Diversity in meiotic spindle origin and determination of cytokinetic planes in sporogenesis of complex thalloid liverworts (Marchantiopsida)

As the earliest divergent land plants, bryophytes (mosses, hornworts, and liverworts) provide insight into the evolution of the unique plant process of sporogenesis by which meiosis results in heavy walled spores. New immunohistochemical data on microtubules and γ-tubulin in four genera of complex thalloid liverworts combined with previously published data on another four genera demonstrate grades in the evolution of spindle organization in meiosis. We have discovered that all recognized forms of microtubule organizing centers (MTOCs) in plant cells (plastid MTOCs, spheroid cytoplasmic MTOCs, polar organizers, and nuclear envelope MTOCs) occur in organization of the meiotic spindle of complex thalloid liverworts. In addition, all aspects of pre-meiotic preparation for quadripartitioning of the sporocyte into a tetrad of spores occur, with the exception of pre-meiotic wall precursors found in certain simple thalloids. The preparation includes morphogenetic plastid migration, cortical bands of microtubules that mark future cytokinetic planes in pre-meiosis, quadrilobing of the cytoplasm during meiotic prophase, and quadripolar microtubule systems that are transformed into functionally bipolar metaphase I spindles. Quadripolar spindle origin is typical of bryophyte sporogenesis even though the MTOCs involved may differ. However, in certain crown taxa of complex thalloids the spindle develops with no traces of quadripolarity and placement of intersporal walls is determined after meiosis, as is typical of higher plants.     

The quadripolar microtubule system in lower land plants

The quadripolar microtubule system (QMS) is a complex array that is associated with predivision establishment of quadripolarity in sporocytes of lower plants (bryophytes and lycopsids). The QMS unerringly predicts the polarity of the two meiotic divisions and plays a central role in development of both the mitotic apparatus (MA) and cytokinetic apparatus (CA) which together accomplish quadripartitioning of the sporocyte into four haploid spores. The QMS is typically, but not exclusively, associated with monoplastidy and precocious quadrilobing of the cytoplasm. In early meiotic prophase the single plastid divides and the resultant plastids migrate so that either the tips of two plastids or the four plastids resulting from a second division are located in the future spore domains. Microtubules that emanate from the plastid tips or from individual plastids in the spore domains interact in the future planes of cytokinesis and give rise to the QMS. The QMS, which encages the prophase nucleus, consists of at least four and usually six (when spore domains are in tetrahedral arrangement) bipolar spindle-like arrays of microtubules presumably with minus ends at plastids in spore domains and plus ends interacting in the future plane of cytokinesis. Each of the six arrays is essentially like the single axial microtubule system (AMS) that intersects the division site and is transformed into the spindle in monoplastidic mitosis in hornworts. As comparative data accumulate, it appears that the AMS is not unique to monoplastidic cell division but instead represents a basic microtubule arrangement that survives as spindle and phragmoplast in cell division of higher plants.     

ULTRASTRUCTURE OF SPOROGENESIS IN THE MOSS,AMBLYSTEGIUM RIPARIUM I. MEIOSIS AND CYTOKINESIS

Emphasis is placed on three aspects of meiosis in the moss Amblystegium riparium (Hedw.) BSG: 1***) nature of the sporogenous layer; 2) prophasic microtubules and polarity; and 3) cleavage pattern. Spore tetrads develop while still encased by archesporial cell walls. The cellular nature of the sporogenous layer differs from the more usual occurrence of free sporocytes released into a common spore sac. Two important events mark the establishment of sporocyte polarity during meiotic prophase: 1) migration of the four plastids to the distal tetrad poles (telophase II poles); and 2) ingrowth of the sporocyte wall in eventual cleavage planes between the tetrad poles. An extensive, plastid-based microtubule system is associated with organelle migration during the establishment of sporocyte polarity in meiotic prophase. Disruption of the nuclear envelope in prometaphase I occurs at sites opposite the four plastids where microtubules extend from plastid envelope to nuclear envelope. Formation of a cell plate following the first meiotic division results in a dyad, whereas in many mosses meiosis is completed in the undivided sporocyte and is followed by simultaneous cleavage into a spore tetrad. Spore cleavage is accomplished by vesicular coalescence resulting in septa that coincide with the prophasic wall ingrowths.     

