Serial Development of Foliar Gemmae inTortula(Pottiales, Musci), an Ultrastructural Study

Annals of Botany 78 : 305–315, 1996

Serial Development of Foliar Gemmae in Tortula (Pottiales, Musci), an Ultrastructural Study R O B E R T O L I G R O N E†, J E F F R E Y G. D U C K E TT‡ and R A F F A E L E G A M B A R D E L L A* * Dipartimento di Biologia Vegetale, UniŠersita[ di Napoli, Via Foria 223, I-80139 Napoli, Italy, † Facolta[ di Scienze Ambientali del Secondo Ateneo Napoletano, Šia Arena 22, 81100 Caserta, Italy and ‡ School of Biological Sciences, Queen Mary & Westfield College, Mile End Road, London E1 4NS, UK Received : 19 October 1995

Accepted : 6 March 1996

The development and liberation mechanism of foliar gemmae have been studied by electron microscopy in two mosses, Tortula latifolia Bruch and Tortula papillosa Wils. The gemmae develop on the adaxial surface of mature leaves from single initial cells on both the lamina and costa in T. latifolia but only on the costa in T. papillosa. Elongation of the initial cell is associated with the deposition of a highly extensible new wall whilst the old wall and cuticle in the apical dome rupture. The first division is transverse and separates a short basal cell embedded in the foliar tissue and a distal cell, or gemma primordium, protruding from the leaf surface. Subsequent divisions of the gemma primordium give rise to a six-to-eight-celled globose gemma with mucilaginous outer walls. During gemma development the basal cell produces a new wall and elongates again whilst the common wall with the gemma splits apart centripetally along the boundary between the old and new wall in the basal cell ; plasmodesmal connections are gradually severed and eventually the young gemma remains connected to the basal cell only by mucilage. After separation of the first-formed gemma, the basal cell may expand and produce a second gemma by the same mechanism. The whole process may be repeated several times resulting in the formation of a chain of gemmae stuck together by mucilage and which are liberated only when the leaves are fully hydrated. Accumulation of abundant lipid deposits in the gemmae after symplasmic isolation reflects considerable photosynthetic autonomy. # 1996 Annals of Botany Company Key words : Abscission, bryophytes, cell wall formation, plasmodesmata, vegetative reproduction.

INTRODUCTION Development of new, physiologically independent plants by separation of younger gametophyte sectors and}or regeneration from mature cells (Giles, 1971 ; Longton and Miles, 1982 ; Chopra and Kumra, 1988) are mechanisms essential for the maintenance and expansion of already established bryophyte colonies. By contrast diaspores, i.e. biological units of dispersal, are needed for colonization of new habitats. Diaspore formation by the sporophyte usually covers only a fraction of the life cycle and, moreover, is seasonal and subject to severe ecological and physiological constraints (Longton and Schuster, 1983 ; Mishler, 1988 ; Longton, 1990). For this reason reproduction by asexual diaspores, produced more or less continuously by the gametophyte, is of utmost importance for population spread in numerous bryophyte taxa (Longton and Schuster, 1983 ; Mishler, 1988). Asexual diaspores in bryophytes range from caducous or fragile stems, leaves or perianths, often barely distinguishable from normal vegetative organs, to highly specialized structures such as bulbils, tubers and gemmae (Buch, 1911 ; Correns, 1899 ; Cavers, 1903 ; Schuster, 1966, 1984 ; Duckett and Ligrone, 1992). As initially pointed out by Correns (1899) and reviewed more recently by Duckett and Ligrone (1992), diaspore liberation in mosses may occur either by a schizolytic mechanism, i.e. detachment along cell walls, or a 0305-7364}96}090305­11 $18.00}0

