Sexual expression and genetic diversity in populations of Cryptogramma crispa (Pteridaceae)

The reproductive biology of Cryptogramma crispa, a tetraploid species with a broad circumboreal and alpine distribution, growing mainly in siliceous boulder fields and crevices, was studied in the laboratory by growing gametophytes in plates with both solidified agar media and sterilized soil. In addition, an electrophoretic study of isozymes was carried out on frond samples from five natural populations, as an additional source of evidence concerning the breeding system and the genetic structure of sporophyte populations. Populations throughout the Iberian range of the species were selected for this study, and a Scottish population was included to represent plants from outside our local area and ecology. The morphological development of gametophytes is of the Adiantum type. All multispore cultures developed into a bigametophytic system, consisting in most cases of male and female prothalli. This pattern of sexual expression provides evidence for outcrossing as the main breeding system in this species. Moreover, there is good evidence that the species possesses an antheridiogen system to promote outcrossing. The long time needed by gametophytes to produce gametangia, and afterwards to fertilize and produce sporophytes, might be the primary reason why so few young sporophytes are found in the wild. The values of the percentage of polymorphic loci and the similarity levels obtained from the isozyme analyses indicate a level of genetic variability that would be expected in an outcrossing species. All these characteristics are usually associated with diploid fern species rather than polyploid species.     

环境因子对蕨类植物孢子萌发的影响

蕨类植物通过孢子萌发形成独立生活的配子体,配子体能够形成精子器和颈卵器,进而通过受精作用形成新的孢子体。孢子萌发是蕨类植物生活史过程中配子体世代向孢子体世代转变的关键步骤。同时,此过程不仅受到多种环境因子的影响,也是研究细胞核极性移动、细胞不对称分裂、假根极性生长等独特的细胞学事件的良好模型。迄今为止,人们已经研究发现多种环境因子对约200余种蕨类植物孢子萌发有影响。总结了环境因子对蕨类植物孢子萌发影响的规律如下:(1)孢子萌发除了受到光照强度影响外,主要受光质的影响,光质的影响主要表现为4种方式:①孢子萌发受红光刺激与远红光抑制像开关一样调控;②孢子萌发不受远红光抑制;③孢子萌发受蓝光抑制;④孢子只能在黑暗条件下萌发。(2)重力作用会影响孢子细胞核移动,进而影响孢子细胞发育的极性。(3)赤霉素(GA)能增加孢子萌发率或帮助孢子打破休眠。成精子囊素与GA作用相似,启动或促进孢子萌发。而脱落酸(ABA)、茉莉酸(JA)和乙烯等其它激素对孢子萌发的影响相对较小。(4)不同植物孢子有着各自最适的萌发培养基条件,如不同种类孢子对MS培养基中无机盐含量、蔗糖含量、pH值的要求不同。孢子外被中的Ca2+、Mn2+和Mg2+,培养基中的Cd2+和La3+,以及孢子接种密度、萌发空间CO2含量也会对孢子萌发造成影响。(5)多数蕨类植物孢子在15-30℃可以萌发,最适萌发温度为25℃。(6)4℃和液氮储藏可以延长孢子寿命并保持较高萌发率。     

Natural soil spore banks — can they be used to retrieve lost ferns?

Some fern species form spore banks — reservoirs in the soil of viable spores which remain dormant while buried but germinate in light if brought to the surface. The recently discovered characteristics of these spore banks are described. Enough is now known to suggest that they might have a role in the conservation of endangered fern species as alternatives toex situ collections of sporophytes, gametophytes and spores, the relative merits of which are also considered. Mature sporophytes of several British species have now been raised from natural spore banks in soil samples; if this proves to be possible also for endangered species, some interesting options become available. The possibilities are discussed of augmenting surviving populations and increasing their genetic diversity, even perhaps of retrieving lost populations, by reintroduction of spore bank-derived plants or by stimulating regeneration from spore banksin situ. Botanic gardens are well placed to provide the further research, the regular monitoring of endangered populations, and the taxonomic and horticultural support required to realise these possiblities.     

