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Fertile Prototaxites taiti: a basal ascomycete with inoperculate, polysporous asci lacking croziers
Associated Data
Abstract
The affinities of Prototaxites have been debated ever since its fossils, some attaining tree-trunk proportions, were discovered in Canadian Lower Devonian rocks in 1859. Putative assignations include conifers, red and brown algae, liverworts and fungi (some lichenised). Detailed anatomical investigation led to the reconstruction of the type species, P. logani, as a giant sporophore (basidioma) of an agaricomycete (= holobasidiomycete), but evidence for its reproduction remained elusive. Tissues associated with P. taiti in the Rhynie chert plus charcoalified fragments from southern Britain are investigated here to describe the reproductive characters and hence affinities of Prototaxites. Thin sections and peels (Pragian Rhynie chert, Aberdeenshire) were examined using light and confocal microscopy; Přídolí and Lochkovian charcoalified samples (Welsh Borderland) were liberated from the rock and examined with scanning electron microscopy. Prototaxites taiti possessed a superficial hymenium comprising an epihymenial layer, delicate septate paraphyses, inoperculate polysporic asci lacking croziers and a subhymenial layer composed predominantly of thin-walled hyphae and occasional larger hyphae. Prototaxites taiti combines features of extant Taphrinomycotina (Neolectomycetes lacking croziers) and Pezizomycotina (epihymenial layer secreted by paraphyses) but is not an ancestor of the latter. Brief consideration is given to its nutrition and potential position in the phylogeny of the Ascomycota.
This article is part of a discussion meeting issue ‘The Rhynie cherts: our earliest terrestrial ecosystem revisited’.
1. Introduction
The genus Prototaxites, whose fossils extend from Silurian (Ludlow) to Upper Devonian strata (Famennian) [1,2], remains one of the most debated conundra in the Palaeozoic record. Some Prototaxites ‘logs’ reached more than 1 m in diameter and several metres in height, the maximum being 8.8 m; they were the largest terrestrial organisms of their time [1]. Some, but not all Prototaxites axes reveal irregular growth rings. Its habitat has been much debated [3,4], but based on sedimentological studies Prototaxites was found to be truly terrestrial [5]. Being part of terrestrial food chains, its axes have been invaded by boring terrestrial arthropods [6].
Approximately 14 species have so far been described. These have been collected in North America [1,7–10], northern Europe (UK [3,11,12], Germany [1,13–17]), North Africa (Libya [18,19]), western Asia (Saudi Arabia [20,21]) and Australia [1]. Prototaxites spp. were locally abundant, as concluded from allochthonous charcoalified fragments in Lower Devonian fluvial rocks in the Welsh Borderland, where Prototaxites often makes up a high percentage of fossil remains. Eroded, fragmented silicified samples have been found as pebbles in fluvial deposits, e.g. in gravel quarries (Middle Eocene [22] from the Eifel area to coastal Belgium and Netherlands [23]). However, only very rarely were Prototaxites samples found with an intact surface layer, exceptions being a few permineralized fossils in the Rhynie chert palaeoecosystem, and fertile parts have been largely missing [10].
As the etymology of the name suggests, its originator Dawson [7] initially identified the large ‘trunks’ of Prototaxites as a conifer with anatomy superficially similar to that in the wood of the extant genus Taxus (yew) with its tertiary spirals. Such assignation was subsequently challenged by the phycologist Carruthers [24] who, illegitimately based on precedence, renamed the Lower Devonian Gaspé fossils as Nematophycus, here with connotations to brown algal affinity and resemblance to the laminarialean genus Lessonia. Subsequent authors (e.g. Penhallow) [25] supported an algal affinity, while Dawson [26] changed the name to Nematophyton, a name, although again illegitimate, which was used by a number of authors, including Kidston & Lang [3] (N. taiti). Later a red algal affinity was postulated [27], and recently Prototaxites axes have been interpreted as rolled mats of liverworts (marchantiophytes), which had rolled down a slope and stabilized themselves with their own rhizinae [28,29]; this interpretation has been rejected [30,31].
An early dissenting voice had been that of Church [32] who saw resemblances with fungi, a relationship reinforced by Hueber [1]. In a very detailed reinvestigation of Dawson's type, P. logani, and more recently acquired material, Hueber described the organism as possessing three forms of interactive hyphae (plectenchyma as opposed to embryophyte parenchyma) with similarities to other Prototaxites species.
Definitive evidence for the fungal status of Prototaxites and then its position in the fungal clades requires information on its reproduction. Hueber [1] described structures on the margin of a growth increment in P. logani, which he interpreted as remnants of a hymenium on a previous outer surface in which dendritically branching hyphae (dendrophyses) were associated with ‘apparent remnants of primitive basidia in the form of a closely linear or clustered individually inflated sterigmata, each with a prominent speculum’ (p. 145; Plate VII). Neither spores nor basal metabasidia were preserved. On such somewhat equivocal evidence, Hueber concluded that a hymenium originally present on the outermost surface of the presumed sporophore (trunk) supported basidiomycete affinity.
