Comparative morphology of the gametophytes of Equisetum subgenus Hippochaete and the sexual behaviour of E. ramosissimum subsp. debile, (Roxb.) Hauke, E. hyemale var. affine (Engelm.) A.A., and E. laevigatum A. Br.

Gametophytes of Equisetum , subgenus Hippochaete , species E. hyemale, E. ramosissimum, E. laevigatum, E. variegatum and E. scirpoides are morphologically distinct. Lamellae from female branches and the archegonial neck cells provide a range of diagnostic features but antheridia are uniform throughout this subgenus, unlike the subgenus Equisetum , in which antheridial morphology is the most reliable criterion for specific identification of gametophytes. The classification of Equisetum into two subgenera based on sporophytic features is supported by gametophyte morphology: column lamellae and sunken antheridia with two opercular cells occur in Hippochaete whereas plate lamellae and projecting antheridia prevail in subgenus Equisetum. The absence of well-marked discontinuities in gametophyte morphology in Hippochaete reinforces cytologica! and hybridization data indicating that the taxa are more closely interrelated man in the subgenus Equisetum. No differences in gametophyte morphology were detected at the intraspecific level. Neither gametophyte morphology nor sexuality provide any definitive data to support me theory that Hippochaete contains the most primitive extant horsetails.
Sexuality in E. ramosissimum subsp. debile, E. hyemale var. affine and E. laevigatum is far more labile than in other taxa of Equisetum. Both male and female secondary gametophytes may be derived from initially male or female individuals as a result of lamellar proliferation.     

Sexual behaviour of the genus Equisetum, subgenus Equisetum

Gametophytes of five species of Equisetum that were studied in axenic single spore cultures are initially male or female, and the first antheridia appear on the males before the first arche-gonia on the females. The males never produce archegonia, but the females all produce antheridia later in progressively increasing numbers. The proportions of male and bisexual gametophytes are relatively constant within each species but vary greatly between species. Likewise between species there is great variation in the rate at which females later produce antheridia. Male gametophytes are smaller and grow more slowly than females, but individuals of both sexes live for the same length of time. After prolonged culture, growth rates fall to very low levels and there is necrosis of the older parts of the gametophytes; but sex organs are still produced. Initially female gametophytes have much sexually uncommitted tissue, whereas males are composed almost entirely of antheridial branches. The absence of archegonium formation on males may well be related to this lack of sexually undifferentiated tissue and the fact that the growth of the males is intimately connected with antheridium production.     

Antheridogen activity in the fern Ceratopteris thalictroides (L.) Brongn.

Spores of the homosporous fern Ceratopteris thalictroides , in multispore culture, initially produce spatulate gametophytes bearing only antheridia (males) and cordate gametophytes bearing both antheridia and archegonia (hermaphrodites). When multispore cultures are sampled, the ratio of male to hermaphroditic gametophytes is a constant for each population examined. Four possible causes of such a sex ratio (cytoplasmic inheritance, nuclear inheritance, incipient heterospory and an antheridogen) are investigated. Evidence presented indicates that an antheridogen causes the existence of two gametophyte types, while one or more cytoplasmic units are the probable cause of the sex ratio. The activity of the antheridogen is to cause potentially hermaphroditic plants to become male. This activity was elucidated in monospore culture. Populational differences in antheridogen activity are also demonstrated. The significance of antheridogens is discussed in relation to the mating system of these plants.     

不同环境因子对珍稀观赏蕨类金毛狗配子体性别分化的影响

金毛狗[Cibotium barometz(L.)J.Sm.]是珍稀观赏蕨类的重要类群,为国家二级重点保护野生植物。该研究以金毛狗孢子为试验材料,探究培养密度、外源赤霉素以及光质等不同环境因子对金毛狗配子体性别分化的影响,为金毛狗人工繁育和蕨类植物配子体性别决定机制研究提供技术支持。结果表明:(1)低配子体培养密度(1个/cm2和5个/cm2)有利于颈卵器和雌配子体形成,随着配子体培养密度增加,颈卵器平均数量及雌配子体比率下降,精子器平均数量以及雄配子体和两性配子体比例增加,但配子体培养密度过高(80个/cm2)会导致大量无性配子体产生。(2)不同配子体培养密度下,随着培养时间延长,两性配子体比率均有增加,且增加幅度基本一致。(3)外源GA4显著抑制颈卵器和雌配子体形成,并显著促进精子器和雄配子体形成;外源GA3对金毛狗配子体性别分化没有显著影响。(4)白光、红光、蓝光等不同光质对金毛狗配子体性别分化未产生显著影响,但会影响配子体的发育和形态建成。     

