Mycologia, 105(3), 2013, pp. 521–529. DOI: 10.3852/12-272 2013 by The Mycological Society of America, Lawrence, KS 66044-8897 # Paleomycology of the Princeton Chert I. Fossil hyphomycetes associated with the early Eocene aquatic angiosperm, Eorhiza arnoldii Ashley A. Klymiuk1 Thomas N. Taylor Edith L. Taylor INTRODUCTION Although several Eocene deposits are known from western Canada’s Okanagan Highlands, the majority of these sites contain fossil plants preserved only as compressions. By contrast, the fossil plants of the Princeton Chert have been anatomically preserved (such that a cellular level of detail is available for study) within a succession of silicified, coal-forming peats. Because microbial biota associated with plant tissues also are subject to permineralization (Taylor et al. 2005, Dotzler et al. 2008), the Princeton Chert constitutes not only a pre-eminent paleobotanical locality but also an important opportunity to examine a microbial assemblage in the context of a well described and highly diverse Eocene flora. The floristic components of the peat-forming Princeton mire have been intensively documented over the past 30 y, and the described flora includes several filicalean ferns (Basinger and Rothwell 1977, Stockey et al. 1999, Karafit et al. 2006, Smith et al. 2006) and three conifers, two of which have been reconstructed as whole plants (Rothwell and Basinger 1979, Stockey 1984, Klymiuk et al. 2011). Angiosperms, however, comprise most of the taxonomic diversity and include fruits, seeds and vegetative organs attributed to basal angiosperms and magnoliids (Cevallos-Ferriz and Stockey 1989, 1990a; Smith and Stockey 2007; Little et al. 2009), monocots (Erwin 1987; Cevallos-Ferriz and Stockey 1988a; Erwin and Stockey 1989, 1991, 1994; Smith and Stockey 2003) and core eudicots (Basinger 1976, Cevallos-Ferriz and Stockey 1988b, 1990b, 1991; Erwin and Stockey 1990; Cevallos-Ferriz et al. 1993; Pigg et al. 1993; Stockey et al. 1998; Little and Stockey 2003). Several flowering plants also cannot be confidently placed in systematic context, including the rhizomatous vegetative axes of an emergent or aquatic dicot, Eorhiza arnoldii Robison et Person (1973. Stockey and Pigg 1994). In contrast to the flora, fungal diversity within the chert has been less comprehensively assessed; most were recognized due to their symbiotic or pathogenic relationships with vascular plants. The coralloid roots of the two dominant conifers hosted arbuscular mycorrhizae and ectomycorrhizae (LePage et al. 1997, Stockey et al. 2001) and several fungi parasitized angiosperms. These include a tar-spot infestation on leaves of the palm, Uhlia allenbyensis Erwin et Stockey (Currah et al. 1997), loculate pseudoparenchymatous mycelia associated with sepals, seeds, and University of Kansas, Department of Ecology & Evolutionary Biology, and Biodiversity Institute, Lawrence, Kansas 66045-7534 Michael Krings Department für Geo- und Umweltwissenschaften, Paläontologie und Geobiologie, Ludwig-MaximiliansUniversität, and Bayerische Staatssammlung für Paläontologie und Geologie, 80333 Munich, Germany Abstract: The Eocene (, 48.7 Ma, Ypresian–Lutetian) Princeton Chert of British Columbia, Canada, has long been recognized as a significant paleobotanical locality, and a diverse assemblage of anatomically preserved fossil plants has been extensively documented. Co-occurring fossil fungi also have been observed, but the full scope of their diversity has yet to be comprehensively assessed. Here, we present the first of a series of investigations of fossilized fungi associated with the silicified plants of the Princeton Chert. This report focuses on saprotrophic, facultative-aquatic hyphomycetes observed in cortical aerenchyma tissue of an enigmatic angiosperm, Eorhiza arnoldii. Our use of paleontological thin sections provides the opportunity to observe and infer developmental features, making it possible to more accurately attribute two hyphomycetes that were observed in previous studies. These comprise multiseptate, holothallic, chlamydospore-like phragmoconidia most similar to extant Xylomyces giganteus and basipetal phragmospore-like chains of amerospores like those of extant Thielaviopsis basicola. We also describe a third hyphomycete that previously has not been recognized from this locality; biseptate, chlamydosporic phragmoconidia are distinguished by darkly melanized, inflated apical cells and are morphologically similar to Brachysporiella rhizoidea or Culcitalna achraspora. Key words: Brachysporiella, Culcitalna, Eocene, fossil fungi, Princeton Chert, Thielaviopsis, Xylomyces Submitted 26 Jul 2012; accepted for publication 13 Oct 2012. 