In Press at Mycologia, preliminary version published on February 27, 2015 as doi:10.3852/14-270
Short title: New species of fossil Meliolaceae A new species of Meliolinites associated with Buxus leaves from the Oligocene of Guangxi, southern China Fu-Jun Ma Bai-Nian Sun1 Qiu-Jun Wang Jun-Ling Dong Guo-Lin Yang Yi Yang School of Earth Sciences & Key Laboratory of Mineral Resources in Western China (Gansu Province), Lanzhou University, Lanzhou 730000, China Abstract: A new species of Meliolinites (fossil Meliolaceae), M. buxi sp. nov., is reported from the Oligocene Ningming formation of Guangxi, South China. The fungus has hyphopodia characteristics of extant Meliolaceae, such as thick-walled, branching hyphae with appressoria and phialides. However, these fossils entirely lack mycelial or perithecial setae and have only a few phialides, thereby distinguishing the new species from most known species. The fungus was discovered on the adaxial and abaxial cuticles of several fossilized Buxus leaves. Thickening and twisting of cell walls in the Buxus leaf cuticle, along with the parasitic feeding strategy of the extant Meliolaceae, suggest that a parasitic interaction between Buxus and M. buxi seems feasible. The distribution of modern Meliolaceae suggests that they live in warm, humid subtropical-tropical climates. It is possible that the presence of M. buxi indicates a similar climatic condition. The co-occurrence of large-leaf Buxus and floristic comparisons of the Ningming assemblage also corroborate this conclusion. Key words: Buxaceae, fossil Meliolaceae, palaeoecology, parasitism
Copyright 2015 by The Mycological Society of America.
INTRODUCTION To date 1980 species are classified in the family Meliolaceae, widely distributed in the tropics and subtropics (Kirk et al. 2008). They are obligate plant parasitic fungi and usually parasitic on adult leaves. These fungi infect plant species in numerous families and cause diseases that are typically benign and commonly known as black or dark mildews. Species of this family are characterized by superficial, dark, thick-walled and branching hyphae with appressoria and phialides. In China 341 species and varieties belonging to seven genera of modern Meliolaceae are reported (Song et al. 2002). The largest genus is Meliola Fr. with 239 species and varieties, followed by Asteridiella McAlpine with 54, Appendiculella Höhn. with 22, Irenopsis F. Stevens with 16, Armatella Theiss. & Syd. with five, Prataprajella Hosag. with three and Amazonia Theiss. with two (Hu 1996, Song et al. 2002). The genus Armatella is characterized by hyphae with no or few phialides (Hu 1996), distinguishing it from the other six genera. Only Asteridiella lacks setae in the Meliolaceae (Hu 1996). In contrast with the high diversity of extant Meliolaceae, there are few fossil representatives. Some records are palynological, focusing exclusively on morphology of fungal spores (Sen and Banerjee 1990, Yeloff et al. 2007, Kar et al. 2010), while other reports emphasize similarities to living forms but lack detailed information about hosts or patterns of infection (Daghlian 1978, Mandal et al. 2011). Relatively few studies investigate the relationships of fossil fungi with their hosts (Dilcher 1965, Selkirk 1975, van Geel et al. 2006) or use Fungi as paleoenvironmental indicators (Dilcher 1963, Daghlian 1978). In the present paper we present several epiphyllous fungi associated with leaf macrofossils of Buxus collected from the Oligocene Ningming formation of Ningming,
Guangxi, southern China. A new fossil species of Meliolaceae is described on the basis of a detailed study using light microscopy (LM) and scanning-electron microscopy (SEM). We studied the host plant leaves in detail and discuss the interaction between the fungus and its host plant. Finally the distribution of extant Meliolaceae let us make inferences about the palaeoecology of Ningming during the Oligocene. MATERIAL AND METHODS Host leaves were collected from the Ningming formation in western Ningming County (22°08′20.6″N, 107°01′47.3″E), Guangxi, southern China. The Ningming Formation is most likely the Oligocene (33.9–23.03 MYA), on the basis of palynostratigraphy (Wang et al. 2003), fishes (Chen and Chang 2011) and plant macrofossils (Li et al. 2003), although absolute dating is lacking. Host leaves were photographed, first with a Sony Super Steady Shot DSC-T70 camera for general morphology, and then with a Leica MZ 12.5 stereoscope to identify areas with fungal colonies; the leaves then were marked. Next 5% hydrogen peroxide was dripped evenly on the marked surface of the leaf compression to remove the leaf from the matrix. Leaf fragments were immersed in a 10% HCl solution before treatment with 50% HF solution. They were placed in a 5% KOH solution for 1–7 d. When the dark brown leaves changed to light brown, samples were removed from the solution. Without further treatment the fungi could then be clearly observed on the fossil cuticles under LM and were photographed with a Leica MZ 12.5 stereoscope. Next a drop of 5% NH4OH was added until the adaxial and abaxial cuticles separated. Samples were mounted on stubs, coated with gold and examined under SEM (JEOLJSM-6380LV) at Lanzhou University, China. For comparison leaves of extant Buxus is collected from the Herbarium of School of Life Sciences, Lanzhou University. The cuticles of extant specimen were prepared following the experimental treatments described by Li et al. (2010). All specimens, cuticle slides and stubs for SEM are deposited at the Institute of Paleontology and Stratigraphy, Lanzhou University, Gansu Province, China. For descriptions of Meliolaceae we followed the format of Hu (1996). For illustrations of host leaf morphology we followed the Leaf Architecture Working Group (Ellis et al. 2009), while terminology for cuticles follow Dilcher (1974). Specimens are deposited in the Institute of Paleontology and Stratigraphy, Lanzhou University, China collection (LDGSW).
TAXONOMY Meliolinites buxi Fu-Jun Ma et Bai-Nian Sun sp. nov.