{gamma}-Tubulin and microtubule organization during meiosis in the liverwort Ricciocarpus natans (Ricciaceae)

Extant liverworts are "living fossils" considered sister to all other plants and as such provide clues to the evolution of the microtubule organizing center (MTOC) in anastral cells. This report is the first on microtubule arrays and their γ-tubulin-nucleating sites during meiosis in a member of the Ricciales, a specialized, species-rich group of complex thalloid (marchantioid) liverworts. In meiotic prophase, γ-tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci give rise to a multipolar prometaphase spindle. By metaphase I, the spindle has matured into a bipolar structure with truncated poles. In both first and second meiosis, γ-tubulin forms box-like caps at the spindle poles. γ-Tubulin moves from spindle poles to the proximal surfaces of telophase chromosomes where interzonal microtubules are nucleated. Although a phragmoplast is organized, no cell plate is deposited, and second division occurs simultaneously in the undivided sporocyte. γ-Tubulin surrounds each of the tetrad nuclei, and phragmoplasts initiated between both sister and nonsister nuclei direct simultaneous cytokinesis. The overall pattern of meiosis (unlobed polyplastidic sporocytes, nuclear envelope MTOC, multipolar spindle origin, spindles with box-like poles, and simultaneous cytokinesis) more closely resembles that of Conocephalum than other marchantiod liverworts.     

MONOPLASTIDIC CELL DIVISION IN LOWER LAND PLANTS

In many bryophytes and vascular cryptogams mitosis and/or meiosis takes place in cells containing a single plastid. In monoplastidic cell division plastid polarity assures that nuclear and plastid division are infallibly coordinated. The two major components of plastid polarity are morphogenetic plastid migration and microtubule organization at the plastids. Before nuclear division the plastid migrates to a position intersecting the future division plane. This morphogenetic migration is a reliable marker of division polarity in cells with and without a preprophase band of microtubules (PPB). The PPB, which predicts the future division plane before mitosis, is a characteristic feature of land plants and its insertion into the cytokinetic apparatus marks the evolution of a cortical microtubule system and a commitment to meristematic growth. Microtubule systems associated with plastid division, the axial microtubule system (AMS) in mitosis and the quadripolar microtubule system (QMS) in meiosis, contribute to predictive positioning of plastids and participate directly in spindle ontogeny. Division polarity in monoplastidic sporocytes is remarkable in that division sites are selected prior to the two successive nuclear divisions of meiosis. Plastid arrangement prior to meiosis determines the future spore domains in monoplastidic sporocytes, whereas in polyplastidic sporocytes the spore nuclei play a major role in claiming cytoplasmic domains. It is hypothesized that predivision microtubule systems associated with monoplastidic cell division are early forming components of the mitotic apparatus that serve to orient the spindle and insure equal apportionment of nucleus and plastids. “Can it be supposed that cytoplasm would be intrusted with so important a task as the preparation of a chloroplast for each of the four nuclei that are later to preside over the spores before there is any indication that such nuclear division is to take place?” Bradley Moore Davis, 1899     

Induced apogamous sporophytes inPteris dispar andP. semipinnata,and the meiotic behavior in their sporocytes

Spores ofPteris dispar andP. semipinnata were aseptically cultured in flasks for apogamous sporophyte induction. Calli or cell colonies similar to calli were induced in cultures supplemented with hormones. Sporophytic leaves subsequently developed from them in hormone-free medium and the young sporophytes were raised into plants with sporangia. Since the wild-type plants having 116 chromosomes are tetraploid, the sporophytic plants originating from spores would appear to be diploid (dihaploid). In induced sporophytes ofP. semipinnata, non-homologous chromosomes (58 univalents) were found during the meiotic process in sporocytes. InP. dispar, however, the meiotic cells showed many bivalent chromosomes (maximum 29ll). These results suggest thatP. semipinnata is allotetraploid, whereasP. dispar is autotetraploid.     

