Heterozygosity for recessive lethals in homosporous fern populations: Thelypteris palustris and Onoclea sensibilis

Sporophytes from natural populations of Thelypteris palustris and Onoclea sensibilis were subjected to breeding tests to reveal the presence of recessive lethal alleles in their genotypes. All sporophytes were found to be heterozygous for recessive sporophytic lethals. Despite the capability for producing completely homozygous genotypes by intragametophytic self-fertilization, ferns in natural populations seem to retain considerable genetic loads of deleterious recessive alleles. It is suggested that natural selection operates to strongly favour heterozygous rather than homozygous sporophytes under most conditions.     

POPULATIONAL AND GENETIC STUDIES OF A HOMOSPOROUS FERN—OSMUNDA REGALIS

The mating system and the genetic system of the homosporous fern Osmunda regalis were investigated. Seven populations from western Massachusetts were sampled. All the sporophytes investigated were found to be heterozygous for zygotic lethals. Morphological studies of the gametophytes indicated an intergametophytic mating system when the gametophytes were spatially and chronologically situated to exchange male gametes. Genetic studies evidenced a genetic system based upon duplicate loci.     

EVIDENCE FOR GENETIC HETEROZYGOSITY IN A HOMOSPOROUS FERN

Homosporous fern sporophytes from natural populations exhibited heterozygous electrophoretic patterns for several enzyme systems. Genetic tests utilizing individual gametophytes demonstrate that the observed heterozygosity is coded by alleles at single loci. This simple procedure makes it possible to distinguish segregating from fixed or phenotypic heterozygosity, previously a problem in homosporous vascular plants.     

Parthenogenesis in the brown alga Lessonia nigrescens (Laminariales,Phaeophyceae) from central Chile1*

Parthenogenesis, the development of female gametes without fertilization, is relatively common in brown algae, although limited quantitative information on the phenomenon is available. Its occurrence is reported for the first time in Lessonia nigrescens Bory, a member of the Laminariales and a key ecological component of the benthic algal communities along the Chilean coast. Isolated female gametophytes developed into parthenosporophytes throughout the year, with a maximum in spring to early summer. Isolated male gametophytes, on the other hand, never developed fronds. Parthenosporophytes obtained in the laboratory developed normally when cultivated under greenhouse conditions, and the resulting individuals were indistinguishable in size, shape, texture, and color from heterozygous sporophytes. Quantification of DNA of various tissues demonstrated that early during their development, parthenosporophytes duplicated their DNA content, displaying levels similar to heterozygous sporophytes and almost twice the level found in gametophytes. One out of 45 individuals from a field population yielded only female gametophytes, strongly suggesting that parthenogenesis does occur in wild stands of L. nigrescens.     

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.     

An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines

? The sequencing of the Physcomitrella patens genome, combined with the high frequency of gene targeting in this species, makes it ideal for reverse genetic studies. For forward genetic studies, experimental crosses and genetic analysis of progeny are essential. ? Since P. patens is monoicous, producing both male and female gametes on the same gametophore, and undergoing self-fertilization at a high frequency, the identification of crossed sporophytes is difficult. Usually spores from many sporophytes from a mixed culture must be tested for the production of recombinant progeny. ? Here, we describe the use of transgenic lines that express a fluorescent transgene constitutively, to provide a direct visual screen for hybrid sporophytes. ? We show that segregations in crosses obtained with this technique are as expected, and demonstrate its utility for the study of the rate of outcrossing between three isolates of P. patens.     

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.     

羊栖菜幼孢子体对不同N水平生长条件和阳光紫外辐射的响应

探讨了太阳紫外辐射对两种N水平生长条件下羊栖菜幼孢子体光化学特性的影响及其恢复。结果显示,在高的光辐射下羊栖菜藻体的有效光化学效率和相对电子传递速率急剧下降,在全波长太阳辐射条件下它们的下降幅度要比仅在可见光处理下的藻体更大,2种N水平条件下藻体的光化学活性下降趋势相似,但是N加富的生长条件使得藻体具有更高抵御紫外辐射的能力,这可能是与N加富生长条件下的藻体中含有较高含量的紫外吸收物质和类胡萝卜素有关。结果表明羊栖菜的幼孢子体具有比成体更强抵御紫外线的能力,这主要体现在藻体受到紫外辐射损伤后的修复上。     

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.     