lysigenic mechanism involving the rupture of a specialized abscission or tmema cell. In contrast, only the schizolytic mechanism has been reported in liverworts (Hughes, 1971 a, b ; Duckett and Ligrone, 1994). Until very recently the cytological and physiological mechanisms underlying development, liberation and germination of asexual diaspores in bryophytes have been virtually ignored. Studies of the mosses Calymperes (Duckett and Ligrone, 1991 ; Ligrone, Duckett and Egunyomi, 1992), Funaria (Bopp et al., 1991 ; Sawidis et al., 1991 ; Schnepf and Sawidis, 1991 ; Schnepf, 1992) and Bryum (Goode et al., 1993) have revealed two basically different patterns of tmema cell (TC) formation and breakage. In Calymperes the TC originates by tip growth, whilst in Funaria and Bryum it is formed by unequal intercalary division. In Funaria and Calymperes a new wall is built beneath the original wall but whilst in Funaria the TC breaks off by elongation and subsequent lysis of the new wall, TC breakage in Calymperes results from sliding apart of the old and new walls. As pointed out in a recent survey of asexual diaspores in mosses (Duckett and Ligrone, 1992), these are but two examples of an impressive variety of mechanisms that are still very poorly understood. Thus the aims of this paper are to provide the first documentation of the ultrastructural events associated with schizolytic gemma formation and liberation in mosses and to compare these with the ontogeny and separation of # 1996 Annals of Botany Company

306

Ligrone et al.—Foliar gemmae in Tortula

catenate foliar gemmae in liverworts. The selected species, Tortula papillosa and T. latifolia are both characterized by the continuous copious production of foliar gemmae in nature (Smith, 1978).

MATERIALS AND METHODS Plants of Tortula latifolia were collected from Acer roots by the River Mole, Boxhill, Surrey, UK, whilst the material of T. papillosa was from Tilia trunks, Roscigno, Salerno, Italy. Gemmiferous leaves of different ages were selected and fixed with 2 % glutaraldehyde­1 % formaldehyde­0±5 % tannic acid in 0±05  Na-phosphate buffer, pH 7±0, for 2 h at room temperature, followed by 1 % osmium tetroxide in the same buffer, pH 6±8, overnight at 4 °C. The samples were then dehydrated through a graded series of ethanol to propylene oxide and embedded in Spurr’s resin. Thin sections, cut with a diamond knife, were sequentially stained with 3 % methanolic uranyl acetate and lead citrate. The periodic acid}thiocarbohydrazide}silver proteinate (PATAg) test was performed according to Roland and Sandoz (1969) on sections collected on gold grids. Either periodic acid or thiocarbohydrazide treatment was omitted as controls. The ultrastructural observations were performed with a Jeol 100C or a Philips CM12 electron microscope. For scanning electron microscopy, specimens fixed as above were dehydrated with ethanol, critical point dried, coated with a layer of gold about 20 nm thick and observed with a Jeol JMS 35 scanning electron microscope.

OBSERVATIONS The adaxial surface of mature leaves of both Tortula species bears numerous globular gemmae ; these are restricted to the nerve in T. papillosa (Fig. 1 A, C) or scattered on both the monolayered lamina and nerve in T. latifolia (Fig. 1 B). Fully developed gemmae comprise six to eight cells with numerous starch-containing chloroplasts (Fig. 1 D). Primary gemmae arise from mature photosynthetic leaf cells. If present on the costa, these are symplasmically connected to underlying food conducting cells (deuters). The entire sequence of development and liberation is illustrated in Fig. 7. The first visible indication of the transformation of mature leaf cells into gemma initials is a change in the disposition of the chloroplasts (Fig. 1 E, F). From a predominantly peripheral location along the periclinal walls these become randomly arranged with the consequent displacement of the central aggregation of vacuoles (Fig. 2 A). The initial cells expand outwards and breakage of the adaxial wall and overlying cuticle is associated with the deposition of new wall material, initially in the apical dome (Fig. 2 B, C) and subsequently throughout the cell (Fig. 2 D). As the initial cell grows the new wall expands whilst the broken ends of the old wall are left behind (Fig. 2 D). During this cellular expansion the nucleus migrates from the lower part of the cell to a more central position, whilst the chloroplasts rapidly increase in number from the four to six

