps. 1, 2; Table 3.1), each set of canopymodules and controls were separated from the corresponding target clover clone with atransparent Plexiglas sheet (Fig. 2. ib). This was an attempt to minimize the differencesin temperature, humidity, C02 or other factors created by the different canopies (grass orcontrol) beyond what the air-circulation would accomplish. The transparent barrierswould keep environmental conditions more uniform between treatments, while allowinglight signals to pass between grass canopies or controls and clover. In this arrangement,the influence on light conditions that grass canopies had on target clover plants would beemphasized even further.412.2.2. PARTIAL SHADE (PARTIAL CANOPY)In this set of experiments (Chapter 4) canopy modules of Lolium, Holcus,Dactylis, and controls were positioned to the east and west of clover clones (Fig. 2.2).This arrangement resulted in the clovers and grasses being physically separated bothabove and below ground, preventing root competition yet allowing interference withregard to light. The orientation of canopy modules allowed light to be transmittedthrough the grass canopies while also allowing a substantial amount of unaltered light (aswell as reflected light) to reach clover clones. This is a pattern experienced by a cloverplant in the field, when at different times of the day, a plant might receive unequal lightconditions. The gap created directly over the clover clones allowed a period of direct sunduring the middle of the day, and on overcast days, a patch of high R:FR light would beexperienced while receiving light filtered by grasses. This mimics the field situationwhere a clover stolon is in a small gap, but is near grass neighbors on at least two sides.Each treatment consisted of two north-south rows of canopy modules with twoflats containing clover clones placed in the same orientation (north-south) between therows. Each row of canopy modules was 100 cm long, consisting of 10 individual potsplaced side-by-side, the rows containing grass forming a uniform continuous hedge. Thespacing of the rows created an aisle of standard width, and because all grasses wereclipped to 15 cm above the pot (or 17 cm above clover ground level), these rows formedan aisle of standard height. The dimensions chosen (15 cm wide x 17 cm tall) created agap that allowed equal exposure to the sky from any point on a line in the middle of thefloor of the gap. From any of the points on this line, a “corridor” of sky running north tosouth, directly overhead, would never be obstructed.The primary stolon of each clover clone was oriented so it grew from north tosouth on the line down the middle of each tray, beginning at a point near the north end of42each flat. This kept each primary stolon apex under relatively constant conditionsthroughout the experiment (because it generally grew straight down this line) and assuredthat the environment was symmetrical from the point of view of the clones, i.e. havingconditions imposed equally from the left or from the right.2.2.3. LOCALIZED SHADE (LOCALIZED CANOPY)In the localized shade experiments (Chapter 5) grass canopy modules werelocated to the south of clones which were divided into two portions (Fig. 2.3). Thesecanopies provided shade in a natural pattern, with sunflecks and natural scatteringthrough the canopies of grass while maintaining below-ground separation. The shadewas imposed on one or both portions of a clone, independently, by physically separatingthe two neighborhoods with opaque barriers (see below). The intent was to present tworegions of a clover clone with distinct conditions, formed by shade that mimics neighborsgrowing in the field. This allows an evaluation of the independence of the responses ofthe two regions of the clover, by testing the conditions experienced by one region for aneffect on the other region of the clone.The designs of the neighborhoods used in these experiments mimic a fieldsituation where a single clover stolon has portions experiencing different environmentalconditions. In the field, as the apex of a stolon advances into a gap it experiences highlight conditions, while the basal portion of that stolon might remain within a clump ofgrass. The reverse is also common in the field, where a stolon apex invades a clump ofgrass from a gap and experiences shade, while the basal portion remains in the gap. Inthe field, the two portions clearly do not experience their own conditions independently.The design of these experiments, with separation between the two neighborhoods, allowsan evaluation of the independence of two regions of a clone under different sets ofconditions. A response in the remote portion of a plant would have to come from an43intra-plant signal rather than from the direct effects of the distant canopy, effects whichcould not be disentangled without isolation of the two regions.Clover clones were divided between basal and apical regions of a primary stolon,and all four combinations of two canopies (formed by grass or control canopy modules)and two regions (apical or basal) were used in the course of four experiments. Only twoarrangements were used in each experiment, due to limitations of space and the need togrow replicates of each treatment. In the first two experiments, the effect of apical-regionshading on the growth of basal regions of clover clones is investigated, first with thebasal regions themselves in the open (Fig. 2.3a), and second, with the basal regionsshaded by a grass canopy (Fig. 2.3b). In these experiments, the manipulation is doneonly to the apical regions of clones, and the response of only basal regions is examined.Differences in the basal regions of clones having their apical regions manipulateddifferently, is taken as evidence of physiological integration within the plant. In thesecond two localized canopy experiments, the effects of a basal-region canopy on thegrowth of apical regions of clones is examined, first with the apical regions in the open,(Fig. 2.3c) and second, with the apical regions shaded by a grass canopy (Fig. 2.3d). Inthese experiments, it is the response of apical regions, themselves growing underidentical conditions, which determine the existence of physiological integrationthroughout the plant.To summarize, in three sets of experiments, three different types ofneighborhoods were designed from pots of grass or control pots and flats of cloverclones. Each of these presented a unique environment which was established by thepresence of grass in the canopy, and these all simulate conditions white clover couldexperience in the field. The canopies of grass, however, could cause no correspondingbelow-ground interaction, so the responses of clover clones could only have been due to44detection of changes in above-ground conditions, perhaps primarily through changes inlight conditions.2.3. EXPERIMENTAL CHAMBERS- DETAILS OF A ‘BOX’ AND FLAT OFCLOVERIn each experiment, regardless of type, it was necessary to isolate treatments inthe greenhouse to prevent the canopy of one treatment from influencing the conditions ofother clover clones. A difficulty in experiments of this type is ensuring that each canopyonly exerts influence on its neighboring clover clone. The distance needed to effectivelyisolate different treatments of this type without an opaque barrier is unknown, andpossibly quite large. Space was limited in the greenhouse, and compartment wallsallowed treatments to be adjacent, maximizing the number of replicate treatments thatcould be grown. These “boxes” were designed within practical constraints, e.g. cost,transportability, reusability and are described below.Each treatment, consisting of a canopy and a single clone of clover (see above),was placed within a standard wooden compartment (box) designed to isolate thetreatments and to standardize conditions. These boxes ensured that the light conditionsexperienced by a single clover clone were affected only by grasses immediatelyneighboring that clone. The main features that these boxes had in common were theopaque walls and the uniformity imposed through the height of the walls and theirorientation in the same direction. The specific design of the boxes used in each type ofexperiment is described in more detail below.Walls were always high enough to maintain isolation to the point where thecanopy of an adjacent treatment could have no effect on any other clover clone.However, the height of the walls was limited due to their blocking of the sun and sky, andthe corresponding reduction in the quantity of light. It was important to grow clover45clones with quantities of light that were typical of the field, in order to be able to applyclover response to the field.The orientation of the boxes remained consistent throughout each experiment.Within each experiment, since all boxes were arranged in the same direction and hadwalls of the same height, the sun tracked identically from any particular coordinate withinthe boxes. In all cases they were aligned so that they were open (i.e. widest exposure) tothe south. This allowed as much light as possible into the boxes and allowed the sun totrack symmetrically through the boxes, spending an equal time left and right of center.2.3.1. REFLECTIONThe boxes used in reflection experiments were designed so that the grass canopiesadded reflected light without impeding the photosynthetic light available for cloverclones. The boxes had three walls, as compared to a complete box having six; they wereopen to the top, bottom, and south (Fig. 2. ic; Plate 1). The side walls (east, west) werehigher towards the north where the grass canopy modules were situated, although the restof the walls were tall enough to obscure clover clones from grass or clover in adjacentboxes. The north wall was the same height as the higher portion of the side walls. Thisextra height towards the north prevented the sun (or sky) from appearing through thecanopy, limiting light transmission through the canopies, and reducing the PPFDavailable from behind control canopies. The only light which could originate frombehind the canopies would be the reflection off the inside walls of the boxes, and thiscould be reduced to a minimum. This design allowed as much of the available light aspossible into the box, increasing PPFD (towards field level) and maximizing the exposureof grass canopies.The walls of the boxes were painted with a matte black finish (CIL indoor latexmatte-black) to reduce reflection within the boxes. It was especially important to reduce46the amount of reflection from the northern wall of the box, behind the barriers, so thatexperimental treatments, having grass canopies which obscured the wall, containedroughly the same amount of PPFD as controls, which did not obscure the (upper portionof the) walls. In this arrangement then, experimental and control clover clones receivedequal light from the south, however, experimental clones in addition received reflectedlight from the grass canopies to the north, and control clones in addition receivedreflected light from the matte-black-painted northern wall of the box.2.3.2. PARTIAL SHADEExperimental compartments used in this experiment were also designed to preventlight passage between treatments and allow equal exposure to the sky. These wereconstructed from separate wooden boards (120cm long x 10cm high x 1cm wide) paintedwith matte-black paint (Cit indoor latex matte-black) and placed N-S on stands to formeast and west walls (Fig. 2.2; Plate 3). The height of the stands could be adjusted, andthey were placed at a standard height (30 cm) in each box, high enough to never allow agrass leaf from one treatment to be exposed to a neighboring treatment. Black darkroomcurtains were draped downward from the boards, so the height of the wall could beextended while maintaining isolation down to the level of the pots. These boards withcurtains made opaque walls which restricted the differences in light conditionsexperienced by clover clones to the canopy present in its own compartment. Thetreatments were placed adjacent so that one of these opaque walls served as a wall in twoadjoining treatments. Twenty compartments were thus created, and with the walls placed45 cm apart, each compartment had enough space behind the rows of canopy modules toallow access to the “rear” of the pots. This space allowed water to be applied to the grasson the side away from clover clones, creating minimal alteration of the light conditionsby matting of grass and minimal splashing off the canopy modules. The space alsoallowed the grasses to grow within the treatments without experiencing too much shade47from the barriers, which may have caused yellowing and loss of leaves from the pots ofgrass.As in reflection experiments, the walls were matte-black to reduce reflection fromthe barriers, because this would focus the differences between treatments to influences ofthe grass rather than influences of the wooden barrier. With dark rather than bright walls,control clones, which had no grasses obscuring the walls, would receive a more similarPPFD to the experimental clones, which have grasses obscuring the walls.2.3.3. LOCALIZED SHADEIn localized shade experiments the standard wooden compartments (boxes) inwhich each clone grew were each two-chambered, which allowed the independentmanipulation of light conditions experienced by the two inter-connected parts of eachclone. In these experiments, localized portions of experimental clover clones weresubjected to shade from a grass canopy. To isolate the direct effects of this canopy to oneportion of the plant, an opaque wall was present between the two portions of each plant,and the second portion would then occupy a separate chamber.The boxes used were the same as the ones used in reflection experiments (Fig.2.1; Plate 4). These boxes originally had been constructed in pairs, i.e. two boxes werealways side-by-side, sharing the middle wall (this made them easier to construct andtransport). In these experiments, a single two-chambered box was used for each cloverclone (Fig.2.3). An extension to the lower part of the middle wall, made of 2 mm-thickhigh-impact polystyrene, was installed, dividing the two chambers just after the plantswere placed with their primary stolon apices extending into the second chamber. Thislower-wall addition was necessary to fully isolate the two neighborhoods right down tosoil level while allowing the primary stolon through. A small gap (2mm wide x 5mmhigh) was cut into the bottom edge of this wall at the point where the primary stolon48crossed, so the wall could be pressed into the soil around the stolon until it fit snugly intothe gap. Thick white tape was placed around the top of the gap, so that light transmissionwould be blocked without applying damaging pressure from the top of the gap down onthe clover stolon. Light conditions in the two chambers could then be independentlymanipulated.In an attempt to increase similarity to the field, all of the walls within thechambers used for localized shade experiments were painted white, which would increasethe overall illumination. It was thought that with maximally-reflecting barriers,conditions within the boxes would have remained more similar to field light conditions.These treatments, therefore, would be indicative of a more common situation in the field,i.e. where there are better and worse light-resource patches due to neighboring grass,rather than having patches with light present and light absent. In addition, with whitewalls inside the boxes, the advantage that open neighborhoods have over shaded onesincreases, perhaps promoting separation of the responses to the different localizedcanopies.2.4. MEASUREMENT OF WHITE CLOVERThe piece of stolon that was taken from the stock material always continued itsdevelopment throughout the experiments, and this was considered the primary stolon. Itdeveloped secondary stolon branches at nearly every node. In nearly all experiments,these secondary stolons also branched to produce tertiary stolons.A single clover ramet in these experiments, consisted of a leaf, the root from itsnode, the axillary bud in the node, and the internode just basal to the leaf (Fig. 1.1). Theplants’ total number of ramets considered only ramets with the leaf petiole > 2.0cm,which was approximately the same time that leaf lamina unfolded, while the total lengthof stolon incorporated all stolon length, right up to each apex. A young leaf was49considered to be part of a new ramet when its petiole length reached 2.0 cm, otherwise itwas considered as part of the expanding apex. A branch stolon was considered to havebeen produced when the axillary bud produced its first leaf petiole > 2.0 cm. In localizedshade experiments, since it is only the response of one region of the plant that isnecessary to determine the existence of intra-plant integration, only the measurementsmade on the appropriate region in each experiment are presented (in two experiments it isthe apical region, in the other two it is the basal region).Clover clones were measured in detail at the final harvest of each experiment aswell as monitored over time in all of the experiments. At the final harvest of eachexperiment, the length, the number of ramets, and the number of branches on each stolonwere recorded. Also, five fully expanded petioles, leaf lamina, and internodes wererecorded from the primary stolon of each plant, and in the partial shade experiment, infour clones from each treatment, all petiole lengths and leaf areas were recorded. Fromthese the following variables were measured or calculated:Total stolon lengthTotal number of rametsTotal number of branchesPrimary stolon-% of nodes branchingSecondary stolon-% of nodes branchingWhole plant-% of nodes branchingPrimary stolon-age to first branch (number of nodes more distal without branches)Secondary stolons-age to first branchSecondary stolons-mean internode lengthTertiary stolons-mean internode lengthWhole plant-mean internode lengthMean primary stolon internode length (n=5)Mean primary petiole length (n=5)Mean primary leaf lamina area (n=5)Whole plant-leaf areaWhole plant-mean petiole lengthBranch stolon length ratio (ratio of branch stolon length to whole plant length)Branch ramet number ratio (ratio of branch ramet number to whole plant length)At the final harvest of each experiment, plants were divided into leaf lamina, petioles andstolons on each particular order of branching (primary, secondary, and tertiary) and driedat 60°C for 96 hours. These measurements allowed the determination of the following:50Total above ground dry weightPetiole weight ratio (percentage allocation to petioles, i.e. petiole dryweight/whole plant dry weight)Stolon weight ratio (percentage allocation to stolons)Leaf lamina weight ratio (percentage allocation to leaf lamina)Branch weight ratio (percentage allocation to branchesRatio of petiole/stolon weightMean weight per rametWhole plant-stolon specific length (length per unit weight)Whole plant-petiole specific lengthWhole plant-leaf lamina specific areaIn the reflection experiments, in addition, the newest five ramets on primary stolons ofclones were harvested, measured (leaf area, petiole length, and stolon length) and driedseparately from the rest of the primary stolon. In partial-canopy experiments, all leaflamina, petioles, and stolon internodes on the primary stolon were measured and dried.From these measurements of length or area and the corresponding weights, the followingwere calculated:Primary stolon-specific stolon length (length per unit weight)Primary stolon-specific petiole length (length per unit weight)Primary stolon-specific leaf area (area per unit weight)In the localized shade experiments, only the stolon length associated with a particularweight was known, so only the specific stolon lengths (whole plant and primary stolon)were determined.Measurements over time did not involve destructive sampling, so only the lengthof stolon, the number of ramets, and the number of branches could be recorded over time.From these the following were calculated:Mean absolute growth rate (AGR) of stolon lengthMean absolute growth rate (AGR) of ramet numberMean absolute growth rate (AGR) of the number of branchesMean relative growth rate (RGR) of stolon length (change in natural logarithm ofstolon length over time)Mean relative growth rate (RGR) of ramet numberMean relative growth rate (RGR) of number of branchesRatio of primary stolon length growth to whole plant stolon length growthRatio of primary stolon ramet number growth to whole plant ramet numbergrowthRatio of branch number growth to primary stolon length growth51In two of the reflection experiments (#‘s 2,5; Table 3.1) and in all localized shadeexperiments, the length of expanding petioles was monitored over the 8 days from thepetiole first appearing. When recording stolon length and number, it was not difficult tomeasure the length of the newest petiole and a few of the next older petioles. Whenmeasurements of this type were made frequently enough (every 2 days) the growth ratecould be determined for these petioles. The lengths of successive petioles on each plantwere then grouped according to the number of days since first appearing, 2-4, 4-6, and 6-8 days, and the growth rates during these intervals were compared between treatments,disregarding any ontogenetic effects on petiole growth rate.In reflection and partial shade experiments, the position of leaves on the primarystolon was sampled on overcast days during the experiments, and the following weredetermined:Height of leaf above potting-medium surfaceLateral displacement from the stolon (excluding leaves that crossed the stolon)The side on which the leaf originatedThe side on which the leaf lamina was currently positionedThe angle normal to the lamina as viewed from the basal portion of the clones(elevation angle).In reflection experiments, the lateral displacement of the primary stolon apex was alsorecorded. This was a measurement of the displacement north/south from the center of theflat indicating if the stolon grew straight down the middle of the flat. In the localizedshade experiment, the height of the highest leaf lamina and the number of leaves crossingthe primary stolon was also recorded.2.5. DATA ANALYSESThe specific methods for data analysis depended on the type of data collected, andthis differed slightly between experiments. In many of the experiments, a few plants,which were inhibited by some uncontrolled factor, were visually detectable as smaller,52slower-growing plants,. and these differed from the overall group mean by at least 4standard deviations in at least one of the