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Iso&euml, tes sabatina is an aquatic quillwort endemic to Italy. It is one of the rarest quillworts in Europe, and is critically endangered due to restricted range and to the continuous decline of both population and habitat quality. This study aims to develop an optimized protocol to reproduce and grow I. sabatina sporelings. Mature and immature megaspores were mixed with mature microspores to evaluate the influence of the developmental stage on germination and sporeling development. Two substrates, distilled water and water-agar medium, were tested for germination and sporeling emergence, and three substrates, sand, lake sediment and water-agar, were tested for transplants. A high percentage of megaspore germination (a total of 79.1%) was obtained in both substrates, higher for mature than immature spores. A total of 351 sporelings were produced in distilled water and water-agar cultures, with similar percentages (64.5% and 69.6%, respectively). The development stage of the megaspores affected both germination and sporeling development. Sporeling emergence showed significantly higher percentages in mature megaspores than immature ones (69.6% vs. 11.6%, respectively), with 85% of germinated spores developing sporelings. Only transplants over water-agar medium were successful. This protocol could be useful for the propagation of sporelings as the key step towards the planning of in situ actions to save this Mediterranean quillwort from extinction.

In bryophytes, establishment can occur by a sexual or asexual process, but the production of spores enables colonization of a wider range of habitats and substrates than can asexual propagules. Successful germination is critical for establishment in a new environment. This paper addresses germination and sporeling development in Frullania ericoides, a leafy liverwort species. Fresh spores were inoculated in vitro in different culture strengths of Knop’s nutrient solution (one-fourth strength, half strength, full strength, one and a half strength and double strength), in order to evaluate the effects of this solution on spore germination and on the development of external protonema. On the first assessment, spore germination was observed at all the concentrations. Germination was endosporic, with cell division and proliferation, resulting in a globular protonema, within the spore wall. Beginning at the fourth week, the development of tightly concave primordial leaves was observed in all but the double-strength medium. Throughout the period of study, the treatments with lower concentrations exhibited external protonema with greater lengths. The double-strength treatment was statistically different from other treatments in at least two parameters. The results of this study demonstrate the potential of in vitro culture techniques for bryophyte spore studies and germplasm preservation.

The liverwort species, Marchantia polymorpha, shows environment-dependent morphological plasticity throughout its life cycle. Thalli, representing the predominant body form throughout most of this bryophyte's life cycle, grow with repeated dichotomous branching at the apex and develop horizontally under sufficient light intensity. Spores, after germination, produce a mass of cells, called sporelings, which then grow into thalli. Both thalli and sporelings, if grown under weak light conditions, form narrow shapes, and their apices grow toward the light source. These phototropic responses are specific to blue light and dependent on the blue-light receptor phototropin. This chapter provides several basic procedures, along with some tips, for designing and performing experiments with M. polymorpha to observe their phototropic responses, as well as methods for observing the localization of the phototropin "Mpphot" with a fluorescent protein tag.

Gracilariopsis lemaneiformis is an economically important red alga worldwide, and is currently cultured by vegetative propagation. However, information related to the practice of spore culturing in red alga, especially for this species, is limited. Here, we describe a successful pilot study that used tetraspores and carpospores for the industrial cultivation of G. lemaneiformis in Jiaozhou Bay, Qingdao, China. Morphological observation and culture studies of G. lemaneiformis spores were carried out in the laboratory and field. Related spore parameters of daily output, germination rate, survival number, and growth rate were determined. The results showed that the maximum daily output occurred on the 6th day for tetraspores, with a value of 5.71 ± 0.68 × 105 g− 1 fresh weight, and on the 4th day for carpospores, with the value of 1876 ± 153 carpospores per cystocarp. During early culturing, the development and growth of tetraspores and carpospores were very similar. Inoculation density variation had significant effects on germination rate, survival number, and growth rate, the highest values of which were recorded at medium density, high density, and medium density inoculation, respectively. After 3 weeks in the field, the tetrasporelings and carposporelings were ~ 3 cm in length. Different heights below sea level also had a notable effect on sporeling growth, and the optimal condition was 0.5 m below sea level. After further culturing, the sporelings showed fast adaptation to the sea with average growth rates of 6.21 ± 0.48% day− 1 for tetrasporophytes and 4.21 ± 0.36% day− 1 for gametophytes. This study has demonstrated that the mass cultivation of G. lemaneiformis using tetraspores and carpospores under controlled culture conditions is feasible, and has also provided valuable information for improving the only available culture method (vegetative propagation) for G. lemaneiformis in the field.

