Generating Autotetraploid Sporophytes and Their Use in Analyzing Mutations Affecting Gametophyte Development in the Fern Ceratopteris

The haploid gametophytes of the fern Ceratopteris richardii are autotrophic and develop independently of the diploid sporophyte plant. While haploid genetics is useful for screening and characterizing mutations affecting gametophyte development in Ceratopteris, it is difficult to assess whether a gametophytic mutation is dominant or recessive or to determine allelism by complementation analysis in a haploid organism. This report describes how apospory can be used to produce genetically marked polyploid sporophytes whose gametophyte progeny are heterozygous for mutations affecting sex determination in the gametophyte and a known recessive mutation affecting the phenotype of both the gametophyte and sporophyte. The segregation ratios of wild-type to mutant phenotypes in the gametophyte progeny of polyploid sporophyte plants indicate that all of the mutations examined are recessive. The presence of many multivalents and few univalents in meiotic chromosome preparations of spore mother cells confirm that the sporophyte plants assayed are polyploid. The DNA content of the sperm of their progeny gametophytes was also found to be approximately twice that of sperm from wild-type haploid gametophytes.     

THE OCCURRENCE AND SIGNIFICANCE OF EPISTATIC VARIANCE FOR QUANTITATIVE CHARACTERS AND ITS MEASUREMENT IN HAPLOIDS

Epistatic genetic variance for quantitative traits may play an important role in evolution, but detecting epistasis in diploid organisms is difficult and requires complex breeding programs and very large sample sizes. We develop a model for detecting epistasis in organisms with a free-living haploid stage in their life cycles. We show that epistasis is indicated by greater variance among families of haploid progeny derived from individual diploids than among clonally replicated haploid sibs from the same sporophyte. Simulations show that the power to detect epistasis is linearly related to the number of sporophytes and the number of haploids per sporophyte in the dataset. We illustrate the model with data from growth variation among gametophytes of the moss, Ceratodon purpureus. The experiment failed to detect epistatic variance for biomass production, although there was evidence of additive variance.     

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.     

ALTERNATION OF GENERATIONS IN LAND PLANTS: NEW PHYLOGENETIC AND PALAEOBOTANICAL EVIDENCE

Current ideas on the evolution of alternation of generations in land plants are reviewed in the context of important recent advances in plant systematics and the discovery of remarkable new palaeobotanical evidence on early embryophyte life cycles. An overview of relationships in major groups of green plants is presented together with a brief review of the early fossil record as a prelude to discussing hypotheses of life cycle evolution. Recent discoveries of life cycles in the early fossil record are described and assessed. The newly discovered gametophyte and sporophyte associations are based on exceptionally well-preserved material from the Rhynie Chert, Scotland (Middle Devonian: 380–408 Myr) and compression fossils from other Devonian localities. These data document diplobiontic life cycles in plants at the ‘protracheophyte’ and early tracheophyte level of organization. Furthermore, the early fossils have a more or less isomorphic alternation of generations, a striking departure from life cycles in extant embryophytes. This unexpected similarity between gametophyte and sporophyte calls for a cautious approach in identifying ploidy level in early groups. Viewed in a systematic context, the neontological and palaeontological data contribute towards the formulation of a coherent hypothesis of life cycle evolution in major, early embryophyte groups. Evidence from extant groups strongly supports a single direct origin of the diplobiontic life cycles of land plants from haploid, haplobiontic life cycles in ancestral ‘charophycean algae’. The interest of the new palaeobotanical data lies in its relevance to life cycle evolution at the restricted level of vascular plants rather than at the more general level of embryophytes (vascular plants plus ‘bryophytes’). The occurrence of morphologically complex, axial gametophytes in early vascular plants is consistent with the moss sister-group proposed in some cladistic analyses. Similarities of moss gametophytes to fossils in the vascular plant stem-group are discussed, and it is argued that the late appearance of mosses in the macrofossil record may be due to the problem of recognizing stem-group taxa. The new palaeobotanical evidence conflicts with previous hypotheses based on extant groups that interpret morphological simplicity as the plesiomorphic condition in the gametophytes of vascular plants. These new data indicate that a significant elaboration of both gametophyte and sporophyte occurred early in the tracheophyte lineage, and that the gametophytes of extant ‘pteridophytes’ are highly reduced compared to those of some of the earliest ‘protracheophytes’. Vestiges of this early morphological complexity may remain in the gametophytes of some extant groups such as Lycopodiaceae.     