Here we describe a different type of hymenium associated with Prototaxites taiti in the Rhynie chert [3], which offers an alternative solution to relationships within the fungi. Although the hymenial layer of the type specimen is not in organic continuity with the axis, we are convinced, as were Kidston & Lang [3], that it was neither a parasite nor a saprotorophic degrader, but part of P. taiti. The new material studied here shows a similar juxtapositioning with P. taiti and contains medullary spots. Peels of P. taiti comprise detached fragments of the hymenial layer and a larger fragment whose entire surface is covered by a hymenium. Similar organization detected in charcoalified fragments from the Welsh Borderland leads to the inference that some members of the Nematothallus complex, which were common elements of terrestrial ground covers in the Late Silurian–Early Devonian [33], also belong to Prototaxites.
2. Material and methods
Anatomy was studied using petrographic thin sections and cellulose acetate peels of the silicified matrix from Rhynie chert:
(1) Figured specimen, i.e. thin section no. 2525 from the Hunterian Museum, is part of the original Kidston's collection (Plate X, figs 113, 115, 116, 118 in [3]); this section was made by W Hemingway. Courtesy Dr Neil Clark.
(2) One section and numerous peels from Block 149 in the Lyon Collection at Aberdeen University; thin section 149/CT/B had been prepared by AG Lyon in Cardiff and is now in the Aberdeen collection. Courtesy Prof. Nigel Trewin.
Sections had been prepared by standard petrographic techniques. Peels had been prepared by AG Lyon by etching smooth surfaces of chert with HF before washing and applying a superficial sheet of cellulose acetate moistened with acetone.
Peels and thin sections were examined in transmitted light using a LEICA DMR microscope fitted with LEICA LAS software, and a Nikon Eclipse LV100ND compound microscope. Confocal images from the thin sections were acquired with a Nikon A1-Si laser-scanning confocal microscope. Autofluorescence of the sample was excited with four laser lines. Autofluorescence signal was collected with four photomultiplier detectors with the following wavelength emission windows: 425–475 nm for the 405 nm laser, 500–550 nm for the 488 nm laser, 570–620 nm for the 561 nm laser and 675–725 nm for the 640 nm laser. Samples were visualized using a 29.9 µm (1.2 airy units) confocal pinhole and a number of z-stacks (typically between 100 and 400) with optical thickness between 200 and 300 nm each were acquired. The fluorescence signal from each z-stack was then projected onto a maximum projection image.
For SEM investigations coalified material collected at Ludford Corner (Přídolí) and North Brown Clee Hill (Lochkovian) in the Welsh borderland was isolated from the matrix using HCl and HF (protocol in [33,34]). Selected specimens were mounted on stubs and examined using an environmental scanning electron microscope (ESEM-FEG, Philips XL 30, with field emission gun; FEI).
3. Results
(a) Light microscopy preparations from Rhynie chert fossils
(i) Figured specimens in same block as the type specimens of Prototaxites taiti
Slide no. 2525 of the Kidston collection, the petrographic thin section with the type specimen of P. taiti Kidston & Lang 1921 [3], contains an isolated fragment interpreted by Kidston and Lang as its peripheral layer (figure 1a). This was based on the presence of medullary spots (figure 1b–d) noted in P. taiti and common in other Prototaxites species; the presence of ‘tubes’ within this region thought equivalent to those in the tissues of Prototaxites, although with smaller diameter in the peripheral tissue (figures 1a and and22e); and the close proximity to P. taiti in the same block. According to Kidston & Lang [3] the peripheral layer, which ‘has a thickness of about 1 mm’ (p. 888), is built up by ‘tubes’ arranged in parallel at right angles to the surface with homogeneous brown contents and fine hyphae in between these (p. 886). They describe a narrow outer zone above the summits of the vertical tubes as a ‘structureless, and perhaps mucilaginous, layer during life. It has no sharply defined outer limit, though it is marked as a dark line owing to a granular deposit’.
Petrographic thin section of Prototaxites taiti in bright field (a) and confocal laser microscopy (b,c), and peel in bright field microscopy (d). (a–c) The type specimen of P. taiti from the Rhynie cherts, new photograph of slide no. 2525 of the Kidston Collection. (a) This fragment is depicted in black and white in Kidston & Lang [3] in fig. 113, with the following legend: ‘View of one of the sections of the second fragment which shows the peripheral tissues'. Here these peripheral tissues are interpreted as the hymenial layer of the ascoma of an ascomycete, the section plane being slightly tangential. Note the innumerable silicate crystals, best visible outside the tissues. (b–d) Medullary spots: in confocal laser microscopy (b,c), (slide no. 2525), intense wefts of hyphae in close contact with a population of minute cells visible in bright field microscopy (d), (slide 149 B 3).
Slides of a petrographic thin section and a series of peels of Prototaxites taiti, from Rhynie chert as prepared by Prof. AG Lyon. (a) Overview of petrographic thin section 149 CT B (see details in figures figures33–5) and serial peels in slides 149/B 2, 3, 8 & 9. (b) Detail of slide 149/B 8, the position of figures (c) and (d) being marked. (c) Cross-section of a cord (or a very young axis?) with a somewhat denser peripheral zone. (d) Slightly tangential section of the peripheral layer of a ‘fleshy’ axis which, in the present study, is interpreted as the hymenial layer of an apothecium-bearing ascomycete. (e) Confocal laser micrograph of the hymenial layer of the type specimen of P. taiti (slide no. 2525 of the Kidston and Lang collection; see figure 1a), showing the tubes, i.e. asci with ascospores, in the hymenial layer.
