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.     

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.     

LIFE HISTORY AND SYSTEMATIC POSITION OF AKKESIPHYCUS LUBRICUS (PHAEOPHYCEAE)1

The life history in culture of Akkesiphycus lubricus Yamada et Tanaka, an alga which has been placed in the Coilodesmaceae or the Punclariaceae, Dictysiphonales, was studied. In culture the species alternates between a microscopic filamentous gametophyte and a macroscopic polystichous sporophyte, a pattern common to the Dictyasiphonales and Laminariales. However, it has a unique anisogamous dioecious gametophyte. Fusions between mac-ro-gametes and micro-gametes were not observed, Macro-gametes or zygotes germinated, mostly developing into sporophytes that formed unilocular sporangia and the rest developed into reduced gametophytic flaments again. The gametophyte matures in 50C short-day conditions, corresponding to winter in Hokkaido. The sporophyte develops normally and matures only in low-temperature conditions irrespective of daylength. In regard to iits systematic position, Akkesiphycus lubricus is considered to have a closer relationship with the Laminariales than with the Dietyosiphonales in the following characters; lack of pyrenoids; early stages of parenchyma formation in the sporophyte; direct development of sporophytes from gametes or zygotes without forming a besal system zoospore becomes almost empty after germination by the migration of cell contents into a germ lube; formation of macro-gametangia by direct conversions of mother cells of mother cells of fertile branches; and micro-gametangia formed in clusters showing closeresemblance to the antheridia of Pseudochorda nagii (Tokida) Inagaki.     

African Hepatics: VI. Euosmolejeunea

Abstract

(1) The ratio of ¹⁴CO₂ uptake of fully expanded capsules to that of corresponding gametophytes in the species studied was about 1 : 15 in Mnium hornum, 1 : 35 in Pleurldium acuminatum and around 1 : 1 in Funaria hygrometriea.

(2) Translocation of photosynthate from gametophyte to sporophyte took place within 1 to 4 days of exposure to ¹⁴CO₂; in expanded capsules the final activity was at least equal to that remaining in the gametophyte.

(3) In M hornum there was no evidence of reverse translocation from sporophyte to gametophyte.

(4) Translocation from gametophyte continued at a high level throughout the expansion and differentiation of the capsule in M. hornum. The activity reaching the sporophyte was approximately proportional to its fresh weight.

(5) Both in situ photosynthesis and translocation to the capsule of F. hygrometrica rose to a maximum during the latter part of capsule expansion and ensuing differentiation, subsequently declining as the green tissues of the capsule senesced.

(6) Fully expanded (but premeiotic) capsules of M. hornum contributed approximately 20% of the assiinilate necessary for their growth. The corresponding figure for P. acuminatum was about 10%, and for F. hygrometrica about 50%.     

Squash Techniques in the Cytological Investigation of Mosses

Abstract

Photosynthetic activity of attached sporophytes is very low (a few per cent or less) compared with that of associated gametophyte structures (perianth, bracts and uppermost leaves) in Cephalozia bicuspidata and Lophocolea heterophylla, or with even small areas of thallus in Pellia epiphylla. Photosynthetic uptake of ¹⁴CO₂ by developing sporophytes of P. epiphylla, C. bicuspidata and L. heterophylla is at most a few per cent of the ¹⁴C translocated subsequently from the gametophyte, and could be negligible. In L. heterophylla, the perianth, bracts and uppermost leaves appear to play only a limited role in nutrition of the sporophyte, the leafy shoots making a major contribution. In C. bicuspidata the perianth and leaves of the short archegonial shoot may provide a substantial part of the nutrition of the sporophyte. There is some indication in all three species that translocation from the gametophyte is most active when the sporophyte reaches full size but is still green, declining in the final stages of maturation of the capsule.     

Development of microsatellite markers in the green algae Enteromorpha intestinalis (Chlorophyta)

The fouling green algae Enteromorpha intestinalis is a cosmopolitan benthic species, which causes green tides in many coastal areas and is used as an indicator species for eutrophication in the Baltic Sea area. The life cycle of E. intestinalis alternates between two morphologically identical reproductive stages, a haploid gametophyte phase and a diploid sporophyte phase. However, it also reproduces through asexual propagation. The reproductive cycles of E. intestinalis in the Baltic Sea and elsewhere are largely unknown. Here we report five polymorphic microsatellite markers developed from enriched genomic libraries. The number of alleles per locus ranged from 7 to 25.     

