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.     

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.     

Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization

Background

There is a heteromorphic alternative life in the brown seaweed, Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders ( = Laminaria japonica Aresch.), with macroscopic monoecious sporophytes and microscopic diecious gametophytes. Female gametophytes are genetically different from males. It is very difficult to identify the parent of a sporophyte using only routine cytological techniques due to homomorphic chromosomes. A sex-specific marker is one of the best ways to make this determination.

Methodology/Principal Findings

To obtain clear images, chromosome preparation was improved using maceration enzymes and fluorochrome 4′, 6-diamidino-2-phenylindole (DAPI). The chromosome number of both male and female haploid gametophytes was 31, and there were 62 chromosomes in diploid sporophytes. Although the female chromosomes ranged from 0.77 µm to 2.61 µm in size and were larger than the corresponding ones in the males (from 0.57 µm to 2.16 µm), there was not a very large X chromosome in the females. Based on the known female-related FRML-494 marker, co-electrophoresis and Southern blot profiles demonstrated that it was inheritable and specific to female gametophytes. Using modified fluorescence in situ hybridization (FISH), this marker could be localized on one unique chromosome of the female gametophytes as well as the sporophytes, whereas no hybridization signal was detected in the male gametophytes.

Conclusions/Significance

Our data suggest that this marker was a female chromosome-specific DNA sequence. This is the first report of molecular marker localization on algal chromosomes. This research provides evidence for the benefit of using FISH for identifying molecular markers for sex identification, isolation of specific genes linked to this marker in the females, and sex determination of S. japonica gametophytes in the future.     

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。     

Gametogenesis and chromosome number in Postelsia palmaeformis (Laminariales, Phaeophyceae)

Gametophytes of the ‘sea palm’, the kelp Postelsia palmaeformis Ruprecht, produced gametes whether or not chelated iron was supplied in the culture medium, in contrast to the inhibition of gametogenesis seen with the absence of iron in many other kelps. As gametogenesis proceeded, every cell of the gametophytes was converted into a gamete so that the gametophytes did not continue to grow vegetatively. The portion of the life history from spore release through germination, gametophyte growth, gametogenesis, fertilization and growth of the young sporophyte was completed in 9–10 days under laboratory conditions. Chromosome counts showed that sporophytes had a diploid number of 26–34 chromosomes while sporangia and gametophytes had a haploid number of 14–17 chromosomes, indicating a typical haplodiplophasic life history as seen in other Laminariales.     

GENOTYPIC VARIATION AND ALTERNATING DNA LEVELS AT CONSTANT CHROMOSOME NUMBERS IN THE LIFE HISTORY OF THE BROWN ALGA HAPLOSPORA GLOBOSA (TILOPTERIDALES)1

The brown algal order Tilopteridales contains three monospecific genera with reduced life histories, Which are assumed to have been derived form ancestors with oogamous reproduction and alternation of generations. The Newfoundland population of Haplospora globosa Kjellman still shows an alternation of gametophytes and sporophytes, but the chromosome Numbers remain equal because of parthenogenesis and apomeiosis, However, DNA fluorometry showed that the DNA level is twice as high in the Sporophytes as in the gametophytes, The DNA variation at constant chromosome numbers is presumably due to endomitosis combined with a law degree of polyteny. A genotypic variant of Haplospora is represented by the population at Helgoland (F.R.G.) where only sporophytes exist, Spores develop into sporophytes instead of gametophytes, and the plants have reduced chromosome number but the same DNA level as the Newfoundland sporophytes     

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.     

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.     

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.     

Biological and nutritional aspects involved in fern multiplication

Gametophytes of several species of ferns were mechanically triturated and the resulting homogenates cultured in vitro for propagation purposes. Differences in the time period from spore culture to sporophyte development were perceivable between species. For those species with a fast life cycle and high sporophyte production such as Woodwardia virginica and Dryopteris affinis sp. affinis, homogenization of gametophytes can be considered to be excellent method for propagation, yielding hundreds of sporophytes in a short period of time. Sporophyte formation was inhibited in O. regalis by the succesive application of homogenization to gametophytes regenerated by this technique. The effect of the culture medium composition on fern production was also studied in O. regalis and P. ensiformis gametophytes. In these species, sporophyte formation increased when the gametophytes were cultured in a medium containing water+0.7% agar. Addition of sucrose inhibited gametophyte development and induced their necrosis. The 1/2 dilution of Murashige and Skoog basal medium, without sucrose, favoured leaf expansion in P. ensiformis sporophytes. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Multiple molecular forms of serum α1-antitrypsin

Total soluble proteins, peroxidase, and peroxidase isozymes were examined in polyploid series of fern gametophytes and sporophytes. A distinctive pattern of protein bands was associated with gametophytes and sporophytes and the pattern did not vary within each phenotype with increases in the genome. Peroxidase activity per cell increased in direct proportion to increases in the genome and was determined to be gene dosage related. Slight differences in the patterns of peroxidase isozyme bands were associated with increases in the chromosome complement in both series of plants, but major variations were found between gametophyte and sporophyte. Quantitative analysis of peroxidase activity in each band revealed both increases and decreases in individual isozymes as ploidy increased. These findings suggest the involvement of regulatory mechanisms controlling isozyme activity.     

