Morphological diversity of fungal reproductive units in the Lower Devonian Rhynie and Windyfield cherts, Scotland: a new species of the genus Windipila

Michael Krings & Carla J. Harper

PalZ Paläontologische Zeitschrift

ISSN 0031-0220

PalZ DOI 10.1007/s12542-019-00507-5

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PalZ https://doi.org/10.1007/s12542-019-00507-5

RESEARCH PAPER

Morphological diversity of fungal reproductive units in the Lower Devonian Rhynie and Windyfeld cherts, Scotland: a new species of the genus Windipila

Michael Krings1,2,3 · Carla J. Harper1,3,4

Received: 9 October 2019 / Accepted: 7 November 2019 © Paläontologische Gesellschaft 2019

Abstract The Lower Devonian Rhynie and Windyfeld cherts from Scotland contain a remarkable diversity of microscopic fungal prop- agules and reproductive units; however, only relatively few of these fossils are described. One of them is Windipila spinifera, an unusual reproductive unit from the Windyfeld chert that consists of a walled spheroid (~ 100 µm in diam.) surrounded by a mantle of interlaced hyphae; prominent spines and otherwise shaped projections, produced by these hyphae, extend out from the mantle. Here, we present W. wimmervoecksii sp. nov., also from Windyfeld, which difers from W. spinifera in that the mantle hyphae produce vesicle-like infations instead of spines. Narrow processes arising from the infations connect the persistent inner mantle tier with a probably ephemeral outer tier of irregularly infated hyphae. There is some evidence to suggest that W. wimmervoecksii was a member of the Glomeromycota; however, the precise systematic afnities remain unresolved. This discovery broadens our understanding of the patterns of mantle formation that were present in fungi by the Early Devonian.

Keywords Fossil fungi · Fungal paleodiversity · Hyphal mantle · Glomeromycota · Reproduction · Subtending hypha

Introduction important proxy indicator of fungal diversity in the Rhynie paleoecosystem is the abundance and morphological diver- The Early Devonian Rhynie chert Konservat-Lagerstätte sity of microscopic propagules and reproductive units, which (here including the Rhynie and Windyfeld cherts) in Aber- occur in virtually all thin sections of the chert (Kidston and deenshire, Scotland, has provided detailed insights into the Lang 1921; Krings et al. 2017a). However, dealing with diversity and biology of fungi in a continental paleoeco- these remains is notoriously difcult because they usu- system approximately 410 Ma old (Taylor et al. 2015). One ally occur detached from the systems on or in which they were produced, and thus do not provide a complete range of structural features necessary to determine their systematic Handling Editor: Mike Reich. afnities (Krings and Harper 2017). Only a few forms occur in characteristic confgurations or possess special features * Michael Krings [email protected] that make it possible to recognize distinctiveness and some- times even reveal afnities (e.g., Dotzler et al. 2009). Conse- 1 SNSB-Bayerische Staatssammlung für Paläontologie und quently, one important frst step in assessing fungal diversity Geologie, Richard‑Wagner‑Straße 10, 80333 Munich, in the Rhynie paleoecosystem is to report the occurrence Germany 2 of distinctive morphologies and document their features as Department für Geo‑ und Umweltwissenschaften, thoroughly as possible (Krings et al. 2017b). Paläontologie und Geobiologie, Ludwig-Maximilians-Univer sität, Richard‑Wagner‑Straße 10, 80333 Munich, Germany An ancillary covering in the form of a hyphal investment or mantle characterizes several types of Rhynie and Windy- 3 Department of Ecology and Evolutionary Biology, Natural History Museum and Biodiversity Institute, University feld chert fungal reproductive units. Mantle morphology of Kansas, Lawrence, KS 66045‑7534, USA varies considerably among the diferent types, and thus ren- 4 Botany Department, School of Natural Sciences, Trinity ders them easy to distinguish from one another (Kidston College Dublin, Dublin 2, Ireland and Lang 1921; Krings et al. 2014, 2016, 2017b; Krings

