BUILDING A SPECIES PHYLOGENY FOR THE GENUS COLEOCHAETE (COLEOCHAETALES) USING THE GENES RBCL AND ATPB

The freshwater green alga Coleochaete Breb. (Coleochaetaceae; Coleochaetales) is a key streptophyte genus and is important to the understanding of the evolutionary origin of embryophytes (land plants). To date only a few species have been available from public culture collections. To facilitate research on this genus we have isolated 17 previously uncultured species of Coloechaete from material collected in the United States, Puerto Rico, and the Dominican Republic. Sequences for the genes rbcL and atpB were determined for these new isolates of Coleochaete (and for existing cultures) and combined with sequences from representative other streptophytes. Phylogenetic analyses indicate that Coleochaete, along with Chaetosphaeridium and Chara, are closely related to embryophytes and constitute a ‘higher streptophyte’ clade. At least four well‐supported lineages exist within Coleochaete. Characteristic growth forms have been identified for these four lineages, with important characters including aspects of thallus establishment, thallus habit, zygote development and hair sheath position. These data provide an improved understanding of species diversity and character evolution in the genus Coleochaete, and facilitate examination of hypotheses concerning character evolution in the streptophytes.     

COLEOCHAETE AND THE ORIGIN OF LAND PLANTS

For a century the green alga Coleochaete has figured prominently in considerations of the origins of land plants (embryophytes). Certain of its advanced features contributed to Bower's (1908) theories on the origin of the land plant sporophyte by intercalation. Though Bower's ideas were disputed in later years, recent investigations of Coleochaete and other green algae have lent strong support to them. At present it appears that further study of Coleochaete and other charophycean algae may contribute much to our understanding of how a number of plant features, including reproductive ones, originated.     

PHYLOGENY OF THE GENUS COLEOCHAETE (COLEOCHAETALES,CHAROPHYTA) AND RELATED TAXA INFERRED BY ANALYSIS OF THE CHLOROPLAST GENE rbcL1

The genus Coleochaete Bréb. is considered to be a key taxon in the evolution of green algae and embryophytes (land plants), but only a few of the approximately 15 species have been studied with molecular phylogenetic methods. We report here the sequences of the gene rbcL from six new cultures of Coleochaete and two of Chaetosphaeridium Klebahn. These sequences were combined with 32 additional sequences, and phylogenetic analyses were performed with maximum likelihood, distance optimality, and parsimony methods. Important subgroups within Coleochaete include two primary lineages, one marked by fully corticated zygotes and the other by naked or weakly corticated zygotes. In the first lineage there is a subclade with tightly joined filaments and distinctive (“T‐shaped”) cell division, an assemblage of strains that resembles the endophytic species Coleochaete nitellarum Jost, and a clade with loosely joined filaments and “Y‐shaped” cell divisions. Consistent with recent multigene phylogenies, these analyses support the monophyly of the Coleochaetales, place the Charales as the sister taxon to land plants, and indicate that Chaetosphaeridium is far more closely related to Coleochaete than to Mesostigma Lauterborn.     

Occurrence and evolutionary significance of RESISTANT CELL WALLS IN CHAROPHYTES AND BRYOPHYTES