variables examined (95% confidence intervaln=6, Glantz, 1987). The experimental group. of clones was always compared to thecontrol group after these plants (if any) were removed. The remaining plants were usedto compute the treatment-specific means and standard deviations for each of thesevariables. Homogeneity of variance between groups was tested using a Bartlett test and ifp<0.05, the data were transformed as follows: log10 (of size measurements), arcsine (ofproportions), and square-root (of internode length and age to branch). If after suchtransformations the data were still heteroscedastic, a non-parametric test was used.Means were compared with Student’s t-tests, or ANOVA and Tukey HSD when multiplecomparisons were to be made (Systat, 1990), and non-parametric Mann-Whitney orKruskal-Wallis rank-sum tests were used when the variances of the groups to becompared were significantly different (Bartlett test p<O.O5). All tests used were two-tailed.In reflection experiments, the following ANOVA’s were conducted:Leaf position data were compared using ANOVA model:Data = Constant + Side of plant + Treatment + (Side of plant x Treatment) +error.Data collected at several intervals during one experiment were analyzed using a repeatedmeasures ANOVA model.:Data = Constant + Interval + Treatment + (Interval x Treatment) + error.Some of the measurements in reflection experiments were available from severaldifferent experiments, and the treatment effects were analyzed between as well as withinexperiments:Data = Constant + Experiment + Treatment + (Experiment x Treatment) + error.53Leaf position data that were available across several experiments, were analyzed usingANOVA model:Data = Constant + Experiment + Side of plant + Treatment + (Side of plant xTreatment) + (Experiment x Side of plant x Treatment) + error.In partial shade experiments, differences among variables were examined with aone-way ANOVA using all treatments (3 species of grass and the control) as the effectusing the model:Data=Constant + Treatment + error.Tukey HSD multiple-comparison tests were then conducted to determine whichtreatments had different effects.In the localized shade experiments, one or two of the largest plants reached theedges of the flat well before the others, and when this occurred, an equal number of plantsfrom each treatment was harvested early. For the remaining plants, means of final-harvest measurements were compared using Student’s t-tests if the variances betweengroups were not significantly different (Bartlett test p>O.O5)and the observations wereapproximately normally distributed. If these conditions were not met, non-parametricMann-Whitney or Wilcoxon signed-rank tests were used instead. In cases where thepairing of individuals allowed, Wilcoxon signed-rank tests included the pairs harvestedearly. For the measurements made over time, the early-harvested pairs were excludedfrom the analyses. These repeated-measures ANOVA’s were conducted using the model:Data = Constant + Interval + Treatment + (Interval x Treatment) + error.2.6. MICROCLIMATIC MEASUREMENTSWhile detailed measurements of microclimate were not attempted, prudent use ofcrude measurements was used to describe these conditions. The temperature and SEDwere measured under different conditions. As resources permitted, measurements were54made under extremes of conditions, under various weather conditions, and at differenttimes of the day and of the season. This would allow better comparison with otherexperiments, and it would describe the extremes that clover clones had to deal with.These data were either collected directly in the experiments or under simulations of theexperiments which could provide more standardized conditions than were possible duringthe experiment itself. The measurements were not intended to quantify differencesbetween treatments, but merely to establish if there was a difference, and if so, itsdirection.2.6.1. SPECTRAL ENERGY DISTRIBUTIONSimulations of the experiments constructed in summer 1991 were used to producespectroradiometric data representative of these experiments. A LICOR LI- 1800Spectroradiometer (Lincoln, Nebraska) with a cosine-corrected sensor on a fibre-opticcable was used to collect and analyze spectral information. Scans were made at severallocations within one compartment, under simulations of treatment and controls. All scansrecorded were means of three consecutive scans. These data were collected outdoors,since in the greenhouse the length of time it took to scan from 300 to 1100 nm in eachsimulation corresponded with changes in ambient light, e.g., the sun moving behind asupport beam. Some measurements also were made in the greenhouse to compareoutdoor and greenhouse light conditions. Spectroradiometric scans were made underuniform sky conditions, i.e., clear sky or overcast, at various times of the day includingsolar noon, morning and afternoon. Using arrangements identical to the experiment, potsof grass and pots of soil could be alternated without disturbing the sensor to produce twospectra, differing only in the presence of live grass. Any significant differences in thespectra were considered as indicative of spectral differences between the two treatmentsof the experiment. The R:FR ratio and PPFD were calculated from these scans.552.6.2. TEMPERATURETemperature was monitored in several experiments by simultaneously recordingin the different treatments. Resistors calibrated for temperature were placed inexperimental compartments, either taped to the wall of the compartment with the resistorsupported 3cm away from the wall (used when the sun was not able to heat the resistoritself), in a small white tent 3cm above the potting-medium surface, or buried 1cm belowthe surface. The sensors were mounted in place and left to stabilize. The temperaturewas then recorded by moving the monitor (ohmmeter calibrated for temperature) betweentreatments while leaving the sensors in place. This was repeated a minimum of threetimes as rapidly as possible, and these were averaged. This was done at representativetimes (e.g. near noon, early evening, late evening), throughout the course of each set ofexperiments to give a general picture of the temperature regime in all treatments.(a)NI•ii•rn••iii•.uJ.••‘‘‘‘‘I’ll.—I >1 I >1Pots of neighboringgrassPots of soil withno grass(b)(c)iiiiiiiii•iI >1Flats with cloverBlack plywoodpartitionPlexiglaspartitionScale: .—10 cnI >160Fig. 2.1. Treatments used in four reflection experiments in 1990 showing the arrangement of the modular grass canopy. (a)shows the design without using Plexiglas dividers between the regions occupied by the grass canopies and the cloverclones, while (b) shows the design with a Plexiglas barrier. (c) is an enlarged side-view of (b).ww•Pots of neighboringgrassPots of soil withno grassFlats with cloverBlack plywoodpartitionsScaie:10cmI IIFigure 2.2. Design used in partial shade experiment. Canopy modules were pure stands of either Dactylis glomerata,Holcus lanatus, or Lolium perenne, and pots of potting-medium only were used as the control. Clover clones grew for 25days.S Pots of neighboringgrassj Pots of soil withno grassFlats with cloverN Black plyvoodpartitionsScele: ,—i10cmI I111111AVBRI 1R RI11111111 1111551Neighborhood 1 Neighborhood 2 Neighborhood 1 Neighborhood 2OPEN-OPEN OPEN- CLOSEDFigure 2.3a. Design of localized canopy experiment where canopy variation is presented to apical regions of clover clones,while basal regions remain in the open. Growth of basal portion in A: Neighborhood 1 was compared to growth of thesame portion in B: Neighborhood I.I 11111_AVBI I S• n •5151111 55555Neighborhood 1 Neighborhood 2 Neighborhood I Neighborhood 2CLOSED -OPEN CLOSED - CLOSEDFigure 2.3b. Design of localized canopy experiment where canopy variation is presented to apical regions of clover clones,while basal regions receive closed canopies. Growth of basal portion in A: Neighborhood 1 was compared to growth of thesame portion in B: Neighborhood 1.‘ I11111 I_AVBII 1R ftI__•RiiII LililiNeighborhood 1 Neighborhood 2 Neighborhood 1 Neighborhood 2CLOSED-OPEN OPEN-OPENFigure 2.3c Design of localized canopy experiment where canopy yariation is presented to basal regions of clover clones,while apical regions remain in the open. Growth of apical portion in A: Neighborhood 2 was compared to growth of thesame portion in B: Neighborhood 2.I I•I I I I_• ) • El •• • Elliii ••INeighborhood 1 Neighborhood 2 Neighborhood I Neighborhood 2CLOSED-CLOSED OPEN -CLOSEDFigure 2.3d. Design of localized canopy experiment where canopy variation is presented to basal regions of clover clones,while apical regions receive a closed canopy. Growth of apical portion in A: Neighborhood 2 was compared to growth ofthe same portion in B: Neighborhood 2.59Chapter ThreeDOES LIGHT REFLECTED FROM NEIGHBORS (NORTHERN CANOPY)AFFECT THE GROWTH AND MORPHOLOGY OF WHITE CLOVERCLONES?3.1. INTRODUCTIONThe importance of the ability to sense and respond to neighboring plants beforethere is resource limitation has been recognized recently (see Chapter 1). There isconvincing evidence that neighboring plants can be detected through changes in the R:FRof light before there is photosynthetic limitation by neighbors (Ballare et al., 1987, 1988,1989, 1990; Novoplansky et al., 1990; Smith, 1990). These changes in the lightenvironment are primarily through additions of FR light from reflection off neighboringplants. The ability to successfully avoid photosynthetic limitation by neighbors mayallow a plant to achieve success in a competitive environment, and this might be a traitthat is under genetic control, hence, a population can be under selective pressure from itshabitat to improve in this regard (Ballare et al., 1992b; Ballare, 1994; Bell andLechowicz, 1994).The responses of plants to photosynthetic changes in the light environmentbrought about by neighboring grasses are relatively well studied (see Chapter 1). Theseresponses, which typically include increased internode length, reduced branching, greaterspecific leaf area, and increased biomass allocation to stems, largely match the responsesto artificially imposed conditions that establish differences in both the quantity andquality of light. The responses in orthotropic plants to neighbors through nonphotosynthetic changes in the light environment have included increased internodelength, reduced branching, and a greater proportion of biomass allocated to stem (Ballare,1987, 1988; Kasperbauer, 1987; Kasperbauer and Hunt, 1992). In plagiotropic plants, the60responses to non-photosynthetic changes in the light environment are less well studied,but can include differential horizontal placement of ramets in response to reflection fromneighbors (Novoplansky et al., 1990; Novoplansky, 1991). In white clover this has not asyet been studied, although its response to neighboring vegetation through changes in theoverall light environment include reduced branching and alteration of petiole andinternode elongation (Solangaarachchi and Harper, 1987; Thompson and Harper, 1988;Thompson, 1993). In these studies, the R:FR along with the PPFD were altered byneighboring plants. It seems that the responses to alterations in R:FR depend on theavailable PPFD. At relatively high PPFD (at levels typical of light shade from neighborsin the field) internode and petiole elongation are increased, and branching is reduced.However, the responses to changes in the R:FR at PPFD levels typical of relatively openconditions or when unfiltered light is also received, has not been studied in white cloveror other plagiotropic plants. For example, it is unknown if the reception of light reflectedfrom grass neighbors (lowered R:FR at high PPFD) has an influence on white clover.Using natural vegetation to alter the light conditions, the hypothesis could be tested thatlight reflected off grass neighbors alters the growth and morphology of white cloverclones.3.2 METHODSFive experiments were designed to assess the effects of light reflected fromgrasses on the growth and form of white clover clones (Table 3.1). The experiments haveunique features, but they are otherwise similar in many aspects. For example, thelocation (indoors or outdoors) and the time of year created different backgroundconditions, so the experiments are not true repetitions, but many of the primarytechniques are similar, such as the treatment of the plant material, the design of thecanopies and barriers (see Fig. 2.1; Plate 1), the type of data collected, and the dataanalysis (Chapter 2). In four of the experiments, all conducted in 1990 (Exps. 1-4, Table613.1), whole clones were exposed to light reflected from neighbors, while, in the fifth,conducted in 1992 (Exp. 5, Table 3.1), it was only the apical region of clover clones thatwas exposed to reflected light, basal regions remaining untreated. In the fifth experimentalso, control “canopies” were formed by hedges of bleached grass, rather than pots withonly potting-medium, so the apical regions in these plants received reflection from non-living neighbors. These and other distinguishing features are described here as asupplement to the descriptions in Chapter 2.Preparation of plant materialGrasses and clover clones were prepared as described in Chapter 2.1. The speciesof grass chosen for these experiments was Dactylis glomerata (Dactylis), and the grasswas clipped to 20cm above the pot, 22cm above the clover medium surface. In all of theexperiments, an excess of these and control pots was prepared. In the first fourexperiments, control pots contained only potting medium making a “canopy” of “empty”pots, while in the fifth, control pots contained bleached grass forming a hedge similar tolive grasses. The bleached-grass control pots were formed by spraying paraquat (0.01mgactive ingredient per pot) on live Dactylis canopy modules. After a few weeks, these hadcompletely yellowed, and along with a set of the same Daclylis which was leftunsprayed, made up the control and canopy modules used in this experiment.Clones of white clover were taken from stock material (Chapter 2.1.1). Thecuttings were then selected for uniformity from an excess, placed into experimentalcompartments and allowed to acclimate, in the first four experiments, for a further 2-4days before treatments began, and in the fifth experiment, for 43 days before treatmentsbegan. In the latter experiment, unlike the earlier four, only the apical region (of aprimary stolon) of clones received light reflected from neighbors. For there to be asubstantial amount of basal-region growth in these clones, extra time spent before62applying the treatment was needed. During this period, the position of clones wasrerandomized every four days. To begin this experiment, the clones were repositionedwith only the apical-most ramet protruding into the second neighborhood, where grasscanopies were positioned two days later. The experimental chambers used in thisexperiment were similar to the ones used in localized shade experiments (Chapter 2.3.3)except that they were painted black in the region where grass canopies were situated andwhite in front (to the south) of this. Eight replicate clones were used in each treatment(grass canopies or controls) in the first four experiments, and six replicates were used inthe fifth experiment. Treatments were randomly assigned to compartments, and this waschanged every four days at the same time that clones were rerandomized.Arrangement of canopy modulesThe arrangements of canopy modules used in these experiments are described inChapter 2.2.1. In Experiment #5 (Table 3.1), bleached grasses were used instead ofcontrol pots with potting-medium only.Treatment of plant materialClover clones during the experiments received treatment as described in Chapter2.1.1.3. In Experiments #2-4 (Table 3.1), the orientation of the clover clones was rotated1800 at the same time as rerandomizing (every 4 days). This was done to avoid theasymmetry which developed in all clones including controls in preliminary experimentsand Experiment #1. This type of asymmetry, which inhibited growth on the northern sideof all clones, could have prevented reflection from this direction from having anyadditional effect.on clover clones. By rotating the clones, the darkening from the thenorthern side of the experimental compartments would come from no particular direction,nor would the reflection from neighboring grasses. In addition, a description of thetreatment of clover clones in Experiment #5 is needed. In this experiment, a small apical63portion of the primary stolon was the only region of the clones that received reflectedlight. These portions were fixed in location once treatments began, so they could not bererandomized during the experiment. The two portions of each clone were treated as one,both receiving irrigation and pesticide applications at the same time.Grass canopy modules and controls were treated as described in Chapter collectionTechniques for spectroradiometric measurement are described generally inChapter 2.6.1., and are detailed further here. Under as many different ambient conditionsas possible, simulations of the reflection experiments were arranged outside thegreenhouse. One experimental compartment with a flat of potting-medium and canopy-forming pots (Dactylis or controls) was placed on a table at the same height as thebenches in the greenhouse. The sensor was placed horizontally in the middle of the flat,in place of a clover plant, and this was mounted securely so it could not move betweenmeasurements. Then, the only changes that were made between spectroradiometermeasurements were the type of canopy modules used, and the measurement made withcontrol canopy modules present became the standard to which the, other scans werecompared. All combinations of canopy type and Plexiglas presence were placedindividually within the compartment, and the global radiation (300-1100 nm) under eacharrangement was measured. Next, a clover plant was placed into position, and the sensorwas remounted on the potting-medium surface, directly in the shade of one of the cloverclon&s primary leaves. This was a mid-sized clone (the same as the one used in allexperiments) with several large primary leaves and few secondary branches. The canopywas then rearranged while the sensor remained in the same position. Measurements werealso made with the sensor mounted vertically 7 cm above the potting medium (with noclover clone present), facing the northern wall of the compartment. This greatlyenhanced the emphasis of horizontally impinging radiation, and would allow the sensor to64detect conditions similar to those experienced by upright clover structures such aspetioles (Ballare et al., 1987). The different canopy arrangements were then placed in thecompartment and measured as above. In sets of scans made at a later date using similartechniques, bleached grass plants were used as the controls, and several of the samearrangements as above were used.Temperature was monitored using techniques described in Chapter 2.6.2. Anoutdoor reflection experiment (Exp. 2; Table 3.1) was used to determine the effects ofPlexiglas on the temperature just above the potting-medium surface (sensor 3cm above).This was recorded around 1640 hrs (PDT) under a clear sky with a 8-15 kph wind by firstrecording with the Plexiglas present, and then again after the Plexiglas was removed.Indoors, (in Exp.3; Table 3.1) two sensors were mounted in each compartment, one 3cmabove the potting-medium surface and another 15cm above, mounted 3cm away from theeastern side wall of the compartment. From these locations the temperature was recordedjust before noon (solar time) and several hours after noon (around 1830 hrs PDT), afterthe sun ceased to directly affect the plants or the compartments.The collection of data on clover clones is described in Chapter 2.4. This additiondescribes the techniques for measuring primary leaf position in these experiments. Leafposition measurements were taken on overcast days at least once during each experiment.These were made near solar noon and always when the plants were relatively turgid. Atthese times, the position of the oldest two of the newest three primary leaves wasdescribed in terms of: (i) height of lamina above potting-medium surface, (ii) lateraldistance from the lamina to the stolon, (iii) the side on which it originated, (iv) the side onwhich its lamina was presently positioned, and (v) the angle normal to the larnina asviewed from the basal portion of the clones (i.e. looking east or west) giving the anglewith respect to north/south. While the height of leaves was easy to record within 0.5 cm,65and it was easy to tell whether or not a leaf was on the same side of the stolon on which itoriginated, the leaf lamina angle was determined visually with a protractor and pencil,and therefore this measurement was only accurate to ca. DATA ANALYSISThese are described in Chapter 2.53.3. RESULTSLight MeasurementsSpectroradiometric measurements made under simulations of the experimentsindicate that there was a detectable increase in FR wavelengths in treatments with grassbarriers (Fig. 3. la,b,d,e). These differences in reflected FR light led to decreases in theglobal R:FR, as detected by the horizontally-positioned sensor which was either in fullsunlight or in the shade of a clover leaf (Table 3.2) The presence of a grass canopyincreased the PPFD observed in the open but not beneath a clover leaf. Beneath the coverof a clover leaf, the increase in FR due to neighboring grasses is noticeable both in termsof absolute increase and the resulting decrease in R:FR (Fig. 3. ib, Table 3.2). Plexiglashad no observable effect on light quality, but the quantity of PPFD was increased in theexperimental compartments with Plexiglas (Fig. 3. ic, Table 3.2).With the sensor placed directly in front of the canopy of empty pots or live grassand oriented vertically facing the canopy, the relative influence of the canopy over thelight conditions detected is increased. Fig. 3. le and Table 3.3 describe the changes in thelight scattered horizontally, due to the presence of live grass neighbors. Thesemeasurements indicate a strong increase in FR and resulting decrease in the R:FR due toneighboring grasses. There was also a noticeable increase in PPFD in the horizontaldirection due to a canopy of live grass. Readings from the vertical-orientation of the66sensor (Fig. 3.1 a, Table 3.2) indicate that this led to an overall increase in PPFD of lessthan 2%. Horizontally-scattered radiation from bleached grass (Fig. 3. le, Table 3.3) washigher in R:FR, similar to controls using empty pots, and was higher in PPFD, similar