Results

3.4.1.3.1 Description of the fossil ectomycorrhizas

Approximately 20 unramified, cruciform and monopodialpinnate ectomycorrhizas are fossilized adjacent to rootlets of up to 180 μm in diameter (Fig. 14a–e). The nonmycorrhizal parts of the absorbing roots are 300 μm to 8 mm in length and 60–130 μm in diameter. Unbranched mycorrhizas are 320 μm to 1.9 mm long and 90–140 μm in diameter (Fig. 14b). Cruciform ectomycorrhizal systems (Fig. 14c) are 200–310 μm (rarely up to 700 μm) long, and their two branches are 120–220 μm (rarely 500 μm) long and 70–100 μm in diameter. Monopodial-pinnate ectomycorrhizal systems (Fig. 14a,d,e) mostly

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range between 350 and 550 μm in length, and their finger-like branches are 100–300 μm (rarely up to 530 μm) long and 60–90 μm wide. The monopodial-pinnate system in amber fragment TAD 248b (Fig. 14e) is 1.3 mm in length and bears five finger-like branches, in which both of the most basal branches are bifurcated.

Figure 14: Light-microscopic photomicrographs of the ectomycorrhizal systems of Eomelanomyces cenococcoides gen. et spec. nov. from Eocene amber of India. (a) Overview of a rootlet with three monopodial-pinnate ectomycorrhizal systems and one cruciform system (located on the right) with surrounding mycelium (TAD 248a). The system in the middle right represents the holotype (shown in greater detail in Fig. 14(d)). (b) An unramified ectomycorrhiza which has been later removed from the amber for ultrastructural and Raman analyses (see Figs. 15 and 16; TAD 248a). (c) Cruciform ectomycorrhizal system (TAD 248a). (d) Monopodial-pinnate ectomycorrhizal system forming microsclerotia. This system represents the holotype of E. cenococcoides gen. et spec. nov. (TAD 248a). (e) Large monopodialpinnate ectomycorrhizal system with five finger-like branches forming microsclerotia. The arrowheads show additional furcation of the basal branches (TAD 248b). (f) Microsclerotia formed at the surface of the basal branch of the system shown in the lower left of Fig. 14(a) (TAD 248a). (g) Large microsclerotium exposed at the surface of the system shown in Fig. 14(e) (TAD 248b). (h) Dark hypha with two septa (arrowheads) extending from the system shown in Fig. 14(b) (TAD 248a). Bars, 500 μm (a); 100 μm (b–e); 20 μm (f–h).

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Different developmental stages of the ectomycorrhizas are preserved in the piece of amber. Young ectomycorrhizas show dark pseudoparenchymatous mantles from which numerous irregularly septate dark pigmented hyphae of 1.2– 3.3 μm in diameter extend (Figs. 14b,c,h, 15a–c,). Their walls are 0.2–0.3 μm thick, and iris diaphragms are possessed at the septa (Fig. 15g) whereas clamp connections are absent. Compounds of melanins were detectable in these dark hyphae using Raman spectroscopy. Some of the peaks were assignable to the key monomers of eumelanin: hydroquinone, indolequinone and semiquinone (Fig. 16). Some of these hyphae form chlamydospore-like inflated distal hyphal ends which are clavate or broad fusiform to lemon-shaped and 12–16 μm long and 6.5–9.7 μm wide (Fig. 15c,d). Short forked flat hyphae 7–15 μm long, 2.4–5 μm wide and c. 0.5 μm thick (Fig. 2e), as well as short young hyphae 2.5–3 μm in diameter, are sometimes visible at the surface of the pseudoparenchymatous mantle. Dense hyphal systems extend in all directions into the clear translucent amber (Fig. 14a,c), suggesting that some ectomycorrhizas were still alive when initially embedded. Sometimes several hyphae form simple rhizomorphs that are mostly c. 10 μm in diameter or thinner, seldom reaching 75 μm (Fig. 17a,b). Generally, hyphae exhibit thick cell walls (Fig. 15f,g) and are frequently coated by a tolueneinsoluble substance.

