The first fossil record of the anamorphic genus Zygosporium Mont. from the Oligocene of Csolnok (N Hungary)

Remains of a fungus with unique morphological characters were found on the leaf cuticle of a fossil leaf preserved in Oligocene deposits from Csolnok, Hungary. Vesicular conidiophores with characteristic, darkly pigmented, incurved vesicles were compared with those of the modern representatives of the anamorphic genus Zygosporium. Based on the fossil find, a new fossil-species, Zygosporium oligocenicum G. Worobiec sp. nov., having vesicular conidiophores that arise directly from the mycelium, was described. The fossil Zygosporium oligocenicum presumably preferred warm climate and, similarly to most modern members of the genus, was a saprophyte on fallen, decaying leaves.


Introduction
Fungi is one of the kingdoms of eukaryotic organisms that presumably appeared in the Neoproterozoic (Loron et al. 2019;Bonneville et al. 2020). Their species richness is estimated between 2 and 4 million species (Hawksworth and Lücking 2017). There are nine major lineages (phyla) of fungi including Opisthosporidia, Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Zoopagomycota, Mucoromycota, Glomeromycota, Ascomycota, and Basidiomycota (Naranjo-Ortiz and Gabaldón 2019). They reproduce both sexually and asexually and, as a result, have different reproductive stages (morphs). In the phyla Ascomycota and Basidiomycota, the sexual morphs are named teleomorphs, and asexual morphs that mitotically sporulate, are classified as anamorphs or conidial stage (previously called Fungi imperfecti or Deuteromycetes). They are divided into the artificial groups Hyphomycetes and Coelomycetes (Seifert et al. 2011). Most of the anamorphic fungi are asexual stages of Ascomycota and rarely of Basidiomycota and comprise about 25 000 modern species (Kirk et al. 2008). Nowadays, fungi are classified not only on the basis of their morphology and life cycle, but also based on their phylogenies inferred using DNA sequence analysis (Spatafora et al. 2017;Voigt et al. 2021;Wijayawardene et al. 2022). In the fossil state, however, only morphological characters are available to researchers and their analysis is the key to assess their taxonomy and to reconstruct the evolutionary history of fungi (Le Renard et al. 2020). Fossil forms of anamorphic fungi have repeatedly been recorded (Kalgutkar and Jansonius 2000;Taylor et al. 2015;Worobiec et al. 2022). One of the most common fossil fungal anamorphs is the sporodochium of the epiphyllous Neomycoleptodiscus pertusus (Dilcher) G. Worobiec (Worobiec et al. 2020), but on the other hand, epiphyllous hyphomyces are very rarely described in the fossil state. For this reason, the survey of new fossil forms of epiphyllous anamorphic fungi seems to be of great importance for understanding the fossil history of this group of fungi.
During a survey of the cuticles isolated from the fossil leaf compressions preserved in Oligocene deposits from Csolnok, Hungary, Erdei and Wilde (1998) recognized abundant fungal colonization on the surface of leaf cuticles. Afterwards, Worobiec et al. (2020) identified the sporodochia of Neomycoleptodiscus pertusus (Dilcher) G. Worobiec among the epiphyllous fungi from Csolnok. Another mitosporic fungus, so far not reported in the fossil record, was found on leaf Section Editor: Roland Kirschner * Grzegorz Worobiec g.worobiec@botany.pl cuticles and based on its unique morphological characters (vesicular conidiophores with characteristic, darkly pigmented, incurved vesicles) was considered to be morphologically identical to modern representatives of the genus Zygosporium Mont. A new fossil-species, Zygosporium oligocenicum G. Worobiec sp. nov., was described, and it was compared with the present-day species of Zygosporium. The modern distribution and ecology of the genus Zygosporium and the assumed palaeoecology of the fossilspecies, Zygosporium oligocenicum, are discussed.

Geological setting
The investigated remains of Zygosporium were found on leaf cuticles that were isolated from fossil leaf compressions unearthed from Oligocene clayey sediments of Csolnok, Hungary. Leaf specimens were recovered from a mine shaft near Csolnok, N Hungary (47°41' 37" N 18°42' 18''E) during the 1950s. The settlement is located in the Dorog Basin, where brown coals of Eocene and Oligocene age had long been excavated (Siposs 1968). The stratigraphic sequence of the Csolnok region is composed of Triassic, Paleogene, and Pleistocene strata. Triassic limestones are unconformably overlain by Eocene clays, freshwater limestones, and brown coal. Overlying Oligocene strata are represented by both terrestrial and marine sediments like clay, brown coal, and sandstone. The top of the sequence is formed by Pleistocene loess (Siposs 1968;Nagy-Gellai 1973). The exact stratigraphic position of the fossils, however, was not documented (see Erdei and Wilde 2004). Based on the mollusc and microfossil content of the sediments, the fossiliferous layers were deposited during the Oligocene. The overall character of the plant assemblage also corroborates an Oligocene age of the sediments (Erdei and Wilde 2004).