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M. Krings, C. J. Harper and Taylor 2013, 2014, 2015a, b; Krings and Harper 2017, cherts are regarded as broadly coeval, i.e. 411.5 ± 1.3 to 2018a). The most unusual of these fossils, Windipila spin- 407.6 ± 2.6 million years old according to Mark et al. (2011, ifera M. Krings et C. J. Harper, possesses a mantle of inter- 2013) and Parry et al. (2011), which is in broad agreement laced circumferential hyphae, and prominent, thin-walled with spore assemblages indicating that the cherts date from spines and otherwise shaped projections extending out from the middle Pragian to lower Emsian stages of the Lower the mantle (Krings and Harper 2017, 2018a). A similar man- Devonian according to Wellman (2006, 2017) and Wellman tle morphology occurs in Windipila pumila M. Krings et et al. (2006). For details on the geology, development, and C. J. Harper; however, this fossil is only half the size of W. paleontology of the Rhynie chert Lagerstätte, refer to Trewin spinifera, and the projections are less prominent (Krings and and Kerp (2017), Edwards et al. (2018), and Garwood et al. Harper 2018a). (2019). This paper describes Windipila wimmervoecksii sp. nov., a reproductive unit from the Windyfeld chert that corre- sponds to W. spinifera in basic organization, but difers in Materials and methods that the circumferential mantle hyphae are characterized by prominent, vesicle-like infations from which may extend The specimens used in this study were identified in a delicate, needle-like processes. Moreover, the longest of series of nine thin sections prepared from a single block of these processes connect the persistent inner mantle tier with Windyfeld chert by cementing wafers of the chert to glass a probably ephemeral outer tier of multi-branched, irregu- slides, and then grinding the rock slices until they were thin larly infated and interwoven hyphae. The new fossil con- enough to transmit light but still between 90 and 120 µm tributes to our knowledge of the morphological diversity of thick. While this may not be ideal for the analysis of plant fungi in the Early Devonian Rhynie ecosystem. tissues, it is generally advantageous for the study of fungi since thicker thin sections contain a higher percentage of complete individuals that provide information on the exact Geological setting size and shape of the organisms (Krings et al. 2010; Tay- lor et al. 2011). The block and slides are deposited in the The Rhynie chert Lagerstätte is located in the northern part SNSB-Bayerische Staatssammlung für Paläontologie und of the Rhynie outlier of the Lower Old Red Sandstone in Geologie (SNSB-BSPG) in Munich, Germany, under acces- Aberdeenshire, Scotland, within an extensive sequence of sion number SNSB-BSPG 2017 XXVI. Slides were studied sedimentary and volcanic rocks. The chert deposits occur in using normal transmitted light microscopy; digital images the Rhynie Block of the Dryden Flags Formation, located were captured with a Leica DFC-480 camera and processed northwest of the village of Rhynie. The Lagerstätte con- in Adobe Photoshop CS5. For comparison, specimens of sists of fossiliferous beds containing lacustrine shales and Windipila spinifera from the holdings of the SNSB-BSPG chert that have been interpreted as a series of ephemeral (accession number SNSB-BSPG 2016 VII) have also been freshwater pools within a geothermal wetland (Trewin and illustrated. Slide numbers for the specimens illustrated are Kerp 2017), with alkali-chloride hot springs that were part included in the fgure captions. of a complex hydrothermal system in a region afected by volcanic activity (Trewin and Fayers 2016; Channing 2017). Preserved within the chert are both aquatic (freshwater) Description facies from the pools and subaerial soil/litter horizons with in situ plants that are hypothesized to have grown along the Context margins of the pools. Preservation of the biota is thought to have taken place as a result of temporary fooding of silica- Three of the nine thin sections prepared from Windyfeld rich water, or by groundwater high in silica percolating to chert block SNSB-BSPG 2017 XXVI have yielded a total the surface (Powell et al. 2000). of 12 well preserved and > 10 incomplete and/or (partially) The Windyfeld chert occurs some 700 m to the north- degraded specimens of the fungal reproductive unit that is east of the original Rhynie chert site (Fayers 2003). It was described in the following paragraphs. Specimens occur deposited in an area of hot-spring feeder activity based on singly or in loose groupings of 2–4 individuals (arrows the hydrothermally altered nature of its associated fuvio- in Figs. 1a, c, 2a–c) within a dense accumulation of land lacustrine shales and sandstones; paleoenvironments ranged plant (likely Nothia aphylla Lyon ex El-Saadawy et Lacey) from terrestrial, vegetated aprons of laminated and brecci- axes (“lpa” in Fig. 1a) in which the xylem strand (“xy” in ated sinter to low-temperature pools and marginal aquatic Fig. 1b, c) and cuticle (“cu” in Fig. 1b) are still partially settings (Trewin and Rice 1992; Rice et al. 2002; Fayers intact and in place, while the epidermis and cortical tissues 2003; Fayers and Trewin 2004). The Rhynie and Windyfeld (“co” in Fig. 1b) are (largely) lost. The reproductive units

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Morphological diversity of fungal reproductive units in the Lower Devonian Rhynie and Windyfeld cherts, Scotland