We interpret this peripheral zone of P. taiti as the hymenial layer of an apothecium-bearing ascomycete, the section plane being slightly tangential, the tubes with granular contents being asci with large numbers of small ascospores and the fine hyphae between them as paraphyses. The thin, amorphous and pigmented outermost layer is presumably an amorphous epihymenial layer; the granular deposits therein might have been secondary metabolites, as secreted by the tip cells of the paraphyses prior to crystallization. Such gelatinous, often brightly pigmented epihymenial layers are common and widespread in the apothecia of extant non-lichenized and lichenized Pezizomycotina.
(ii) Slide no. 149/CT/B, petrographic thin section
In this section (figures 2a and and33–5), several detached fragments of the presumed hymenial layer of P. taiti are preserved. Most of them are tangentially sectioned, but a comparatively large fragment of a median section can be explored (marked in figure 3, white arrow). The hymenial layer is approximately 1100 µm thick. Moreover, there are two dark areas contained in this slide with hundreds of sporangia and resting sporangia of a zoosporic fungus [35]. This large assembly probably developed on detritus, as accumulated in the chert palaeoecosystem.
Petrographic thin section of Prototaxites taiti from Rhynie chert specimen no. 149 CT B shown in figure 2a. The white arrow points to a median-longitudinally sectioned fragment of the presumed hymenial layer, black arrows to additional hymenial fragments; framed is a thin area of which photographs in figures figures44 and and55 had been taken. The asterisk refers to part of a thin, cross-sectioned axis of Prototaxites with a simple peripheral zone. White circles refer to areas with hundreds of sporangia of a zoosporic fungus (insert: resting sporangium; see [35]). Photo: Thomas Honegger.
Prototaxites aff. taiti from Rhynie chert, details from slide no. 149 CT B; (a,b) confocal laser microscopy, (c–e) bright field light microscopy. (a) Detail of the hymenial layer with asci, paraphyses and invasive, contaminating fungal filaments. (b) Spores inside an ascus. (c) Contaminating fungal and bacterial filaments growing below the hymenial layer. (d) Laminal view, and (e) tangential (left) and cross-section (right) of detached fragments of the epihymenial layer, with tip cells of paraphyses and open ascus tips, the interspace being filled with dark pigments.
In the thinner part of the median section of the hymenium of P. taiti, the asci, paraphyses and epihymenial layers were resolved (figures 4 and and55a,b). At maturity the ascus tip reached the surface of the hymenium. The delicate paraphyses (approx. 5–6 µm thick) are septate (figure 4) and end with a globose to pyriform tip cell (figure 4a,d); anastomoses occur (figure 4e). The asci contained more than 100, at presumed maturity, well-defined spores with thin, translucent walls measuring approximately 6 µm in diameter (figure 4a–c); these are best visible in confocal micrographs (figure 5a,b), the chemical composition of the autofluorescent material being unknown, fluorescence resulting either from mineral or organic preservation. Some of the asci had already released their spores.
(a–e) Prototaxites aff. taiti from Rhynie chert, details from the longitudinal section in slide no. 149 CT B (figure 3). Polysporous asci and thin-walled, delicate paraphyses. The darkly pigmented epihymenial layer is best visible in (d). The arrow in (e) points to anastomosing paraphyses. Same magnification in (a–e).
The amorphous epihymenial layer of P. taiti, with wall fragments of tip cells of the paraphyses and granular deposits embedded therein, was locally peeling off the hymenial surface and detached fragments were found in the surroundings of the hymenia (figure 5d,e). The apical region of asci after spore release appears as holes in these detached epihymenial layers.
Various hyphae of fungal and bacterial invaders are seen below and partly within the hymenial layer of P. taiti (figure 5c). They might have grown into the detached hymenial fragments post-mortem. Moreover, there are many silicate crystals, often rosette-like, around and within the fossils (figure 1a).
(iii) Slides 149/B 2, 3, 5, 8 & 9 with peels of P. taiti
Serial peels of a block containing P. taiti (figure 2a–d) contained several detached fragments of hymenial layers in various section planes (marked with pen in figure 2a), and a near-median section of the hymenial layer covering the surface of an axis (figure 2b,d). A cross-section of presumed cords are seen (figure 2b,c), but not in conjunction with the axes.
In these serial peels of P. taiti two large, dark fragments and a distinctly brighter one are seen (figure 2a,b). The dark fragments lack a peripheral layer, whereas the surface of the bright fragment is covered by an extensive hymenium, the latter being tangentially sectioned. Although this fertile fragment reveals the anatomy characteristic of Prototaxites it seems to be more ‘fleshy’. With high probability the detached hymenial fragments had peeled off such fertile parts when these were either senescent or otherwise damaged. The large number of detached hymenial fragments, as found in these Rhynie chert specimens, infers an abundance of fertile parts.
(b) Scanning electron micrographs of charcoalified fragments from the Lochkovian Welsh Borderland
(i) Prototaxites sp. (HD(L)171/03), figures figures66 and and77
SEM micrographs of a charcoalified fragment of the hymenial layer of Prototaxites sp. (HD(L)171/03) from Welsh Borderland. (a,b, d,e) Laminal view of the surface. (a) Overview; (b) detail of the hymenial surface. The asterisks mark an ascus apex prior to dehiscence. Thick arrows point to asci shortly after ascospore release, the inoperculate ascus tips having slightly expanded and opened with a slit. Thin arrows point to empty asci whose wall sank back into the hymenial layer. (c) Lower surface of the specimen. (d) Arrows point to shrivelled, presumed ascospores after landing on the hymenial surface. (e) Presumed ascospores having germinated next to the apex of the empty ascus.
