J.H. de Lange

Autecological studies on Audouinia capitata (Bruniaceae). I. Plant-derived smoke as a seed germination cue.

Seeds of Audouinia capitata , a threatened fynbos species, are known to germinate under natural conditions only after fires. Experimental results are presented which demonstrate that seed germination is initiated by chemical factor(s) found in smoke, derived from burning fynbos plant material.

Autecological studies on Audouinia capitata (Bruniaceae). 8. Role of fire in regeneration

In A. capitata the protracted juvenile phase, the longevity of plants and their effective vegetative fire regeneration, are all possibly adaptively linked to poor sexual reproduction. After a fire-free period of more than ten years, averages of only 4.7 and 0.4 seedlings per plant were recruited following summer and winter burns, respectively. The positive correlation between the prevalence of fynbos fires from January to March and the germination response of soil-borne seeds when treated with smoke at different times of the year, suggest that the species has evolved in a fire-prone vegetation in a climate with a seasonal rainfall distribution similar to the present one. The vegetative vigour of shoots, the size and numbers of inflorescences, and seed set were increased by fire. The continuation of a policy of late autumn and winter burns may be fatal for survival of the species. Plants responded favourably to fire at a post-fire age of 11 years. Some populations, however, were still vigorous and sexually productive at a post-fire age of 20 years.

Autecological studies on Audouinia capitata (Bruniaceae). 4. Seed production, seed banks and seedling recruitment

Audouinia capitata (L.f.) Brongn. has an average annual production of 98 seeds per plant, determined from a study on approximately 800 of the 1200 plants in 25 known populations. Most of the buried seeds occurred within a radius of 2 m from the parent, in the top 20 mm of soil. Following exposure to plant-derived smoke, 13% of the fresh seeds and more than 80% of the oldest seeds germinated. Thirty-nine percent of the germinated seeds were unable to emerge from a mere 5 mm soil depth under optimal laboratory conditions. Seventeen to 31 months after a burn, 148 seedlings were recorded in seven populations containing 332 plants, from an estimated total annual production of approximately 40 000 seeds. The natural occurrence of small numbers of seedlings is only partially explained by factors investigated in this study. Seed set in at least 14 of the populations, having less than approximately 10 mature plants each, is probably too low to sustain future population expansions or even to maintain present levels.

Autecological studies on Audouinia capitata (Bruniaceae). 2. Insects as pollen vectors

The role of insects as pollen vectors of Audouinia capitata was studied. The frequency of insect visits to flowers and pollen load counts showed that small beetles (Nitidulidae) should be the best insect pollen vectors. Pollen dispersal by these beetles, however, was limited to short distances, which reduced their beneficial impact on the plants. It is suggested that poor pollen transfer may be one of the reasons for the scarcity of A. capitata.

Autecological studies on Audouinia capitata (Bruniaceae). 5. Seed development, abortion and pre-emergent reproductive success

Ovule development, megasporogenesis, megagametogenesis, embryogenesis, endosperm development and seed germination of Audouinia capitata are described. In sharp contrast to the relatively high incidence of abortion and non-viability of microspores demonstrated previously, no pre-anthesis aberrations occur in the megagametophyte. In many fertilized ovules the primary endosperm nuclei remain undivided, enlarge considerably for up to five weeks and eventually degenerate, accompanied by abortion of the zygote. A low incidence of abortion is evident after the fifth week. Abortions after 11 weeks of development are identifiable in mature fruits. A substantial number of mature seeds are devoid of, or contain low densities of, protein bodies. After germination, the radicles of these seeds stop growing soon after emergence from the fruits. It is suggested that abortion is regulated by inter-ovular competition for available nutrients, or by a strongly fixed, genetically controlled system. This strategy could have been selected to allow for the economization of reserves in the 6-ovuled, indehiscent ovaries that act as dispersal units. Possible explanations are suggested for abortion rates in excess of those implied by a fixed general system of one seed per indehiscent fruit. When accepting a seed:ovule ratio of 1:6 as full realization of the seed-setting potential, and ignoring all nonsexual factors, the pre-emergent reproductive success is only 9%. This deficiency is a contributing factor in the rarity of the species.