同形鳞毛蕨成精子囊素系统的初步研究

研究了同形鳞毛蕨成精子囊素对该种和水蕨孢子萌发和配子体发育的影响,结果表明：同形鳞毛蕨配子体能产生成精子囊素,该成精子囊素能抑制同种孢子的萌发,抑制作用随配子体成熟度的增加而增强;同形鳞毛蕨成精子囊素还可促进同种孢子发育为雄配子体;光照条件下,同形鳞毛蕨成精子囊素对水蕨孢子萌发和配子体发育影响不大,黑暗条件下,同形鳞毛蕨成精子囊素能显著的促使水蕨孢子提早萌发,但都不影响其孢子最终萌发率和配子体的性别分化,表明同形鳞毛蕨和水蕨的成精子囊素不属于同一系统。     

狭眼凤尾蕨配子体发育及其成精子囊素对水蕨配子体发育的影响

对狭眼凤尾蕨（Pteris biaurita）配子体发育特征及其外源成精子囊素对模式植物水蕨（Ceratopteris thalictroides）在黑暗和光照条件下孢子萌发和配子体发育的影响进行了研究。结果表明：（1）狭眼凤尾蕨孢子深褐色,三裂缝,孢子萌发为书带蕨型,原叶体发育为水蕨型,无毛状体产生;培养发现,其配子体能产生精子器,但不产生颈卵器,当接种密度适中时,可进行无配子生殖。（2）在光照和黑暗条件下狭眼凤尾蕨成精子囊素有促进和抑制水蕨孢子萌发的作用,但效果均不显著。（3）在光照条件下,狭眼凤尾蕨成精子囊素可以延迟水蕨心脏形配子体分生组织缺刻的形成,但对其配子体形态和性别分化无明显影响;而在黑暗条件下狭眼凤尾蕨成精囊素对水蕨长条形配子体的形态发育具有一定影响,与对照组相比其顶端分生组织发达,整体呈长楔形,对性别分化影响不显著。可见,狭眼凤尾蕨和水蕨不具有同种成精子囊素系统。     

Genetics, phylogenetics, and biogeography: Considering how shifting paradigms and continents influence fern diversity

About 25 years ago, a revolution began in evolutionary studies of seed-free vascular plants. Whereas common wisdom and laboratory-based observations had averred that minute spores and inbreeding of individual bisexual gametophytes diminished barriers to long distance migration, genetic analyses of sporophyte populations demonstrated outcrossing breeding systems that required two spores for each successful migration event. After those population-based discoveries, the processes controlling biogeographic patterns of ferns appeared to resemble those of seed plants, and vicariance took on renewed significance. More recently, data from DNA sequencing predicted that some of the most diverse extant fern families originatedafter the isolation of major land masses, and these new hypotheses also demanded fresh consideration of biogeographic assumptions. The Polypodiaceae yield phylogenetic insights through integration of DNA sequence analysis and biogeography. New evidence shows separate yet simultaneous radiations in the New and Old Worlds. By combining sequence data, vicariance, and a reassessment of morphological features, new family and generic boundaries are obtained. Contributors to this symposium discovered similar patterns in the systematics and biogeography of the seed-free vascular plants they studied. Although long distance migration remains an important factor in explaining fern distributions, local and recent radiations that result in species complexes are also significant in explaining fern biogeography.     

The Spontaneous Formation of Antheridia in Onoclea is not Blocked by Light

Onoclea sensibilis gametophytes were grown from spores on ashedsoil and agar to determine if the spontaneous formation of antheridiacan be blocked by light. Under most conditions, dark-grown gametophytesformed antheridia later than or at the same time as gametophytesgrown in the light. Under no circumstances was there a rapidonset of maleness in the dark. These results contradict thehypothesis that, in Onoclea, antheridiogen is required to inducemaleness because light inhibits the formation of antheridia.In the light, antheridia formed on heart-shaped thalli. In darkness,antheridia formed on filamentous gametophytes. The timing ofonset of maleness was affected by temperature and the presenceof sucrose. The effect of sucrose on the comparison betweenlight and dark treatments depended on both substrate and temperature Onoclea sensibilis, L., sensitive fern, fern gametophytes, sexuality, light-induced block     

DIFFERENTIAL ANTHERIDIOGEN RESPONSE AND EVOLUTIONARY MECHANISMS IN CYSTOPTERIS

It has been demonstrated that antheridiogens determine gametangial initiation in cultures of some homosporous fern gametophytes. Studies of natural populations have implicated these compounds as breeding system controllers. In Cystopteris, response to antheridiogen varied significantly among species. While Cystopteris protrusa responded to exogenous antheridiogen, C. bulbifera did not. The allopolyploid derivative of these two species, C. tennesseensis, had an intermediate level of response. This marked physiological difference among morphologically similar taxa is valuable in delineating species and suggests a mechanism that may be associated with interspecific hybridization.     