Arsenic hyperaccumulation in gametophytes of Pteris vittata. A new model system for analysis of arsenic hyperaccumulation

The sporophyte of the fern Pteris vittata is known to hyperaccumulate arsenic (As) in its fronds to >1% of its dry weight. Hyperaccumulation of As by plants has been identified as a valuable trait for the development of a practical phytoremediation processes for removal of this potentially toxic trace element from the environment. However, because the sporophyte of P. vittata is a slow growing perennial plant, with a large genome and no developed genetics tools, it is not ideal for investigations into the basic mechanisms underlying As hyperaccumulation in plants. However, like other homosporous ferns, P. vittata produces and releases abundant haploid spores from the parent sporophyte plant which upon germination develop as free-living, autotrophic haploid gametophyte consisting of a small (<1 mm) single-layered sheet of cells. Its small size, rapid growth rate, ease of culture, and haploid genome make the gametophyte a potentially ideal system for the application of both forward and reverse genetics for the study of As hyperaccumulation. Here we report that gametophytes of P. vittata hyperaccumulate As in a similar manner to that previously observed in the sporophyte. Gametophytes are able to grow normally in medium containing 20 mm arsenate and accumulate >2.5% of their dry weight as As. This contrasts with gametophytes of the related nonaccumulating fern Ceratopteris richardii, which die at even low (0.1 mm) As concentrations. Interestingly, gametophytes of the related As accumulator Pityrogramma calomelanos appear to tolerate and accumulate As to intermediate levels compared to P. vittata and C. richardii. Analysis of gametophyte populations from 40 different P. vittata sporophyte plants collected at different sites in Florida also revealed the existence of natural variability in As tolerance but not accumulation. Such observations should open the door to the application of new and powerful genetic tools for the dissection of the molecular mechanisms involved in As hyperaccumulation in P. vittata using gametophytes as an easily manipulated model system.     

CONTROL OF SEX RATIOS IN HAPLOID POPULATIONS OF THE MOSS,CERATODON PURPUREUS

Female biased sex ratios occur in a number of unrelated mosses. Such ratios refer to the relative numbers of male and female gametophytes in moss populations and are therefore more comparable to the numbers of pollen grains and ovules in populations of seed plants than to the numbers of male (microsporangiate) and female (megasporangiate) sporophytes. A survey of 11 populations of the moss, Ceratodon purpureus, showed that sex ratios are heterogeneous, but that female biases occur in more than half the populations. One hundred and sixty single spore isolates representing 40 sporophytes from one population demonstrated that female gametophytes outnumbered males by a ratio of 3:2 at the time of germination. Female gametophytic clones formed significantly more biomass than male clones, and individual female shoots were more robust. Male clones, however, produced more numerous stems. These sexually dimorphic traits may be related to life history differences between male and female gametophytes since females must provide nutritional support to the “parasitic” sporophyte generation, a burden that males do not share.     