tolive grass canopies. Global (horizontally-detected) PPFD, however, was approximately10% higher in treatments with bleached controls than treatments with live grass (Fig.3.ld), whereas in controls using empty pots, PPFD was <2% higher than in treatmentswith live grass (Fig. 3.la).Spectroradiometric measurements also were made under several other ambientconditions (e.g. different time of day, sky conditions, etc.). The results (data not shown)follow a similar pattern, i.e. a decrease in R:FR and a slight increase in PPFD wasdetected in the presence of live grass neighbors. Measurements were also made usingtwo other species of grass, Holcus lanatus and Lolium perenne (also used in Chapter 4),to see how the influence of Dactylis compared to other common neighbors of whiteclover. These indicate that while the influence of the grasses on the light conditions isqualitatively similar, Dactylis is a relatively strong FR-reflecting grass (data not shown).TemperatureExperiments conducted outdoors clearly experienced lower temperatures thanexperiments conducted inside the greenhouse (Tables 3.4, 3.5). Under all conditions, thetemperatures measured within the experimental compartments were elevated over theambient greenhouse or outside air temperature. While outdoors with a slight breeze (8-10kph) Plexiglas warmed the area occupied by the clover clones, it did so equally in bothtreatments. The increase over ambient temperature was more or less equal in bothtreatments under all conditions measured, except late in the afternoon inside thegreenhouse, where, after the influence of the sun’s direct beam no longer directly affectedthe compartments, the air in treatments having grass barriers was warmer (Table 3.5).67Under overcast conditions, both indoors and outdoors, no difference between treatmentswas detected.Clover growth and morphologyIn the five reflection experiments, there were few detectable differences betweenclover clones growing next to barriers of live grass and control clones in measurementsmade at the final harvest of each experiment (Table 3.6). Measurements differingsignificantly in one particular experiment generally did not show a significant differencein the other experiments. The ANOVA on these measurements using all fiveexperiments, reveals that mean primary leaf area was the only measurement that differedsignificantly between treatments over all experiments (Fig. 3.2). It appears that clonesnext to live grass had a larger mean primary leaf area than clones next to empty pots. Inthis ANOVA, also, there was a significant “experiment” effect, showing thatmeasurements differed between experiments, but there was no significant “treatment xexperiment” interaction, which was common in the other measurements.Despite this consistent lack of response for so many characters acrossexperiments, a few observations are worth noting. No differences between clonesdeveloped in the two experiments without Plexiglas. The experiment in which most ofthe significant differences developed was the one in which whole plants were oriented inthe same direction (growing east to west) for the entire experiment. In this experiment(Exp. 1), clover clones in front of live grass grew somewhat larger with longer stolons,more above-ground biomass, larger primary leaves, and longer internodes on theirsecondary branches. In the other experiment in which clones’ orientation remainedconstant (Exp. 5), the neighbor-treatment was given only to the apical region of theplants, and this region also produced longer stolons with more ramets and a reducedpercentage allocation of biomass to leaf lamina. In all five experiments, there was no68evidence of increased allocation specifically to petioles, nor of differences in specificlength of petioles and stolons, or the specific area of leaf lamina. None of the branchingmeasurements indicated any significant differences in branching between treatments inany of the experiments. Mean internode lengths did not differ consistently, and petiolemeasurements indicated no significant differences in length. Petiole growth rates in Exp.5, measured at 2-day intervals during the twelve days after appearing, also indicated nodifferences between treatments (data not shown). In addition, neither the mean AGR(analyzed by repeated-measures ANOVA) nor the mean RGR throughout the wholeexperiment (in stolon length, ramet number, or number of branches) differed betweentreatments, even in the experiments where differences in size developed in the experiment(data not shown).A summary of the variation in measurements made on primary leaf positionduring each experiment is shown in Table 3.7. In Exp. 1, w,here the clones’ orientationwas not rotated, the height at which leaves were displayed varied significantly betweenthe two sides of the primary stolon on which the leaf could have originated. Primarystolon leaves were displayed at a higher elevation if the leaf was toward the north,regardless of treatment. Leaves towards the grass neighbors were displayed at a higherelevation when the clones’ orientation was reversed, in Exp. 3 and 4, i.e. leaves thatoriginated and were situated on the side of the primary stolon towards the north, weredisplayed at a higher elevation above the soil than leaves on the side away from grasses(Fig. 3.3; Plate 2). This occurred at consecutive measurements, when a different side ofthe clone was toward the grass. In control clones, the leaves towards the south were oftenat least as high (or higher) than leaves toward the wall.In one of the experiments where the orientation of the clover clones was rotated(Exp. 4), the lateral distance of primary stolon leaf lamina from the stolon was reduced in69clones growing in front of grass barriers on both sides of the plant (Fig. 3.4). Thisindicates that these leaves on clover clones in front of grass were kept at a greaterdistance from the canopy, and that the leaves retained this posture when they were awayfrom the barrier. A similar effect on lateral placement of primary stolon leaves,especially if the leaves were currently towards grass, was noticeable but not statisticallysignificant in the other experiments where clover orientation was rotated.In Exp. 1, where clover orientation was not changed, all plants regardless oftreatment had several primary leaves cross the primary stolon from north to south andvery few cross from south to north (Yates’-corrected Chi-square=5.45, p<O.O5). In one ofthe experiments where clones were rotated, there was a greater number of leaves crossingthe primary stolon from north to south, regardless of treatment. Observations of leavescrossing the stolon over consecutive periods in one experiment, when different leavesoriginated towards the north, show that a leaf that was once crossing the stolon fromnorth to south, after rotation will often re-cross the stolon and reside on the other side ofthe stolon after it is rotated (usually within one day). Although the number and directionof leaves crossing the stolon seemed to be unaffected by the canopy of grass to the northin the first four experiments, primary stolon leaves on clones growing in front of livegrass in Exp. 5 crossed the stolon significantly more frequently from north to south thanon clones in front of bleached grass (Yates’-corrected Chi-square=4.7, p<O.O5). In thisexperiment, the orientation of the clover clones was unchanged throughout theexperiment.There was no evidence that the angle at which the primary stolon leaf lamina wasdisplayed was affected by its side of origin on a plant or by a grass canopy, in any of theexperiments.70The above data also support the visual evidence of the leaves and primary stolonrolling away from the north, and this happened more or less regardless of treatment andregardless of which side of the clone was toward the north, i.e., in experiments withclonal orientation reversed, the stolon rolled in the opposite direction after each rotation.In one of the experiments where the clones’ orientation was not reversed (Exp. 5), therewas a statistically significant deflection of the stolon apices towards the south, regardlessof treatment (i.e., the mean distance south from the middle of the flat was significantlydifferent from 0 and always positive; n=12, p<0.05, Sign test, Systat, 1990), and therewas no evidence that this differed between treatments. There was no asymmetry detectedin the secondary branch growth in these clones under this condition. In the experimentswhere the clones had their orientation reversed, there was no significant deflection of theprimary stolon apices from the middle of the flat in any of the treatments. In theseclones, the older portions of the primary stolon appeared to zig-zag down the flat,indicating that while stolon apices generally proceeded down the middle of the flat, therewas tendency to orient the stolon tip away from the northern barrier, regardless of itsdesign.3.4. DISCUSSIONThe microclimatic measurements indicate that the presence of grass neighbors tothe north under controlled conditions influenced the microclimate, including the lightconditions, in ways detectable to instruments, although differences between thesemeasurements were unreliable predictors of the response in nearby clover. Themeasurements of light conditions on the surface of an open flat (Figs. 3.la, d, e) arerepresentative of the conditions in the experimental chambers before considering theinfluence of the clover itself. These measurements also would be indicative of conditionspresent early in the experiment (when clover clones were small) as well as of conditionsabove the clover leaf canopy. Measurements made under the shade of a clover leaf (Fig.713. ib) are more indicative of the conditions experienced by a clover stolon or petiolebelow the cover of leaf lamina. In the shade of a leaf, the increase in FR due toneighboring grasses is perhaps more noticeable to the clover, since the resulting decreasein R:FR (Table 3.2) is in the range where there may be more sensitive phytochromephotomorphogenic responses (Smith and Holmes, 1977; Morgan, 1981). However, noeffect on petiole or internode extension was observed in these experiments.The vertical orientation of the spectroradiometer sensor enhances the contributionof horizontally-scattered radiation to the measured SED, and this would be morerepresentative of the light conditions received by vertical structures such as cloverpetioles (Ballare, 1987). With this sensor orientation, it was clear that there was anincrease in the horizontally-scattered FR due to live neighboring grasses. There was alsoan increase in the PPFD in front of neighbors when compared to “empty” controls. Inthis northern orientation, a grass canopy such as this may provide conflicting signals to aclover plant. On the one hand, it provides low R:FR light, while at the same timeprovides additional PPFD, even though the overall increase in global (sensor horizontal)PPFD was small (<2%) (Table 3.2).Temperatures taken at representative times can give only a partial description ofthe temperature regimes under the different treatments. Few differences betweentreatments were detected, so the effects of Plexiglas with respect to decreasing betweentreatment temperature variation in the region occupied by clover clones could not bedetermined. The difference detected late on a clear day (Table 3.5) suggest that there wasa difference in the way grass barriers retained and emitted heat after the sun went belowthe “horizon”. A Plexiglas barrier was not in place, and therefore its effect under theseconditions could not be tested. Under late-afternoon clear conditions, the northern andeastern edges of a gap might retain more heat (in the soil and surrounding air) under72calm conditions (Bazzaz and Wayne, 1994). There is also likely to be long-waveradiation emanating from the leaves of live grasses at this time of day (Smith, 1982).Increases in temperature have been reported to increase stolon extension rate, leaf size,and petiole length in white clover (Turkington and Burdon, 1983; Frame and Newbould,1986), and in these experiments the presence of inconsistent temperature regimes waslikely, perhaps causing the increased stolon extension observed in experiments 1 and 5.Humidity differences are likely to be reflected in these temperature measurements. In thepresence of grass, there will be a slight increase in water vapor pressure in the air near thegrass neighbors, especially under relatively calm conditions. The higher water-vaporpressure at similar temperatures (as detected at mid-day) would give the air in thesetreatments a smaller vapor pressure deficit (higher relative humidity), and this couldpromote growth in white clover when under low-humidity conditions, e.g. bright sun orinside the heated greenhouse (Frame and Newbould, 1986). There were times whenclover clones were not completely turgid, and after application of water to the floors andradiators in the greenhouse, clover clones would rapidly regain turgidity. A slightincrease in local humidity in treatments with neighboring grass could have reduced thetime spent in these water deficits, and hence could have increased clonal growth as seenin experiments 1 and 5. Another response to increased humidity which might have beenobserved in the clones was increased leaf size, however most responses to increasedhumidity are dependent on other factors, e.g. light, temperature, water status of roots(Grantz, 1990). The higher temperature reading found late in the day in front of livegrass also could have been caused by increased latent heat, a characteristic of more humidair.The measurements of clover clones themselves should indicate whetherconditions with neighbors to the north were different from conditions in controltreatments. Few consistent differences arose in the morphological characteristics of73clones measured in different experiments conducted at different times of the year, indifferent locations, with and without Plexiglas. Only one variable compared across allexperiments varied significantly due to a treatment effect, mean leaf area. This normallyis seen as a response to leaf shade (Smith, 1982) or to a reduction in light quantity(Morgan and Smith, 1981; Caradus and Chapman, 1991; Dong, 1993), while in this caseit could not have been caused by shade, but perhaps by another alteration to the lightconditions, e.g., R:FR (Solangaarachchi and Harper, 1987), or an effect of humidity. Thelack of response in petiole length to the substantial increase in horizontally-impingingFR, is surprising because it is known that petioles of white clover can respond quitesensitively to R:FR under uniform natural-canopy conditions (Solangaarachchi andHarper, 1987; Thompson, 1989; Thompson, 1993a). It seems that for this response inwhite clover, light with high PPFD and R:FR might override the effects of low PPFD andR:FR light. One effect of the northern grass barriers seemed to be an increase to clone’ssize in front of grass neighbors, which appeared in experiments where the clones’orientation was not reversed (Exp. 1, 5; Table 3.6). However, there was no detectableeffect on internode length or branching patterns in any of the experiments, indicating thatthere was little if any selective placement of ramets, i.e. no foraging was exhibited.Selective placement of ramets in white clover can be affected by physical displacement ofthe stolon in addition to changes in internodes and branching. The newest node or two oneach stolon does not root for several days after appearing, and the potential exists forlateral movement before these nodes root. Phototropic bending away from strong FRsources has been reported in shoots of orthotropic plants (Ballare et al., 1992), and thedirection in which plagiotropic shoots of Portulaca oleracea seedlings grow is affectedby the direction of a strong FR source (Novoplansky et al., 1990). In the presentexperiment, it appears that the primary stolon in white clover was affected in this way byneighbors, and in these clones this had a stronger effect on ramet placement than differentinternode extension or branching patterns74Foraging in white clover can also be considered with respect to placement ofresource-gathering organs, leaf lamina, as leaves grow, e.g., through differences in petioleelongation, and in the shorter term, through the proximal positioning of the leaf lamina atany given time. There was evidence that the positioning of leaf lamina could change overtime, even after the petioles have stopped elongating. Under the experimental conditions,leaves were allowed to adopt their own position. In experiments where the clone’sorientation was reversed, data on leaf position indicate that leaves can adopt a positionsuitable for brief periods of growth. Changes occurring at four-day intervals can be“tracked” by these leaves, and this phenomenon was affected by the presence of livegrasses to the north. In the field, clover leaves are often intermingled with neighboringgrasses (Haynes, 1980), not allowing them as much flexibility in positioning as seen here.These results suggest that in a gap, clover leaves are able to alter their position daily (andperhaps during the day) using phototropic responses to differences in FR.In clones having their orientation reversed every four days (Exp.2, 3, 4), thereseemed to be fewer differences between clones under the different treatments than in theexperiments where the clones’ orientation was not reversed (Exp. 1, 5). It appears thatlight reflected from neighbors that does not consistently originate in a particular directionmight cause little response in white clover. This suggests that there may be some side-dependent effects that develop when one side of the plant remains in closer proximity toneighbors. It has been suggested that in some plants this might be accomplished throughan integrated system able to compare conditions between interconnected portions and“choose” between them (Novoplansky, 1991). Solangaarachchi and Harper (1989) foundthat clover clones growing near neighboring clones produced greater growth on the sideaway from the neighbors than the corresponding side of an isolated clone. This wouldsuggest the possibility of asymmetric growth in experiments with unrotated clones. In75one of these two experiments (Exp. 5), no asymmetry was found, so even though clonesin front of grass neighbors produced more stolon length in this experiment, stolons onboth sides of the clone were larger. In the other experiment where clones were notrotated (Exp. 1), asymmetry was found, but the side of clones towards the barriers wasthe larger, regardless of treatment. The asymmetry in these clones could have been dueto the Plexiglas barriers. The presence of Plexiglas in these chambers increased thePPFD in full sun by more than 10% (Table 3.2), provided that the reflection of the sunwas cast onto the detector. During the time the experiment was conducted, ending justbefore the summer solstice, the reflection from the sun in the 30cm-tall Plexiglas sheetwould reach approximately 13 cm out onto the flat containing clover clones, orapproximately half-way. This would have created the conditions where branches to thenorth were under greater PPFD for most of the day. Reversing the orientation of theclones eliminates the possibility of an effect developing from the interaction between oneside receiving a particular condition and the barrier itself. For example, if the side thatalways remained near the neighbors displayed leaves higher, this could alter the way theclone responded, e.g., by reducing mutual shading in clover (shading of northern cloverleaves by southern ones would be reduced). In clones that were rotated, the number ofsignificantly different morphological characteristics dropped to almost nil. The effects onprimary stolon lateral displacement were similar, this being eliminated when theorientation of the plants was periodically reversed. In clones that were not rotated, andperhaps clover in the field with uni-directional reflection from neighbors, the combinationof greater stolon extension along with lateral movement of stolon apices may increase thespeed with which a plant diminishes the effects of neighboring plants. Perhaps in clonesthat were reversed, placement of ramets was unaffected, since there was no directionalityto the signal of neighbors and hence, no clear signal of where they could best be avoided.Finally, if clones under different treatments in experiments without Plexiglasbarriers (Exps. 3, 4, 5) had more obvious differences than experiments with Plexiglas76(Exps. 1, 2), the suggestion might have arisen that factors other than light wereinfluencing clover clones in front of neighboring grasses. Experiments without Plexiglasshowed few detectable differences between clones (Table 3.6), while more differenceswere detected in experiments with Plexiglas, so no such suggestion about the effects ofgrass neighbors can arise from these data.In conclusion, under this canopy configuration, possible interactions through theradiation environment were confined largely to reflection off the neighboring grasses, andit appears that this potential signal of neighboring grasses, whether directional or not,initiates little change in the growth and morphology of clover clones. Other than meanprimary leaf lamina area (n=5 leaves/plant), it appears that few morphological characterswere affected by the presence of neighboring grasses. It appears that the grass neighborswere detected by the clover clones however, because leaf and stolon apex positions werealtered by neighboring grasses. The movement of these structures, presumablyphototropic, placed them in positions where the effects of neighbors was reduced, andthis could have been involved in the lack of further morphological response in the clones.Initially, in the presence of neighbors, behavioral responses such as these mayalleviate the need for morphological changes. Perhaps, if these initial responses areinadequate, then morphological modifications might be expected. Variable sensitivity ineither of these responses to neighbors could regulate an individual’s response toheterogeneous conditions, and a wide range of these may be present in a population.77Table 3.1. Summary of five reflection experiments conducted at different times in 1990and 1992.Neighbors used Plexiglas Location TimeExp. 1 Dacrylis glomerata yes indoor Spring, 1990empty potsExp. 2 Dactylis glomerata yes outdoor Autumn, 1990empty potsExp. 3 Dacrylis glomerata no indoor Summer, 1990empty potsExp. 4 Dactylis glomerata no outdoor Summer, 1990empty potsExp. 