Fig. 1 Thin section SNSB-BSPG 2017 XXVI 3. a Accumulation of (xy) and cuticle (cu) still partially intact and in place, and epidermis land plant axes (lpa) containing Windipila wimmervoecksii sp. nov. and cortical tissues (co) largely lost; scale bar 400 µm. c Detail of (arrows); subl substrate layer traversed by land plant axes; scale bar a, W. wimmervoecksii sp. nov. in plant axis, located close to vascular 5 mm; b detail of a, plant axis (transverse section) with xylem strand strand (xy), within what used to be cortical tissue; scale bar 200 µm are located in the plant axes, typically in regions of the axis contains a large spheroidal body (~ 94 µm in diameter) with that used to be cortex, and often in close proximity to vas- a wrinkled and blurred outer demarcation. Degraded speci- cular strands (white arrows in Figs. 1c, 2a, b). Co-occurring mens difer from the intact ones in that the mantle is col- with the land plant axes are various types of fungal hyphae, lapsed or partially missing (Fig. 4a, b). scattered land plant spores, unidentifable organic matter, and abundant sediment particles. The axes containing the Mantle fungal reproductive units rest upon a substrate layer that also contains degraded (and usually somewhat compressed) land Circumferential mantle hyphae (Figs. 2f, 3b, c; black plant axes (“subl” in Fig. 1a). All fossils are preserved as arrows in Fig. 3h, i) are 3–6(–6.5) μm wide and relatively three-dimensional petrifactions. thick-walled (wall thickness up to 1 µm); they are circular, oval, triangular, or dome-shaped in cross section if located Overall morphology directly adjacent to the cavity wall, and mostly circular else- where. Septa are relatively common (e.g., arrows in Figs. 2f, The reproductive units (Figs. 1c, 2a–e, g, 3a, k, l) are sphe- 3b). Emerging from the mantle are prominent projections roidal, 120–160 μm in diameter (including infations but in the form of blister-like vesicles (up to 23 µm high and excluding processes and outer mantle tier; see below), and < 10–18 µm in diameter in top view; see Figs. 2f, 3a1, j, composed of a central cavity up to 122 µm in diameter that k), which are derivatives of the circumferential hyphae is bounded on the outside by a wall 2–7 µm thick (“cw” in that develop as intercalary and terminal hyphal infations Fig. 2g). The cavity is enveloped in a prominent mantle that (Figs. 2e1,2, 3d). Projections are loosely to densely spaced, occurs in the form of a one- or two-layered (rarely three- regularly distributed over the entire surface of the reproduc- layered) system (up to 30 µm thick; “it” in Fig. 3h, i) of tive unit, and highly variable in size and shape; they are interlaced septate hyphae (arrows in Figs. 2f, 3b), which all predominantly near-spherical, prolate or oblate spheroidal extend along the circumference of the structure; moreover, or ellipsoidal, drop-shaped, or irregular, but some may also hyphal infation and subsequent formation of prominent pro- be rod- to column-like or, rarely, conical (black arrow in jections represents a major component of the mantle forma- Fig. 3a2). The conditions of the infations vary from dis- tion process. All specimens are empty or contain amorphous tended (e.g., Fig. 2e1) to somewhat faccid (e.g., Fig. 3l), matter, with the exception of the one shown in Fig. 2g that probably refecting diferent levels of turgescence of these

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Fig. 2 Windipila wimmervoecksii sp. nov.—morphology; slide f Top view of mantle, showing septate circumferential mantle hypha SNSB-BSPG 2017 XXVI 3. a, b Specimens in degraded land plant (arrows) and several vesicle-like infations; scale bar 50 µm. g Speci- cortical tissue; white arrows indicate remains of vascular tissue, men showing cavity wall (cw) and mantled subtending hypha in cross black arrows indicate subtending and associated hyphae; scale bars section (arrow); scale bar 50 µm. h1 Mantled hypha (h) that forks 100 µm. c Specimens conjoined by hyphal meshwork (arrow); scale into a subtending hypha (sh; continuation of the subtending hypha bar 100 µm. d1,2 Detail of a, holotype in two diferent focal planes; denoted “sh” in d1) and a prominent branch (h; continuation of the sh subtending hypha, h prominent hyphal branch (details of hyphal branch denoted “h” in d2); scale bar 50 µm. h2 Detail of h1, subtend- attachment in h1,2); black arrow indicates infation containing ovoid ing hypha (sh) and hyphal branch (h); scale bar 10 µm. i, j Portions inclusions; scale bars 50 µm. d3 Detail of d1, infation with inclu- of mantled hyphae; arrow in j indicates septum; scale bars 10 µm. k sions; scale bar 10 µm. e1,2 Specimen with large infations, many of Hypha surrounded by mantle with infations in cross section; scale which bearing distal processes (especially in e2); scale bars 50 µm. bar 10 µm

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Morphological diversity of fungal reproductive units in the Lower Devonian Rhynie and Windyfeld cherts, Scotland

structures at the time of fossilization. Some of the infations not always; e.g., see “h” in Fig. 2d2) enveloped in a mantle, possess a rounded tip, while others are mucronate (Fig. 2d2, which consists of tenuous, colorless hyphae that are septate ­e2, g). Still others give of one or two distally tapering pro- (arrow in Fig. 2j) and characterized by prominent, sac- or cesses (prolongations), which typically are distinctly thinner- vesicle-like infations (Fig. 2i–k). The infations are densely walled than the structure from which they arise (Figs. 2e2, spaced, up to 16 µm high, and vary in shape from sphe- 3e, f); a septum usually occurs at the base of the process roidal or bulb-shaped to irregular (in section view); they (e.g., arrow in Fig. 3f). Most processes are simple, straight, are generally smaller than the infations produced by the (slightly) bent, or kinked, needle-like structures, 2.3 µm hyphae sheathing the reproductive unit. No evidence has wide proximally and up to 14 µm long; some, however, been found of pointed tips or elongate processes in any of are considerably longer (up to 35 µm), one to several times these infations. septate, and may branch distally (Fig. 3g; white arrows in Fig. 3i). These latter processes in vivo gave rise to (or were Additional observation part of) a second, outer mantle tier (10 to > 25 µm thick) constructed of loosely interwoven, multi-branched and A few of the infations (< 10) contain single specimens or irregularly infated hyphae (“ot” in Fig. 3h, i), which were small clusters of thin-walled ovoid inclusions ~ 4.5 × 6 in probably ephemeral because the available evidence of the size (Fig. 2d3; black arrows in Figs. 2d1, 3c). outer mantle tier in these otherwise so well preserved struc- tures is exceedingly scanty and limited to small patches in two diferent specimens. Two of the reproductive units that Systematic paleomycology are located directly adjacent to one another are conjoined by a confuent, gleba-like hyphal meshwork extending from Fungi incertae sedis one mantle to the other (white arrows in Figs. 2c, 3l). The meshwork is very similar, if not identical in morphology to Genus Windipila M. Krings et C. J. Harper, 2017 (MB the outer mantle tier seen in two specimens occurring singly. 821334)