SEM micrographs of the charcoalified fragment of the hymenial layer of Prototaxites sp. (HD(L)171/03) from Welsh Borderland, lateral view. (a) The dense epihymenial layer is partly peeling off (figure 5a,b). (b). Detail of the hymenial layer with immature asci. Arrows point to presumed paraphyses and/or fungal or bacterial invaders. (c) Arrows point to remains of a few thin-walled paraphyses. The asterisk points to an ascus with superficial impressions of lost paraphyses.
This small Lochkovian fragment reveals an intact, hummocky surface of the hymenial layer (figure 6a,b) comprising well-spaced structures interpreted as asci. Various developmental stages of asci embedded in the amorphous epihymenial layer indicate that they were ripening at different times (figure 6b). Still closed ascus apices appear near others, which slightly bulge above the surface prior to opening with a slit. After ascospore release the wall of the empty ascus sunk back into the hymenial layer (figure 6b), leaving a roundish slit before getting covered by mucilage. Groups of ovoid, somewhat shrivelled presumed ascospores were repeatedly seen lying on the hymenial surface (figure 6d); their size (approx. 5.5 µm long) corresponds to those seen in the silicified specimens (figure 4). Some of these presumed ascospores seemed to germinate (figure 6e), but cannot be distinguished from contaminant fungal spores. Both landing of ascospores and their germination on the hymenial surface occur frequently in extant ascomycetes. However, crystals of the same size and shape as the presumed ascospores are frequently found on this type of material.
On the lower surface of this fragment are medullary spots embedded within hyphae (figure 6c). The epihymenial layer has locally peeled off (figure 7a), revealing a reticulum characteristic of cuticles of the Nematothallus complex. The probably largely immature asci are well preserved (figure 7a–c), but most of the delicate paraphyses were lost, probably during charcoalification, leaving some impressions on the surface of the asci (figure 7c). Presumed paraphyses are seen in figure 7c. The amorphous epihymenial layer is well preserved, revealing impressions of tip cells of paraphyses on its inner surface; these are visible at sites where the epihymenial layer detaches (figure 7a). A subhymenial layer with crozier formation was not seen here nor in the silicified material examined by light microscopy. As described by Kidston & Lang [3] for the latter ‘the tubes bend outwards and branch, and in the superficial layer … stand parallel at right angles to the surface’ (p. 888). This specimen was invaded by either fungal or bacterial contaminants (figure 7b).
(c) A charcoalified apothecial fragment of a presumed pezizomycete from the Late Silurian Shropshire
The unnamed specimen LL03/02 from Ludford Lane, Shropshire (figure 8), is interpreted as a fragment of at least two apothecia, which had been growing side by side (figure 8a,b,e). In contrast to the hymenium of P. taiti, a dense subhymenial layer is seen with presumed crozier formation (figure 8c). The apothecia have a thin margin built up by only one cell layer (figure 8b,e). The apex of the polysporous ascus reached the hymenial surface at maturity (figure 8e,f). Some of the tip cells of the septate paraphyses bend and branch (figure 8f,g), a feature which is seen also among some extant ascomycetes. This specimen, which was unfortunately lost while being turned over for additional fracturing and investigations, represents the first apothecium-bearing member of the Pezizomycotina so far found.
SEM micrographs of a charcoalified apothecial fragment from the Late Silurian (LL03/02) from Ludford Lane, Shropshire. (a) Upside-up, and (b, e) upside-down positional views of the specimen. (c) Presumed crozier in the basal part of the hymenial layer. (d) The longitudinally fractured hymenial layer is built up by slim, unbranched paraphyses. The numerous ascospores within the fractured, polysporous ascus reveal irregular outlines, i.e. are deformed. Arrows point to bacterial contaminants which had grown post-mortem over the fragment. (e) Detail of surface in (b): the thin apothecial margin is built up by parallel hyphae. (f) The rounded tip cells of paraphyses are grouped around presumed ascus apices. (g) Septa are visible in fractured paraphyses.
4. Discussion
(a) Prototaxites taiti: an ascomycete with extensive hymenial layer
A striking structural similarity of the ascomata of Prototaxites taiti with the sori (meiosporangia-bearing zones) of the large sporophytes of marine brown algae (kelps) of the order Laminariales (Phaeophyceae, Heterokonta) is evident. Sori of Laminariales are formed as patches on the surface of the phylloids (‘blades’); they are built up by sterile filaments (paraphyses) interspersed with elongate, ascus-shaped meio-sporangia comprising large numbers of biflagellate zoospores. The morphological similarity of Prototaxites stems with cauloids and of their basal part with holdfasts of Laminariales was reported by earlier investigators [13,17,18,24,25]. However, the meristem-derived tissues of laminarialean sporophytes differ anatomically from the plectenchyma of Prototoaxites spp. Fossil records are largely missing. Based on multigene phylogenetic analyses the branching time of Laminariales from Ectocarpales (filamentous brown algae) is estimated around 100–90 Ma [36]. Thus a laminarialean affiliation of the genus Prototaxites can be excluded.