FIGS 1, 2, 3
MycoBank MB811012 Typification: CHINA: southern China. Guangxi, Ningming County, Ningming Formation (Oligocene), Fu-Jun Ma, 16 Aug 2013 (holotype LDGSWF2013021). Paratype: Same locality, collector and dates (LDGSWF2013022, LDGSWF2013023, LDGSWF2013024). Etymology: The specific epithet buxi is derived from the fossil host. Diagnosis: Hyphae septate, thick-walled, bearing many appressoria and occasional phialides. Hyphal cells cylindrical, thick-walled, 14.0–20.0 × 5.0–8.0 μm. Appressoria bicellular; stalk cells cylindrical, 5.0–7.5 × 8.0–9.0 µm; head cells globose, pyriform, rounded-angulose or 2–4-lobed, 13.5–16.5 × 13.5–20.5 µm. Phialides ampulliform, 12.5–20.5 × 6.5–14.5 µm, mixed with appressoria. Perithecia round in outline, with crenate to crenulate edges, 150.0–230.5 μm diam. Mycelial and perithecial setae absent. Description: Colonies amphigenous (FIG. 3A–D), dense (FIG. 2A) or scattered (FIG. 2B), dark brown to black (FIG. 2A–B), 1.0–2.0 mm diam (eight measurements). Vegetative hyphae dark brown to black (FIG. 2B–D), septate (FIG. 3B, G), thick-walled (FIG. 3B, G), straight to slightly undulate, branching unilateral or opposite (FIGS. 2D, 3A–D) at 40°–80° (25 measurements), bearing many appressoria and occasional phialides (FIGS. 2D, 3A–D). Hyphal cells cylindrical, thick-walled (FIG. 3B, G), 14.0–20.5 μm × 4.9–8.0 μm (25 measurements). Appressoria always branching alternately while sometimes appearing unilateral (FIGS. 2D, 3A–D); each appressorium comprising two cells, a short stalk cell and a capitate head cell (FIGS 2D, 3A–D, H, I); stalk cells dark brown (FIG. 2D), cylindrical (FIGS. 2D, 3A–D, H, I),
5.1–7.6 × 7.8–9.1 µm (25 measurements), sometimes short and inconspicuous (FIG. 2D). Head cells dark brown (FIG. 2D), 13.5–16.5 × 13.5–20.5 µm (20 measurements), thick-walled (FIG. 3I), globose or pyriform (FIGS. 2D, 3C, D), often irregularly rounded-angulose (FIG. 3A, B), sometimes 2–4-lobed (FIG. 3H). Phialides sparse (FIGS. 2D; 3A, C), brown (FIG. 2D), mixed with appressoria, opposite to alternate, ampulliform (FIGS. 2D, 3A, C), 12.5–20.5 × 6.5–14.5 µm (SIX measurements). Perithecia dark brown to black, scattered, round in outline (FIG. 2B, C), ~ 150.0–230.0 μm diam (five measurements); hyphae radiating from the perithecia (FIG. 2C). No mycelial setae, perithecial setae, ascospores or conidia observed. Host plant: Leaves ovate (FIG. 4A). Apex reflex and convex terminating with a retuse tip (FIG. 4A). Base with obtuse angle, convex (FIG. 4A). Margin entire (FIG. 4A). Leaf venation pinnate, major secondary cladodromous (FIG. 4A). Secondaries together with higher-order veins, fused into a strong continuous marginal secondary (FIG. 4A, C). A range of variation in the higher order venation can be observed in the host leaves, from almost no recognizable secondaries to weakly organized secondaries (FIG. 4A, C). Leaf epidermal cells generally pentagonal and hexagonal (FIG. 3F). Anticlinal walls 1.0–2.0 μm thick (30 measurements), straight or rounded (FIG. 3F). Stomata anomocytic, rounded to broadly ovate (FIG. 4E, G), with a prominent outer ring (FIG. 4E), ~ 6.0 μm thick (30 measurements). The host fossil leaf is similar to extant Buxaceae in gross morphology. Buxaceae is a small family comprising five genera Buxus, Notobuxus, Sarcococca, Pachysandra and Styloceras (Min and Brückner 2008). These genera are different in venation pattern. Buxus is characterized by pinnate venation, whereas Sarcococca and Pachysandra always display triplinerved venation (Min and Brückner 2008). Also Notobuxus, endemic to Africa and Styloceras restricted in Ecuador, are different from
the present fossil in the venation patterns. The examples are available on http://specimens.kew.org/herbarium/K000353978 and http://specimens.kew.org/herbarium/K000573598. In addition, the current species and Buxus are identical in stomatal structure with the prominent outer ring on guard cells (Kvaček et al. 1982). Fossil leaves are similar to extant Buxus in architectural (FIG. 4A–D) and cutical features (FIG. 4E–H). Therefore the host leaves are consistent with classification in the genus Buxus (Buxaceae). A detailed taxonomy of these host leaves is addressed in another study. DISCUSSION The fossil fungus that we discovered on the Oligocene Buxus leaves in southern China exhibit hyphopodia characteristics of extant Meliolaceae, but it lacks mycelial and perithecial setae. This combination of characters is unusual in extant Meliolaceae, except for the genus Asteridiella. Furthermore, these fossils have a few phialides, a feature observed only in species of Armatella. Because our fossils combine characters not seen in extant genera, it is most appropriate to assign them to the fossil genus Meliolinites, erected for fossil epiphyllous fungi lacking setae but otherwise possessing the general characters of the Meliolaceae (Selkirk 1975, Daghlian 1978). The fossil record of the Meliolaceae epiphyllous fungi is limited and is restricted to fossils of angiosperm leaves. Köch (1939) described material belonging to Meliolaceae from the Eocene of Germany. However, comparisons of his descriptions with M. buxi are difficult because his illustrations are incomplete. Two distinct species of Meliolaceae, Meliolinites spinksii (Dilcher) Selkirk and M. anfractus (Dilcher) Kalgutkar & Janson., were discovered from the Eocene of Tennessee (Dilcher 1965, Selkirk 1975, Kalgutkar and Jansonius 2000). M. spinksii