环境因子对中华双扇蕨孢子萌发的影响

为了解珍稀濒危植物中华双扇蕨(Dipteris chinensis)濒危原因,研究了温度和湿度对其孢子萌发的影响.结果表明,中华双扇蕨孢子萌发能力较强,萌发方式为Vittaria型,配子体萌发为Marattia型.中华双扇蕨孢子繁殖不受温度影响,而湿度显著影响孢子繁殖过程,湿润环境中孢子正常萌发,并形成心形配子体,顺利...     

Antheridiogen production and response in Polypodiaceae species

Antheridiogen chemicals secreted by living fern gametophytes have been shown to influence production of male gametangia and thus mating systems in a large number of terrestrial fern species. Antheridiogens have not previously been thought to be prevalent in the Polypodiaceae, a large family composed mostly of tropical epiphytes. This study presents bioassay methods more sensitive than previously used to detect antheridiogen and demonstrates that antheridiogens are also operative in the Polypodiaceae and in epiphytic species. Seven species in six genera (Campyloneurum angustifolium, C. phyllitidis, Lepisorus thunbergianus, Microgramma heterophylla, Phlebodium aureum, Phymatosorus scolopendria, and Polypodium pellucidum) were tested for the presence of an antheridiogen system. All species tested except P. aureum were induced to produce antheridia precociously by their own antheridiogen and by that of Pteridium aquilinum (APt). Phlebodium aureum responded to APt and promoted antheridium formation in Onoclea sensibilis but did not respond to its own antheridiogen. Spores of all species except P. aureum were induced to germinate in darkness by antheridiogen of the same species and by APt and to form antheridia in the dark, further enhancing the possibility of intergametophytic mating.     

Characterization and genetic analysis of antheridiogen-insensitive mutants in the fern Ceratopteris

WARNE, T. R., HICKOK, L. G. & SCOTT, R. S., 1988. Characterization and genetic analysis of antheridiogen-insensitive mutants in the fern Ceratopteris . The pheromone antheridiogen mediates the differentiation of male gametophytes in the fern Ceratopteris . Mutants insensitive to antheridiogen were isolated using an in vitro selection procedure. Antheridiogen-insensitive mutants exhibited partial or complete insensitivity to antheridiogen, but were normal in all other respects. Two mutants were completely insensitive to antheridiogen, whereas, another mutant was insensitive to supplemented antheridiogen, but produced male gametophytes in multispore cultures. Genetic analysis suggested a single gene basis for each mutant.     

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.     

Morphological functions of gibberellins in leptosporangiate fern gametophytes: insights into the evolution of form and gender expression

In addition to genetic load, the induction of maleness in leptosporangiate gametophytes by the pheromone antheridiogen may facilitate rates of outcrossing similar to those found in angiosperms. The antheridiogens that have been chemically identified are similar to gibberellins and probably evolved from this common plant hormone. The purposes of this study were to determine the functions of endogenous gibberellins in morphological development and gender expression in leptosporangiate fern gametophytes and to elucidate how antheridiogens may have evolved from gibberellin precursors. We grew gametophytes of Osmunda regalis and Athyrium filix‐femina on nutrient agar enriched with APOGEE, which blocks gibberellin synthesis. Osmunda regalis is a member of Osmundaceae, the only family in Osmundales, the sister group of all remaining extant leptosporangiate ferns. This family possesses a male‐first gender in isolation and lacks any known antheridiogen systems. In contrast, A. filix‐femina is a member of a derived family, Woodsiaceae, which possesses antheridiogen systems. Disruption of gibberellin synthesis retarded notch development and expression of both maleness and femaleness (i.e. gender status) in both species. On the basis of these results, we offer a simple model of gametophyte evolution driven by gender‐based fitness gain curves and the influence of exogenous gibberellins. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 599–615.     

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.

     

HAPLOID PARTHENOGENETIC SPOROPHYTES OF LAMINARIA JAPONICA (PHAEOPHYCEAE)1

Parthenogenetic sporophytes were obtained from three strains of Laminaria japonica Areschoug. These sporophytes grew to maturity in the sea, producine spores that all grew into female gametophytes. These female gametophytes gave rise to another generation of parthenogenetic sporophytes during the next year, so that by the year 1990 parthenogenetic sporophytes had been cultivated for 12, 9, and 7 generations, respectively, for the three strains. When female gametophytes from parthenogenetic sporophytes were combined with normal male gametophytes, normal sporophytes that reproduced and gave rise to both female and male gametophytes were obtained. The parthenogenetic sporophytes were shorter and narrower than the normal sporophytes of the same strain. Chromosome counts on mature sporophytes showed that normal sporophytes (from fertilized eggs) were diploid (2n = approximately 40) and that the spores they produced were haploid (n = approximately 20), while nuclei from both somatic and sporangial cells in parthenogenetic sporophytes were haploid. All gametophytes were haploid. Young sporophytes derived from cultures with both female and male gametophytes were diploid, while young, sporophytes obtained from female gametophytes from parthenogenetic sporophytes had haploid, diploid, or polyploidy chromosome numbers. Polyploidy was associated with abnormal cell shapes. The presence of haploid parthenogenetic sporophytes should be use in breeding kelp strains with useful characteristics, since the sporophyte phenotype is expressed from a haploid genotype which can be more readily selected.     