An experimental study of the reproductive biology and hybridization in the European and North American species of Equisetum

Sporophytc production was investigated in the eleven North American and European species of Equisetum by means of fertilization experiments on both intact and subcultured gametophytes. Comparison of intra- and inter-gametophytic selfing with intergametophytic crossing failed to reveal any evidence of recessive sporophytic lethals, leaky lethals or deleterious recessive genes. Fertilization in Equisetum depends solely on the availability of ripe archegonia. Despite the absence of detectable genetic load in Equisetum , intergametophytic mating is ensured by gametophyte dioecism in the wild. All populations tested were freely interfcrtile indicating a high degree oi genetic con-specihcity, even between North America and Europe. Low sporophyte frequencies in European E. scirpoides are attributed to defective spermatozoids. Polyembryony occurs. Cessation in gametophyte development is mediated by allelopathic compounds liberated from the young sporophytes.
Reciprocal crosses resulted in the synthesis of 12 hybrids (six in subgenus Equisetum and six in subgenus Hippochaete plus abortive zygotes in the cross E. ramosissimum female x E. scirpoides male), 11 of which had previously been reported in nature. No intersubgeneric hybrids were produced, indicating that both subgenera are natural groups only distantly related to one another. The putative wild combinations E. pratense x E. atvense and E. variegatum x E. scirpoides were not synthesized, but a new hybrid from the allopatric E. ramosissimum and E. laevigatum arose. Reciprocal crosses within the subgenus Hippochaete usually yielded equal frequencies of hybrids, but in the subgenus Equisetum the majority of successful crosses were unidirectional. All taxa oi Hippochaete are linked by hybrids whereas in subgenus Equisetum, E. pratense and E. sylvatkum form an isolated pair, Hybridization studies, and evidence from gametophyte morphology support the traditional sectional classification of Equisetum.     

Comparative morphology of the gametophytes of the genus Equisetum, subgenus Equisetum

When grown under a variety of axenic conditions gametophytes of E. arvense, E. telmateia, E. sylvaticum, E. palustre , and E. fluviatile are all morphologically distinct. Lamellae from archegonial branches provide several diagnostic features but lamellae associated with antheridia are much less distinct. The archegonia of all five species have two neck canal cells and three tiers of neck cells and are of limited use taxonomically. Although antheridium development is basically similar in all species, during the metamorphosis of the spermatocytes specific differences arise in the shape of the cavity, degree of emergence and the number of opercular cells. A taxonomy of the gametophytes based on antheridium morphology corresponds very closely to the classification of the subgenus Equisetum into three sections based on the coning behaviour of the sporophytes.     

Triploidy in Equisetum subgenus Hippochaete (Equisetaceae, Pteridophyta)

BACKGROUND AND AIMS: The genus Equisetum is cytologically uniform, having a base chromosome number of x = 108. All previously known species and hybrids that have been counted represent diploids with a sporophytic chromosome number of 2n = 216. Biosystematic studies on Equisetum subgenus Hippochaete revealed evidence that triploids occur in nature. The objective of this study was to confirm that triploid plants exist in the natural environment. METHODS: Flow cytometry was used to establish nuclear DNA values and cytological investigations of meiosis were carried out to obtain information on chromosome number and pairing behaviour. KEY RESULTS: Triploidy exists in three morphologically different hybrid taxa. Two of these are morphologically intermediate between a primary diploid hybrid and a parent, while the third apparently combines genomes from all three Central European Hippochaete species. Nuclear 1C DNA values for the four European Hippochaete species range from 21.4-31.6 pg. For the hybrids, the 1C DNA values not only occupy the same range as the species, but their total DNA amounts agree closely with values predicted by adding the 1C DNA values of each parental genome. Chromosome counts confirm diploidy in the species E. hyemale and E. variegatum and in the hybrid E. xtrachyodon (= E. hyemale x E. variegatum). For the triploids (2n approximately 324), cytological information is presented for the first time. CONCLUSIONS: Triploid taxa may have originated by backcrossing or by crossing of a diploid hybrid with an unrelated diploid species. As tetraploid plants are unknown, these crossings probably involve diploid gametophytes that developed from unreduced diplospores. By repeated crossing events or backcrossing, reticulate evolution patterns arise that are similar to those known for a number of ferns and fern allies.     

