Diverse Epiphyllous Fungi on the Cunninghamia Leaves from the Oligocene of South China and Their Paleoecological and Paleoclimatic Implications

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Jianhua Jin ORCID iD: 0000-0002-1486-8631

Research Article

Diverse epiphyllous fungi on the Cunninghamia leaves from the Oligocene of South China and their paleoecological and paleoclimatic implications

Natalia P. Maslova 1, 2, Aleksandra B. Sokolova 2, Тatiana M. Kodrul 3, Anna V. Tobias 4, Natalia V. Bazhenova 2, Xin-Kai Wu 1, and Jian-Hua Jin 1*

1 State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China

2 Borissiak Paleontological Institute, Russian Academy of Sciences, Moscow 117647, Russia

3 Geological Institute, Russian Academy of Sciences, Moscow 119017, Russia

4 Saint-Petersburg State University, St. Petersburg 199034, Russia

*Author for correspondence. E-mail: [email protected], Tel.: 86-20-84113348

Received 3 March 2020; Accepted 19 June 2020

Running title: Fungi on the Oligocene Cunninghamia leaves

Abstract The unique co-occurrence of thyriothecia belonging to three fossil genera of epiphyllous fungi, Stomiopeltites Alvin & Muir (Micropeltidaceae), Callimothallus Dilcher, and Trichothyrites Rosendahl (Microthyriaceae), are reported on the leaves of the same host plant, Cunninghamia shangcunica Kodrul, Gordenko & Sokolova from the Oligocene Shangcun Formation of the Maoming Basin, South China. In China, Stomiopeltites is identified for the first time, Callimothallus is known from the Oligocene and Miocene of

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jse.12652.

Accepted Article Accepted

Guangxi and Zhejiang provinces, and Trichothyrites previously has been found only in the Eocene palynological assemblages of the Maoming Basin. The presence of abundant and diverse epiphyllous micromycetes, together with the taxonomic composition of the Shangcun megaflora and pollen assemblage, as well as quantitative climatic estimates obtained using Climate Leaf Analysis Multivariate Program (CLAMP), confirm the existence of a warm and humid climate in this region during the late early Oligocene. The geographic and stratigraphic distributions, comparisons with extant analogues, as well as ecological and

paleoclimatic implications of the fossil fungi are discussed.

Graphical Abstract

The unique co-occurrence of thyriothecia belonging to three fossil genera of epiphyllous fungi are reported on the leaves of the same host plant Cunninghamia from the Oligocene of South China, confirming together with the taxonomic composition of the megaflora and pollen assemblage, as well as quantitative climatic estimates, the existence of a warm and humid climate in this region during the late early Oligocene.

Key words: Ascomycota, host plants, Microthyriaceae, Micropeltidaceae, paleoecology, thyriothecia 1 Introduction

Extant fungal epiphytes represent a geographically widespread polyphyletic group with a great diversity of life cycles and morphology (Wu et al., 2011, 2014; Hyde et al., 2013; Tripathi, 2015). The phylum Ascomycota is the largest and most diverse group of extant fungi with 6600 genera (Wijayawardene et al., 2018). Its two families, Microthyriaceae and Micropeltidaceae, have the same type of fruiting bodies (thyriothecia) but differ in many morphological characters (Wu et al., 2011; Hyde et al., 2013; Hongsanan et al., 2015; Hongsanan & Hyde, 2017). Microthyriaceous fungi are characterized by ostiolate or non- ostiolate thyriothecia, which are usually globose or disk-shaped bodies. Thyriothecium upper walls are composed of radiating rows of mycelial cells, and bitunicate asci contain two-celled Accepted Article Accepted

ascospores. Micropeltidaceous fungi possess flattened thyriothecia that easily fall off the host surface. Thyriothecia with a prominent ostiole are made up of irregular hyphae forming a plectenchyma and the ascospores are hyaline, trans-septate, usually with more than two septa.

Due to their characteristic morphology, microthyriaceous and micropeltidaceous thyriothecia are easily distinguished and thus can be identified accurately in palynological

assemblages and on the various plant surfaces. Owing to their chitin composition (a resistant material of fungal fruiting bodies as well as of spores and hyphae), microfungi are often well preserved and resist chemical treatment of samples during cuticle maceration. Hence, fungal remains comprising spores, hyphae and fruiting bodies are well represented both in palynological assemblages and on fossil host plants (Taylor et al., 2015). Spores associated with fossil fruiting bodies are rarely found impeding their reference to extant genera. However, in most cases, distinctive morphological features of fossil thyriothecia facilitate their comparison with extant relatives.

The earliest record of microthyriaceous thyriothecia is dated to the Late Permian of India (Jha & Aggarwal, 2011), and the earliest micropeltidaceous thyriothecia are known from the Jurassic of Argentina (García-Massini et al., 2012). Numerous types of epiphyllous fungi of both families growing on leaf surfaces have been described from Cretaceous and Cenozoic deposits worldwide (Cookson, 1947; Dilcher, 1965; Alvin & Muir, 1970; Ding et al., 2011; Du et al., 2012; Phadtare, 1989; Phipps & Rember, 2004). Microthyriaceous fungi are probably the most studied fossil fungi, and fossil records indicate that their morphological diversity considerably increased during the Cenozoic (Cookson, 1947; Elsik, 1978; Kalgutkar & Jansonius, 2000; Tripathi, 2009; Saxena & Tripathi, 2011). Dilcher (1965) supposed that microthyriaceous fungi had a fairly modern aspect by the Eocene. Fossil micropeltidaceous fungi have been described from the Jurassic to the Miocene (García-Massini et al., 2012; El Atfy et al., 2013), but they are less diverse and rarer.

Occurrences of fossil micromycetes in association with different host plant organs are of great interest (Cookson, 1947; Dilcher, 1965; Alvin & Muir, 1970; Phipps & Rember, 2004; Shi et al., 2010; Ding et al., 2011; Du et al., 2012). Such studies, unlike those of micromycetes in palynomorph assemblages, allow detailed determining of their interaction with the host plant and other micromycetes, examination of their co-evolutionary Accepted Article Accepted

relationships as well as interpretations of their paleoecological and paleoclimatic significance.

Recently, the new species Cunninghamia shangcunica Kodrul, Gordenko & Sokolova was described from the Oligocene of the South China based on a combination of leaf morphological and epidermal characters (Kodrul et al., 2018). Here we assess the diversity of microfungi on C. shangcunica leaves. Three different types of fruiting bodies co-occurred on

both surfaces of infected leaves and we have assigned them to three genera: Stomiopeltites Alvin & Muir (Micropeltidaceae), Callimothallus Dilcher (Microthyriaceae), and Trichothyrites Rosendahl (Microthyriaceae).

2 Material and methods

The compressions/impressions of four leafy shoots of Cunninghamia shangcunica, of which two contained the fungal remains, were collected from the Shangcun Formation, Maoming Basin, in the southwest of Guangdong Province, China. The Shangcun Formation consists mostly of mudstones, silty shales, and siltstones with minor intercalations of oil shales and coal seams in the lower part (Nan & Zhou, 1996; Ye et al., 1997). Fossiliferous greyish brown lacustrine mudstones were recovered from the Lishan opencast mine (21°50′ 39.22″ N; 110° 46′ 42.8″ E) located approximately 25 km northwest of Maoming City. Plant-bearing deposits have been dated to the second half of the early Oligocene based on the palynological data (Herman et al., 2017). To date identified genera from a diverse plant megafossil assemblage include Equisetum (Equisetaceae), Osmunda (Osmundaceae), Pinus (Pinaceae), Calocedrus, Cunninghamia (Cupressaceae), Quercus subgen. Cyclobalanopsis (Fagaceae), Burretiodendron (Malvaceae s.l.), Calophyllum (Calophyllaceae), Palaeocarya (Juglandaceae), Myrica (Myricaceae), Ailanthus (Simaroubaceae), and Sabalites sp. (Palmae) (Liu et al., 2019; Wu et al., 2019).

