ECOPHYSIOLOGY OF LEUCOSPERMUM R. BR. SEED GERMINATION IN FYNBOS by Town G.J. BRITS Cape of Thesis submitted for the degree of Doctor of Philosophy in the Department of Botany at the University of Cape Town University June 1996 .P::J:;·.:·. ·: .~··~T·}··:·: ·~".] .·-.· - -. ; ;.;~ ' '·-'1 ;_,~· _: .. • The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgementTown of the source. The thesis is to be used for private study or non- commercial research purposes only. Cape Published by the University ofof Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University Statement The conception, planning, execution and writing of this study was my own except in the instances mentioned below. Some of the chapters are adapted from published papers which were coauthored with my supervisors, Neville Brown and John Manning, as well as Jonathan Cutting, Prof Hannes van Staden and my statistical consultant Frikkie Calitz. Their contributions were mainly complementary to the relevant main studies. i Four months after fire: seedlings of Leucospennum cordifolium emerge from under a stone, in Strandveld fynbos, presumably from an abandoned ant nest. ii I . \. ABSTRACT A primary goal in the study of natural seed regenerating systems is to understand how seed dormancy and germination is controlled by the environment. The aim of this thesis was to develop a general model for major seed anatomical, physiological and ecological factors interacting in Leucospemium R.Br in fynbos. The work is a collation of studies on several seed biological aspects and Leucospennum species with the main focus on the horticulturally used Leucospennum cordifolium (Salish. ex Knight) Fourcade. The study thus eclectically investigates diverse study fields and plant material. The result, as far as the practical limits of such a project allowed, is a first integrated ecophysiological model for seed regeneration in a group of fynbos species. Seed ontogeny. Anatomy of the developing L. cordifolium seed was studied as a background and to search for possible links between seed structure and function. The outer integument was shown to form the seed coat: the outer epidermis becomes tanniniferous and th<; inner epidermis develops into a crystalliferous palisade. Examination of the literature reveals that the crystal palisade layer of the outer integument has been erroneously assumed to constitute an endocarp. This finding indicates that a re-interpretation of all published information on the seed coat in indehiscent Proteaceae is necessary. Germination requirements. The first objective, of characterization and quantification of germination requirements in Leucospermum, was to achieve full control of germination of the notoriously unresponsive seeds. In two studies to maximize germination in L. cordifolium a tetrazolium viability test indicated that all viable seeds germinated. A combination of treatments was subsequently tested to achieve complete germination in six species from contrasting habitats (L. cordifolium, L cuneifonne, L. erubescens, L. glabrum, L. reflexum and L. vestitum). The most effective treatment combination stimulated all viable and a significant proportion of subviable achenes to germinate. Treatment effects on germination percentage were cumulative in most species and also resulted in similar germination rate patterns, suggesting common physiological mechanisms for germination in this species group. The second objective was to relate experimental Leucospermum germination requirements to nature. Scarification and stratification requirements of L. cordifolium seeds were investigated. It was found that prolonged stratification at sub-optimal germination temperature does not play a role in fynbos, but that scarification of the seed coat layers does. Temperature requirements in Leucospe1mum seed germination were studied using the same species as before. Leucospermum seeds generally required alternating temperatures for successful germination but the optimum low and high temperature iii requirements of individual species differed markedly. These requirements correlated positively with mean late autumn air temperatures representative of the natural habitats of species. It was concluded that diurnal alternating temperature requirement is a character syndrome in Leucospermum in which the constituent base and ceiling temperature requirements are adaptive, independent and genetically stable characters. The concept is proposed of diurnal bi-temperature requirement in Leucospermum, as distinct from optimal temperature amplitude requirement. Hormonal regulation of seed dormancy and germination in relation to the environment. Seed hormonal changes were explored in relation to known dormancy and germination characters in a difficult-to-germinate species, Leucospermum glabrum. The endogenous levels of gibberellins (GAs) and cytokinins (CKs) changed phasically during normal germination under a single alternating temperature regime. The early increases found in GAs are thought to control the induction of the germination process. The CK pattern, correlated with known ultrastructural and morphological changes in the embryo, suggests that CKs control at least three major processes of germination sensu stricto following induction: 1) early mobilization of protein and lipid reserves in the axis and later in cotyledons, 2) cotyledon expansion which causes the endotesta to split permitting radicle protrusion and 3) later, radicle growth. A correlated experiment was performed with exogenously applied GAs and CKs under different dormancy-enforcing conditions. It was concluded that synthesis of or tissue sensitizing to, both hormone classes GAs and CKs depends on moderately low temperature as the primary environmental requirement, in the presence of oxygen. For GA synthesis a secondary, daily pulse of high temperature is also required. Inhibiting hormones, specifically ABA, appear not to play a role. Ecological aspects. This chapter foremostly addressed the question - why is there a germination flush in fynbos after fire? Experiments were conducted to test the reaction of germinating Leucospermum seeds to smoke treatment. Negative results suggested that smoke does not play a role in Leucospermum seed germination in nature. A new seed biological phenomenon in Leucospermum was investigated. This is the extensive breaking, following desiccation treatment, of the seed testa layers which in intact, soil-stored seeds impose dormancy on the embryo by means of oxygen exclusion. Heat-desiccation- treatments resulted in extensive breaking of the exo- as well as the endotesta of seeds. The results suggest that in nature desiccation by fire could break the exotesta and the endotesta as well, if fire is followed soon by rain, wetting being required in addition to fracture the desiccated endotesta. The viability of embryos was not affected by heat. A positive relationship is therefore predicted between heat intensity of fire and emergent seedling numbers. The promotive influence of heat desiccation was iv found to be similar for three species tested (L. cordifolium, L. glabmm, L. reflexum) and this could not be ascribed to physiological changes caused by high temperature per se. In a seed storage study low temperature-stored seeds maintained a high viability and vigour for c. two years but ambient temperature storage led to a marked decline after 1 year ending in almost complete mortality after 4 years of shelf storage. By contrast a seed batch stored for 4 years in the soil maintained a reasonably high viability and vigour of 60% of the fresh-seed response. This first experimental demonstration of longterm seed survival during soil-storage suggests a form of cellular repair during periodic wet periods as a survival mechanism in Leucospermum. In a study of germination extending over seasons, seed bed emergence was recorded in the first and four subsequent winters. Sporadic germination occurred in all treatments, only during autumn and early winter each year, and more pronounced in intact (non-oxygenated) seeds. The model of long-living, soil-stored seeds, in which the testa is gradually scarified and which leads tQ the extension of germination in an erratic emergence pattern, is supported. Seed structure and ecological function. In order to synthesize an ecophysiological model from the above results, the development and fate of structures with ecological function was studied, using light microscopy and SEM. The model developed related to the individual seed as well as the recruiting population in fynbos. The elaiosome was shown to be instrumental in myrmecochory only and the seed is therefore the true germination unit in Leucospermum. The woody endotesta protects the embryo indefinitely in the soil. The ecological roles of individual testa sublayers were elucidated. The effects of repeated desiccation/hydration cycles during soil storage cause the testa layers of the long-lived seeds of previous flowering seasons to become fully scarified. In contrast seeds of the latest flowering season will be in various stages of incomplete scarification, especially the exotesta, leading to differences in degree of embryo oxygenation. This appears to regulate extended (polymorphic) germination of some seeds in the young­ seed component of the seed bank. During germination the endotesta breaks in a specific pattern along weak structural lines which facilitates emergence of the radicle.