Reproductive biology and population dynamics of wild gametophytes ofEquisetum

This is d study of the reproductive biology of wild gametophytes of Equisetum arvense L., E. fluviatile L., and E. palustre L., which form extensive populations during the summer months on previously inundated bare mud along the margins of reservoirs and lakes in Northern England and Wales. The garnetophytes have all the characteristics of pioneer species: they rapidly attain sexual maturity and are adversely affected by competition. Early in the summer mixtures of male and female gametophytes are produced. The former are smaller and their frequency (which is always less) diminishes throughout the season as a result of bryophytic competition. The rarity of bisexual, initially female, gametophytes, in wild populations is related to the absence of metabolite accumulation which mediates this change in rulture. Significantly different sex ratios between populations and species underlines the labile nature of the sex-determining mechanism in Equisetum. The high frequency of females bearing sporophytes indicates that intergarnetophytic fertilization is highly effective in nature. Absence of any correlation between the incidence of fertilized females and the proportions of males, in conjunction with a consideration of the male gamete dispersal distance, suggests that sporophyte formation is restricted by the availability of ripe archegonia. Sexual reproduction in Equisetum is probably limited by the narrow range of conditions under which gametophytes can become established rather than availability of water for fertilization. The majority of females, which bear one sporophyte, are smaller than unfertilized or polyembryonic females. Their small size results from nutrient demands and allelopathic compounds from the sporophytes which also probably prevent the establishment of gametophytes within mature stands of the parent species. Correlations between female diameters and frequencies of males suggest that gametophytes are more likely to produce archegonia under favourable conditions. The natural reproductive biology of Equisetum is in accord with predictions based on an understanding of the mechanism of sex determination in axenic culture. Several striking parallels between sexuality in Equisetum and dioecious angiosperms are revealed. The absence of winter flooding at two of the gametophyte sites led to the establishment of mature rtands of Equisetum, which produced cones after 3–4 years; two hybrids, E. fluviatile×E. arvense and E. fluviatile×E. palustre, were detected.     

The effects of methionine and cobalt ions on sex expression and development of <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

One of the prime precursor for ethylene synthesis — L-methionine and the inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) — Co²⁺-were tested for their effects on sex expression and development of Anemia phyllitidis fern gametophytes. Five concentrations of both chemicals (0, 10, 25, 50, 100 μM) were analysed with reference to antheridia and archegonia formation, number and size of cells as well as thalli length using the three-zone model of gametophyte structure. Both substances, however at different concentrations, enhanced the number of GA₃-induced antheridia and similarly stimulated the cell number and inhibited thalli length. Both of them at 100 μM concentrations without GA₃ induced meristematic area formation while methionine also induced archegonia in the apical parts of gametophytes. These findings correspond with the previous observations concerning the important role of ethylene synthesis precursor (ACC) in controlling gibberellic acid-induced male sex expression in ferns and broaden the knowledge about the mechanisms of fern gametophyte development.     

Towards an understanding of sex determination in Equisetum: an analysis of regeneration in gametophytes of the subgenus Equisetum

Equisetum gametophytes possess extensive powers of regeneration without showing any signs of diminished vigour or abnormal development. Isolated lamellae, pieces of antheridial and archegonial branches were used to analyse gametophyte morphogenesis and sex determination. Intact individuals and subcultured sexual branches never revert to a vegetative state. Transplantation of apical pieces of archegonial branches on to fresh medium delays the onset of antheridium formation indicating phenotypic control. On the other hand, male branches invariably continue producing antheridia. Experimentally induced adventitious branches on lamellae are interpreted as the result of physiological isolation from inhibitors. In intact individuals these adventitious branches are always antheridial: after subculturing, archegonial branches are also produced, demonstrating that the gametophytes of Equisetum are all potentially bisexual. Isolated lamellae regenerate secondary gametophytes, the sexual ratios between which vary between species, but lamellae from archegonial tissues invariably produce more female individuals, probably reflecting the different sizes of the respective lamellae. The view that mixtures of intact male and bisexual Equisetum gametophytes result from inherent physiological differences between the spores is not supported.     

Gibberellins and antheridiogen on sex in Blechnum spicant L.