Based on the present findings P. taiti is interpreted as a basal ascomycete; in the fertile state a hymenial layer comprising polysporous asci and delicate, septate paraphyses covered the surface of either part of its axis or of fertile, presumably lateral and/or terminal outgrowths. It is not clear how the hymenial fragment of the type specimen of P. taiti (figure 1a) was connected to the axis; the ‘fleshy’ fertile fragment in the peels (figure 2a,b) might represent a fruit body as a lateral outgrowth of the axis. Further material is needed to answer these questions.
The oldest ascoma of an ascomycete so far described are the beautifully preserved perithecia of Palaeopyrenomycites devonicus [37,38], a presumed sordariomycete, parasite or epibiont of Asteroxylon mackiei. However, the most basal taxa among the extant Pezizomycotina (i.e. the Peziomycetes and Orbiliomycetes; [39,40]) produce neither ostiolate peri- or pseudothecia, nor closed cleisto- or gymnothecia, but apothecia, the former ascomal types having evolved independently in diverse taxa [41–44].
The dimensions of the hymenial layer of P. taiti differ considerably from extant ascomycetes, both in circumference and height. Very large apothecial surfaces are found, e.g. among the earth tongues (Geoglossaceae, Geoglossomycetes; figure 9b), the elfin saddles (Helvellaceae, Pezizomycetes) and the morels (Morchellaceae, Pezizomycetes). Their comparatively soft, large ascomata are relatively short-lived (few weeks) and die off after ascospore release, all asci reaching maturity at almost the same time. In contrast, the tough, club-shaped dead moll's finger (Xylaria longipes) and related taxa among the Xylariaceae as well as caterpillar fungi (Cordiceps spp., Sordariomycetes) have no superficial hymenial layer, but large numbers of minute perithecia peripherally inserted in their robust sclerotia. Long-living hymenial layers are found in the apothecia of the majority of lichen-forming ascomycetes, especially in the Lecanoromycetes (e.g. Cladonia spp., Cladoniaceae, or Baeomyces spp., Baeomycetaceae; figure 9c,d), their fruit bodies producing asci over many months to a few years. However, their apothecia are minute compared with the aforementioned taxa.
Extant ascomycetes with clavate to roundish sporophores (ascomata). (a) The presumably saprotrophic Neolecta vitellina, one out of four species of Neolectomycetes, the only Taphrinomycotina with ascomata. The extensive hymenial layer is bright yellow. Photo courtesy of Raymond McNeil. (b) The saprotrophic dark-purple earth tongue (Geoglossum atropurpureum, syn. Thuemenidium a.). Photo courtesy of Knud Knudsen. (c) The stalked, non-lichenized ascomata of Dibaeis baeomyces (syn. Baeomyces roseus) growing out of the crustose lichen thallus. (d) Convex ascomata of Cladonia bellidiflora with red coloration (due to crystals of the anthraquinone bellidiflorin in the epihymenial layer) on an erect podetium whose surface is covered by dorsiventrally organized, lichenized squamules.
The hymenial layer of P. taiti is approximately 1100 µm high and thus more than twice the size as reached by extant apothecia-bearing ascomycetes. The length of the mature ascus, as recorded in the taxonomic literature, does not necessarily reflect the full height of the hymenium; the apex of the mature ascus may be below the hymenial surface and the epihymenial layer often accounts for additional micrometres. In extant pezizomycetes, ascus lengths around 300–380 µm are found in morels (Morchella spp.), up to 390 µm in cedar cup (Sepultaria sumneriana), and up to 450 µm in midnight disco (Pachyella violaceonigra), all representatives of the Pezizales/Pezizomycetes (data from [45]).
(b) The oldest fossil apothecia
This is the first report on fossil apothecia, the characteristic ascomata as formed by numerous taxa within the subphylum Pezizomycotina, which are considerably older than those found in Cenozoic amber. So far a striking discrepancy between the presumed age of the earliest apothecia-bearing ascomycetes (Ordovician), as concluded from molecular datasets [38,39], and the fossil record was evident. So-called calicioid ascomata (a paraphyletic assemblage of taxa with stalked apothecia and superficial mazaedium, i.e. a powdery spore mass, as released by deliquescent asci [46,47]), were found in Cenozoic amber [48–51].
Extant representatives of the Pezizomycotina have a haploid vegetative mycelium. Ascoma formation is initiated by dikaryon formation, either via spermatisation of ascogonia (the contact sites being thin-walled trichogynes, which grow out of the ascogonia) or via fusion of non-differentiated vegetative hyphae (somatogamy). Upon successful dikaryotisation a system of dikaryotic hyphae grows towards the future hymenial layer of the ascoma. The haploid hyphal system in close contact with the ascomal primordium builds up the ascoma with characteristic margin and the hymenial layer with paraphyses, which, in many groups, secrete the hydrophilic hymenial gelatin and the epihymenial layer plus the pigments located therein. In the majority of Pezizomycotina, crozier formation with nuclear migration into the penultimate cell along the dikaryotic ascogenous hyphae, precedes karyogamy; this allows the maintenance of the dikaryotic state with nuclei of opposite mating types.
(c) Neolectomycetes (Taphrinomycotina): basal Ascomycota with club-shaped ascomata
The Taphrinomycotina, formerly termed Archaeascomycetes, is the most basal out of three subphyla of the Ascomycota; it comprises the classes Taphrinomycetes (plant pathogens), Schizosaccharomycetes (fission yeasts), and the monotypic class Neolectomycetes (with one order, one family and one genus, Neolecta Speg., comprising four presumably saprotrophic species) [52]; these are the only extant representatives of the Taphrinomycotina which form ascomata.