INTERGENERIC HYBRIDIZATION AMONG FIVE GENERA OF THE FAMILY LESSONIACEAE (PHAEOPHYCEAE) AND EVIDENCE FOR POLYPLOIDY IN A FERTILE PELAGOPHYCUS×MACROCYSTIS HYBRID

Hybridization was attempted by combining gametophytes between intergeneric pairs among the following taxa in the Lessoniaceae: Macrocystis pyrifera ( L.) C. Agardh , M. integrifolia Bory, M. angustifolia Bory , Pelagophycus porra ( Leman) Setch ., Nereocystis luetkeana ( Mert.) Post & Rupr ., Dictyoneurum californicum Rupr ., and Dictyoneuropsis reticulata ( Saud.) Smith. Hybrid sporophytes were produced in some combinations involving Macrocystis × Pelagophycus and Macrocystis × Dictyoneurum, and in all combinations of Dictyoneuropsis × Dictyoneurum. This is the first report of intergeneric hybrids involving Dictyoneurum. Gametophytes of P. porra had 16–24 chromosomes. Gametophytes from a fertile Macrocystis-Pelagophycus hybrid were crossed with Macrocystis and Pelagophycus gametophytes. Hybrid male gametophytes and Pelagophycus female gametophytes produced sporophyte progeny, but hybrid males with Macrocystis females did not. A single hybrid female gametophyte did not produce gametophytes in combination with hybrid males , Pelagophycus males or Macrocystis males. The hybrid gametophytes had approximately 30 chromosomes. It is hypothesized that the hybrid is an alloploid, containing a complete set of Macrocystis and Pelagophycus chromosomes, which may have allowed meiosis and sporogenesis to proceed normally in the hybrid sporophyte found in the sea. Thus, reproductive isolating mechanisms appear to operate at both pre- and postzygotic stages, and both can be overcome in intergeneric hybrids .     

Life history characteristic ofCyrtomium falcatum around the natural northern boundary in Hokkaido,with reference to the alternation of generations

To understand the life history characteristic for expanding the distributional area to colder climates, developmental age structure of population ofCyrtomium falcatum was observed along southwestern coasts of Hokkaido at the natural northern boundary of its distribution, with reference to the alternation of generations. The length and number of pinna of fertile leaves ofCyrtomium falcatum decrease towards the northern part of Japan. In southwestern Hokkaido, typically dwarf fertile leaves and gametophytes were observed growing together on cliffs nearby the sea. To estimate the developmental ages of small and dwarf leaves, the number of venation (NV: branching number of vein from midrib) of leaves was counted on each sporophyte. The sporophyte with leaves at the simple pinna stage ranging from 0–25NV, is predominant in the population of southwestern coasts of Hokkaido. The fertility of the sporophyte seems to be achieved more than five years after the germination. The gametophytes were also observed at the location to be almost equal in number to sporophytes. The number of gametophytes and sporophytes decreases with advancement of developmental stages. In the same location at Okushiri Isl. with slight gradiency of humidity, the gametophyte is predominant on the drier cliff, while the sporophyte is predominant on the humid hole. The population ofCyrtomium falcatum at the natural northern boundary in Hokkaido, seems to have the life history characteristic with alternation of generations. Contribution No. 2557 from the Inst. of Low Temp. Sci.     

Sporophyte and gametophyte generations differ in their thermotolerance response in the moss Microbryum

BACKGROUND AND AIMS: Actively growing post-embryonic sporophytes of desert mosses are restricted to the cooler, wetter months. However, most desert mosses have perennial gametophytes. It is hypothesized that these life history patterns are due in part to a reduced thermotolerance for sporophytes relative to gametophytes. METHODS: Gametophytes with attached embryonic sporophytes of Microbryum starckeanum were exposed whilst desiccated to thermal episodes of 35 degrees C (1 hr), 55 degrees C (1 hr), 75 degrees C (1 hr) and 75 degrees C (3 hr), then moistened and allowed to recover for 35 d in a growth chamber. KEY RESULTS: All of the gametophytes survived the thermal exposures and produced protonemata, with the majority also producing shoot buds. Symptoms of gametophytic stress (leaf burning and discoloration of entire shoots) were present in lower frequencies in the 55 degrees C exposure. Sporophyte resumption of growth and maturation to meiosis were significantly negatively affected by thermal treatment. Not a single sporophyte exposed to the two higher thermal treatments (75 degrees C for 1 h and 75 degrees C for 3 h) survived to meiosis, and those sporophytes exposed to 75 degrees C that survived to the post-embryonic phenophase took significantly longer to reach this phase. Furthermore, among the thermal treatments where some capsules reached maturity (35 degrees C and 55 degrees C), maternal shoots that produced a meiotic capsule took longer to regenerate through protonemata than maternal shoots aborting their sporophyte, suggestive of a resource trade-off between generations. CONCLUSIONS: Either (1) the inherent sporophyte thermotolerance is quite low even in this desert moss, and/or (2) a gametophytic thermal stress response controls sporophyte viability.     