in mature leaf cells. The first division produces a basal cell embedded in the leaf tissue and a distal cell, or gemma primordium, protruding from the leaf surface (Fig. 2 E). The gemma primordium undergoes further expansion and then divides by a slightly oblique or longitudinal septum forming two cells that in turn divide into three to four cells. Depending on the orientation of the first septum, the gemma is connected to the basal cell by either one or two cells. While proliferative divisions take place in the developing gemma, the basal cell, initially rather short (Fig. 2 E), elongates markedly. Elongation involves deposition and subsequent expansion of a new wall layer, first at the upper corners of the cell (Fig. 3 A, B, C), later all over the distal wall, i.e. the wall common to the developing gemma (Fig. 3 D). When the basal cell has elongated by 3–6 µm, i.e. about one-third the original height of the initial cell, the distal wall begins to split. Usually by this stage the gemma has not yet completed its development. Separation starts from the margins and proceeds centripetally along the boundary between the old and new wall of the basal cell (Figs 3 D, E and 4 A). This is particularly evident in T. latifolia where the middle lamella, i.e. the boundary between the gemma and basal cell, is relatively clearly discernible (Fig. 4 B, C). During separation a new wall layer is also deposited in the gemma cell(s) adjacent to the basal cell. Plasmodesmata connecting the gemma to the basal cell are not obliterated by the additional wall layer on either side of the septum (Fig. 4 B) and persist until they are disrupted as a result of the splitting of the wall (Fig. 4 C). Electron-opaque material, probably artifactual in origin, is visible occasionally in the cytoplasmic channel of the plasmodesmata (Fig. 4 B). Before separation is complete the last proliferative divisions occur within the gemma. Following liberation the old wall between the gemma and the basal cell, i.e. the wall formed during the first division in the initial cell, is sloughed off and becomes mucilaginous (Fig. 4 D). The basal cell, now free from the original gemma (Fig. 4 E), continues growing and becomes the initial of a second gemma. A new wall is soon deposited beneath the old outer wall which partially degenerates. Different from a primary initial, the outer wall is not invested by cuticle (Fig. 2 A) and contains remnants of plasmodesmata truncated during the separation of the first-formed gemma (Fig. 4 F). During cellular expansion the old wall is sloughed off from the underlying new wall (Fig. 5 A) and eventually disrupted. Subsequent development follows the same pattern as described above, resulting in the formation of a second gemma. The process can be repeated several times (Fig. 5 B) thus producing a chain of gemmae stuck to each other by mucilage (Fig. 6 A, B). The ends of the broken walls form a concentric series surrounding the initial}basal cell (Fig. 5 B, C). PATAg staining reveals a distinct multilayered structure in the lateral walls of initial}basal cells (Fig. 5 D), reflecting successive cycles of gemma formation. After a number of cycles, usually three to six, the initial cell ceases activity. Inactive initials in T. latifolia may be lost with the last-formed gemma, leaving a cavity on the surface of the leaf (Fig. 5 E). Each cycle of gemma formation, except the first one, involves the deposition and subsequent rupture of

Ligrone et al.—Foliar gemmae in Tortula

307

F. 1. A–C, Scanning electron micrographs of the adaxial face of gemmiferous leaves in Tortula papillosa (A, C) and T. latifolia (B). The gemmae are restricted to the costa in the former but are scattered throughout the surface in the latter. C, The rough surface of the gemmae of T. papillosa (at higher magnification) is due to the presence of investing mucilage. D, Transmission electron micrograph of a maturing gemma in T. latifolia. The arrowheads in (C) and (D) indicate the first-formed septum during proliferative divisions of the gemma primordium. E, F, Mature leaf cells of T. latifolia and T. papillosa, respectively. Note the peripheral location of the chloroplasts (c) around the central vacuoles (v).

308

Ligrone et al.—Foliar gemmae in Tortula

F. 2. Primary gemma development in Tortula latifolia. A, Mature superficial cell of the leaf costa becoming transformed into a gemma initial. The outer adaxial wall, covered with a cuticle (arrowheads) is bulging outwards and the chloroplasts (c) have lost their peripheral disposition. n, nucleus. B, At a slightly later stage in gemma development the old outer adaxial wall and cuticle have ruptured (arrowhead) and a new wall is being deposited underneath. n, Nucleus. C, Detail of (B) showing the broken cuticle (cu) and old wall (ow), and the expanding new wall (nw). D, Initial cell during elongation. Remnants of the old wall are visible (arrowheads) around the expanding dome. The nucleus (n) retains its basal location. E, The first division of the gemma initial separates a short basal cell (bs) and a gemma primordium (gp) that swells markedly. Note the much more numerous chloroplasts (c) than in mature leaf cells.

two walls, the first during the expansion of the initial cell preceding division, the second during the expansion associated with gemma liberation (Fig. 7). During the first cycle, the first wall that is broken is the original wall of the leaf cell. Nascent gemmae contain numerous chloroplasts with a well-developed thylakoid system but little or no starch (Figs

