Megaspore Apparatuses Research Articles

Water fern spores (Salviniales) from the Late Cretaceous of Patagonia, Argentina

We describe a water fern spore assemblage from the Maastrichtian La Colonia Formation, Chubut Province, Patagonia, Argentina. The assemblage includes three species of Azolla (A. andreisii sp. nov., A. coloniensis, and A. sp. 1), two species of Azollopsis (A. intermedia and A. tomentosa), Crybelosporites pannuceus, Gabonisporis cristata, Ghoshispora sp., Grapnelispora loncochensis, two species of Molaspora (M. lobata and M. reticulata), and Paleoazolla patagonica. A. tomentosa, A. intermedia, G. cristata, and M. reticulata are recorded for the first time in the Southern Hemisphere. Ghoshispora sp. constitutes the southernmost record of the genus and its first mention for Argentina. In addition, the genus Azollopsis is emended to include megaspore apparatuses with more than one megaspore. This water fern spore assemblage is one of the most diverse so far known for South America and substantiates the significant radiation that aquatic ferns underwent worldwide at the end-Cretaceous. Furthermore, it also implies that the presence of widespread suitable paleoenvironments allowed the establishment and rapid evolution of these plant communities in Patagonia.

Azolla Sporophytes and Spores from the Late Cretaceous and Paleocene of Patagonia, Argentina

Premise of research. While Azolla has a rich fossil record based on dispersed megaspore apparatuses and microspore massulae, fossil sporophytes are relatively rare. In this contribution, we describe two fossil Azolla species based on both sporophytes and spores from Chubut Province, Patagonia, Argentina: Azolla coloniensis De Benedetti & Zamaloa, emend. Hermsen et al., and A. keuja Jud et al., sp. nov. Azolla coloniensis and A. keuja are the first fossil species of Azolla to be represented by vegetative structures (i.e., leaves, stems, and roots) from both South America and the Southern Hemisphere.Methodology. We examined sporophyte material of A. coloniensis from the Cañadón del Irupé locality, Upper Cretaceous, La Colonia Formation, and A. keuja from the Palacio de los Loros locality PL-2, Paleocene, Salamanca Formation. Spores of A. keuja were obtained from a sporophyte specimen and its surrounding rock matrix. Material was studied using standard light microscopy, epifluorescence microscopy, and scanning electron microscopy. Fossils are held at the Museo Paleontológico Egidio Feruglio, Trelew, Chubut Province, Argentina.Pivotal results. Azolla coloniensis produced many-floated megaspore apparatuses and microspore massulae with anchor-tipped glochidia, placing it in the fossil Azolla section Florschuetzia. Azolla keuja sporophytes are structurally similar to those produced by the extant African species A. nilotica and the Late Cretaceous–Paleocene North American species A. schopfii in overall size, growth form, leaf structure, and production of fascicled roots; while all three taxa produce similar microspore massulae, the structure of their megaspore apparatuses differ. Azolla keuja cannot be assigned to any section of Azolla.Conclusions. Azolla coloniensis and A. keuja are important because they provide two new organismal concepts for extinct species of Azolla. Our inability to fully classify A. keuja to section, in combination with the great morphological diversity of fossil Azolla, indicates that a comprehensive reevaluation of phylogeny and taxonomy that incorporates both extant and fossil species is needed.

Reinterpretation of Azolla primaeva (Azollaceae, Eocene, Canada) using electron microscopy and X-ray tomographic microscopy

