OBLIGATE OUTCROSSING IN A HOMOSPOROUS FERN: FIELD CONFIRMATION OF A LABORATORY PREDICTION

While homosporous ferns are potentially capable of producing totally homozygous sporophytes in one generation via selfing of their bisexual gametophytes, laboratory analyses indicate that a variety of mechanisms promote gametophytic outcrossing. The operation of these mechanisms in natural sporophyte populations, however, has not been previously demonstrated. Laboratory analyses of gametophyte ontogeny show that Bommeria hispida is obligately outcrossing. Electrophoretic data presented here indicate that individuals from natural sporophyte populations of this species are highly heterozygous. Electrophoretic data, therefore, corroborate evidence from the in vitro analysis of gametophyte development and demonstrate that sporophytes of B. hispida in nature typically are products of outcrossing between genetically different gametophytes. Extrapolations from the literature, together with our findings, indicate that outcrossing mechanisms may operate frequently in ferns, thereby maintaining genetic variability between individuals within populations. This evidence questions whether most ferns are highly inbred and therefore predominantly homozygous.     

Gametophyte contribution to sporophyte growth on the basis of carbon gain in the fern <Emphasis Type="Italic">Thelypteris palustris</Emphasis>: effect of gametophyte organic-matter production on sporophytes

At an early stage of growth gametophytes support the sporophytes of ferns. Young sporophytes become independent of gametophytes when the first leaves develop. Although large fern gametophytes produce multiple archegonia simultaneously, only one sporophyte is typically established on one gametophyte. The number of sporophytes is believed to be controlled in two possible directions, from gametophyte to sporophyte or from preceding sporophyte to another sporophyte. To investigate the effects of gametophytes on their sporophytes, we studied the relationship between organic matter production by gametophytes and the growth of young sporophytes of Thelypteris palustris. We cut gametophytes in half (CGs) to reduce the gametophytes’ production of matter. There was no significant difference between the growth of sporophytes on intact gametophytes (IGs) and that on CGs. According to our estimates, based on the rate of organic matter production, the large gametophyte was able to produce two or more sporophytes. The resources required for CGs to make similar-sized sporophytes was twice that for IGs. In polyembryony each of the multiple sporophytes was similar in size to the single sporophytes. Resource limitation does not seem to explain why fern gametophytes establish single sporophytes.     

GAMETOPHYTE ONTOGENY,SEX EXPRESSION,AND GENETIC LOAD AS MEASURES OF POPULATION DIVERGENCE IN BLECHNUM SPICANT

Early gametophyte ontogeny was quantitatively distinct for Olympic Peninsula, Alaskan, and disjunct Idaho populations of the homosporous fern Blechnum spicant (L.) J. Sm. Although variable, gametophyte sex expression was shown to have a genetic component. Statistically different patterns of sex expression characterize each population. The Olympic Peninsula populations were distinct from each other but consistent in having a predominantly unisexual pattern. The disjunct Idaho population was predominantly bisexual at the time when comparable field collected gametophytes bear sporophytes. Preliminary experiments suggest that an antheridogen operates in this species. Increased sowing density favors maleness, and an extract from soil cultures of gametophytes shifts cultures to an exclusively male pattern after a dramatic suppression of growth. Mating experiments revealed that all populations are interfertile, although fertility was highest when the test Idaho population underwent intergametophytic-selfing. The Idaho population evidenced a low level of genetic load consistent with predictions based on its sex expression. Although Olympic Peninsula populations evidenced apparent high genetic load in some experiments, failure to produce abundant sporophytes in other experiments suggested that additional cultural factors operated to reduce sporophyte formation. Moderate density mating experiments produced single sporophytes that were comparable to field collections. Isolated gametophytes underwent polyembryony after a time delay and gametophyte proliferation. Cultural conditions which allow sporophyte formation on isolated gametophytes without this delay or proliferation must be sought before further genetic analysis is undertaken.     