1 Corresponding author. E-mail: klymiuk@ku.edu 521 522 MYCOLOGIA fruits of Decodon allenbyensis Cevallos-Ferriz & Stockey and an Ascochyta-like pycnidial fungus found within some fruits and seeds of Princetonia allenbyense Stockey (LePage et al. 1994). Studies also indicated the presence of a smut associated with floral remains (Currah and Stockey 1991, LePage et al. 1994), but the putative teliospores are now recognized as pollen of Saururus tuckerae Smith et Stockey (Saururaceae; Smith and Stockey 2007). Less emphasis has been placed on fungi occupying predominantly saprotrophic niches, although LePage et al. (1994) proposed that dense sclerotia observed in seeds of the nymphaeaceous dicot Allenbya collinsonae Cevallos-Ferriz et Stockey (1989) might have affinities with Alternaria Nees, an anamorph genus that includes both parasitic and saprotrophic species. These authors also suggested that fungi inhabiting the aerenchymatous tissues of Eorhiza arnoldii were saprotrophic, citing the presence of several species as an indication that the host tissue was moribund. Conidia occurring in E. arnoldii were the first fungi described from the Princeton Chert (Robison and Person 1973) and originally were identified as septate hyphae that formed arthric conidia and phragmospores. LePage et al. (1994) interpreted the former as pleurogenous ‘‘cercosporoid’’ phragmospores and did not differentiate them from the second conidial morphology observed by Robison and Person (1973). By examining additional specimens of Eorhiza rhizomes, we are able to elucidate further details of growth and development for both microfungi, allowing a more confident attribution of these fossils to extant lineages. We also recognize a third hyphomycetous anamorph that has not been previously observed in the Princeton Chert. These microfungi indicate that E. arnoldii was colonized by several fungi before permineralization, and they provide new insight into both the early diagenesis of this fossil plant and its paleoecological context, in addition to expanding our understanding of fungal diversity during the early Eocene. MATERIALS AND METHODS Fungi described in this study occur within cortical tissues of the extinct aquatic angiosperm Eorhiza arnoldii, which occurs within many of the individual bedding planes that comprise the Princeton Chert locality of southern British Columbia, Canada (UTM 10U 678057 5472372; 49u229400N, 120u329480W). The locality is a single inclined exposure that crops out along the east bank of the Similkameen River and is composed of at least 49 layers of chert interbedded with sub-bituminous coal and carbonaceous shale. The 7.5 m thick deposit occurs within the informally named Ashnola Shale, the uppermost unit of the Allenby Formation (Fm) of the Princeton Group (Read 1987, 2000; Mustoe 2011). A volcanic ash within Layer No. 22 of the chert has been radiometrically dated as 48.7 Ma (Smith and Stockey 2007); the age of the locality is therefore latest Ypresian or earliest Lutetian. Slabs of chert containing Eorhiza specimens were selectively sectioned into 3–5 cm2 samples, which were mounted on glass slides with Hillquist Two Part mounting medium (Hillquist, USA). Serial paleontological thin sections were cut with a Buehler PetrothinH; sections were 50–150 mm thick. Photomicrographs were captured directly from the rock surface under oil immersion, with a Leica DC500 CCD attached to a Leica DM5000B transmitted-light compound microscope. Serial photomicrographs of the same specimen at different focal planes were compiled into composite focal-stacked images, produced by selectively erasing specific areas to reveal three dimensionality of the specimen as is visible under transmitted light (after Bercovici et al. 2009). Image processing was performed in Adobe Photoshop CS5 12.1. Specimens and slides are deposited in the Paleobotanical Collections, Natural History Museum and Biodiversity Institute, University of Kansas (Lawrence) (KUPB), under specimen accession numbers 17030 Cbot 001, 17035 Etop # 001, 002; 17035 Ebot #001 and 17037 Fbot #001. RESULTS Type I.—Conidia of a hyphomycetous anamorph have developed preferentially within locules, or intercellular spaces, of cortical aerenchyma, (FIG. 1A), often with more than 50 individual mitospores in similar orientation. The macroconidia are dematiaceous or darkly pigmented smooth-walled, cylindrical and phragmosporic, 75–125 mm long and 7–10 mm diam, with as many as 30–35 transverse septa (FIG. 1A, B). Observation of subtending hyphae at conidial apices and bases (FIG. 1B, at arrows) indicates that conidiogenesis was holothallic. Although some conidial cells exhibit constriction or contraction of the conidial wall (FIG. 1C, at arrow), these features are not regular and do not occur in most conidia (FIG. 1B, D). Furthermore, because constricted intercalary cells exhibit walls that are otherwise of similar thickness to adjacent cells, it is probable that constriction represents a preservational effect, as opposed to indicating alternate-arthric conidiogenesis. Instead, conidiogenesis appears to have been thallic solitary (FIG. 1D, E, F), with individual multiseptate chlamydosporous conidia produced from sparsely branched, 2.5–3.0 mm diam, micronematous conidiophores (FIG. 1E, F, G), indistinct from mycelial hyphae (FIG. 1F). Conidial secession is rhexolytic (FIG. 1E, F), and dispersed conidia may bear remnants of the subtending cell (FIG. 1F, lower arrow). Type II.