Azolla primaeva (Penhallow) Arnold fertile whole plants from the lower Eocene of Driftwood Creek, Canada have been examined using LM, SEM, TEM and SRXTM methods on hand specimens and sieved residues. The new data have resulted in an emended diagnosis. The megaspore is partly covered by filosum and the wall lacks excrescences. The exoperine is highly variable with nodular, to clavate, baculate and tabulate elements, which fuse to form a foveolate to reticulate megaspore surface, which is complex and contorted. Glochidia on the microspore massulae are sometimes septate and the flukes on their anchor-shaped tips are variable, some being long, narrow and wavy, others shorter and broad and a few are recurved. Hairs are lacking on the massula surface and glochidia. The organization of the float zone of the megaspore apparatus has always been controversial with 0, 1, 3 or 9 floats proposed. The new results show that there are no pseudovacuolate floats. By contrast, the ‘float’ zone consists of bundles of modified granular and filamentous endoperine elements surrounded by bundles of broader suprafilosum hairs. The absence of pseudovacuolate floats in A. primaeva is suggested to be an important character for phylogenetic analyses. The high variability in glochidia tips, glochidia septa and exoperine organisation is unusual within the genus Azolla. The new data on A. primaeva are compared with the other two whole plant Azolla species from Canada (Azolla stanleyi and Azolla schopfii) and with dispersed Azolla reproductive structures from Canada including the Arctic. This confirms that all are distinct species.

Salviniales from the Late Cretaceous of the Golfo San Jorge Basin

The freshwater ferns (Salviniales) are well represented in the Maastrichtian deposits of Cerro de los Fragmentos in the headwaters of the Río Chico, Golfo San Jorge Basin. The fossil material of Salviniaceae includes complete megaspore apparatuses with attached microspore massulae and dispersed megaspores, float systems and microspore massulae of Azolla. The new species Azolla colhuehuapensis displays distinctive morphological characters including relative small megaspore apparatuses with easily detached floats and usually attached eglochidiate massulae. The Marsileaceae are represented by the megaspore Molaspora lobata, microspores of Crybelosporites and vegetative remains of Marsileaceaephyllum sp. The assemblage also includes zygospores of the green filamentous algae Zygnemataceae, spores of Marchantiophyta and Monilophyta (Schizaeales), megaspores of Isoetalean affinity and Palm-type angiosperm pollen grains. All these organic microfossils and the associated palynomorphs indicate the presence of a freshwater environment where abundant water ferns were developing and reproducing.

The freshwater fern Azolla (Azollaceae) from Eocene Arctic and Nordic Sea sediments: New species and their stratigraphic distribution

Three new species of the freshwater fern Azolla are described from Eocene marine deposits of the Arctic and Nordic seas, bringing the total number of species now documented from these areas to five. Azolla arctica Collinson et al., Azolla jutlandica Collinson et al., Azolla nova sp. nov. and Azolla nuda sp. nov. are known from both megaspore apparatuses and microspore massulae, and occur at more than one site. Their microspore massula characters are sufficiently distinctive to allow their recognition in palynological preparations. Presence or absence of hairs on the microspore massula surface and glochidia bases is shown to be a useful species diagnostic character and is worthy of greater attention in Azolla taxonomy. The fifth species, Azolla astroborealis sp. nov., is only known from a few megaspore apparatuses in the Northstar 1 Well in the western Arctic, but these are strikingly different from those of the other four species e.g., in having nine distinct pseudovacuolate floats.Samples from cores and most individual sites contain more than one Azolla species. This indicates that conditions were particularly favourable for growth of Azolla for a prolonged time interval in the latest early and possibly earliest middle Eocene (latest Ypresian and earliest Lutetian) around the western Arctic and the Norwegian–Greenland Sea.

Megaspores from the upper Maastrichtian of the eastern Spanish Pyrenees and their biostratigraphic, palaeogeographic and palaeoenvironmental significance

Maastrichtian sediments from the eastern Spanish Pyrenees have yielded a variety of mesofossils. The megaspores and megaspore apparatuses are referred to Minerisporites succrassulus Tschudy, Ghoshispora epispinosa sp. nov., Ghoshispora sp., Molaspora lobata (Dijkstra) Hall and Parazolla vallcebrensis sp. nov. Also recovered were specimens of uncertain affinity, some of which are possibly immature P. vallcebrensis, and massulae clusters that might be attributable to either this species or Azolla Lam. Minerisporites succrassulus and megaspores referable to Parazolla have not been recorded previously from beyond North America, and hitherto only one European occurrence of Ghoshispora has been noted. Hence, these new records indicate that the parent-plant groups concerned were more widespread during the Late Cretaceous than previously recognised. It is likely that all of them lived in or close to bodies of fresh water, reflecting a wetland habitat at the time of deposition.