MORPHOLOGY AND ANATOMY OF NEW SPECIES OF SCHIZAEA AND ACTINOSTACHYS

The gametophyte, old embryo, and sporophyte of Schizaea pseudodichotoma sp. nov., sporophyte and female parental gametophyte of S. diversispora hybr. nov. (S. pseudodicholoma X probably S. dichotoma), sporophyte of S. rhacoindusiata sp. nov., and gametophyte, old embryo, and sporophyte of Actinostachys macrofunda sp. nov. are described. The taxonomy of Schizaea is discussed and the system of Diels is strongly supported. The two sectional names used by Diels, Euschizaea Hook, and Lophidium Rich, are replaced by Pectinatae Prantl and Schizaea respectively. The prime morphological significance of Schizaea pseudodicholoma lies in its leafless embryo and its simple leaf differing from other species in its section, and that of Actinostachys macrofunda lies in its reduction to nearly complete heterotrophic existence and its frequent multiple annulus. Fungal hyphae have been traced from Schizaea and Actinostachys through the substratum and into root nodules of Casuarina and into roots of two other angiosperms.     

THE DEVELOPMENT AND REPRODUCTIVE MORPHOLOGY OF HALOSACCION AMERICANUM I. K. LEE (RHODOPHYTA,PALMARIALES)1

Halosaccion americanum, a member of Palmariaceae, was grown in culture from spores and the life history was critically examined by the use of scanning EM and light microscopy. A mature tetrasporangium of H. americanum produces four spores that germinate to form two male and two female gametophytes. The male gametophytes grow to maturity in approximately eight months and macroscopically resemble the tetrasporophyte. Following the first division of the tetraspore, the two-celled female gametophyte consists of a vegetative cell and a carpogonium with trichogynes. Fertilization is accomplished by spermatia from male plants of the preceding generation, as male plants of the same season are immature. Spermatia are formed in a continuous layer over the surface of the mature male gametophytes and, when released, are entrained in long strands of mucous. Spermatia adhere to and fuse with trichogynes and, nuclear fusions presumably follow. The carposporophyte is absent; the new tetrasporophyte develops directly from the fertilized carpogonium. Growth of the sporophyte eventually obliterates the female gametophyte, and development into a mature tetrasporophyte proceeds over a period of approximately eight months. The development of H. americanum, with its extremely abbreviated female gametophyte stage and direct development of the tetrasporophyte from the zygote, indicates that this rhodophyte has the same life history as reported for other members of the Palmariales.     

Hybridization involving independent gametophytes in the Vandenboschia radicans complex (Hymenophyllaceae): a new perspective on the distribution of fern hybrids

To test our hypothesis of hybrid formation involving the ‘independent gametophyte’ phenomenon in ferns, we identified the genomic formulae and ploidy level of gametophytes of the Vandenboschia radicans complex at the periphery of a sporophyte population. We identified haploid gametophytes of V. kalamocarpa (one of the two putative parents of V.×stenosiphon) in a hybrid sporophyte population in Japan that lacks fertile non‐hybrid individuals. Furthermore, diploid sporophytes of the species were not found within a 50‐km radius. This finding supports a hypothesis of hybridization involving the ‘independent gametophyte’ phenomenon and provides a new perspective on the geographical distribution of fern hybrids.     

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.     