Subtending hypha Type species. Windipila spinifera M. Krings et C. J. Harper, 2017 (MB 821335) The reproductive units are positioned terminally on rela- tively thick-walled (wall thickness up to 2 µm), tubular Windipila wimmervoecksii sp. nov. hyphae, which are 4.5–12 µm in diameter beneath the sphe- Figures 1c, 2, 3, 4a, b, 5 roid (Fig. 4a; “sh” in Figs. 2d1, ­h1,2, 4b4); some are char- acterized by a conspicuous kink (black arrows in Figs. 3l; Mycobank. MB 832439 4a, ­b4). It is often difcult to trace the point of origin and length of the subtending hypha, as well as the confgura- Etymology. The epithet is proposed in honor of Messrs tion of the hyphal nexus on which the reproductive unit is Edgar Wimmer and Helmut Voecks of Munich, Germany, borne, because the subtending structures are buried within, for their continued support of our work on the Rhynie chert. and hence obscured by, the mantle (e.g., white arrow in Fig. 2d1, g; black arrows in Fig. 3l). Several of the speci- Holotype. Specimen illustrated in Fig. 2d (details of hyphal mens suggest that the subtending hypha proper (e.g., “sh” in attachment in Fig. 2h–j); in slide SNSB-BSPG 2017 XXVI Figs. 2d1, ­h1,2, 3a2) either represents a short lateral branch or 3, SNSB-Bayerische Staatssammlung für Paläontologie und outgrowth of a tubular running hypha (e.g., “h” in Figs. 2d2, Geologie, Munich, Germany. ­h1,2, 3a1–3), or gives of one prominent lateral branch imme- diately beneath (or at some distance away from) the terminal Type locality. Windyfeld chert site (National Grid Refer- spheroid. In addition, one partially degraded specimen lack- ence NJ 4977 2825), ~ 700 m NE of original Rhynie chert ing part of the mantle (Fig. 4b1–5) shows a subtending hypha site of Special Scientifc Interest, Aberdeenshire, Scotland (“sh” in Figs. 4b5, 5a) arising from the immediate vicinity (National Grid Reference NJ 494276). of an H-connection (“Hb” in Figs. 4b2, 5a) and what most likely is an anastomosis (“A” in Figs. 4b4, 5a [contact area Age. Early Devonian; early (but not earliest) Pragian to ear- enlarged in Fig. 5b]) between two hyphal segments (“h1” liest Emsian (Wellman 2006, 2017), 411.5 ± 1.3 Ma (Parry and “h2” in Figs. 4b1,3, 5a). Subtending hyphae (e.g., “sh” et al. 2011), 407.1 ± 2.2 Ma (Mark et al. 2011). in Fig. 2d1; white arrow in Fig. 2g; black arrows in Figs. 3l, 4a), as well as their associated running hyphae (e.g., black Diagnosis. Reproductive unit > 100 and < 200 µm in diam- arrows in Fig. 2a, b, h–k; “h” in Fig. 3a3), are usually (but eter (including inner mantle tier); cavity wall distinct; inner

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mantle tier up to 30 µm thick, composed of 1–2(–3) lay- reproductive unit, variable in size and shape, often with one ers of interlaced, regularly septate hyphae characterized by or two thin-walled, simple, straight or bent needle-like pro- prominent projections in the form of intercalary and termi- cesses; longest processes septate, often distally branched, nal blister- or vesicle-like infations; infations loosely to connecting inner mantle tier with probably ephemeral outer densely spaced, regularly distributed over entire surface of mantle tier (not normally preserved) of multi-branched,

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Morphological diversity of fungal reproductive units in the Lower Devonian Rhynie and Windyfeld cherts, Scotland