Undaria pinnatifida is an annual brown kelp growing naturally in coastal areas as a major primary producer in temperate regions and is cultivated on an industrial scale. Kelps have a heteromorphic life cycle characterized by a macroscopic sporophyte and microscopic sexual gametophytes. The sex-dependent effects of different environmental factors on the growth and maturation characteristics of the gametophyte stage were investigated using response surface methodology. Gametophytes were taken from three sites in Japan: Iwate Prefecture, Tokushima Prefecture, and Kagoshima Prefecture in order to confirm the sexual differences in three independent lines. Optimum temperature and light intensity were higher for males (20.7&ndash, 20.9 &deg, C and 28.6&ndash, 33.7 &micro, mol m&minus, 2 s&minus, 1, respectively) than females (16.5&ndash, 19.8 &deg, C and 26.9&ndash, 32.5 &micro, 1), and maturity progressed more quickly in males than females. Optimum wavelengths of light for growth and maturation of the gametophytes were observed for both blue (400&ndash, 500 nm, &lambda, max 453 nm) and green (500&ndash, 600 nm, &lambda, max 525 nm) lights and were sex-independent. These characteristics were consistent among the three regional lines. Slower growth optima and progress of maturation could be important for female gametophytes to restrict fertilization and sporophyte germination to the lower water temperatures of autumn and winter, and suggest that the female gametophyte may be more sensitive to temperature than the male. The sexual differences in sensitivity to environmental factors improved the synchronicity of sporeling production.

Notothylas vitalii is an endemic Brazilian hornwort species, easily identifi ed by the absence of pseudoelaters and columella, and the presence of yellow spores. Plant material was collected in Recife, Brazil, and the spores were sown onto Knop’s medium, germinating after thirty days only with the presence of light. Germination occurred outside the exospore, and only after the walls had separated into three or four sections did a globose sporeling initiate its development. Following longitudinal and transversal divisions, the initial loose mass of cells became a thalloid gametophyte, subsequently developing into a rosette-like juvenile thallus with fl attened lobes. Additional information concerning sporeling types in key genera of hornworts, such as and FoliocerosPhymatoceros, will be crucial for inferring the possible ancestral type and the evolution of this trait among hornworts. Our study supports the necessity of supplementary studies on sporeling development, combined with morphological and phylogenetic investigations, to help elucidate the evolution of the Anthocerotophyta and their distribution patterns.

Silvetia babingtonii is a potentially economic brown alga for sources of food and high-value added utilization. So far, sporeling nursery and field cultivation has not been successful. The lack of knowledge on development and life cycle of this alga hinder the development of techniques for the sporeings and cultivation. In this study, internal structure of oogonium and antherium of S. babingtonii was observed with hematoxylin and eosin staining and through microscope. Meanwhile, early development from zygotes to juvenile sporelings was studied at 20 degrees C under 60-100 mu mol photons m(-2) s(-1). Zygotes germinated and divided into thallus and rhizoid cells. The larger thallus cells further divided and developed into juvenile sporelings; while the smaller rhizoid cells divided and elongated into rhizoid hairs. These findings documented the life cycle of S. babingtonii and provided fundamental knowledge for sporeling nursery in the near future.

Dolichomitriopsis diversiformis is an endemic and threatened moss in eastern Asia. In vitro culture and light microscopic observation were employed to study its developmental process from spore germination to the formation of young gametophyte, as well as effects of light and temperature on its spore germination and protonemal development. Microscopic observations revealed that its spores were positively photoblastic with exosporous germination, and sporelings were classified as the bryum-type. Light and dark conditions were compared to understand their effects on spore germination. In 24 h continuous illumination, all spores germinated in both 20 ℃ and 25 ℃. In contrast, in darkness at 20 ℃ from one to 30 days, spores did not germinate. However, when dark-cultured spores transferred to continuous light, they started to geminate in 48 hours. In addition, effects of 20 ℃ and 25 ℃ on sporeling were compared. In a continuous light condition, sporelings grew longer and developed more protonema branches in the 20 ℃ treatment than in the 25 ℃ treatment. It was interesting that a phenomenon of slow and hysteretic spore germination and protonematal development was observed in natural light and room temperature (5-10℃). These observations suggest that spore germination and sporeling development is regulated by light and temperature.