can be easily distinguished from the new species because of its opposite appressoria and the shape of its head cells. Head cells of M. spinksii are oblong to ovoid (Dilcher 1965, Kalgutkar and Jansonius 2000). Whereas M. buxi has globose, pyriform, rounded-angulose or 2–4-lobed head cells. M. anfractus displays mycelial setae arising directly from hyphal cells (Dilcher 1965, Kalgutkar and Jansonius 2000) and thus obviously differs from the present species. In addition, head cells of M. anfractus are always lobate (Dilcher 1965, Kalgutkar and Jansonius 2000), which are different from that of M. buxi. M. dilcheri Daghlian, from the early Eocene of Texas (Daghlian 1978), can be easily distinguished from the present fungus by the absence of phialides in its colonies. M. nivalis Selkirk with typical spores, mycelia and hyphopodia, was reported from the Miocene of New South Wales, Australia (Selkirk 1975). It clearly differs from the present species in its shape of appressoria and its hyphal features. Hyphal cells of M. nivalis are 19.0–34.0 µm long (Selkirk 1975), which are longer than that of the new species. Head cells of M. nivalis are irregularly globose (Selkirk 1975), while head cells of M. buxi are various, from globose, pyriform, to rounded-angulose or 2–4-lobed. M. siwalika Mandal, Samajpati & Bera was discovered from the Miocene in West Bengal, India (Mandal et al. 2011). It differs by the longer shape of the appressoria and the much larger hyphal cells. Meliola ellis Roum. from the Holocene of northern England (van Geel et al. 2006) can be easily distinguished from the present species by its distinct setae. Fossil species often are inferred to exhibit similar living strategies as their nearest living relatives (Mosbrugger 1999). Extant fungi in Meliolaceae demonstrate a limited range of biological interactions and are obligate parasites on plant leaves and stems. Therefore we speculate that M. buxi was parasitic on Buxus. In addition the observation of well-preserved appressoria, whose main function is to penetrate a host
(Emmett and Parbery 1975), may be another indicator of parasitism. While characterizing interactions among fungi and host plants, the presence or absence of a host response provides a wealth of information for understanding the interaction (Taylor et al. 2004). Evidence that the host of M. buxi was alive at the time of infection includes the thickening and twisting of epidermal cell walls in the Buxus cuticle. The walls of uninfected leaf epidermal cells are straight or rounded and are typically 1.0–2.0 μm thick (FIG. 3F). However, infected leaf epidermal cells appear under or near the fungal colonies and are more curved and much thicker, 5.0–6.0 μm (FIG. 3E). These thickened and twisted leaf epidermal cell walls represent a clear host response. Similar responses to fungal invaders are common in extant plants (Taylor and Osborn 1996). Fossil fungi can provide significant information about the palaeoecology and past habitats (Lange 1978, van Geel et al. 2003, Chambers et al. 2012). Species of extant Meliolaceae are distributed mainly in tropical to subtropical zones (Kirk et al. 2008). Hence the presence of M. buxi is generally indicative of a warm and humid tropical to subtropical climate in the area during deposition. Preliminary floristic study of the Ningming flora reached a similar conclusion. The fungus was found in a forest assemblage dominated by angiosperms, including Fabaceae, Lauraceae, Fagaceae, Moraceae, Arecaceae, Hamamelidaceae, Betulaceae, Anacardiaceae, Simaroubaceae, Juglandaceae, Ulmaceae and Sapindaceae (Shi 2010). A preliminary study indicates that the Ningming flora represents a tropical-subtropical forest (Shi 2010). Kvaček et al. (1982) noted that the genus Buxus as a whole ranges from subtropical to warm temperate and mesic to even xerophilous (although rarely) in ecological preferences. But the large-leaf Buxus in Asiatic taxa are associated with tropical-subtropical conditions. The Buxus illustrated in this paper is about 45.0 mm long (FIG. 4A), which
fit in the living large-leaf forms. The occurrence of large-leaf Buxus may also might be indicative of a tropical-subtropical condition. In addition, based on palynological studies of Buxus from the early Paleogene to the late Miocene of Europe, Kvaček et al. (1982) suggested that Buxus was a component of tropical-subtropical flora. These analyses all corroborate our conclusion that the fossil M. buxi was deposited and lived in a warm and humid tropical to subtropical climate. ACKNOWLEDGMENTS We thank Executive Editor Dr Seifert, Associate Editor Conrad Schoch and two anonymous reviewers for their invaluable suggestions and thoughtful comments for improving the manuscript. We also thank Ru-Yong Zheng from the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, for her advice on the paper. We also thank David L. Dilcher from the Departments of Geology and Biology, Indiana University, and Subir Bera from the Centre of Advanced Studies, Department of Botany, University of Calcutta, for their kindness in providing scientific literature on fossil Meliolaceae. This work is supported by the National Basic Research Program of China (973 program number 2012CB822003), the National Natural Science Foundation of China (Grants 41172022, 41272026) and the Fundamental Research Funds for the Central Universities (No.lzujbky-2014-285).
LITERATURE CITED Chambers FM, Booth RK, de Vleeschouwer F, Lamentowicz M, le Roux G, Mauquoy D, Nichols JE, van Geel B. 2012. Development and refinement of proxy-climate indicators peats. Quat Int 268:21–33.
Chen GJ, Chang MM. 2011. A new cyprinin from Oligocene of south China. Sci China Earth Sci 54:481–492.
Daghlian CP. 1978. A new melioloid fungus from the early Eocene of Texas. Palaeontology 21:171–176.
Dilcher DL. 1963. Eocene epiphyllous fungi. Science 142:667–669.
———. 1965. Epiphyllous fungi from Eocene deposits in western Tennessee, USA. Palaeontogr Abt B 116:1–54.
———. 1974. Approaches to the identification of angiosperm leaf remains. Bot Rev 40:1–157.
Ellis B, Daly DC, Hickley LJ, Johnson KR, Mitchell JD, Wilf P, Wing SL. 2009. Manual of Leaf Architecture. New York: Cornell Univ. Press. 190 p.
Emmett RW, Parbery DG. 1975. Appressoria. Annu Rev Phytopathol 13:147–165.
Hu YX. 1996. Flora Fungorum Sinicorum. Vol. 4. Meliolales I. Beijing: Science Press. 270 p. (in Chinese)
Kalgutkar RM, Jansonius J. 2000. Synopsis of fossil fungal spores, mycelia and fructifications. Dallas, Texas: AASP Contrib Ser 39 AASP Found.
Kar R, Mandaokar BD, Kar RK. 2010. Fungal taxa from the Miocene sediments of Mizoram, northeast India. Rev Palaeobot Palynol 158:240–249.
Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi.10th ed. Walingford, UK: CAB International.784 p.
Köch C. 1939. Fossile Kryptogamen aus der eozänen Braunkohle des Geiseltales. Nova Acta Acad Lepop Carol 6:333–359.
