Mycorrhizas are mutualistic associations between plants and fungi; > 90% of embryophytes are capable of forming symbioses of this type. The fungus uses the host as a source of carbon, while the host is supplied with mineral elements by the fungus. Endomycorrhizal fungi associated with prostate axes of Aglaophyton major (paramycorrhizas sensu Strullu-Derrien & Strullu, 2007) from the Lower Devonian Rhynie chert represent the oldest occurrence of mycorrhizas (Remy et al., 1994; Taylor et al., 2005). The fungi involved in this and other mycorrhizal associations from the Rhynie chert belong to the Glomeromycota, a fungal phylum established by Schüßler et al. (2001) using molecular data. Evidence from these plant-bearing deposits indicates that all main spore types in the Glomeromycota were in existence before the evolution of true roots (Dotzler et al., 2008).
Extensive collections of thin sections of petrified plant material were manufactured during the early twentieth century. These collections are an invaluable source of information about associations between plants and microorganisms (Krings et al., 2007). Our study focuses on fungal associations in permineralized Radiculites rootlets of the Radiculites-type (assigned to cordaitalean reticulatus) from the flora from Grand’Croix (France) based on the original slides contained in the Lignier, Florin and Carpentier collections. The thin sections were prepared in the early twentieth century from material collected from the Late Pennsylvanian (Upper Carboniferous) ‘Poudingue Mosaïque’ of Grand’Croix, which belongs to the Saint Etienne coal basin (Massif Central, central France). This basin is situated c. 50 km southwest of the town of Lyon. Information about the geological setting of Grand’Croix can be found in Doubinger et al. (1995). Thin sections were prepared according to standard techniques. A piece of silicified rock was cemented to a glass slide and then ground to a thickness sufficiently thin to allow for examination in transmitted light. Slides were studied using dry or oil immersion objectives. The Lignier slide collection is today housed in the Herbarium (C.N.) of the University of Caen (France), the Carpentier slides are kept in the collections of Lille Catholic University (France) and the Florin collection is housed in the Natural History Museum of Stockholm (Sweden).
Characterization of the rootlets
The rootlets are up to 0.65 mm in diameter. In transverse section, they appear well-delimited by an epidermis composed of dark cells (Fig. 1). An exodermis occurs beneath the epidermis; it is one or two layers in thick and consists of cells characterized by thickenings on the periclinal walls. The cells of the outermost exodermal layer are brown in colour. Root-hairs were not observed. The cortex is composed of parenchymatous cells that become more elongated toward the centre of the root. The cells of the cortex are characterized by phi thickenings (Fig. 1b,c), which are prominent in cells located close to the vascular cylinder and gradually become smaller towards the periphery. The name phi thickenings, based on the resemblance of these structures in cross-section to the Greek letter phi, was given by Russow (1875; cited in van Tieghem, 1888). The thickenings form a frame that corresponds to the frame of the adjoining cell (Fig. 1c). An endodermis with a distinct casparian strip on the anticlinal walls separates the cortex from the vascular cylinder. The rootlets generally possess a diarch primary xylem.
The cortical network described above is obvious in all specimens studied and shows a pattern similar to that seen in the rootlets of extant Cupressaceae (s.l.) (Gerrath et al., 2002). There are distinct differences with regard to the shape of the thickenings, which may be linked to habitat because the specimens come from three different localities (Grand’Croix, Cuzieu and Assailly) within the ‘Poudingue mosaïque’ (Fig. 1b,c).