Practical methodology for gametophyte proliferation and sporophyte production in green penny fern (Lemmaphyllum microphyllum C. Presl) using mechanical fragmentation

Propagation of gametophytes and sporophytes using mechanical fragmentation has been considered a suitable method for mass production of ferns. This study aimed to develop a practical propagation method for Lemmaphyllum microphyllum C. Presl, which is a fern of significant ornamental and medicinal value. Gametophytes were obtained through in vitro spore germination and used for propagation experiments. The gametophyte was mechanically fragmented using a scalpel into small fragments, which were then used to investigate gametophyte proliferation. In addition, the gametophyte was fragmented using a blender and then used to study sporophyte formation. Optimal proliferation conditions of the gametophyte were determined using Murashige and Skoog (MS) basal medium (double-, full-, half-, quarter-strength), Knop medium, and medium components (sucrose, nitrogen sources, activated charcoal), at various concentrations. The fresh weight of the gametophyte was 14-fold higher than that of gametophytes (300 mg) used as culture material, when cultured on double-strength MS. Moreover, 1 g of the gametophyte fragmented in 25 mL of distilled water formed more than 430 sporophytes in a soil mixture in an area of 7.5 cm². The sporophytes were successfully cultivated in the greenhouse after acclimation. A large-scale production method for L. microphyllum that can be easily implemented in a fern production farm is outlined.

     

A methodology for large-scale Athyrium sheareri gametophyte proliferation and sporophyte production using tissue culture

Tissue culture methods using gametophytes are considered the easiest ways to mass-produce fern sporophytes. The aim of this study was to develop a practical propagation method for the ornamental fern, Athyrium sheareri. The gametophytes obtained from in vitro spore germination were used as experimental materials. We used the chopping method to investigate the culturing conditions for proliferating gametophytes and the blending method for evaluating the mass production of sporophytes in mixed soil. Gametophyte proliferation was determined via Knop medium, various concentrations of Murashige and Skoog (MS) basal medium (1, 1/2, 1/4), and media components (sucrose, nitrogen source, and activated charcoal). The fresh weight of the gametophytes increased by more than 24-fold in 1/2 MS medium. In addition, 1 g of gametophyte could produce a maximum of 255.3 sporophytes in a mixed soil of 7.5 cm² area. Treating gametophytes with exogenous plant growth regulators promoted the formation and growth of sporophytes. The cultivated young sporophytes were acclimated and successfully grown in greenhouses. We developed a mass production protocol for A. sheareri sporophytes suitable for field application, which is expected to have commercial value.

     

Antheridiogen,dark spore germination,and outcrossing mechanisms in Bommeria (Adiantaceae)

Isozymic analyses of the patterns of genetic variability in sporophyte populations have demonstrated that most fern species have outcrossing breeding systems. However, because fertilization takes place during the ephemeral, diminutive gametophyte generation, direct observation of breeding systems in nature has not been possible. Recent discoveries of soil-bound spore banks suggested that genetic diversity could be stored beneath the surface and subsequently released by appropriate chemical cues. Previous studies demonstrated that Bommeria sporophytes are the product of outcrossing, that their gametophytes carry high levels of genetic load, and that they produce and respond to antheridiogen. Research reported here demonstrated that Bommeria spores can survive long-term storage but will not germinate in the dark. Antheridiogen, however, will release spores from this light requirement and stimulate germination. Higher concentrations of antheridiogen result in higher germination rates. Gametophytes grown in the dark on antheridiogen-enriched agar form antheridia and release actively swimming sperms. Thus, spores housed beneath the soil surface could remain dormant until stimulated to germinate by antheridiogen secreted by surface-dwelling, archegoniate gametophytes. Sperm released from these subterranean gametophytes could fertilize eggs on the surface. Because spores housed in the soil are likely to be genetically different than those at the surface, heterozygous sporophytes would be more likely to result. Discovering that Bommeria species contain all of the prerequisites for this proposed outcrossing mechanism provides an explanation for the maintenance of genetic diversity in some fern populations.     