Kvaček Z, Bužek Č, Holý F. 1982. Review of Buxus fossils and a new large-leaved species from the Miocene of central Europe. Rev Palaeobot Palynol 37:361–394.
Lange RT. 1978. Southern Australian Tertiary epiphyllous fungi, modern equivalents in the Australasian region and habitat indicator value. Can J Bot 56:532–541.
Li HM, Chen YF, Chen GJ, Kuang GD, Huang ZT. 2003. Tertiary fossil winged fruits of Palaeocarya from Ningming of Guangxi, S. China. Acta Palaeontol Sin 42:537–547. (in Chinese with English abstract)
Li XC, Sun BN, Xiao L,Wu JY, Lin ZC. 2010. Leaf macrofossils of Ilex protocornuta sp. nov. (Aquifoliaceae) from the Late Miocene of east China: implications for palaeoecology. Rev Palaeobot Palynol 161:87–103.
Mandal A, Samajpati N, Bera S. 2011. A new species of Meliolinites (fossil Meliolales) from the Neogene sediments of sub-Himalayan West Bengal, India. Nova Hedwigia 92:435–440.
Min TL, Brückner P. 2008. Buxaceae. In: Wu ZY, Raven PH, Hong DY, eds. Flora of China. Vol. 11, Science Press, St Louis: Missouri Botanical Garden Press. p 321–328.
Mosbrugger V. 1999. The nearest living relative method. In: Jones TP, Rowe NP, eds. Fossil plants and spores: Modern techniques. London: Geological Society. p 261–265.
Selkirk DR. 1975. Tertiary fossil fungi from Kiandra, New South Wales. Proc Linn Soc NSW 100:70–94.
Sen PK, Banerjee M. 1990. Palyno-plankton stratigraphy and environmental changes during the Holocene in the Bengal Basin, India. Rev Palaeobot Palynol 65:25–35.
Shi GL. 2010. Fossil plants from the Oligocene Ningming Formation of Guangxi,and a preliminary palaeoclimatic reconstruction of the flora [doctoral dissertation]. Nanjing Institute of Palaeontology and Geology: Chinese Academy of Sciences. (in Chinese with English abstract)
Song B, Li TH, Shen YH. 2002. Ecological and floristic analyses of Meliolales (Fungi) in China. J Trop Subtrop Botany 10:118–127.
Taylor TN, Klavins D, Krings M, Taylor EL, Kerp H, Hass H. 2004. Fungi from the Rhynie chert: a view from the dark side. Trans R Soc Edinburgh. Earth Sci 94:457–473.
———, Osborn JM. 1996. The importance of fungi in shaping the paleoecosystem. Rev Palaeobot Palynol 90:249–262.
van Geel B, Aptroot A, Mauquoy D. 2006. Sub-fossil evidence for fungal hyperparasitism (Isthmospora spinosa on Meliola ellisii, on Calluna vulgaris) in a Holocene intermediate ombrotrophic bog in northern England. Rev Palaeobot Palynol 141:121–126.
———, Buurman J, Brinkkemper O, Schelvis J, Aptroot A, van Reenen G, Hakbijl T. 2003. Environmental reconstruction of a Roman-period settlement site in Uitgeest (the Netherlands), with special reference to coprophilous fungi. J Archaeol Sci 30:873–883.
Wang WM, Chen GJ, Chen YF. 2003. Tertiary palynostratigraphy of the Ningming Basin, Guangxi. J Stratigr 27:324–327.
Yeloff D, Charman D, van Geel B, Mauquoy D. 2007. Reconstruction of hydrology, vegetation and past climate change in bogs using fungal microfossils. Rev Palaeobot Palynol 146:102–145.
LEGENDS FIG. 1. Meliolinites buxi (holotype), showing hyphae with many appressoria and occasional phialides; bar = 50 μm. FIG. 2. Mycelial colonies of M. buxi under LM. A. Mycelial colony, LDGSWF2013022. B. Mycelial colony, LDGSWF2013021. C. Magnified view of B, showing perithecium. The black arrow indicates radiating hyphea. D. Magnified view of B, showing hyphae bearing appressoria and phialides. The white arrow indicates an appressorium with cylindrical stalk cells; the black arrow indicates an appressorium with short and inconspicuous stalk cells; the black arrowhead refers to an ampulliform phialide. Bars: A = 1 cm, B = 500 μm, C = 200 μm, D = 50 μm. FIG. 3. Detailed characters of Meliolinites buxi and indications of reactions to the parasitism on the host leaf under SEM. A–B. Hyphae on the surface of adaxial leaf cuticles mainly bear appressoria, LDGSWF2013023. Black arrows indicate ampulliform phialides. C–D. Hyphae on the surface of abaxial leaf cuticle, indicated by the occurrence of stomata with prominent outer rings, LDGSWF2013024. The black arrow indicates an ampulliform phialide. E. Thickened and twisted epidermal cell walls in the Buxus leaf cuticle. The black arrow indicates the thickened and twisted epidermal cell wall. F. Uninfected epidermal cells in the Buxus leaf cuticle. The black arrow indicates the uninfected epidermal cell wall. G. Magnified view of B. The black arrow indicates a septum in a hyphal cell. H. Magnified view of A, showing the shape of appressoria. The white arrow indicates a 2–4-lobed head cell; white arrowhead indicates a cylindrical stalk cell. I. Amplification of B, showing the characters of appressoria. The white arrow indicates a globose and thick-walled head cell; the black arrow indicates a cylindrical stalk cell; bars: A, C, E, F = 50 μm;. B = 20 μm; D = 100 μm; G–I = 5 μm. FIG. 4. A, C, E, G. Fossil leaf of Buxus; B, D, F, H. Extant Buxus sp. A. Fossil leaf of Buxus. B. Extant Buxus sp. for comparison with the fossil. C. Details of fossil specimen, showing vein architecture. D. Details of extant Buxus sp., showing vein architecture. E. Stomatal apparatus of the fossil leaf, outer surface. F. Stomata apparatus of Buxus sp., outer surface. G. Stomatal apparatus of the fossil leaf, inner surface. H. Stomatal apparatus of Buxus sp., inner surface. Bars: A–B = 5 mm. C–D = 2 mm. E–G =5 μm. H = 10 μm.
FOOTNOTES Submitted 15 Oct 2014; accepted for publication 28 Jan 2015.