The success of seaweed cultivation depends on the scientific control of the tolerance limits and the optimal physiological conditions that affect the spore germination and the early development of algal species. In order to establish cultivation techniques for spores of Hidropuntia caudata (J. Agardh) Gurgel & Fredericq, the effects of irradiance, salinity, and temperature on the carpospore germination and carposporeling development were evaluated under laboratory conditions. Five photon flux densities (PFD, from 18 to 200 µmol photons m-2s-1), six salinity values (from 7 to 55 psu), and four temperatures (from 20 ºC to 35 ºC) were investigated. The level of irradiance caused significant differences in the growth, in the following order: 200±5 > 100±5 62.5±2.5 > 30±1.5 > 18±1 µmol of photons m-2s-1, but they did not inhibit the carposporeling development. Maximum growth occurred under 35 psu, while at 15 psu the formation of carposporeling erect axis was limited. The optimal temperature for growth was 25 ºC, while at 35 ºC the spores died. These results show the importance of previous knowledge on the tolerance limits and optimal conditions for sporeling development of H. caudata for the implementation of an aquaculture program.

Spore germination characteristics of Azolla filiculoides Lamarck, a recent invader of the Donana Mediterranean wetlands, have been quantified experimentally. Batches of spores were subjected to different storage conditions, salinity concentrations, phosphorus availability and drought effects. Cold pre-treatment did not stimulate spore germination, whereas drought, a characteristic different from Azolla 's native habitat, reduced it (40 ± 7 vs 13 ± 5). Germination declined with the increase of salinity (r = −0.50, notably above 5000 μS cm −1 ), whereas phosphorus availability had no significant effect.

Saccharina japonica is a brown alga that has been commercially cultured on a large scale in China. Integrating the light condition under seawater and the adaptation of Saccharina to this condition, it is expected that blue light would be beneficial to Saccharina culture system. Consequently, the detailed effect of blue light on the key stages during indoor seedling culture of S. japonica was investigated in this study. Irradiances and light qualities had little effect on zoospore attachment and germination. Egg formation occurred sooner under blue light than white light. Under optimum irradiances, 95 ± 4% female gametophytes gave rise to eggs in 6 d under blue light, while it took 12 d for over 90% formation of eggs under white light. Over a culture period of 3 weeks, mean sporeling length and width under blue light was 1.39 and 1.56 times of that under white light, respectively, while the mean sporeling size obtained under red light was only 25% of that under white light. The higher growth rate under blue light was largely due to higher photosynthetic efficiency, as indicated by chlorophyll fluorescence of photosystem II. In addition, the mean ratio of sporeling width to length under blue light was significantly higher than that under white light. These results suggest that blue light would be superior to white light for indoor seedling culture of S. japonica. Based on these findings, an improved S. japonica seedling culture system is discussed.

Sporeling development in Cavicularia densa was determined using controlled culture and light microscopy techniques. Ontogeny is typified by endosporic germination followed by early protonematal development via the production of a terminal quadrant of four cells which ruptures the spore wall distally. Sporeling production is triggered by the delimitation of a cuneate apical cell in one of the quadrants. Regular apical cell segmentation produces derivatives and adult merophytes that ultimately result in a juvenile Cavicularia gametophyte. The fundamental sporeling pattern exhibited by Cavicularia is shared only with that of its sister genus Blasia. Moreover, the patterns of sporeling and gemma/gemmaling ontogeny in Cavicularia and Blasia share quadrant systems of precise uniformity, thus reinforcing the close relationship between the blasialean genera.

The fine structure of released, attached, and germinating carpospores of Porphyra variegata (Kjellm.) Hus is described. Adhesive vesicles, formed during sporogenesis and discharged upon settling of the spore, produced a layer of adhesive mucilage around the spore and filled a deep imagination on the spore's ventral side. The mucilage layer was punctured by the emergence of a germ tube. Both spore and germ tube were lined by newly deposited cell wall. Germination was accompanied by vacuolation and starch mobilization. The morphological development of the sporeling was not noticeably influenced by the great variability of the timing, location, and orientation of septum formation. The attached carpospore possessed a plastid like that of gametophyte cells: stellate with one large central pyrenoid and no peripheral encircling thylakoids. Cells of mature vegetative cells of the conchocelis had plastids that were elongate and parietal and had multiple pyrenoids and encircling thylakoids. Most stages in the transition between the two forms of plastids occurred during carpospore germination.

