Arbuscular mycorrhizal‐like fungi in Carboniferous arborescent lycopsids

Approximately 300 million yr ago, during the Carboniferous, vast areas of what today are Europe and North America were covered by extensive swamp forest ecosystems. Many of these ecosystems were dominated by arborescent lycopsids (members of the Lepidodendrales), distant relatives of present-day herbaceous club mosses, quillworts, and spike mosses. Some of the Carboniferous lycopsids were in excess of 30 m tall, with stems up to 1 m in diameter (Taylor et al., 2009). While the above-ground parts show some degree of variability, the below-ground parts of all of these plants (i.e. the rhizomorphic system) are essentially the same and are assigned to the fossil morphogenus Stigmaria; they consist of extensive shallow branch systems that functioned as roots in providing anchorage and uptake of water and nutrients. While it might be argued that the rhizomorphic system represents some intermediate stage in root evolution, the anatomy and development (including embryology) of these peculiar below-ground organs indicate that the rooting structures of all rhizomorphic lycopsids represent shoots (Stubblefield & Rothwell, 1981), in which the ultimate units (termed appendages or rootlets) are greatly modified leaves that develop exogenously (Rothwell & Erwin, 1985). Stigmarian appendages are up to 40 cm long and typically < 0.5–1 cm wide (Taylor et al., 2009). They consist of a central vascular strand that exhibits bilateral symmetry, like the veins in leaves, surrounded by a parenchymatous cortex composed of three zones, i.e. an outer zone of thin-walled isodiametric cells, a middle zone that disintegrates as the appendage matures, and thus is usually represented by a void, and an inner zone of small-sized cells (Stewart, 1947). All of the arborescent lycopsids produced simple leaves termed microphylls. Although the total leaf surface area therefore was relatively small, it has been suggested that the plants grew rapidly and attained maximum height in c. 10–15 yr (Phillips & DiMichele, 1992). Colonization by mycorrhizal fungi has been observed in most modern relatives of the arborescent lycopsids (e.g. Boullard, 1979; Winther & Friedman, 2008; Sudováet al., 2011). It has been speculated that mycorrhizal associations, the mutually beneficial nutritional partnerships between a fungus and a plant in which the fungus facilitates uptake of water and minerals for the plant, while receiving carbon from photosynthesis by the host (Parniske, 2008), may have also occurred in arborescent lycopsids of the Carboniferous (Wagner & Taylor, 1981), but incontrovertible fossil evidence of these associations has been lacking. Here we present the first evidence of an arbuscular mycorrhizal (AM)-like fungus comprised of hyphae, arbuscule-like structures, vesicles, and spores within the cortex of stigmarian appendages from the Carboniferous of Great Britain. Structurally preserved stigmarian appendages containing an AM-like endophytic fungus have been discovered in thin-section preparations of coal ball material from the Carboniferous of Great Britain. The coal balls come from the Union Seam at Dulesgate (Lancashire) and the Halifax Hard Seam at Halifax (Yorkshire). Both seams have been dated as Langsettian (=Westphalian A) (Bashkirian/Early Pennsylvanian, Carboniferous). Thin sections were prepared according to standard procedures. A piece of the coal ball was cemented to a glass slide and subsequently ground with an abrasive until it was thin enough to be examined in transmitted light. All material is deposited in the Bavarian State Collection for Palaeontology and Geology (Munich, Germany) under acquisition number BSPG 1964 XX. The slides were analysed using normal transmitted light microscopy equipment; digital images were captured with a Leica DFC-480 camera. All components of the fungus (i.e. hyphae, spores, vesicles, putative arbuscules) occur together in two appendages preserved in close proximity in one slide; 27 slides contain appendages displaying some complement of these structures. It should be noted that, although stigmarian appendages are generally abundant in these coal balls, the chance of obtaining a perfect longitudinal section through the growing tip of an appendage is very rare due to the manner in which the thin sections are prepared. The fungus occurs near the tip of the appendages, where the middle cortex is still partly intact, and occupies a well-defined zone around the vascular strand (Fig. 1). This zone is located within the inner portion of the middle cortex which is composed of elongate, relatively narrow parenchyma cells. We have seen no evidence of the fungus in the outer cortex or in the matrix surrounding the appendages, which might suggest that inoculation occurred at a very early stage in appendage formation and that the fungus maintained continuity by endophytically following the growth of the appendage. The fungus comprises hyphal threads (4–8 μm in diameter) that grow along the long axis of the appendage. Extending from these trunk hyphae are narrower branches that may produce large vesicles (Fig. 1a–c) or spores (Fig. 1f). Other branches penetrate individual cells of the cortex to form multi-branched structures (Fig. 1b,d,e) that we interpret as arbuscules based on their intracellular formation on longitudinal hyphae and diffuse appearance of hyphal remains, both of which are characteristic features of arbuscules seen in modern AM associations. In modern AM symbioses, arbuscules