1
Corresponding author. E-mail: [email protected]
Short title: New species of fossil Meliolaceae A new species of Meliolinites associated with Buxus leaves from the Oligocene of Guangxi, southern China Fu-Jun Ma Bai-Nian Sun1 Qiu-Jun Wang Jun-Ling Dong Guo-Lin Yang Yi Yang School of Earth Sciences & Key Laboratory of Mineral Resources in Western China (Gansu Province), Lanzhou University, Lanzhou 730000, China Abstract: A new species of Meliolinites (fossil Meliolaceae), M. buxi sp. nov., is reported from the Oligocene Ningming formation of Guangxi, South China. The fungus has hyphopodia characteristics of extant Meliolaceae, such as thick-walled, branching hyphae with appressoria and phialides. However, these fossils entirely lack mycelial or perithecial setae and have only a few phialides, thereby distinguishing the new species from most known species. The fungus was discovered on the adaxial and abaxial cuticles of several fossilized Buxus leaves. Thickening and twisting of cell walls in the Buxus leaf cuticle, along with the parasitic feeding strategy of the extant Meliolaceae, suggest that a parasitic interaction between Buxus and M. buxi seems feasible. The distribution of modern Meliolaceae suggests that they live in warm, humid subtropical-tropical climates. It is possible that the presence of M. buxi indicates a similar climatic condition. The co-occurrence of large-leaf Buxus and floristic comparisons of the Ningming assemblage also corroborate this conclusion. Key words: Buxaceae, fossil Meliolaceae, palaeoecology, parasitism
Copyright 2015 by The Mycological Society of America.
INTRODUCTION To date 1980 species are classified in the family Meliolaceae, widely distributed in the tropics and subtropics (Kirk et al. 2008). They are obligate plant parasitic fungi and usually parasitic on adult leaves. These fungi infect plant species in numerous families and cause diseases that are typically benign and commonly known as black or dark mildews. Species of this family are characterized by superficial, dark, thick-walled and branching hyphae with appressoria and phialides. In China 341 species and varieties belonging to seven genera of modern Meliolaceae are reported (Song et al. 2002). The largest genus is Meliola Fr. with 239 species and varieties, followed by Asteridiella McAlpine with 54, Appendiculella Höhn. with 22, Irenopsis F. Stevens with 16, Armatella Theiss. & Syd. with five, Prataprajella Hosag. with three and Amazonia Theiss. with two (Hu 1996, Song et al. 2002). The genus Armatella is characterized by hyphae with no or few phialides (Hu 1996), distinguishing it from the other six genera. Only Asteridiella lacks setae in the Meliolaceae (Hu 1996). In contrast with the high diversity of extant Meliolaceae, there are few fossil representatives. Some records are palynological, focusing exclusively on morphology of fungal spores (Sen and Banerjee 1990, Yeloff et al. 2007, Kar et al. 2010), while other reports emphasize similarities to living forms but lack detailed information about hosts or patterns of infection (Daghlian 1978, Mandal et al. 2011). Relatively few studies investigate the relationships of fossil fungi with their hosts (Dilcher 1965, Selkirk 1975, van Geel et al. 2006) or use Fungi as paleoenvironmental indicators (Dilcher 1963, Daghlian 1978). In the present paper we present several epiphyllous fungi associated with leaf macrofossils of Buxus collected from the Oligocene Ningming formation of Ningming,
Guangxi, southern China. A new fossil species of Meliolaceae is described on the basis of a detailed study using light microscopy (LM) and scanning-electron microscopy (SEM). We studied the host plant leaves in detail and discuss the interaction between the fungus and its host plant. Finally the distribution of extant Meliolaceae let us make inferences about the palaeoecology of Ningming during the Oligocene. MATERIAL AND METHODS Host leaves were collected from the Ningming formation in western Ningming County (22°08′20.6″N, 107°01′47.3″E), Guangxi, southern China. The Ningming Formation is most likely the Oligocene (33.9–23.03 MYA), on the basis of palynostratigraphy (Wang et al. 2003), fishes (Chen and Chang 2011) and plant macrofossils (Li et al. 2003), although absolute dating is lacking. Host leaves were photographed, first with a Sony Super Steady Shot DSC-T70 camera for general morphology, and then with a Leica MZ 12.5 stereoscope to identify areas with fungal colonies; the leaves then were marked. Next 5% hydrogen peroxide was dripped evenly on the marked surface of the leaf compression to remove the leaf from the matrix. Leaf fragments were immersed in a 10% HCl solution before treatment with 50% HF solution. They were placed in a 5% KOH solution for 1–7 d. When the dark brown leaves changed to light brown, samples were removed from the solution. Without further treatment the fungi could then be clearly observed on the fossil cuticles under LM and were photographed with a Leica MZ 12.5 stereoscope. Next a drop of 5% NH4OH was added until the adaxial and abaxial cuticles separated. Samples were mounted on stubs, coated with gold and examined under SEM (JEOLJSM-6380LV) at Lanzhou University, China. For comparison leaves of extant Buxus is collected from the Herbarium of School of Life Sciences, Lanzhou University. The cuticles of extant specimen were prepared following the experimental treatments described by Li et al. (2010). All specimens, cuticle slides and stubs for SEM are deposited at the Institute of Paleontology and Stratigraphy, Lanzhou University, Gansu Province, China. For descriptions of Meliolaceae we followed the format of Hu (1996). For illustrations of host leaf morphology we followed the Leaf Architecture Working Group (Ellis et al. 2009), while terminology for cuticles follow Dilcher (1974). Specimens are deposited in the Institute of Paleontology and Stratigraphy, Lanzhou University, China collection (LDGSW).
TAXONOMY Meliolinites buxi Fu-Jun Ma et Bai-Nian Sun sp. nov.