A new species of the freshwater fern Azolla (Azollaceae) from the Eocene Arctic Ocean

A new fossil species of the freshwater fern Azolla (Azollaceae, Salviniales) is described from an unusual setting of high palaeolatitude in the Arctic Ocean. Azolla arctica sp. nov., (lower Middle Eocene, Lomonosov Ridge) is represented by fully developed megaspore apparatuses with attached microspore massulae and clustered and dispersed microspore massulae. These abundant co-occurring fossils, combined with their associated biota, demonstrate that Azolla was growing and reproducing on a freshwater surface of the Eocene Arctic Ocean. Azolla arctica is compared with other fossil Azolla species, especially those from around Arctic and Nordic Seas. It documents new characteristics for the genus. The megaspore apparatus is small with a thin megaspore wall and a distinctive exoperine where nodular exoperinal masses fuse at several levels resulting in a rugulate, undulating, punctate to foveolate exoperine surface. Microspore massulae have two size classes of glochidia, short (< 25 µm) and long (> 55 µm), the anchor-shaped tips lack recurved flukes. These distinctive characters provide the potential to recognise Azolla arctica as fragmentary remains in palynological preparations from drill cores. Therefore, future comparisons with other fossils will reveal if a single species grew across the Arctic Ocean and if freshwater spills from the Arctic spread into the Nordic Seas.

Revision of species of Minerisporites, Azolla and associated plant microfossils from deposits of the Upper Palaeocene and Palaeocene/Eocene transition in the Netherlands, Belgium and the USA

Species of the megaspore genus Minerisporites Potonié, megaspore apparatuses of species of the water fern Azolla Lamarck, and some associated organic-walled microfossils recovered from deposits of the Upper Palaeocene and Palaeocene/Eocene transition in the southern part of the Netherlands and neighbouring Belgium are redescribed on the basis of an examination of specimens under scanning and transmission electron microscopes. Originally studied about 40 years ago by S.J. Dijkstra, the re-examination has enabled emended diagnoses to be produced for six taxa: Minerisporites glossoferus (Dijkstra) Tschudy, M. mirabilis (Miner) Potonié, M. mirabilissimus (Dijkstra) Potonié, Azolla schopfii Dijkstra, A. teschiana Florschütz, and A. velus (Dijkstra) Jain and Hall. In addition, a revised description is provided for massulae of Salvinia Séguier that were originally thought to be megaspores and, hence, named by Dijkstra as Triletes? exiguus. The gross morphology and construction of the exospore of the species of Minerisporites are similar, but nevertheless sufficiently distinct for them to be maintained as separate taxa. Monolete microspores are preserved in hollows in the reticulate surface of some of the specimens of M. mirabilissimus. This is consistent with the presumed isoetalean affinity of Minerisporites. An apparent stratigraphic morphocline from M. glossoferus to M. mirabilis, suggested previously, is confirmed following our reassessment of their characteristics. The species of Azolla are all multi-floated, but they differ from each other in several ways, in particular with respect to the ultrastructure of the megaspore wall. They are also distinct from all other species that have been considered in sufficient detail for satisfactory comparisons to be made. The massulae of A. teschiana are described for the first time. The floats in A. velus are attached to the proximal part of the megaspore only by suprafilosal hairs. There are no maniculae. It is argued that these structures do not exist in either this or any other species of Azolla. Whole fertile fossil remains that demonstrate the nature and habitat of the living plants are known for all three genera. Hence, their reproductive structures can be used with confidence in palaeoenvironmental interpretation. The overall assemblage, which also includes a few specimens of the marsilealean megaspore Molaspora lobata (Dijkstra) Hall, undoubtedly reflects the presence of lowland aquatic and semi-aquatic vegetation.