The stipitate, clavate ascomata of Neolecta spp. are covered by a coloured hymenial layer (figure 9a), no ascomal margin being differentiated. The vegetative hyphae are binucleate, the ascogenous hyphae multinucleate, the nuclei being haploid [53]. When asci are to be formed in Neolecta spp. pairs of nuclei, presumably of opposite mating types, are formed and segregated via septum formation from the rest of the hypha [53]; a dense subhymenial layer is missing and no croziers are formed. The mating system of Neolecta spp. has not yet been genetically analysed, and it is not known how the pairing of nuclei is achieved prior to karyogamy and meiosis. In the more advanced, plant pathogenic Taphrinomycetes (Taphrina spp.) the dikaryon is formed soon after ascospore germination via fusion of budding cells, only the dikaryotic (bi-nucleate) mycelium being infective. Based on molecular datasets [54,55] Neolecta (i.e. the Neolectomycetes) was referred to as ‘a fungal dinosaur’ [56].
(d) Prototaxites taiti: a basal ascomycete
According to the present findings P. taiti represents a basal ascomycete which combines characteristic features of Neolectomycetes/Taphrinomycotina, such as asci without crozier formation (in addition to the SEM specimen several millimetres of hymenial length were studied in petrographic thin sections), their inoperculate apex opening with a slit, and of Pezizomycotina, i.e. a system of paraphyses which secrete mucilage into the hymenial and epihymenial layer. However, Upper Silurian and Lower Devonian Prototaxites spp. were unlikely ancestors of the Pezizomycotina since they were contemporaries of apothecia-bearing ascomycetes with more advanced character traits. The unnamed Upper Silurian apothecial fragment (LL03/02) with a thin apothecial margin (figure 8b,e), distinct paraphyses and polysporous asci, reveals a dense subhymenial layer (figure 8d) and presumed croziers (figure 8c), both being characteristic features of Pezizomycotina. No croziers could be resolved in Palaeopyrenomycites devonicus [37,38].
How does our interpretation of P. taiti as a basal ascomycete correlate with Francis Hueber's [1] emendation of the genus Prototaxites as a representative of the class Agaricomycetes (=Holobasidiomycetes)? Based on detailed investigations of P. loganii samples, Hueber interpreted the tangentially sectioned peripheral layer as a hymenial layer and some of the structures therein as sterigmata of presumed basidia. Neither basidia proper nor basidiospores were seen in this sample. The presumed sterigmata could be remains of ascus walls in the tip region after spore discharge. Median sections are required to solve this problem.
In the neotype of P. loganii Hueber [1] described a cluster of very thin, finely branched presumed dendrophyses (= dendrohyphidia), a type of sterile hyphae as found in the hymenia of some basidiomycetes. No comparable structures were found in P. taiti. Since the hymenium, as the peripheral layer, is also easily accessible to fungal and bacterial colonizers during lifetime and post-mortem (figure 5c) and crystallization products of minerals may have developed during the fossilization process on or near the hymenial surface, a more detailed analysis of the cellular structure of the dendrophyses would be of interest.
The septal pore apparatus, a phylogenetically informative character trait, has been investigated in Prototaxites southworthi with TEM techniques [57]. It shows a quite complex central pore with surrounding material, but not a dolipore as typically found in extant Agaricomycetes. A surprisingly similar situation was observed in Neolecta vitellina [58], whose septal pore apparatus differs structurally and biochemically from the one as typically found in Pezizomycotina. The HEX-1 protein, as contained in the membrane-bound Woronin bodies which are plugging the septal pore of Pezizomycotina [59], is missing in Neolecta [60], but a unique type of material is present. Healy et al. [58] conclude that the septal pore-occluding structure in Neolecta might have evolved independently.
(e) Polyspory
With regard to the current discussion about the evolution of polyspory among the Pezizomycotina [61] it is particularly interesting to see polysporous asci in the perithecia of Palaeopyrenomycites devonicus (up to approx. 16 ascospores per ascus; [37,38]), in the earliest yet found apothecium from the Upper Silurian (present study) and in P. taiti (present study).
The majority of extant representatives of the Pezizomycotina contain eight ascospores per ascus (octospory), these being the result of one mitosis following meiosis. However, polyspory is widespread, some taxa containing more than 100 spores per ascus. Examples are more than 500, as in Melanophloea (lichenized ascomycete incertae sedis; [62]), or more than 1000 as in Brigantiaea (Lecanorales) and Gyalidea (Ostropales; both Lecanoromycetes). Among extant Pezizomycotina polyspory occurs in Eurotiomycetes, Dothideomycetes, Lecanoromycetes, Leotiomycetes, Lichinomycetes, Orbiliomycetes, Sordariomycetes, often punctually (one or few polysporous among many octosporous species per genus), but sometimes prominent, with the majority of species per genus or even family being polysporous. Polyspory is also widespread within the Taphrinomycotina, the most basal of all subphyla of Ascomycota.
Some authors distinguish between true polyspory, when asci contain more than 100 spores, and non-true polyspory for taxa with more than eight and less than 100 spores per ascus [61]. Polyspory results from either several mitoses following meiosis (probably the most common case), from multiple budding (e.g. in various Taphrina sp., sometimes also in Neolecta spp; [53,63]), from conidium formation by ascospores within the ascus (e.g. in Rhamphoria [Sordariomycetes], Brigantiaea and Gyalidea [Lecanoromycetes]), or from breaking apart of multicellular ascospores within the ascus (e.g. various species in the genera Claussenomyces, Tympanis [Leotiomycetes] or Cordyceps [Sordariomycetes]).
