The discovery of arbuscules in Aglaophyton major, an Early Devonian land plant, provides unequivocal evidence that mycorrhlizae were estabised >400 million years (Fig. 1), even though the intraradical mycelium is extensively developed within the intercellular spaces in the remaining cortical tissues. The nonseptate hyphae range up to 4.0 ,um in diameter and often show both Y-and H-shaped anastomoses. In extant VAM fungi arbuscules are formed when intercellular hyphal branches penetrate the cell wall of the host but do not rupture the plasmalemma (10). The hyphal trunk ofthe arbuscule is =1.3 Am wide (Fig. 2) and branches repeatedly to form a "bush-like" structure within the cell (Fig. 3). Secondary branches range from 0.7-2.0 pum wide, and the ultimate ones are beyond the resolving power of light microscopy (in the size range 0.2-0.5 pum). Fig. 4 shows the top of an arbuscule indicating the configuration ofthe more distal branches. In the arbuscules of extant VAMs, the walls are osmiophilic and decrease in thickness to <20 nm near the tips. In some arbuscules the tips may be slightly swollen, and this condition also occurs in the fossils.In extant VAMs, the arbuscules are ephemeral and begin to break down at the tips ofthe smallest branches in 4-6 days, ultimately forming an amorphous mass within the cell (11). We have no way of gauging the longevity of the fossil arbuscules except to note that various stages of arbuscule morphology are present in a single section, including those that have collapsed and deteriorated. Boullard (12) suggested that in some living ferns the frequent presence of clumps indicates a short functional span of an arbuscule. Several authors working with modern VAM fungi reported the formation of a subapical septum that separates the functional and nonfunctional portion of an arbuscule during deterioration. We have not observed this structure in any of the fossil arbuscules to date. The fossil arbuscules appear morphologically identical to those of many extant VAMs (13). There is far less information available about the structural and physAbbreviation: VAM, vesicular arbuscular mycorrhiza.fTo whom reprint requests should be addressed. 11841The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Summary• The Early Devonian Rhynie chert has been critical in documenting early land plant-fungal interactions. However, complex associations involving several fungi that enter into qualitatively different relationships with a single host plant and even interact with one another have not yet been detailed.• Here, we studied petrographic thin sections of the Rhynie chert plant Nothia aphylla.• Three fungal endophytes (co)occur in prostrate axes of this plant: narrow hyphae producing clusters of small spores; large spherical spores/zoosporangia; and wide aseptate hyphae that form intercellular vesicles in the cortex. Host responses on attack include bulging of infected rhizoids, formation of encasement layers around intracellular hyphae, and separation of infected from uninfected tissues by secondarily thickened cell walls.• A complex simultaneous interaction of N. aphylla with three endophytic fungi was discovered. The host responses indicate that some of the mechanisms causing host responses in extant plants were in place 400 million yr ago. Anatomical and life history features of N. aphylla suggest that this plant may have been particularly susceptible to colonization by fungi.

No abstract

The 400 million-year-old Rhynie chert has provided a wealth of information not only of early land plants, but also of the fungi that inhabited this paleoecosystem. In this paper we report the first unequivocal evidence of arbuscules in an endomycorrhizal symbiosis. A new genus, Glomites, is characterized by extraradical, aseptate hyphae with a two-parted wall, and an intraradical, highly branched network of thin-walled hyphae. Hyphal branches produce terminal, elongate-globose multilayered spores that lack a basal septum. Other hyphae penetrate cell walls and form arbuscules. Arbuscules are morphologically identical to those of living arbuscular mycorrhizae (AM) in consisting of a basal trunk and highly dichotomous distal branches that form a bush-like tuft. Arbuscules are confined to a narrow band of specialized thinwalled cells in the outer cortex that continue to be meristematic. Features of the fossil biotroph are compared with those of extant arbuscular mycorrhizae. Although interpretations regarding the evolution of mycorrhizal mutualisms continue to be speculative, the demonstration of arbuscules in the Early Devonian indicates that nutrient transfer is an ancient phenomenon that may have been in existence when plants invaded the land.

The ca. 400-million-year-old Rhynie chert biota represents a benchmark for studies of early terrestrial ecosystems. The exquisite preservation of the organisms documents an ancient biodiversity that also includes various levels of biological interaction. Absent from the picture until recently has been detailed information about the development of the gametophyte phase and the alternation of generations of the macroplants in this ecosystem. Here, we trace the development of the gametophyte phase of Aglaophyton, an early land plant with an unusual complement of structural and morphological characters. Mature gametophytes consist of a fleshy protocorm attached to the substrate by basal rhizoids; arising from the upper surface are one to several upright gametangiophores bearing multiple gametangia. Stomata are present on the upper surface of the protocorm and gametangiophore, and endomycorrhizal fungi extend throughout the gametophyte. Gametophytes are unisexual, producing either antheridiophores or archegoniophores. There is no evidence that gametophytes later become hermaphroditic. The sexual dimorphism of the Rhynie chert gametophytes is inconsistent with theoretical ideas about the haploid phase of early land plants. The gametophyte phase of early land plants can now be considered within an ecological and evolutionary framework that, in turn, can be used to develop hypotheses about some aspects of the population dynamics and growth of these early land plants.Early Devonian ͉ fossil gametophytes ͉ Rhynie chert ͉ spore development ͉ alteration of generations