5 Dactylis glomerata no indoor Spring, 1992bleached grass78Plate 1. Layout of reflection experiments: (top) Experiment 2 near the beginning of theexperiment; (bottom) Experiment 4 near the final harvest.77:‘I(- ____-IrJdjA, -rI 1 V I -1(e)Aii—’.—I “1:1,.. I, • II •jlj A1600 700 800 900 1000 110Wavelength (rim)Fig. 3.1. Spectroradiometric measurements made using a LI-1800, portablespectroradiometer (LICOR, Lincoln, Nebraska) under simulations of the experimentsnear solar noon. Each measurement was the average of three consecutive scans. (a) theeffect of a live reflecting barrier of Dactylis glomerata measured on an open flat, (b)same as (a) but measured beneath a clover leaf, (c) the effect of Plexiglas in a controltreatment, (d) the effect of live reflecting barrier of Dactylis go?nerata compared to acontrol using bleached grass, and (e) sensor vertical to detect horizontally-impinginglight.‘S500 600 70070-- (a)-&fl- 4-r\A/.I II3_fl-2.0—-1.0--0 i4(A) 5(10 (.00 7007_li - - (c):: --y::: ‘1.1)-0. 0 I,‘0 5130 600 70(1Grass presentControlGrass presentControl-Bleached grassGrass presentControl3.0—a,a, 2.0--a,zC—C1.0—0.0-400without Plexiglaswith Plexiglas3.0—2.5-2.0-1.5-1.0-0.5-0.0--Control-Empty potsGrass presentControl-Bleached grass(d)300 400 500 600 700 800 900 1000 110Wavelength (rim)1.8-1.8-1.4—1.2—1.0-0.8-0.6—0.4-0.2-300 400 50081Table 3.2. R:FR (quantum ratio 655-665:725-735) and PPFD (i.tmolm2s-’) derived fromspectroradiometric scans (using LI-1800, LICOR, Lincoln, Nebraska) made at solar noonon a clear day. Scans were made (i) on the surface of an open flat (containing pottingmedium but no clover) with Plexiglas, (ii) on the surface of an open flat withoutPlexiglas, and (iii) on the surface of a flat in the shade of a clover leaf without Plexiglas.Each measurement was the average of three consecutive scans. (Uncertainties:R:FR<0.01, PPFD<5)Canopy type ConditionsOpen, no Plexiglas Open, Plexiglas Under clover leaf,no PlexiglasR:FRControl - empty pots 1.20 1.20 0.55Dactylis glomerata 1.12 1.14 0.46PPFDControl - empty pots 1542 1723 280Dactylisgiomerata 1561 1739 273Table 3.3. R:FR (quantum ratio 655-665:725-735) and PPFD (!i.molm2s’)derived fromspectroradiometric scans (using LI- 1800, LICOR Lincoln, Nebraska) with the sensorvertical, facing the canopy to the north, to detect horizontally-scattered radiation.Canopy typeR:FR PPFDDactylis glomerata 0.42 106Control - bleached grass 0.94 140Control - empty pots 1.19 5482Table 3.4. Mean (±SE) of temperature measurements (°C) made outdoors in reflectionexperiments with and without Plexiglas (Wind speed 8-10 kph, clear sky, air temp. 13.5OC). Each individual compartment was measured three times and averaged beforedetermining the mean for all treatments of that type. Shown below are the significancelevels for the ANOVA effects of canopy type, Plexiglas, and their interaction. nsnotsignificant at p<O.05.Canopy type with Plexiglas without PlexiglasDactylis glomerata 18.1 (0.3) 16.0 (0.5)Control 17.6 (0.4) 16.2 (0.4)ANOVA results:Canopy type nsPlexiglas p<0.5Interaction nsTable 3.5. Mean (±SE) of temperature (OC) measurements made under differentconditions in the greenhouse. Measurements were made on a clear day around noon(greenhouse temperature 26.0°C) or late in the afternoon, after the sun no longer directlyaffected the plants (greenhouse temperature 23.0°C). Shown below the column headingis the significance of the difference between means of each measurement made under thetwo treatments. ns=not significant at p<O.05.Noon Late PMCanopy type Soil surface Air Soil surface Air(ns) (ns) (ns) (p<O.05)Dactylis 32.0 (0.7) 28.5 (0.5) 23.4 (0.4) 22.5 (0.3)glomerataControl 32.9 (1.3) 28.2 (0.4) 22.6 (0.4) 20.3 (0.3)83Table 3.6. Summary of differences between morphological measurements of cloverclones made in five reflection experiments. If significant, the relative size of themeasurement between treatments is shown. C=Control, D=Dactylis glomerata, * p<005,** p<O.Ol, ns = not significant.Exp. 1 Exp. 2 Exp. 3 Exp.4 Exp. 5Indoor, Outdoor, Indoor, Outdoor, Indoor,with with without without Control isPlexiglas Plexiglas Plexiglas Plexiglas bleachedgrassC (n=7) C (n=5)Variable (n=5) D (n=6) (n=7) (n=7) D (n=6)Total stolon length * (D>C) ns ns ns * (D>C)Total number of ramets ns ns ns ns * (D>C)Total number of branches ns ns ns ns nsTotal above-ground dry weight * (D>C) ns ns ns nsMean weight/ramet ns ns ns ns nsPetiole weight ratio ns ns ns ns nsStolon weight. ratio ns ns ns ns nsLeaf lamina weight. ratio ns ns ns ns * (D<C)Ratio: petiole/ stolon weight ns ns ns ns nsMean primary petiole length ns ns ns ns nsMean primary leaf lamina area * (D>C) ns ns ns nsMean primary stolon internode ns ns ns ns nslengthRatio: primary petiole/ stolon ns ns ns ns nslengthSecondary stolons- internode ** (D>C) ns ns ns nslengthTertiary stolons- internode length ns * (C>D) ns ns nsWhole plant- internode length ns ns ns ns ns84Table 3.6. cont.Variable Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5Primary stolon- specific leaf area ns ns ns ns 115Primary stolon-- specific petiole ns ns ns ns nslengthPrimary stolon--specific stolon ns ns ns ns nslengthSecondary stolons-ratio: new ns * (D>C) ns ns flSpetiole/ new internodePrimary stolon-% of nodes ns ns ns ns nsbranchingSecondary stolons-% of nodes ns ns ns ns nsbranchingWhole plant-% of nodes branching ns ns ns ns nsPrimary stolon- age to first branch ns ns ns ns flSSecondary stolons- age to first ns ns ns ns nsbranchBranch weight ratio ns ns ns ns nsBranch stolon length ratio ns ns ns ns nsBranch ramet number ratio ns ns ns ns nsMean RGR-total stolon length ns ns ns ns nsMean RGR-total ramet number ns ns ns ns nsMean RGR-total number of ns ns ns ns nsbranches85• ControlDactylisFig. 3.2. Mean (±1 SE) of five primary leaves per white clover plant under the twotreatments (reflecting bathers of live Dactylis glomerata (Dactylis) or control pots(Control)) for the five reflection experiments. Shown below is a summary of theANOVA of mean primary leaf area by treatment (Neighbor) and experiment.Source of variation Sum of Squares Degrees of freedom ProbabilityExperiment 138.0 4 0.000Neighbor 2.3 1 0.039Exp. X Neighbor 1.0 4 0.750error 26.3 52Mean primaryleaf area (cmxcm) 2 3 4 5Experiment #86Table 3.7. Summary of ANOVAs of white clover primary leaf position measurements made onboth sides of the primary stolon during reflection experiments. Treatments had neighbors ofeither live Dactylis glomerata (D) or controls (C). The significance of the various effects(T=treatment, S=side of plant, i.e. towards grass or away from grass) is shown: *= p<O.O5,ns=not significant.Primary leaf Bxp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5positionmeasurementHeight of leaf T ns ns ns ns nsS * ns ns ns nsTxS ns ns * *Lateral distance T ns 115 fl5 * nsfrom stolon S ns ns ns ns ns(excluding leaves TxS ns ns ns ns nsthat crossed stolon)Number of leaves T ns ns ns ns *crossing stolon 5 * ns * fl5 *(using Yates- TxS ns ns ns ns nscorrected Chisquare)Lamina angle T ns ns ns ns nsS ns ns ns ns nsTxS ns ns ns ns ns87Fig. 3.3. Mean (1± SE) height of white clover primary leaves (Exp. 3) on two sides of theprimary stolon, one towards the north (Towards), the other towards the south (Away).ANOVA results; “treatment” ns; “side” ns; “treatment x side” p.<0.05. (Dactylis Towardsn=6, Away n=4; Control Towards n=6, Away n=7 leaves)Lateral distancefrom stolon (cm)• TowardsD AwayFig. 3.4. Mean (±1 SE) lateral distance of white clover primary leaves (Exp. 4) on twosides of the primary stolon, one towards the reflecting wall (north), the other away fromthe wall (south). ANOVA results: “treatment”, p<O.05; “side”, ns; “treatment x side”, ns.(Dactylis Towards n=7 Away n=7; Control Towards n=7, Away n=7 leaves)Height of primaryleaves (cm)5.0 -4.0 -3.0 -2.0 -1.0 -0.0 -• TowardsD AwayDactylis ControlDactylis Control88Plate 2. Side-view of clover (from east looking west) after the last four days spent withcontrol pots (top) or live grasses (bottom) to the north (i.e., immediately to the right).Photographs were taken near noon on an overcast day.90Chapter FourTHE EFFECTS OF PARTIAL SHADE FROM THREE SPECIES OF GRASSNEIGHBORS ON THE GROWTH AND MORPHOLOGY OF WifiTE CLOVERCLONES4.1. INTRODUCTIONInteractions between a plant and its neighbors can occur through severalphysiological systems simultaneously. Competition from neighbors is, by definition,ascribed to limiting resources either above- or below-ground. This experiment wasdesigned to allow interaction only above-ground and to assess the effects of interferencefrom neighboring grasses on the growth and form of a white clover clone.This experiment mimics a field situation where an individual clone or seedling ofwhite clover experiences a small gap in the canopy. As a clover clone grows through agrass sward, it encounters a range of biotic and abiotic microenvironments. Thesepatches of microhabitat are often imposed by neighboring grasses or by gaps within thesward. Such gaps can be created in many ways, e.g. patchy grass-growth, hoof-marks,non-uniform grazing, and deposition of dung pats (Parish, 1987). At different times ofthe day, different light conditions are experienced by plants in these gaps, depending onthe location within the gap, the height of the neighbors, and the solar angle. In mostsituations when a clover plant is in a gap, the clover would have extended into the openarea from a neighboring region having different conditions, and this is likely to affect itsgrowth response in its new gap environment (Pitelka and Ashmun, 1985; Hutchings andde Kroon, 1994; and see Chapter Five). The experiment described in this Chapterexamined the effects of shade from grass on clones of clover that were located entirelywithin gaps.91Attempts have been made to duplicate sward light conditions under controlledconditions in the greenhouse. For example, in studies by Solangaarachchi and Harper(1987) and Thompson and Harper (1988), target plants were arranged in such a way as toprevent root competition from neighbors. These target plants were shaded by canopieswhich were formed by healthy green leaves of neighbors that were either detached andfloating in a transparent water tray above them (Solangaarachchi and Harper, 1987), orattached and held in position by wire frames (Thompson and Harper, 1988). A uniquefeature of the conditions imposed by these canopies was that, while preventing below-ground interaction, they mimicked shade cast in the field, having the precise spectralqualities of natural conditions because it was being made by competitors’ leaves andcontained many small sunflecks. This shade was presented uniformly, along with tinysunflecks, obstructing the entire patch of available light above experimental clover plants.This shading pattern creates conditions which are more likely to be experienced by anunderstory plant in a dense woodland than a white clover plant in a pasture. The fieldconditions experienced by clover are commonly far more patchy. In the presentexperiment, shade from different species is presented in a more natural way, with cloverclones continuing to receive large patches of unfiltered light.Gaps in a pasture will provide light with higher R:FR and PPFD than the lightfiltered through neighbors (Holmes and Smith, 1977a,b; McCartney and Unsworth,1975). Under such a canopy, the overall R:FR would depend on the size of the availablepatch. In laboratory experiments, it has been shown that changes in the overall R:FRthrough the addition of incandescent light from individual bulbs can cause changes inplant response (Morgan and Smith, 1981). It is unknown how the reception of a distinctpatch of higher R:FR and PPFD light modulates the response to patches of low R:FR andPPFD light. On sunny days, even within a tall sward, there are likely to be periods whendirect sun is available for a duration longer than a large sunfleck (Parish, 1987). It isknown that short periods of direct light can have profound morphological effects on92otherwise shaded plants under experimental conditions (Morgan and Smith, 1978; Pearcyet al., 1994), although it is unknown how this would modulate the response of clover toneighboring grasses. The present experiment was designed to present clover clones withshade from neighbors for only part of the day, to determine the effects of shade whensome direct (unfiltered) light (much bigger than sunflecks) is also received by cloverclones.In this study the different treatments were imposed by canopies of three differentspecies of grass neighbors arranged in a standard design (see Fig. 2.2; Plate 3). Thisdesign created a gap among the grasses which presented all clover clones with equalexposure to direct light. The clones were placed in the same orientation within the gap.The design allowed different clover clones to receive shade from different species ofneighbors for the same part of each day, while all clones continued to receive relativelyhigh levels of PPFD. Thus the experiment investigates the effects of shading by differentgrass neighbors on the growth of white clover clones under conditions more typical of thefield, primarily by presenting the various light stimuli in patches and allowing clones toreceive PPFD typical of the level experienced by clover in the field. The clones underdifferent grass canopies were compared to each other and to clones growing under nograss canopies, which served as controls for this experiment.4.2. METHODSPreparation of plant materialClover clones and pots containing Dactylis, Holcus, and Lolium were prepared forthe experiment as above (see Chapter 2). For this experiment, the grasses were clipped to15 cm above the pot every four days. Due to natural variation in the density of leaves andtillers and in the architecture inherent in a canopy of each species, the precise nature ofthe shade cast by different grasses was expected to differ. The light transmitted and93reflected by these canopies was considered to be representative of the field, wheredifferent species of grass form clumps of varying density, SED, and competitiveness towhite clover.Arrangement of canopy modulesEach treatment in this experiment consisted of two rows of canopy modules withtwo flats containing clover clones placed in the same orientation (north-south) betweenthe rows (see Chapter 2.2.2, Fig. 2.2, Plate 3). Each aisle of standard width (15 cm widex 17 cm tall) created a gap that allowed a “corridor” of sky running north to south,directly overhead, to never be obstructed. The amount of potential direct sunshineavailable to clover clones can be calculated from these dimensions and astronomicalformulae for the position of the sun at given times on given days. This is shown in Fig.4.1, and this was used to calculate the daily PPFD and average R:FR in the differenttreatments.Treatment of plant materialSee Chapter for treatment of white clover clones, and Chapter fortreatment of grass canopy modules.Data collection and analysisClover clones were measured as described in Chapter 2.4. Temperature andspectroradiometric measurements were also conducted as described in Chapter 2.6, withthe following exception. Because the sun moved behind the walls of grass, casting adifferent pattern of shade and sunflecks, three such sets of scans were made at slightlydifferent times during this period, in order to produce a better representation of theconditions. Analysis of variance between species could then be conducted between thesemeasurements of PPFD and R:FR. In this experiment also, the daily PPFD and mean94daily R:FR could be calculated as a weighted sum or average, respectively, using the timespent at each level (from Fig. 4.1) as the weighting factor. In addition to thesemeasurements, the density of grass tillers in pots of different species forming the canopieswas determined shortly after the experiment. A transect was placed along a row of potsof each type at a standard height within the canopy of grass (2 cm below the clippingline). The number of different tillers touched by the transect was recorded, and this linearmeasurement was converted to the number of tillers per square centimeter. Also, onceduring the experiment, the height of regrowth in the four days between clippings wasrecorded for the three species of grass used to form the canopies. Data analysis for thisexperiment are described in Chapter RESULTS4.3.1. MICROCLIMATIC MEASUREMENTSSpectral Energy DistributionLight measurements taken under simulations of the experiment indicate that therewere large differences in the quantity and quality of light under the various types ofcanopy (Fig. 4.2). Data from these spectroradiometric scans indicate that all treatmentsreceived the highest PPFD and R:FR during the middle part of the day (Fig. 4.3, Table4.1). At times when sunlight was filtered by grasses, which occurred equally in themorning and afternoon, the PPFD under the grass canopies fell to less than 14% of fullsun (see “Morning, Afternoon” in Table 4.1). At the same time under grass canopies, theR:FR fell below 0.50, while the R:FR under control canopies remained above 1.1. Atmid-day the PPFD and R:FR were slightly reduced by grass canopies (see “Morning,Afternoon” in Table 4.1). These effects were due to the increase in “blue” radiation incontrols (more blue sky exposed) and the increase in radiation above 700 nm from95reflection in treatments with grass (Fig. 4.2c,d). With the sun less than 100 from thehorizon, and blocked .by the wall of the experimental compartment, (see “Dawn, Dusk” inTable 4.1), grass canopies also reduced the PPFD and R:FR, although overall PPFDlevels were quite small (<1% of full), and the time spent under this condition was limited(less than 2 hrs/day). At the canopy dimensions chosen, direct sunlight was available forapproximately 4 hours during the middle of the day (at the ending date of the experiment)in treatments with grass canopies, while controls had 7 hours and 20 minutes of possibledirect sunshine (Fig. 4.1). Using this and the above observations of PPFD, the cloverclones under grass canopies received approximately 70% of the full total daily PPFDreceived by controls (Table 4.2).Although the effects of the different grass canopies on PPFD and R:FR werequalitatively similar, these observations indicate small differences among the grassspecies. In the mornings and afternoons when the sun was obstructed by either the east orwest wall of the grass aisle respectively, Dactylis lowered the PPFD significantly morethan canopies of Lolium but not more than canopies of Holcus (Table 4.1, Fig. 4.2c,d).Dactylis canopies also reduced the R:FR significantly more than canopies of both Holcusand Lolium at these times, and it appears that this was due to both an increase in PFD atwavelengths above 700 nm and a decrease below 700 nm (Fig. 4.2d). At times when thesun was within 100 of the horizon (see “Dawn, Dusk” in Table 4.1), Holcus canopiesreduced the PPFD and R:FR more than canopies of the other two species, while Loliumcanopies reduced the PPFD more than Dacrylis canopies.TemperatureTemperature measurements made under various conditions indicate no differencesbetween treatments, except in the afternoon on sunny days (Table 4.3). Control clonesexperienced temperatures 1 20 C higher than clones under grass canopies at these times(and perhaps in the morning when sun was blocked). These were times when direct96sunlight would reach the space occupied by control clover clones, while shade was castthrough grass canopies. The weather during the experiment was frequently sunny, socontrols would have experienced elevated temperatures on several occasions. Therelative humidity was likely to have been higher on these occasions under grass canopiesdue to the lower temperatures and proximity of the grass. At mid-day in the sun, thehumidity could also have been higher, since the temperatures experienced at this timewere quite similar. The same is true of overcast conditions, when the temperaturesobserved under different canopies were not significantly different, however there was stilllikely to be an elevation of water-vapor content due to the proximity of live grass.4.3.2. GRASS CANOPY MEASUREMENTSCanopies of Lolium had a lower tiller density than the other two grass canopies(Table 4.4). The density of leaves would have been 2-3 times higher than the density of.tillers, as each species had approximately 2-3 leaves per tiller (data not shown). Since thewidth of leaves of these three species were quite different (approximately 4.0, 3.0,2.Omm:Dactylis, Holcus, Lolium respectively), the leaf area clearly differed, withDactylis having the most, followed by Holcus and then Lolium. Leaf growth in the fourdays between clippings, was the greatest in Dactylis, then Holcus and Lolium (Table 4.4).This observation again indicates that Dactylis glomerata canopies had the greatest leafarea followed by Holcus lanatus and then Lolium perenne.4.3.3. CLOVER MEASUREMENTSYieldAll but one of the 32 clones (four treatments X eight replicates=32 plants) grewfor the duration of this experiment without flowering. The one that flowered was acontrol clone, growing in the open, and it was excluded from any further data analysis.97The remainder of the plants were vigorous and grew with their primary stolonapproximately down the center of the flat.By the end of the experiment, clones growing in control treatments (n=7) hadproduced a mean (±1 SE) of 48.7 (±1.6) cm of stolon, 35.0 (±1.0) ramets, 9.9 (±0.6)branches, and a biomass (when dried at 60° C for 96 hours) of 1.85 (±0.09) g. Each ofthese determinants of yield was substantially reduced by partial shading from all threespecies of grass (p<O.Ol; Figs. 4.4a, b, c, d).Differences in these yield variables among grass treatments were not as obviousas the differences between grass and control treatments. The Tukey multiple pair-wisecomparisons indicate that, while there was no evidence of different amounts of reductionin the total stolon length and dry weight between different grass species, the differentgrass canopies caused different amounts of reduction in the number of ramets andbranches produced by clover clones. Canopies of Holcus reduced the number of ramets(Fig. 4.4b) significantly more than canopies of Lolium, and canopies of Dactylis andHolcus reduced the number of branches significantly more than canopies of Lolium.There was no evidence of significant differences in any of the yield measurements madeon clones under canopies of Holcus and Dactylis. However, the effects under canopiesformed by Dact-ylis were always intermediate between the other two grasses. Althoughthe effects of different grass canopies on clover stolon length and biomass were notsignificantly different, these values suggest the same