Among the lichen-forming Pezizomycotina polyspory is widespread [61,62]. Few families such as the Ascosporaceae, Biatorellaceae (Lecanoromycetes) and Thelocarpaceae (incertae sedis) and at least 16 genera therein are largely polysporous, while 38 (unrelated) genera comprise only few polysporous species (representatives of Lecanoromycetes, Lichinomycetes, Eurotiomycetes). Polyspory was assumed to have evolved independently at least 57 times among extant lichen-forming ascomycetes [62]. However, the possibility of polyspory being an ancestral trait and repeatedly lost during evolution cannot be excluded. Larger sets of molecular data and distinctly more fossil records are required.
(f) The detached epihymenial layer of apothecia: a type of ‘dispersed cuticle’
In the palaeobotanical literature the term cuticle is used for (usually fragmented) surface layers of various origins: arthropods, plants or thalli of presumably lichenized fungi. Dispersed cuticles of nematophytes had so far been interpreted as the top layer of vegetative thalli [64,65]. Here we present the first examples of dispersed cuticles as a detached epihymenial layer of apothecia in permineralized Prototaxites aff. taiti from Rhynie chert material and from charcoalified Prototaxites sp. from the Lochkovian of the Welsh Borderland. Apothecia might be more common in the fossil record than previously assumed, e.g. among nematophytes with distinct palisade zone below the peripheral layer. Asci differ in their cell-wall structure, composition and function from paraphyses and vegetative hyphae (figure 10a–c) and thus might often have been lost during fossilization.
Light micrographs of iodine-stained free-hand sections of the ascoma (apothecium) of extant Peltigera spp. (lichenized ascomycetes) whose ascus wall differs structurally and chemically from ascospores, paraphyses and vegetative hyphae. (a) Detail of the hymenial layer of P. canina and (b) mature ascus with eight spindle-shaped, three-septate ascospores, both stained with Lugol's solution (IKI). (c) Detail of the hymenial layer and adjacent margin of P. venosa subjected to the van Wisselingh test for chitin (red coloration of chitosan after hydrolytic transformation of chitin). Red coloration, indicative of chitin, is discernible in the walls of vegetative hyphae of the apothecial margin, of paraphyses and of the ascospores. In both preparations, the ascus wall stains blue with iodine due to amyloid compounds (presumably lichenin-like glucans with β-1,3 and β-1,4 linkage bonds).
Looking at extant apothecia-bearing ascomycetes one should keep in mind that their vegetative mycelium is either hidden in the substrate (saprotrophs) or in a host tissue (parasites) or ensheathing plant root systems (mycorrhizae), only their ascomata being visible above ground. On suitable substrates, apothecia are locally abundant and appear in large and often colourful clusters (e.g. in the genera Sepultaria, Peziza, Helotium, Hymenoscyphus, etc., examples in [45]). Upon suitable fossilization, fragments of apothecial discs should be detectable in the fossil record. Exceptions are the lichenized taxa; these have to expose their photobiont cell population to adequate illumination and therefore differentiate their vegetative thallus at or above ground (rock, soil, bark, etc.), fossilized remains of their vegetative thallus having been found from the mid-Palaeozoic onwards [33,34], but fertile fragments remain to be found.
(g) Nutritional aspects
Assignation to the heterotrophic fungi of organisms with such enormous body mass (‘trunks’ of Saudi Arabian Prototaxites attained a length of 8.8 m and a diameter of 1.2 m, fig. 14 in [1]) raises questions on sources of nutrients which Hueber suggested were acquired via a hypothetical extensive soil mycelium exploiting decaying organic material. Our present data do not contribute substantially to our understanding of its nutritional strategies. There is no evidence for underground structures in Prototaxites, although the basal part of P. hefteri resembles the basal part of Laminaria sp. whence rhizoids derive [17]. Edwards and coworkers described putative cords or rhizomorphs of Nematosketum, closely allied to Prototaxites [66], and presumed rooting structures of Prototaxites sp. in rocks of the Early Devonian Anglo-Welsh Basin [67]; similar structures likely occur alongside sections of P. taiti (this investigation, figure 2c). The authors speculated that such organs might have translocated soluble carbohydrate produced by leakage from biofilms or their decay. A similar source has been suggested from δ13C data from Prototaxites and coeval vascular plants [68]. The former showed a considerable range in values (−15.6% to −26.6%) allowing the inference of heterotrophic nutrition involving a number of isotopically distinct substrates. The values determined by Graham et al. [29] were more consistent with those obtained experimentally from Marchantia, but no allowance was made for differences in atmospheric δ13C in Devonian times. They used such data to support their hypothesis that Prototaxites represented accumulations of concentric layers of thalloid hepatic mats, bound together by rhizoids, produced on rolling down a slope. Both isotopic data and marchantialean affinity have subsequently been questioned and concluded unlikely on sedimentological, anatomical and geochemical grounds (e.g. [30,31,68,69]).
(h) Saprotrophic, lichenised or mixed nutritional lifestyle?