The role of gibberellins (GAs) in determining sex in the gametophyte of the fern Blechnum spicant L. was studied through (a) the effect of exogenous GA₄₊₇ and GA₃ (b) quantitation of the endogenous levels of GA_1, GA₃, GA₄, GA₇, GA₉, and GA₂₀ in male and female gametophytes, and (c) the effect of flurprimidol, a GAs biosynthesis inhibitor of the steps of oxidation of ent-kaureno to ent-kaurenoic acid. Our results show that GA₄₊₇ had a slight effect of inducing either male or female sexual organs, antheridia and archegonia, respectively. The endogenous GAs content was not significantly different between sexes, but the GA₄, GA₇, and GA₂₀ levels were raised above those of the other GAs in both sexes. Neither antheridiogen biosynthesis nor antheridia formation was inhibited by flurprimidol. Gametophytes regenerated from homogenized mature gametophytes (HG) show a different physiological behavior than spore-derived gametophytes. In the first case, gametophytes are males and synthesize antheridiogen before they attain maturity, in contrast to what occurs in spore-derived gametophytes which are females and synthesize antheridiogen when mature.     

EFFECTS OF BENLATE ON FERN GAMETOPHYTE DEVELOPMENT

The widely used fungicide, benlate, was tested for its effect(s) on gametophyte development in the fern Ceratopteris thalictroides (L.) Brongn. The active ingredient of benlate, benomyl, represents 50% of the fungicide by weight. Seven concentrations of benomyl were tested on C. thalictroides gametophytes: 0, 0.001, 0.01, 0.1, 1.0, 10.0 and 100 mg/l. Five developmental stages were observed for possible effects of benomyl. These were 1) germination, and the initiation of 2) antheridia, 3) notch meristems, 4) archegonia, and 5) sporophytes. Overall inhibition was greatest at 100 mg/l benomyl. At 10 mg/l and 100 mg/l, sporophyte initiation was completely blocked. This was probably the consequence of two characteristics found only on gametophytes growing on medium containing 10 mg/l and 100 mg/l benomyl. These characteristics were lack of sperm motility and production of callus growths in the areas proximal to the notch meristems, just proximal to the younger archegonia. Besides blocking the completion of sexual reproduction, the highest concentrations tested also produced smaller (cell number) and chlorotic gametophytes (especially at 100 mg/l). The bigametophyte population (made up of hermaphroditic and male gametophytes) was changed from 51% hermaphrodites (at 0 mg/l benomyl) to 26% hermaphrodites at 100 mg/l. This would, since only hermaphrodites possess archegonia, also decrease the potential for the production of sporophytes.     

OCCURRENCE OF INTERCALARY AND UNINTERRUPTED MERISTEMS IN EQUISETUM

Quantitative data on cell length and mitotic index in elongating internodes of seven species of Equisetum are presented as evidence for the occurrence of two patterns of internode development in rhizomes of different species. In rhizomes of three species of subgenus Equisetum (E. arvense, E. diffusum, E. telmateia) uninterrupted meristems are present, characterized by acropetal internode maturation. In rhizomes of four species of subgenus Hippochaetae (E. hyemale, E. variegatum, E. scripoides, and E. laevigatum) intercalary meristems are present. The number and locations of intercalary meristems are described for an aerial shoot of E. diffusum. The absence or diminution of intercalary meristems from rhizomes of a variety of vascular plants with intercalary meristems in aerial shoots is discussed from the standpoint of adaptive significance.     

麦秆蹄盖蕨（Athyrium fallaciosum）配子体的发育规律及播种密度对配子体性别分化的影响

在组织培养条件下,对麦秆蹄盖蕨（Athyrium fallaciosum）配子体发育的连续过程进行了详细观察。结果表明:麦秆蹄盖蕨孢子为四面体型; 孢子萌发为书带蕨型（Vittaria-type）;原叶体发育为铁线蕨型（Adiantum-type）,成熟原叶体为对称的心形;精子器近圆球形,成熟颈卵器细长,常向原叶体基部倾斜或弯曲。常规播种条件下,发现麦秆蹄盖蕨配子体有雌配子体、雄配子体、雌雄同体配子体和无性配子体类型。配子体的性别随密度不同而呈现一定的变化趋势,雄配子体随密度增大呈上升趋势;雌配子体随密度增大先上升后下降;雌雄同体配子体和无性配子体随密度变化不大。雌配子体和雌雄同体配子体具颈卵器数目一般为10～15个;精子器数目随密度的增大逐渐减少,雄配子体中具有约50个精子器,雌雄同体配子体具有约20个精子器。     