Family affiliation,sex ratio and sporophyte frequency in unisexual mosses

Patterns of sex expression and sex ratios are key features of the life histories of organisms. Bryophytes are the only haploid‐dominant land plants. In contrast with seed plants, more than half of bryophyte species are dioecious, with rare sexual expression and sporophyte formation and a commonly female‐biased sex ratio. We asked whether variation in sex expression, sex ratio and sporophyte frequency in ten dioecious pleurocarpous wetland mosses of two different families was best explained by assuming that character states evolved: (1) in ancestors within the respective families or (2) at the species level as a response to recent habitat conditions. Lasso regression shrinkage identified relationships between family membership and sex ratio and sporophyte frequency, whereas environmental conditions were not correlated with any investigated reproductive trait. Sex ratio and sporophyte frequency were correlated with each other. Our results suggest that ancestry is more important than the current environment in explaining reproductive patterns at and above the species level in the studied wetland mosses, and that mechanisms controlling sex ratio and sporophyte frequency are phylogenetically conserved. Obviously, ancestry should be considered in the study of reproductive character state variation in plants. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 163–172.     

Alternation of generations – unravelling the underlying molecular mechanism of a 165‐year‐old botanical observation

Characteristically, land plants exhibit a life cycle with an ‘alternation of generations’ and thus alternate between a haploid gametophyte and a diploid sporophyte. At meiosis and fertilisation the transitions between these two ontogenies take place in distinct single stem cells. The evolutionary invention of an embryo, and thus an upright multicellular sporophyte, in the ancestor of land plants formed the basis for the evolution of increasingly complex plant morphologies shaping Earth's ecosystems. Recent research employing the moss Physcomitrella patens revealed the homeotic gene BELL1 as a master regulator of the gametophyte‐to‐sporophyte transition. Here, we discuss these findings in the context of classical botanical observations.     

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 GENETIC STRUCTURE OF SPOROPHYTIC AND GAMETOPHYTIC POPULATIONS OF THE MOSS,FUNARIA HYGROMETRICA HEDW

Patterns of phenotypic and genotypic variability in two populations of the moss, Funaria hygrometrica, were investigated using measurements of gametophytic and sporophytic morphology, sporophytic reproductive output, spore germination, gametophytic growth rates and tolerances of copper, cadmium, and low nutrient conditions, and electrophoretically detectable enzyme variation. The two populations differed in all traits measured, but complete monomorphism within populations at 14 enzyme loci suggested that each represented a single clone. Variability in gametophytic growth rates and responses to different experimental media, however, occurred among haploid sib families (families of meiotic progeny derived from the same sporophyte) and among sibs within families within both populations, suggesting high levels of genetic variability. Low mean reproductive output and a high level of variability among sporophytes in a mine site population probably reflected heavy metal toxicity. Based on this study, in combination with previous work on F. hygrometrica (Shaw, 19906), somatic mutation and/or nongenetic effects appear to contribute significantly to phenotypic variability in natural populations.     

Sexual conflict and the alternation of haploid and diploid generations

Land plants possess a multicellular diploid stage (sporophyte) that begins development while attached to a multicellular haploid progenitor (gametophyte). Although the closest algal relatives of land plants lack a multicellular sporophyte, they do produce a zygote that grows while attached to the maternal gametophyte. The diploid offspring shares one haploid set of genes with the haploid mother that supplies it with resources and a paternal haploid complement that is not shared with the mother. Sexual conflict can arise within the diploid offspring because the offspring's maternal genome will be transmitted in its entirety to all other sexual and asexual offspring that the mother may produce, but the offspring's paternally derived genes may be absent from these other offspring. Thus, the selective forces favouring the evolution of genomic imprinting may have been present from the origin of modern land plants. In bryophytes, where gametophytes are long-lived and capable of multiple bouts of asexual and sexual reproduction, we predict strong sexual conflict over allocation to sporophytes. Female gametophytes of pteridophytes produce a single sporophyte and often lack means of asexual reproduction. Therefore, sexual conflict is predicted to be attenuated. Finally, we explore similarities among models of mate choice, offspring choice and segregation distortion.     

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.     

Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.Key words: cytokinin, cytokinin receptor, female gametophyte, male gametophyte, stigma     

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.     