1 D and 6 A, B, C). After symplasmic isolation the gemmae enter a maturation phase characterized by an initial cellular expansion due to enlargement of the vacuolar system (Fig. 6 A). Subsequently the vacuoles reduce in size and abundant lipid reserves accumulate in the cytoplasm (Fig. 6 B, C). Electron-opaque vacuolar deposits are sometimes present in mature gemmae of T. latifolia (Fig. 6 D). During maturation

Ligrone et al.—Foliar gemmae in Tortula

309

F. 3. Gemma liberation in Tortula papillosa. A, Detail of a basal cell (bc) showing the deposition of new wall material around its upper margins. The dotted lines indicate the boundaries between the original outer wall of the leaf cell (1), the second wall formed during expansion of the gemma initial (2) and a third developing wall layer (3). g, Developing gemma. B, Basal cell which has started elongation prior to producing a second gemma. The arrowheads indicate where the second wall layer has broken. C, Detail from (B) at higher magnification, showing the new expanding inner wall layer (3) and the broken old wall strata (1 and 2). D, Splitting of the wall (arrowheads) between the basal cell (bc) and gemma (g). nw, New wall ; ow, old wall. E, Higher magnification from (D). nw, New wall ; ow, old wall.

brown pigmentation of the walls of the gemmae is associated with progressively increasing electron-opacity (Fig. 6 B, C). Frequently the outer walls are sloughed off and converted

into mucilage whilst new wall layers are deposited inside them (Fig. 6 E). The mucilage maintains the attachment of the maturing gemmae to each other and to the parent leaves.

310

Ligrone et al.—Foliar gemmae in Tortula

F. 4. Gemma liberation in Tortula latifolia. A, Splitting of the wall (arrows) between the basal cell (bc) and gemma (g). B, C, Details from (A) showing an uninterrupted plasmodesma in the mid region of the wall (B) and truncated plasmodesmata (arrowheads) along the splitting edge of the wall. ml, Middle lamella ; ow, old wall ; nw, new wall. D, Advanced stage in gemma liberation. Symplasmic connections have all been severed and the new walls (nw) in the basal cell (bc) and gemma (g) have thickened, whilst the old wall (ow) is becoming disorganized. E, Expanding basal cell after liberation of the first gemma. F, Detail of the outer wall of a basal cell at the same stage as shown in (E), PATAg staining. The old degenerating wall contains remnants of plasmodesmata (arrowheads). The new expanding wall (nw) is less reactive than the old (ow) to the PATAg staining.

Ligrone et al.—Foliar gemmae in Tortula

311

F. 5. Development of secondary gemmae in Tortula latifolia. A, Fully expanded basal cell. The old wall, now mucilaginous (arrowheads) invests the expanded new wall (nw). Arrows indicate plasmodesmata in the basal and lateral walls. B, A transverse division produces a new basal cell (bc) and a new gemma primordium (gp). The spindle-shaped nucleus (n) in the gemma primordium is about to undergo mitosis. C, D, Details from (B), showing a concentric series of broken walls. D, Detail of the lateral wall of a basal cell after PATAg-staining showing at least five distinct strata. E, A cavity in the leaf surface produced by detachment of a basal cell.

312

Ligrone et al.—Foliar gemmae in Tortula

F. 6. Maturation of gemmae in Tortula latifolia. A, Detail of young gemmae during their initial expansion. v, Vacuole ; c, chloroplast. The arrowheads point to mucilage connecting sister gemmae. B, Maturing gemmae showing the accumulation of lipid (arrowheads) between the vacuoles and increased electron-opacity of the walls. C–E, Details of gemmae at an advanced stage of maturation. C, Abundant lipid accumulation and deposition of additional wall layers. D, Electron-opaque vacuolar deposits (arrowheads) and cytoplasmic lipid droplets. E, Detail from (C) showing a new wall layer (nw) deposited beneath the old wall (ow) that is becoming mucilaginous. The outermost mucilaginous layer (arrow) derives from the expanded wall of the initial cell.