Material and methods
Fungal remains in the Csolnok assemblage were first reported by Erdei and Wilde (1998 Diagnosis. Setae or setae-like hyphae structures sterile, becoming thinner towards the apex. Vesicular conidiophores in small to larger groups, with the cylindrical stalk cell arising probably directly from the mycelium and with dark brown, thick-walled, curved and swollen vesicles. Description. Preserved vesicular conidiophores and remains of hyphae and setae or setae-like structures on the surface of leaf epidermis. Setae or setae-like hyphae structures sterile, up to 55 μm long and 2.5-5.0 μm wide near the base, becoming thinner towards the apex. Vesicular conidiophores in small to larger groups (up to several dozens of conidiophores), with the cylindrical stalk cell arising directly from the mycelium and with dark brown, thick-walled, curved and swollen vesicles. Stalk cell up to 12 μm long and about 2-3 μm wide. Vesicles 12-15 μm long and 8-10 μm wide. Conidiogenous cells and conidia not preserved.

Generic classification
The vesicular conidiophores with characteristic, darkly pigmented, incurved vesicles are identical to the representatives of the modern anamorphic genus Zygosporium Mont. The genus has a set of unique morphological characters, i.e., dark, incurved vesicular cells from which the ampulliform conidiogenous cells are formed, which in turn produce aseptate, globose or ellipsoid, smooth or ornamented conidia (Hughes 1951;Whitton et al. 2012;Li et al. 2017). No other modern or fossil fungal genera have similar vesicular conidiophores. Contrary to many modern Zygosporium species, the vesicles of which are borne from the side of a setiform conidiophore (Whitton et al. 2012), the stalk cells of the vesicles of the Zygosporium from Csolnok arise presumably directly from the mycelium and surely not from the accompanying setae or setae-like structures. Thus, these setae or setae-like hyphae structures are similar to "true setae" observed in some modern members of Zygosporium (Whitton et al. 2003(Whitton et al. , 2012. In the case of the Zygosporium from Csolnok, conidiogenous cells and conidia are not preserved. In present-day Zygosporium species, conidiogenous cells are usually hyaline and delicate and thus they presumably have low fossilization potential. Due to the absence of the conidiogenous cells and conidia, a detailed comparison of the fossil Zygosporium with the modern representatives of the genus is not possible. Therefore, the fossil hypomycete from Csolnok has been described as a new fossil-species, Zygosporium oligocenicum. Fossil forms of the anamorphic genus Zygosporium have not been recorded so far. Fungal structures, almost identical to the conidiophores of Zygosporium, were reported from the late Neogene lignites of the Sindhudurg Formation of Maharashtra, west coast of India, as Cirrenalia pygmea Kohlmeyer (Kumaran et al. 2004, fig. 2: M). Contrary to the remains from Csolnok,  (Whitton et al. 2003(Whitton et al. , 2012Li et al. 2017;MycoBank 2022 (Crous et al. 2020). Zygosporium is characterised by a single, darkly pigmented, incurved, and swollen vesicles that may be stalked or sessile, borne from the side of a setiform conidiophore, or may arise directly from the mycelium (vesicular conidiophores). Ampulliform conidiogenous cells produce aseptate, usually ellipsoid or globose, smooth or variously ornamented conidia (Mason 1941;Hughes 1951;Whitton et al. 2003Whitton et al. , 2012Li et al. 2017 (Hughes 1951;Ellis 1971Ellis , 1976Pirozynski 1972;Thakur and Udipi 1976;Subramanian and Bhat 1987;Whitton et al. 2003Whitton et al. , 2012Manoharachary et al. 2006;McKenzie et al. 2007;Dubey 2014;Lucena and Fernández-Valencia 2017;Crous et al. 2018Crous et al. , 2019Khalkho et al. 2020). Among the listed taxa only Zygosporium deightonii has setae and vesicular conidiophores that arise directly from the mycelium (Ellis 1976). The same set of features characterizes the fossil-species Zygosporium oligocenicum. Therefore, one might suppose that the fossil Z. oligocenicum could be closely related to Z. deightonii. The modern species, however, differs from Z. oligocenicum in the dimension of the setae or setae-like structures, which in Z. deightonii is twice as long (60-130 μm) (Ellis 1976) as in Z. oligocenicum (up to 55 μm long). Moreover, Z. oligocenicum lacks conidiogenous cells and conidia aiding a proper comparison. Since the close comparison of the two taxa could not be performed, the relation of the fossil-species, Z. oligocenicum, and the modern species, Z. deightonii, could not be confirmed.
Representatives of Zygosporium are predominantly saprotrophs, found usually on dead plant parts, such as leaves, twigs, decaying wood of mainly angiosperms, but also on some gymnosperms and ferns (Ellis 1971;Whitton et al. 2003Whitton et al. , 2012Li et al. 2017). Sometimes, fungi belonging to the genus Zygosporium are found as elements of the mycobiota of soils (Majumder and Shukla 2008;Yabuki et al. 2014). Exceptionally, Z. masonii was also reported from the dry-walls or wall-papers of indoor environments in the USA (Li and Yang 2004) and from biofilms developing on stone chambers from Japan (Sugiyama et al. 2009). An unidentified species of Zygosporium was also found as partly responsible for the bio-deterioration of archival photographic materials (Bučková et al. 2014). These records infer Zygosporium as a saprobe on organic material. Dreyfuss and Petrini (1984) found Zygosporium cf. echinosporum as a root endophyte of Kalanchoe sp. (Crassulaceae). Some species of Zygosporium, however, were reported as biotrophic growing as parasites on living plants. Pirozynski (1972) found Z. majus as causing brown leaf spots. Z. gibbum was reported as an agent causing leaf spots on bean from the Maldives (Hunter and Shafia 2000) and on Mangifera indica L. from Pakistan (Abbas et al. 2011). The same species was found to be fungicolous on rust Puccinia oxalidis Dietel & Ellis (Carreón and Romero 1999), rust Coleosporium plumeriae Pat. (Manimohan and Mannethody 2011), and on Cladosporium cladosporioides (Fresen.) G.A. de Vries, Alternaria sp. and Papulospora sp. developing on decaying oak leaves (Gupta and Upadhyaya 2015). Khalkho et al. (2020) reported Z. chinensis as infecting both alive and decaying dead leaves of Litchi chinensis Sonn. Crous et al. (2019) found Z. pseudomasonii as a cause of leaf spots on the palm, Serenoa repens (W. Bartram) Small, and Dubey (2014) reported Z. dilleniae occurring on living leaves of Dillenia pentagyna Roxb.
Species of Zygosporium inhabit mostly terrestrial habitats, but some species have been reported from aquatic environment, both in freshwater (Barbosa and Gusmão 2011;Fiuza et al. 2019;Carvalho and Pereira 2021) and in the coastal marine environment of mangroves (Devadatha et al. 2021) and sabkha (Mahasneh et al. 2006), probably as facultative aquatic hyphomycetes.