This study integrates landings statistics and biological studies of the red algaGigartina skottsbergii Setchell & Gardner. The analysis of the landings and carrageenan production in Chile suggeststhat this resource will suffer a strong harvesting pressure during the nextyears. Biological results on sporulation, germination, sporeling growth and survivorship in laboratory,indoor tanks and field conditions, indicated that cultivation of this species istechnically feasible, as spores can be seeded on ropes and other substrata. Vegetative propagation of this species through tissue fragmentationis also possible. Vegetative fragments of this carrageenophyte have 20 to30% higher growth rates than whole fronds in suspended culture systems. Protoplast production can be also explored for bypassing restrictions inspore availability. Major advantages that encourage the cultivation of G. skottsbergii include its gel quantity and quality, its pathogen-freecondition, a high reproduction potential and its regeneration capacity. Onthe other hand, the major constraints are related to its relatively slowgrowth as compared to other carrageenophytes, limited availability ofspores and high mortality during juvenile stages.

A seedling and sporeling emergence assay was conducted on 50 soil cores taken from withinCarex stricta tussocks (intra-tussock) and from paired, adjacentinter-tussock areas in the Hawkins Conservation Area, South Hadley, Massachusetts, USA, to test the hypothesis that local heterogeneity in water levels alters seed bank composition and subsequent seedling emergence patterns. Soil cores were sliced into 2.5-cm-thick sections and split into flooded and drawn-down treatments. Germination of buried seeds and spores was assessed in these treatments under greenhouse conditions for 6 months. Eighteen species emerged from both treatments combined: 17 frominter-tussock samples and 12 fromintra-tussock samples. Pooled across sample depths and watering treatments, more species of grasses, forbs (exclusive ofImpatiens capensis), and woody plants germinated fromintra-tussock samples, while more pteridophytes germinated from theinter-tussock samples. Emergence of pteridophytes, grasses, and forbs was associated significantly with sample depth. More pteridophytes germinated from samples close to the swamp surface, while grasses and forbs were most abundant in samples 10–20 cm below the surface. In the drawn-down treatment, pteridophyte and forb seedlings were more plentiful frominter-tussock samples. In contrast, more forb seedlings emerged fromintra-tussock samples in the flooded treatment. This variation in forb emergence seems to reflect differences in species composition within and between tussocks. Twenty-two species occurred in the standing vegetation of the study area, but only nine of these also occurred in the seed bank. The composition of standing vegetation atop and between tussocks did not differ significantly. Grasses, which dominated the seed bank, were absent from standing vegetation. Grasses clearly represent a persistent population of seeds in the seed bank, while forbs are more transient within the seed bank. The depth-stratified species composition of the seed bank also suggests patterns of temporal succession in the aboveground vegetation of this New England tussock swamp.

Sporulation in the floating fern Azolla filiculoides Lam. is both frequent and widespread in Britain and might therefore play a greater part in the population dynamics of the species than has been suggested by earlier reports. In laboratory experiments, increasing plant density and/or phosphorus supply resulted in increased sporulation. It was estimated that a thick mat of 8 kg m2 fresh biomass can produce 380000 microsporocarps and 85000 megasporocarps per m2.Light and temperatures >10°C were necessary for sporocarp germination. Sporocarps could survive exposure to both low temperatures (5°C for at least 3 months) and sub-zero temperatures (−10°C for at least 18 d). Sporocarps were found to survive storage in water for 3 yr and to germinate from mud samples collected in the field. In laboratory culture, sporeling growth and survival were optimal at 15°C.There is some evidence that A. filiculoides might have adapted to the British climate since its introduction.

The increased incidence of UV-B radiation, particularly in southern South America, has sparked more interest in its effects on marine organisms. Here we describe the effects of UV-B on the early life stages of Gelidium floridanum, an agar-producing species abundant on rocky coasts in southern Brazil. Treated samples were submitted to 0.12 W m−2 of artificial UV-B radiation for 2 h per day over a 15-day period. UV-B exposure induced impairment of tetraspore germination and germling development. We observed a delay in germination, a reduced growth rate of tetrasporelings, and morphological changes, such as a different length-to-width ratio, twisted thallus, loss of pigmentation, and differentiation of more than one apical cell, all leading to a different growth pattern in sporelings that survived treatment. Results indicated a great susceptibility of early-developing Gelidium to UV-B, even at low irradiance and short exposure time.

The sexually dimorphic liverwort, Sphaerocarpos texanus, can be used to study changes in sex ratios in a cohort of individuals during different life stages, from spore development to germination to senescence. Recent attempts to germinate spores under controlled conditions have been successful. This study used laboratory-derived spores to detect subtle sex-specific differences in germination in a cohort of spores across different dormancy breaking temperatures. Sex ratios were determined for subsets of sporelings held at 35/20, 30/15, and 25/15°C for one to eight wk. Sex ratios were female biased for spores that lost dormancy in the 25/15°C and when the data were pooled across the three dormancy breaking thermoperiods. This pattern coupled with the potential for spore banks formation in S. texanus can lead to a single cohort of spores influencing the sporeling sex ratios across seasons.