Autecological studies on Audouinia capitata (Bruniaceae). 7. Nutritional aspects in the mature seed

Lipids and proteins are the major storage compounds present in the starchless endosperm of dormant A. capitata seed. Calcium oxalate crystals are present in protein bodies of the epidermal endosperm, and in the pericarp cells. The globoid inclusions of protein bodies are a major source of P, K, Cl and Mg and the epidermal endosperm crystals, a major source for Ca. No crystalloids are found in the protein bodies. The seeds store N and P. They also contain substantial concentrations of Cu, Zn and Mn. Lower amounts of these elements are present in the pericarp. Nutrients in the pericarp of the indehiscent fruit are probably utilized by the developing seedling. The high N, P and lipid content in the seed, and also the distribution pattern of calcium oxalate crystals in the seed and pericarp, seem to be beneficial evolutionary adaptations. Lipids in seeds from the soil seed bank differ quantitatively from those in fresh seeds. Low densities of protein bodies are found in many seeds resulting in deficiencies of proteins and mineral elements. This may partially explain high incidence of pre-emergent seedling mortality contributing to the rarity of the species.

Autecological studies on Audouinia capitata (Bruniaceae). 3. Pollination biology

Flower production in Audouinia capitata is highest during the first post-fire season. Self-pollinated flowers produced a mean of only 0.8% fruits bearing seeds. This seed set was increased 17-fold by natural insect-mediated cross-pollination. Hand cross-pollination induced an approximately 5-fold increase in seed set when compared to natural cross-pollination. Germination and initial pollen tube growth are normal following self- as well as cross-pollination. Selfing, however, is characterized by abnormal pollen tube growth at the stylar base, resulting in cessation of growth except in the case of occasional pollen tubes. By one week after cross-pollination, pollen tubes have penetrated the egg apparatus. Average in vitro germination of pollen and frequencies of small underdeveloped microspores in 20 populations varied from 2.5 to 28.4% and from 2.0 to 23.7%, respectively, with large interplant variations within populations in both germination (0 – 79.6%) and underdeveloped microspores (0 – 61.0%). The prevalence of non-viable pollen and insufficient insect-mediated transfer of pollen between the plants which have an insignificant ability to produce seed following self-pollination, are limiting factors in reproduction.

Autecological studies on Audouinia capitata (Bruniaceae). 6. Nutritional aspects of the developing ovule

The present investigation provides a basis for studies about morphological and nutritional aspects involved in late abortion of embryos and nutrient-linked, pre-emergent mortality of seedlings in this threatened species. The nucellus is digested completely during megagametogenesis and the integument likewise during embryogenesis, except for a diminutive testa. Mass starch deposition is found in the mature embryo sac. Involvement of the persistent synergid in nutrition of the zygote and the 2-celled proembryo until the fifth week, is suggested. This function is taken over by the suspensor which initially channels integumentary reserves to the developing proembryo. From the eighth week, spherical protein bodies are evident in the endosperm. At this stage, the embryo proper is still surrounded by free nuclear endosperm, and the suspensor of the linear proembryo mobilizes, in addition to integumentary reserves, also adjacent cellular endosperm as well as the micropylar layer of cellular endosperm. The embryo proper of the more advanced stages mobilizes nutrients directly from the adjacent cellular endosperm. The seed enters dormancy at the heart-shape stage of embryo development.

Stimulation of in vitro pollen germination in Audouinia capitata by the simultaneous use of several carbohydrates

Sucrose, fructose, galactose, raffinose, mannitol and sorbitol, when used as single exogenous carbohydrate sources, resulted in hardly any in vitro germination of Audouinia capitata pollen. Germination was increased significantly using either glucose or glycerol, the latter being the most effective carbohydrate. Sucrose : fructose : glycerol used in a 65 : 10 :25 molar ratio with a total molarity of 0.3, resulted in good germination of pollen. Compared to the levels of germination when using fructose, sucrose and glycerol alone, the composite medium produced approximately 30-, 20- and 2.5- fold increases, respectively. The composite pollen germination medium provides a useful means to survey pollen fertility of individual plants.