FIGS 1, 2, 3
MycoBank MB811012 Typification: CHINA: southern China. Guangxi, Ningming County, Ningming Formation (Oligocene), Fu-Jun Ma, 16 Aug 2013 (holotype LDGSWF2013021). Paratype: Same locality, collector and dates (LDGSWF2013022, LDGSWF2013023, LDGSWF2013024). Etymology: The specific epithet buxi is derived from the fossil host. Diagnosis: Hyphae septate, thick-walled, bearing many appressoria and occasional phialides. Hyphal cells cylindrical, thick-walled, 14.0–20.0 × 5.0–8.0 μm. Appressoria bicellular; stalk cells cylindrical, 5.0–7.5 × 8.0–9.0 µm; head cells globose, pyriform, rounded-angulose or 2–4-lobed, 13.5–16.5 × 13.5–20.5 µm. Phialides ampulliform, 12.5–20.5 × 6.5–14.5 µm, mixed with appressoria. Perithecia round in outline, with crenate to crenulate edges, 150.0–230.5 μm diam. Mycelial and perithecial setae absent. Description: Colonies amphigenous (FIG. 3A–D), dense (FIG. 2A) or scattered (FIG. 2B), dark brown to black (FIG. 2A–B), 1.0–2.0 mm diam (eight measurements). Vegetative hyphae dark brown to black (FIG. 2B–D), septate (FIG. 3B, G), thick-walled (FIG. 3B, G), straight to slightly undulate, branching unilateral or opposite (FIGS. 2D, 3A–D) at 40°–80° (25 measurements), bearing many appressoria and occasional phialides (FIGS. 2D, 3A–D). Hyphal cells cylindrical, thick-walled (FIG. 3B, G), 14.0–20.5 μm × 4.9–8.0 μm (25 measurements). Appressoria always branching alternately while sometimes appearing unilateral (FIGS. 2D, 3A–D); each appressorium comprising two cells, a short stalk cell and a capitate head cell (FIGS 2D, 3A–D, H, I); stalk cells dark brown (FIG. 2D), cylindrical (FIGS. 2D, 3A–D, H, I),
5.1–7.6 × 7.8–9.1 µm (25 measurements), sometimes short and inconspicuous (FIG. 2D). Head cells dark brown (FIG. 2D), 13.5–16.5 × 13.5–20.5 µm (20 measurements), thick-walled (FIG. 3I), globose or pyriform (FIGS. 2D, 3C, D), often irregularly rounded-angulose (FIG. 3A, B), sometimes 2–4-lobed (FIG. 3H). Phialides sparse (FIGS. 2D; 3A, C), brown (FIG. 2D), mixed with appressoria, opposite to alternate, ampulliform (FIGS. 2D, 3A, C), 12.5–20.5 × 6.5–14.5 µm (SIX measurements). Perithecia dark brown to black, scattered, round in outline (FIG. 2B, C), ~ 150.0–230.0 μm diam (five measurements); hyphae radiating from the perithecia (FIG. 2C). No mycelial setae, perithecial setae, ascospores or conidia observed. Host plant: Leaves ovate (FIG. 4A). Apex reflex and convex terminating with a retuse tip (FIG. 4A). Base with obtuse angle, convex (FIG. 4A). Margin entire (FIG. 4A). Leaf venation pinnate, major secondary cladodromous (FIG. 4A). Secondaries together with higher-order veins, fused into a strong continuous marginal secondary (FIG. 4A, C). A range of variation in the higher order venation can be observed in the host leaves, from almost no recognizable secondaries to weakly organized secondaries (FIG. 4A, C). Leaf epidermal cells generally pentagonal and hexagonal (FIG. 3F). Anticlinal walls 1.0–2.0 μm thick (30 measurements), straight or rounded (FIG. 3F). Stomata anomocytic, rounded to broadly ovate (FIG. 4E, G), with a prominent outer ring (FIG. 4E), ~ 6.0 μm thick (30 measurements). The host fossil leaf is similar to extant Buxaceae in gross morphology. Buxaceae is a small family comprising five genera Buxus, Notobuxus, Sarcococca, Pachysandra and Styloceras (Min and Brückner 2008). These genera are different in venation pattern. Buxus is characterized by pinnate venation, whereas Sarcococca and Pachysandra always display triplinerved venation (Min and Brückner 2008). Also Notobuxus, endemic to Africa and Styloceras restricted in Ecuador, are different from
the present fossil in the venation patterns. The examples are available on http://specimens.kew.org/herbarium/K000353978 and http://specimens.kew.org/herbarium/K000573598. In addition, the current species and Buxus are identical in stomatal structure with the prominent outer ring on guard cells (Kvaček et al. 1982). Fossil leaves are similar to extant Buxus in architectural (FIG. 4A–D) and cutical features (FIG. 4E–H). Therefore the host leaves are consistent with classification in the genus Buxus (Buxaceae). A detailed taxonomy of these host leaves is addressed in another study. DISCUSSION The fossil fungus that we discovered on the Oligocene Buxus leaves in southern China exhibit hyphopodia characteristics of extant Meliolaceae, but it lacks mycelial and perithecial setae. This combination of characters is unusual in extant Meliolaceae, except for the genus Asteridiella. Furthermore, these fossils have a few phialides, a feature observed only in species of Armatella. Because our fossils combine characters not seen in extant genera, it is most appropriate to assign them to the fossil genus Meliolinites, erected for fossil epiphyllous fungi lacking setae but otherwise possessing the general characters of the Meliolaceae (Selkirk 1975, Daghlian 1978). The fossil record of the Meliolaceae epiphyllous fungi is limited and is restricted to fossils of angiosperm leaves. Köch (1939) described material belonging to Meliolaceae from the Eocene of Germany. However, comparisons of his descriptions with M. buxi are difficult because his illustrations are incomplete. Two distinct species of Meliolaceae, Meliolinites spinksii (Dilcher) Selkirk and M. anfractus (Dilcher) Kalgutkar & Janson., were discovered from the Eocene of Tennessee (Dilcher 1965, Selkirk 1975, Kalgutkar and Jansonius 2000). M. spinksii