The origin of the sporophyte shoot in land plants: a bryological perspective

Background

Land plants (embryophytes) are monophyletic and encompass four major clades: liverworts, mosses, hornworts and polysporangiophytes. The liverworts are resolved as the earliest divergent lineage and the mosses as sister to a crown clade formed by the hornworts and polysporangiophytes (lycophytes, monilophytes and seed plants). Alternative topologies resolving the hornworts as sister to mosses plus polysporangiophytes are less well supported. Sporophyte development in liverworts depends only on embryonic formative cell divisions. A transient basal meristem contributes part of the sporophyte in mosses. The sporophyte body in hornworts and polysporangiophytes develops predominantly by post-embryonic meristematic activity.

Scope

This paper explores the origin of the sporophyte shoot in terms of changes in embryo organization. Pressure towards amplification of the sporangium-associated photosynthetic apparatus was a major driver of sporophyte evolution. Starting from a putative ancestral condition in which a transient basal meristem produced a sporangium-supporting seta, we postulate that in the hornwort–polysporangiophyte lineage the basal meristem acquired indeterminate meristematic activity and ectopically expressed the sporangium morphogenetic programme. The resulting sporophyte body plan remained substantially unaltered in hornworts, whereas in polysporangiophytes the persistent meristem shifted from a mid-embryo to a superficial position and was converted into an ancestral shoot apical meristem with the evolution of sequential vegetative and reproductive growth.

Conclusions

The sporophyte shoot is interpreted as a sterilized sporangial axis interpolated between the embryo and the fertile sporangium. With reference to the putatively ancestral condition found in mosses, the sporophyte body plans in hornworts and polysporangiophytes are viewed as the product of opposite heterochronic events, i.e. an anticipation and a delay, respectively, in the development of the sporangium. In either case the result was a pedomorphic sporophyte permanently retaining juvenile characters.     

TRANSITION TO A LAND FLORA: PHYLOGENETIC RELATIONSHIPS OF THE GREEN ALGAE AND BRYOPHYTES

Abstract— Separate cladistic analyses of the green algae, liverworts, and hornworts are presented. Classificatory and evolutionary implications of these analyses, in addition to our previously published cladistic analyses of mosses and the embryophytes as a whole, are discussed. The embryophytes are monophyletic, and are part of a larger monophyletic group that includes some of the green algae (the "charophytes"). Important evolutionary transformations in the early phylogeny of the land plants include: (1) retention of the zygote on the haploid plant (gametophyte), with the sporophyte generation arising de novo by delaying meiosis, (2) independent elaboration of an elongate sporophyte in some liverworts, some hornworts, and in the moss-tracheophyte clade, (3) independent origin of radial (axial) symmetry in the gametophyte in some liverworts and in the moss-tracheophyte clade, (4) independent origin of leaves on the gametophyte in some liverworts and in mosses, and (5) the unique development of a branching sporophyte with multiple sporangia in the tracheophytes.     

Sporogenesis in Bryophytes: Patterns and Diversity in Meiosis

Meiosis in bryophytes retains unusual features that provide clues to the innovation of sporogenesis in early land plants. Sporocytes are typically quadrilobed before nuclear division and the meiotic spindle is quadripolar with poles in the four future spore domains. Whereas seed plants consistently have anastral spindles arising from γ-tubulin in the perinuclear area, bryophytes have spindles organized at POs, plastids, or nuclear envelope. All of these MTOCs are significantly different from centrosomes of the algal ancestors. Mosses and hornworts have quadrilobed sporocytes with meiotic spindles organized at plastids. Meiosis in liverworts is extremely varied. Sporocytes of Jungermanniopsida are deeply quadrilobed and have microtubule bands marking division planes prior to cytoplasmic shaping. Spindles are organized at POs or nuclear envelope. Sporocytes of Marchantiopsida are quadrilobed to apolar with spindles organized at plastids, POs, or nuclear envelope. Pre-meiotic bands have been reported in only one marchantiod, the early divergent Blasia. An atlas of cytological data on 13 liverworts, 3 mosses and 2 hornworts is presented and analyzed.     