◂Fig. 3 Windipila wimmervoecksii sp. nov.—Morphology; slide and Tayler 2012; Krings et al. 2012, 2013b), while most do SNSB-BSPG 2017 XXVI 3 unless otherwise stated. a1–3 Specimen not permit further delimitation of their systematic position. in three focal planes, from top view to median cross section; note Moreover, the complex patterns of mantle formation that mantled hypha (“h” in a ) that forks into a short subtending hypha 1 Helmutella devonica (sh) and a prominent branch (left letter “h” in a2), the continuation occur in some of the fossil types (e.g., of which is denoted “h” in a3; black arrow in a2 shows rare conical M. Krings et T. N. Taylor [2014: fg. 4], Mycocarpon asteri- infation; scale bar 50 µm. b Circumferential mantle hypha with septa neum T. N. Taylor et J. F. White [1989: fg. 15]) are not (arrows); scale bar 10 µm. c Elongate infation with ovoid inclusion (arrow), and several circumferential mantle hyphae in cross section; known in present-day zygomycetes or Glomeromycota. scale bar 10 µm. d Detail of Fig. 2e1 (slightly diferent focal plane), circumferential mantle hypha with infations; scale bar 10 µm. e, f Comparisons and attribution Infations bearing needle-like processes; note septum (arrow) at base of process in ; scale bars 10 µm. Long, distally branched process; f g The Windyfeld chert fossils presented in this study difer scale bar 10 µm. h, i Portions of mantle in which also the outer tier is preserved; black arrows indicate mantle hyphae in cross section, in mantle morphology from all previously described fos- white arrows show long process connecting inner and outer mantle sil mantled fungal reproductive units; however, a high level tiers; ru reproductive unit, it inner mantle tier, ot outer mantle tier; of morphological congruence exists to Windipila spinifera slide SNSB-BSPG 2017 XXVI 1; scale bars 10 µm. Top view of j (Fig. 4c–g), the largest mantled reproductive unit hitherto specimen in a (magnifed), showing densely spaced infations; scale bar 10 µm. k Detail of Fig. 2c (diferent focal plane), reproductive known from the Rhynie and Windyfeld cherts (Krings and unit in top view; scale bar 50 µm. l Detail of Fig. 2c (diferent focal Harper 2017, 2018a). Several morphological features of the plane), reproductive unit in median section view, and gleba-like new fossil correspond to key traits defning the genus Win- meshwork (white arrows); black arrows indicate subtending hypha dipila W. spinifera immersed in mantle of reproductive unit; scale bar 50 µm and its type species, , including (1) the mantle of interlaced septate hyphae extending along the cir- cumference of the structure; (2) the prominent projections, irregularly infated and interwoven hyphae; clustered speci- which develop as intercalary and terminal infations and lat- mens may be embedded in confuent hyphal meshwork; eral outgrowths of the circumferential mantle hyphae and subtending hypha tubular, sometimes with a conspicuous represent the most important element in the visual appear- kink; subtending hypha and their associated running hyphae ance of the mantle; (3) narrow processes or branches arising usually (but not always) surrounded by mantle composed of from the projections; and (4) the tubular subtending hypha thin-walled septate hyphae with sac- or vesicle-like interca- sheathed (at least in part) by a mantle (Table 1). lary infations; pointed tips or elongate processes absent in The new form difers from Windipila spinifera chiefy in these infations. that the projections produced by the mantle hyphae typically occur in the form of vesicle- or blister-like infations, while they are mostly spine-like or otherwise elongate structures Discussion in W. spinifera (Fig. 4c–e). However, projections identical to the ones characteristic of the new form can, very rarely, The fossil record of mantled fungal reproductive units cur- also occur in W. spinifera (Fig. 4g; Krings and Harper 2017: rently includes seven diferent types from the Lower Devo- fg. 1E, M) and vice versa (e.g., black arrow in Fig. 3a2). nian Rhynie and Windyfeld cherts, seven or eight from the This raises the question as to whether both forms were Carboniferous of Europe and North America, and at least perhaps morphological variants of one biological species? three from the Triassic of Antarctica (surveyed in Krings Nothing is known about the range of morphological plastic- et al. 2013a, 2017a; Taylor et al. 2015). Mantled reproduc- ity of fossil fungal reproductive units. However, W. spinifera tive units resembling these fossils in varying degrees occur occurs in the chert matrix (Krings and Harper 2017, 2018a), in two extant fungal groups, namely in the form of invested/ while all specimens of the new form are located within land mantled zygosporangia (and azygosporangia) in the zygo- plant axes. It is, therefore, possible that mantle morphology mycete fungi (e.g., Bucholtz 1912; Yao et al. 1996), and in appears as a function of the surrounding in which the struc- the form of mantled chlamydospores in the Glomeromycota ture develops, and that the surrounding plant tissue perhaps (or AMF = arbuscular mycorrhizal fungi; e.g., Błaszkowski prohibited the formation of elongate spines in the new form 2012). Consequently, the vast majority of fossil types, or made them unnecessary. including Windipila (see Krings and Harper 2017, 2018a), Vesicle-like infation of circumferential mantle hyphae have also been interpreted as belonging to either of these has also been observed in two other Rhynie chert fungal groups of fungi; however, only a few (e.g., Halifaxia taylorii reproductive units, namely Zwergimyces vestitus (Kidston et M. Krings, J. F. White, Dotzler et C. J. Harper, Jimwhitea W. H. Lang) M. Krings et T. N. Taylor (Krings and Taylor circumtecta M. Krings et T. N. Taylor) have been assigned 2013: pl. I, 6) and an unnamed form that occurs in small with confdence based on the presence of structures indica- clusters in land plant tissue (Krings and Taylor 2015b: tive of gametangial fusion (White and Taylor 1989; Krings fig. 1Ia). However, these fossils are both considerably