can be easily distinguished from the new species because of its opposite appressoria and the shape of its head cells. Head cells of M. spinksii are oblong to ovoid (Dilcher 1965, Kalgutkar and Jansonius 2000). Whereas M. buxi has globose, pyriform, rounded-angulose or 2–4-lobed head cells. M. anfractus displays mycelial setae arising directly from hyphal cells (Dilcher 1965, Kalgutkar and Jansonius 2000) and thus obviously differs from the present species. In addition, head cells of M. anfractus are always lobate (Dilcher 1965, Kalgutkar and Jansonius 2000), which are different from that of M. buxi. M. dilcheri Daghlian, from the early Eocene of Texas (Daghlian 1978), can be easily distinguished from the present fungus by the absence of phialides in its colonies. M. nivalis Selkirk with typical spores, mycelia and hyphopodia, was reported from the Miocene of New South Wales, Australia (Selkirk 1975). It clearly differs from the present species in its shape of appressoria and its hyphal features. Hyphal cells of M. nivalis are 19.0–34.0 µm long (Selkirk 1975), which are longer than that of the new species. Head cells of M. nivalis are irregularly globose (Selkirk 1975), while head cells of M. buxi are various, from globose, pyriform, to rounded-angulose or 2–4-lobed. M. siwalika Mandal, Samajpati & Bera was discovered from the Miocene in West Bengal, India (Mandal et al. 2011). It differs by the longer shape of the appressoria and the much larger hyphal cells. Meliola ellis Roum. from the Holocene of northern England (van Geel et al. 2006) can be easily distinguished from the present species by its distinct setae. Fossil species often are inferred to exhibit similar living strategies as their nearest living relatives (Mosbrugger 1999). Extant fungi in Meliolaceae demonstrate a limited range of biological interactions and are obligate parasites on plant leaves and stems. Therefore we speculate that M. buxi was parasitic on Buxus. In addition the observation of well-preserved appressoria, whose main function is to penetrate a host
(Emmett and Parbery 1975), may be another indicator of parasitism. While characterizing interactions among fungi and host plants, the presence or absence of a host response provides a wealth of information for understanding the interaction (Taylor et al. 2004). Evidence that the host of M. buxi was alive at the time of infection includes the thickening and twisting of epidermal cell walls in the Buxus cuticle. The walls of uninfected leaf epidermal cells are straight or rounded and are typically 1.0–2.0 μm thick (FIG. 3F). However, infected leaf epidermal cells appear under or near the fungal colonies and are more curved and much thicker, 5.0–6.0 μm (FIG. 3E). These thickened and twisted leaf epidermal cell walls represent a clear host response. Similar responses to fungal invaders are common in extant plants (Taylor and Osborn 1996). Fossil fungi can provide significant information about the palaeoecology and past habitats (Lange 1978, van Geel et al. 2003, Chambers et al. 2012). Species of extant Meliolaceae are distributed mainly in tropical to subtropical zones (Kirk et al. 2008). Hence the presence of M. buxi is generally indicative of a warm and humid tropical to subtropical climate in the area during deposition. Preliminary floristic study of the Ningming flora reached a similar conclusion. The fungus was found in a forest assemblage dominated by angiosperms, including Fabaceae, Lauraceae, Fagaceae, Moraceae, Arecaceae, Hamamelidaceae, Betulaceae, Anacardiaceae, Simaroubaceae, Juglandaceae, Ulmaceae and Sapindaceae (Shi 2010). A preliminary study indicates that the Ningming flora represents a tropical-subtropical forest (Shi 2010). Kvaček et al. (1982) noted that the genus Buxus as a whole ranges from subtropical to warm temperate and mesic to even xerophilous (although rarely) in ecological preferences. But the large-leaf Buxus in Asiatic taxa are associated with tropical-subtropical conditions. The Buxus illustrated in this paper is about 45.0 mm long (FIG. 4A), which
fit in the living large-leaf forms. The occurrence of large-leaf Buxus may also might be indicative of a tropical-subtropical condition. In addition, based on palynological studies of Buxus from the early Paleogene to the late Miocene of Europe, Kvaček et al. (1982) suggested that Buxus was a component of tropical-subtropical flora. These analyses all corroborate our conclusion that the fossil M. buxi was deposited and lived in a warm and humid tropical to subtropical climate. ACKNOWLEDGMENTS We thank Executive Editor Dr Seifert, Associate Editor Conrad Schoch and two anonymous reviewers for their invaluable suggestions and thoughtful comments for improving the manuscript. We also thank Ru-Yong Zheng from the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, for her advice on the paper. We also thank David L. Dilcher from the Departments of Geology and Biology, Indiana University, and Subir Bera from the Centre of Advanced Studies, Department of Botany, University of Calcutta, for their kindness in providing scientific literature on fossil Meliolaceae. This work is supported by the National Basic Research Program of China (973 program number 2012CB822003), the National Natural Science Foundation of China (Grants 41172022, 41272026) and the Fundamental Research Funds for the Central Universities (No.lzujbky-2014-285).
LITERATURE CITED Chambers FM, Booth RK, de Vleeschouwer F, Lamentowicz M, le Roux G, Mauquoy D, Nichols JE, van Geel B. 2012. Development and refinement of proxy-climate indicators peats. Quat Int 268:21–33.
Chen GJ, Chang MM. 2011. A new cyprinin from Oligocene of south China. Sci China Earth Sci 54:481–492.
Daghlian CP. 1978. A new melioloid fungus from the early Eocene of Texas. Palaeontology 21:171–176.
Dilcher DL. 1963. Eocene epiphyllous fungi. Science 142:667–669.
———. 1965. Epiphyllous fungi from Eocene deposits in western Tennessee, USA. Palaeontogr Abt B 116:1–54.
———. 1974. Approaches to the identification of angiosperm leaf remains. Bot Rev 40:1–157.
Ellis B, Daly DC, Hickley LJ, Johnson KR, Mitchell JD, Wilf P, Wing SL. 2009. Manual of Leaf Architecture. New York: Cornell Univ. Press. 190 p.
Emmett RW, Parbery DG. 1975. Appressoria. Annu Rev Phytopathol 13:147–165.
Hu YX. 1996. Flora Fungorum Sinicorum. Vol. 4. Meliolales I. Beijing: Science Press. 270 p. (in Chinese)
Kalgutkar RM, Jansonius J. 2000. Synopsis of fossil fungal spores, mycelia and fructifications. Dallas, Texas: AASP Contrib Ser 39 AASP Found.
Kar R, Mandaokar BD, Kar RK. 2010. Fungal taxa from the Miocene sediments of Mizoram, northeast India. Rev Palaeobot Palynol 158:240–249.
Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi.10th ed. Walingford, UK: CAB International.784 p.
Köch C. 1939. Fossile Kryptogamen aus der eozänen Braunkohle des Geiseltales. Nova Acta Acad Lepop Carol 6:333–359.