The evolution of root hairs and rhizoids

Background

Almost all land plants develop tip-growing filamentous cells at the interface between the plant and substrate (the soil). Root hairs form on the surface of roots of sporophytes (the multicellular diploid phase of the life cycle) in vascular plants. Rhizoids develop on the free-living gametophytes of vascular and non-vascular plants and on both gametophytes and sporophytes of the extinct rhyniophytes. Extant lycophytes (clubmosses and quillworts) and monilophytes (ferns and horsetails) develop both free-living gametophytes and free-living sporophytes. These gametophytes and sporophytes grow in close contact with the soil and develop rhizoids and root hairs, respectively.

Scope

Here we review the development and function of rhizoids and root hairs in extant groups of land plants. Root hairs are important for the uptake of nutrients with limited mobility in the soil such as phosphate. Rhizoids have a variety of functions including water transport and adhesion to surfaces in some mosses and liverworts.

Conclusions

A similar gene regulatory network controls the development of rhizoids in moss gametophytes and root hairs on the roots of vascular plant sporophytes. It is likely that this gene regulatory network first operated in the gametophyte of the earliest land plants. We propose that later it functioned in sporophytes as the diploid phase evolved a free-living habit and developed an interface with the soil. This transference of gene function from gametophyte to sporophyte could provide a mechanism that, at least in part, explains the increase in morphological diversity of sporophytes that occurred during the radiation of land plants in the Devonian Period.     

Phylogenetic analyses of morphological evolution in the gametophyte and sporophyte generations of the moss order Hookeriales (Bryopsida)

Morphological characters from the gametophyte and sporophyte generations have been used in land plants to infer relationships and construct classifications, but sporophytes provide the vast majority of data for the systematics of vascular plants. In bryophytes both generations are well developed and characters from both are commonly used to classify these organisms. However, because morphological traits of gametophytes and sporophytes can have different genetic bases and experience different selective pressures, taxonomic emphasis on one generation or the other may yield incongruent classifications. The moss order Hookeriales has a controversial taxonomic history because previous classifications have focused almost exclusively on either gametophytes or sporophytes. The Hookeriales provide a model for comparing morphological evolution in gametophytes and sporophytes, and its impact on alternative classification systems. In this study we reconstruct relationships among mosses that are or have been included in the Hookeriales based on sequences from five gene regions, and reconstruct morphological evolution of six sporophyte and gametophyte traits that have been used to differentiate families and genera. We found that the Hookeriales, as currently circumscribed, are monophyletic and that both sporophyte and gametophyte characters are labile. We documented parallel changes and reversals in traits from both generations. This study addresses the general issue of morphological reversals to ancestral states, and resolves novel relationships in the Hookeriales.     

NORTHERN AND SOUTHERN HEMISPHERE HYBRIDS OF MACROCYSTIS (PHAEOPHYCEAE)1

All combinations of individuals of Macrocystis pyrifera (L.) C. Agardh, M. integrifolia Bory, and M. angustifolia Bory hybridized. Gametophyte isolates obtained from 18 individuals were used, including M. pyrifera and M. integrifolia from the extremes of their Northern Hemisphere ranges along the Pacific Coast of North America and M. pyrifera and M. angustifolia from Tasmania, Australia. All combinations of gametophytes produced sporophytes of normal morphology, with the exception of crosses involving three gametophyte isolates. One female (M. integrifolia) and two male (M. pyrifera and M. angustifolia)gametophyte isolates were unable to produce normal sporophytes in combination with gametophytes of the opposite sex. Some cultures of female gametophytes produced abnormally shaped parthenogenetic sporophytes. Gametophytes and sporangia of M. pyrifera had n= 16 chromosomes. The M. integrifolia female gametophyte that was unable to produce normal sporophytes had n = ca. 32 chromosomes. These results show that these species of Macrocystis have not become reproductively isolated. Although these species may be considered conspecific according to the biological species concept, we recommend that they continue to be recognized as separate species based on morphological differences.     