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Fig. 4 Windipila wimmervoecksii sp. nov.—hyphal attachment; of an H-connection (Hb) between two parallel hyphae ­(h1, ­h2) and slide SNSB-BSPG 2017 XXVI 3; and W. spinifera M. Krings et C. a putative anastomosis (A); scale bars 50 µm. c–g Windipila spinif- J. Harper—morphology; slide SNSB-BSPG 2016 VII 82 unless era. c Median section view of reproductive unit, scale bar 50 µm. d otherwise stated. a, b Windipila wimmervoecksii sp. nov. a Detail Mantle hypha (arrow) and spine-like projection; scale bar 10 µm. e of Fig. 2b, showing kinked subtending hypha enveloped in color- Spine-like projection with blunt apex giving of narrow, distal process less mantle hyphae with infations; scale bar 50 µm. b1–5 Partially (arrow); scale bar 10 µm. f Mantle hypha with two septa (arrows); degraded specimen in fve diferent focal planes; reproductive unit is scale bar 10 µm. g Mantle hyphae (section views) and vesicle-like positioned terminally on kinked subtending hypha (sh), which arises infation; slide SNSB-BSPG 2016 VII 117; scale bar 10 µm from a hyphal nexus (graphic presentation in Fig. 5a, b) composed smaller, and the infations in the unnamed form are exclu- indicative of interspecifc variability, and hence a diagnostic sively terminal. Moreover, the infations occur scattered structural diference, or is due to the ephemeral nature, and and do not dominate the visual appearance of the mantle. thus a low preservation potential, of the peripheral mantle Finally, there is no evidence that narrow processes extended portions. For example, the outer mantle tier and confuent from any of the infations. On the other hand, Z. vestitus is hyphal meshwork were perhaps components of the juvenile the only fossil mantled fungal reproductive unit besides the or developing reproductive unit and normally disintegrated new form and W. spinifera in which the subtending hypha upon maturation. Support for the latter conjecture is the fact is sometimes also sheathed by a mantle (Krings et al. 2016: that ephemeral outer mantle tiers and confuent, gleba-like pl. I, 3a). hyphal meshworks are occasionally present also in several Several specimens of the new form provide evidence other fossil mantled fungal reproductive units, including of the presence in vivo of a second, outer mantle tier of Zwergimyces vestitus from the Rhynie chert (Krings et al. interwoven and irregularly swollen hyphae (Fig. 3h, i) or 2016: pl. II, 1), the Carboniferous Mycocarpon pachyderma a hyphal meshwork extending around and between adja- (Williamson) S. A. Hutchinson (McLean 1922: pl. IX, 12) cent individuals (Fig. 3l). Conversely, no evidence of addi- and M. fexus B. H. Davis et Leisman (Stubblefeld et al. tional, peripheral mantle layers or hyphal meshworks has 1983: fg. 1), as well as an unnamed form, also from the been found in Windipila spinifera (Krings and Harper 2017, Carboniferous (Krings and Taylor 2012: pl. I, 7–10). An 2018a). It is impossible to determine whether the absence outer mantle layer roughly similar to the outer mantle tier of evidence of these mantle components in W. spinifera is of the new Windyfeld form occurs in the Mississippian

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Morphological diversity of fungal reproductive units in the Lower Devonian Rhynie and Windyfeld cherts, Scotland

Fig. 5 Windipila wimmervoeck- sii sp. nov.—reconstructions. a Graphic presentation of hyphal nexus fgured in several focal planes in Fig. 4b1–5 (for explanation of abbreviations, see Fig. 4b). b Close-up of puta- tive anastomosis (denoted “A” in a). c Synoptic reconstruction of reproductive unit, showing central cavity (ca), cavity wall (cw), and hyphal mantle com- posed of persistent inner (it) and portion of probably ephemeral outer (ot) tiers in paramedian section view, as well as kinked subtending hypha and portion of mycelium composed of hyphae partially enveloped in mantle (based on specimens fgured in Figs. 2, 3, and 4a, b)

reproductive unit Roannaisia bivitilis T. N. Taylor, Gal- component of the zygomycete life cycle that can be used to tier et Axsmith (Taylor et al. 1994: pl. II, 2). However, this positively identify a fossil member in this group of fungi form is up to 600 µm in diameter and characterized by what (Krings et al. 2013a). Windipila wimmervoecksii cannot appears to be a non-hyphal outer covering, which has not be positively identifed as a zygomycete because there is been observed in any other mantled fungal reproductive unit. no evidence of gametangia and/or suspensors. Rather, the Moreover, the outer mantle tier in R. bivitilis is believed to occurrence terminally on a simple, tubular hypha or hyphal be persistent, not ephemeral. branch suggests that this reproductive unit, precisely as W. Based on the comparisons and refections presented in spinifera (Table 1; see Krings and Harper 2017), formed the preceding paragraphs, we assign the new Windyfeld asexually by blastic infation and thickening of a hyphal tip, chert fossils to the genus Windipila as defned by Krings rather than sexually and following gametangial fusion, and and Harper (2017), and interpret them as a distinct species, for this reason might represent a glomoid chlamydospore. for which the name W. wimmervoecksii sp. nov. is proposed, One of the partially degraded specimens of Windipila albeit with the understanding that they might represent the wimmervoecksii provides rare insights into the organiza- same biological species as W. spinifera, but which is impos- tion of the parental mycelium, which may also be relevant sible to determine from the material at hand. to the assessment of the systematic afnities. This fossil, fgured in several focal planes in Fig. 4b, is physically con- Systematic afnities nected via a kinked subtending hypha (“sh” in Figs. 4b5, 5a) to a small hyphal nexus (graphic presentation in Fig. 5a, Like most other fossil mantled fungal reproductive units, b) composed of an H-connection (“Hb” in Figs. 4b2, 5a) Windipila wimmervoecksii was probably produced by a resulting in two parallel hyphae (“h1” and “h2” in Figs. 4b1,3, member of the zygomycete fungi or Glomeromycota (see 5a), and a hyphal bridge that most likely represents an anas- Taylor et al. 2015). One of the key features distinguishing tomosis extending from one of these hyphae to the other these two groups of fungi is the sexual stage of the life cycle, (“A” in Figs. 4b4, 5a; details in Fig. 5b). H-connections (or which occurs in zygomycetes as a result of zygosporogenesis H-branching) are typical of the mycelium in Glomeromy- following gametangial fusion, but has not been observed cota, and hyphal anastomoses have been reported frequently to date in Glomeromycota (Benjamin 1979; Benny et al. in several glomeromycotan lineages (Walker et al. 2018). 2001; Benny 2012). Mature zygosporangia or zygospores Conversely, anastomoses are believed to be lacking or rare with attached gametangia and/or suspensors (for structural in zygomycete fungi (Gregory 1984; Glass and Fleissner nomenclature, see Edelmann and Klomparens 1995: fg. 1; 2006; Ivarsson et al. 2015). Hyphal anastomoses play key Krings et al. 2012: fg. 3) are, therefore, the most important roles in the formation of efcient, interconnected AMF