Evidence for endomycorrhizal colonization in the rootlets of Cordaites
Lignier (1906) was the first to report endophytic fungi in Radiculites reticulatus cordaitalean rootlets and referred to them as mycorrhizas. Zimmermann (1933), who also studied material from Grand’Croix, suggested that mycorrhizal infection was restricted to a few large cells that lack a casparian strip. In fact, the occurrence of mycelial hyphae was only documented in the outer cortex. This meant that the mycorrhizal status of R. reticulatus, as well as that of other supposedly mycorrhizal cordaitalean rootlets (e.g. Amyelon radicans; see Osborn, 1909; Halket, 1930), was contested (Cridland, 1962). Cridland suggested that the fungus in Lignier's material represents a parasite or saprotroph, rather than a mutualist. He also restudied Carpentier's material, but from the Lower Permian, while we focused on specimens from the Upper Pennsylvanian of Grand’Croix (Carpentier, 1932). However, the cortical network that characterizes the Radiculites-type rootlets has not been reported for Amyelon-type rootlets (Cridland, 1964). As a result, these two types of rootlets appear to belong to different genera of Cordaites. The colonization of Amyelon radicans by endophytic fungi has not yet been reinvestigated.
The relationship of R. reticulatus rootlets to the Cordaites was established by Lignier (1911). Attachment of the Radiculites-type rootlets to larger cordaitalean roots have been observed in three of the Lignier's slides. Mycorrhizal associations occur in the rootlets that correspond to the primary state of growth of cordaitalean roots (Fig. 1a). The best preservation occurs in the material from the Lignier and Carpentier collections, where many rootlets show evidence for mycorrhization. Rootlet diameters vary from 0.5 to 0.65 mm. The fungus colonizes a discontinuous fungal zone in the central layers of the cortex (Fig. 2a,e, arrows). Colonization is characterized by the absence of intercellular phase and by the development of intracellular coiled hyphae (Fig. 2f), which spread from cell to cell. While vesicles have not been observed in the cordaitalean rootlets, small arbuscules occur in some of the cortical cells. The arbuscules are most easily recognized in longitudinal sections (Fig. 2b–d). As in living plants, the cell wall shows a slight thickening, called an apposition, that forms at the point of entry of the fungal hypha (Fig. 2b). Arbuscules (Fig. 2b–d) originate from the coiled hyphae and are morphologically identical to those seen in arbuscular mycorrhizas of extant plants. The hyphal trunk of the arbuscule is 2 µm wide and branches repeatedly to form a bush-like tuft within the cell (Fig. 2c,d). Moreover, a few of the cortical cells appear to be filled with material similar in appearance to the amorphous masses that are the result of arbuscule degeneration in the cells of living plants (Fig. 2a). Based on the evidence assembled, we suggest that the AM association in R. reticulatus is of the Paris type (Strullu, 1985). The endophyte is only associated with ontogenetically young axes. Additional details of the association are difficult to resolve, owing primarily to the prominence of the cortical thickenings in the rootlets; a similar masking of fine details of the mycorrhiza by cortical cell thickenings has been recorded for extant plants with Paris-type mycorrhizas (cf. Thuya occidentalis, Brundrett et al., 1990).
In a recent survey (Strullu-Derrien & Strullu, 2007), we reported the distribution of mycorrhizal associations in fossil and extant plants. The oldest fossil evidence for the existence of mycorrhizas occurs in Aglaophyton major from the Lower Devonian Rhynie chert (Remy et al., 1994; Taylor et al., 2005). Robust arbuscules and vesicles have been found in Antarcticycas schopfii from the Triassic (Stubblefield et al., 1987) and mycorrhizal associations have been described in Lower Cretaceous and Middle Eocene conifer roots (Stockey et al., 2001). The colonization of the Carboniferous cordaitalean R. reticulatus rootlets described here is the oldest unequivocal fossil evidence for eumycorrhizas (i.e. fungal–plant root associations) and the oldest evidence for mycorrhizal associations in the conifer clade.
The authors thank Prof. M. Krings for useful analysis of the manuscript. C.S-D. gratefully acknowledges Prof. E. M. Friis for her welcome in the Department of Palaeobotany in the framework of the Synthesys program and thanks her collaborators for Laboratory assistance. The authors extend their gratitude to Dr D. Brice for making the slides from the collection Carpentier available. They also thank Dr P. Gerrienne and C. Ferrier for their cooperation and the anonymous reviewers for their constructive suggestions. This work was partly funded by Synthesys support made available by the European Community-Research Infrastructure Action under the FP6 ‘Structuring the European Research Area Programme’, SE-TAF 3444.