Variations in Azolla section Azolla megaspore apparatus and their implications for palaeotaxonomy and European Pleistocene biostratigraphy

Fossil remains of the aquatic pteridophyte Azolla filiculoides Lamarck have always aroused great interest. However, a number of workers have questioned if all the European Pleistocene fossil remains in the section Azolla Meyen (taxa whose megaspore apparatus possess three floats) originate from Azolla filiculoides. This has important implications not only for palaeotaxonomy and the understanding of evolutionary trends in this section through the Pleistocene, but also for European Pleistocene biostratigraphy. The discovery of a large fossil population of Azolla megaspore apparatus in Hoxnian Stage sediments at Tye Green, Stansted, Essex, England allowed the detailed description and measurement of this fossil population and its comparison with modern Azolla filiculoides megaspore apparatus populations. The results show variation in size, shape and external morphology of the megaspore apparatus in both the Tye Green fossil and modern populations. Even so, the Tye Green fossils are confidently identified as Azolla filiculoides because the general features of the megaspore apparatus match those of modern Azolla filiculoides, and the glochidia stalks exhibit regular terminal septation and mostly only have two barbs. This variation should be taken into account when considering the creation of new extinct Azolla species in the section Azolla using megaspore apparatus characteristics and may prompt the re-examination of a previously described extinct species.

The Azolla–Anabaena symbiosis: endophyte continuity in the Azolla life‐cycle is facilitated by epidermal trichomes †

SUMMARYLight, scanning electron, and transmission electron microscopy were used to document events tending to the re‐establishment of the Azolla mexicana–Anabaena azollae symbiosis during sexual reproduction. In mature megasporocarps. Anabaena akinetes occupy the space between the indusium and apical membrane, termed the inoculation chamber. Formation of the megagametophyte within the megaspore apparatus extends the floats but does not trigger akinete germination. The tatter is contingent upon embryogenesis. The embryo develops a cotyledonary leaf primordium bearing epidermal trichomes while within the archegonial chamber. Incipient akinete germination is discernible before the cotyledonary leaf with its four seriate trichomes, the cells of which exhibit TCU (transfer cull ultrastructure), and the shoot apex penetrate the apical membrane to enter the inoculation Chamber. Rapid development of the vase‐like cotyledonary leaf maintains and fosters the initial association of the Anabaena population, which includes akinetes, germinating akinetes and germlings, with the cotyledonary hairs and the shoot apex. It engulfs the Anabaena in the inoculation chamber as it surrounds the four trichomes and the shoot apes with its developing true leaves. The latter soon arch over the apes to further confine the Anabaena. The first four or five true leaves are unlobed. The first differentiates two multibranched epidermal trichomes while each of the others differentiate one. The cells of these distinctive trichomes exhibit TCU. They occur in a cavity‐like structure which forms in the unlobed leaves and are associated with Anabaena cells which appear to be degenerating. Oilier Anabaena cells entrapped in the region of the apical meristem yield vegetative filaments which provide the inoculum for the developing cavities of the normal, bilobed leaves. The possibility that akinete germination is initially triggered by diffusion of embryo‐produced oxygen and water vapour into the inoculation chamber is discussed as is the possibility that those Anabaena cells entrapped in the unlobed leaves arc cannabalized by the fern.

RE-ESTABLISHMENT OF SYMBIOSIS TO Anabaena-FREE Azolla

Symbiosis to Anabaena-free Azolla has been re-established by placing indusium containing Anabaena onto the Anabaena-free megasporocarps. Anabaena-free megasporocarps were obtained by removing the indusium and the apical membrane of the megaspore apparatus of normal Azolla. The recovery of symbiosis in artificially reconstituted Azolla-Anabaena association was confirmed using the scanning electron microscope, monoclonal antibody test (McAb-C16), and nitrogen fixation capacity (ARA). Laboratory-grown Anabaena azollae cells inoculated subsequently to the decapitated megaspore apparatus were observed to have entered the leaf cavities of sporophyte, although some portions of Anabaena filaments were found outside the leaf cavity. Plants invaded by artificially inoculated Anabaena did not have sufficient N2-fixing activity to allow the growth in the N-free medium.