Hyphal penetration of a root epidermal cell by a vesicular-arbuscular endophyte was considered by Nicolson (1959) and Mosse (1959b) to be preceded by hyphal swelling and formation of an appressorium. Observation of early mycorrhizal infections of adventitious roots of leek seedlings obtained using one sporeling of Glomus monosporum Gerdemann & Trappe as inoculum (San Antonio, unpubl. data) indicated that the shape of some appressoria were reminescent of the spherical spore-like structures characteristically present on external mycelium of G. monosporum (Fig. 1) described by Nicolson (1959) and Mosse (1959a). Warner and Mosse (1980) reported that such spore-like structures never became detached naturally from the mycelium, and when deliberately detached, neither germinated nor grew in culture. Warner and Mosse's comment that "it is not known whether root infection can

Spores from herbarium specimens of Braunia secunda, Hedwigia ciliata, Hedwigidium integrifolium, Pseudobraunia californica, and Rhacocarpus purpurascens were cultured on nutrient agar to observe germination and protonemal development. Germination in the five species was exosporic, and two protonemal types were observed. The protonema in Rhacocarpus consists of branched filaments composed of short oblong, or rectangular cells, and is, therefore, of the generalized Macromitrium-type. The protonema in Braunia, Hedwigia, Hedwigidium, and Pseudobraunia is globular, a type not known elsewhere in mosses. Two different developmental modes result in the globular protonema. Hedwigia and Hedwigidium illustrate one mode, in which the earliest cell divisions directly produce a spherical group of cells. The second mode is found in Braunia and Pseudobraunia, in which an early short filamentous germ tube later becomes globular. Differences in sporeling developmental patterns, as well as in other characters of the mature gametophytes and sporophytes, suggest that Rhacocarpus should be excluded from the Hedwigiaceae. The globular protonema is interpreted as a synapomorphy that helps to define the Hedwigiaceae as a monophyletic group, including only Hedwigia, Hedwigidium, Braunia, and Pseudobraunia. The application of developmental data to sys- tematic and phylogenetic problems is not free of controversy. Most of the controversy is fo- cused on whether ontogeny, under certain cir- cumstances, can help in assessing evolutionary polarity of characters (Humphries 1988; Kluge and Strauss 1985; Lundberg 1973; Nelson 1978

The early stages of development of the carpospores and tetraspores of Hypnea muscilormis (Wulfen) Lamouroux have been investigated. Both types of spores germinated immediately after liberation. The spores segmented repeatedly into, 2, 3, 4 cells, etc. until a multicellular ball of cells was produced. The germination pattern was thus of the discal type, i.e., Typus discalis mediatus (Inoh, 1947) or the “Dumontia-type” in the terminology of Chemin (1937). Subsequently, a relatively expansive attachment holdfast was produced from each sporeling. This was followed by the development of up to 4 or more shoot axes arising from the basal disc. These findings are discussed with reference to an earlier study by the senior author on the developmental biology of other species of Hypnea Lamouroux.