Sporophyte formation in experimentally-induced unisexual female and bisexual gametophytes ofLygodium japonicum

Unisexual female and male and bisexual gametophytes were experimentally induced inLygodium japonicum. A single bisexual gametophyte was isolated in a dish and a female gametophyte was paired with a male one to allow intragametophytic selfing and intergametophytic mating, respectively. About 30% of the females formed sporophytes but no bisexual gametophytes formed them.     

Growth and reproductive characteristics in artificially formed clonal gametophytes ofDryopteris filix-mas (Dryopteridaceae)

Artificially formed clonal gametophytes ofDryopteris filix-mas (L.)Schott were able to grow and reproduce, although growth rates, proportions of hermaphrodites and reproductive efforts were low. Variable density and nutrient levels appeared to affect gametophytic life histories and they continued to influence the viability of the developing sporophyte generation. Differences between populations and sporophytes were discovered in life histories. Hermaphroditic gametophytes were considerably larger and possessed higher viability than did males. Clear differences between clones in the level of phenotypic plasticity in gametophyte size were observed. No neighbor effect on gametophyte sex expression was detected with the densities and nutrient levels used.     

THE INITIATION OF SPOROPHYTES BY OBLIGATE APOGAMY IN CHEILANTHES CASTANEA

The initiation of apogamous sporophytes in Cheilanthes castanea was recorded by daily photography of individual gametophytes. Whereas an ordinary embryo arises from a zygote, apogamous embryos of C. castanea originate from one to three initial cells which occur just behind the apical region of the prothallus. The initial (or initials) produce cells with small chloroplasts behind the sinus of the gametophyte. The appearance of cells with smaller chloroplasts than those normally found in gametophytes is the first indication that apogamy is occurring. The cells with small plastids produce a group of densely-cytoplasmic meristematic cells. The size of the meristematic mass increases until shoot and root apices of the apogamous embryo are organized.     

Interploidal hybridization and mating patterns in the Sphagnum subsecundum complex

Polyploidization is thought to result in instant sympatric speciation, but several cases of hybrid zones between one of the parental species and its polyploid derivative have been documented. Previous work showed that diploid Sphagnum lescurii is an allopolyploid derived from the haploids S. lescurii (maternal progenitor) and S. subsecundum (paternal progenitor). Here, we report the results from analyses of a population where allodiploid and haploid S. lescurii co-occur and produce sporophytes. We tested (i) whether haploids and diploids form hybrid triploid sporophytes; (ii) how hybrid and nonhybrid sporophytes compare in fitness; (iii) whether hybrid sporophytes form viable spores; (iv) the ploidy of any viable gametophyte offspring from hybrid sporophytes; (v) the relative viability of sporelings derived from hybrid and nonhybrid sporophytes; and (vi) if interploidal hybridization results in introgression between the allopolyploid and its haploid progenitor. We found that triploid hybrid sporophytes do occur and are larger than nonhybrid sporophytes, but exhibit very low germination percentages and produce sporelings that develop more slowly than those from nonhybrid sporophytes. All sporophytes attached to haploid gametophytes were triploid and were sired by diploid males, but all sporophytes attached to diploid gametophytes were tetraploid. This asymmetric pattern of interploidal hybridization is related to an absence of haploid male gametophytes in the population. Surprisingly, all sporelings from triploid sporophytes were triploid, yet were genetically variable, suggesting some form of aberrant meiosis that warrants further study. There was limited (but some) evidence of introgression between allodiploid and haploid S. lescurii.     