A survey of charophycean green algal and bryophyte taxa revealed the frequent occurrence of vegetative cell walls that were characterized by a specific form of autofluorescence and resistance to high temperature acid treatment (acetolysis). The time of production and the location of resistant, autofluorescent cell walls varied among charophyte and bryophyte taxa in patterns that suggest that bryophytes inherited the capacity to produce such walls from charophyte ancestors. A number of charophytes produced resistant walls in response to desiccation stress, suggesting an evolutionarily early adaptive response. Coleochaete was unique among charophytes, but similar to all bryophytes tested in that sexual reproduction induced autofluorescence in cell walls of well-hydrated tissues at the placental junction. Maternal tissues in apical portions of the pseudoseta bearing Sphagnum sporophytes were characterized by autofluorescent, acetolysis-resistant cell walls similar to those observed in maternal cells adjacent to Coleochaete zygotes. These observations suggest that cell–cell stimulus–response interactions regulate deposition of autofluorescent compounds in placental cell walls, and that this characteristic may have been shared by the earliest embryophytes and their charophyte ancestors. Various bryophytes deposit autofluorescent, acid-resistant compounds at other adaptively significant sites including sporangial epidermis, spiral thickenings of elaters, rhizoids, and leaves in the special case of Sphagnum moss. Sphagnum and liverwort sporangial epidermis, which had been subjected to acetolysis or strong acid procedures commonly used to release microfossils from rock matrices, resembled published photographs of Ordovician–Devonian microfossils consisting of cellular scraps that have been attributed to earliest land plants. Our work suggests that at least some of these fossils, previously thought to represent “dispersed cuticles,” could be reinterpreted as earliest known remains of plant sporophytic tissues, and that they may be homologous with resistant sporangial epidermis of modern bryophytes. In general, the patterns of occurrence of resistant, autofluorescent cell walls in charophytes and bryophytes suggest repeated exaptation. Regulation of deposition appears to have been modified through time, so that resistant wall compounds have had a sequence of functions: desiccation resistance and/or microbial resistance in lower charophytes, a role in embryogenesis in Coleochaete and embryophytes, and finally, decay resistance in innovative structures that characterize bryophytes, such as rhizoids, sporangial epidermis, and elaters.     

PYRENOID FORMATION ASSOCIATED WITH THE CELL CYCLE IN THE BROWN ALGA,SCYTOSIPHON LOMENTARIA (SCYTOSIPHONALES,PHAEOPHYCEAE)1

Vegetative cells of the brown alga Scytosiphon lomentaria (Lyngbye) Link characteristically have only one chloroplast with a prominent protruding pyrenoid, whereas zygotes have both paternal and maternal chloroplasts. In zygotes, before cell and chloroplast division, each chloroplast has an old and a new pyrenoid. In this study, we raised a polyclonal antibody to RUBISCO and examined the distribution of RUBISCO by immunofluorescence microscopy, focusing on new pyrenoid formation in vegetative cells of gametophytes and zygotes in Scytosiphon. In interphase, only one old pyrenoid was positively indicated by anti‐RUBISCO antibody in vegetative cells of gametophytes. From mid‐S phase, small fluorescence aggregates reflecting RUBISCO localization started to appear at stroma positions other than adjacent to the old protruding pyrenoid. The fluorescent spots eventually coalesced into a protrusion into the adjacent cytoplasm. We also used inhibitors to clarify the relationship between the cell cycle and new pyrenoid formation, using zygotes after fertilization. When DNA replication was blocked by aphidicolin, new pyrenoid formation was also inhibited. Washing out aphidicolin permitted new pyrenoid formation with the progression of the cell cycle. When mitosis was prolonged by nocodazole, which disrupted the spindle microtubules, the fluorescent masses indicating RUBISCO localization continued to increase when compared with pyrenoid formation in untreated zygotes. During treatment with chloramphenicol, mitosis and cytokinesis were completed. However, there was no occurrence of new RUBISCO localization within the chloroplast stroma beyond the old pyrenoid. From these observations, it seems clear that new pyrenoid formation in the brown alga Scytosiphon depends on the cell cycle.     

OCCURRENCE AND PHYLOGENETIC SIGNIFICANCE OF “SPECIAL WALLS” AT MEIOSPOROGENESIS IN COLEOCHAETE

Within germinating zygotes of Coleochaete pulvinata, meiospores are individually surrounded by chamber walls which are ultrastructurally and chemically different from vegetative cell walls of the same species. Meiospore chamber walls exhibit the staining reactions typical of callose. They thus resemble the “special walls” present during sporogenesis in embryophytes. Their presence suggests that the charophycean green algal ancestors of land plants may have possessed spore development preadaptations influential in the evolution of walled spores, an important plant adaptation to terrestrial life.     