Examination of macerated leaf cuticles of Cunninghamia shangcunica revealed fungal hyphae and small fungal spores, isolated or in clusters, as well as epiphyllous fungal fruiting

bodies. The leaf cuticles were prepared by maceration in Schulze’s solution (70% HNO3,

saturated with KClO3), followed by a treatment with 5–10% KOH and washing in distilled water. Isolated cuticles were mounted on slides with glycerin jelly and examined using an Axioplan 2 (Carl Zeiss) light microscope (LM); images were captured with a Leica DFC420 digital camera at the A.A. Borissiak Paleontological Institute (PIN RAS). Cuticles for SEM Accepted Article Accepted

study were mounted on aluminium stubs, coated with palladium and examined using a Tescan Vega II XMU scanning electron microscope (SEM) at PIN RAS. All photographs were optimized for size, color and contrast using Adobe Photoshop CC 2018.

All specimens are curated at the Museum of Biology of Sun Yat-sen University, Guangzhou, China. The abbreviations MM3A and MMB denote the sites in the vicinity of Lishan Village, where the specimens were collected.

3 Systematics

Phyllum Ascomycota

Class Dothideomycetes O.E. Eriksson & Winka, 1997

Order Incertae sedis

Family Micropeltidaceae Clements & Shear, 1931 Genus Stomiopeltites Alvin & Muir, 1970 Species Stomiopeltites shangcunicus N. Maslova & A. Tobias, sp. nov.

Diagnosis Thyriothecia up to 360 µm, spheroidal to slightly ellipsoidal with central area thicker than the periphery, ostiolate. Thyriothecium wall plectenchymatous. Central part of mature thyriothecium falls off leaving thin external contour of stroma. Free hyphae septate, spores unknown.

Holotype Specimen MMB-013a (shoot), FCun1 (stub for SEM), mature ostiolate thyriothecium, designated here (Figs. 5B, 5E, 5F).

Derivation of name From the Shangcun Formation.

Type locality Lishan Village vicinity, about 25 km northwest of Maoming City, the Maoming Basin, Guangdong Province, South China.

Stratigraphic horizon Shangcun Formation, lower Oligocene.

Repository The Museum of Biology of Sun Yat-sen University, Guangzhou, China.

Description Thyriothecia are irregularly distributed on both adaxial (Figs. 1A–1F; Figs.

Accepted Article Accepted 2C, 2D; Fig. 3F) and abaxial (Figs. 2B, 2F; Figs. 3A–3E) leaf surfaces. Thyriothecia are

spheroidal to slightly ellipsoidal in shape, with a diameter ranging from 25 to 360 µm. The central area is thicker than the peripheral part (Figs. 1A, 1B; Figs. 2A–2E; Figs. 3A, 3B, 3D, 3F). The upper surface of the thyriothecium consists of septate hyphae, 3–5 µm in diameter (Figs. 1B, 1D, 1F; Figs. 3B, 3D, 3F; Figs. 4A–4D; Figs. 5D, 5E, 5F; Figs. 6A, 6E, 6F). The septa are distributed 7–10 µm apart and towards the thyriothecium edge they become more elongate and radially arranged (Figs. 2A, 2D, 2E). The thyriothecium wall is composed of several layers of irregular hyphae forming a plectenchyma. The mature thyriothecium has a

single central ostiole18–20 µm in diameter with a central pore 7–10 µm in diameter (Figs. 5A–5F). Occasionally the central part of the mature thyriothecium falls off (Fig. 2F; Figs. 3C, 3E; Figs. 6B, 6C; Figs. 7A, 7B), leaving a thin external contour of the stroma up to 100 µm wide (Fig. 1F; Fig. 7A). Young thyriothecia are globous, with the minimum visible diameter of 25 µm, consisting of irregularly arranged hyphae (Figs. 4A, 4D–4F; Fig. 5B). Free hyphae are numerous, septate, somewhat sinuous, with septa 3–4 µm wide and up to 10 µm long (Figs. 1A, 1C, 1E; Figs. 2A–2F; Figs. 3A, 3C). Spores are unknown.

Comparison The genus Stomiopeltites includes three species: S. cretacea Alvin & Muir from the Lower Cretaceous of Isle of Wight, (Alvin & Muir, 1970) and the Cenomanian of France (Pons & Boureau, 1977), S. plectilis (Dilcher) Kalgutkar & Jansonius from the Eocene of Tennessee, USA (Dilcher, 1965; Kalgutkar & Jansonius, 2000), and S. amorphos Phipps & Rember from the lower Miocene of Clarkia, Idaho (Phipps & Rember, 2004). The new species differs by exhibiting a larger size of mature thyriothecia (up to 360 µm), smaller septae within the thyriothecia, and by the presence in the mature thyriothecium of a relatively wide and thin peripheral area of radiating hyphae, some of which are ramified and form a network outside the fruiting body.

Specimens of Cunninghamia shoots with Stomiopeltites shangcunicus thyriothecia MM3A-250; MMB-013a, b.

Order Microthyriales G. Arnaud, 1925

Family Microthyriaceae Saccardo, 1883

Genus Callimothallus Dilcher, 1965

Species Callimothallus pertusus Dilcher, 1965 Accepted Article Accepted

Description Thyriothecia are located on both abaxial and adaxial leaf surfaces, irregularly distributed, solitary (Fig. 7E; Figs. 8B–8F; Figs. 9A–9E) or in groups (Figs. 7A–7C; Fig. 8A). Young thyriothecia are rounded (Figs. 8C–8F), about 20 µm in diameter; mature ones are broadly ovate in shape, lobed, non-ostiolate, with sinuate margins and with a diameter ranging from 30 to 75 µm (Figs. 8A, 8B; Figs. 9A–9E). The thyriothecium wall consists of rectangular cells in radiating rows. The cells extend outwardly from the center of the thyriothecium; individual cells are 2–6 μm wide and 3–8 μm long. Most cells of the

thyriothecium have a small rounded or elliptic pore, 1–2 μm in diameter (Fig. 9F). Free hyphae are absent; no spores were found.

Specimens of Cunninghamia shoots with Callimothallus pertusus thyriothecia MMB- 013a, b.

Genus Trichothyrites Rosendahl, 1943

Species Trichothyrites padappakarensis (Jain & Gupta) Kalgutkar & Jansonius, 2000

Description Thyriothecia are irregularly distributed mainly on the abaxial (Figs. 10A, 10B, 10E, 10F), more rarely on the adaxial (Figs. 10C, 10D) leaf surface, solitary, discoid, rounded in outline, flattened, 60–80 µm in diameter, ostiolate, with entire (Figs. 10A, 10B) to sinuous (Figs. 10C, 10D; Figs. 11A, 11B) margin. The thyriothecium wall consists of rectangular cells 2–11 μm long and 2–4 μm wide in radiating rows becoming more elongated towards the periphery (Figs. 10A, 10C–10F; Fig. 11D). Tangential walls of peripheral cells are strongly thickened. Cell walls are generally thicker on the lower surface of the thyriothecium while on the upper surface they are more delicate (Figs. 10D, 10F). Thyriothecia have a single ostiolar collar about 20 µm in diameter with a central pore 7–10 µm in diameter, either centrally (Figs. 10A, 10B; Figs. 11A, 11B) or eccentrally (Figs. 11D, 11F) placed and elevated. The ostiolar collar is formed by thick-walled papilla-like cells, 1.5–2.5 µm in diameter (Figs. 10A, 10B; Fig. 11B). Free hyphae are absent. Spores are unknown.

Specimens of Cunninghamia shoots with Trichothyrites padappakarensis thyriothecia MMB-013a.

Germlings of microthyriaceous fungi Accepted Article Accepted

Only one young germling of the microthyriaceous fungus was found (Fig. 11C). The germling is disc-shaped, slightly lobed, about 12 µm in diameter.

Mycelium under the leaf cuticle

Occasionally, remains of a ramified mycelium confined to the periclinal wall of the cells are preserved on the cuticle inner surface (Fig. 11E). Hyphae do not cross the anticlinal walls

of the cells. The hyphae are 1–2 µm in diameter, septae were not detected.