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。     

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.     

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.     

ENVIRONMENTAL CONTROL OF GAMETOGENESIS IN LAMINARIA SACCHARINA. IV. IN SITU DEVELOPMENT OF GAMETOPHYTES AND YOUNG SPOROPHYTES1,2

Meiospores of Laminaria saccharina labeled with a fluorescent stain were put into the sea and recovered at frequent intervals. They developed into gametophytes which produced gametes throughout the year. Antheridia were produced earlier than oogonia. Gametogenesis was slowest during the late autumn and early winter. On the basis of nutritional studies and the seasonal distribution of nutrients, it was concluded that the rate of gametogenesis was not due to nutrient deficiency but most likely to light. Production of macroscopic sporophytes appeared to be limited by survival of microscopic sporophytes rather than gametogenesis. Macroscopic sporophytes became evident during the late winter and autumn. The greater light and perhaps higher temperatures of summer were suggested as agents limiting macroscopic sporophyte production during this period.     

Auxin regulation of axial growth in bryophyte sporophytes: its potential significance for the evolution of early land plants

To identify developmental mechanisms that might have been involved in the evolution of axial sporophytes in early land plants, we examined the effects of auxin-regulatory compounds in the sporophytes of the hornwort Phaeoceros personii, the liverwort Pellia epiphylla, and the moss Polytrichum ohioense, members of the three divisions of extant bryophytes. The altered growth of isolated young sporophytes exposed to applied auxin (indole-3-acetic acid) or an auxin antagonist (p-chlorophenoxyisobutyric acid) suggests that endogenous auxin acts to regulate the rates of axial growth in all bryophyte divisions. Auxin in young hornwort sporophytes moved at very low fluxes, was insensitive to an auxin-transport inhibitor (N-[1-naphthyl]phthalamic acid), and exhibited a polarity ratio close to 1.0, implying that auxin moves by simple diffusion in these structures. Emerging liverwort sporophytes had somewhat higher auxin fluxes, which were sensitive to transport inhibitors but lacked any measurable polarity. Thus, auxin movement in liverwort sporophytes appears to result from a unique type of apolar facilitated diffusion. In young Polytrichum sporophytes, auxin movement was predominantly basipetal and occurred at high fluxes exceeding those measured in maize coleoptiles. In older Polytrichum sporophytes, acropetal auxin flux had increased beyond the level measured for basipetal flux. Insofar as acropetal and basipetal fluxes had different inhibitor sensitivities, these results suggested that moss sporophytes carry out bidirectional polar transport in different cellular pathways, which resembles the transport in certain angiosperm structures. Therefore, the three lineages of extant bryophytes appear to have evolved independent innovations for auxin regulation of axial growth, with similar mechanisms operating in moss sporophytes and vascular plants.     

Interploidal hybridization and mating patterns in the Sphagnum subsecundum complex

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

Growth of Ecklonia cava (Laminariales, Phaeophyta) sporophytes transplanted to a locality with different temperature conditions

Transplanting experiments were carried out to determine whether the small type sporophytes with short stipe of Ecklonia cava Kjellman (Laminariales, Phaeophyta) growing in a locality with warm temperatures, change into larger type with a long stipe when transplanted to a locality with cooler temperatures. Juvenile E. cava sporophytes, having a stipe shorter than 5 cm long were collected from Tei in Tosa Bay (southern Japan) (seawater temperature 15–29°C) and transplanted to Nabeta Bay (central Japan) (seawater temperature 13–25°C), where larger type E. cava sporophytes characterized by long stipe (ca 1 m) grow. They were attached to artificial reefs at the sea bottom (9 m depth) in Nabeta Bay to monitor their growth. For comparison, juvenile E. cava sporophytes of almost similar size growing in Nabeta Bay were also transplanted in the same way to the same experimental site. Observations of growth of sporophytes from Tei and Nabeta were carried out monthly for 2 years from November 1995 to October 1997. The transplanted Tei and Nabeta sporophytes showed an increase in stipe length and diameter from winter to spring, whereas almost no increase was observed during summer and autumn. At the end of the study period, the stipe of Nabeta sporophytes reached 25.6 cm in length and 17.0 mm in diameter, whereas that of Tei sporophytes reached 11.1 cm in length and 11.2 mm in diameter. The primary blade length was 16.0 cm in Nabeta sporophytes, whereas it was 5.5 cm in Tei sporophytes. Thus, Tei sporophytes still remained smaller than Nabeta sporophytes even under the same environmental conditions.     