Modelling reproductive effort in sub-and maritime Antarctic mosses

A comparison is made of the reproductive effort (RE), considered as the investment in sporophyte relative to gametophyte biomass, of eight species of moss occurring at sub-and maritime Antarctic sites. Six of the species showed smaller sporophytes and game-tophytes at the climatically more extreme maritime Antaretic sites and one species showed no size difference between regions. The remaining species, although showing no regional difference, showed some evidence of a reverse pattern, with higher altitude samples having greater biomass than lower altitude samples. Spore counts indicated a measure of compensation in maritime Antarctic samples, with no significant decrease in spore output in several species despite smaller sporophyte biomass. The relationship between sporophyte (S) and gametophyte (G) biomass within samples was described by an allometric curve (S=aG ^b ) which gave a better fit than a straight line for six species. This form of model allows comparisons of patterns of RE to be made between samples with non-or partially overlapping size distributions, even when the relationship involves size-dependence. An allometric curve was not appropriate for describing samples of one species (Andreaea regularis), and insufficient data were available to identify any relationship in Polytrichum alpinum. The exponent (b) differed between species, but there were no statistically significant differences between exponents from samples of the same species. Samples of two species could further be described by the same coefficient (a), indicating that they lie on the same curve. However, samples of three species from sub-Antarctic South Georgia gave significantly higher coefficients, indicating increased RE relative to maritime Antarctic populations.     

An in vitro whole plant selection system: paraquat tolerant mutants in the fern Ceratopteris

Summary A whole plant selection system using the haploid gametophyte generation of the fern Ceratopteris richardii has been developed to select for mutations that confer resistance or tolerance to various selection pressures. The expression of the mutations can be analyzed and characterized in both the haploid gametophyte and diploid sporophyte generations. Genetic analyses are facilitated by the fern's rapid life cycle and the ease of manipulating the gametophyte generation. Selection for tolerance to the herbicide paraquat has yielded two mutants which have an increased tolerance to the herbicide in both the gametophyte and sporophyte generations. Both mutants exhibit single nuclear gene inheritance patterns and appear to be closely linked or allelic.     

Genetic regulation of life cycle transitions in the brown alga Ectocarpus

The life cycle of an organism is one of its most elemental features, underpinning a broad range of phenomena including developmental processes, reproductive fitness, mode of dispersal and adaptation to the local environment. Life cycle modification may have played an important role during the evolution of several eukaryotic groups, including the terrestrial plants. Brown algae are potentially interesting models to study life cycle evolution because this group exhibits a broad range of different life cycles. Currently, life cycle studies are focused on the emerging brown algal model Ectocarpus. Two life cycle mutants have been described in this species, both of which cause the sporophyte generation to exhibit gametophyte characteristics. The ouroboros mutation is particularly interesting because it induces complete conversion of the sporophyte generation into a functional, gamete-producing gametophyte, a class of mutation that has not been described so far in other systems. Analysis of Ectocarpus life cycle mutants is providing insights into several life-cycle-related processes including parthenogenesis, symmetric/asymmetric initial cell divisions and sex determination.     

ROLE OF SETTLEMENT DENSITY ON GAMETOPHYTE GROWTH AND REPRODUCTION IN THE KELPS PTERYGOPHORA CALIFORNICA AND MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Laboratory studies were used to examine how variation in the density of spore settlement influences gametophyte growth, reproduction, and subsequent sporophyte production in the kelps Pterygophora californica Ruprecht and Macrocystis pyrifera (L.) C. Ag. In still (non-aerated) cultures, egg maturation in both species was delayed when spores were seeded at densities 300 · mm^?2. Although the density at which this inhibition was first observed was similar for both species, the age at which their eggs matured was not. P. californica females reached sexual maturity an average of 4 days (or ～ 30%) sooner than did M, pyrifera. As observed previously in field experiments, per capita sporophyte production was negatively density dependent for both species when seeded at spore densities of 10 · mm^?2. Total sporophyte production (i.e. number · cm^?2) for both species, however, was greatest at intermediate densities of spore settlement (～ 50 spores · mm^?2). In contrast, total sporophyte production by P. californica steadily increased with increasing spore density in aerated cultures; highest sporophyte density was observed on slides seeded at a density of 1000 spores · mm^?2. Preliminary experiments with P. californica involving manipulation of aeration and nutrients indicate that inhibition of gametophyte growth and reproduction at higher densities of spore settlement in non-aerated cultures was probably caused by nutrient limitation.     