4 Discussion

4.1 Systematic position of the described fungi

Extant microthyriaceous and micropeltidaceous fungi were traditionally classified on the basis of morphological features such as cell development patterns of the fruiting body surface, the mode of the thyriothecium dehiscence, and characters of the mycelia and ascospores. Recently, molecular data have begun to play an essential role in classifications of fungi, revealing possible new relationships between families and genera (Kirk et al., 2008; Wijayawardene et al., 2018). In the latest version of Ascomycota classification (Wijayawardene et al., 2018), which considers the discovery of many novel species and inclusion of DNA sequence data to provide a more natural system, the family Microthyriaceae is placed in the order Microthyriales (class Dothideomycetes), while the family Micropeltidaceae occupies an uncertain position within the Dothideomycetes. Lumbsch & Huhndorf (2010) treated Microthyriaceae and Micropeltidaceae as families incertae sedis within the Dothideomycetes, but Index Fungorum (http://www.indexfungorum.org/names/Names.asp), MycoBank (http://www.mycobank.org/MycoTaxo.aspx), and The Kalgutkar and Jansonius Database of Fossil Fungi (https://advance.science.sfu.ca/fungi/fossils/Kalgutkar_and_Jansonius) proposed the use of Microthyriales.

Identification of fossil microfungi causes some taxonomic and nomenclatural difficulties because of their frequent incomplete preservation or lack of diagnostic characters. Taylor et al. (2015) provided an analysis of classifications of fossil fungi, in particular, of microthyriaceous and micropeltidaceous micromycetes. One of the major difficulties in identification of the fossil Dothideomycetes is related to the lack of reliable taxonomic

Accepted Article Accepted criteria based only on morphology. Some authors suggested using recent genera for naming

the fossil microthyriaceous and micropeltidaceous fungi as they are closely related to the extant taxa (Dilcher, 1965; Phipps & Rember, 2004). Other authors named the fossils by adding the suffix “-ites” to the name of extant genera to show the close similarities with living forms (Edwards, 1922; Rosendahl, 1943; Cookson, 1947; Alvin & Muir, 1970). However, most of the diagnostic features of the extant taxa concern ascus and spore structure, which may be difficult to observe in fossil fungi. Reynolds & Gilbert (2005) noted that the taxonomy of epiphyllous fungi has been impaired by an underestimation of

intraspecific variations, while Ellis (1977) asserted that both the host substrate and ascospore structure are necessary for assignment to extant genera.

The main morphological characters considered in the classifications of fossil microthyriaceous and micropeltidaceous ascocarps (Elsik, 1978; Tripathi, 2015) are the shape, margin and surface cell arrangement of the fruiting body, mode of the fruiting body dehiscence, and the presence or absence of pores in individual cells. Based on these criteria, we identified epiphyllous microfungi on the Cunninghamia shangcunica leaves as belonging to three extinct genera, Stomiopeltites (Micropeltidaceae), Callimothallus (Microthyriaceae), and Trichothyrites (Microthyriaceae).

4.2 Fossil microthyriaceous and micropeltidaceous fungi: geographic and stratigraphic distribution and сomparison with extant species

Stomiopeltites The genus Stomiopeltites has been proposed for fossil microfungi with an ostiolate thyriothecium consisting of irregularly arranged cells; the spores are unknown (Alvin & Muir, 1970). Some authors (Dilcher, 1965; Phipps & Rember, 2004; Van Geel & Aptroot, 2006) suggested that Stomiopeltites is closely related to the extant genus Stomiopeltis Theissen (Theissen, 1914). Stomiopeltis is characterized by ostiolate thyriothecia composed of a pseudoparenchyma with irregularly arranged cells, becoming plectenchymatous at the margin and with asci grouped in locules containinig hyalodidymous ascospores. The central part of a mature thyriothecium of Stomiopeltis can eventually separate from the leaf leaving only the ring-like peripheral area on the plant surface (Luttrell, 1946).

The extant genus Stomiopeltis has been identified in the fossil record by Dilcher (1965), who described S. plectilis Dilcher from the Eocene of Tennessee. However, most of the significant diagnostic morphological features of the extant Stomiopeltoideae concern ascus Accepted Article Accepted

characters (Batista, 1959; Phipps & Rember, 2004), which may be difficult to observe in the fossils. Spores are unknown for S. plectilis, hence, an assignment of this fossil to the extant genus is doubtful. For this reason, S. plectilis has been considered as Stomiopeltites plectilis (Kalgutkar & Jansonius, 2000), so making a total of three species of this fossil genus known from the Lower Cretaceous to Miocene.

The earliest Stomiopeltites-like rounded ostiolate thyriothecia with plectenchymous tissue

consisting of irregularly arranged cells were recorded from the Jurassic San Agustín hot spring deposits, Patagonia, Argentina (García-Massini et al., 2012). Taylor et al. (2015) believe that another extant ascomycete, Muyocopron Speg, which based on genetic analysis belongs to the Muyocopronales (Tibpromma et al., 2016), has some similarities to Stomiopeltites in terms of the fruiting body but free hyphae are absent. Thyriothecia of Stomiopeltites were identified also in the palynomorph assemblages from the uppermost Oligocene–lower Miocene Nukhul Formation, Egypt (El Atfy et al., 2013) and now here the genus Stomiopeltites is recorded for the first time from China.

Callimothallus The genus Callimothallus was established for fungal fossils with a rounded stroma of porate cells without central dehiscence (Dilcher, 1965). Perforated cells forming rounded thyriothecia are typical only for this genus. The first fossil record of the genus Callimothallus was documented from the Maastrichtian of North and South America. The species C. irregularis (Martínez-Hernández & Tomasini-Ortiz, 1989) Kalgutkar & Jansonius and C. ruedae (Martínez-Hernández & Tomasini-Ortiz) Kalgutkar & Jansonius were recorded from Piedras Negras, Coahuila State, Mexico (Kalgutkar & Jansonius, 2000; Martínez-Hernández & Tomasini-Ortiz, 1989), and the species C. corralesensis Doubinger & Pons was described from Corrales mine, Boyacá Basin, Colombia (Doubinger & Pons, 1975; Kalgutkar & Jansonius, 2000). These Cretaceous species are characterized by a rounded stroma with outer walls composed of porate cells, which makes these fossils undoubtedly referable to the genus Callimothallus. However, an undifferentiated ostiole in the center of the thyriothecium was noted for the species C. irregularis (Martínez-Hernández & Tomasini- Ortiz, 1989), while the diagnosis of the genus Callimothallus specifies an absence of an ostiole. No free hyphae are known for C. corralesensis (Doubinger & Pons, 1975), although these thyriothecia are associated with other fungal colonies and it is difficult to distinguish to which fungi the hyphae belong. Accepted Article Accepted

Several species of Callimothallus have been reported from the Cenozoic of India, USA, Canada, Argentina, China, Australia, and New Zealand. Most of the Cenozoic species of this genus are known from India. Callimothallus assamicus Kar, Singh & Sah was described from the Eocene Tura and Siju formations of Garo Hills, Assam and Meghalaya provinces (Kar et al., 1972; Saxena & Sarkar, 2000; Sarkar et al., 2014; Samarakoon et al., 2019), and the upper Miocene Middle Siwalik of Bagh Rao, Uttar Pradesh (Sarkar et al., 1994). Callimothallus senii (Venkatachala & Kar) Kalgutkar & Jansonius was documented from the

Eocene of Matanamadh, Kutch, Gujarat, western India (Venkatachala & Kar, 1969; Kalgutkar & Jansonius, 2000). Miocene species, C. dilcheri Rao & Ramanujam (Rao & Ramanujam, 1976) and C. quilonensis Jain & Gupta (Jain & Gupta, 1970) are known from Quilon, southern India. Callimothallus raoi Ramanujam & Rao was documented from the upper Miocene Warkalli lignite of Kerala, southwestern India (Ramanujam & Rao, 1973), and Miocene–Pliocene species C. ramanujamii (Saxena & Singh) Kalgutkar & Jansonius was described from the Hospiarpur-Una Road section, Punjab (Saxena & Singh, 1982; Kalgutkar & Jansonius, 2000).