trend, with canopies of Holcushaving the largest effect, while those of Lolium had the smallest effect (Figs. 4.4 a,c).GrowthEvery clone, regardless of canopy treatment, developed a highly symmetricalbranching pattern with branches growing from successive nodes. Measurements made98over time indicate a regular and predictable increase in the total length of stolon, thenumber of ramets, and the number of branches (Fig. 4.5).The total number of ramets and total stolon length increased more or lessexponentially in all treatments (Figs. 4.5a, b). In both measurements, the three grasscanopies induced a significantly slower mean relative growth rate (i.e. the change innatural logarithm of the measurement per day, RGR) than control canopies, but underdifferent grass canopies these were not significantly different. While there is a strongindication of an exponential increase in the number of branches on control clones (Fig.4.5c;R2=.994), the number of branches on clones under grass canopies increased muchmore linearly (R2=.995, .999, .996; Dactylis, Holcus, Lolium). The increase in theabsolute rate of appearance of branches (as is necessary for exponential increase), wasseen somewhat in clones under control canopies before Day 25, when branches fromsecondary stolons first appeared. This early exponential increase in control clones musthave been due to an increasing rate of appearance of new branches from the primarystolon. In the clover clones under grass canopies, secondary stolons rarely branched (seebelow), so it was only the primary stolon that produced branches. In these clones the rateof branch production (entirely by the primary stolon) was more or less constant,producing one new branch approximately every 3.6 days.Results of the measurements made over a two-day period during the experiment,where the primary petiole and internode on the expanding ramet of primary stolons weremonitored, are shown in Table 4.5. While there was no indication of different extensionrates for petioles in these clones, control clones and clones under Lolium had more rapidinternode extension than clones under Dactylis and Holcus canopies. On clones underHolcus and Dactylis over the two-day period, the expanding petiole produced 3 to 4 timesas much length as the expanding internode, while in control clones and clones under99Lolium, the expanding petiole produced less than twice as much length as the internode inthe same period.The above measurements of clover growth support the observations of final yield,which suggest that while all of the grass canopies restricted growth relative to controls,clones under Lolium were inhibited least by the canopy. Short-term evaluation of therates of primary internode and petiole extension also indicate that clones grown undercanopies of Lolium more closely resembled control clones with rapid internode and slowpetiole extension, than clones grown under Holcus or Dactylis. Similar to final harvestmeasurements, the effects of Daclylis and Holcus canopies were not distinguishable.Morphological measurementsThere are significant differences in several measurements describing biomasspartitioning and organ dimensions (Tables 4.6, 4.7 and Fig 4.6). The most consistentdifferences are between clones under grass canopies and clones under control canopies,although there are some differences between clones under the canopies of the differentgrass species.Under all canopies approximately half of the clones’ biomass was allocated to leaflamina (Fig. 4.6b; Table 4.6), although this was reduced under canopies of grass. Oncontrol clones the remaining half of the biomass was shared approximately equally bypetioles and stolons, while on clones under grass, petioles retained approximately 60% ofthe remaining biomass, and stolons, 40%. Clones under Lolium had a significantlygreater proportion of their remaining biomass in stolons than clones under the other twograsses, and a smaller percentage of biomass in petioles than clones under Dactylis. Theratio of petiole to stolon weight in clones under Lolium was significantly lower thanclones under the other two grasses (Table 4.7). These large differences in biomass100partitioning between aerial structures were achieved at a relatively constant meanweight/ramet in clones under the different canopies (Table 4.7).Significant differences in internode length were observed only on the secondarystolons (first-order branches) of different clones, where controls developed longerinternodes than clones under all of the grass canopies (Table 4.7). Although the meaninternode length on secondary stolons were shorter on clones under grass, the meanpetiole length for the whole clone (n=4; Table 4.7), and the ratio of petiole to internodelength were significantly higher on clones under grass canopies than on control clones(Table 4.7), as was the case when considering only primary stolon structures (data notshown).The specific length (or specific area) of the three aerial primary ramet structures isshown in (Table 4.6). All clones under grass canopies produced more primary leaf areaper unit weight than control clones, while clones under different species of grass did notdiffer. Primary stolons had longer specific length under Dactylis and Holcus than undercontrols, and primary stolons under Dactylis were longer on the same weight as underLolium. While there were clear differences in mean petiole length due to partial shade,there was no indication of significant differences in the length per unit weight of petioles.Grass canopies caused an increase in biomass allocation to petioles and a decreaseto stolons (Fig. 4.6). The functional size of stolons (length) seems to have beenmaintained through increases in the specific length. The measurements of biomasspartitioning and organ dimensions also suggest the same trend as yield and growthmeasurements, with the morphology of clones under Lolium canopies intermediatebetween control clones and clones under the other two grasses.101BranchingTable 4.8 contains several measurements of clover clonal branching. The numberof new branches, or apices, produced was substantially decreased by grass canopies,while branch production by clones under Lolium canopies was intermediate betweenclones under control canopies and clones under the other two grasses. The primarystolons on all clones regardless of treatment produced roughly the same proportion ofnodes with branches, and the age of the node having most recently branched on thisstolon did not differ between treatments. Secondary stolons, on the other hand, hadproduced branches by the end of the experiment on control clones, whereas secondarystolons on clones under grass canopies scarcely branched. This led to a greater overallbranching percentage on control clones over all others but to no differences in branchingpercentage between clones under different grass canopies.Branching in white clover can also be described by determining the proportion ofthe whole plant (in terms of dry weight, stolon length, number of ramets) found in branchstructures as opposed to on the primary stolon (branch weight, length, and ramet numberratios; Table 4.8). The higher this value, the weaker the suppression of branches by theprimary stolon. On control clones branching described in this manner was increased,with a greater percentage of each plant’s total stolon length, ramet number, and biomassaccounted for by secondary branches. Again, Lolium canopies produced clones that wereintermediate between control clones and clones under Holcus or Dactylis canopies.Since branch growth was followed over time, the strength of the suppression ofsecondary length and ramet production could be determined at specific times during theexperiment (Table 4.9). The proportion of each plant’s growth allocated to the primarystolon declined throughout the experiment. There was greater suppression of branchlength growth in clones under grass than in controls, and the level of suppression inclones under Lolium was intermediate between control clones and clones under Dactylis102or Holcus. Even though suppression of branch length growth occurs under grasscanopies, there was no clear indication of suppression in branch ramet production undergrass canopies.Leaf PositionMany leaves that originated on one side of a stolon were displayed in their finalposition on the other side of the stolon (Table 4.10). In control clones fewer primaryleaves crossed the primary stolon than in clones under grass canopies. The height atwhich primary leaves were displayed during the experiment was also greater on clonesunder grass canopies than in controls (Table 4.10). Even though the mean primarypetiole lengths were greater under grass canopies, the primary leaves on these cloneswere displayed at a steeper angle from their point of origin on the stolon, at a height thatapproached the height of the grass neighbors.SummaryClones under the different canopies had visually distinct forms, ranging from themost branched with many ramets and thick leaves in controls, to fewer branches andfewer ramets in Lolium treatments, to very few branches, few ramets, and a largeallocation to petioles in clones under Dactylis and Holcus. Table 4.11 shows a summaryof the analyses of variance of the effects of the three grass canopies on several plantmeasurements. This indicates the measurements in which clones were the most distinct,and also indicates which clones differed among the groups under the three grass canopies.Measurements concerned with branching, petiole vs. stolon production, and clonal size interms of ramet number and dry weight, differed the most between clones under canopiesformed by different species of grass.In nearly all measurements, clones grown under canopies of Lolium wereintermediate between clones shaded by Dactylis or Holcus and control clones. While103clones under Lolium were occasionally different from clones under Dactylis, they weremore consistently different from clones under Holcus, and in none of these measurementswere clones under canopies of Holcus different from clones under canopies of Daclylis.4.4. DISCUSSIONThe effects on morphology observed in clover clones under partial shade in thisexperiment (reduced yield, increased petiole length, decreased branching) are generallyconsistent with the effects of complete shade at a wide range of daily PPFD(Solangaarachchi and Harper, 1987; Thompson and Harper, 1988; Thompson, 1993).Therefore, the effects of spending part of the day in full sunlight does not entirelyalleviate the influence of shade received at other times during the day. This supportswhat is known about white clover’s demands for light (Haynes, 1980; Frame andNewbould, 1986) and suggests that small portions of the day with shade have an effect onmorphology, illustrating the strength of the “shade avoidance” responses (Grime, 1981).The species of grass neighbor forming the small gap in this experiment also had avariable influence on the growth of clover clones within, suggesting that clover clones inthe field may be able to adjust their phenotypes to the neighboring vegetation underpartial-shade conditions and in the absence of below-ground interaction. The responsesto neighboring grasses in the field are likely to involve responses to similar alterations inthe light conditions, i.e. to shade interrupted by periods of direct sun, rather than toconsistent day-long shading.The selective placement of ramets (foraging) in clones under natural partial shadein this experiment involved changes in ramet production, branching, and partitioning ofbiomass, rather than through modification of internode extension. Differences were alsoevident in resource-gathering structures, leaf lamina and petioles, suggesting that bothtypes of modifications are adopted by white clover in response to partial shade from theirneighbors. Clones beneath Lolium canopies were nearly always intermediate in104phenotype between control clones and clones under the other two grass canopies, whichsuggests that many of the same changes in morphology can occur in response torelatively small changes in conditions due to different species of neighbors.Clones beneath all grass canopies produced internodes of similar length to controlclones, however they were lighter per unit length (higher specific length), and in thisregard stolon extension was increased in shaded clones. Leaf lamina size and specificarea were also increased beneath canopies of grass. Petioles, on the other hand, werelonger with proportionately more biomass (equal specific length), and they accounted fora greater proportion of total dry weight in clones under partial shade. Stolons and leaflamina in these clones accounted for a lower percentage of total biomass, suggesting thatwhile extension of stolons, larnina, and petiole were maximized, petiole extension mayhave had the highest priority in these clones. Among the different grass treatments,although there was a visual indication of longer petioles when observing clones grownunder Holcus and Dactylis next to clones grown under Lolium, this was not statisticallysignificant. The proportion of biomass that went into petioles, and the ratio of petiole tostolon biomass, however, was greater under Dactylis and Holcus, indicating more intensedemands of this type under canopies of Dactylis and Holcus than Lolium.Branching in clones under grass was greatly limited, while the primary stolonshad similar length and ramet number as in control clones (data not shown). Theproportion of the plant accounted for by branches (Table 4.8), and the observations ofgrowth of branches (compared to the primary stolon, in Table 4.9), describes the amountof branch inhibition caused by the primary axis, and under grass canopies a greater levelof suppression led to more linear extension. While the restriction of length growth toprimary stolons was greater in clones under grass at all three intervals, measurements oframet number show no such suppression in branch ramet number production. Foragingin this regard does not appear to be conducted through modulation in the rate of105appearance of branch ramets, but rather through the suppression of growth in length ofbranch internodes. In support of this, secondary internodes were found to be shorter inclones under grass (Table 4.7). Among grass canopies, this type of foraging difference ismost clearly seen between clones under Holcus and clones under Lolium canopies.Canopy modules of Lolium developed a lower tiller density than the other twograsses, even though grown under the same fertilizer and spacing conditions. However,the observed effects on temperature and light conditions beneath canopies of Lolium andHolcus were nearly indistinguishable. The effects of Daclylis and Holcus canopies on theclones growing beneath them were very similar (Holcus seemed to have a strongereffect), yet for much of the afternoon, Dactylis lowered the R:FR significantly more thancanopies of Holcus. The effect of these reductions in R:FR beneath Dactylis could havebeen alleviated or offset by conditions at some other part of the day, or perhaps by someother (unmeasured) feature of the canopies. The conditions observed at Dawn, Dusk(Table 4.1), indicate that Holcus had the largest effect on PPFD and R:FR at this time,and this is consistent with the observed effects of Holcus canopies on clonal morphology.Light conditions at the end of the day, especially R:FR, can have large effects on plantmorphology (Kasperbauer, 1971; Smith, 1982; Casal and Smith, 1989b), and this mayaccount for the strong response beneath Holcus canopies.In Thompson and Harper (1988), growth of clover clones also was affected moresubstantially beneath canopies of Holcus than canopies of Lolium, and this occurred at aconstant PPFD, while the shade differed in R:FR. In the present experiment, shade castfrom these canopies reduced PPFD and R:FR equally for a large majority of the day. Thedifference between these effects of the canopies on the light measurements might havebeen caused by the presence of leaf pubescence, which, it has been suggested, increasesits reflection of red light (Thompson and Harper, 1988). With only small gaps betweenleaves in a canopy of Holcus, as in Thompson and Harper (1988), there would be no106source of light rich in red wavelengths to be reflected from leaf hairs. With a relativelylarge gap in the canopy, as in the present experiment, and the sun shaded, there wouldstill be a source of light relatively rich in red light (unfiltered diffuse sky or clouds)available for reflection from the leaf hairs, and the R:FR detected beneath “equally” denseshade would be increased.Since all grasses were clipped to 15cm for all spectroradiometric measurements,the growth of grasses that took place between clippings in the experiment is not reflectedin these measurements. Each centimeter of additional height in the grasses had thepotential to interfere with sunlight for several minutes each morning and afternoon, andthe differences in height growth between species were significant (Table 4.4). WhileHolcus grew taller than Lolium, and this may explain the differences between growthunder these two canopies, Dactylis grew significantly taller than Holcus betweenclippings, yet there was little difference in the resulting clover morphology. From thedata on light conditions, grass tiller densities, leaf widths, and height growth betweenclippings, it was expected that canopies of Lolium would be the least influential, that itseffects would resemble the effects of Holcus canopies more closely than Dactyliscanopies, and that Dactylis canopies would be the most influential. There seems to be aquality of Holcus canopies (perhaps its effects on end-of-day R:FR) that gives it arelatively strong influence for its size. This result may reflect the strong competitiveability of Holcus observed in the field towards many of its neighboring species (Haynes,1980; Solangaarachchi, 1985; Turkington et al., 1991).There is another possible explanation for the strong effect of Holcus on thegrowth of clover in these experiments. Between clippings (every four days in the presentexperiment), grass grew into the space formed within the corridor of canopy modules.Little intermingling with clover leaves occurred, but small differences in the width of thecorridor could have affected the conditions experienced by clover clones within. This107temporary encroachment was not measured, but it appeared that Holcus canopiesextended most obviously into this area between clippings. So, even though clipped to thesame dimensions every four days, its effective size may have been larger than Dactyliscanopies, which primarily grew upwards. Regrowth between clipping beneath Holcuscanopies may have had an influence on the morphologies observed in Thompson (1987)(7 days) and Thompson and Harper (1988) (3 days), as well as in the present experiment.The vigorous lateral growth of Holcus lanatus has been attributed with a role in itssuccess in the field (Solangaarachchi, 1985).The foraging expressed in clones under partial natural shade in this experimentinvolved changes in ramet placement through changes in ramet production, branching,and partitioning of biomass, and modification of resource-gathering structures throughchanges in biomass partitioning, leaf lamina and petioles. A similar pattern ofmodifications to phenotype occurred under Lolium canopies when compared to clonesunder Dactylis and Holcus, which may suggest that many of the same changes tomorphology occur in response to slight alterations in conditions due to different speciesof neighboring grass. Finally, many of the characters shown to respond most sensitivelyto these small differences in partial-shade conditions might be likely to respond to subtlechanges in the light conditions, e.g., increased FR from reflection off neighboring grass(Chapter 3).It appears that a particular phenotype in white clover can be developed inresponse to the species of neighbor growing around a gap. Success of clover in the fieldmight rely on its ability to alter its morphology when growing with different grasses(Evans and Turkington, 1988). An appropriate morphology might be necessary for thesuccessful capture of resources, allowing persistence under particular conditions. Theseresults show that the different species of grass can induce different morphologies in108clover clones under standard, partially-shaded conditions. This stresses the importance inplants of responding to relatively small differences in natural light conditions.109Plate 3. Layout of partial shade experiment showing canopy modules, flats of clover, andopaque barriers.111waflof vaflof 15cmgrass 47.6 grass17cm 66.2________pot of pot ofsoil flatof clover soil 10cm________10cm15cm8070a.)I — — — — - Elevation needed-grass50 Elevation needed-control04030 Sun-April 1Sun-May 120100Hours from mid-dayFigure. 4.1. above) The canopy dimensions used in the partial shade experiment. In thiscross-section from point A in the middle of the flat of clover, the angle perpendicular tothe wall of grass is 66.2 degrees (vertical: 17.0cm, horizontal: 7.5cm). Using the fact thatthe further south along the top of the wall that the sun crosses it, the lower the elevationangle needed, the potential hours of sunshine during the experiment can be estimated forgrass “corridors” and controls (perpendicular angle to experimental compartment wall38.7 degrees; vertical: 20.0cm, horizontal; 25cm, not shown). below) are the elevationangles needed to clear the grass barriers and experimental compartment walls dependingon the time of day (time angle) at which the sun would cross, and the plots of the sun’selevation by the time of day, near the starting (April 1) and ending points (May 1) of theexperiment.0 1 2 3 4 5 6112.D. glomerata(a)H. lanatus6.0—- . L. perenne5.0—./__jS/\ J’ f Control— -._.‘/‘___0 4.O-IVia) I ‘7D‘3.0qoo:H I400 500 600 700(b)6.03.0—2.0—1.00.0— I I650 670 690 710 730 750Wavelength (nm)Fig. 4.2. Spectroradiometric measurements made on a clear day at solarnoon (a) and (b), and three hours after solar noon (c) and (d) undersimulations of the experiment. Note that (b) is a closer view of the red andfar-red wavelengths in (a), and (d) is a closer view of the red and far-redwavelengths in (c).D. glomerata(c)H. lanatus4.0L. perenneControl3.0—C)a2‘4-- 0 .,E01.0-.400 500 600 700(d)3.0—20—650 670 590 710 730 750Wavelength (nm)Fig. 4.2 Spectroradiometric measurements made on a clear day at solarnoon (a) and (b), and three hours after solar noon (c) and (d) undersimulations of the experiment. Note that (b) is a closer view of the red andfar-red wavelengths in (a), and (d) is a closer view of the red and far-redwavelengths in (c).1141300“ 1200110010009008007006005004003002001000(b)R:FR1. 