—This hyphomycetous anamorph exhibits propagation via unequally pigmented, apically dema- KLYMIUK ET AL.: EOCENE HYPHOMYCETES 523 FIG. 1. Type I fossil hyphoymycete. A,B. Holothallic multiseptate chlamydospores occurring within intercellular spaces in cortical aerenchyma of the Middle Eocene vascular plant Eorhiza arnoldii. Note subtending hyphae in B (arrows). C. Chlamydospores attached to hyphae; an intercalary cell (arrow) exhibits shrunken internal cell walls. D–G. Chlamydospores attached to branching mycelia; arrows indicate sites of rhexolytic secession and remnant of torn hyphal cell. A, G: 17037 Fbot #001; B, C, D, E, F: 17035 Ebot #001. Bars 5 25 mm. tiaceous chains of amerospores resembling clavate phragmospores; basipetal holoblastic chains are 15– 25 mm long and 8 mm diam, with 3–6 transverse septa (FIG. 2A, B). Simple septal pores are present between successive cells (FIG. 2B, inset). The conidiogenous cells are 4 mm diam and elongated, 5–9 mm long; some are ampulliform (FIG. 2A, at left arrow), although most are doliiform. Secession is schizolytic, sometimes occurring at the base of conidiogenous cells, which may remain attached to the dispersed spores (FIG. 2A, at right arrow). The full extent of conidiophore morphology is not visible, but conidia may have been borne from a number of short, terminal branches, an inference supported by the distribution of conidia preserved in or near growth position (FIG. 2A, C). It also appears that multiple conidia were produced from the same conidiogenous cell (FIG. 2C, at arrow), and the conidiogenous locus is therefore indeterminate. Type III.—The 20–25 mm long biseptate, dematiaceous, pyriform phragmospores of this hyphomycetous anamorph (FIG. 2D–G) are characterized by an apical cell that is deeply pigmented when mature (FIG. 2D, cf. F), and markedly inflated, up to 15 mm diam. 524 MYCOLOGIA oriented toward associated assimilative mycelia, the hyphal diameters are 2.5–3 mm (FIG. 2G, at arrow). DISCUSSION In their description of the aquatic angiosperm Eorhiza arnoldii, Robison and Person (1973) noted that the material contained abundant fungal remains, including assimilative mycelia and conidia. Subsequent to these early investigations, the cellulose acetate peel technique was modified for use with hydrofluoric acid (Basinger and Rothwell 1977), permitting rapid and extensive exploration of the flora. This may have occurred at the cost of observing the full extent of paleomicrobial diversity, because the peel technique is not optimal for recovery of fungal remains (Taylor et al. 2011). Our re-investigation of Eorhiza arnoldii with paleontological thin sections supports previous assessments of microbial diversity in the Princeton Chert (Robison and Person 1973, LePage et al. 1994) and has made it possible to elucidate developmental features of anamorphs previously known only from dispersed conidia. Although fossil conidia are frequently attributed to palynological form genera, this practice has been criticized for Cenozoic specimens, because many can be attributed to extant lineages (Pirozynski 1976, Pirozynski and Weresub 1979). Therefore, the aim of this study is twofold: In addition to describing these anamorphs in more detail than previously possible, we also seek to identify their probable context among extant fungi. FIG. 2. A–C. Type II fossil hyphomycete. Apically pigmented, phragmospore-like aleuriospores with simple pores (B, inset, at arrow) produced from typically doliiform but occasionally ampulliform (A, at left arrow) conidiogenous cells. Dispersed chains of conidia can appear caudate as a result of schizolytic secession at the base of conidiogenous cells (A, at right arrow). Some conidiogenous cells appear indeterminate (C, arrow). D–G. Type III fossil hyphomycete. Apically inflated, biseptate phragmosporic chlamydospores produced from gracile conidiophores (D, F, at arrows); note possible ‘‘rhizoidal’’ growth of associated hypha (G, at arrow). A: 17035 Etop #002; B, C: 17030 Cbot #001; D, E, F, G: 17035 Ebot #001. Bars 5 25 mm. Conidiogenesis is acrogenous and monoblastic. Conidiogenous cells are isodiametric, 5 mm wide, globose (FIG. 2D, F), and are retained at the base of the dispersed conidium (FIG. 2E) as a consequence of schizolytic secession from the micronematous conidiophore (FIG. 2D, F at arrows). Some hyphae in close association with spores produce curved branches Affinities of type I.