The type species of the genus, C. pertusus, first described from the lower Eocene of Western Tennessee, USA (Dilcher, 1965) has been recorded in India from the lower Eocene Cambay Shale of the Cambay Basin (Rao et al., 2013; Monga et al., 2015). This species was also reported from the upper Miocene Shengxian Formation of Zhejiang Province, East China (Ding et al., 2011; Du et al., 2012), as well as from the Oligocene of Guangxi Province, South China (Shi et al., 2010). Ding et al. (2011) described the presence of free hyphae and spores associated with stromata of C. pertusus, while Dilcher (1965) defined the genus Callimothallus as lacking free hyphae and spores. Free septate hyphae of C. pertusus, composed of rectangular cells with a distinct pore in most of them, were also described by Selkirk (1975). Free hyphae surrounding the thyriothecia of C. pertusus on the leaves of Cunninghamia shangcunica are also observed (Fig. 7A; Figs. 8A, 8B), but it is possible that these hyphae belong to another epiphyllous fungi on this host plant.

Dispersed thyriothecia of C. pertusus were found in palynological assemblages from the Paleocene Calumbi Formation of the Sergipe Basin, Brazil (Ferreira et al., 2005), Eocene deposits of Golden Grove, South Australia (Lange, 1978), Eocene Youganwo and Huangniuling formations of the Maoming Basin, South China (Aleksandrova et al., 2015), the Miocene Cullen Formation in Tierra del Fuego, Argentina (García-Massini et al., 2004), Accepted Article Accepted

Miocene deposits of Bełchatów Lignite Mine, Central Poland (Worobiec & Worobiec, 2017), the Eocene Tura Formation of Garo Hills, Assam, India (Kar et al., 1972), the Paleogene Iceberg Bay Formation, Axel Heiberg Island, Canada (Kalgutkar, 1997), and Cenozoic deposits of Gorgan's area, Iran (Velayati, 2013).

Another Cenozoic species, C. australis R.T. Lange, was described from the Eocene deposits of Golden Grove, South Australia. Apart from the central knotted hyphae attached to

the thyriothecia, this fossil is similar to C. pertusus. Both C. australis and C. pertusus were found on the same leaf surface (Lange, 1978).

Callimothallus funingensis Song from the Paleogene Funing Formation, Subei Basin, eastern China (Song et al., 1999) was not validly published as there was no Latin or English description or diagnosis. This micromycete differs from C. pertusus in having smaller cells making up the fruiting body walls and in the development of distinct radial ribs (Song et al., 1999). Thyriothecia assigned to the genus Callimothallus were also isolated from the upper Eocene carbonized leaf litter associated with the Pikopiko Fossil Forest, New Zealand (Bannister et al., 2016; Conran et al., 2016).

Cenozoic species differ in the topography of the pore distribution on the cell walls. Callimothallus dilcheri is characterized by a proximally located pore in each cell (Rao & Ramanujam, 1976). C. assamicus shows close affinity to C. pertusus in shape and size range of the fruiting body but bears small circular pores only in the central, and few peripheral, cells (Kar et al., 1972). In C. quilonensis only peripheral cells of the thyriothecium are porate (Jain & Gupta, 1970), and in C. raoi only few cells in the central and peripheral regions possess pores (Ramanujam & Rao, 1973). Our studies revealed a variety of porate cell distributions in the thyriothecia of C. pertusus. Porate cells are distributed over the whole surface of the fruiting body except for the very peripheral areas (Figs. 7D, 7F; Figs. 9A–9F), or are aggregated only in the central part of the thyriothecium mainly in smaller, probably younger, fruiting bodies (Figs. 8C–8F).

It is important to note that the SEM images of thyriothecia are the most appropriate to reliable documentation of porate cells in Callimothallus species. Taylor et al. (2015) have noted that some of fossil epiphyllous fungi are morphologically and structurally similar to the fossil chlorophycean green alga Ulvella Crouan & Crouan. Thus, it is essential to study such fossils in detail, especially if they are dispersed and not associated with the host plant. Accepted Article Accepted

Trichothyrites The fossil genus Trichothyrites has been interpreted as morphologically similar to extant Trichothyrina (Petr.) Petr. (Petrak, 1950). The genus Trichothyrites represents a disc-shaped thyriothecia of approximately square, radiating cells and a well- defined ostiolar collar (Rosendahl, 1943). The thyriothecial outline is usually smooth, but may appear lobate. Spores of Trichothyrites are unknown. Kalgutkar & Jansonius (2000) considered species of Notothyrites Cookson within the genus Trichothyrites.

The first appearance of the genus Trichothyrites was documented from the Upper Permian (Jha & Aggarwal, 2011), while the widest geographic and stratigraphic distribution was reported for T. hordlensis Smith. This fossil is known from the Permian Barakar and Raniganj horizons of the Godavari Graben, India (Jha & Aggarwal, 2011), the Cenozoic deposits of Gorgan's area, Iran (Velayati, 2013), the upper Eocene Lower Headon deposits, Hordle Cliff, Hampshire, England (Smith, 1980), and the Miocene of Central Poland (Worobiec & Worobiec, 2017).

Thyriothecia belonging to the genus Trichothyrites were found in the palynomorph assemblages of the Upper Cretaceous Milk River Formation, southern Alberta, Canada (Kalgutkar & Braman, 2008), the Cenozoic Tervola deposits of northern Finland (Eriksson, 1978), the upper Paleocene–lower Eocene Iceberg Bay Formation, Canada (Kalgutkar, 1997), the upper Eocene Youganwo and Huangniuling formations of the Maoming Basin, South China (Aleksandrova et al., 2015), the lower Oligocene Krabbedalen Formation of central East Greenland (Worobiec & Worobiec, 2013; the uppermost Oligocene–lower Miocene of the Nukhul Formation, Egypt (El Atfy et al., 2013), the Miocene Cullen Formation, Argentina (García-Massini et al., 2004), the Miocene deposits of Bełchatów Lignite Mine, Poland (Worobiec & Worobiec, 2017), the upper Miocene of Sopron-Piusz Puszta, Hungary (Erdei & Lesiak, 1999–2000), the Neogene of Jharkhand, India (Singh & Chauhan, 2008), the Holocene of Tripura state, India (Prasad, 1986), and the upper Quaternary of the Kerala Basin, India (Limaye et al., 2007).

Four species of these micromycetes are known from Australia and New Zealand. Trichothyrites airensis (Cookson) Kalgutkar & Jansonius, T. ostiolatus (Cookson) Kalgutkar & Jansonius, and T. setifer (Cookson) Saxena & Misra were described from the lower Oligocene–upper Miocene of Australia, New Zealand and the Kerguelen Archipelago (Cookson, 1947; Saxena & Misra, 1990; Kalgutkar & Jansonius, 2000). Recently, T. airensis

Accepted Article Accepted was recorded also from the carbonaceous shales of the Permian Raniganj Formation,

Godavari Graben, India (Jha & Aggarwal, 2011). Trichothyrites kiandrensis (Selkirk) Kalgutkar & Jansonius was documented from the lower Miocene of Kiandra, New South Wales (Selkirk, 1975; Kalgutkar & Jansonius, 2000). Another species of this genus, T. hordlensis, was described from the upper Eocene Lower Headon deposits, Hampshire, England (Smith, 1980). This species is also known from the Permian Barakar Formation, Godavari Graben, India (Jha & Aggarwal, 2011). The type species of the genus, T. pleistocaenicus Rosendahl was described from the lower Pleistocene of Springfield,

Minnesota, USA (Rosendahl, 1943).