Homologous Versus Antithetic Alternation of Generations and the Origin of Sporophytes

The late-nineteenth/early-twentieth century debate over homologous versus antithetic alternation of generations is reviewed. Supporters of both theories, at first, used Coleochaete as a model for the origin of land-plant life cycles. The early debate focused on the morphological interpretation of the sporophyte and on whether vascular cryptogams had bryophyte-like ancestors. The terms of the debate shifted after the discovery that the alternation of morphological generations was accompanied by an alternation of chromosome number. Supporters of homologous alternation now promoted a model in which land plants had been derived from an algal ancestor with an isomorphic alternation of haploid and diploid generations whereas supporters of antithetic alternation favored a model in which land plants were derived from a haploid algal ancestor with zygotic meiosis. Modern evidence that embryophytes are derived from charophycean green algae is more compatible with an updated version of the antithetic theory.     

MOLECULAR AND MORPHOLOGICAL DATA IDENTIFY A CRYPTIC SPECIES COMPLEX IN ENDOPHYTIC MEMBERS OF THE GENUS COLEOCHAETE BRÉB. (CHAROPHYTA: COLEOCHAETACEAE)1

The genus Coleochaete Bréb. is a relatively small group of freshwater microscopic green algae with about 15 recognized species. Although Coleochaete has long been considered to be a close relative of embryophytes, a comprehensive study of the genus has not been published since Pringsheim's 1860 monograph. As part of a systematic study of Coleochaete, we investigated four accessions of the genus that are morphologically similar to the endophytic species C. nitellarum Jost. Each of the four cultures was determined to be capable of endophytic growth in Nitella C. A. Agardh, a member of the closely related order Charales. Maximum likelihood and maximum parsimony analyses were performed on nucleotide data from the chloroplast genes atpB and rbcL that were sequenced from 16 members of the Coleochaetales and from other members of the Charophyceae, embryophytes, and outgroup taxa. These analyses indicate that the Coleochaetales are monophyletic and that the endophytic accessions are members of the scutata group of species. In addition, cell size and nucleotide data suggest that at least three different endophytic species may be represented. Herbivory, nutritional benefits, and substrate competition are three hypotheses that could explain the evolution and maintenance of the endophytic habit in Coleochaete. These data also imply that diversity in the genus may be markedly underestimated.     

THE ULTRASTRUCTURE OF MULTILAYERED STRUCTURES ASSOCIATED WITH FLAGELLAR BASES IN MOTILE CELLS OF TRENTEPOHLIA AUREA1

An ultrastructural study of motile cell development in the green alga Trentepohlia aurea has revealed the presence of multilayered structures (MLS) associated With flagellar bases. These MLS are ultrastructurally similar to MLS described in pteridophyte and bryophyte sperm and in the zoospore of the green algae Coleochaete and Klebsormidium. However, 2 MLS are found in each biflagellate motile cell of T. aurea, while other previously described MLS occur singly in biflagellate motile cells. In addition, the MLS of T. aurea consist of fewer microtubules and are structurally simpler than most other MLS described. The MLS of Trentepohlia may represent a stage in the evolutionary development of the MLS of land plants. The presence or absence of the MLS in motile cells of green algae may be a useful character in phylogenetic studies.     

Cyanolichens can have both cyanobacteria and green algae in a common layer as major contributors to photosynthesis

Background and Aims

Cyanolichens are usually stated to be bipartite (mycobiont plus cyanobacterial photobiont). Analyses revealed green algal carbohydrates in supposedly cyanobacterial lichens (in the genera Pseudocyphellaria, Sticta and Peltigera). Investigations were carried out to determine if both cyanobacteria and green algae were present in these lichens and, if so, what were their roles.