Ligrone et al.—Foliar gemmae in Tortula

A

B

C

D

E

F

G

H

I

F. 7. Diagrammatic representation of gemma development and liberation in Tortula. Thick black lines indicate newly formed expanding walls, and partial shading mucilaginous walls. The circle in the cells represents the nucleus. A, Mature superficial cell of the leaf. B, Onset of dedifferentiation and deposition of a new wall beneath the outer periclinal wall. C, Extension of the new wall towards the base of the cell, during the onset of cellular expansion, and breakage of the old wall and cuticle. D, Swelling of the initial cell, now completely surrounded by the new wall. E, Transverse division producing a basal cell and a gemma primordium. The basal cell is becoming the initial of a second gemma. F, Elongation of the basal cell associated with deposition of a new wall and breakage of the old wall. Expansion of the gemma and the onset of separation. G, Separation is complete. The oldest wall layers are now mucilaginous. Deposition of a new wall has started in the apical dome of the basal cell. H, Bulging of the new initial cell with expansion of the new wall and disruption of the old. F, Formation of a second gemma primordium. The first-formed gemma has been omitted in (H) and (I).

DISCUSSION The pattern of gemma formation and liberation in Tortula is a striking example of percurrent proliferation (Hughes, 1971 b) involving dedifferentiation of mature cells and subsequent redifferentiation. As a mechanism of multiple formation and liberation of asexual diaspores, percurrent proliferation is common in fungi and algae but among land plants has been described only in mosses (Hughes, 1971 b ; Wang, 1990 ; Duckett and Ligrone, 1992). As in protonemal gemmae in Funaria (Schnepf, 1992) and Calymperes (Ligrone et al., 1992), the elongation of the initial cell of foliar gemmae in Tortula involves the deposition and expansion of a new wall combined with rupture of the old wall. This is probably a consequence of the fact that the wall of the mature, fully differentiated cells whence the initials arise, have very limited extensibility. A similar mechanism has been observed during side branch formation from protonemal subapical cells in Funaria (Schmiedel and

313

Schnepf, 1979 ; Schnepf, 1982). In several other instances, e.g. the foliar gemmae in Calymperes (Ligrone et al., 1992) or in the liverwort Odontoschisma (Duckett and Ligrone, 1995), the gemma initial is an undifferentiated cell and expands and elongates without deposition of new layers of wall material nor rupture of the original walls. During the dedifferentiation phase the initial cell of gemmae in Tortula produces a new wall, then elongates and divides, whilst during the short redifferentiation phase the newly produced wall is stabilized and becomes relatively rigid so that it is disrupted during the following expansion cycle. Because of the absence of a tmema cell, the foliar gemmae in Tortula are perhaps most appropriately referred to as sessile gemmae. From first hand observations on over 20 species in the Pottiales (Duckett, Matcham and Ligrone, unpubl. res.) it would appear that such gemmae are a characteristic of this order in contrast to the closely allied family, the Encalyptaceae, where liberation involves tmema cells. Elsewhere in mosses sessile foliar and}or protonemal gemmae are found in Diphyscium (Duckett, 1994), Mniaceae (Duckett and Ligrone 1994), Cryphaea, Leptodon, Homalia, Myrinia and Grimmiaceae and less commonly than those with tmema cells in the Dicranales and Orthotrichaceae. The deposition of a new wall beneath the pre-exisiting one plays a major role in the liberation of the gemma. This occurs by schizolytic separation along a weak area at the boundary between the newly deposited wall and the overlying wall in the basal cell. Separation is probably induced by expansion of both the basal cell and adjoining gemma cell(s) and seems not to involve extensive wall degradation as is the case in abscission and fruit maturation in higher plants (Sexton and Roberts, 1982 ; Zanchin et al., 1993, 1994). The deposition of a new wall preceding separation of the gemma from the basal cell does not interrupt the plasmodesmata in the common septum. In contrast severance of plasmodesmata by deposition of a new wall layer has been reported in stomatal guard cells (Wille and Lucas, 1984), in cells adjoining tmema cells in Funaria protonemata (Schnepf and Sawidis, 1991 ; Schnepf, 1992) and in the symplasmic isolation of the initial cell of the subsequently multicellular endogenous gemmae in metzgerialean liverworts (Ligrone and Duckett, 1993). In Tortula the persistence of supposedly functional plasmodesmata probably permits physiological interaction until separation is complete and may perhaps be associated with the gemmae of Tortula developing as determinate structures, i.e. becoming dormant at the eightcelled stage. In contrast in Metzgeria the gemmae continue to grow in situ forming juvenile thalli whilst still attached to the parent plants. The symplasmic connection with foodconducting cells (Ligrone and Duckett, 1994) in the leaf nerve may account for the particularly copious production of gemmae in T. papillosa. During maturation the gemmae undergo marked cytological changes. The initial expansion, probably due to water uptake from the outside, facilitates the detachment of the gemma from the basal cell. The subsequent reduction in the vacuolar system reflects dehydration preparatory to dormancy. As a rule asexual diaspores of bryophytes accumulate abundant reserves before detachment from the