Most species of Trichothyrites have been described from India. Trichothyrites amorphus (Kar & Saxena) Saxena & Misra was documented from the Paleocene of the Bhuj-Lakhpat Road section, Matanomadt Village, Gujarat (Kar & Saxena, 1976) and the Neogene Ratnagiri beds, Amberiwidi section, Maharashtra (Saxena & Misra, 1990). Trichothyrites ovatus (Venkatachala & Kar) Kalgutkar & Jansonius is known from the Eocene of Bore-hole No. 14, Matanamadh, Gujarat (Venkatachala & Kar, 1969; Kalgutkar & Jansonius, 2000). First described in Australia and New Zealand, T. setifer (Cookson) Saxena & Misra was found subsequently in the Oligocene–Miocene Ratnagiri beds, Amberiwidi section, Sindhu Durg District, Maharashtra (Saxena & Misra, 1990), and the upper Eocene Kopili Formation, North Cachar Hills, Assam (Saxena & Trivedi, 2009). Trichothyrites siwalikus Phadtare was described from the middle Miocene Lower Siwaliks, near Tanakpur (Phadtare, 1989), while Trichothyrites denticulatus (Ramanujam & Rao) Kalgutkar & Jansonius was recorded from the Miocene Neyveli lignite Formation, Tamilnadu (Elayaraja & Kumarasamy, 2016) and the upper Miocene Warkalli lignite of Kerala State (Ramanujam & Rao, 1973; Kalgutkar & Jansonius, 2000). Additionally, two other species, T. echinatus (Rao & Ramanujam) Kalgutkar & Jansonius and T. keralensis (Rao & Ramanujam) Kalgutkar & Jansonius, were also reported from the upper Miocene Warkalli lignite (Rao & Ramanujam, 1976; Kalgutkar & Jansonius, 2000). Trichothyrites sastryi Patil & Ramanujam is known from the Miocene of Tonakkal, Kerala (Patil & Ramanujam, 1988), and the lower Eocene Cambay Shale of the Cambay Basin, Gujarat (Samant & Tapaswi, 2000).

Trichothyrites padappakarensis identified on the leaves of Cunninghamia shangcunica from the Maoming Basin was first described from the upper Miocene of Padappakkara, Quilon, Western Ghats, South India (Jain & Gupta, 1970). This species is most similar to T. denticulatus, T. hordlensis, and T. keralensis in the size of the thyriothecia; other species Accepted Article Accepted

have larger thyriothecia (up to 330 µm in T. kiandrensis). Trichothyrites denticulatus differs from T. padappakarensis in having teeth-like (denticular) projections, often slightly reflexed, protruding into the ostiole cavity (Ramanujam & Rao, 1973; Kalgutkar & Jansonius, 2000). In contrast to T. hordlensis (Smith, 1980), T. padappakarensis has smaller and more elongated thyriothecium cell walls. Trichothyrites keralensis is characterized by a solely centric ostiole (in T. padappakarensis the ostiole is centric as well as eccentric), and also differs in displaying a unique character – the presence of hyphopodiate mycelial shreds in the

vicinity of the ostiole border (Rao & Ramanujam, 1976; Kalgutkar & Jansonius, 2000). In T. amorphus, hyphae are radially arranged but do not anastomose to form distinct pseudoparenchymatous cells (Kar & Saxena, 1976). In T. setifer, some of the cells that make up the collar have delicate bristle-like setae (Saxena & Misra, 1990). Trichothyrites echinatus differs from other known species in having prominent spinous projections protruding into the ostiole (Rao & Ramanujam, 1976; Kalgutkar & Jansonius, 2000). The main distinction of T. airensis is the narrower (up to 8 µm) conical ostiole (Cookson, 1947; Kalgutkar & Jansonius, 2000), while in T. ostiolatus the ostiole is wider (up to 13 µm). Trichothyrites pleistocaenicus is characterized by short setae on the marginal cells of the ostiole, and by the thyriothecial outline which is usually smooth, but may appear lobate (Rosendahl, 1943). In T. sastryi, the ostiole is slightly raised above the thyriothecium surface and marginal layers of cells are thicker than in other regions, forming an uneven margin (Patil & Ramanujam, 1988). T. siwalikus differs in having eccentric, deeply-lobed thyriothecia (Phadtare, 1989).

In China, the fruiting bodies of Trichothyrites were previously identified only in palynomorph assemblages from the upper Eocene Youganwo and Huangniuling formations of the Maoming Basin, Guangdong (Aleksandrova et al., 2015).

Germlings of microthyriaceous fungi and dispersed spores Germlings of microthyriaceous fungi have been described in detail by Dilcher (1965). He supposed that simple spore-like bodies increase in size and develop undulations and invaginations around the margin because of differential growth rates of the walls. Dilcher has shown that spore- like forms are identical in their early stages of development, but develop into quite different mature forms. They are capable of developing into various forms of fungi belonging to different systematic groups, so it is impossible to determine which microthyriaceous fungi the germlings would eventually become. Dilcher (1965) has assumed that young stages of microthyriaceous fruiting bodies found on Sapindus leaves could be assigned to two taxa, Accepted Article Accepted

Callimothallus pertusus or Trichopeltinites fusilis Dilcher, and regarded them as a separate group without a generic name but referred to only as “germlings of microthyriaceous fungi”.

Fossil germlings of microthyriaceous fungi are known from various localities around the world (Selkirk, 1975; Sherwood-Pike & Gray, 1988; Carpenter et al., 1994; Greenwood, 1994; Conran et al., 2016). Similar developmental stages from spore germination to mature perithecia in other ascomycetes such as Asterina kosciuskensis Selkirk have been reported by

Selkirk (1975) on lower Miocene leaves from Australia. Sherwood-Pike & Gray (1988) described abundant germlings of microthyriaceous fungi on Cephalotaxus leaves from the Miocene of Northern Idaho. Lange (1976) has divided germlings into five grades: from the beginning of the sequence, being simple, spore-like bodies (grade I) to the largest, most complex germlings (grade 5) and provided modern equivalents. It was shown that germlings should become larger and more morphologically complex (higher grades) on mature leaves, but newly formed leaves would have mostly grade I germlings.

Germlings of microthyriaceous fungi were also documented in paleopalynological assemblages (Jain & Gupta, 1970; Ramanujam & Rao, 1973; Sahay et al., 2016). Bannister et al. (2016) believed that these structures have different origins.

Examination of numerous leaf cuticles of Cunninghamia shangcunica revealed the only germling of microthyriaceous fungi (Fig. 11C), probably representing one of the first developmental stages. Apparently, at the moment of fossilization the formation of fruiting bodies was mostly finished and no new fruiting bodies were formed reflecting a seasonal state.

None of the microfungi described here contains in situ spores, and no information on the in situ spores of studied three genera has been published so far. Solitary spores (Figs. 1E, 1F; Fig. 4E; Fig. 6E; Fig. 7A; Fig. 12F) and their clusters (Figs. 12C, 12E) were found on the cuticle surface of C. shangcunica. The spore size varies from 7 to 10 µm. The spore shape is mostly spheroidal or slightly oval, non-septate, one- or two-septate. It is impossible to refer these spores to a definite type of fungus. Dilcher (1965, Plate IV, Figure 18) illustrated similar rounded spore-like form as the first developmental stage of germlings of various microthyriaceous fungi.

Accepted Article Accepted

4.3 Host plants

Not all of the descriptions of Stomiopeltites, Callimothallus, and Trichothyrites species include data on their host plants. These descriptions mostly deal with findings of fungal fruiting bodies in palynomorph assemblages (Kar et al., 1972; Lange, 1978; Kalgutkar, 1997; García-Massini et al., 2004, 2012; Jha & Aggarwal, 2011; Worobiec & Worobiec, 2017). Occurrences of micromycetes on the leaf surface of fossil plants allow a more reliable

identification due to the possible observation of different stages of fungal development and the type of connection to the substrate. As the completion of the life cycle of an epiphyllous fungus may take a long time, the presence or absence of epiphyllous fungi may be an additional indicator distinguishing an evergreen from a deciduous habit within a given fossil plant genus (Sherwood-Pike & Gray, 1988). Extant epiphyllous fungi prefer coriaceous leaves of evergreen plants, as they apparently provide a better substrate for fungi to complete their life cycle (Eriksson, 1974; Kirk & Spooner, 1989; Flessa et al., 2012; Worobiec & Worobiec, 2013). The majority of extant microthyriaceous and micropeltidaceous fungi live on broad-leaved species of angiosperms (Taylor et al., 2015).