安徽大龙山国家森林公园东亚小金发藓配子体与孢子体个体性状及其相互关系

植物性状是植物对环境变化响应和适应的综合反映。目前对苔藓植物个体水平功能性状的研究较为匮乏。以安徽大龙山国家森林公园分布的东亚小金发藓（Pogonatum inflexum）雌株为例,在其孢子体成熟期测定地上部分孢子体和配子体的形态特征及生物量,系统分析了植株性状变异特征、异速生长关系及协变（整合）格局。结果表明,东亚小金发藓配子体形态性状变异性高于孢子体,且二者生物量变异系数最大。孢子体高度是配子体的2倍,但其生物量仅占地上部分的23%。孢子体和配子体功能性状之间有一定相关性;形态性状（Y）与生物量（X）之间多为指数<1.0异速生长关系,而孢子体和配子体生物量之间为等速生长关系（指数为1.135±0.158）。孢子体生物量分配比例随个体增大而显著减小,体现出显著的负的个体大小依赖。主成分分析表明,东亚小金发藓孢子体和配子体主要个体性状具有不同的协变方向（即两个不同的性状群）,其中孢子体生物量是两类性状群的关键结点。综合来看,东亚小金发藓个体性状间具有与维管植物相似的协变关系。     

Nuclear behavior, cell polarity, and cell specification in the female gametophyte

In flowering plants, the haploid gamete-forming generation comprises only a few cells and develops within the reproductive organs of the flower. The female gametophyte has become an attractive model system to study the genetic and molecular mechanisms involved in pattern formation and gamete specification. It originates from a single haploid spore through three free nuclear division cycles, giving rise to four different cell types. Research over recent years has allowed to catch a glimpse of the mechanisms that establish the distinct cell identities and suggests dynamic cell–cell communication to orchestrate not only development among the cells of the female gametophyte but also the interaction between male and female gametophytes. Additionally, cytological observations and mutant studies have highlighted the importance of nuclei migration- and positioning for patterning the female gametophyte. Here we review current knowledge on the mechanisms of cell specification in the female gametophyte, emphasizing the importance of positional cues for the establishment of distinct molecular profiles.     

FERTILIZATION SUCCESS CAN DRIVE PATTERNS OF PHASE DOMINANCE IN COMPLEX LIFE HISTORIES1

Many algal life cycles alternate between two free‐living generations. Life histories in which the two generations look identical (isomorphic) are common, particularly in the Rhodophyta. Reports of natural populations dominated by one generation of the life history have sought explanation in terms of phase‐specific differences in mortality and reproductive output, yet in many cases identification of these adaptations has been elusive or inconsistent with predictions. We hypothesized that the gametophyte‐to‐sporophyte ratio of ecologically equivalent isomorphs could result from variation in fertilization rate. We developed two models to test this hypothesis: one representing a generalized isomorphic life history and the other specific to red algae with a Polysiphonia‐type life history. Fertilization rate affected the gametophyte‐to‐sporophyte ratio, especially at low fertilization rates. In the general model, gametophytes dominated the population regardless of fertilization rate unless egg production greatly exceeded meiospore production. In the red algal model, phase dominance depended on the combination of fertilization rate and the number of carpospores produced per fertilization. The generational composition of model multiphasic algal populations results from their inherent reproductive characteristics and the dynamic environment to which fertilization and mortality rates are tied.     

GENETIC LOAD IN OSMUNDA REGALIS POPULATIONS

Osmunda regalis sporophytes form haploid spores which develop into functionally hermaphroditic gametophytes. The self-fertilization of such gametophytes results in zygotes which are completely homozygous. Spore samples collected from sporophytes in natural populations were used to establish gametophyte cultures. The majority of these gametophytes were unable to form viable embryos when only self-fertilization was possible. Controlled selfing and crossing experiments revealed that the inability of these homozygous embryos to develop normally is attributable to the presence of recessive lethals. To account for this genetic load, an hypothesis is proposed integrating the morphology and ecology of the gametophyte generation with the polyploid genetic system of the sporophyte generation.