ABNORMAL REDUCTIONAL AND NONREDUCTIONAL MEIOSIS IN CERATOPTERIS: ALTERNATIVES TO HOMOZYGOSITY AND HYBRID STERILITY IN HOMOSPOROUS FERNS

Natural and synthesized hybrids of Ceratopteris were investigated to determine the effect of hybridization on the genetic system. Studies indicated that the hybrids exhibited massive spore abortion and pairing abnormalities at meiotic prophase, characteristic of “sterile diploids and triploids” reported in hybridization studies of other fern genera. However, a small percentage of viable spores also was produced by the hybrids. Cytological investigations indicated the presence of previously unreported meiotic adaptations that allowed the production of unreduced spores and reduced spores exhibiting chromatid heterozygosity. The reduced spores allow haploid gametophytes to form heterozygous zygotes in spite of intragametophytic selfing. The unreduced spores were shown to be responsible for the fertility of the “sterile” hybrid and allowed the subsequent production of up to three generations of sporophytes. The literature suggests that these meiotic adaptations are present in other fern genera and may play a significant role in evolution through hybridization.     

The composition of lipids and fatty acids determined at various stages of haploid and diploid generations in the fern Adiantum capillus-veneris

The composition of lipids and fatty acids was determined at various developmental stages of gametophytes and sporophytes in the fern Adiantum capillus-veneris L. When the lipid content was expressed per g fresh weight, triacylglycerol was a major reserve lipid in spores and remained the dominant lipid in the gametophytes up to the 15th day after germination, but it was a minor one in the pinnae of sporophytes. In contrast, triterpenoids were abundant in sporophytes but not detected in gametophytes. When the content of fatty acids was expressed in percentage of the total fatty acids, the content of arachidonic acid in phosphatidylcholine and linolenic acid in diacyglyceryltrimethylhomoserine and phosphatidylcholine was significantly higher in both pinnae and petioles of sporophtes than in gametophytes. Significant differences in fatty acid composition of phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and triacylglycerol were also noted between gametophytes and pinnae of sporophytes. All these differences were attributed to the difference of generation rather than tissue differences or age.
The content of monogalactosyldiacylglycerol, hexadecatrienoic acid in monogalactosyldiacylglycerol, and 3- trans hexadecenoic acid in phosphatidylglycerol was higher in the pinnae of sporophytes than in gametophytes or in young leaves of sporophytes. Electron microscopy of the ultrastructure of chloroplasts indicated that the high content of these components was consistent with the development of chloroplasts in pinnae.     

Efficient induction of apospory and apogamy in vitro in silver fern (Pityrogramma calomelanos L.)

Silver fern (Pityrogramma calomelanos L.) is a terrestrial or lithophytic herbaceous fern used for ornamental and medicinal purposes. In its farina it produces the cytotoxic and anticancer compound dihydrochalcone. In vitro induction of apospory and apogamy, and direct field establishment of aposporous gametophytes and subsequent sporophyte development has been accomplished. Half-strength Murashige and Skoog (MS) medium with 3.33 μM N⁶-benzyladenine (BA) and 2.32 μM kinetin (Kn) showed earlier development and produced higher numbers of aposporous gametophytes than half-strength MS basal medium. Crozier explants developed higher numbers (mean value 29.2) of gametophytes, but were slower than frond explants (mean value 23.2). The gametophytes originated from the epidermal hairs progressed from uniseriate filamentous to cordate through bi-, tri- and multiseriate and spatulate stage with the development of antheridia. Reduction in the nutrient and sucrose concentrations in the media favoured apogamy. Sucrose-free 1/10 strength MS medium and agar plates developed a mean of 30.4 and 29.9 sporophytes, respectively in the light. The greenhouse-established gametophytes developed sporophytes. The established sporophytes ex vitro showed 95% survival rate. Apogamous sporophytes and the source plant showed the same chromosome numbers (2n=116). The established protocol accomplishes apogamy and apospory in silver fern, and the aposporous gametophytes can be used for genetic transformation and development of transgenic silver fern.     

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.