Methods

The types of photobiont present were determined by light and fluorescence microscopy. Small carbohydrates were analysed to detect the presence of green algal metabolites. Thalli were treated with selected strengths of Zn²⁺ solutions that stop cyanobacterial but not green algal photosynthesis. CO₂ exchange was measured before and after treatment to determine the contribution of each photobiont to total thallus photosynthesis. Heterocyst frequencies were determined to clarify whether the cyanobacteria were modified for increased nitrogen fixation (high heterocyst frequencies) or were normal, vegetative cells.

Key Results

Several cyanobacterial lichens had green algae present in the photosynthetic layer of the thallus. The presence of the green algal transfer carbohydrate (ribitol) and the incomplete inhibition of thallus photosynthesis upon treatment with Zn²⁺ solutions showed that both photobionts contributed to the photosynthesis of the lichen thallus. Low heterocyst frequencies showed that, despite the presence of adjacent green algae, the cyanobacteria were not altered to increase nitrogen fixation.

Conclusions

These cyanobacterial lichens are a tripartite lichen symbiont combination in which the mycobiont has two primarily photosynthetic photobionts, ‘co-primary photobionts’, a cyanobacterium (dominant) and a green alga. This demonstrates high flexibility in photobiont choice by the mycobiont in the Peltigerales. Overall thallus appearance does not change whether one or two photobionts are present in the cyanobacterial thallus. This suggests that, if there is a photobiont effect on thallus structure, it is not specific to one or the other photobiont.     

Microspore ofSecale cereale as a transfer cell type

Summary In the mature microspore ofSecale cereale, a set of wall ingrowths deposited as the first (outer) intine layer between exine and the microspore plasma membrane, are revealed by electron microscopy. The wall ingrowths form a girdle in the vicinity of the apertural region at the external pole of microspore which is in contact with the tapetum, so the microspore can be considered as a transfer cell which is polarized. After microspore division the second (inner) intine layer is deposited by the vegetative cell and forms a labyrinth of branched wall ingrowths. As a result, the periphery of a vegetative cell is also irregular and appears as very thin plasmatubules or evaginations delimited by plasma membrane and penetrating the pollen wall.The possible functions of the microspore as a transfer cell and the wall-membrane system of the vegetative cell are discussed.     

CYANOPHYTE CEPHALODIA IN THE LICHEN GENUS NEPHROMA

The lichens, Nephroma expallidum (Nyl.) Nyl. and N. arcticum (L.) Torss., consistently have at least two symbionts in a single thallus: a green alga in the algal layer and a blue-green alga in the internal cephalodia. The cephalodia originate from algal cells in contact with the lower surface of the lichen, in the zone of rhizine formation. The rhizines surround the epiphytic algal colony and form a second cortical layer; following dissociation of the original lower cortex, further growth of the two organisms results in the cyanophyte colony being enveloped by a compact layer of fungal tissue and positioned in the lichen medulla. The colony may eventually assume a superior or inferior position in relation to the lichen thallus, depending in part on the lichen species. Nephroma anticum may have two distinct morphological forms of blue-green algae in the same thallus and occasionally in the same cephalodium. It appears that the relationship that exists between the cephalodial algae and the lichen thallus is antagonistic and results, in some cases, in the exclusion of the green algal layer and death to the cephalodial cyanophytes.     

EVIDENCE FOR SEXUAL REPRODUCTION IN THE PRIMITIVE GREEN ALGA NEPHROSELMIS OLIVACEA (PRASINOPHYCEAE)1

Evidence for a specialized sexual process in Nephroselmis olivacea Stein is presented. This alga is a member of the Prasinophyceae, which is regarded by some as the most primitive Class of green plants. N. olivacea has a heterothallic type of mating system. Plus and minus gametes were morphologically similar but showed different behaviors during the mating process. The minus gamete settled to the substratum, attaching by its ventral side. The plus gamete attached to the dorsal side of the minus gamete by the region near the flagellar bases of the plus gamete. The mature zygotes were spherical and strongly adhered to the substratum. After zygote germination, two biflagellate daughter cells, each with two pyrenoids were liberated. These cells divided, resulting in four vegetative cells, each with a single pyrenoid.     