LIFE HISTORY OF COLPOMENIA SINUOSA (SYCTOSIPHONACEAE,PHAEOPHYCEAE) IN THE AZORES1

Colpomenia sinuosa (Mertens ex Roth) Derbès and Solier (Scytosiphonaceae, Phaeophyceae) is a common species on the rocky intertidal shores of the Azores, where reproductive gametophytes occur throughout the year. Life‐history studies of this species were carried out in culture, and both sexual and asexual reproduction were observed. Anisogamous gametes fused to form zygotes. The zygotes gave rise to a filamentous prostrate sporophyte generation bearing unilocular sporangia, under both short‐day and long‐day conditions at 15 and 22°C, and to both unilocular and plurilocular sporangia, under the lower temperature condition. Unispores developed into gametophytes, and plurispores gave rise to filamentous sporophytes. Asexual reproduction was carried out by unfused female gametes and asexual plurispores produced from the same gametophyte. Unfused gametes developed into filamentous prostrate sporophytes producing unilocular sporangia in both culture conditions, and unispores released from the sporangia gave rise to gametophytes. Asexual plurispores from field gametophytes, under both culture conditions, developed directly into new gametophytes. The species exhibited three types of life history: a heteromorphic, diplohaplontic; a heteromorphic, monophasic (both with alternation between the erect and filamentous prostrate thalli); and a monomorphic, monophasic.     

In vitro polyploidization of mature ostrich fern sporophytes through rejuvenation

An in vitro method is described for producing ostrich fern (Matteuccia struthiopteris (L.) Todaro) polyploids from mature sporophytes as a possible means of plant improvement in this economically important fern species. The procedure is based on rejuvenating adult sporophytes (2n) to enable the aposporous production of diploid (2n) gametophytes, and then mating the gametophytes to produce tetraploid (4n) sporophytes. The adult sporophytes were rejuvenated by culturing excised shoot tips for a minimum of three months in a liquid medium (Murashige and Skoog salts) under conditions of extreme carbohydrate deprivation (0.01% sucrose). Apospory was induced by culturing leaves excised from the rejuvenated shoots for two months on a semi-solid medium lacking sucrose, resulting in the production of diploid gametophytes. The gametophytes were transferred to fresh medium and grown to sexual maturity for one or two months, then floated on the surface of a liquid medium containing 0.01% sucrose for up to two months to promote opening of the sex organs. Subsequent self-fertilization resulted in the successful production of tetraploid sporophytes in 11 of the 14 diploid clones in which polyploidization was attempted. Tetraploids (4n=156) were confirmed by cytological examination. This method permits polyploidization of mature, fully characterized plants.     

In vitro regeneration patterns of Platycerium bifurcatum leaf cell suspension culture

A simple suspension culture system of Platycerium bifurcatum was developed where sporophytes could be regenerated directly from leaf cells or indirectly through an aposporous gametophyte stage under the same culture conditions. Single cells and aggregates of up to 100 cells developed aposporous gametophytes which later gave rise to sporophytes. Such gametophytes started apogamy when they were mostly less than 0.7 mm in length, bearing only rhizoids. In most cases, only one sporophyte was regenerated from one gametophyte. Aggregates of 500–1000 or more cells, on the other hand, regenerated sporophytes directly. Intercellular interaction was considered to be the physiological cause, and the separation of leaf cells to a certain degree drove the cells to embark on different regeneration paths. It is suggested that the possible existence of a threshold size of cell aggregates separates the two regeneration patterns. Received: 3 March 1997 / Revision received: 11 April 1997 / Accepted: 3 June 1997     

Effect of temperature and photon fluence rate on gametophytes and young sporophytes of Laminaria ochroleuca Pylaie

We analysed the effects of temperature and photon fluence rate on meiospore germination, growth and fertility of gametophytes, and growth of young sporophytes of Laminaria ochroleuca. Maximum percentages of germination (91–98%) were obtained at 15°C and 18°C, independent of photon fluence rate. Optimal development of female gametophyte and maximum fecundity and reproductive success of gametophytes occurred at 15°C and 18°C and at 20 and 40 μmol m^–2 s^–1. Maximum relative growth rate of young sporophytes after 2 weeks of culture was achieved under the same conditions. L. ochroleuca gametophytes cannot reproduce and growth of its sporophytes is not competitive at temperatures close to 10°C. Received in revised form: 31 August 2001 Electronic Publication     