C3 photosynthesis in the gametophyte of the epiphytic CAM fern Pyrrosia longifolia (Polypodiaceae)

Sporophytes of some epiphytic species in the fern genus Pyrrosia exhibit Crassulacean acid metabolism (CAM), generally considered to be a derived physiological response to xeric habitats. Because these species alternate between independent sporophytic and gametophytic generations yet only the sporophyte has been characterized physiologically, experiments were conducted to determine the photosynthetic pathways present in mature sporophytes, immature sporophytes, and gametophytes of Pyrrosia longifolia. Diurnal CO₂ exchange and malic acid fluctuations demonstrated that although the mature sporophytes exhibited CAM, only C₃ photosynthesis occurred in the gametophytes and young sporophytes. Consideration of the above results and those from previous studies, as well as the life cycle of ferns, indicates that the induction of CAM probably occurs at a certain developmental stage of the sporophyte and/or following exposure to stress. Elucidation of the precise mechanisms underlying this C₃-CAM transition awaits further research.     

Generational differences in response to desiccation stress in the desert moss Tortula inermis

BACKGROUND AND AIMS: Active growth in post-embryonic sporophytes of desert mosses is restricted to the cooler, wetter months. However, most desert mosses have perennial gametophytes. It is hypothesized that these life history patterns are due, in part, to a reduced desiccation tolerance for sporophytes relative to gametophytes. METHODS: Gametophytes with attached post-embryonic sporophytes of Tortula inermis (early seta elongation phenophase) were exposed to two levels of desiccation stress, one rapid-dry cycle and two rapid-dry cycles, then moistened and allowed to recover, resume development, and/or regenerate for 35 d in a growth chamber. KEY RESULTS: Gametophytes tolerated the desiccation treatments well, with 93 % survival through regenerated shoot buds and/or protonemata. At the high stress treatment, a significantly higher frequency of burned leaves and browned shoots occurred. Sporophytes were far more sensitive to desiccation stress, with only 23 % surviving after the low desiccation stress treatment, and 3 % surviving after the high desiccation stress treatment. While the timing of protonemal production and sporophytic phenophases was relatively unaffected by desiccation stress, shoots exposed to one rapid-dry cycle produced shoots more rapidly than shoots exposed to two rapid-dry cycles. CONCLUSIONS: It is concluded that sporophytes of Tortula inermis are more sensitive to rapid drying than are maternal gametophytes, and that sporophyte abortion in response to desiccation results from either reduced desiccation tolerance of sporophytes relative to gametophytes, or from a termination of the sporophyte on the part of the gametophyte in response to stress.     

Evidence of genetic segregation in the apogamous fern species Cyrtomium fortunei (Dryopteridaceae)

In apogamous ferns, all offspring from a parent are expected to be clonal. However, apogamous 'species' frequently demonstrate a large amount of morphological and genetic variations. Cyrtomium fortunei composed of four varieties (C. fortunei var. fortunei, var. clivicola, var. intermedium, and var. atropunctatum), is all reported to be apogamous triploids, but demonstrates large and continuous morphological variation. In previous studies, we showed that considerable genetic diversity was observed in many local populations of the apogamous fern 'species'. We hypothesized that genetic segregation has occurred, because neither sexual type nor intraspecific polyploidy have been observed in C. fortunei in Japan. Of 732 progeny examined (250 gametophytes and 482 sporophytes), obtained from a parental sporophyte whose pgiC genotype was estimated as aab, 11 (4.4%) gametophytes and 8 (1.7%) sporophytes showed a different genotype (aaa) from that of the parent sporophyte. We showed that genetic segregation occurs in apogamous C. fortunei in relatively high frequency. Moreover, we could first show that the segregation frequency in gametophytes is significantly higher than that in sporophytes of the next generation (χ (2)?=?4.90, P?=?0.027). It may suggest the existence of deleterious genes, which are expressed during the morphogenesis and growth of sporophytes.     

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.