314

Ligrone et al.—Foliar gemmae in Tortula

parental plant, in the form of starch or lipid and protein, the last two being typical of hornworts and Blasia, taxa associated with nitrogen-fixing cyanobacteria (Verdus, 1978, 1983 ; Ligrone and Lopes, 1989 ; Ligrone et al., 1992 ; Ligrone and Duckett, 1993 ; Duckett and Renzaglia, 1988). The gemmae of Tortula are unusual because they lack reserves immediately after severance of the symplasmic connections with the plant but subsequently accumulate abundant lipid deposits. This indicates that during maturation the gemmae possess considerable photosynthetic activity whose products are mainly converted into lipid. Although the foliar gemmae of Tortula and jungermannialean liverworts are both produced in chains they have little else in common either developmentally or in the manner in which they are dispersed ; they are yet another example in the ever growing catalogue of developmental analogues found in bryophytes (Crandall-Stotler, 1984 ; Duckett and Renzaglia, 1988 ; Renzaglia and Duckett, 1988). Both in Tortula and in jungermannialean liverworts the gemmae develop by endogenous proliferative division(s) of unicellular primordia formed exogenously by formative division of an initial cell. [For a detailed consideration of formative and proliferative divisions see Gunning (1982) ; the former, usually not definite in number, produce undifferentiated, but often determinate cells, the latter are definite in number and immediately precede the final differentiation of tissues and organs.] However, whilst in Tortula and other moss taxa the chains of gemmae arise from regular alternation of formative and proliferative divisions, in jungermannialean liverworths an acropetal sequence of formative divisions of the initial cell produces a branched chain of gemma primordia that subsequently undergo an endogenous proliferative divison, or sometimes more than one, in basipetal succession (Duckett and Ligrone, 1995 ; Hughes, 1971 a ; Schuster, 1966, 1984). Suppression of the proliferative division leads to the liberation of unicellular gemmae in certain taxa, e.g. Scapania (Schuster 1966, 1984). Conversely, in metzgerialean liverworts the initial cell has no formative activity and after symplasmic isolation it divides endogenously producing an isolate multicellular gemma (Ligrone and Duckett, 1993). In jungermannialean liverworts, where the original walls of the gemma primordium remain intact, shallow detachment scars are visible on both gemmae and parent leaf cells ; in Tortula, where the walls become mucilaginous and expansion during maturation is more pronounced, these scars are absent. Whereas in liverworts the walls of the maturing gemmae become firmly textured and water repellant, the increasingly mucilaginous walls in Tortula become progressively looser in texture and hydrophilic. These different properties almost certainly relate to the conditions under which the gemmae of the two groups are liberated and dispersed. In liverworts the foliar gemmae, which are exposed at all times, may be dispersed either floating on water films under wet conditions, or by wind when the plants are dry. In Tortula gemma liberation and dispersal occurs only when the plants are fully hydrated since in the dry state appression and incurving of the leaves

(Smith, 1978) shuts off their gemma-producing adaxial surfaces. It is also noteworthy that gemmae adhesion to new substrata will be maximal when the investitive of mucilage is fully hydrated. Mature gemmae of both Tortula latifolia and T. papillosa can survive drought for several months in nature and at least 2 years in laboratory and germinate to produce protonemata within 48 h after rehydration (Duckett and Ligrone, 1992). These are properties shared with the protonemal brood cells in a further pottialean genus, Aloina (Goode et al., 1994), both kinds of diaspores possessing thick multistratose walls and abundant lipid reserves. Extreme resistance to drought, immediate germination, abundant lipid reserves and continuous production throughout the vegetative season, whenever the gametophyte is fully hydrated, indicate that the gemmae of Tortula are highly effective propagules for rapid and successful colonization of new habitats.