—The cylindrical macroconidia redescribed here originally were identified as thallicarthric conidia (Robison and Person 1973). In their review of Princeton Chert fungi, LePage et al. (1994) suggested that the long, multiseptate conidia were produced pleurogenously, and they attributed these conidia to the genus Cercospora Fres. It is now apparent that the conidia were produced via holothallic conidiogenesis, wherein existing hyphae are transformed into conidia by production of transverse septa, enlargement and subsequent melanization. Several conidia exhibit attachment immediately adjacent to branches in the subtending hyphae (e.g. FIG. 1E, F), and some also exhibit attachment to distal hyphae (e.g. FIG. 1B, at arrows). Although Robison and Person (1973) suggested that these spores might disaggregate as arthrospores, there is evidence only for rhexolytic secession of the entire conidium from the subtending hypha. Dispersed spores are common within the aerenchyma of Eorhiza and are invariably long, with no evidence for subsequent alternate-arthric disaggregation. Consequently, it is unlikely that these fossils have close KLYMIUK ET AL.: EOCENE HYPHOMYCETES affinity with extant genera like Eriocercosporella Rak. Kumar, A.N. Rai et Kamal ex U. Braun, in which some schizolytically abscising thalloblastic conidia subsequently break into smaller arthrospores (Braun 1998). Similarly, although developing conidia of Ampulliferina B. Sutton resemble the fossil spores, they subsequently break into oblong didymospores (Ellis 1971). Rhexoampullifera P.M. Kirk also produces short, cylindrical conidia through thallic-arthric conidiogenesis, but in this genus intercalary cells within a conidial chain remain thin-walled and act as the site of rhexolytic secession (Kirk 1982). Elongate, cylindrical phragmoconidia with darkly pigmented, smooth surfaces are typically attributed to the palynological form genus Scolecosporites Lange & Smith (Lange and Smith 1971, Kalgutkar and Jansonius 2000). A number of extant genera produce conidia consistent with this morphology but can be differentiated from the fossils by morphological variations in their conidiogenous cells and conidiophores, which reflect developmental sequences incongruent with those of these fossils. For instance, conidiogenesis in Gangliophora Subram. and Phragmoconidium G.F. Sepúlveda, Pereira-Carvalho & Dianese occurs at fixed, enteroblastic loci (Subramanian 1992, Pereira-Carvalho et al. 2009) , as do phragmoconidia of Fusichalara Hughes & Nag Raj (1973), which are further distinguished by extremely long ‘‘collarettes’’ of hyphal cell wall surrounding the conidiogenous locus. The holothallic conidia described here are most appropriately attributed to the ascomycete Xylomyces Goos, Brooks & Lamore (Dothideomycetes: Jahnulales: Aliquandostipitaceae), a genus of saprotrophic aquatic hyphomycetes that produce thick-walled, dematiaceous, multiseptate chlamydospores (Goos et al. 1977, Goh et al. 1997, Sivichai et al. 2011). The resistant spores of Xylomyces are produced by intercalary hyphal septation, which we infer to have been the mode of conidiogenesis in these fossil fungi (FIG. 1B), and subsequent melanization. Of the eight described species of Xylomyces, most occur in freshwater and produce 3–7 septate conidia (Goos et al. 1977, Goh et al. 1997, Hyde and Goh 1999). However, the chlamydospores of X. chlamydosporus Goos, Brooks, & Lamore may have 14 septa, while those of X. giganteus Goh, Ho, Hyde & Sui possess up to 26 septa (Goos et al. 1977, Goh et al. 1997). The fossils described here are most similar to X. giganteus, although we have not been able to observe irregular longitudinal striations that typically occur on chlamydospore surfaces (Goh et al. 1997) owing to opacity of the chert matrix; nor have we observed intercalary germination in the specimens presently available to us. 525 Affinities of type II.—Although Robison and Person (1973) observed these conidia, they grouped them with the Type I (Xylomyces giganteus-like) chlamydospores and attributed them to the palynological form genus Multicellaesporites Elsik (Sheffy and Dilcher 1971). Because more specimens are now available for study, it is apparent that the Type II conidia are distinct from the Type I chlamydospores, in that conidiogenesis in Type II is holoblastic and the branching conidiogenous cell is terminal upon micronematous conidiophores. Several extant genera produce terminal cylindrical to clavate phragmospores from sympodial conidiogenous cells. It is possible to exclude Marielliottia Shoemaker, because the conidiogenous cells are cicatrized (Shoemaker 1998, Ellis 1971). Rhodoveronaea Arzanlou, W. Gams & Crous and Eriocercospora Deighton have hyaline to lightly pigmented conidia (Deighton 1969, Ellis 1971, Arzanlou et al. 2007), and while the conidia of Brachysporiellina Subram. & Bhat (Subramanian and Bhat 1987, Leão-Ferreira et al. 2008) are dematiaceous, they also are inflated apically, and the indeterminate conidiogenous cells are denticulate. As such, the fossil spores described here are clearly not attributable to these genera. Instead, we consider these fossil fungi to be most similar to Thielaviopsis Went, in which basipetal chains of doliiform amerospores are produced from a weakly sympodial or branching conidiogenous cell (Ellis 1971). A synanamorph frequently found in close spatial association (sometimes even arising from the same mycelium) produces narrow, doliiform to cylindrical enteroblastic