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。     

蕨类植物性别分化对环境的响应

蕨类植物是维管植物中唯一的孢子体和配子体都能独立生活的类群.同型孢子蕨类配子体的性别分化受到激素和环境因子的影响.生理学研究表明,成精子囊素与赤霉素能诱导雄配子体发育,抑制雌配子体发育;脱落酸阻止成精子囊素诱导的精子器形成;乙烯合成前体ACC促进赤霉素诱导的精子器形成,而乙烯合成抑制因子AOA通过抑制细胞分化来抑制精子器形成.光照对不同种类蕨类配子体分化的影响存在差异.糖类能够促进雄配子体形成,并可加速成熟雌配子体向两性分化.钙离子、钴离子和甲硫氨酸等分别参与了蓝光和赤霉素对配子体性别分化的调控过程.培养密度影响配子体生长及性别表达,高密度下雄性和无性配子体居多,而低密度下两性和雌性配子体居多.近年来的突变体表型分析与分子生物学研究表明,成精子囊素通过影响ANI1、HER、TRA、FEM和MAN等基因的表达调控配子体性别分化.综述了蕨类植物性别分化对环境响应的研究进展.     

Antheridia and Archegonia of the Apogamous Fern Pteris cretica

The external appearance of gametophytes and gametangia of Pteriscretica closely resembled those of sexually reproducing ferns.Antheridia were fully functional but archegonia were not. Despitethe formation of egg, ventral and neck canal cells, the flask-shapedarchegonia failed to open and Pteris spermatozoids failed toexhibit a chemotactic response to them. Cryo SEM revealed thatthe topmost cells of mature archegonia had collapsed, and thisis thought to account for the loss of archegonial function. Pteris cretica, fern, apogamy, antheridia, archegonia, cryo SEM     

Gametophytic phase of Alsophila odonelliana (Cyatheaceae)

The gametophytic morphology and development of Alsophila odonelliana (Alston) Lehnert, have been studied through in vitro cultures. This species grows in southern Bolivia and northwestern Argentina. The spores are uniform in structure, but not in size; a certain percentage being smaller than the average. 16 spores per sporangium were found. The germination is of the Cyathea type. It was found that spores stored at 4°C can maintain their viability for over two years. The maximum value of germination depends on spore age. The filamentous gametophytes are 4–16 cells long. Young gametophytes have 1–2 branches that give rise to new gametophytes. Male, female, bisexual and neuter gametophytes were found. Propagules were frequently found in neuter gametophytes, and female and bisexual gametophytes were found to have chlorophyll containing scales. The antheridia are made up of five cells and produce non‐viable spermatozoids. The archegonia have necks formed by four columns with four cells each. Most of the gametophytic phase is documented with photomicrographs.     

GEMMIFEROUS FERN GAMETOPHYTES—VITTARIACEAE

Approximately 10 % of all fern species reproduce vegetatively in the gametophytic stage by means of gemmae. Gametophyte morphologies in these species depart radically from the commonly figured heart-shaped type and expand considerably the opportunities for physiological and morphological studies utilizing fern gametophytes. Original observations on four species of vittarioid ferns are presented and compared with earlier observations on gametophytes of this family. Vittarioid gametophytes grow from a discontinuous marginal meristem which results in a much branched thallus of indeterminant growth. Aerial branches of the gametophytes terminate in gemma production, which proceeds by a regular and predictable sequence of events. The sequence may differ considerably among species but is remarkably constant within species. Archegonia are produced on short ventral branches, and antheridia are produced primarily on germinating gemmae. Ananthacorus angustifolius is the only known member of the Vittariaceae which does not produce gemmae and is considered to represent the primitive condition. In this species antheridia are scattered over the thallus, suggesting that a change in the mode of control of antheridium production may have evolved in the family along with gemma production.     

Evidence for antheridogen production and its mediation of a mating system in natural populations of fern gametophytes

Gametophytes of Asplenium pimpinellifolium Fee and Lygodium heterodoxum Kze., occurring as natural populations in Veracruz, Mexico, were studied with respect to their spacing, size, and number of antheridia and archegonia. Small gametophytes, 0.3-2.2 mm in width and growing in colonies, usually had high numbers of antheridia. Gametophytes of the same size, growing at least 1 5 cm from the nearest neighbour, had few antheridia. In the colonial gametophytes there was a strong correlation between heavily antheridiate ones and their proximity to large archegoniate gametophytes. The data are the first to suggest the presence of an antheridogen system operating in nature, with concomitant opportunities for cross-fertilization. The rôle of polyploidy in storing genetic variation and the rôle of antheridogen-mediated out-crossing in releasing variation are seen as co-adaptive phenomena in the ferns.