A C K N O W L E D G E M E N TS This study was supported by CNR as a part of a Bilateral Research Project Italy}United Kingdom. R. Ligrone thanks Queen Mary & Westfield College for laboratory facilities during his sabbatical leave in 1992–3. The observations were in part performed at the CIRVB (University of Naples ‘‘ Federico II ’’, Italy).

LITERATURE CITED Bopp M, Quader H, Thoni C, Sawidis T, Schnepf E. 1991. Filament disruption in Funaria protonemata : formation and disintegration of tmema cells. Journal of Plant Physiology 137 : 273–284. Buch H. 1911. UX ber die Brutorgane der Lebermoose. Helsingfors : J. Simelii Arvingars Boktryckeriaktiebolag. Cavers F. 1903. On asexual reproduction and regeneration in Hepaticae. New Phytologist 2 : 121–133, 155–165. Chopra RN, Kumra PK. 1988. Biology of bryophytes. New Delhi : Wiley & Sons. Correns C. 1899. Untersuchungen uX ber die Vermehrung der Laubmoose durch Brutorgane und Stecklinge. Jena : G. Fisher. Crandell-Stotler B. 1984. Musci, hepatics and anthocerotes—an essay on analogues. In : Schuster RM, ed. New manual of bryology Vol. 2. Nichinan, Japan : Hattori Botanical Laboratory, 1093–1129. Duckett JG. 1994. Studies of protonemal morphogenesis in mosses. V. Diphyscium foliosum. (Hedw). Mohr (Buxbaumiales). Journal of Bryology 18 : 223–238. Duckett JG, Ligrone R. 1991. Gemma germination and morphogenesis of a highly differentiated gemmiferous protonema in the tropical moss Calymperes (Calymperaceae, Musci). Cryptogamic Botany 2/3 : 219–228. Duckett JG, Ligrone R. 1992. A survey of diaspore liberation mechanisms and germination patterns in mosses. Journal of Bryology 17 : 335–354. Duckett JG, Ligrone R. 1994. Studies of protonemal morphogenesis in mosses. III. The perennial gemmiferous protonema of Rhizomnium punctatum (Hedw). Kop. Journal of Bryology 18 : 13–26. Duckett JG, Ligrone R. 1995. The formation of catenate foliar gemmae and the origin of oil bodies in the liverwort Odontoschisma denudatum (Mart.) Dum. (Jungermanniales) : a light and electron microscope study. Annals of Botany 76 : 405–419. Duckett JG, Renzaglia KS. 1988. Cell and molecular biology of bryophytes : ultimate limits to the resolution of phylogenetic

Ligrone et al.—Foliar gemmae in Tortula problems. In : Newton ME, Wanstall PJ, eds. Bryology : modern research and ways forward. Botanical Journal of the Linnean Society 98 : 225–246. Giles KL. 1971. Dedifferentiation and regeneration in bryophytes : a selective review. New Zealand Journal of Botany 9 : 689–694. Goode JA, Alfano F, Stead AD, Duckett JG. 1993. The formation of aplastidic abscission (Tmema) cells and protonemal disruption in the moss Bryum tenuisetum Limpr. is associated with transverse arrays of microtubules and microfilaments. Protoplasma 174 : 158–172. Goode JA, Stead AD, Ligrone R, Duckett JG. 1994. Studies of protonemal morphogenesis in mosses. IV. Aloina (Pottiales). Journal of Bryology 18 : 27–41. Gunning BES. 1982. The root of the water fern Azolla–cellular basis of development and multiple roles of cortical microtubules. In : Subtelny S, ed. DeŠelopmental order : its origin and regulation. Symposium for the Society of DeŠelopmental Biology 40 : 379–422. New York : AR Liss. Hughes SJ. 1971 a. On conidia of fungi, and gemmae of algae, bryophytes, and pteridophytes. Canadian Journal of Botany 49 : 1319–1339. Hughes SJ. 1971 b. Percurrent proliferations in fungi, algae and mosses. Canadian Journal of Botany 49 : 215–231. Ligrone R, Duckett JG. 1993. Formation of endogenous gemmae in the simple thalloid liverworts Metzgeria and Riccardia (Metzgeriales). Giornale Botanico Italiano 127 : 328–330. Ligrone R, Duckett JG. 1994. Cytoplasmic polarity and endoplasmic microtubules associated with the nucleus and organelles are ubiquitous features of food-conducting cells in bryoid mosses (Bryophyta). New Phytologist 127 : 601–614. Ligrone R, Duckett JG, Egunyomi A. 1992. Foliar and protonemal gemmae in the tropical moss Calymperes (Calymperaceae) : an ultrastructural study. Cryptogamic Botany 2 : 317–329. Ligrone R, Lopes C. 1989. Ultrastructure, development and cytochemistry of storage cells in the ‘ tubers ’ of Phaeoceros laeŠis Prosk. (Anthocerotophyta). New Phytologist 112 : 317–325. Longton RE. 1990. Sexual reproduction in bryophytes in relation to physical factors of the environment. In : Chopra RN, Bhatla SC, eds. Bryophyte deŠelopment : physiology and biochemistry. Boca Raton, USA : CRC Press, 139–166. Longton RE, Miles CJ. 1982. Studies on the reproductive biology of mosses. Journal of the Hattori Botanical Laboratory 52 : 219–240. Longton RE, Schuster RM. 1983. Reproductive biology. In : Schuster RM, ed. New manual of bryology Vol. 1. Nichinan, Japan : Hattori Botanical Laboratory, 386–462. Mishler BD. 1988. Reproductive ecology of bryophytes. In : Lovett Doust J, Lovett Doust L, eds. Plant reproductiŠe ecology : patterns and strategies. New York : Oxford University Press, 285–305.