Observations on the fine structure of spermatozoids and vegetative cells of the green alga Golenkinia

Spermatozoids and vegetative cells of the green alga Golenkinia minutissima Iyengar et Balakrishnan have been examined by light and electron microscopy. The biflagellate spermatozoids are of a somewhat specialised type, elongated with the nucleus attached to the flagellar bases, and containing a reduced chloroplast without pyrenoid or eyespot. The flagellar apparatus and root system has been examined in detail and is compared with that found in other green algae. The flagella are of a previously unknown type; they contain only one central microtubule—possibly non-functional—but they move in an apparently normal way. Present knowledge about flagellar roots in green algae has been assembled in a table, showing that the cruciate root has now been found in 10 genera, belonging to almost as many families. Exceptions are Oedogonium, which contains a modification of this type, and the Charales, which are very different. During spermatogenesis in Golenkinia each spermatozoid is surrounded by a wall which disappears at maturity. This fact may prove to be of taxonomic value.

The spines on the vegetative cells are composed of regularly arranged longitudinal fibrils, possibly cellulose, attached to the inner part of the two-layered cell wall. The content of the vegetative cell is typically chlorococcalean.     

THE OCCURRENCE,EVOLUTION, AND PHYLOGENETIC SIGNIFICANCE OF PARENCHYMA IN COLEOCHAETE BRÉB. (CHLOROPHYTA)

Thalli of four species of the charophycean green alga Coleochaete Bréb.; C. irregularis, C. soluta, C. orbicularis, and C. scutata, were examined using light and electron microscopy. The parenchymatous nature of the plant bodies of C. orbicularis and C. scutata became apparent when the degree of cellular association in these species was compared with that of parenchymatous land plant tissues, the pseudoparenchymatous thalli of C. soluta, and the branched filaments of C. irregularis. Coleochaete soluta was shown to be morphologically and phylogenetically transitional between the more primitive branched filamentous species (C. irregularis) and the more advanced discoid species (C. orbicularis and C. scutata). The branching patterns observed in C. soluta suggested a possible pathway for the evolutionary development of land plant parenchyma from branched, filamentous charophycean green algae.     

Polarized microtubule deposition coincides with wall ingrowth formation in transfer cells ofVicia faba L. cotyledons

Summary The epidermal transfer cells in developingVicia faba L. cotyledons are highly polarized. Extensive wall ingrowths occur on their outer periclinal walls and extend part way down both anticlinal walls. This ingrowth development serves to increase the surface area of the plasma membrane and thus maximize porter-dependent uptake of sugars from the seed apoplasm. In contrast, the inner periclinal walls of these transfer cells do not form wall ingrowths. We have commenced a study of the mechanisms responsible for establishing this polarity by first analysing the microtubule (MT) cytoskeleton in developing transfer cells. Thin sections of fixed cotyledons embedded in methacrylate resin were processed for immunofluorescence microscopy using monoclonal anti--tubulin and counterstained with Calcofluor White to visualize wall ingrowths. In epidermal cells of young cotyledons where wall ingrowths were yet to develop, MT labelling was detected around all cortical regions of the cell. However, in cells where wall ingrowths were clearly established, MT labelling was detected almost exclusively in cortical regions adjacent to the wall ingrowths. Little, if any, MT labelling was detected on the anticlinal or inner periclinal walls of these cells. This distribution of MTs was most prominent in cells with well developed wall ingrowths. In these cells, a subpopulation of MTs were also detected emanating from the subcortex and extending towards the wall ingrowth region. The possible role of MT distribution in establishing transfer cell polarity and wall ingrowth formation is discussed.Abbreviations MT microtubule     

Fine structure of the vegetative thallus of the lichen,Peltigera polydactyla

Summary An investigation was made of the vegetative thallus of the lichen Peltigera polydactyla. Using a modified embedding technique it was shown that the ultrastructure of the mycobiont was not radically different from that of nonlichenized Discomycetes, and that the ultrastructure of the phycobiont was like that of the blue-green alga Nostoc. In addition to what were considered healthy blue-green algal cells other cells were present which obviously were breaking down. Well defined heterocysts were also present. No haustoria were found in the thallus.     