function as physiological exchange sites. The fossil arbuscule-like structures appear in longitudinal section in 1–2 rows (Fig. 1a–c). Most are more or less isodiametric (up to 40 μm in diameter) (Fig. 1c,d), but some are wider than high (Fig. 1e). The full complement of features of the fungal endophyte, including hyphae and putative arbuscules, is only found in close proximity to the tip region of the appendage. As development continues and the middle cortex of the appendage disintegrates (mature appendages show a characteristic void in this region), the fungus is represented only by vesicles and spores, and occasional trunk hyphae that remain in the void. This appears to be consistent with one of the key points in Brundrett's (2004) definition of mycorrhizas, namely synchronized plant–fungus development. As is the case with modern AM, the active association is confined to the growing tips of the appendages, while the more proximal (mature) regions only contain trunk hyphae, spores, and vesicles. The second key element used by Brundrett (2004) to define AM associations, i.e. the fungi being dual soil and root inhabitants, however, cannot be documented at present based on the fossil material at hand. While the spatial arrangement and morphology of the fungal endophyte in the stigmarian appendages certainly is identical to those of modern AM fungi, because these are fossils one cannot unequivocally state how this fungus functioned. Arbuscular mycorrhizal (AM)-like fungi in Early Pennsylvanian stigmarian appendages from Great Britain. (a) Longitudinal section through central region of appendage (distal, but not tip region; most of the middle cortex no longer present), showing vascular bundle (centre) surrounded by inner cortex, and AM-like fungus just to the outside of inner cortex; note large vesicles (left and right), and single row of small arbuscule-like structures (arrows). Bar, 100 μm. (b) Detail of Fig. 1(a), showing vesicle and row of putative arbuscules. Bar, 50 μm. (c) Trunk hyphae, intercalary vesicle (left), and putative arbuscules (right). Bar, 50 μm. (d) Two arbuscule-like structures arising from common trunk hypha. Bar, 20 μm. (e) Putative arbuscule; note slight constriction of parental hypha at point of entry into host cell (arrow). Bar, 20 μm. (f) Thick-walled spore. Bar, 40 μm. Arbuscular symbioses are ancient mutualistic associations that are hypothesized to have been involved in the transition of plants onto land (e.g. Bonfante & Selosse, 2010). Today, the arbuscular mycorrhiza is probably the ecologically and evolutionarily most important symbiosis in nature (Brachmann & Parniske, 2006). It appears to occur in the majority of vascular plants, including ferns and lycopsids (e.g. Boullard, 1979), regardless of environment, and arbuscular mycorrhizal fungi have also been reported in hornworts, liverworts, and mosses (Pressel et al., 2010). Evidence for mycorrhizal associations from the fossil record, however, is exceedingly rare. The oldest fossil arbuscules come from the Lower Devonian (c. 408 million yr ago) Rhynie chert, where they occur in a specific zone of the cortex of the rootless and leafless prostrate and upright axes of the sporophytes and gametophytes of Rhynia gwynne-vaughanii and Aglaophyton major (Taylor et al., 2009). Similar arbuscule-like structures have also been reported to occur in the cortex of Radiculites-type cordaitalean rootlets from the Carboniferous of France (Strullu-Derrien et al., 2009). In studies of extant plants, arbuscular mycorrhizas have been shown to be involved in a number of important biological and ecological functions, including above-ground biodiversity, carbon partitioning, and immobile nutrient uptake, and as stabilizing agents in the formation and maintenance of soil structure (references in Koltai & Kapulnik, 2010). Although the occurrence of mycorrhizal fungi in leaves has sporadically been reported in extant plants (e.g. in scale leaves of ginger, see Taber & Trappe, 1982), they do not appear to form arbuscules and there is no evidence of a mutualistic interaction. This report of an AM-like fungus in the below-ground organs of arborescent lycopsids, which are highly modified leaves, may strengthen the concept that it is not the type of plant organ dictating the establishment of a mycorrhizal association, but rather the functional environment that the plant organ provides (see Brundrett, 2002; Bonfante & Genre, 2008). Although today mycorrhizal fungi in vascular plants are found almost exclusively in roots, there appears to be a greater diversity of location within the plant in the fossil record (Strullu-Derrien & Strullu, 2007). Mycorrhizal plants through time demonstrate an incredible change of structure, internal organization, reproductive mode, and habitat, whereas the morphology of the fungal partners appears to have remained relatively unchanged. A contributing factor to the morphological stasis exhibited by mycorrhizal fungi for > 400 million yr may have been that terrestrial photosynthetic organisms require some form of absorbing structure and it is the continuous presence of these organs that has enabled mycorrhizal fungi to become established and remain successful through time. Funds were provided by the National Science Foundation (EAR-0949947 to TNT and MK) and the Alexander von Humboldt-Foundation (V-3.FLF-DEU/1064359 to MK). We thank four anonymous referees and Marc-André Selosse for their constructive comments and suggestions.

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