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M. Krings, C. J. Harper 2018a ) This study Krings and Harper ( 2018a ) Krings and Harper ( 2017 , Reference(s) wide, often with a kink; by enveloped usually with prominent hyphae infations - wide; distal portion envel hyphae narrow oped by Tubular hyphae up to 12 µm up to hyphae Tubular Unknown Tubular hypha up to 8 µm up to hypha Tubular Hyphal attachment - hyphae with prominent hyphae intercalary and terminal may which infations from - pro narrow extend distally cesses connecting the per inner mantle tier with sistent outer mantle an ephemeral preserved) normally tier (not infated and of irregularly hyphae interwoven hyphae giving of outwardly giving of outwardly hyphae spine-like usually directed, projections - giving of promi hyphae directed nent, outwardly spine-like and usually may which from projections extend or laterally distally or hyphal processes narrow branches Interlaced circumferential circumferential Interlaced Interlaced circumferential circumferential Interlaced Interlaced circumferential circumferential Interlaced Mantle organization 25 µm (outer tier) 30 µm (inner tier); > 10 µm 50 µm Maximum mantle thickness Maximum mantle thickness and (including projections infations) of interior sphere 3.5 µm) 7 µm < 1 µm (wall 3.5 µm Maximum of thickness wall cavity 122 µm 48.8 µm 105 µm Maximum diam - cavity eter Windyfeld Rhynie Windyfeld Provenance Windipila units of the genus reproductive mantled fungal Devonian of Early characters Synopsis of key mervoecksii sp. nov. Windipila wim - Windipila Windipila pumila Windipila Windipila spinifera Windipila Taxon 1 Table

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Morphological diversity of fungal reproductive units in the Lower Devonian Rhynie and Windyfeld cherts, Scotland mycorrhizal networks, which are fundamental to AMF sur- Provided that the Windipila wimmervoecksii hyphal man- vival, to plant/soil nutrient fow, and to the maintenance of tle remained (partly) in place even upon maturation of the genetic diversity (e.g., Chagnon 2014; Giovannetti et al. propagule (= spore) and liberation of the reproductive unit 1999, 2015; de Novais et al. 2013, 2017). In addition, anas- from its host plant, it is also conceivable that this structure tomoses between extraradical mycelia originating from the accommodated certain microorganisms (Krings et al. 2016) root systems of diferent host species allow for the formation which were benefcial to the development of the propagule, of wide hyphal networks interconnecting an indefnite num- in some way stimulated propagule germination (for similar ber of plants living in the same ecosystem (de Novais et al. relationships in modern Glomeromycota, see Roesti et al. 2017). Thus, the hyphal nexus co-occurring with one of the 2005; Bonfante and Desirò 2017), or promoted mycorrhizal W. wimmervoecksii specimens supports the hypothesis that activity and optimized plant performance (see Frey-Klett this type of reproductive unit was produced by a member of et al. 2007; Agnolucci et al. 2015, 2019). Support for this the Glomeromycota. hypothesis is molecular evidence showing that the symbio- The third species in Windipila, W. pumila, is based on a sis between AMF and certain endobacteria dates back at single specimen lacking evidence of the subtending structure least to the time when AMF formed the ancestral symbio- (Krings and Harper 2018a: fg. 2); it difers from the other sis with early land plants (Naumann et al. 2015; Naito and species not only in regard to size, but also in that it con- Pawlowska 2016). Moreover, dissemination of propagules tains a single interior sphere (Table 1). The cavity wall in W. together with their benefcial microorganisms (“piggyback pumila has been suggested to represent a sporangium wall, transportation”) might have been advantageous because perhaps comparable to the zygosporangium (or azygospo- this strategy ensured the presence of suitable microbiota rangum) wall in extant zygomycetes or the outmost layer(s) in the place of germination. One could even go so far as of glomeromycotan chlamydospore walls, while the interior to speculate that these complex mantled fungal reproduc- sphere would accordingly be a zygospore (or azygospore) or tive units were “seed-like” in function and served to vec- the chlamydospore wall proper (Krings and Harper 2018a). tor the fungus around in the environment. Many modern The spheroidal structure that occurs in the cavity of one of seeds host bacteria and fungi on their surfaces and interi- the W. wimmervoecksii specimens (Fig. 2g) is more irregu- ors that the seedling will need to establish itself in a new lar in outline, and its outer boundary is blurred, and thus site, procure nutrients, and ward of disease agents (e.g., probably represents a preservation artifact resulting from Verma et al. 2017; Shahzad et al. 2018; Verma and White water loss and condensation of the cavity contents during 2018; White et al. 2019). Some of the hyphal infations in fossilization. W. wimmervoecksii could have been containers for nutrients that fed the surface-vectored bacterial community. On the Mantle function other hand, the chance of reaching a suitable microhabitat for germination is an important parameter determining the Whether the hyphal mantles in Windipila wimmervoecksii likelihood for fungal dispersal units to establish new repro- and other fossil fungal reproductive units served a specifc ductive individuals (Calhim et al. 2018). It is, therefore, also biological function is difcult to answer, due in part to a possible that hyphal mantles somehow facilitated arrival of lack of knowledge of the functions of hyphal mantles in the reproductive units on a specifc substrate. For example, extant zygomycete fungi and Glomeromycota. Krings and it has been shown that fungal spore surface structures in Harper (2017) suggested that hyphal mantles were perhaps certain zygomycetes and higher fungi dictate where in the efective as modulating interfaces or protective shields. soil column they are preferentially deposited (Calhim et al. For example, the mantle of W. wimmervoecksii might have 2018: fg. 2; Hemkemeyer et al. 2019). developed early and pushed the surrounding cells of the host plant cortex to the side to make space for the expanding reproductive unit. Another scenario views the mantle as a Conclusions barrier against mycoparasitic fungi, which were superabun- dant in the Rhynie paleoecosystem (Hass et al. 1994; Krings The many diferent fungal propagules and reproductive and Harper 2018b); the living hyphae could actively have units that are preserved in the Early Devonian Rhynie and degraded invaders that penetrated into the mantle. As a mat- Windyfeld cherts have recently begun to receive attention, ter of fact, none of the W. wimmervoecksii specimens show and their morphology and distribution are now being more symptoms of fungal colonization of the reproductive unit, systematically documented (e.g., Krings et al. 2017b; Krings and evidence of intruders in the mantle is exceedingly rare and Harper 2017, 2018a). These accounts represent valuable (Figs. 2d3, 3c). It has also been speculated that surface struc- resources that can be used to assess paleodiversity and biotic tures protect reproductive units and propagules from UV-B complexity, and thus contribute to our ability to ultimately radiation and can prevent desiccation (Norros et al. 2015). understand some of the biological processes that made the