315

Renzaglia KS, Duckett JG. 1988. Different developmental processes underlie spermatozoid architecture in mosses, hepatics and hornworts. Journal of the Hattori Botanical Laboratory 64 : 219–235. Roland JC, Sandoz D. 1969. De! tection cytochimique des sites de formation des polysaccharides pre! -membranaires dans les cellules ve! ge! tales. Journal de Microscopie 8 : 263–268. Sawidis T, Quader H, Bopp M, Schnepf E. 1991. Presence and absence of the preprophase band in moss protonemata : a clue to understanding its function. Protoplasma 163 : 156–161. Schmiedel G, Schnepf E. 1979. Side branch formation and orientation in the caulonema of the moss, Funaria hygrometrica : normal development and fine structure. Protoplasma 100 : 367–383. Schnepf E. 1982. Morphogenesis in moss protonemata. In : Lloyd CW, ed. The cytoskeleton in plant growth and deŠelopment. London : Academic Press, 321–344. Schnepf E. 1992. Structure and development of tmema cells in protonemata of Funaria hygrometrica (Bryophyta). Cryptogamic Botany 3 : 35–39. Schnepf E, Sawidis T. 1991. Filament disruption in Funaria protonemata : occlusion of plasmodesmata. Botanica Acta 104 : 98–102. Schuster RM. 1966. The Hepaticae and Anthocerotae of North America Vol. 1. New York : Columbia University Press. Schuster RM. 1984. Comparative anatomy and morphology of the Hepaticae. In : Schuster RM, ed. New manual of bryology Vol. 2. Nichinan, Japan : Hattori Botanical Laboratory, 760–891. Sexton R, Roberts JA. 1982. Cell biology of abscission. Annual ReŠiew of Plant Physiology 33 : 133–162. Smith AJE. 1978. The moss flora of Britain and Ireland. Cambridge : Cambridge University Press. Verdus MC. 1978. E! tude ultrastructurale des propagules de Campylopus introflexus (Hedw.) Brid. (Bryopsida, Dicranales). Bulletin de la SocieteU botanique de France 31 : 33–40. Verdus MC. 1983. Ultrastructure des propagules protonematiques de Dicranoweisia cirrata (Hedw.) Lindb. Journal of the Hattori Botanical Laboratory 54 : 95–105. Wang CJK. 1990. Ultrastructure of percurrently proliferating conidiogenous cells and classification. Studies in Mycology 32 : 49–64. Wille AC, Lucas WJ. 1984. Ultrastructural and histochemical studies on guard cells. Planta 160 : 129–142. Zanchin A, Bonghi C, Casadoro G, Ramina A, Rascio N. 1993. Abscission in leaf and fruit explants of Prunus persica (L.) Batsch. New Phytologist 123 : 555–565. Zanchin A, Bonghi C, Casadoro G, Ramina A, Rascio N. 1994. Cell enlargement and cell separation during peach fruit development. International Journal of Plant Science 155 : 49–56.