Based solely on energy relationships, Selosse [70,71] had suggested a lichen-type association in Prototaxites, an affinity reinforced on very tenuous anatomical grounds by Retallack & Landing [10], but a sufficiently large and productive photobiont cell population has not yet been convincingly demonstrated. However, as in the other genera of nematophytes [33], the photobiont cell population might not have been preserved during fossilization. Almost all green algal and cyanobacterial cells in the Lower Devonian lichens Chlorolichenomycites salopensis and Cyanolichenomycites devonicus were missing, but mucilaginous cyanobacterial sheaths were well preserved [34].
When part of the Prototaxites axes were covered by a hymenial layer, as concluded from the present findings, lichenized zones might have been either interspersed with fertile areas, or restricted to either the surface proper or lateral outgrowths of a non-fertile basal part, as in podetia of cup lichens (Cladonia spp., figure 9d). Alternatively the basal cords of Prototaxites might have connected the fertile stems with the common and widespread, possibly lichenized nematophytes with dorsiventrally organized thalli, common and widespread organisms in the groundcover of terrestrial ecosystems of the Mid-Palaeozoic [33,66]. This situation, although in miniature format, is found in extant Dibaeis and Baeomyces spp. (figure 9c), but also in lichenized basidiomycetes (Agaricomycotina) with non-lichenized basidiomata (e.g. Lichenomphalia spp. [72]). However, a superficial hymenial layer and an underlying, photosynthetically active photobiont cell population are not mutually exclusive; this situation occurs in innumerable representatives of Lecanoromycetes with a lichenized, so-called thallus margin around the apothecial disc (e.g. Parmeliaceae, Xanthoriaceae), photobiont cells living not only in the lateral margin, but also underneath the hymenium, the latter allowing sufficient light transmission in the hydrated state, especially when ascospores are hyaline. If originally present below the hymenial layer, traces of green algae or cyanobacteria should have been fossilized in the Rhynie chert material of P. taiti.
It is also possible that Prototaxites had a mixed nutritional lifestyle with simultaneous saprotrophic and symbiotic acquisition of fixed carbon. The majority of extant lichen-forming ascomycetes are physiologically facultatively biotrophic and thus can be axenically cultured in the aposymbiotic state. In nature many lichenized taxa likely derive nutrients not only as soluble, mobile photosynthates from their photoautotrophic partner, but also saprotrophically, e.g. by enzymatic degradation of organic matter, such as the cellulosic mother cell walls of green algal photobionts after autospore formation or decaying plant material in the substratum. Hyphae of Icmadophila ericetorum, a lichen-forming lecanoromycete growing on rotten wood (lignin having largely been removed by white rot fungi), were shown to grow through the cell walls of woody tissues which had been partially degraded [73]; quantitative data are missing. The large axes of Prototaxites spp. certainly carried a diverse and probably at least partly beneficial microbiome which remains to be circumscribed, epi- and endolichenic bacteria and fungi having been found in the Lower Devonian Chlorolichenomycites salopensis [74].
(i) Fossil records and the calibration of the molecular clock
Prior to the advent of molecular genetic tools, the shape and ontogeny of the fruiting bodies, together with ascus types, were prime characters in ascomycete classification. However, in molecular phylogenies both character sets turned out to be homoplaseous; thus, groupings such as discomycetes (comprising apothecia-bearing taxa), pyrenomycetes (comprising perithecia-bearing taxa), etc., as found in the older literature, became obsolete [41–44]. Phylogenetic datasets indicate that ascomycetes derived from other fungal lineages in the Proterozoic, estimates ranging between 1490 and 600 Ma, depending on calibration [40,46,75–81]. Unfortunately the fruiting bodies of the majority of ascomycetes lack robust structures capable of withstanding microbial degradation and thus are very rarely found in fossil records. Well-preserved, fertile fungal fossils are urgently needed for calibrating the molecular clock. To conclude: the beautifully preserved Rhynie chert material and the charcoalified specimens from siltstone of the Welsh Borderland, as investigated in this study, give new insights into the taxonomic affiliation of the genus Prototaxites.
Acknowledgements
Our sincere thanks are due to Dr Neil Clark (Hunterian Museum, Glasgow) and to Prof. Nigel Trewin (Aberdeen University) for providing specimens for investigation. We gratefully acknowledge the technical support by Dr Tomasz Goral (confocal microscopy; NHM London) and by Peter Fisher (SEM; Cardiff University). Raymond McNeil and Knud Knudsen provided images of extant ascomycetes. We thank Marc-André Selosse and an anonymous reviewer for their comments on the manuscript.
Data accessibility
This article has no additional data.
Authors' contributions
R.H. did the light and most of the scanning electron microscopy and mycological interpretation, and wrote most of the manuscript; D.E. was initially investigating specimens of Prototaxites with intact surface layer from various slide collections, wrote part of the manuscript, provided the material and infrastructure, organized financial support and helped draft the manuscript. L.A. prepared the specimens for SEM analysis, helped with LM and SEM investigations and contributed to the manuscript. C.S.-D. did the confocal microscopy and contributed to the manuscript. All authors gave final approval for publication.
Competing interests
We declare we have no competing interests.
Funding
D.E. is grateful for financial support from the Leverthulme Trust and the Gatsby Charitable Foundation. C.S.-D. thanks the European Commission, Programme FP7-People-2011(SYMBIONTS 298735) and the Paleontological Association, UK (grant no. PA-RG201602) for financial support.
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