The tentative palaeoecology of the fossil-species Zygosporium oligocenicum
Although modern representatives of the genus Zygosporium inhabit tropical to temperate regions, they seem to be more common in the tropics than in temperate areas (McKenzie et al. 2007). It is especially true considering the European range of Zygosporium, namely, the highest species diversity is recorded in areas with warm, Mediterranean climate (Rambelli 2011, see above). The climatic requirements of the fossil-species, Zygosporium oligocenicum, however, should be reconstructed with caution, considering the fact that its modern counterpart has not yet been established. Nonetheless, the climatic assessment of Z. oligocenicum can be drawn from the results of the investigations of the accompanying fossil leaf flora. The high percentage of entiremargined leaves along with the presence of the members of the family Lauraceae suggest subtropical to warm temperate climate Wilde 1998, 2004). Moreover, besides Z. oligocenicum the sporodochia of another fossil mitosporic fungus, Neomycoleptodiscus pertusus (Dilcher) G. Worobiec were also reported from Csolnok. Its presence supports warm climatic conditions (Worobiec et al. 2020). The above data suggest that Zygosporium oligocenicum preferred warm climate conditions. But further findings of the fossil fungus are necessary to verify this hypothesis. The fossil-species Zygosporium oligocenicum was most probably a saprophyte on fallen, decaying leaves (leaf litter), similarly to most modern members of the genus Zygosporium (Ellis 1971;Whitton et al. 2003Whitton et al. , 2012Li et al. 2017).

Conclusions
We document the first fossil record of the anamorphic Zygosporium genus. Biostratigraphic control of the fossiliferous sediments (planktonic microfossils, molluscs, accompanying flora) suggests that the flora of Csolnok must have flourished at least 23 Ma before present (geochronological upper boundary of the Oligocene Epoch; Cohen et al. 2013, updated). Fungal remains, found on cuticles isolated from the fossil leaf compressions in the Oligocene deposits of Csolnok, Hungary, were described as a new fossil-species, Zygosporium oligocenicum G. Worobiec. Z. oligocenicum most probably was a saprophyte on decaying leaf litter and preferred warm climate conditions. These results may be essential for the calibration of divergence time estimations of fungal lineages (Samarakoon et al. 2019).
Acknowledgements We are grateful to Prof. Dr. Volker Wilde for his help and useful suggestions when starting fossil fungi studies. Two anonymous reviewers are acknowledged for detailed revision and valuable suggestions that improved our manuscript. This study contributes to NECLIME-an open international network of scientists working on the Cenozoic climate evolution and related changes in continental ecosystems.
Author contribution All authors contributed to the study conception and design. Material preparation and description of geological setting were performed by B. Erdei. Fungal remains were analyzed and interpreted by G. Worobiec. The first draft of the manuscript was written by G. Worobiec and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding This work was supported by the W. Szafer Institute of Botany, Polish Academy of Sciences, through its statutory funds to Grzegorz Worobiec and by the National Research, Development and Innovation Office (NKFIH-OTKA 120123) and German Academic Exchange Service (DAAD) to Boglárka Erdei.
Data availability The data generated during the current study are available from the corresponding author on reasonable request.

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Competing interests The authors declare no competing interests.
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