Fungal diseases affecting leaf and shoot tissue of extant Cunninghamia lanceolata (Lamb.) Hook are shoot dieback as well as needle, twig, and terminal bud blights. The fungus Pestalotia apiculata T.Z. Huang (Amphisphaeriales, Xylariomycetidae) causes shoot dieback affecting seedlings and young trees (Huang, 1983). The fungus Bifusella cunninghamiicola Korf & Ogimi (Rhytismatales, Leotiomycetidae), which causes needle blight, has been recorded on C. lanceolata in Okinawa, Japan (Ogimi & Korf, 1972) and in Chiba near Tokyo (Saho & Zinno, 1975). The fungus Discosia pini Heald (Amphisphaeriales, Xylariomycetidae) associated with needle blight was reported from Tokyo (Kobayashi & Zhao, 1987), while Botryosphaeria cunninghamiae T.Z. Huang (Botryosphaeriales, Dothideomycetes) is known as twig blight pest (Huang, 1977). Terminal bud blight can also be caused by Phomopsis sp. (Diaporthales, Diaporthomycetidae) (Su et al., 1981). There is no evidence of Microthyriaceae or Micropeltidaceae fungi on the leaves of extant species of Cunninghamia.

Stomiopeltites Dilcher (1965), analyzing data from Luttrell (1946), showed that most species of the extant genus Stomiopeltis (to which a fossil genus Stomiopeltites is morphologically related) can be found on the upper as well as on the lower epidermis of host leaves, on stems,

Accepted Article Accepted or may lack any specific habitat on the host plant. Stomiopeltis aspersa (Berk.) Theiss. is the

only extant species that is selective to the lower leaf epidermis of its host plant, a species of Lauraceae from India (Luttrell, 1946). Deighton (1965) showed that extant members of Micropeltidaceae occurring in tropical Africa can be found on a wide range of hosts, particularly on evergreens with waxy leaves, and usually they are more numerous on the upper leaf surface than on the lower. At the same time, Holm & Holm (1984) observed S. dryadis (Rehm) L. Holm on dry leaves of Dpyas octopetala L. (Rosaceae) from Iceland.

Thyriothecia of Stomiopeltites have been found on leaves of different fossil angiosperms and on an extinct member of the Pinaceae. Alvin & Muir (1970) described S. cretacea from leaves of Early Cretaceous undescribed conifer resembling Frenelopsis Schenk (Cheirolepidiaceae), where the thyriothecia are mostly confined to stomata. Thyriothecia of Eocene S. plectilis (Dilcher, 1965) occur on the abaxial epidermis of Sapindus Tourn. ex L. leaves. Miocene S. amorphos has been reported on Betula L., Smilax L, and Zizyphoides Seward & Conway leaves (Phipps & Rember, 2004), but the authors did not specify the occurrence of fruiting bodies on the leaf surface. Here, we report for the first time the presence of the genus Stomiopeltites on Cunninghamia leaves.

Callimothallus Dilcher (1965) did not find any close similarity between the fossil genus Callimothallus and any extant genera of micromycetes. Without reliable similar extant analogues of Callimothallus, we are unable to carry out a comparative analysis of their host plants. Callimothallus thyriothecia have been found attached to the leaves of fossil conifers and angiosperms. Fruiting bodies of Callimothallus were detected on the abaxial leaf surface of Smilax (Smilacaceae) from the upper Miocene of East China (Ding et al., 2011), and on Diospyros L. leaves (Ebenaceae) from the Miocene of India (Phadtare, 1989). The thyriothecia on the leaves of Diospyros are closely associated with costal zones of the leaf surface. Shi et al. (2010) have reported Callimothallus thyriothecia on the leaves of Cephalotaxus Siebold & Zucc. ex Endl. (Cephalotaxaceae) from the Oligocene of Guangxi Province, South China.

Two fossil species of Cunninghamia, C. shangcunica from the Oligocene of Guangdong Province, South China (Kodrul et al., 2018) and C. praelanceolata Du & Sun (Du et al., 2012) from the upper Miocene of Zhejiang Province, East China served as a host plants for Callimothallus pertusus. These two conifer species differ in morphological and epidermal characters, as well as in their temporal and spatial distribution, yet both fossil Cunninghamia

Accepted Article Accepted species were infected by the same micromycete species, C. pertusus, that may indicate an

early, at least Oligocene, age for co-evolutionary relations between these conifers and epiphyllous micromycetes. Du et al. (2012) showed that the long axis of the Callimothallus thyriothecia is arranged parallel or slightly oblique to the long axis of the leaf, whereas the thyriothecia on the leaves of Cunninghamia shangcunica are oriented randomly. Since the relationship between the host plant Cunninghamia and microfungi Callimothallus lasted for millions of years, the fungi seem to have had the opportunity to develop a specialized morphology/physiology on the same host genus. Dilcher (1965) assumed that fungal

migration probably followed very closely the migration of host plants. It should be noted that only one type of fruiting body belonging to the genus Callimothallus has been documented for Cunninghamia praelanceolata, while C. shangcunica was affected by fungi belonging to three genera of epiphyllous micromycetes.

Trichothyrites The fossil genus Trichothyrites is related to the extant genus Trichothyrina. Ellis (1977) showed that species of Trichothyrina occur on dead leaves and stems of various grasses (T. alpestris (Sacc.) Petrak, T. ammophilae J.P. Ellis, T. nigro-annulata (Webster) J.P. Ellis), on leaves and fruits of woody angiosperms (T. alpestris, T. nigro-annulata, T. cupularum J.P. Ellis, T. norfolciana J.P. Ellis, T. parasitica (Fabre) von Arx, T. salicis J.P. Ellis), and on conifer leaves (T. fimbriata J.P. Ellis, T. pinophylla (von Hohn.) Petrak).

Thyriothecia of Trichothyrites have been found on fossil leaves of both angiosperms and conifers. Trichothyrites ostiolatus was observed on the upper epidermis of Oligocene– Miocene Oleinites willisii Cookson (Cookson, 1947; Kalgutkar & Jansonius, 2000), and Trichothyrites sp. on leaves of Pleistocene Buxus pliocenica Saporta & Marion (Erdei & Lesiak, 1999–2000). Thyriothecia of Trichothyrites pleistocaenicus are known only on the conifer Picea mariana (Mill.) B.S.P. (black spruce) from the Pleistocene of Minnesota, USA (Rosendahl, 1943). The occurrence of Trichothyrites on Cunninghamia leaves is recorded here for the first time.

4.4 Ecological interpretations

Phadtare (1989) analyzed patterns of vertical epiphyllous fungal distribution in extant tropical and subtropical forests. He showed that these microfungi grow mainly on the lower leaf surfaces in the basal part of the upper canopy and in the upper part of the middle canopy. At the same time, they grow on both leaf surfaces in the lower and central parts of the middle canopy. Phadtare attributed these microfungal distribution patterns to atmospheric Accepted Article Accepted

factors such as light and rain fall. Both direct sunlight and open aeration may prevent the growth of epiphyllous fungal germlings, and thus fungal colonization. Based on these data, it can be assumed that the type of microfungal distribution on the upper or lower leaf surface is due to their location in a forest canopy or tree crown rather than an essential systematic character. Epiphyllous fungi are much more common and diverse in the understory than in the canopy (Gilbert et al., 2007). These authors showed that environmental conditions differed between the canopy and understory, and this defined a strong vertical stratification

of abundance and diversity of epiphyllous fungi. Based on these data, we infer that the host plant Cunninghamia shangcunica occupied the middle level of the forest canopy or that the studied shoots were located in the middle of the tree crown where the upper leaf surface was hidden from the sunlight and rain fall so that various species of fungi could thrive.