Single hyphae within the rhizomorphs are 1–3 μm wide. The dark hyphae of the mycelium are often coated by light circular structures possessing a rough surface. Hyphae are absent around older ectomycorrhizal systems; instead, numerous spherical to ovoid microsclerotia (hardened mycelia serving as dormant stages) are formed at their surface (Fig. 14d–g). The microsclerotia are mostly 35–40 μm long and 25–30 μm wide, sometimes reaching 55–60 μm in length and 50 μm in width. Small ones are only 15–20 μm in size. Microsclerotia are also formed in the nearby hyphal systems (Fig. 17d,). Clavate short hyphal ends 15–43 μm long and 5.5–6.5 μm wide are regularly formed in the mycelium (Fig. 17c). Sometimes they appear at regular distances of c. 450–550 μm apart at the supporting hyphae. The otherwise thick walls of the hyphae become thinner and almost disappear in these branches (Fig. 17c).

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Figure 15: Scanning electron micrographs of Eomelanomyces cenococcoides gen. et spec.

nov. from Eocene amber of India (TAD 248a). (a,b) Fragments of the pseudoparenchymatous mantle of the ectomycorrhiza shown in Fig. 14(b). (c) Surface of the pseudoparenchymatous mantle showing extending hyphae with initial formation of chlamydospore-like inflated distal hyphal ends. (d) Lemon-shaped inflated distal hyphal end of a short hypha extending from the mantle. (e) Surface of the pseudoparenchymatous mantle with short forked flat hyphae. (f) Hypha showing thick walls at cross-break. (g) Broken hypha exposing a septum with an iris diaphragm. Bars, 20 μm (a,b); 5 μm (c–e); 1 μm (f,g).

Preservation of the ectomycorrhizas is excellent, allowing description of the mycobiont as Eomelanomyces cenococcoides gen. et spec. nov. (see the next section, ‘Taxonomic summary’). E. cenococcoides is a fungus containing melanin and developing ectomycorrhizas as black pseudoparenchymatous mantles on the surface of absorbing roots of the host. Hyphae with iris diaphragms at the septa extend outward from this mantle. In this regard, the

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fossil is similar to the recent anamorphic genus Cenococcum, but distinguished by the high variability in the branching of the ectomycorrhizal systems and by the regular formation of microsclerotia.

3.4.1.3.2 Taxonomic summary

Eomelanomyces cenococcoides Beimforde, Dörfelt et A. R. Schmidt gen. et spec. nov. (Figs. 14, 15 and 17).

Descriptio: Fungus anamorphus cum substantia ‘melanin’ et ectomycorrhizam formans in plantis. Systema mycorrhizas non ramosa vel cruciformis aut monopodialiter pinnata. Rami frequenter situ in dextero angulo. Tunica mycorrhizae in superficie est pseudoparenchymatica cum cellulis planis, 60–

140 μm in diametro, frequenter cum hyphis ramosis, coloratis, non regularibus septis, 1.2–3.3 μm in diametro. Ex tunica pseudoparenchymaticae hyphae eminentes cum septis. Septa cum simplicibus centralibus cavis ut in genere recentem Cenococcum. Nonnullae hyphae apices formantes ad similitudinem chlamydosporibus, usque ad 8 x 5 μm in diametro. Hyphae conjunctae in chordam myceliae ut in simplicibus rhizomorphis. In aetate mycorrhizae sine vividis ramosis hyphis eminentibus autem cum multis microsclerotiis ovoideis, c.

35–50 x 25–35 μm in diametro.

Typus: In resina fossile ex India, collectio numerous AMNH TAD 248; Systema ectomycorrhizae est spectata in Fig. 14(d) est holotypus.

Etymologia: Eo, eos: Eocaen; melanos: nigrum. Epitheton speciei propter similitudinem cum recenti genere anamorpho Cenococcum.