hyaline amerospores from obvious phialides (Nag Raj and Kendrick 1975). The dematiaceous conidia, in comparison, are aleuriosporic, and because the cells do not readily undergo schizolytic secession, they often remain attached to hyphae in chains resembling phragmospores (Ellis 1971). Conidiogenesis of aleuriospores from indeterminate loci in Thielaviopsis closely resembles the condition seen in some fossil specimens (e.g. FIG. 2C). Of the four extant species of Thielaviopsis that produce aleurioconidia, the fossils most closely resemble T. basicola (Berk. & Br.) Ferr., because aleurioconidia of other species are globose and solitary (Nag Raj and Kendrick 1975, Paulin-Mahady et al. 2002). However, there is as yet no evidence of synanamorph phialides or endoconidia in association with the fossils, and secession of the individual conidia from basipetal chains has not been observed. Affinities of type III.—The final hyphomycete described in this study has not been previously recognized within the Princeton Chert. It is similar to the palynological form genus Brachysporisporites 526 MYCOLOGIA Lange & Smith (Lange and Smith 1971, Kalgutkar and Jansonius 2000). Relatively few extant fungi produce phragmospores that are as distinctively inflated and apically pigmented as the fossils described here. Some species of Brachysporiellina and Acaracybiopsis J. Mena, A. Hern. Gut. & Mercado are morphologically similar, but in the former conidia are produced from acropleurogenous or sympodial conidiogenous cells, and in the latter conidiogenous cells are percurrent (Subramanian and Bhat 1987, Mena-Portales et al. 1999, Leão-Ferreira et al. 2008), while the fossils are solitary and terminal. The apical inflation of these fossil conidia is similar to that of both Brachysporiella rhizoidea (V. Rao & de Hoog) W.P. Wu, and B. setosa (Berk. & M.A. Curtis) M.B. Ellis. Lengthy percurrent conidiophores like those of B. setosa have not been observed in the fossils, but one specimen (FIG. 2G) may exhibit ‘‘rhizoidal’’ mycelial branching similar to B. rhizoidea (Rao and de Hoog 1986), although Wu and Zhuang (2005) consider this character to be of minor taxonomic value. Although suggestive, the fossils currently available to us are not oriented in such a way as to allow observation of the entire conidiophore, and basal cells of the fossil conidia are more inflated than those of B. rhizoidea. In this latter respect, the fossils are more comparable to chlamydospores of Culcitalna achraspora Meyers & R.T. Moore (Sordariomycetes: Microascales: Halosphaeriaceae). Culcitalna achraspora produces 2–3-septate phragmosporic chlamydospores, in which each cell is inflated, and the most distal cell is deeply pigmented (Meyers and Moore 1960). The phragmospores are borne on micronematous conidiophores that are typically so highly reduced that spores can appear to be borne from hyphae, although longer conidiophores can occur (Meyers and Moore 1960, Seifert et al. 2011). Because the chlamydospores of Culcitalna can exhibit intercalary branching, observation of this character in a fossil specimen would let us more conclusively attribute this hyphomycete to the genus. Although Culcitalna is often regarded as a marine hyphomycete (e.g. Abdel-Wahab 2011), Meyers and Moore (1960) indicated there was no difficulty culturing it on artificial medium prepared with distilled water. Therefore, the occurrence of a Culcitalna-like hyphomycete within the tissues of Eorhiza, which had an aquatic or emergent habit, would not be particularly surprising. This preliminary investigation of fungal diversity within the exquisitely preserved plants of the Princeton Chert indicates that this Eocene mire will prove a significant resource for paleomycologists. By preparing samples of chert in paleontological thin section, we have been able to observe developmental features of several anamorphic fungi preserved within the cortical aerenchyma of Eorhiza arnoldii. As a result, we have been able to better attribute two hyphomycetes described in Robison and Person et al. (1973) and LePage et al. (1994) and have observed chlamydospores not previously identified within the Princeton Chert. All three are attributable to extant lineages, and one appears morphologically congruent with an extant species. The fossil chlamydospores that we suggest are most similar to Xylomyces giganteus are of particular interest as calibration points in molecular divergence hypotheses. Because a holomorphic concept linking the teleomorph Jahnula aquatica (Kirschst.) Kirschst. with its anamorph, X. chlamydosporus, has been established (Sivichai et al. 2011), it is probable that X. giganteus also has its teleomorph among the , 15 species of Jahnula Kirschst. (Hyde and Wong 1999, Pang et al. 2002, Pinruan et al. 2002, Raja and Shearer 2006, Raja et al. 2008, Sivichai and Boonyeun 2010) or else within closely related members of the Jahnulales (Pang et al. 2002). If so, the presence of an X. giganteus-like species within the early Eocene provides a