Fossil microfungi are often referred to as nonparasitic because it is presumed that they do not derive their nutrition from the host (Taylor et al., 2015). Convincing evidence of parasitic micromycetes on fossil plants is quite rare. There is no direct evidence of parasitism on fossil Cunninghamia host leaves neither at the early stages of fungal development nor in the mature fungi studied here. The only indication of possible parasitic penetration of a micromycete into the leaf tissue is in the form of very rarely observed branched hyphae on the inner periclinal wall of epidermal cells (Fig. 11E). However, no other fungal internal absorption apparatus was detected, and it is impossible to attribute this mycelium to any genus studied here.

Most species of extant genus Stomiopeltis have been reported as pathogens on fruits (Gleason et al., 2011; Mayfield et al., 2013; Ajitomi et al., 2017). This genus is one of three prevalent taxa of sooty blotch and flyspeck (SBFS) fungi. SBFS fungi colonize the surface wax layer of the fruits and also grow on the surface of plant stems, twigs, and leaves, but cause no physiological damage to the host plants (Gleason et al., 2011).

Dilcher (1965) showed that Stomiopeltites plectilis mainly develops on lower leaf surfaces. He reported the infection (apparently entering) of a few stomata and hair bases by free hyphae of S. plectilis, but he found no haustorial penetration into the epidermis of the host leaf. No such infection was found for S. shangcunicus, which is mainly confined to the upper leaf surface. We found no evidence of hyphal penetration into the stomata or anywhere else in the leaf tissue. Free hyphae are numerous and often entangle across almost the whole

Accepted Article Accepted upper leaf surface (Figs. 1A, 1C, 1E; Figs. 2A–2F; Figs. 3A, 3C; Figs. 7A, 7E; Figs. 8A, 8B;

Figs. 10A, 10C, 10E; Figs. 11A, 11C; Figs. 12A, 12C), without visible damage to the integrity of the plant tissues. Occasionally the central part of the mature thyriothecia falls off, sometimes with an attached leaf cuticle fragment (Figs. 3C, 3E; Figs. 6B, 6C), leaving a thin external contour of the stroma on the leaf surface. Episodic occurrence of large (mature) fruiting bodies within leaf tissues, in cuticle breaks (Figs. 12A, 12B, 12D) serve as an indirect proof of a possible parasitism by S. shangcunicus on the plant.

Dilcher (1965) also did not reveal any evidence of host leaf infection by germling stages or by mature Callimothallus pertusus. He illustrated a cross section of the thyriothecium attached to the cuticle, demonstrating close association between the fruiting body and leaf cuticle. However, no erosion or penetration into the cuticle or epidermal cells, which are in contact with the thyriothecium, was shown. We observed several thyriothecia of Callimothallus pertusus developed within the exterior contour of the stromata of Stomiopeltites shangcunicus that remained on the leaf surface after abscission of the central part of the mature Stomiopeltites thyriothecia (Figs. 7A–7C). Apparently, life cycles of the studied fungi do not coincide in time and C. pertusus formed thyriothecia after destruction of the fruiting body of S. shangcunicus. Fragments of free hyphae are preserved within the external contour of the Stomiopeltites thyriothecia, but it is impossible to assign these hyphae to any studied genus. However, it should be noted that thyriothecia of the C. pertusus are always surrounded by free hyphae (Figs. 7A, 7C, 7E; Figs. 8A, 8B).

Many extant trichothyriaceous fungi are often hyperparasites growing in close association with other epiphyllous microfungi, but are also found on non-fungal, vegetative substrates (Hughes, 1953; Ellis, 1977). Hyphae of these parasitic fungi form fruiting bodies over the host hyphae or a reticulum over the leaf surface between the host hyphae. The thyriothecia of Trichothyrites padappakarensis are also associated with clusters of free hyphae on the leaf surface (Figs. 10A, 10C, 10E; Fig. 11A), but no clear certainty exists regarding the interaction of Trichothyrites padappakarensis and its host plant, or other epiphyllous microfungi.

4.5 Paleoclimatic implications

Fungi are significant and essential members of terrestrial plant ecosystems. Epiphyllous microfungi develop in close association with host plants. The fossil record indicates similar relationships between fungi and plants in the geological past (Saxena & Tripathi, 2011). Accepted Article Accepted

Stubblefield & Taylor (1988) mentioned that extant fungi are mostly well-known from temperate areas, while fossil fungi are generally known from semitropical and tropical regions. Recent researches have shown that extant epiphyllous fungi are more frequent and diverse in regions characterized by a humid climate with stable temperatures throughout the year (Reynolds & Gilbert, 2005; Thaung, 2006; Hofmann, 2010; Piepenbring et al., 2011). High annual rainfall and humidity are the most important factors for their successful growth (Edwards, 1922; Last & Deighton, 1965; Selkirk, 1975; Lange, 1976; Elsik, 1978; Johnson &

Sutton, 2000; Limaye et al., 2007). However, some extant microthyriaceous fungi do not require warm conditions and grow even in high latitudes with a wet climate (Holm & Holm, 1984). Hofmann (2010) revealed that extant microthyriaceous plant-parasitic species generally occur in subtropical and tropical areas, while saprotrophic and hyperparasitic species are found in more temperate regions. Phadtare (1989) supposed that humidity, temperature and humus were critical for abundance of saprophytic fungi, while the distribution of parasitic and symbiotic fungi is controlled by their hosts.

Both modern and fossil germlings of microthyriaceous fungi are often considered to be climate indicators (Lange, 1976; Wells & Hill, 1993; Carpenter et al., 1994; Greenwood, 1994; Saxena & Tripathi, 2011; Conran et al., 2016). These studies showed the abundant occurrence of microthyriaceous germlings on plants growing in moist tropical habitats. Similarly, fossil microthyriaceous thyriothecia are generally considered to be reliable indicators of the paleoenvironment. Occurrences of microthyriaceous fungal thyriothecia in various developmental stages are correlated with humid, tropical to subtropical climates (Dilcher, 1965; Kalgutkar & Jansonius, 2000; Tripathi, 2009; Du et al., 2012; Paruya et al., 2017). Sherwood-Pike & Gray (1988) believed that the abundance and diversity of epiphyllous fungi could be a more sensitive indicator of paleoclimate than fossil plant species lists, pollen diagrams or foliar physiognomy. Dilcher (1965) argued that any paleoecological and paleoclimatic conclusions have to be based on the ecological analysis of both extant and related fossil epiphyllous fungi as well as other associated fossils (palynomorphs, leaves, fruits, seeds, wood). More recently, integrated studies of climatic conditions often include the technique known as Climate Leaf Analysis Multivariate Program (CLAMP) (Yang et al., 2011) and the study of epiphyllous microfungi on a variety of angiosperm leaves, together with CLAMP analysis, were used for paleoclimatic reconstructions by Conran et al. (2016). Accepted Article Accepted

The early Oligocene Shangcun megaflora is composed mainly of warm temperate to tropical plant taxa. The taxonomic composition of this flora and occurrence of diverse epiphyllous micromycetes on the Cunninghamia shangcunica leaves are consistent with the previously published quantitative climatic estimates obtained using CLAMP analysis (Herman et al., 2017; Spicer et al., 2017) that determined that the prevailing climate experienced by the Shangcun flora was humid subtropical.

5 Conclusions

Three different types of fruiting bodies of epiphyllous micromycetes are here referred to three genera of two families, Stomiopeltites (Micropeltidaceae), Callimothallus, and Trichothyrites (Microthyriaceae), found associated on the leaves of the Oligocene conifer Cunninghamia shangcunica (Cupressaceae). Such fungal diversity on the same fossil host leaves are here described for the first time. While Callimothallus pertusus is relatively widely distributed in the Oligocene–Miocene of China (Shi et al., 2010; Ding et al., 2011; Du et al., 2012), dispersed thyriothecia referable to the genus Trichothyrites have so far only been described in China in palynomorph assemblages from the upper Eocene Youganwo and Huangniuling formations of the Maoming Basin (Aleksandrova et al., 2015). The fossil genus Stomiopeltites is here revealed in China for the first time.

Considering that fruiting bodies of micromycetes were developed on both upper and lower leaf surfaces, we suppose that microclimatic conditions for these leaves were more or less similar, likely within the understory, or in the middle part of the forest canopy, or by the shoot location inside tree crown.