Kvaček Z, Bužek Č, Holý F. 1982. Review of Buxus fossils and a new large-leaved species from the Miocene of central Europe. Rev Palaeobot Palynol 37:361–394.
Lange RT. 1978. Southern Australian Tertiary epiphyllous fungi, modern equivalents in the Australasian region and habitat indicator value. Can J Bot 56:532–541.
Li HM, Chen YF, Chen GJ, Kuang GD, Huang ZT. 2003. Tertiary fossil winged fruits of Palaeocarya from Ningming of Guangxi, S. China. Acta Palaeontol Sin 42:537–547. (in Chinese with English abstract)
Li XC, Sun BN, Xiao L,Wu JY, Lin ZC. 2010. Leaf macrofossils of Ilex protocornuta sp. nov. (Aquifoliaceae) from the Late Miocene of east China: implications for palaeoecology. Rev Palaeobot Palynol 161:87–103.
Mandal A, Samajpati N, Bera S. 2011. A new species of Meliolinites (fossil Meliolales) from the Neogene sediments of sub-Himalayan West Bengal, India. Nova Hedwigia 92:435–440.
Min TL, Brückner P. 2008. Buxaceae. In: Wu ZY, Raven PH, Hong DY, eds. Flora of China. Vol. 11, Science Press, St Louis: Missouri Botanical Garden Press. p 321–328.
Mosbrugger V. 1999. The nearest living relative method. In: Jones TP, Rowe NP, eds. Fossil plants and spores: Modern techniques. London: Geological Society. p 261–265.
Selkirk DR. 1975. Tertiary fossil fungi from Kiandra, New South Wales. Proc Linn Soc NSW 100:70–94.
Sen PK, Banerjee M. 1990. Palyno-plankton stratigraphy and environmental changes during the Holocene in the Bengal Basin, India. Rev Palaeobot Palynol 65:25–35.
Shi GL. 2010. Fossil plants from the Oligocene Ningming Formation of Guangxi,and a preliminary palaeoclimatic reconstruction of the flora [doctoral dissertation]. Nanjing Institute of Palaeontology and Geology: Chinese Academy of Sciences. (in Chinese with English abstract)
Song B, Li TH, Shen YH. 2002. Ecological and floristic analyses of Meliolales (Fungi) in China. J Trop Subtrop Botany 10:118–127.
Taylor TN, Klavins D, Krings M, Taylor EL, Kerp H, Hass H. 2004. Fungi from the Rhynie chert: a view from the dark side. Trans R Soc Edinburgh. Earth Sci 94:457–473.
———, Osborn JM. 1996. The importance of fungi in shaping the paleoecosystem. Rev Palaeobot Palynol 90:249–262.
van Geel B, Aptroot A, Mauquoy D. 2006. Sub-fossil evidence for fungal hyperparasitism (Isthmospora spinosa on Meliola ellisii, on Calluna vulgaris) in a Holocene intermediate ombrotrophic bog in northern England. Rev Palaeobot Palynol 141:121–126.
———, Buurman J, Brinkkemper O, Schelvis J, Aptroot A, van Reenen G, Hakbijl T. 2003. Environmental reconstruction of a Roman-period settlement site in Uitgeest (the Netherlands), with special reference to coprophilous fungi. J Archaeol Sci 30:873–883.
Wang WM, Chen GJ, Chen YF. 2003. Tertiary palynostratigraphy of the Ningming Basin, Guangxi. J Stratigr 27:324–327.
Yeloff D, Charman D, van Geel B, Mauquoy D. 2007. Reconstruction of hydrology, vegetation and past climate change in bogs using fungal microfossils. Rev Palaeobot Palynol 146:102–145.
LEGENDS FIG. 1. Meliolinites buxi (holotype), showing hyphae with many appressoria and occasional phialides; bar = 50 μm. FIG. 2. Mycelial colonies of M. buxi under LM. A. Mycelial colony, LDGSWF2013022. B. Mycelial colony, LDGSWF2013021. C. Magnified view of B, showing perithecium. The black arrow indicates radiating hyphea. D. Magnified view of B, showing hyphae bearing appressoria and phialides. The white arrow indicates an appressorium with cylindrical stalk cells; the black arrow indicates an appressorium with short and inconspicuous stalk cells; the black arrowhead refers to an ampulliform phialide. Bars: A = 1 cm, B = 500 μm, C = 200 μm, D = 50 μm. FIG. 3. Detailed characters of Meliolinites buxi and indications of reactions to the parasitism on the host leaf under SEM. A–B. Hyphae on the surface of adaxial leaf cuticles mainly bear appressoria, LDGSWF2013023. Black arrows indicate ampulliform phialides. C–D. Hyphae on the surface of abaxial leaf cuticle, indicated by the occurrence of stomata with prominent outer rings, LDGSWF2013024. The black arrow indicates an ampulliform phialide. E. Thickened and twisted epidermal cell walls in the Buxus leaf cuticle. The black arrow indicates the thickened and twisted epidermal cell wall. F. Uninfected epidermal cells in the Buxus leaf cuticle. The black arrow indicates the uninfected epidermal cell wall. G. Magnified view of B. The black arrow indicates a septum in a hyphal cell. H. Magnified view of A, showing the shape of appressoria. The white arrow indicates a 2–4-lobed head cell; white arrowhead indicates a cylindrical stalk cell. I. Amplification of B, showing the characters of appressoria. The white arrow indicates a globose and thick-walled head cell; the black arrow indicates a cylindrical stalk cell; bars: A, C, E, F = 50 μm;. B = 20 μm; D = 100 μm; G–I = 5 μm. FIG. 4. A, C, E, G. Fossil leaf of Buxus; B, D, F, H. Extant Buxus sp. A. Fossil leaf of Buxus. B. Extant Buxus sp. for comparison with the fossil. C. Details of fossil specimen, showing vein architecture. D. Details of extant Buxus sp., showing vein architecture. E. Stomatal apparatus of the fossil leaf, outer surface. F. Stomata apparatus of Buxus sp., outer surface. G. Stomatal apparatus of the fossil leaf, inner surface. H. Stomatal apparatus of Buxus sp., inner surface. Bars: A–B = 5 mm. C–D = 2 mm. E–G =5 μm. H = 10 μm.
FOOTNOTES Submitted 15 Oct 2014; accepted for publication 28 Jan 2015.
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