stratigraphically well constrained minimum calibration record for this order of lignicolous freshwater saprotrophs. The three hyphomycetes illustrated in this study also provide additional insight into the paleoecological and taphonomic context of the Eorhiza plant. LePage et al. (1994) suggested that many Eorhiza specimens were moribund, in that several fungal anamorphs are present within most specimens. In addition, we have observed extensive mycelial proliferation, both inter- and intracellularly, with no evidence of host response. The presence of Thielaviopsis-like conidia suggests that Eorhiza might have been infected by a pathogenic fungus in life, because these fungi commonly occur as root pathogens (Paulin-Mahady et al. 2002). We suggest that the other two hyphomycetes are most comparable to genera that are facultative aquatic hyphomycetes and consistently occur on submerged substrates (Meyers and Moore 1960, Rao and de Hoog 1986, Goh and Hyde 1996, Shearer et al. 2007), indicating postmortem colonization of Eorhiza in an inundated setting. Because fossil conidia were produced preferentially within intercellular spaces of the cortical aerenchyma, the host tissue probably was colonized quickly before becoming so degraded as to be waterlogged. By inference, this also suggests that the earliest stages of subsequent permineralization likewise occurred within a short temporal span. Continuing investigations of mycological diversity in association with the silicified plants of this Eocene mire are likely to provide additional specimens of the KLYMIUK ET AL.: EOCENE HYPHOMYCETES fungi described here. In addition to providing calibration points, the discovery of fossil exemplars of extant lineages will continue to expand our understanding of microbial contributions to the paleoecology of the Princeton Chert. ACKNOWLEDGMENTS This research was supported in part by the National Science Foundation (EAR-0949947 to T.N.T. and M.K.; OPP0943934 to E.L.T. and T.N.T.) and the Department of Ecology and Evolutionary Biology, University of Kansas. We gratefully acknowledge Drs. Ruth A. Stockey and Gar W. Rothwell, who made samples of Princeton Chert available to us, and sincerely appreciate advice offered by two anonymous referees, whose suggestions have markedly improved this contribution. LITERATURE CITED Abdel-Wahab MA. 2011. Marine fungi from Sarushima Island, Japan, with a phylogenetic evaluation of the genus Naufragella. Mycotaxon 115: 443– 456, doi:10.5248/115.443 Arzanlou M, Groenewald JZ, Gams W, Braun U, Crous PW. 2007. Phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera. Stud Mycol 58:57– 93, doi:10.3114/sim.2007.58.03 Basinger JF. 1976. Paleorosa similkameenensis gen. et sp. nov. permineralized flowers (Rosaceae) from the Eocene of British Columbia. Can J Bot 54: 2293– 2305, doi:10.1139/b76-246 ———, Rothwell GW. 1977. Anatomically preserved plants from the Middle Eocene (Allenby Formation) of British Columbia. Can J Bot 55: 1984– 1990, doi:10.1139/b77-223 Bercovici A, Hadley A, Villaneuva-Amadoz U. 2009. Improving depth of field resolution for palynological photomicrography: Palaeontologica electronica 12. http:// palaeo-electronica.org/2009_2/170/ Braun U. 1998. A monograph of Cercosporella, Ramularia and allied genera (phyopathogenic Hyphomycetes). Vol. 2. Eiching, Germany: IHW-Verlag. 493 p. Cevallos-Ferriz SRS, Stockey RA. 1988a. Permineralized fruits and seeds from the Princeton Chert (Middle Eocene) of British Columbia: Araceae. Am J Bot 75: 1099–1113, doi:10.2307/2444092 ———. 1988b. Permineralized fruits and seeds from the Princeton Chert (Middle Eocene) of British Columbia: Lythraceae. Can J Bot 66:303–312, doi:10.1139/b88-050 ———. 1989. Permineralized fruits and seeds from the Princeton Chert (Middle Eocene) of British Columbia: Nymphaeaceae. Bot Gaz 150:207–217, doi:10.1086/337765 ———. 1990a. Vegetative remains of the Magnoliaceae from the Princeton Chert (Middle Eocene) of British Columbia. Can J Bot 68:1327–1339, doi:10.1139/b90-169 ———. 1990b. Permineralized fruits and seeds from the Princeton Chert (Middle Eocene) of British Columbia: Vitaceae. Can J Bot 68:288–295, doi:10.1139/b90-039 527 ———. 1991. Fruits and seeds from the Princeton Chert (Middle Eocene) of British Columbia: Rosaceae (Prunoideae). Bot Gaz 152:369–379, doi:10.1086/337899 ———, Erwin DM, Stockey RA. 1993. Further observations on Paleorosa similkameenensis (Rosaceae) from the Middle Eocene Princeton Chert of British Columbia, Canada. Rev Palaeobot Palynol 78: 277– 291, doi:10.1016/0034-6667(93)90068-6 ———, Stockey RA, Pigg KB. 1991. The Princeton Chert: evidence for in situ aquatic plants. Rev Palaeobot Palynol 70:173–185, doi:10.1016/0034-6667(91)90085-H Currah RS, Stockey RA. 1991. A fossil smut fungus from the anthers of an Eocene angiosperm. Nature 350:698–699, doi:10.1038/350698a0 ———, ———, LePage BA. 1997. An Eocene tar spot on a fossil palm and its fungal hyperparasite. Mycologia 90: 667–673, doi:10.2307/3761225 Crane JL, Dumont KP. 1978. Two new Hyphomycetes from Venezuela. Can J Bot 56:2613–2616, doi:10.1139/ b78-313 Deighton FC. 1969. Microfungi IV. Some hyperparasitic hyphomycetes, and a note on Cercosporella uredinophila Sacc. Mycol Pap 118:1–41. Dotzler N, Krings M, Agerer R, Galtier J, Taylor TN. 2008. Combresomyces cornifer gen. sp. nov., an endophytic peronosporomycete in Lepidodendron from the Carboniferous of central France. Mycol Res 112:1107– 1114, doi:10.1016/j.mycres.2008.03.003 Ellis MB. 1959. Clasterosporium and some allied Dematiaceae-Phragmosporae II. Mycol Pap 72:1–75. ———. 