PARASITIC INTERACTIONS AND VEGETATIVE ULTRASTRUCTURE OF CHOREOAEMA THURETII (CORALLINALES,RHODOPHYTA)1

The monotypic coralline red alga, Choreonema thuretii (Bornet) Schmitz (Choreonematoideae), grows endophytically within three geniculate genera of the Corallinoideae. Although the thallus of Choreonema is reduced, lacks differentiated plastids, and is endophytic except for its conceptacles, its status as a parasite has been questioned because cellular connections to the host had not been ob served. Transmission electron microscopy, however, disclosed a previously undescribed type of parasitic interaction in which Choreonema interacts with its host through specialized cells known as lenticular cells. These small, lens-shaped cells are produced from the single file of host-penetrating vegetative cells. Pit plug morphology between vegetative and lenticular cells is polarized. Plug caps facing the vegetative cell have normal coralline morphology, while those facing the lenticular cell are composed of three layers. Regions of lenticular cells near host cells protrude toward the host cell; upon encountering the host cell wall, the prolrusion produces numerous finger-like fimbriate processes that make cellular connections with the host cell. Lenticular cells may extend several protrusions toward a host cell or penetrate more than one host cell; two or more lenticular cells may also penetrate the same host cell. The lack of secondary pit connections, cell fusions, and passage of parasitic nuclei suggest that this parasitic relationship may be evolutionarily older than previously reported cases of parasitism in red algae.     

ULTRASTRUCTURE OF TETRASPOROGENESIS IN THE MARINE RED ALGA PTILOTA HYPNOIDES1

Both tetrasporangia and dormant apical cells of short vegetative filaments of the marine red alga Ptilota hypnoides have been examined by electron microscopy. Various cytoplasmic inclusions readily distinguish the vegetative apical cells from the reproductive apical cells which become tetrasporangial mother cells. The transformation of tetrasporangial mother cells into mature tetrasporangia involves a series of cytoplasmic changes which can be correlated with specific changes in the investing wall layers. The extracellular changes provide the basic criteria for the division of tetrasporogenesis into 3 successive stages. The ultrastructure of each stage is described and discussed in relation to the current knowledge of red algal cytology. In addition, a possible mechanism for the liberation of spores and gametes of red algae is proposed.     

SINGLE-CELL PIGMENTATION OF PORPHYRA LINEARIS ANALYZED BY FOURIER TRANSFORM MULTI-PIXEL SPECTROSCOPY AND IMAGE ANALYSIS1

The novel method of Fourier transform multi-pixel spectroscopy was used for the nondestructive analysis of and comparison of pigmentation in different regions of live thalli of the red alga Porphyra linearis. Because the thallus in this alga consists of a monolayer of nonoverlapping cells, we were able to analyze the pigmentation of single cells by combining light absorbance with natural fluorescence data. From the image of each cell in the vegetative male and female reproductive and holdfast regions, more than 4 ± 10⁴ fluorescence and absorbance spectra were obtained. Specific pigments in the different regions were localized by the use of a software program of similarity mapping followed by image construction. The reconstructed images revealed subcellular localization of each pigment according to specific spectroscopic fingerprints. The results showed that the vegetative and female reproductive cell types had a significantly higher content of phycoerythrin than of phycocyanin, and quite similar chlorophyll a levels. Most of the holdfast cells were poorly pigmented, but had more chlorophyll a than phycoerythrin or phycocyanin. The male reproductive cells contained only traces of pigments. Thus, by using Fourier transform multipixel spectroscopy, we were able to characterize the pigmentation of different regions of the thallus and follow the distribution patterns of the different pigments on the subcellular level along the differentiation gradient of the alga.