ULTRASTRUCTURE OF MEIOSIS IN DASYA BAILLOUVIANA (RHODOPHYTA). I. PROPHASE I1

This first of two papers on ultrastructural observations of meiosis in the red alga Dasya baillouviana (Gmelin) Montague describes stages of prophase I of meiosis. Although the five stages of prophase were originally derived from light microscopic studies, the same stages were utilized for this study based on the developmental sequence of the synaptonemal complex, which has the same morphology and mode of development as those reported for other red algae. The cytoplasm in early prophase sporocytes was typically less electron dense than either vegetative cells or sporocytes in later stages of meiosis. The reduction in density suggests clearing of ribosomes and other cytoplasmic components prior to conversion from sporophyte to gametophyte control. Leptotene cells often had an amorphous, chromatin-free area, function unknown, which was not obviously associated with any specific nuclear region. Diplotene cells were characterized by nuclei containing prominent ring-shaped nucleoli composed of a dark staining ring of material surrounding an electron-translucent “vacuole.” Packets of electron-dense, fibrillar material were often noted in the cytoplasm of late prophase cells. These packets are thought to he “nuage,” a term applied to large cytoplasmic aggregations of RNA in germ cells of several other phyla. It is suggested that nuage may represent a new infusion of ribosomal and messenger RNA for post-meiotic development. The division pales are established by late prophase and a single polar ring is found within each large “exclusion zone” in close association with a pore-free area of nuclear envelope. Both annulate lamellae and small, numerous vesicles are located in the exclusion zones. The significance of the various aspects of prophase I is discussed with the overall observation that this phase of meiosis in red algae is very similar to the process in higher plant and animal cells.     

Phylogeny and diversification of bryophytes

The bryophytes comprise three phyla of embryophytes that are well established to occupy the first nodes among extant lineages in the land-plant tree of life. The three bryophyte groups (hornworts, liverworts, mosses) may not form a monophyletic clade, but they share life history features including dominant free-living gametophytes and matrotrophic monosporangiate sporophytes. Because of their unique vegetative and reproductive innovations and their critical position in embryophyte phylogeny, studies of bryophytes are crucial to understanding the evolution of land plant morphology and genomes. This review focuses on phylogenetic relationships within each of the three divisions of bryophytes and relates morphological diversity to new insights about those relationships. Most previous work has been on the mosses, but progress on understanding the phylogeny of hornworts and liverworts is advancing at a rapid pace. Multilocus multigenome studies have been successful at resolving deep relationships within the mosses and liverworts, whereas single-gene analyses have advanced understanding of hornwort evolution.     

Polar organizers and girdling bands of microtubules are associated with γ-tubulin and act in establishment of meiotic quadripolarity in the hepatic Aneura pinguis (Bryophyta)

Summary. Meiosis in Aneura pinguis is preceded by extensive cytoplasmic preparation for quadripartitioning of the diploid sporocyte into a tetrad of haploid spores. In early prophase the four future spore domains are defined by lobing of the cytoplasm and development of a quadripolar prophase spindle focused at polar organizers (POs) centered in the lobes. Cells entering the reproductive phase become isolated and, instead of hooplike cortical microtubules, have endoplasmic microtubule systems centered on POs. These archesporial cells proliferate by mitosis before entering meiosis. In prophase of each mitosis, POs containing a distinct concentration of γ-tubulin appear de novo at tips of nuclei and initiate the bipolar spindle. Cells entering meiosis become transformed into quadrilobed sporocytes with four POs, one in each lobe. This transition is a complex process encompassing assembly of two opposite POs which subsequently disperse into intersecting bands of microtubules that form around the central nucleus. The girdling bands define the future planes of cytokinesis and the cytoplasm protrudes through the restrictive bands becoming quadrilobed. Two large POs reappear in opposite cleavage furrows. Each divides and the resulting POs migrate into the tetrahedral lobes of cytoplasm. Cones of microtubules emanating from the four POs interact to form a quadripolar microtubule system (QMS) that surrounds the nucleus in meiotic prophase. The QMS is subsequently transformed into a functionally bipolar metaphase spindle by migration of poles in pairs to opposite cleavage furrows. These findings contribute to knowledge of microtubule organization and the role of microtubules in spatial regulation of cytokinesis in plants. Correspondence and reprints: Department of Biology, University of Louisiana-Lafayette, Lafayette, LA 70504-2451, U.S.A.     