APOGAMY AND SOMATIC RESTITUTION IN THE FERN CERATOPTERIS

A mutant stock of the fern Ceratopteris has been derived from an inbreeding study following an interspecific hybridization between two diploid species. The mutant is characterized by gametophytes that produce non-functional spermatozoids and are incapable of selfing. Sporophytes develop apogamously from the mutant gametophytes and, although they are initially haploid and sterile, portions of the fronds later become doubled somatically and behave like tissues of sexually derived homozygous sporophytes. The mutant segregates from sporophytes in a 1:1 ratio when crosses are made with wild type gametophytes. Certain aspects of the behavior are similar to those seen in some naturally occurring apomictic ferns.     

RADIOACTIVE TRACER STUDY OF SPOROPHYTE NUTRITION IN HEPATICS

Sporophytes of Fossombronia foveolata, Lophocolea heterophylla, Pellia epiphylla, Ptilidium pulcherrimum, and Riella affinis were surgically isolated from host gametophyte tissues and treated with ¹⁴CO₂. Sporophytes of all five species are capable of fixing CO₂ in the light. Sporophyte/gametophyte ratios for ¹⁴CO₂ fixation/mg fresh weight range between 0.12 and 0.39. Corresponding ratios for chlorophyll content are 1.07 to 3.30. Of the total ¹⁴CO₂ fixed by excised Lophocolea sporophytes, 40% can be attributed to the photosynthetic activity of haploid spores. Enveloping gametophytic tissues (calyptra and pseudoperianth) inhibit photosynthesis of attached sporophytes by as much as 50%. For sustained growth, sporophytes rely on organic nutrients supplied by the gametophyte: radioactivity of Lophocolea sporophytes increases significantly after application of ¹⁴C-glucose to host gametophytes. Surgically isolated sporophytes develop slowly in mineral culture, without significant increase in dry weight. The assumption that hepatic sporophytes are at least partly autonomous with respect to organic nutrition (an assumption that figures prominently in speculation on the evolutionary origin of the sporophyte) is confirmed.     

Effects of temperature and nutrients on microscopic stages of the bull kelp (Nereocystis luetkeana,Phaeophyceae)

Warming ocean temperatures have been linked to kelp forest declines worldwide, and elevated temperatures can act synergistically with other local stressors to exacerbate kelp loss. The bull kelp Nereocystis luetkeana is the primary canopy-forming kelp species in the Salish Sea, where it is declining in areas with elevated summer water temperatures and low nutrient concentrations. To determine the interactive effects of these two stressors on microscopic stages of N. luetkeana, we cultured gametophytes and microscopic sporophytes from seven different Salish Sea populations across seven different temperatures (10–22°C) and two nitrogen concentrations. The thermal tolerance of microscopic gametophytes and sporophytes was similar across populations, and high temperatures were more stressful than low nitrogen levels. Additional nitrogen did not improve gametophyte or sporophyte survival at high temperatures. Gametophyte densities were highest between 10 and 16°C and declined sharply at 18°C, and temperatures of 20 and 22°C were lethal. The window for successful sporophyte production was narrower, peaking at 10–14°C. Across all populations, the warmest temperature at which sporophytes were produced was 16 or 18°C, but sporophyte densities were 78% lower at 16°C and 95% lower at 18°C compared to cooler temperatures. In the field, bottom temperatures revealed that the thermal limits of gametophyte growth (18°C) and sporophyte production (16–18°C) were reached during the summer at multiple sites. Prolonged exposure of bull kelp gametophytes to temperatures of 16°C and above could limit reproduction, and therefore recruitment, of adult kelp sporophytes.