The effect of pH 5-8 and lichen acid concentration gradients (2.7 x 10-3 2.7 x 1o-6 M) on the toxicity of the following lichen acids: usnic, lecanoric, evernic, vulpinic, stictic, fumarprotocetraric, psoromic, and atranorin, on spores of Funaria hygrometrica was tested. Percent germination and sporeling growth were used as indicators of toxicity. None of the lichen acids were significantly toxic, for either percent germination or sporeling growth at concentrations equal to or below 2.7 x 10-5 M at pH 7.0, but many of the lichen acids which increased in toxicity at values different from pH 7 may have been toxic at lower concentrations if a different pH was used for the assay. Lichen acid toxicity showed a good correlation with pH for the parameter of spore germination, or sporeling growth, or both. Some lichen acids did not inhibit germination but were effective in retarding sporeling growth, or vice versa. This observation is discussed in relation to changing fatty acids and other lipid composition as germination occurs. Two of the three O-methylated lichen acids (evernic and psoromic) were among the most effective in inhibiting growth over all, but at lower pH values these were less effective than non-O-methylated lichen acids. Stictic, which is also an O-methylated lichen acid, was the least effective inhibitor over all the pH values for both parameters, while vulpinic was the most toxic over all the pH values. The order of relative toxicity for the lichen acids is different, depending on the pH and concentration at which they are tested and depending on the parameter measured. Thus, in an ecological sense, it is difficult to evaluate the adaptive significance of a particular compound or group of compounds without knowing what factors influence the toxicity of those compounds and how these factors vary in the organism's habitat. LICHENS produce a number of chemically diverse secondary products known as lichen acids, of which many are unique to lichenized fungi. These can be found in concentrations as high as 25% of the dry weight of the lichen thallus but more typically 5-10% of the dry weight. A single lichen thallus may produce several different acids. The majority of the compounds are depsides and depsidones but other classes of compounds also exist (Culberson, 1969). Lichen acids are found crystallized on the outer surface of the medullary hyphae of the mycobiont in the lichen thallus. In this position they are readily available to the environment, as well as to systematists who wish to extract them in a simple quick manner. Aside from their usefulness in systematics, lichen acids have been postulated to have several natural roles. These roles include biochemical weathering of rock surfaces, acting as sun-screens to protect the phycobiont from 1 Received for publication 20 December 1979; revision accepted 8 April 1980. We would like to thank Drs. Page W. Morgan and Robert S. Egan for helpful suggestions during the conduction of the experiments and for critical reading of the manuscript. Reprint requests should be sent to the second author. intense radiation, and acting as allelopathic agents against both plants and animals (cf. review by Rundel, 1978). Many studies have dealt with the toxic potential of lichen acids. Some have been shown to retard the growth of gram-positive bacteria (Vartia, 1973), several pathogenic and decay fungi (Henningsson and Lundstrom, 1970; Vartia, 1973), and mycorrhizal-forming fungi (Brown and Mikola, 1974). Symbiont and non-symbiont algae, as well as growth of vascular plants, also are retarded by lichen acids (Follmann, 1965; Follmann and Villagran, 1965; Kinraide and Ahmadjian, 1970; Huneck and Schreiber, 1972; Lawrey, 1977). While most lichen acids are effective in most assays conducted, differing reports of relative toxicity have emerged from the data. Where comparisons were attempted, the relative toxicity of many of the lichen acids was different from assay to assay and changed as concentration was changed. Recently, Lawrey (1977) suggested that certain lichen acids (0-methylated), which are considered evolutionarily advanced, are of greater ecological significance than the hydroxylated counterparts. The data on toxicity of lichen acids to bacteria would bear out such a hypothesis (with notable ex

Apogamy was induced in the fern Ampelopteris prolifera by culturing the gametophytes on mineral nutrients supplemented with various concentrations of sucrose. Higher concentrations (5–8%) of sucrose were detrimental to prothallial growth, while in lower concentrations (2–3 %) apogamy was delayed. Gametophytic callus was induced from the germinating spores by culturing them on 2,4-D rich (3–5 mg/1) media. The differentiation of this gametophytic callus was conditioned by sucrose and auxin concentrations of the medium. In the presence of sucrose, calli responded like prothalli, while in the presence of 2,4-D, differentiation was delayed or completely inhibited. Apospory was induced on the sexual cotyledonary and juvenile sporeling leaveS. Leaves with petiole, excised from aseptically raised plants from excised cultured buds, also exhibited apospory, while no success was achieved with the excised leaves of the parent plantS. Rhizome segments of various length were cultured on media containing different concentrations of sucrose. The differentiation of rhizome segments into gametophytes or sporophytes was conditioned by the length of the rhizome segments and the sucrose concentration of the medium. The possible significance of all the results is discussed.

The rapid spread of bracken in some parts of the country has led to considerable investigation into the habits of the plant. But in almost all cases it is the spread of the adult sporophyte which has been described, and little attention has been paid to the insignificant, complementary half of the life cycle—the prothallus. Nor does any attempt appear to have been made to assess the rate of development of the prothallus and the young sporeling. A recent attempt to collect records of the occurrence of prothalli and young sporophytes in the field has shown how seldom they are found, and has emphasised the fact that little is known of the extent to which the plant spreads by the initiation of new colonies from spores (White, 1930).

1. The nucleus of the gamete-bearing plants contains 22 chromosomes and the male and female gametes contain the same number. 2. In the union of the gametes the number is doubled, and 44 chromosomes appear in the fertilized sporeling, which develops into the Zanardinia plant containing 44 chromosomes. 3. The nucleus of the zoospore-producing plants contains 44 chromosomes, and the number is reduced in zoospore formation,the zoospore containing 22 chromosomes. The zoospore with the reduced number of chromosomes germinates and develops into an individual with 22 chromosomes. 4. It is evident that the gamete-bearing plants come from zoospores and that the zoospore-bearing plants come from fertilized gametes, so that the two generations alternate in the life history. 5. The female gamete of Zanardinia may germinate apogamously. There is no irregularity in the mitotic process, 22 chromosomes being invariably present. The individual produced shows external morphological characters similar to those of the product...

