Study of the independent gametophytes found on Jeju Island in South Korea and the first record of the obligate independent gametophyte of Antrophyum obovatum Baker

Fern gametophytes have often been neglected in research; however, studies on gametophytes are crucial for a better understanding of the evolution of ferns. During their life cycle, some gametophytes produce large and long‐lived populations without producing sporophytes and reproduce independently through asexual means, such as through the formation of gemmae. In this study, we investigated independent gametophytes on the Jeju Island of Korea, which was located on the land bridge between East China and Japan during the glacial periods. Fourteen gametophyte populations were collected from seven sites, of which 13 populations were clearly identified as belonging to four fern species known to occur in Jeju Island with BLAST searches using rbcL and trnL‐F sequences. Surprisingly, the last remaining population constituted undescribed taxa in Korea. We presented the first report of the independent gametophytes of Antrophyum obovatum Baker which has not been previously recorded in Korea. It has been supposed that many ferns sought suitable habitat throughout the land bridge between China and Japan. However, Jeju Island might be unsuitable for vittarioid ferns that prefer a tropical or subtropical environment. Consequently, only two species of vittariod ferns (A. obovatum and Haplopteris flexuosa (Fée) E.H. Crane) in the form of a gametophyte and sporophyte, respectively, exist on Jeju Island. Therefore, this gametophyte population must be protected and managed from a conservation perspective. In the case of the independent gametophyte of Hymenophyllum wrightii Bosch, haplotype analysis was conducted based on the rbcL sequences and the result supported that the North American populations were migrated from Japan through land bridge during the glacial periods and Jeju populations were recently established by long‐distance dispersal of the Japanese populations.     

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.     

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.     

A method to establish an “immortalized F2” sporophyte population in the economic brown alga Undaria pinnatifida (Laminariales: Alariaceae)

Studies of quantitative trait loci based on genetic linkage maps require the establishment of a mapping population. Permanent mapping populations are more ideal than temporary ones because they can be used repeatedly. However, there has been no reported permanent sporophyte population of economically important kelp species. Based on the characteristics of the kelp life cycle, we proposed a method to establish, and then constructed experimentally, an “immortalized F₂” (IF₂) population of Undaria pinnatifida. Doubled-haploid “female” and “male” sporophytes were obtained through the parthenogenesis of a female gametophyte clone and the selfing of a “monoicous” gametophyte clone (originally male), respectively, and they were used as the parents. The F₁ hybrid line was generated by crossing the female and male gametophytes derived from the respective female and male parents. Full-sibling F₂ gametophyte clones, consisting of 260 females and 260 males, were established from an F₁ hybrid sporophyte. Thirty-five females and 35 males were randomly selected and paired to give rise to an IF₂ population containing 35 crossing lines. A parentage analysis using 10 microsatellite markers confirmed the accuracy of the IF₂ population and indicated the feasibility of this method. This proposed method may be adapted for use in other kelp species, and thus, it will be useful for genetic studies of kelp.     

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.     

Nuclear DNA level and life cycle of kelps: Evidence for sex‐specific polyteny in Macrocystis (Laminariales,Phaeophyceae)

Giant kelp, Macrocystis pyrifera (Linnaeus) C. Agardh, is the subject of intense breeding studies for marine biomass production and conservation of natural resources. In this context, six gametophyte pairs and a sporophyte offspring of Macrocystis from South America were analyzed by flow cytometry. Minimum relative DNA content per cell (1C) was found in five males. Unexpectedly, nuclei of all female gametophytes contained approximately double the DNA content (2C) of males; the male gametophyte from one locality also contained 2C, likely a spontaneous natural diploid variant. The results illustrate a sex‐specific difference in nuclear DNA content among Macrocystis gametophytes, with the chromosomes of the females in a polytenic condition. This correlates with significantly larger cell sizes in female gametophytes compared to males and resource allocation in oogamous reproduction. The results provide key information for the interpretation of DNA measurements in kelp life cycle stages and prompt further research on the regulation of the cell cycle, metabolic activity, sex determination, and sporophyte development.     

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.     

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.     

UV‐radiation effects on photosynthesis and photoprotection in gametophytic and sporophytic stages of the freshwater red alga Kumanoa ambigua (Rhodophyta,Batrachospermales)

The aim of this study was to analyze the photosynthetic performance of gametophytic and sporophytic (‘Chantransia’) stages of Kumanoa ambigua in culture under UV radiation. We hypothesized that both life history stages of K. ambigua would exhibit different photosynthetic responses to UVR exposure. Experiments were performed under three conditions: (i) photosynthetically active radiation (PAR) only (400–700 nm), P control; (ii) PAR + UVA (320–700 nm), PA treatment; and (iii) PAR + UVA + UVB (280–700 nm), PAB treatment. The photosynthetic parameters were measured as in vivo chlorophyll a fluorescence. Differences were found between life stages, observing higher values of NPQ and effective quantum yields (ΔF/F_m′) under UVA and PAR in gametophytes compared to sporophytes. One type of mycosporine‐like amino acid (MAA) was detected in the gametophyte in all treatments, but not in the ‘Chantransia’ stage. The increased photosynthetic performance for some parameters and the presence of MAA in gametophyte suggest that it is less sensitive to UV radiation, particularly UVA, in comparison to sporophyte under culture conditions. This approach is relevant for a better understanding of the adaptation and physiological acclimation of freshwater Rhodophyta to varying light climates in terms of global changes.     

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 .     