Major transitions in the evolution of early land plants: a bryological perspective

Background Molecular phylogeny has resolved the liverworts as the earliest-divergent clade of land plants and mosses as the sister group to hornworts plus tracheophytes, with alternative topologies resolving the hornworts as sister to mosses plus tracheophytes less well supported. The tracheophytes plus fossil plants putatively lacking lignified vascular tissue form the polysporangiophyte clade. Scope This paper reviews phylogenetic, developmental, anatomical, genetic and paleontological data with the aim of reconstructing the succession of events that shaped major land plant lineages. Conclusions Fundamental land plant characters primarily evolved in the bryophyte grade, and hence the key to a better understanding of the early evolution of land plants is in bryophytes. The last common ancestor of land plants was probably a leafless axial gametophyte bearing simple unisporangiate sporophytes. Water-conducting tissue, if present, was restricted to the gametophyte and presumably consisted of perforate cells similar to those in the early-divergent bryophytes Haplomitrium and Takakia. Stomata were a sporophyte innovation with the possible ancestral functions of producing a transpiration-driven flow of water and solutes from the parental gametophyte and facilitating spore separation before release. Stomata in mosses, hornworts and polysporangiophytes are viewed as homologous, and hence these three lineages are collectively referred to as the 'stomatophytes'. An indeterminate sporophyte body (the sporophyte shoot) developing from an apical meristem was the key innovation in polysporangiophytes. Poikilohydry is the ancestral condition in land plants; homoiohydry evolved in the sporophyte of polysporangiophytes. Fungal symbiotic associations ancestral to modern arbuscular mycorrhizas evolved in the gametophytic generation before the separation of major present-living lineages. Hydroids are imperforate water-conducting cells specific to advanced mosses. Xylem vascular cells in polysporangiophytes arose either from perforate cells or de novo. Food-conducting cells were a very early innovation in land plant evolution. The inferences presented here await testing by molecular genetics.     

The placenta in Monoclea forsteri Hook. and Treubia lacunosa (Col.) Prosk: insights into placental evolution in liverworts

Placental morphology is remarkably diverse between major bryophyte groups, especially with regard to the presence and distribution of transfer cells in the sporophyte and gametophyte. In contrast, with the exception of metzgerialean liverworts, placental morphology is highly conserved within major bryophyte groups. Here we examine the ultrastructure of the placenta in Monoclea forsteri and Treubia lacunosa, basal members of the marchantialean and metzgerialean liverwort lineages, respectively. In both species several layers of transfer cells are found on both sides of the placenta, with sporophytic transfer cells exhibiting prominent wall labyrinths. Consistent with previous reports of a similar placenta in other putatively basal and isolated liverwort genera such as Fossombronia, Haplomitrium, Blasia and Sphaerocarpos, this finding suggests that this type of placenta represents the plesiomorphic (primitive) condition in liverworts. Distinctive ultrastructural features of placental cells in Monoclea include branched plasmodesmata in the sporophyte and prominent arrays of smooth endoplasmic reticulum, seemingly active in secretion in the gametophyte. These arrays contain a core of narrow tubules interconnected by electron-opaque rods, structures with no precedent in plants. Analysis of the distribution of different types of placenta in major bryophyte groups provides valuable insights into their inter-relationships and possible phylogeny.     

Nuclear cytoplasmic domains,microtubules and organelles in microsporocytes of the slipper orchid Cypripedium californicum A. Gray dividing by simultaneous cytokinesis

Microsporocytes of the slipper orchidCypripedium californicum A. Gray divide simultaneously after second meiosis. The organization and apportionment of the cytoplasm throughout meiosis are functions of nuclear-based radial microtubule systems (RMSs) that define domains of cytoplasm - a single sporocyte domain before meiosis, dyad domains within the undivided cytoplasm after first meiosis, and four spore domains after second meiosis. Organelles migrate to the interface of dyad domains in the undivided cytoplasm after first meiotic division, and second meiotic division takes place simultaneously on both sides of the equatorial organelle band. Microtubules emanating from the telophase II nuclei interact to form columnar arrrays that interconnect all four nuclei, non-sister as well as sister. Cell plates are initiated in these columns of microtubules and expand centrifugally along the interface of opposing RMSs, coalescing in the center of the sporocyte and joining with the original sporocyte wall at the periphery to form the tetrad of microspores. Organelles are distributed into the spore domains in conjunction with RMSs. These data, demonstrating that cytokinesis in microsporogenesis can occur in the absence of both components of the typical cytokinetic apparatus (the preprophase band of microtubules which predicts the division site and the phragmoplast which controls cell-plate deposition), suggest that plant nuclei have an inherent ability to establish a domain of cytoplasm via radial microtubule systems and to regulate wall deposition independently of the more complex cytokinetic apparatus of vegetative cells.     