THE PRESENT investigation resulted from the discovery in California of a species of Riella previously known only from the original description (Thompson, 1940). This species, R. a,flnis, was described by Howe and Underwood in 1903 from material collected five years earlier near Tafira, Grand Canary, by Mrs. 0. F. Cook (Howe and Underwood, 1903). It is the only homothallic species of Riella in which the archegonial involucre is ornamented with eight longitudinal flange-like wings. The California specimens of R. afflnis were found growing in an artificial lake, Lagunita, on the Stanford University campus. The bed of the lake is completely dry for six or seven months each year. With the advent of the rainy season the lake fills rapidly, and the water remains at a uniform level from early in February to early in May. When full the greatest depth of the water is about 35 feet. Between the first of May and the middle of June the water is completely withdrawn from the lake for irrigation purposes and the lake bed is entirely dry from the first of July until it begins to fill late in the fall. At the time when the lake is being emptied, isolated plants of Riella become exposed when the water level has dropped about five feet. As the water level drops further, more and more plants are exposed. A survey of the lake bed after withdrawal of the water reveals that R. affinis grows most abundantly at a depth of 15 to 20 feet. The plants grow in thick clusters and on becoming dry, form very distinctive sods on the exposed lake bed. There are seventeen species of Riella (Wigglesworth, 1937), four of which are South African and eleven Mediterranean. Of the remaining two species, one, R. americana Howe and Und., is known only from the United States and at present only from Texas and possibly South Dakota. R. affinis is now known from two widely separated stations, the Canary Islands and California. METHODS.-Cultures for a study of development in the adult thallus were made by breaking dry Riella sod into an ordinary finger-bowl culture dish and covering with pond water to a depth of an inch or more. The spores germinated within two to three weeks and the resultant thalli developed sex organs about two months after the culture was started. This material was used for study of the living plant and also fixed for embedding and sectioning. 1 Received for publication June 10, 1941. The writer is indebted to Dr. Gilbert Morgan Smith of Stanford University under whose direction this investigation was carried out. He is also indebted to Mrs. E. Bodenstein and Mrs. E. Varneck for aid in the translation of Italian and Jugoslavian papers. Cultures were also started with spore masses, intact within the capsule wall, which had been dissected from the dry plants. These were kept intact and placed in Syracuse watch glasses. Pond water was used for some of these cultures; for others a mixture of equal parts of pond water and filtered soil extract was used. The latter was made by boiling 500 grams of soil from the lake bed in one liter of pond water after the procedure given by Bold (1936). Such extracts have been used with much success in the culture of algae. The spores germinated more rapidly in the soil extract medium than in ordinary pond water and also germinated sooner when kept in masses than when the individual spores were dispersed. It was also found that, while spores from plants of the current season would germinate soon after drying, a much higher percentage of germination was obtained if they had been kept dry for a year or more before a culture was started. It was soon found that the food content, particularly the oil globules, of the germ tube and young sporeling so obscured the nuclei and division walls that an accurate knowledge of early growth could not be obtained from a study of living material. Hence, clearing and staining were necessarv. All material was fixed in chromic anhydride-formalin. That to be sectioned was carried through a tertiary-butyl alcohol dehydrating series and embedded in paraffin. All material was stained in safranin or eosin at some stage in the dehydrating series so that it would be visible in the paraffin. Most material was sectioned at eight microns. The stains used were Heidenhain's iron-alum haematoxylin and gold orange, or safranin and fast green. The materials selected for whole mounts were first stained and then passed from absolute alcohol into terpinol. Material in the stages from germination to young juvenile thalli were taken graduallv into terpinol and from that gradually into pure balsam. Keeping a mass of germinated spores intact facilitated the handling of it during staining and balsam infiltration. The adult thalli were passed from alcohol directly into terpinol and from that directly into pure balsam. It was found most useful to mount the adult thalli between two cover slips so that the entire mount could be turned over and examined on either side. The necessity for mounting germination and earlv growth stages whole is obvious. The whole mounts of all stages of adult thallus growth are of considerable value. They are like thick sections and are exceptionally useful in furnishing a three dimensional picture of structures and aiding in the interpretation of serial sections.