A novel method to produce Anabaena-free Azolla by in vitro fertilization of micromanipulated megasporocarps

In the Azolla-Anabaena azollae symbiotic system, Anabaena akinetes get entrapped between the indusium and the apical cap of the megaspore apparatus during megasporocarp development, thus maintaining the continuity of the cyanobacterial association throughout the life cycle of the fern. The entrapped akinetes serve as the source of inoculum for infecting the new sporophyte when it is emerging from the megaspore apparatus. A procedure to generate Anabaena-free Azolla was developed by fertilizing the germinating megasporocarps in which the indusium along with the akinetes were removed by micromanipulation. This method has the advantage of not requiring drastic treatments of Azolla with antibiotics to eliminate the endosymbiotic cyanobacterial cells. Details of this new method and its usefulness in studies aimed at recombination of Azolla with Anabaena azollae are discussed.

A new method for obtaining Anabaena-free Azolla.

Anabaena azollae can be completely eliminated from megasporocarps of Azolla by the removal of the indusium and apical membrane after horizontally cutting the megaspore apparatus beneath the apical membrane. All subsequent sporelings of Azolla are free from Anabaena. These do not fix N and fail to survive on N2 -free media. They grow normally, however, in N2 -containing liquid medium or in a soil-water system with small amounts of added N and eventually bear sporocarps. Anabaena is again absent in the new megasporocarps and sporelings of Azolla derived from the second generation of megaspores are likewise Anabaena-free.

Megaspore germination, embryo development and maintenance of the symbiotic association in Azolla filiculoides Lam.

Megaspore germination and embryo development in Azolla filiculoides was examined using SEM and thin–sectioning. Within the released megaspore apparatus, resting cells of the endosymbiont Anabaena azollae Stras. arc located distally to the outside of the mcgasporangial wall and adhering to the inside of the megasporocarp wall. Growth of the female gametophyte displaces the floats pushing this part of the wall (the indusial cap) upwards, so providing access to the archegonia for the multifiagellalc spermalozoids. Embryo development and its inoculation with Anabaena involves a subtly–timed sequence of events resulting in the perpetuation of the symbiosis. Growth of the lunnel–shaped cotyledon leaf ruptures the mcgasporangial wall to provide access and a channelled route between the Anabaena and embryo shoot apex; subsequent leaf development severely restricts such access. During this process, the Anabaena is dislodged by the cotyledon leaf and growth of the first leaf traps the now actively–dividing Anabaena colonv; this becomes established around subapical trichomes from where filaments become incorporated into the cavities of developing leaves. The voung sporophyte rises vertically to the water surface as a result of gas accumulation in intercellular spaces; at no stage do floats endow buoyancy.

Quaternary record of Azolla pinnata from China and its sporoderm ultrastructure

Fossil megaspore apparatuses of Azolla pinnata R. Brown are described from the late Quaternary of Zhejiang Province, East China. They prove to be identical in gross morphology and structure with those of the herbarium specimens of the same species. An electron microscopic study shows that the sporoderm ultrastructure is essentially constant in the megaspores of A. pinnata, either fossil or living, except those of some African specimens which have been considered by some authors as either a variety of this species or a distinct species. It is believed that the sporoderm ultrastructure will be found progressively useful in the taxonomic separation of Azolla species.

Classification and phylogeny of living and fossil water ferns of the genus « Azolla »

Summary Opinions on classification and phylogeny of the genus Azolla have been changed by discovery, made since 1965, of new species and genera of Cretaceous Salviniaceae. An account is given of different section in which Azolla species are grouped and of recent hypotheses on its phylogeny: the multifloat megaspore apparatuses should be arisen from three-float megaspore apparatuses, Azollopsis megaspore apparatus should be precursor of Azolla megaspore apparatus, etc. After a short discussion, the term gula is proposed again for internal cone-shaped structure above megaspore body in the genus Azolla.