Along with data on the systematic composition of Shangcun flora and palynomorph assemblages (Aleksandrova et al., 2015), as well as data from CLAMP analysis (Herman et al., 2017; Spicer et al., 2017), the occurrence of abundant and diverse fruiting bodies of epiphyllous micromycetes on Cunninghamia shangcunica leaves serve as an indirect evidence of a warm and humid South China regional climate during late early Oligocene.

Acknowledgements

The authors are deeply grateful to Prof. R. Spicer from the Open University, UK for his helpful comments and suggestions. This study was supported by the National Natural Science

Accepted Article Accepted Foundation of China (Grant nos. 41820104002, 41572011, for JJ, XW), the Russian

Foundation for Basic Research (Grant no. 19-04-00046, for AS, AT, NB, NM, and partly for TK), and by the State program (No. 075-00872-20-00, Geological Institute, Russian Academy of Sci., for TK).

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Figure captions

Fig. 1. Thyriothecia of Stomiopeltites shangcunicus N. Maslova & A. Tobias representing different growth stages on the external surface of macerated adaxial leaf cuticles of Cunninghamia shangcunica. Specimen MMB 013a. A, C, E, LM. B, D, F, SEM. Note numerous free hyphae surrounding the thyriothecia and a single one-septate spore (arrow) in E, F. The same thyriothecia are shown in A and B, C and D, E and F. Scale bars = 20 µm D,

F; 50 µm A–C, E.

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Fig. 2. Thyriothecia of Stomiopeltites shangcunicus N. Maslova & A. Tobias surrounding by free hyphae on the external surface of macerated leaf cuticles of Cunninghamia shangcunica. Specimen MMB 013a, LM. A, C–E, Adaxial leaf surface; B, F, Abaxial leaf surface; D, Enlargement of thyriothecium in C; F, Exterior contour of the mature thyriothecium remaining on the leaf surface after abscission of its central part. Scale bars = 20 µm A, D; 50 µm B, E, F; 100 µm C.

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Fig. 3. Thyriothecia of Stomiopeltites shangcunicus N. Maslova & A. Tobias of different growth stages on the external surface of macerated leaf cuticles of Cunninghamia shangcunica. Specimen MMB 013a. A, C, E, LM; B, D, F, SEM. A–D, F, Adaxial leaf surface. E, Abaxial leaf surface. The same group of thyriothecia is shown in A and B. D, F, Enlargement of thyriothecia in B. C, E, Mature thyriothecia with the central part fallen away. Note the lack of a cuticle fragment at the detachment area in E. Scale bars = 50 µm A, C–F; 100 µm B.

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Fig. 4. Thyriothecia of Stomiopeltites shangcunicus N. Maslova & A. Tobias on the external surface of macerated leaf cuticles of Cunninghamia shangcunica. Specimen MMB 013a, SEM. A, Two thyriothecia of different growth stages. B, C, Mature thyriothecia. D–F, Young thyriothecia. Note single spore (arrow) in E. Scale bars = 20 µm C–F; 50 µm A, B.

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Fig. 5. Thyriothecia of Stomiopeltites shangcunicus N. Maslova & A. Tobias. A, Mature thyriothecium with prominent ostiole on the leaf surface of Cunninghamia shangcunica. Specimen MM3А-250, SEM. B–F, Specimen MMB 013a, macerated cuticles, SEM. B, Young thyriothecium and two mature ostiolate thyriothecia; example at the bottom right is the holotype (arrow). C, Mature thyriothecium with central ostiole. D, Enlargement of mature ostiolate thyriothecium in B. E, F, Enlargement of mature thyriothecium in B possessing a central ostiole, holotype. Scale bars = 10 µm F; 20 µm C; 50 µm B, D, E; 100

µm A.

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Fig. 6. Thyriothecia of Stomiopeltites shangcunicus N. Maslova & A. Tobias on the external surface of macerated leaf cuticles of Cunninghamia shangcunica. A, C, E, F, Specimen MMB 013a, SEM. B, D, Specimen MMB 013b, SEM. A, Mature thyriothecium with partly destroyed central part. B, C, Thyriothecia with central parts fallen away. Fruiting body peripheral parts with radially arranged hyphae are visible. Note the lack of a cuticle fragment at the detachment area in C; molds of epidermal cells are seen. D, Fallen central part of mature thyriothecium. E, F, Thyriothecium peripheral parts composed by radially arranged

hyphae. Note the round or oval hyphal cross sections and single spore (arrow). Scale bars = 10 µm F; 20 µm A, E; 50 µm B–D.

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Fig. 7. Thyriothecia of Callimothallus pertusus Dilcher on the external abaxial surface of macerated leaf cuticles of Cunninghamia shangcunica. A–D, F, Specimen MMB 013a. E, Specimen MMB 013b. A, Thyriothecia occurring inside the peripheral contour of the thyriothecium of Stomiopeltites shangcunicus N. Maslova & A. Tobias remaining on the leaf surface after its abscission; note two-septate spore (arrow), LM. B, The same, SEM. C, Enlargement of the thyriothecia in A, LM. D, F, Enlargement of the thyriothecia in B, SEM. E, Mature thyriothecium, LM. Scale bars = 20 µm C, D, F; 50 µm E; 100 µm A, B.

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Fig. 8. Thyriothecia of Callimothallus pertusus Dilcher on the external surface of macerated leaf cuticles of Cunninghamia shangcunica, SEM. A, C–F, Specimen MMB 013a. B, Specimen MMB 013b. A, Two thyriothecia at different growth stages. B, Mature thyriothecium. C–F, Young thyriothecia with small cell pores arranged mostly in their center part. Scale bars = 10 µm D; 20 µm B, C, E, F; 50 µm A.

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Fig. 9. Mature thyriothecia of Callimothallus pertusus Dilcher on the external surface of macerated leaf cuticles of Cunninghamia shangcunica. A–F, Specimen MMB 013a, SEM. Note broadly ovate, lobed thyriothecium shape and rounded or elliptic cell pores. F, Enlargement of the cell pores in E. Scale bars = 2 µm F; 20 µm A–E.

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Fig. 10. Thyriothecia of Trichothyrites padappakarensis (Jain & Gupta) Kalgutkar & Jansonius on the external surface of macerated leaf cuticles of Cunninghamia shangcunica. Specimen MMB 013a. A, C, E, LM. B, D, F, SEM. A, B, Completely preserved discoid thyriothecium with centrally placed and an elevated ostiolar collar. C–F, Partly destroyed thyriothecia with a sinuous margin. Lower wall of fruiting bodies with radiating rows of rectangular cells is visible. The same thyriothecia are shown in A and B, C and D, E and F. Scale bars = 20 µm B, D–F; 50 µm A, C.

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Fig. 11. Thyriothecia of Trichothyrites padappakarensis (Jain & Gupta) Kalgutkar & Jansonius on the external surface of macerated leaf cuticles of Cunninghamia shangcunica, germling of microthyriaceous fungi, and mycelium remains. Specimen MMB 013a. A, B, Thyriothecium with a sinuous margin and a centrally placed elevated ostiole formed by thick- walled cells, LM and SEM, respectively. D, F, Thyriothecia with eccentrally placed ostiole, SEM. C, Germling of microthyriaceous fungi (arrow) surrounding by numerous free hyphae, LM. E, Remains of ramified mycelium on the inner periclinal cell walls, SEM. Scale bars =

20 µm.

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Fig. 12. Thyriothecia of Stomiopeltites shangcunicus N. Maslova & A. Tobias and dispersed fungal spores. Specimen MMB 013a. A, B, D, Thyriothecia slightly protruding into plant tissue through the cuticle of the leaves of Cunninghamia shangcunica. A, LM. B, D, SEM. C, Cluster of dispersed fungal spores on the external surface of macerated leaf cuticle of Cunninghamia shangcunica, LM. E, Enlargement of the spores in C, SEM. F, Solitary spore, SEM. Scale bars = 5 µm F; 10 µm E; 20 µm C; 50 µm B, D; 100 µm A.

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