Salt Tolerance of Ectocarpus siliculosus (Dillw.) Lyngb.: Comparison of Gametophytes,Sporophytes and Isolates of Different Geographic Origin

Forty Ectocarpus siliculosus isolates from a wide geographical range, including gametophyte and sporophyte plants, have all been acclimated to the same salinity for several years. Their salinity tolerances in respect of cell viability, photosynthesis and dark respiration were evaluated over the salinity range: 8 to 96 ‰. Significant differences in the physiological tolerances to salt stress compared with viability measurements were evident. Genotypic differences in salt tolerances between groupings of the isolates, and also differences in responses of gametophyte and sporophyte generations were found. However, diploid and haploid sporophyte material had similar tolerances. Triploid and tetraploid sporophytes did not have improved tolerances over those of diploid plants. Culture plants originating from low salinities in the Baltic Sea had broader tolerances than field material collected from Baltic waters of similar salinity.     

SPORANGIAL STRUCTURE AND ZOOSPOROGENESIS IN CHORDA TOMENTOSA (LAMINARIALES)1,2,3

Members of the genus Chorda represent the simplest form of sporophyte in the order Laminariales. The present study deals with reproduction in Chorda tomentosa, involving the initiation, growth, and structure of the sporangium and the process of zoosporogenesis. The simple tube-like sporophyte of Chorda tomentosa grows in diameter by means of repeated anticlinal divisions in a superficial meristematic layer known as the meristoderm. The onset of reproduction is marked by the conversion of the meristoderm from contributing cells to the vegetative plant body to producing 2 new cell types: paraphyses and sporangial mother cells. At the time of initiation, sporangial mother cells are crescent shaped and possess a densely staining cytoplasm. Sporangial mother cells increase in size, become ellipsoid, decrease in staining density, and undergo meiosis. After meiosis, sporangia increase in size while their nuclei undergo successive cycles of synchronous mitotic divisions. Sporangia increase to a maximum length of 120 μ;m at which time they possess the characteristic “cap” found in all members of the order studied thus far. At this stage the protoplast of the sporangium is organized into a peripheral layer of nucleus-chloroplast pairs and a central region of vacuoles. Cleavage furrows begin to form at the cell membrane and are met by furrows developing in the interior of the cytoplasm resulting in the division of the entire protoplast into separate units. Each unit is an individual zoospore. The biflagellate zoospores contain a nucleus, one chloroplast with eyespot, perinuclear Golgi, and several bodies of presumed storage carbohydrate. The occurrence of a small population of early developing sporangia is described. In essential details, the origin, development, and structure of sporangia in Chorda tomentosa are identical to all earlier observations in the Laminariales.     

Enhanced sexual reproduction in bryophytes at high latitudes in the maritime Antarctic

Abstract

Contrary to the generally accepted hypothesis that bryophyte fertility decreases with increasing latitude and therefore climatic severity, a detailed study of bryophyte reproductive strategies at sites in the southern maritime Antarctic (68–72°S) has revealed that an unexpectedly high proportion of species is capable of producing sporophytes. Of the regional bryoflora, 43% (19 species; 17 mosses, 2 liverworts) in Marguerite Bay and 47% (17 species; 16 mosses, 1 liverwort) in Alexander Island are known to produce sporophytes, although the number fruiting at comparable latitudes on the colder and more arid Antarctic continent is less (33%). These numbers and proportions are much greater than were previously known at such relatively high southern polar latitudes. Sporophytes of several species are recorded for the first time within the Antarctic biome, while those of two liverworts (Lophozia excisa and Cephaloziella varians) are reported for the first time south of 62°S. High incidence of sporophyte production is attributed to locally favourable microclimatic conditions producing small-scale 'oases'. The large majority of fertile mosses are monoecious short acrocarps growing on rather calcareous soils. Spore production and size data for a number of species are compared with previously published studies of the same or closely related species from the northern maritime Antarctic and sub-Antarctic, but no consistent trends are revealed between species over the latitudinal gradient.