An Escape Mechanism for Spermatozoids in Azolla Massulae

The genus Azolla belongs to the Salviniaceae, a family of heterosporous, freefloating ferns occurring in fresh-water habitats, mainly in warm temperate to tropical regions. Stratigraphically, the genus ranges from the late Cretaceous to the present. Six living species usually are recognized. In recent years, considerable interest has been focused on fossil members (some 48 species have been recorded) which, together with the living species, are placed in six sections of the genus. The morphological and taxonomic diversity of Azolla, particularly of its reproductive structures, coupled with the short stratigraphic ranges of individual fossil species, renders it a potentially valuable stratigraphic indicator. In modern species of Azolla sporocarps are borne in pairs which arise from the ventral lobe of the first leaf of a branch. These paired sporocarps may be of the same sex or they may be male and female, termed microsporocarp and megasporocarp, respectively. The microsporocarp contains numerous microsporangia bearing massulae; within the megasporocarp is a single megasporangium containing a megaspore apparatus. The massula and megaspore apparatus are highly distinctive structures peculiar to Azolla. Probably no group of embryophytic plants has a more complex megaspore. The megaspore apparatus consists of the megaspore, above the proximal pole of which lies a superstructure of hair-like filaments called the columella. The columella, which is continuous with similar filaments on the megaspore wall, bears vacuolate, pseudocellular floats, which number three or nine in modern species. The columella and floats constitute the so-called swimming apparatus. The massula is a pseudocellular structure in which the microspores are embedded; in living species it may be eglochidiate or may have hair-like or anchorshaped processes called glochidia. Interpretation of the development of reproductive structures in Azolla commenced many years ago (Meyen, 1836; Griffith, 1844) and has concentrated on A. filiculoides Lam. (Strasburger, 1873, 1889; Campbell, 1893; Hannig, 1911; Duncan, 1940; and Bonnet, 1957). Azolla caroliniana Willd. was studied by Berggren (1880) and by Pfeiffer (1907), A. pinnata R. Br. by Rao (1935), and A. nilotica Dcne. by Demalsy (1954). Excepting Svenson (1944), little emphasis has been placed on the taxonomic use of anatomical and morphological features of the mature reproductive structures of modern Azolla species. Investigation of the reproductive structures of A. prisca Reid & Chandler, from the Lower Oligocene of the Isle of Wight, southern England, reveals a more

AzollaSpecies from the Pleistocene of the Central North Sea Area

Abstract Reproductive organs of Azolla species (Salviniaceae) from the Lower and Middle Pleistocene of the Dansk Nordsø A-1 and A-2 boreholes, central North Sea area, were studied by means of scanning electron microscopy. A new species, Azolla danica, based on massulae is proposed. The megaspore apparatuses of Azolla filiculoides Lam. and Azolla tegeliensis Florschütz are described and the possible affinity of A. danica to A. tegeliensis is discussed. It is concluded, that the A. danica massulae can be regarded as the previously unknown massulae of A. tegeliensis. A tentative, chronostratigraphical subdivision of the Quaternary section is given. The record of A. tegeliensis is the first proof of the presence of limnic deposits of the Tiglian interglacial within Danish territory.

A CONTRIBUTION TO THE EARLY TERTIARY FOSSIL RECORD OF THE SALVINIACEAE

Megaspores and massulae of Azolla, Azollopsis, and Salvinia are recorded from the Paleocene and Eocene of Montana and the Dakotas. Three new species of Azolla are described and two species of Azolla are redescribed. The columella of the floating apparatus of the megaspore apparatuses of Azolla appears to be a phylogenetic localization of the perispore around the megaspore. A new section of Azolla (section Kremastospora) is created for species whose megaspore apparatuses have many floats and whose massulae have hooked (anchor‐shaped) glochidia. Salvinia preauriculata is based only on leaf remains from the Eocene of North America. Megaspores and massulae which probably are conspecific with the leaf remains are described. The megaspores and massulae of the fossil species are much smaller than those of the living species, S. auriculata, with which S. preauriculata has been compared. The similar size of the megaspores and massulae of the fossil species suggests a less specialized condition than in living species, where these two structures differ greatly in size. Azollopsis tomentosa, previously known from the late Cretaceous, has been found in the Eocene.

Studies on Fossil Azolla: Azolla montana, a Cretaceous Megaspore with Many Small Floats

Azolla montana is a new species from the late Cretaceous; it has a megaspore apparatus with a single large and conspicuous columella which resembles the floats of the megaspore apparatus of many extant species. The columella differs from true floats in being pilose, like the perispore of the megaspore body. The 10–20 small true floats are appressed to the columella, and are difficult to distinguish from it. It is suggested that the floats were derived, phyletically, by segmentation of the columella; the columella represents the primitive “float” of the megaspore apparatus.