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Rhynie paleoecosystem. Windipila wimmervoecksii repre- Calhim, S., P. Halme, J.H. Petersen, T. Læssøe, C. Bässler, and J. Heil- sents the most recent example of the growing number of mann-Clausen. 2018. Fungal spore diversity refects substrate- specifc deposition challenges. Scientifc Reports 8: 5356. mantled fungal reproductive units from the Rhynie and Chagnon, P.L. 2014. Ecological and evolutionary implications of Windyfeld cherts that lead to speculation as to their sys- hyphal anastomosis in arbuscular mycorrhizal fungi. FEMS tematic afnities and biological signifcance. Within these Microbial Ecology 88: 437–444. fossils there are basic similarities in size and organization Channing, A. 2017. A review of active hot-spring analogues of Rhynie: environments, habitats and ecosystems. Philosophical Transac- that suggest most may belong to the zygomycete fungi or tions of the Royal Society London (B: Biological Sciences) 373: Glomeromycota. Like so many aspects of paleomycology, 20160489. one specimen often is the single necessary segment of infor- Dotzler, N., C. Walker, M. Krings, H. Hass, H. Kerp, T.N. Taylor, and mation that helps to clarify the nature and afnities that have R. Agerer. 2009. Acaulosporoid glomeromycotan spores with a germination shield from the 400-million-year-old Rhynie chert. remained elusive (Taylor et al. 2011). This may well also Mycological Progress 8: 9–18. be the trajectory regarding W. wimmervoecksii as we con- Edelmann, R.E., and K.L. Klomparens. 1995. Zygosporogenesis in tinue to screen the Rhynie and Windyfeld cherts for fungal Zygorhynchus heterogamus, with proposal for standardization of Mycologia remains, and new specimens are reported and studied. We structural nomenclature. 87: 304–318. W. wimmervoecksii Edwards, D., L. Dolan, and P. Kenrick (eds.). 2018. The Rhynie cherts: hope that and other recent discoveries our earliest terrestrial ecosystem revisited. Philosophical Trans- of fossil fungi provide a stimulus for a wider appreciation actions of the Royal Society of London (B: Biological Sciences) of the paleodiversity of these organisms and increase the 373: 1–201. The biota and palaeoenvironements of the Windy- interest in questions pertaining to the roles they played in Fayers, S.R. 2003. feld Chert, Early Devonian, Rhynie, Scotland, 1–549. PhD thesis, ancient ecosystems. University of Aberdeen, Aberdeen, Scotland. Fayers, S.R., and N.H. Trewin. 2004. A review of the palaeoenviron- Acknowledgements We thank E. Lange and H. Martin (both Munich, ments and biota of the Windyfeld chert. Transactions of the Royal Germany) for technical assistance, and James F. White Jr. (New Brun- Society of Edinburgh, Earth Sciences 94: 325–339. swick, NJ, USA) and an anonymous referee for insightful comments Frey-Klett, P., J. Garbaye, and M. Tarkka. 2007. The mycorrhiza helper that improved the manuscript. bacteria revisited. 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