Several authors (Scott & Hill, 1900; West & Takeda, 1915; Osborn, 1922; Duthie, 1929) mention that the corms of various species of Isoetes grow beneath the soil surface. Osborn gave the depth at which he found the corms of the adult plants as about two cm; some of the specimens of I. nuttallii supplied to me by Miss Vesta Hesse apparently grew at about the same depth, as indicated by the extent of the non-green, proximal portions of the sporophylls. Duthie reported finding corms at depths as great as 5-6 cm. Osborn and Duthie both worked with terrestrial species; I have found no references in the literature indicating the depth at which the corms of any aquatic species occur. However, the corms of aquatic species of Isoetes (mainly I. tuckermanii) that I collected from several localities in New Jersey, Connecticut, and Massachusetts generally were buried so that the upper surface of the corm was about level with the surface of the substrate. Where the plants grew on fairly coarse gravel, their corms were only partly buried. Observations on spore dispersal and gametophyte growth indicate that the gametophytes of some and probably all Isoetes species live on or near the surface of the substrate, but no suggestions appear in the literature as to how the sporophytes become buried. In some cases sedimentation is apparently partly responsible for the corms becoming buried in the substrate, but this did not appear to be significant around the plants at most of the localities where I collected them. In October 1971 and in September 1972 detached sporophylls of the aquatic I. tuckermanii were seen floating on Ames Long Pond in eastern Massachusetts. The same has been observed with sporophylls of I. bolanderi (Robert Dennis, pers. comm.). It is likely that water movement regularly detaches the bouyant, air filled sporophylls of the aquatic species as they begin to decay at the end of the growing season. As the sporophylls decay further, the sporangia and/or individual spores sink to the bottom, where presumably the gametophytes and sporelings develop. Isoetes tuckermanii sporelings in Ames Long Pond do indeed develop at or near the surface of the substrate, although their exact position was difficult to determine because of the extremely loose nature of the highly organic muck in which the plants were growing. In at least one terrestrial species (I. drummondii), spores are deposited on the soil surface (Osborn, 1922), and therefore it is possible that the gametophytes and sporelings also develop on the surface. Duthie (1929) reports that the spores of terrestrial species are dispersed in the soil by earthworms. Even if the germination of spores originally deposited upon the surface were delayed until they had somehow become passively buried, it seems unlikely that the sporelings of any species could survive at the depths at which adult corms are found, for the limited food reserves of Isoetes megagametophytes requires that the sporelings become nutritionally self-sufficient at an early age. The first few leaves of the sporeling are very

A study has been made of the germination stages and development of the protonema of Chara gymnopitys A. Braun. The development of this Australian species is compared with that previously described for European and Indian species of the Charales. A detailed account of the sequence of cell divisions prior to the first division of the apical cell of the adult plant is given. The dorsiventral development of the protonemal filament and of the "shoot" is noted. The resulting dorsiventrality of the mature plant is discussed. The development and structure of the rhizoids is compared with that of the "stem" portion of the plant. A technique for collecting and germinating Chara spores under sterile conditions is fully described.

Single spore cultures of a mutant strain of Aspergillus amstelodami grew slowly or not at all on media which supported rapid growth of heavy inocula. Retardation was partly due to slowing of the germination process and of the linear rate of sporeling growth, but much more to lack of hyphal branching. A Cellophane technique was used to condition agar media by heavy inocula in such a way that they afterwards supported normal development of single spore cultures. Carbon dioxide, added to the incubation atmosphere, stimulated sporeling branching but did not improve germination or rate of linear growth.

Carbon dioxide is essential for germination and sporeling growth of Chaetomium globosum, probably for vegetative growth, and certainly for the production of perithecia. The characteristic forms assumed by sporelings, 24–120 hr. old, deprived of CO2, are described and illustrated. None of a large number of simple substances tested was able to replace CO2, but certain complex extracts (e.g. jute extract) could do so. The formation of perithecia has been examined by a technique which allows the effect of external additives to be studied. The pathways by which fixation of CO2 takes place have been investigated, and the role of CO2, in metabolism is discussed.

Established cultures of Phytophthora infestans grow well on an agar medium containing KH2PO4, MgSO4, glucose, asparagine, thiamine, yeast extract and casamino acids. Zoospores when germinated on this medium, however, produce a few sparingly branched hyphae only, and very few continue growth to form colonies. The addition of apple pectin induces a much higher percentage of the sporelings to continue growth to form colonies, but even then all sporelings may not continue development, and other factors appear to be involved. The further development of the sporeling was much more readily inhibited by streptomycin and ultraviolet radiation than was germination.