1971. Dematiaceous hyphomycetes. Kew, UK: Commonwealth Mycological Institute. 608 p. Erwin DM. 1987. Silicified palm remains from the Middle Eocene (Allenby Formation) of British Columbia. Am J Bot 74:681. ———. 1990. Sapindaceous flowers from the Middle Eocene (Allenby Fm.) of British Columbia, Canada. Can J Bot 68:2025–2034. ———. 1991. Silicified monocotyledons from the Middle Eocene Princeton Chert (Allenby Fm.) of British Columbia, Canada. Rev Palaeobot Palynol 70:147–162, doi:10.1016/0034-6667(91)90083-F ———. 1994. Permineralized monocotyledons from the Middle Eocene Princeton Chert: Arecaceae. Palaeontographica B. 234:19–40. ———, Stockey RA. 1989. Permineralized monocotyledons from the Middle Eocene Princeton Chert (Allenby Fm.) of British Columbia: Alismataceae. Can J Bot 67: 2636–2645, doi:10.1139/b89-340 Goh TK, Ho WH, Hyde KD, Tsui KM. 1997. Four new species of Xylomyces from submerged wood. Mycol Res 101:1323–1328, doi:10.1017/S0953756297004164 ———, Hyde KD. 1996. Biodiversity of freshwater fungi. J Indust Microbiol 17:328–345, doi:10.1007/BF01574764 Goos RD, Brooks RD, Lamore BJ. 1977. An undescribed hyphomycete from wood submerged in a Rhode Island steam. Mycologia 69:280–286, doi:10.2307/3758653 Hughes SJ, Nag Raj TR. 1973. New Zealand fungi 20. Fusichalara gen. nov. NZ J Bot 11: 661– 671, doi:10.1080/0028825X.1973.10430307 528 MYCOLOGIA Kalgutkar RM, Jansonius J. 2000. Synopsis of fossil fungal spores, mycelia and fructifications. Contributions Series, Am Assoc Stratigraphic Palynologists 39:1–429. Karafit SJ, Rothwell GW, Stockey RA, Nishida H. 2006. Evidence for sympodial vascular architecture in a filicalean fern rhizome: Dickwhitea allenbyensis gen. et sp. nov. (Athyriaceae). Int J Plant Sci 167:721–727, doi:10.1086/501036 Kirk PM. 1982. New or interesting microfungi V. Microfungi colonizing Laurus nobilis leaf litter. Trans Br Mycol Soc 78:293–303, doi:10.1016/S0007-1536(82)80013-2 Klymiuk AA, Stockey RA, Rothwell GW. 2011. The first organismal concept for an extinct species of Pinaceae: Pinus arnoldii Miller. Int J Plant Sci 172:294–313, doi:10.1086/657649 Lange RT, Smith PH. 1971. The Maslin Bay flora, South Australia 3. Dispersed fungal spores. Neues Jahrb Geolog Paläontolog, Monatshefte 11:663–681. Leão-Ferreira SM, Cruz ACR, Castañeda Ruiz RF, Gusmão LFP. 2008. Conidial fungi from the semi-arid Caatinga biome of Brazil. Brachysporiellina fecunda sp. nov. and some new records for Neotropica. Mycotaxon 104:309– 312. LePage BA, Currah RS, Stockey RA. 1994. The fossil fungi of the Princeton Chert. Int J Plant Sci 155:822–830, doi:10.1086/297221 ———, Rothwell GW. 1997. Fossil ectomycorrhizae from the Middle Eocene. Am J Bot 84:410–412, doi:10.2307/ 2446014 Little SA, Stockey RA. 2003. Vegetative growth of Decodon allenbyensis (Lythraceae) from the Middle Eocene Princeton Chert with anatomical comparisons to D. verticillatus. Int J Plant Sci 164:453–469, doi:10.1086/ 367811 Mena-Portales J, Hernández-Gutiérrez A, Mercado-Sierra A. 1999. Acarocybiopsis, a new genus of synnematous hyphomycetes from Cuba. Mycol Res 103:1032–1034, doi:10.1017/S0953756298008041 Meyers SP, Moore RT. 1960. Thalassiomycetes II. New genera and species of Deuteromycetes. Am J Bot 47: 345–349, doi:10.2307/2439220 Miller CN Jr. 1973. Silicified cones and vegetative remains of Pinus from the Eocene of British Columbia. Contrib Mus Paleontol Univ Michigan 24:101–118. Mustoe GE. 2011. Cyclic sedimentation in the Eocene Allenby Formation of south-central British Columbia and the origin of the Princeton Chert fossil beds. Can J Earth Sci 48:25–42, doi:10.1139/E10-085 Nag Raj TR, Kendrick WB. 1975. Monograph of Chalara and allied genera. Waterloo, Ontario: Wilfrid Laurier Univ. Press. 