Growth and survival rates of large-type sporophytes of Ecklonia cava transplanted to a growth environment with small-type sporophytes

Stipe lengths of sporophytes of Ecklonia cava Kjellman have been reported to be longer along the southeast than southwest coast of the Izu Peninsula, central Japan. Two bays in this region that have natural populations of E. cava, but with different stipe lengths, were chosen for transplant experiments to examine if stipe length was an environmentally controlled trait. Transplant experiments were carried out in order to determine whether large-type sporophytes of E. cava with long stipes growing in Nabeta Bay (southeast Izu Peninsula, Japan) would turn into small-type sporophytes with short stipes when transplanted to Nakagi Bay (southwest Izu Peninsula). Ten juvenile sporophytes of E. cava (stipe length < 5 cm) were collected from Nabeta Bay (large-type habitat) and transplanted to Nakagi Bay (short-type habitat) in December 1995. As a transplant control, ten juvenile sporophytes of E. cava growing in Nakagi Bay were also transplanted to the same artificial reefs. Growth and survival rates of the sporophytes were monitored monthly for 3 y until December 1998. The transplanted sporophytes showed an increase in their stipe length and diameter from winter to spring, whereas almost no increase was observed from summer to autumn. However, the elongation was greater in Nabeta sporophytes than in Nakagi sporophytes. The primary blade length increased mainly from winter to early spring and decreased largely in autumn. Average primary blade lengths were similar in both Nabeta and Nakagi sporophytes from the end of the first year of transplanting. Although ca. 70% of both Nabeta and Nakagi sporophytes survived during the first 2 y after transplantation, no Nakagi sporophytes and only two Nabeta sporophytes survived to the end of the 3 y study period. Despite transplantation to Nakagi Bay, where short sitpes are naturally present, the sporophytes from Nabeta Bay persisted in having longer stipes, which suggests that stipe length is genetically, rather than environmentally, controlled.     

MUTATIONAL LOAD IN A FERN POPULATION GROWING IN A POLLUTED ENVIRONMENT

The Osmunda regalis sporophyte is well organized for the storage and perpetuation of post-zygotic mutations. Techniques have been developed to detect sporophytes in which such mutations have occurred. Two populations were screened for sporophytes which are chimeras for gametophytic or recessive sporophytic lethals. The populations had different ecologies; one was in a pristine lake side bog and the other was growing in the waters of a river heavily polluted with industrial wastes. Post-zygotic mutations were detected only in the polluted river population. The level of mutational damage approximated that previously documented from chromosome studies.     

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.     

HOMOEOLOGOUS CHROMOSOME PAIRING AND RESTRICTED SEGREGATION IN THE FERN CERATOPTERIS

The segregation of a marker characterized by pale green gametophytes was monitored within an inbreeding study of the polyploid fern Ceratopteris. Although all of the sporophytes showing segregation were derived from the self-fertilization of haploid gametophytes, a low overall frequency of 2.5% pale gametophytes was observed in the F₃–F₅ generations. A model based upon a duplicated locus and homoeologous chromosome pairing can explain the segregational behavior within the study. The overall level of homoeologous pairing was determined to be 10%. Occasionally, green gametophytes that were presumably heterozygous for the marker contained pale sectors. This behavior may involve mitotic crossing-over between homoeologous chromosomes.