CULTURE STUDIES ON THE LIFE HISTORY OF CHORDARIA LINEARIS (PHAEOPHYCEAE) FROM TIERRA DEL FUEGO,SOUTH AMERICA1

Studies of laboratory cultures of Chordaria linearis (Hooker et Harvey) Cotton from southernmost South America revealed that this species has an obligate sexual life history in which a macroscopic sporophyte alternates with a monoecious microscopic gametophyte. Sexual reproduction is isogamous and under photoperiodic control. Gametes are produced only in short days, whereas in long days, asexual zoospores are formed that recycle the gametophyte generation. Unfused gametes develop into gametophytes, and sporophytes originate only from zygotes. Unlike other sexual members of the Chordariales, gametes of C. linearis have a reduced stigma and do not show phototaxis. They are released at the beginning of the night, not in the morning. In nature, C. linearis seems to be regularly infected by a dictyosiphonalean epiphyte resembling the rare arctic species Trachynema groenlandicum (Lund) Pedersen. The epiphyte is responsible for previous contradictory results obtained in laboratory cultures of C. linearis. This is the first record of Trachynema in the southern hemisphere.     

Origin and taxonomic affinities of Thelypteris (subgen. Stegnogramma) burksiorum (Thelypteridaceae)

This paper examines the ecology, frond and spore morphology, gametophytes, and molecular biology ofThelypteris burksiorum to reveal its taxonomic affinities and phytogeographic origin. The species is endemic to Winston County in northern Alabama (U.S.A.) and only occurs in cave-like rockhouse habitats. A related species,T. pilosa, is widespread in central and southern Mexico, Guatemala, and Honduras.Thelypteris pilosa is extremely variable and our data suggest that current recognition of the varietiesmajor andpilosa is not warranted.Thelypteris pilosa is clearly closely related and likely sister toT. burksiorum. Thelypteris burksiorum exhibits the unusual production of gametophytic propagules. This, in combination with current levels of genetic diversity and reproductive biology, suggests thatT. burksiorum is an ancient Tertiary relict and not an example of recent long distance dispersal. Selection in rockhouse habitats has driven species away from their normal life cycle to partial or complete reliance on the gametophyte generation. The combination of a reduced yet fertile sporophyte and a gametophyte that may rely on gametophytic proliferation placesT. burksiorum in an intermediate evolutionary position relative to other rockhouse pteridophytes.     

Ploidy Distribution of the Harmful Bloom Forming Macroalgae Ulva spp. in Narragansett Bay,Rhode Island,USA, Using Flow Cytometry Methods

Macroalgal blooms occur worldwide and have the potential to cause severe ecological and economic damage. Narragansett Bay, RI is a eutrophic system that experiences summer macroalgal blooms composed mostly of Ulva compressa and Ulva rigida, which have biphasic life cycles with separate haploid and diploid phases. In this study, we used flow cytometry to assess ploidy levels of U. compressa and U. rigida populations from five sites in Narragansett Bay, RI, USA, to assess the relative contribution of both phases to bloom formation. Both haploid gametophytes and diploid sporophytes were present for both species. Sites ranged from a relative overabundance of gametophytes to a relative overabundance of sporophytes, compared to the null model prediction of √2 gametophytes: 1 sporophyte. We found significant differences in cell area between ploidy levels for each species, with sporophyte cells significantly larger than gametophyte cells in U. compressa and U. rigida. We found no differences in relative growth rate between ploidy levels for each species. Our results indicate the presence of both phases of each of the two dominant bloom forming species throughout the bloom season, and represent one of the first studies of in situ Ulva life cycle dynamics.     

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.     

Protein composition of different stages in the life cycle of Ulva mutabilis,Føyn

Summary Soluble protein, about 15% of the total cellular protein, was extracted from different stages in the haplodiplontic life cycle of Ulva mutabilis. The electrophoretic band pattern of the protein extracts from the haploid gametophyte and the diploid sporophyte were found to be the same, except for one slow moving band present in the gametophyte, but lacking in the diploid sporophyte. This band was also missing in the extract from the haploid parthenosporophyte, but is seen in the extract from the zoospores. It was found that the synthesis of the protein in this band occurred during most of the preparation period preceding meiosis. The band is not seen in extracts from gametes, and it is inferred that this protein is broken down during the period preceding the mitotic gametangial division in the gametophyte. So far the protein making up this band behaves as should be required for a factor determining the shift in generation during the life cycle.     

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.