4.3. Photosynthetic photon flux density (PPFD) and the red:far-red ratio (665-665:725-735; R:FR) during three periods on a clear day under Controls (C), Dactylisglomerata (D), Holcus lanatus (H), and Lolium perenne (L). Morning and afternoonvalues are derived from 3 sets of scans (330-1100 nm, using a LICOR LI-1800 portablespectroradiometer) taken at slightly different times one afternoon, hence the error bars(±1 SE). Means at these times with different letters above are significantly different(P<0.05; ANOVA with Tukey multiple comparisons. Times corresponding to theseperiods can be calculated from Figure 4.1. Treatments with grass canopies receivedapproximately 4 hours of sunshine around mid-day and 1 hour 40 minutes of leaf-filteredshade each morning and afternoon.CDHL CDHL CDHLMORNING MID-DAY AFTERNOON115Table 4.1. Photosynthetic photon flux density (PPFD) as a percentage of Control, andthe red:far-red ratio (R:FR, quantum ratio of 665-665:725-735) under four differentcanopies. Measurements were taken on a sunny day at solar noon (Mid-day), 3 hoursafter solar noon (Morning, Afternoon), and <1 hour from sunset (Dawn, Dusk) using aLICOR LI- 1800 portable spectroradiometer. Different letters preceding “Morning,Afternoon” PPFD and R:FR indicate that the measurements under the different canopieswere significantly different (ANOVA with Tukey multiple comparisons)Time of dayMorning, Mid-day Dawn, DuskCanopy type AfternoonDactylis glomerata PPFD a 12.8 98.2 40.6R:FR a 0.35 1.08 0.92Holcus lanatus PPFD a,b 13.1 99.8 20.3R:FR b 0.45 1.09 0.82Loliumperenne PPFD b 14.0 99.1 30.4R:FR b 0.48 1.10 0.91Control PPFD c 100 100 100R:FR c 1.11 1.20 1.26Table 4.2. Total photosynthetic photon flux density per day, and this as a percentage ofControl on a theoretical sunny day calculated from the time spent at “Morning,Afternoon” and “Mid-day” levels (from Fig. 4.1). This is a weighted sum (PPFD) orweighted average (R:FR) from the time spent at each of the observed levels.PPFD/day (molIm2/day) Mean daily R:FRCanopy type and as percentage of controlDactylis glomerata (18.9) 0.7568.9%Holcus lanatus (19.2) 0.8070.1%Lolium perenne (19.2) 0.8270.0%Control (27.4) 1.16100%116Table 4.3. Mean temperatures under the different canopies at two different times on asunny day, and on an overcast day. Means in each column that are preceded by adifferent letter are significantly different (p<O.05; ANOVA with Tukey multiplecomparisons). PDT = Pacific Daylight Time.Temp (°C) Temp (°C) Temp (°C)1320 PDT on a 1600 PDT on a 1300 PDT on anCanopy type sunny day sunny day overcast day(±1 SE)Daclylis glomerata (n=4) a 24.4 (0.3) a 21.0 (0.3) a 20.7 (0.3)Holcus lanatus (n=4) a 24.4 (0.3) a 20.8 (0.2) a 20.2 (0.3)Loliumperenne (n=4) a 24.8 (0.2) a 21.5 (0.2) a 20.4 (0.2)Control (n=4) a 25.0 (0.3) b 22.6 (0.3) a 20.8 (0.2)Table 4.4. The density of grass tillers and the height attained between four-day clippings.Grasses were grown in 10cm pots and used to form the canopies in this experiment.Means in each column that are preceded by a different letter are significantly different(p<O.O5; ANOVA with Tukey multiple comparisons among grasses)Grass tiller density Grass height above clippingCanopy type (tillers/cm2) (cm)Dactylis (n=8) a 1.19 (0.14) a 4.8 (0.2)Holcus (n=8) a 0.97 (0.07) b 3.5 (0.2)Lolium (n=8) b 0.68 (0.04) C 2.4 (0.2)Control (n=7) 0.00 0.060(a)50454035302520151050Total stolonlength (cm)Control Dactylis Holcus LoliumNeighbora11740(b)30Number of 25ramets2015’10’5.NeighborFigure 4.4 (a,b). Mean (±1 SE) yield at final harvest in (a) total stolon length, (b) totalnumber of ramets, (c) total above ground dry weight, and (d) total number of branchesunder four different canopies: Control, i.e. empty pots (n=7), Dactylis glomerata (n=8),Holcus lanatus (n=8), and Lolium perenne (n=8). Bars with different letters above aresignificantly different (p’<0.O5; ANOVA with Tukey multiple comparisons).b,c b CControl Dactylis Holcus LoliumControl, Dactylis Holcus LoliumNeighbor118a b b b(c) above- 1 4ground drywieght (gm) 1.21.0•O.8O. -12 -(d) ii-10 -9.Number of 8:branches54-3210-Control Dactylis Holcus LoliumNeighbora b b c1Figure 4.4 (c,d)lag55(a) 504540ControlTotal stolon 35• Dactylis• Holcuslength (cm)o Lolium1505 10 15 20 25(b) 403530rametsNumber of 251;___________________________________________0 I...., Io 5 10 15 20 25(c) branches 5 10 15 20 25 30DaysFigure 4.5. Increase of mean (±1 SE) in (a) total stolon length, (b) number of ramets, (c)number of branches, in clover clones grown under the four different treatments. Shown isthe best-fitting exponential curve (y=alObX) or linear regression (y=ax+b) for branchnumber in grass treatment.120Table 4.5. Mean (±1 SE) of primary petiole and internode growth and their ratio inclones under four different canopies. Over a two-day period growth of the expandingportion of primary stolons was monitored. Within each column, means preceded by adifferent letter are significantly different (p<O.O5; ANOVA with Tukey multiplecomparisons).Primary petiole Primarygrowth internode growth Petiole!Canopy type (cm/day) (cm/day) internode growthDactylis glomerata (n=8) a 1.55 (0.22) a,bO.61 (0.08) a,b2.90 (0.69)Holcus lanatus (n=8) a 1.57 (0.19) a 0.45 (0.08) a 3.85 (0.82)Loliumperenne (n8) a 1.18 (0.20) b 0.88 (0.12) b 1.62 (0.36)Control (n=7) a 1.35 (0.16) b 0.76 (0.07) b 1.92 (0.35)121Table 4.6. Mean (±1 SE), describing the proportion of total biomass allocated to eachwhite clover structure, primary ramet mean leaf area or petiole and internode lengths, andthe specific area or length of each part on primary stolons of white clover under fourdifferent canopies (D=Dacrylis glomerata, H=Holcus lanatus, L=Lolium perenne,C=control). Values in a column subset preceded by a different letter are significantlydifferent (p<O.05; ANOVA with Tukey multiple comparisons).Primary ramet% of total plant specific leaf areadry weight Primary ramet (mm2l g) orallocated to mean specific lengthPlant character Canopy type character (mm/mg)Leaf lamina D a 48.3 (0.3) a 12.9 (0.2) cm2 a 3.34 (0.05)area H a,b49.3 (0.7) a 13.3 (0.6) a,b 3.23 (0.08)L a 49.0 (0.3) a,b12.2 (0.5) a,b 3.23 (0.11)C b 50.6 (0.1) b 11.0 (0.3) b 2.85 (0.13)Leaf petiole D a 31.4 (0.2) a 13.1 (0.1)cm a 5.29 (0.11)length H a,b30.7 (0.5) a 13.3 (0.3) a 5.31 (0.19)L b 29.4 (0.3) a 12.4 (0.4) a 5.46 (0.16)C c 25.4 (0.2) b 10.8 (0.3) a 5.28 (0.25)Internode D a 20.3 (0.3) a 2.41 (0.05) mm a 1.31 (0.03)length H a 20.0 (0.6) a 2.40 (0.09) a,b 1.25 (0.03)L b 21.7 (0.3) a 2.35 (0.10) b,c 1.19 (0.03)C c 24.1 (0.2) b 2.28 (0.05) c 1.10 (0.03)122Table 4.7. Means (±1 SE) of morphological measurements of clover clones grown underfour different canopies. Values of each measurement preceded by a different letter aresignificantly different (p<O.05; ANOVA with Tukey multiple comparisons).Canopy typeDactylis Holcus 1 Lolium ControlPlant character glomerata anatus perennePrimary stolon- a 24.1 (0.5) a 24.0 (0.9) a 23.5 (1.0) a 22.8 (0.5)internode length(mm)Secondary stolon- a 9.6 (0.4) a 9.0 (0.6) a 10.4 (0.6) b 12.3 (0.4)internode length(mm)Wholeplant- a 14.4(0.3) a 14.3(0.6) al4.4(0.6) a 13.9(0.3)internode length(mm)Mean weight per a 54.5 (1.8) a 57.1 (3.0) a 54.9 (2.5) a 52.9 (2.1)ramet (mg)Petiole weight! a 1.55 (0.03) a 1.54 (0.05) b 1.36 (0.03) c 1.05 (0.02)stolon weightWhole plant-mean a 8.0 (0.2) a 7.8 (0.3) a 7.6 (0.1) b 6.2 (0.1)petiole length!seconday stoloninternode length(n=4)1231.1(a) 55504540Percentage of 35total biomass 302520151050Leaf lamina0 PetioleStolonLeaf laminaPetioleStolonFigure 4.6. Mean (±1 SE) of (a) dry weight per plant of the three above-ground plantstructures and (b) the percentage of above-ground biomass allocated to each of thesestructures under the four different canopies: Control, i.e. empty pots (n=7), Dactylisglomerata (n=8), Holcus lanatus (n=8), and Lolium perenne (n=8). Means of the samestructure with a different letter above are significantly different (p<O.OS; ANOVA withTukey multiple comparisons).baMean dryweight (gm)a aabbControl Dactylis Holcus LoliumNeighborbControl Dactylis Holcus LoliumNeighbor124Table 4.8. Means (±1 SE) of branching measurements made on clover clones grownunder four different canopies. Values of each measurement preceded by a different letterare significantly different (p<O.05; ANOVA with Tukey multiple comparisons).Canopy typeDactylis Holcus LoliumPlant character glomerata lanatus perenne ControlNumber of new a 5.50 (0.19) a 5.38 (0.18) b 6.38 (0.32) c 9.86 (0.55)branches producedPrimary stolon- a 67.7 (2.2) a 69.4(2.1) a 71.4(1.8) a 71.0(2.1)branching (%)Secondary stolon- 0 (-) 0 (-) a 2.6 (1.4) b 16.4 (1.2)branching (%)Whole plant- a 22.6 (0.8) a 24.6 (0.7) a 24.8 (0.7) b32.3 (0.3)branching (%)Branch length ratio a,b 43.9 (1.9) a 40.3 (1.4) b 48.1 (1.4) c 58.5 (0.6)Branch ramet a,b 66.3 (1.5) a 64.4 (0.8) b 68.2 (1.0) c74.6 (0.5)number ratioBranch weight ratio a 49.5 (1.4) a 46.5 (1.3) b 53.6 (0.9) c 61.5 (0.8)125Table 4.9. Percentage of new growth in total stolon length and total ramet number(mean, 1 SE) that occurred on the primary stolon, during three consecutive intervals,under four different canopies. Values at each interval preceded a different letter aresignificantly different (p.<O.O5; ANOVA with Tukey multiple comparisons).Percentage of Percentage ofall new growth all new growthInterval by primary by primaryCanopy type (Period-days) stolon (length) stolon (number)Dactylis glomerata (n=8) 1 a,b38.3 (2.6) a 36.5 (6.3)(6-12)Holcus lanatus (n8) a 45.6 (2.6) a 41.7 (3.2)Loliumperenne (n=8) b 36.6 (2.7) a 35.8 (4.4)Control (n=7) c27.7 (2.6) a 44.6 (4.1)Daclylis glomerata (n=8) 2 a,b17.7 (1.2) a 22.3 (2.0)(12-18)Holcus lanatus (n=8) a 21.4 (1.8) a 26.2 (3.3)Loliumperenne (n=8) b 14.8 (1.1) a 21.7 (1.7)Control (n7) c 10.1 (0.7) a 21.7 (0.7)Daclylis glomerata (n=8) 3 a,b 6.6 (0.6) a 18.7 (1.8)(18-25)Holcus lanatus (n=8) a 7.5 (0.4) a 18.6 (2.1)Loliumperenne (n8) b 6.3 (0.3) a 15.4 (1.6)Control (n=7) c 4.1 (0.1) a 14.1 (1.4)126Table 4.10. Measurements describing the fmal position of primary leaves (mean, 1 SE).A primary leaf crossed the stolon if its lamina was displayed entirely on the side oppositeits origination from the stolon. The height of these leaves was determined by themaximum height of the highest primary lamina on each plant. Means preceded by adifferent letter are significantly different (p<O.O5; ANOVA with Tukey multiplecomparisons).Mean # of primary leaves Mean height of highestCanopy type crossing stolon primary leavesDacrylis glomerata (n8) a 3.6 (0.8) a 14.1 (1.1)Holcus lanatus (n8) a 3.6 (1.4) a 15.3 (0.8)Loliumperenne (n=8) a 3.6 (1.4) a 14.8 (1.4)Control (n=7) b 1.1 (0.8) b 8.8 (1.6)127Table 4.11. Summary of analyses of variance (ANOVA model: Variable = Constant +Treatment + residual) using grass canopies (3 species: D=Daciylis glomerata, H=Holcuslanatus, L=Lolium perenne) as the treatments. (n=8) for all treatments.Comparisons of% sum of means having tVariable squares significance tests (Tukey)between groups with p<0.05Branch weight ratio 45.4 0.002 H<LPetiole/stolon weight 44.1 0.002 D,H>LPetiole weight ratio 42.9 0.003 D,H>LBranch length ratio 36.9 0.008 H<LNumber of branches 32.8 0.015 D,H<LStolon weight ratio 31.9 0.018 H<LNumber of ramets 29.4 0.026 H<LPrimary stolon specific length 26.8 0.03 8 D<LBranch number ratio 21.0 0.084Whole plant-stolon specific length 19.8 0.097Total stolon length 19.5 0.103Total biomass 16.0 0.161Secondary stolon internode length 13.3 0.222Leaf lamina weight ratio 12.0 0.260Mean primary leaf area 11.4 0.279Mean primary petiole length 9.1 0.365Primary leaf specific area 6.4 0.501Primary petiole specific weight 3.0 0.732Primary stolon internode length 1.4 0.857Whole plant internode length 1.0 0.988128Chapter FiveIS THERE AN EFFECT OF REMOTE CANOPY CONDITIONS ON THEGROWTH AND MORPHOLOGY OF LOCAL REGIONS OF WHITE CLOVERCLONES?5.1. INTRODUCTIONClonal plants are widespread in many of our natural and agricultural ecosystems,and this has led to a great increase in studies on how plants respond to heterogeneousconditions around interconnected portions of a single plant. In the field, the extendingapex of a clover stolon occupying a gap in the canopy may reach the edge of the gap andbegin extending into the sward. In these two adjacent “patches”, the apical region will beexperiencing conditions typical of a dense sward, while the rest of the plant remainsunder a far more open canopy. In a white clover plant under such a two-patchenvironment, it may be advantageous for support from basal regions to be reduced, tolimit investment in the apex and focus new growth in the resource-rich patch. On theother hand, support from basal regions under these conditions may be increased in orderto reduce the effects of more intense apical-region competition. The strategies which canbe adopted by clonal plants are numerous and have been discussed in Chapter 1.Under patchy conditions, an ability to regulate the extent and direction ofintegration under different conditions might improve a clover plant’s ability to efficientlyexploit the environment. These are traits which could be selected in white clover if undergenetic control, and/or could develop in the clones and remain “programmed” into theirgrowth patterns (Evans and Turkington, 1988). To establish if these patterns of withinplant integration of ramets are important in white clover, it first would be useful todetermine if, without directly influencing the remote portion of the clone itself,conditions existing in one patch can influence the growth of portions in an adjacent patch.It is useful at first to use “large” patch-differences which are defined so by being near the129extremes of what the plant might encounter in the field. If no effect is found due toconnection with a distant portion of the plant that is manipulated, then the response of alocal portion of a plant in this two-region system would be a local response.A great complication to studying this in the natural habitat of many plants is thatthe conditions existing over one part of the plant are rarely independent of the conditionsexisting over another part. For example, if one part of a plant is next to very tallneighbors, while another is farther away, the influence of the tall neighbors is likely to beexperienced directly by the portion farther away. Under experimental conditions, it isoften difficult to alter the conditions experienced by one portion of a plant withoutdirectly altering the conditions of the interconnected portion. Unless this is satisfied instudies of this type, how much of the observed effect is due to local conditions and howmuch is due to an influence from the conditions on the other portion cannot bedetermined. It is only when the two portions of a plant remain connected but in isolatedenvironments, that these two can be disentangled. Experiments of this design using whiteclover need to pay special attention to isolation of the regions of the plant due to therelatively short distances between interconnected ramets, and this precaution has notalways been observed (e.g. Newton, 1986; Solangaarachchi, 1987; Turkington et al.,1991). White clover has relatively short connections between ramets when compared toother clonal plants used in similar studies, such as Fragaria chiloensis, Glechomahederacea, and Lamiastrum goleobdolon, which have longer stolons between ramets.A series of experiments was designed to determine if there was a response in localportions of white clover clones to the conditions experienced by interconnected portions.The portions of the plant manipulated and examined for response correspond to the field-patch size observed in a pasture with a relatively fine-grained mosaic of species (e.g.,permanent pastures in B.C., Aarssen and Turkington, 1985). These experiments mimicfield situations where an individual white clover plant experiences different conditions130over an interconnected length of one stolon. Are the conditions responded to in isolation,or is there evidence that the particular conditions experienced by the distant portion of theplant can invoke a local response; i.e., are they integrated? If integrated, what types ofpatterns are common; are ramets under poorer conditions supported by ramets underbetter conditions, or are ramets under better conditions supported by ramets under poorerconditions? Do these patterns change under different conditions? A better understandingof these questions should increase our ability to maintain and manage the natural habitatsof clonal plants and to use them efficiently in production systems. The objective of thefollowing experiments is to determine if and under what canopy conditions a localizedregion of a clover clone responds to shading experienced by a remote region of the clone.These experiments subjected clones to two canopy patches along the length of theprimary stolon. The dependence of basal-region growth on apical-region conditions wasexamined by manipulating the neighborhood occupied by the apical region, using shadefrom natural neighbors, and examining for response in the basal region of the clone, (i)under open conditions, and (ii) under naturally shaded conditions. The dependence ofapical-region growth on basal-region conditions was examined by manipulating theneighborhood occupied by the basal region with shade from neighbors and examining theresponse in the apical region of the clone, i) under open conditions, and ii) under shadedconditions. A lack of integration within the plant under all or some of these conditionsmay indicate if and when physiological integration plays a major role in determiningwhite clover morphology and foraging behavior under patchy canopy conditions.5.2. METHODSCanopy modules and clover clones were prepared for these experiments as describedin Chapter 2.1). The arrangements of canopy modules used in these experiments aredescribed in Chapter 2.2.3 (and see Fig. 2.3, Plate 4). In these experiments, an extra length oftime was required to prepare clones for the experiments. During this period, clover clones131were grown under uniform conditions in the experimental chambers without a canopy ofgrass, until they were large enough to have a primary stolon with several nodes (4-7) and arapidly-expanding apex (19-31 days). This was done to ensure that there were patch-sizedportions of clover clones available to experience local canopy conditions. To ensure thatclones were of uniform size and developmental stage when encountering the secondneighborhood, all plants were repositioned simultaneously at the start of each experiment,with only the apex of the primary stolon in the second chamber (see Fig 2.3). The exactposition to which the plants were moved was determined by fixing the barrier at the node ofthe same numbered leaf in each clone (nodes 5 to 8 for the various experiments), and thisalways left the apical portions of each clone with at least one and no more than two ramets inthe second neighborhood at the start of each experiment. In this arrangement then, it was thelength of the segment of primary stolon in the two chambers that differed from clone toclone, rather than the number of branches or ramets. Six clones were randomly assigned tothe two treatments used in each particular experiment. The methods of data collection andanalysis in these experiments are described in Chapter 2.4, 2.5. Details relevant to eachparticular experiment are described below.5.2.1. DOES APICAL-REGION SHADING AFFECT THE GROWTH ANDMORPHOLOGY OF BASAL REGIONS OF CLOVER CLONES? (BASIPETALTRANSFER OF RESPONSE)Basal region openThe canopies designed for this experiment allowed all clones to experience anopen canopy around basal portions, while apical regions of experimental clones grewbehind a canopy formed by live grass plants (see Fig. 2.3a). In controls, the apical regionremained in the open for the duration of the experiment. All clones grew entirely underan open canopy for 18 days before being exposed to the second neighborhood. At this132time all clones were placed under the dividers between the two compartments at the samenode on the primary stolon, leaving four basal nodes with branches, and allowing thenewest one or two nodes to extend into the new treatment neighborhood. The clonesremained like this for two days before the canopies were constructed in the appropriateneighborhoods. Clones grew for 19 days (April 20, 1991 to May 8, 1991) before beingharvested.Basal region shadedThe canopies designed for this experiment allowed apical regions of differentclones to experience different conditions, while basal regions of all clones grew behind agrass canopy (Fig. 2.3b). As above, in control treatments the apical region remained inthe open for the duration of the experiment, while the apical region of the primary stolonin experimental clones grew in the shade of a grass canopy. As in all of the experimentsreported in this chapter, all clones in this experiment grew entirely under an open canopybefore encountering the second neighborhood. After 30 days the clones were largeenough to be placed under the wall, leaving five basal nodes with branches and only themost apical node in the second neighborhood. Two days later, the canopies wereconstructed in the appropriate neighborhoods. Clones grew for 31 days (October 3, 1991to November 3, 1991) before being harvested.5.2.2. DOES BASAL-REGION SHADING AFFECT THE GROWTH ANDMORPHOLOGY OF APICAL REGIONS OF CLOVER CLONES ?