PROBLEMS IN THE IDENTITY AND ORIGIN OF THE APPALACHIAN VITTARIA GAMETOPHYTE,A SPOROPHYTELESS FERN OF THE EASTERN UNITED STATES

Populations of a fern gametophyte presumed to be of the genus Vittaria occur commonly in the uplands of the southeastern United States. The gametophytes occur on non-calcareous rock outcrops of various composition in areas which provide continuous moisture and protection from temperature extremes. Gametophytes in these habitats are robust and long lived, frequently forming the dominant vegetation in areas covering several square feet. Reproduction is exclusively vegetative by production of gemmae. Although sex organs are present in most populations, viable sporophytes are never produced. The gametophytes have previously been considered most likely to be V. lineata, which occurs in Florida, but morphological and physiological comparisons do not support that conclusion. Significant differences between Appalachian and Florida gametophytes occur in growth form, growth rate, cold hardiness, sporophyte production, and patterns of gemma production. The distribution of the Appalachian gametophytes correlates with old, unglaciated land masses suggesting antiquity rather than recent introduction. Present evidence favors the interpretation that the Appalachian Vittaria gametophytes either belong to a tropical American species from which they have long been separated, or that they represent a distinct species of which the sporophyte no longer exists.     

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.     

GAMETOPHYTES AND YOUNG SPOROPHYTES OF OPHIOGLOSSUM CROTALOPHOROIDES WALT.

The lens-shaped, dorsiventral gametophytes of Ophioglossum crotalophoroides are superficially different from the cylindrical gametophytes of other species of Ophioglossum. However, they have the same features as other Ophioglossum gametophytes (fundamentally axial organization, radially symmetrical apical meristem, radial distribution of gametangia) except that the cylindrical axis is reduced in length. Young sporophyte development is unique in the genus: all primary organs clearly arise from the embryo and develop simultaneously. The length of the life cycle of O. crotalophoroicles is considerably shorter than that of some other species in the Ophioglossaceae. A timetable for gametophyte and young sporophyte development is postulated. Spores germinate soon after they are released in the spring, and mature gametophytes develop by the next growing season. Fertilization occurs approximately one year after spore dispersal, and after two years, the photosynthetic first leaf of the young sporophyte emerges.     

Temperature requirements for growth and maturation of the warm temperate kelp Eckloniopsis radicosa (Laminariales,Phaeophyta)

The annual kelp Eckloniopsis radicosa is distributed along Japanese coasts and occurs within the area with a February isotherm ranging 15–18°C and August isotherm ranging 25–28°C. In this study, the effects of temperature on the gametophyte growth and maturation, and the young sporophyte growth of E. radicosa were examined and the results are discussed in relation to the distribution of other warm‐adapted kelp species and the potential effects of climate change. The optimal temperature ranges for growth of male and female gametophytes were 23–27°C and 20–26°C, respectively. The upper survival temperature for gametophyte growth was 31°C for males and 30°C for females, respectively. The optimal temperature range for maturation of female gametophytes was ≤23°C. The optimal temperature range for growth of young sporophytes was 14–22°C. It was clarified that E. radicosa has the most warm‐adapted characteristics for growth and maturation of gametophytes among members of the Laminariales studied so far. The natural seawater temperature ranges during the growth and maturation seasons for gametophytes of E. radicosa, as well as the growth season for young sporophytes near to the northern and southern distribution limits (Izu‐Oshima: 14.9–24.5°C, Ichiki‐kushikino: 17.1–29.6°C), agreed with the experimentally determined temperature requirements. The warm‐adapted gametophyte stage and annual lifecycle are major factors enabling survival of E. radicosa in warm waters near tropical regions along the Japanese coast.     

Allelopathic compounds,thelypterin A and B in the fern Thelypteris normalis

Summary The growth of Thelypteris normalis (C. Chr.) Moxley gametophytes is inhibited under T. normalis sporophytes. Competition for minerals, light, change in pH, or microbial inhibitors were experimentally eliminated as causes of the inhibition. This is the first demonstration of allelopathy between a sporophyte and gametophyte in a fern. Two inhibitors, thelypterin A and B, which were released from the roots of the Thelypteris sporophyte, were isolated and a bioassay for the inhibitors was devised. Thelypterin A gave an Ehrlich-positive reaction indicative of secondary aromatic amines and an ultraviolet absorption spectrum indicative of a heterocyclic ring. The inhibitors affected the growth of Thelypteris, Pteris and Phlebodium gametophytes.     

Gametophyte development and reproduction of <Emphasis Type="Italic">Argyrochosma nivea</Emphasis> (Pteridaceae)

The gametophyte of Argyroschosma nivea was studied, mainly focusing in its morphological development, and in the apogamous production of sporophytes. Some observations on the spores were also made. As far as it is known, this is the second species of the genus whose gametophytes are studied. The germination pattern followed the Vittaria type. The subsequent developmental processes followed the Ceratopteris type. Some of the gametophytes reached an adult stage with a cordate, symmetric shape, but most of them developed as irregular, lobed prothalli. The sporophyte emerged from the anterior part of the prothallus, without formation of gametangia. First, a cell became active and originated a proliferating area of small cells. From this area, long glandular hairs were formed followed by a projected conical cluster of cells. The cluster elongated into a sporophytic structure and its apex became progressively spatulate and finally trilobulate, with marginal, glandular hairs, stomata and tracheids continuously produced. This sporophyte secreted granules of white farina from its beginnings. The production of farina in the sporophyte but not in the gametophyte could help to support the idea of the segregation of this species from its traditional location in Notholaena to Argyrochosma, as farinose gametophytes seem to be a synapomorphy of the notholenoids, group that includes Notholaena but not Argyroschoma.     

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.     

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.     

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.