(ACROPETAL TRANSFER OF RESPONSE)Apical region openThe canopies designed for this experiment presented all clones with an opencanopy over apical portions, while basal regions of experimental clones grew in the shade133of a grass canopy (Fig. 2.3c; Plate 4). In controls, the basal region remained in the openfor the duration of the experiment. All clones grew entirely under an open canopy for 30days before being repositioned, having six basal nodes with branches and their mostapical node in the second neighborhood. Two days later, canopies of grass wereassembled in front of the basal portions of experimental clones (Fig. 2.3c.) Clones grewfor a further 51 days (November 21, 1991 to January 11, 1992) before being harvested.Apical region shadedThe canopies designed for this experiment allowed basal regions of clones toexperience different conditions while all clones grew with apical regions in the shade of agrass canopy. Basal regions of experimental clones grew in the shade of a grass canopy(Fig. 2.3d). As above, in control treatments, the basal region remained in the open for theduration of the experiment. After 27 days of growth behind an open canopy, the cloneswere large enough to be placed with their apex in the new neighborhood, leaving sixbasal nodes with branches. Two days later the appropriate canopies were assembled.The clones grew for 18 days (August 6, 1991 to August 24, 1991) before being harvested.5.3. RESULTS5.3.1. DOES APICAL-REGION SHADING AFFECT THE GROWTH ANDMORPHOLOGY OF BASAL REGIONS OF CLOVER CLONES? (BASIPETALTRANSFER OF RESPONSE)Since in this pair of experiments it is only the response of basal regions that isnecessary for determining the existence of intra-plant integration, results of themeasurements made on this region only are presented.134Basal region openOf the six clones under each treatment, one of the clones with the apical regionunder a closed canopy produced flowers from ramets in the basal region, and it wasexcluded from the analyses. There were no significant differences in any of the clonalmeasurements made on basal regions of the remainder of these plants (Table 5.1).Basal region shadedIn this experiment, one clover clone under each treatment, reached the edges ofthe flats well before the others, and they were harvested early and excluded from furtheranalyses. A photograph of two clones near the final harvest of this experiment is shownin Plate 5. It appears that the clone below (apical region shaded) has a less compactdesign and appears to be slightly smaller. However, few significant differences inmorphological measurements were detected between treatments. There were nosignificant differences in the final yield, branching measurements, or internode lengthsbetween clones having their apical regions under different conditions (Table 5.2) Thedata suggest, however, that there might have been a slight improvement in yield due toapical regions that were unshaded. There was also a strong suggestion that biomassallocation patterns differed between the basal regions of these clones. While theallocation to leaf lamina was similar in the two treatments, of the remaining portion ofabove-ground biomass, a greater proportion was allocated to petioles rather than stolonsin clones which had apical regions under a closed canopy (58.0 ±0.3% apical canopyclosed, 56.5 ±0.5% apical canopy open; p<0.05 Studentst-test). However, there was noevidence of differences in final petiole or stolon internode lengths. The reduction inallocation to stolon weight in basal neighborhoods of clones with their apical regionbehind grass canopies, was also reflected in the mean dry weight of the portion ofprimary stolon that traversed this neighborhood (0.083 ±0.002 g vs. 0.100 ±0.006 g;p<O.05 Student’s t-test). Although this structure had been formed prior to the the135initiation of the canopy treatments, by the final harvest of the experiment it wassignificantly lighter on clones which had their apical region shaded. There were nosignificant differences in repeated-measures ANOVAs on absolute or relative growthrates in stolon length, ramet number, number of branches or petiole length measured overtime in this experiment.Apical region shading appears not to affect growth and morphology in basalregions of clones if basal regions themselves are beneath an open canopy. When basalregions are shaded there was a reduction in primary stolon weight and in the proportionalallocation of biomass to stolons over petioles.5.3.2. DOES BASAL-REGION SHADING AFFECT THE GROWTH ANDMORPHOLOGY OF APICAL REGIONS OF CLOVER CLONES? (ACROPETALTRANSFER OF RESPONSE)Since in these experimental designs it is only a different response by apicalregions that is necessary for determining the existence of intra-plant integration, results ofthe measurements made on this region only are presented.Apical region openIn this experiment, one plant in each treatment reached the edges of the flat morethan two weeks before the rest, and they were excluded from the analyses. This left fivereplicates in each treatment. There were no significant differences in any of the clonalmeasurements made except in the amount of branching that occurred in the apicalneighborhoods. Table 5.3 shows that there was a significant reduction in the number ofbranches, the primary branching percentage, the age to first branch, and percentage oftotal ramets that were on branches in apical regions of clones which had their basal regionshaded by a grass canopy. There were no significant differences in the growth rates ofthe portions of the plants measured over time.136Apical region shadedIn this experiment two clones under each treatment reached the edges of the flatwell before the rest of the clones, and they were excluded from most of the analyses. Ofthe remaining clones (n=4) there was a strong suggestion that a greater dry weight wasproduced in apical regions of clones which had their basal regions under open canopies(Table 5.4; Student’s t-test p<0.063, Mann-Whitney; p<O.O24). Also when the clonesharvested early (above) are paired and considered, apical regions of clones with basalregions unshaded had a larger dry weight than apical regions of clones having a shadedbasal region (p<O.05; Wilcoxon paired-sign test). There were no significant differencesin any other yield measurements. No differences were detected in branching, biomasspartitioning, or internode lengths between the two treatments. Clones with basal regionsshaded, however, produced longer stolons per unit weight in apical regions.Measurements of a more detailed nature in apical regions of these clones did not indicateany significant differences between final primary stolon length, but rather that thisstructure was heavier in clones with basal regions open (0.154 (±0.004) g vs. 0.118(±0.004) g; p.<O.OO1 Student’s t-test).Regardless of local canopy conditions, the growth and morphology of apicalregions of clones responded to basal region shade. When apical regions were in the open,several measurements describing the amount of branching were reduced when basalregions were shaded. When apical regions were shaded, there was a reduction in dryweight, especially in primary stolons, if basal regions were shaded. Even though theweight was reduced, the length of primary stolons was not different, indicating anincrease in specific stolon length.1375.4. MICROCLIMATIC MEASUREMENTS5.4.1 TEMPERATURE MEASUREMENTSMeasurements of temperature were made during one representative experimentwith localized canopies. For these readings two sensors were used, one 3 cm above thesoil surface in a small white tent, the other buried 1cm below the soil surface. Thetemperature at each location was measured three times in rapid succession and averaged,and then the treatment-specific mean and standard deviation were calculated. Thetemperature was measured under different ambient conditions: i) in full sun near mid-day;ii) late in the afternoon on a clear day when direct sunlight was no longer present in thegreenhouse; and iii) under overcast skies near mid-day. Table 5.5 shows the results ofthese measurements and the significance of the differences between treatments usingStudents t-tests. The observed reduction in temperature due to a grass canopy onlyoccurred under clear conditions, and in the soil, the increase carried over until theevening, even though the soil temperature fell to near ambient by this time.5.4.2 SPECTRORADIOMETRIC MEASUREMENTSBehind canopies similar to those used in the experiments, spectroradiometric datawas collected as described in Chapter 2. Light conditions were measured under clearskies near solar noon and again late in the afternoon after the sun’s direct influence wasno longer present in the experimental compartment used for the simulation (more than 6hours after solar noon). Scans were made at several locations behind grass and opencanopies to give a better picture of the range of conditions available within oneneighborhood. Results from noon scans are presented for three locations: 6.5 cm north ofthe canopy, i.e., 6.5 cm nearer to the canopy than the middle of the flat containing clover(Fig. 5.la); 13.0 cm to the north of the canopy, i.e., at the position occupied by theprimary stolon of clover clones (Fig. 5.lb); and 18.5 cm to the north of the canopy, i.e.,1386.5 cm further from the canopy than the middle of the flat containing clover (Fig. 5. ic).The measurement taken nearest the canopy would be representative of conditions nearsecondary stolons growing towards the canopy (south), while the furthest position wouldbe representative of conditions near secondary stolons which were growing away fromthe canopy (north). Due to the height that leaf lamina were elevated, the conditionsobserved at the surface of the flat would be similar to leaves that were closer to thecanopy but located at a higher elevation, at a similar angle from the top of the canopy.The scans presented after the sun’s direct beam was no longer available (Fig. 5.1 d) wererecorded at the midpoint of the flat, 13.0 cm to the north of the canopy, the position thatthe primary stolon of clover clones occupied.The R:FR and PPFD were calculated from these scans and are presented in Table5.6. The PPFD increased as the sensor moved away from the canopy in both treatments,although the increase was more profound behind the canopy of grass. The R:FR followedthe same pattern behind the canopy of grass, but remained stable behind the open canopy.Late in the day, after direct sun was no longer available, presence of the grass canopycontinued to alter both measurements of the light conditions in the location occupied byclover primary stolons.5.5. SUMMARY AND DISCUSSIONThe measurements of temperature indicate that it was not only light conditionsthat were altered by the presence of a grass canopy in these arrangements. The soiltemperature remained elevated beneath the open canopies until late in the day, but it waslikely that overnight, the temperatures equalized under the two canopies. The lower airtemperature beneath the grass canopy would lead to higher relative humidity, especiallywhen the presence of grass itself is likely to increase the amount of water vapor in the air.Also, the differences in soil temperature along with lower humidity, increased the speedwith which the soil dried beneath open canopies, creating larger differences in the water139status of the clones under the two canopies than the air temperature and humidity alonewould have caused. The differences in water status established within one clone withregions under two different canopies might have been involved in the ‘signal” indicatingthe presence or absence of a canopy around one portion of the plant, and this could be animportant indicator of neighboring vegetation in the field.The canopies of grass reduced the PPFD to around 50% of full sun with a R:FRgreater than 1.0, in the middle of the flat of clover, indicating that these conditions wereprobably not as severe as in many previous experiments applying localized shade(Hartnett and Bazzaz, 1983; Slade and Hutchings, 1987b; Solangaarachchi and Harper,1987; Thompson and Harper, 1988; Davies and Evans, 1990). This also suggests thatexperimental conditions providing less than this level of PPFD and a drastically lowerR:FR, e.g. <0.5, might exaggerate the effects of neighboring plants on the light conditionsin a patchy community. In the present experiment, the grass canopies provided cloverclones with a localized region where there was still a reasonable potential for growth,though not as great as in open regions. This type of heterogeneity is likely to be a muchmore common occurrence in the field, where there are numerous gaps, and where cloverleaves are often at or near the top of the sward.In this clover clone, there appears to have been no functional dependence of basalportions on the conditions experienced by apical portions, when basal regions were underopen canopies. In contrast to Solangaarachchi (1987), basal regions of these clones,which had apical regions extend into shaded conditions, did not respond to themanipulation of the apex. It remains uncertain whether this discrepancy is due to adifference in background conditions, e.g. different time of year, use of different clones, orto a difference in experimental techniques, where in the present experiment, no directinfluence of the apical-region canopy on basal regions was allowed. This result agreeswith others demonstrating independence in response between apical and basal portions of140white clover (Kemball et al., 1992), and other species (Slade and Hutchings, 1987b;Dong, 1993)There appears to have been some interdependence on apical conditions whenbasal regions themselves were under closed canopies. The increased allocation ofbiomass to petioles over stolons in basal regions of clones with apical regions shaded, isconsistent with observations on whole plants when they are shaded (Thompson andHarper, 1988; and see Chapter 4; Results). It seems that this effect of shade on biomasspartitioning in basal regions can be alleviated somewhat if the apical region onlyexperiences an open canopy. Some type of signal, perhaps hormonal, might betransmitted from apical to basal regions because photosynthate transfer is generally notexpected from an expanding apex to older basal branches (Harvey, 1970, 1979; Marshall,1990). Since the portion of the stolon which was already formed prior to imposing thecanopies had a smaller weight when apical regions were shaded, it seems likely that thisportion of the stolon provided support for the growth of shaded apical regions (Daviesand Evans, 1990; Chapman and Robson, 1992). Chapman and Robson (1992) alsodemonstrated that old stolon material can have a close link with the expanding apex forseveral nodes along the stolon, through mobilization of existing starch. Although therewere no statistically significant differences in the stolon length, ramet number, total dryweight, and number of branches at the end of the experiment, a closer examination of thedata shows that in the group with apical regions open, where the means of all thesemeasurements are larger, there is a greater variance, due to one small plant. This andvisual evidence tentatively supports the conclusion that a larger yield was produced inbasal regions of these clones relative to clones with apical regions shaded.These observations are consistent with other observed patterns of carbontranslocation in white clover, where there can be movement in the basipetal direction, butonly under special conditions (Harvey, 1970; Ryle et al., 1981; Chapman et al., 1991). It141seems that translocation can occur in this direction (basipetal flow) from an apex torecently formed secondary branches and to defoliated or severely shaded ramets,provided that they are not too distant. In the present experiments, some support may havebeen received from apical regions, and it seemed to be stronger when basal regionsthemselves were shaded. The effects of different background conditions over theobserved portion’s interdependence on the apical region conditions, need to be comparedwith caution however, since the experiments were not conducted simultaneously, andmany differences existed between clones (age, size, pre-treatment conditions) andbetween the grass canopies used to shade apical regions at the different times.There is limited evidence that manipulations of the basal portions of clover clonesaltered the response of apical regions. This indicates that, to some extent, acropetalintegration in a two-patch system such as this can occur in white clover. When apicalregions were open, and perhaps more likely to produce branches themselves (Thompsonand Harper, 1988; Solangaarachchi and Harper, 1987), the amount of branching thatoccurred was strongly dependent on the conditions experienced by the rest of the plant(Table 5.3). In shaded apical regions, when little overall branch production wasobserved, there was no enhancement of branching from interconnection with better-illuminated basal regions. With daughter ramets under sufficiently high resource levels,support from mother ramets may be beneficial to the entire plant, whereas with daughterramets under low resource levels, integration may not be advantageous (Caraco andKelly, 1991). They suggested that with a growth function that is convex-concave (i.e.increase in growth is small with increasing resources at low levels, while increase ingrowth is more rapid with increasing resources at high levels), and daughter ramets undersufficiently high resource levels, support from mother ramets is beneficial to the cloneand hence should occur under these circumstances. This is in agreement with theobservations of branching in the apical regions of the present clover clones. In the apicalregions shaded by grass canopies, the increase in dry weight indicates that a benefit is142obtained through connection to unshaded basal regions. Under shaded conditions theseapical regions can receive support from resource-rich basal regions and continueextension and investment of resources, perhaps at very little cost to these unshaded basalregions (see Results above).Shading of the basal regions seemed to affect apical regions of these clonesregardless of their illumination condition, and this is supported by translocation patternsobserved in white clover, where a stolon apex is a sink that draws from stolon as well asrecent branch-produced carbohydrate (Harvey, 1970; Chapman et al., 1991; Chapmanand Robson, 1992) Shading of older branches has been observed to decrease export tothe main stolon (Kemball et al., 1992). This suggests that in white clover, shading of thebasal portion of a stolon is responded to in a globalized way, regardless of theillumination status of the rest of the plant. This seems to emphasize the importance ofacropetal transport in determining white clover clonal morphology. Support of the stolonapex regardless of its local conditions, serves to support and maintain growth andexpansion of the clone into new environments, even if this currently occupies aninhospitable environment.The measurements on clover clones indicate that while most characteristics weredetermined locally, some responded globally to conditions experienced by other parts ofthe plant, in general agreement with Turkington et al. (1991). The extent of integrationdemonstrated within these clones, however was very limited, in agreement with BulowOlsen et al. (1984) and Solangaarachchi and Harper (1989). This indicates that to a largedegree, portions of one white clover plant in the field growing in two distinct patchesrespond in isolation to their local conditions. When integration between the portions ofthe plant occurred, it was generally seen as some form of support for portions of clonesunder less favorable conditions (i.e., a shaded basal region when the apical region wasopen, or a shaded apical region when the basal region was open). The prediction that143portions of a clone under poorer conditions would receive less from the rest of the plant(or, indeed, shunt some of its own resources to the better-illuminated portion) was notsupported in any of these experiments. This also indicates that an alternative hypothesis,that an increase in branching and ramet production in high-light conditions would occurwhen the remainder of the plant is more carbon-limited, could not be supported in theseclones (Hutchings and de Kroon, 1994).144Plate 4. Layout of localized shade experiments. This shows one experiment, where basalregions (left side of each two-chambered “box”) are given the variable (shaded by grassor open) canopy, while apical regions of all clones remained in the open. Morphologicalcomparisons in this experiment are made between apical regions only.146Table 5.1. Mean (±1 SE) of measurements made at the final harvest on basal regions ofclones with their apical regions under different conditions. In this experiment all basalregions were under an open canopy. Means were compared using Student’s t-test.Plant measurement Apical region Apical region open Probabilityshaded (n=5) (n=6)Total stolon length (cm) 114.4 (7.7) 117.3 (8.9) 0.807Total ramet number 95.0 (5.0) 96.8 (6.9) 0.910Above-ground dry weight (g) 3.83 (0.34) 3.90 (0.33) 0.888Number of branches 22.0 (0.7) 21.2 (0.9) 0.479Branching percentage 67.0 (1.4) 66.2 (1.6) 0.7 13Age to first branch (number of 2.7 (0.1) 2.7 (0.1) 0.954nodes)Percentage of total ramets on 49.5 (2.0) 49.5 (2.9) 0.993branchesPercentage of total length on 65.4 (1.2) 66.4 (2.1) 0.692branches% of aerial dry weight allocated 48.2 (0.7) 48.6 (0.4) 0.588to leaf lamina% of aerial dry weight allocated 25.8 (0.5) 25.4 (0.5) 0.607to petioles% of aerial dry weight allocated 26.1(0.3) 26.0 (0.6) 0.903to stolonsPetiole/stolon weight 0.82 (0.02) 0.83 (0.01) 0.7 17Specific stolon length (cm/mg) 0.127 (0.006) 0.127 (0.005) 0.990Mean internode length (cm) 1.19 (0.05) 1.21 (0.03) 0.784147Plate 5. Two clones growing from right to left, and grown with their apical regions (leftsection) under different canopies; (top) open, (bottom) shaded by grass, while both basalregions (right section) were grown shaded by grass canopies. The morphologicalcomparisons in this experiment were made between the two basal regions.149Table 5.2. Mean (±1 SE) of measurements made at the final harvest on basal regions ofclones with their apical regions under different conditions. In this experiment all basalregions were shaded by a grass canopy. Means were compared using Student’s t-tests.Plant measurement Apical region Apical region open Probabilityshaded (n=5) (n=5)Total stolon length (cm) 97.3 (3.7) 108.8 (8.5) 0.250Total ramet number 110.6 (6.2) 120.8 (10.3) 0.421Above-ground dry weight (g) 2.100 (0.055) 2.380 (0.195) 0.195Number of branches 20.8 (1.8) 24.0 (2.7) 0.355Branching percentage 41.4 (3.0) 46.6 (4.3) 0.345Age to first branch (number of 5.9 (0.3) 5.4 (0.3) 0.268nodes)Percentage of total ramets on 54.3 (1.5) 56.8 (2.9) 0.469branchesPercentage of total length on 24.3 (1.4) 26.4 (3.3) 0.567branches% of aerial dry weight allocated 45.4 (0.3) 45.5 (0.2) 0.911to leaf lamina% of aerial dry weight allocated 31.6 (0.3) 30.8 (0.3) 0.073to petioles% of aerial dry weight allocated 22.9 (0.2) 23.7 (0.3) 0.05 1to stolonsPetiole/stolon weight 1.17 (0.02) 1.09 (0.03) 0.074Specific stolon length (cmlmg) 0.2 10 (0.005) 0.202 (0.003) 0.154Mean internode length (cm) 0.88 (0.03) 0.91 (0.05) 0.674150Table 5.3. Mean (±1 SE) of measurements made at the final harvest on apical regions ofclones with their basal regions under different conditions. In this experiment all apicalregions were under an open canopy. Means were compared using Students t-tests.Plant measurement Basal region Basal region open Probabilityshaded (n=5) (n5)Total stolon length (cm) 21.4 (1.5) 24.3 (2.0) 0.282Total ramet number 39.6 (1.9) 45.6 (2.7) 0.110Above-ground dry weight (g) 0.413 (0.042) 0.469 (0.052) 0.400Number of branches 5.6 (0.4) 7.6 (0.7) 0.035Branching percentage 48.2 (2.9) 65.4 (4.7) 0.014Age to first branch (number of 6.0 (0.3) 4.0 (0.5) 0.013nodes)Percentage of total ramets on 70.5 (1.2) 74.5 (0.7) 0.019branchesPercentage of total length on 32.4 (1.1) 35.4 (1.5) 0.145branches% of aerial dry weight allocated 47.6 (0.5) 47.9 (0.3) 0.650to leaf lamina% of aerial dry weight allocated 27.2 (0.8) 26.9 (0.7) 0.800to petioles% of aerial dry weight allocated 29.3 (1.4) 29.5 (3.2) 0.950to stolonsPetiole/stolon weight 0.94 (0.06) 0.96 (0.13) 0.850Specific stolon length (cm/mg) 0.208 (0.006) 0.209 (0.004) 0.976Mean internode length (cm) 0.54 (0.04) 0.53 (0.06) 0.805151Table 5.4. Mean (±1 SE) of measurements made at the final harvest on apical regions ofclones with their basal regions under different conditions. In this experiment all apicalregions were shaded by a grass canopy. Means were compared using Student’s t-tests.Plant measurement Basal region Basal region open Probabilityshaded (n=4) (n4)Total stolon length (cm) 38.6 (1.2) 43.7 (2.6) 0.124Total ramet number 25.8 (1.8) 30.5 (3.0) 0.216Above-ground dry weight (g) 1.179 (0.041) 1.45 1 (0.112) 0.063Number of branches 7.0 (0.6) 9.0 (1.2) 0.190Branching percentage 30.1 (1.8) 34.5 (3.0) 0.25 1Age to first branch (number of 3.0 (0.4) 3.0 (-)nodes)Percentage of total ramets on 69.1 (2.6) 71.6 (1.9) 0.475branchesPercentage of total length on 50.7 (2.8) 52.3 (2.3) 0.679branches% of aerial dry weight allocated 45.1 (0.7) 45.3 (0.5) 0.762to leaf lamina% of aerial dry weight allocated 28.3 (0.7) 28.9 (0.3) 0.528to petioles% of aerial dry weight allocated 26.6 (0.9) 25.8 (0.6) 0.494to stolonsPetiole/stolon weight 1.07 (0.06) 1.12 (0.04) 0.480Specific stolon length (cm/mg) 0.153 (0.005) 0.127 (0.002) 0.003Mean internode length (cm) 1.51 (0.06) 1.45 (0.06) 0.520152Table 5.5. Mean (±1 SE) of temperature (°C) measurements taken at two locations (Soil),1cm below soil surface, and (Air), 2cm above the soil, under both canopy types underthree different conditions. Shown is the significance of the difference between the pair ofmeans taken under grass and open canopies at each location under each condition. (*p<O.O5, ** p<O.Ol, ns not significant (Student’s t-test)).Soil Air GreenhousetemperatureCanopy typeOpen Grass Open GrassConditionFull sun-midday 25.7 (0.2) 20.3 (0.3)** 25.9 (0.3) 20.8 (0.4)** 21.5(n=6)Overcast-midday 18.3 (0.2) 18.3 (0.1)ns 18.1 (0.1) 18.5 (0.2)ns 18.0(n=6)Clear sky-late PM 19.0 (0.2) 18.4 (0.2)* 18.8 (0.2) 18.6 (0.3)ns 18.0(n=6)153Solar noon Conti-ol(a)D. glomerata6.0— —40 SIX) 600 700Late afternoon(b) (d)400 500 600 700 400 600 600 700Wavelength (na)(c)400 500 600 700Wavelength (nnn)Fig. 5.1. Spectroradiometric measurements made on a clear day at solar noon, (a), (b),and (c), and four hours after solar noon, (d), under simulations of the experiment.Distance of sensor from grass (control) barrier: (a) 6.5 cm north, (b) 13.0 cm north, (c)19.5 cm north, and (d) 13.0 cm north.154Table 5.6. PPFD (.tmolm-2s-1)and R:FR under the two canopy types used in localized-shade experiments at two different times. At solar noon on a clear day, measurementswere made at three different locations within one experimental compartment, and againsix hours after noon at one location. This was after the sun’s beam had left thecompartment. Data are from spectroradiometric scans shown in Figs. 5.la-dNoonLocation of sensor6.5 cm north 13.0 cm north 18.5 cm northCanopy typeGrass PPFD 356 826 1711R:FR 0.88 1.07 1.13Open PPFD 1669 1738 1862R:FR 1.21 1.22 1.221800 hrsGrass PPFD 268R:FR 0.87Open PPFD 399R:FR 1.30155Chapter SixGENERAL DISCUSSIONThe series of experiments reported in this work were designed to investigate therelationship between white clover growth and its light environment. An attempt wasmade to recreate, in as natural a way as possible, the types of light conditions typicallyexperienced by the growing plant in a pasture. As clover grows through a sward, its lightenvironment will be altered by the identity and distance to its neighbors, primarilygrasses. When clover is growing through a gap in the sward, it will receive directsunlight as well as light both reflected from and transmitted through neighboring grasses.Continued growth through the gap will again bring the clover apex into close proximityto neighbors and the relative proportions of the different light sources will change. If thelight conditions are important to its survival, then for continued survival in the sward, theclover must respond to these changes either by altering its growth patterns or form,although the two are intimately linked. Because white clover growth is plagiotropic, inthe field different parts of the same clone will be exposed to different light conditions.An apex that has just invaded a patch of grass might remain connected to another part ofthe plant still growing in the gap. This adds complexity to the responses that can beexpected from white clover in heterogeneous light environments.The results show that in reflection experiments (Chapter 3), except for meanprimary stolon leaf area, growth and morphology of unshaded clover clones was largelyunaffected by reflection from neighboring grasses. Movement of leaves through petiolerepositioning was influenced by neighboring grasses, perhaps through phototropicresponse to increased FR from neighbors. This movement may have lessened the effectson morphological characters of reflection from neighbors. In the partial shadeexperiment (Chapter 4), the effects of spending part of the day in direct sun did noteliminate the effects of shade from neighboring grasses, indicating that white clover has a156low tolerance for shade even when in partial sunshine. The species of grass used to formthe standard gap in this experiment had different effects on the clover clones growingwithin them, and the strength of the effects of different grass species descended fromHolcus lanatus and Dactylis glomerata to Lolium perenne, however; this can be onlypartly attributed to their effects on the light environment. In localized shade experiments(Chapter 5), canopy conditions experienced by apical regions affected basal regionresponse, but only in the experiment where the basal regions were shaded. Canopiesexperienced by basal regions affected the morphological responses of apical regions inboth experiments, where apical regions received different canopies. While generallythere was a reduction in growth due to interconnection with more poorly illuminatedramets, the changes in morphology associated with remote-region shading were notconsistent.The range of phenotypes produced by this single clone of white clover describesthe plasticity with which a clone of clover is endowed, and this undoubtedly plays a rolein its persistence in a heterogeneous community such as a pasture. The various canopysituations presented to this clone reflect some of the variability in the field, where whiteclover will receive light that is transmitted through and reflected from neighbors, as wellas receive different light conditions over interconnected portions of the same plant. Theresponses to the various canopy arrangements indicate that the responses of white cloverare quite complex and can not always be interpreted from an ecological perspectivewithout making large assumptions about what specific changes in morphology do for theplant. By describing the changes to controlled semi-natural conditions we may create anunderstanding of what changes confer an advantage under particular field conditions, butthis should also involve long-term studies to demonstrate an increase in fitness due tothese changes.157Growth of the clones in treatments with neighbors to the north shows that theslight changes in R:FR associated with reflection from neighboring grasses do not causemodifications in the phenotype of the clover directly, but that neighbors are detected andmay be responded to in ways that lead to little difference in observed morphology.Instead, in these clones, movable structures which could respond phototropically (newleaves, stolon apices) did so, perhaps in response to the directional nature of the added FR(Novoplansky et al., 1990; Ballare et al., 1992a). This may have strongly reduced theimpact of neighboring plants on more permanent aspects of the clones’ morphology.Changes in physiological functioning might have also occurred rather than changes inoutwardly-visible (and easily-measured) characteristics. With a slight change to oneprocess, the plant may be able to maintain a constant morphology, and it may be this typeof plasticity that allows plants to be quite similar under different conditions. Theimportance and prevalence of this type of modification to phenotype in determining plantsuccess is unknown (Hutchings and de Kroon, 1994). Also, a lack of detection ofsignificant differences in measurements between these groups, is not necessarily a strongindicator of a lack of real difference between them. If the power of a statistical test(Glantz, 1987) such as a t-test is weak, it will be difficult to detect a difference betweenmeans even if, in actual fact, there were one. With small sample sizes (e.g. <8) in t-tests,the ratio of the differences between means to the pooled standard deviation of the twogroups needs to be greater than one for there to be a 50% chance of detecting a difference(at p<O.O5) when there really is one (Glantz, 1987). If this ratio is less than one, there isa smaller than 50% chance of detecting a difference when there really is one. Data fromthe partial-shade experiment (Chapter 4) indicates that many of the differences in meansbetween pairs of clones were approximately the same magnitude as the pooled standarddeviation. In the other experiments, where the effects of the treatments were expected tobe more subtle, the differences between means are likely to be smaller with roughly thesame magnitude to the pooled standard deviations. This means that, for these158experiments, larger sample sizes would be needed to have at least a 50% chance ofdetecting differences when they they exist and are of this magnitude. The lack ofmorphological response in these experiments, therefore, may be due to the lack ofsufficient replication.Changes in plant morphology do not only involve adaptations to currentconditions, but also involve adaptations to assure continued access to sufficient resources.This may be a particularly important aspect of plant plasticity especially in communitieswith rapidly developing canopies such as in many of our agricultural systems (Ballare,1994). Plants in a monoculture, even if genetically identical, will have differentphenotypic attributes depending on the precise conditions at their particular location. Forexample, the plants near the center of the monoculture are often taller and less branchedthan plants near the edge. In fact, the shape of the canopy attained in such a monocultureis often “dome” shaped, with a regular increase in height towards the center. Thissuggests that each plant senses the height of its neighbors and responds quite accurately,only producing the needed increment of height for successful capture of light resources.The changes that neighbors cause are complex, and this phenomenon indicates the levelof sensitivity with which plants can respond to their environment.Among other features, the existence of small changes in FR associated with theseneighbors is likely to be responsible (Smith et al, 1990; Ballare, 1994). Earlier, it hadbeen suggested that since light with a R:FR over 1.1 has little effect on phytochromephotoequilibrium, changes in this range in the natural environment will not affect plantgrowth through phytochrome (Smith and Holmes, 1977; Smith, 1982). With therecognition of heterogeneity in many plant canopies, a directionality to the variousradiation inputs is likely to occur. This means that the effects of neighboring plants(increased FR or a low R:FR) are also often received in conjunction with both a highPPFD and R:FR. In some of these situations, the globally-detected R:FR might not drop159below 1.1 (Chapter 3, Table 3.2). Horizontally-measured radiation under theseconditions is rich in FR with a R:FR that would alter the state of phytochrome in cellsthat receive this radiation (Ballare et al., 1987; Smith et al., 1990; Chapter 3). Theradiation environment inside upright structures such as stems is remarkably sensitive tothis horizontal radiation (Ballare et al., 1989; Mancinelli et al., 1992). In white clover,there was little permanent response to reflected FR radiation while also receivingunfiltered daylight-level radiation. The difference between these results and those usingorthotropic plants such as Chenopodium sp. (lamb’s quarters), Sinapis alba (whitemustard), and Cucumis sp. seedlings may have been due to the size differences betweentarget plants. In the present experiment, clover clones grew quite large over the course ofeach experiment, and had several times as many leaves as the orthotropic plants usedabove. There would have been considerable amounts of FR in the region occupied by thepetioles and stolons in control clones from the large number of clover leaves present. Theolder leaves of clover may have given the more recently formed leaves on all clones,including controls, a signal similar to neighboring grasses. Perhaps the response toreflection from neighbors in clover clones which are very small, would be easier todetect. The results from the reflection experiments suggest, however, that if a response isdetected when a clover genet is small, these differences might not lead to apparentdifferences later in growth.White clover exhibited little shade tolerance under partial shade conditions.Defining a “shaded” environment as uniformly obstructed precludes the possibility ofsome direct (unfiltered) radiation being received by a plant. In this experiment, “partialshade” meant that sunlight was filtered in a natural pattern (allowing small sunflecksthrough) and that direct sun was also available. The clover clones responded to theshade, and quite sensitively, even though several hours were spent in the direct sun. Theresponse to different species forming the edges of the standard gap varied in severalregards, suggesting that clover clones can detect the species of grass they are neighboring160and respond accordingly, without interaction below ground. The types of morphologicalchanges observed to occur from different species might have been able to predict thetypes of responses to more subtle changes in the light conditions. (However, none ofthese changes occurred consistently in response to treatments with light reflected offneighbors.) The morphologies produced with only shoot interaction support theobservations of the effects on clover morphology when growing associated with thedifferent species of grass in the field, with Holcus lanatus and Daclylis glomerata beingamong the most aggressive, and Lolium perenne being among the least aggressive(Chestnutt and Lowe, 1970; Solangaarachchi, 1985; Frame and Newbould, 1986). Thissuggests that light conditions associated with different neighbors are important in alteringthe phenotype of white clover, allowing it to grow, presumably, in an efficient mannerunder each of the particular conditions. It would be interesting to pre-condition plants bygrowing them with one species of neighbor, allowing it to adopt a suitable morphology,and then transfer them to neighborhoods with different species of grass, or differentheights or densities of the same species. By comparing the long-term success betweenclones that came from different backgrounds, and between similarly pre-conditionedclones which went to different treatments, a possible fitness advantage of having aparticular shoot morphology under each condition could be demonstrated.Under clear skies, more than 90% of the daily PPFD received by partially shadedclones in this experiment occurred during the middle period of the day, when directsunlight was received. In further experiments, it would be interesting to vary the PPFDreceived in this period and monitor the change in response to equal shade at other timesof the day. This may suggest a role for PPFD in modification of the response to lowR:FR. Under uniform natural shade conditions, PPFD and the R:FR would dropsimultaneously no matter what the particular PPFD used, so this can no.t be determined.It may also be interesting to give a range of shade regimes defined by varying the lengthof the shaded period(s) during the day. This may expose some threshold responses,161where below a particular length of time spent shaded, there is no response to shade. Arange of plant species known to vary in their shade tolerance should be examined for this.Shade tolerance in some species might be exhibited by a range of critical lengths ofshading among individuals, perhaps allowing selection for improved shade tolerance. Bytesting a range of genotypes for their response to partial shade given in conjunction withsome unfiltered light, we may be able to more accurately predict responses to shade in thefield, e.g., under higher planting density, growing in companion plantings. Surely the useof more realistic shade in our experimentation on plants should lead to more rapidaccumulation of knowledge on how to manage them.The results of localized shade experiments suggest that physiological integrationcan occur in white clover in a patchy environment. The effects on the plant varieddepending on the direction of the stimulus and the conditions under which the observedportions grew. Shading of the stolon apex did not influence the rest of the plant when itremained unshaded. This suggests that in the field, if the basal regions of a plant are inan opening while an apical region becomes shaded, the basal regions might continuegrowing at the same rate as it would had the apex remained unshaded. This demonstratesa localized response in a region under relatively good conditions, and this would maintaina steady production of ramets on branches. This might occur at the expense of theexpanding apex which receives support from basal regions (Harvey, 1970; Chapman etal., 1991a; Chapter 5, Results). When the basal regions were shaded, more effects ofshading the apical region shade were noticed, describing a more globalized response fromapical region conditions on basal region response, and requiring some sort of basipetaltransport. The globalized response under these conditions suggest that when noalternative is available, an apical region that is under poor conditions might begindrawing at cost from the rest of the plant through mobilization of stored carbohydrate andthrough effects on its morphology and physiology. Also, unshaded apical regionsconnected to shaded basal regions were inhibited from branching, perhaps promoting its162own escape from the area. It appears that an apical region can respond to stimuli(shading) affecting only basal portions, allowing it to increase its distance from thestimulus before the apex itself becomes shaded. It would be interesting to test if the apexincreased its expansion solely due to a signal from neighbors (from reflection) received atbasal regions rather than actual light-resource limitation. The R:FR could be varied overbasal regions without altering PPFD, providing a signal of neighbor presence, while theresponse in apical regions is monitored. The same could be conducted examining thebasal region response to apical region differences in R:FR, but the results above (Chapter5) suggest that apical region conditions would be more likely to have an effect if the basalregions themselves were shaded.What is involved in determining a plant’s morphology to maximize current andcontinued access to sufficient resources is undoubtedly complex, and our ability tointerpret changes in plants with respect to this is rather limited. The experimentsconducted here indicate that even with relatively well understood effects of neighboringplants on the environment of a clone, the morphologies attained are difficult to interpret.Perhaps by describing a range of morphologies attained under particular conditions andlong-term evaluation of the differences in fitness caused by these different morphologies,we might improve our understanding of what particular morphological changes can dofor the plant.The goal of many studies of phenotypic plasticity is the ability to predict andimprove the efficiency with which a crop plant exploits a particular controlled or naturalenvironment. A genetic basis for this level of phenotypic plasticity would allow forselection of types based on the ability to perform to a desired standard. The importanceof this aspect of potential crop improvement has only recently been realized, and theextent of phenotypic plasticity in many of our important crop plants is largely unknown.163There are many potential advantages of a better understanding of how plantsrespond to neighboring vegetation. Having the ability to predict if a particular crop willgrow to satisfactory standards when in a new planting arrangement will clearly bebeneficial. Increased knowledge of the behavior of plants in response to reflected light,for example, could increase the efficiency with which we use plants in our productionfacilities. For example, planting in double- and triple-rows of staggered height, risingfrom the south to north within each row running in an east-west direction, has thepotential for increasing the amount of light intercepted and hence, crop production perunit area. The amount of increase needed in row spacing over single rows of the tallestcrop would not be large enough to offset the benefit of 2-3 times the productivity per row.In order to successfully predict the yields of the shorter crops used in each multiple row,an understanding of these species’ responses to increased FR from light reflected off thetaller companion plants is needed. A similar effect might develop in plantings on asouthern aspect, with increasing elevation to the north and slight increasesin reflectedlight over level plantings. In some species the response to increased FR from reflectionmight affect production, whereas in others, it might not. It seems that breeding for anoptimization of the response to FR reflected from neighbors (in this example perhapseliminating the response), might increase our abilities to use more finely-tunedproduction techniques, which in the long run should have less negative impact on theglobal resources.164LITERATURE CITEDAarssen, L. W. and R. 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