165 p. Pang Kl, Abdel-Wahab MA, Sivichai S, El-Sharouney HM, Jones EBG. 2002. Jahnulales (Dothideomycetes, Ascomycota): a new order of lignicolous freshwater ascomycetes. Mycol Res 106: 1031– 1042, doi:10.1017/ S095375620200638X Paulin-Mahady AE, Harrington TC, McNew D. 2002. Phylogenetic and taxonomic evaluation of Chalara, Chalaropsis and Thielaviopsis anamorphs associated with Ceratocystis. Mycologia 94:62–72, doi:10.2307/ 3761846 Pereira-Carvalho RC, Spúlveda-Chavera G, Armando EAS, Inácio CA, Dianese JC. 2009. An overlooked source of fungal diversity: novel hyphomycete genera on trichomes of cerrado plants. Mycol Res 113:262–274. Pigg KB, Stockey RA. 1996. The significance of the Princeton Chert permineralized floras to the Middle Eocene upland biota of the Okanogan Highlands. Wash Geol 24:32–36. ———, ———, Maxwell SL. 1993. Paleomyrtinaea, a new genus of permineralized myrtaceous fruits and seeds from the Eocene of British Columbia and Paleocene of North Dakota. Can J Bot 71:1–9, doi:10.1139/b93-001 Pinruan U, Jones EBG, Hyde KD. 2002. Aquatic fungi from peat swamp palms. Jahnula appendiculata sp. nov. Sydowia 54:242–247. Pirozynski KA. 1976. Fossil fungi. Annu Rev Phytopathol 14: 237–246, doi:10.1146/annurev.py.14.090176.001321 ———, Weresub LK. 1979. The classification and nomenclature of fossil fungi. In: Kendrick B, ed. The whole fungus, the sexual-asexual synthesis. Vol. 2. Proc. 2nd Int. Mycol. Conf., Univ. Calgary, Kananaskis, Alberta. Natl. Mus. Nat. Sci, p 653–688. Raja HA, Shearer CA. 2006. Jahnula species from North and Central America, including three new species. Mycologia 98:319–332, doi:10.3852/mycologia.98.2.319 ———, Carter A, Platt HW, Shearer CA. 2008. Freshwater ascomycetes: Jahnula apiospora (Jahnulales, Dothideomycetes), a new species from Prince Edward Island, Canada. Mycoscience 49:326–328, doi:10.1007/s10267008-0428-2 Rao VG, de Hoog GS. 1986. New or critical Hyphomycetes from India. Stud Mycol 28:1–84. Read PB. 1987. Tertiary stratigraphy and industrial minerals, Princeton and Tulameen Basins, British Columbia. British Columbia, Ministry of Energy, Minerals and Petroleum Resources. Robison CR, Person CP. 1973. A silicified semi-aquatic dicotyledon from the Eocene Allenby Formation of British Columbia. Can J Bot 51: 1373– 1377, doi:10.1139/b73-172 Rothwell GW, Basinger JF. 1979. Metasequoia milleri n. sp., anatomically preserved pollen cones from the Middle Eocene (Allenby Formation) of British Columbia. Can J Bot 57:958–970, doi:10.1139/b79-118 Seifert K, Morgan-Jones G, Gams W, Kendrick B. 2011. The genera of hyphomycetes. CBS Biodiversity Series 9. CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands. 997 p. Shearer CA, Descals E, Kohlmeyer B, Kohlmeyer J, Marvanová L, Padgett D, Porter D, Raja HA, Schmit JP, Thorton HA, Voglymayr H. 2007. Fungal biodiversity in aquatic habitats. Biodivers Conserv 16:49–67, doi:10.1007/s10531-006-9120-z Sheffy MV, Dilcher DL. 1971. Morphology and taxonomy of fungal spores. Palaeontographica Abt B 133:34–51. Shoemaker RA. 1998. Marielliottia, a new genus of cereal and grass parasites segregated from Drechslera. Can J Bot 76:1558–1569. KLYMIUK ET AL.: EOCENE HYPHOMYCETES Sivichai S, Boonyeun N. 2010. Jahnula morakotii sp. nov. and J. appendiculata from a peat swamp in Thailand. Mycotaxon 112:475–481, doi:10.5248/112.475 ———, Sri-Inrasutdhi V, Jones EBG. 2011. Jahnula aquatica and its anamorph Xylomyces chlamydosporus on submerged wood in Thailand. Mycotaxon 116:137–142, doi:10.5248/116.137 Smith SY, Stockey RA. 2003. Aroid seeds from the Middle Eocene Princeton Chert (Keratosperma allenbyense, Araceae): comparisons with extant Lasioideae. Int J Plant Sci 164:239–250, doi:10.1086/346164 ———, ———. 2007. Establishing a fossil record for the perianthless Piperales: Saururus tuckerae sp. nov. (Saururaceae) from the Middle Eocene Princeton Chert. Am J Bot 94: 1642– 1657, doi:10.3732/ ajb.94.10.1642 ———, ———, Nishida H, Rothwell GW. 2006. Trawetsia princetonensis gen. et sp. nov. (Blechnaceae): a permineralized fern from the Middle Eocene Princeton Chert. Int J Plant Sci 167:711–719, doi:10.1086/501034 Stockey RA. 1984. Middle Eocene Pinus remains from British Columbia. Bot Gaz 145:262–274, doi:10.1086/337455 ———. 1987. A permineralized flower from the Middle Eocene of British Columbia. Am J Bot 74:1878–1887, doi:10.2307/2443970 ———, LePage BA, Pigg KB. 1998. Permineralized fruits of Diplopanax (Cornaceae, Mastixioideae) from the Middle Eocene Princeton Chert of British Columbia. Rev Palaeobot Palynol 103:223–234, doi:10.1016/S00346667(98)00038-4 ———, Nishida H, Rothwell GW. 1999. Permineralized ferns from the Middle Eocene Princeton Chert I. 529 Makotopteris princetonensis gen. et sp. nov. (Athyriaceae). Int J Plant Sci 160:1047–1055, doi:10.1086/ 314191 ———, Pigg KB. 1991. Flowers and fruits of Princetonia allenbyensis from the Middle Eocene of British Columbia. Rev Palaeobot Palynol 70:163–172, doi:10.1016/ 0034-6667(91)90084-G ———, ———. 1994. Vegetative growth of Eorhiza arnoldii Robison and Person from the Middle Eocene Princeton Chert locality of British Columbia. Int J Plant Sci 155:606–616, doi:10.1086/297199 ———, Rothwell GW, Addy HD, Currah RS. 2001. Mycorrhizal association of the extinct conifer Metasequoia milleri. Mycol Res 105: 202– 205, doi:10.1017/ S0953756200003221 Subramanian CV. 1992. A re-assessment of Sporidesmium (Hyphomycetes) and some related taxa. Proc Indian Acad Pl Sci 58:179–190. ———, Bhat DJ. 1987. Hyphomycetes from south India I. Some new taxa. Kavaka 15:41–74. Taylor TN, Hass H, Kerp H, Krings M, Hanlin RT. 2005. Perithecial ascomycetes from the 400 million year old Rhynie Chert: an example of ancestral polymorphism. Mycologia 97:269–285, doi:10.3852/mycologia.97.1.269 ———, Krings M, Dotzler N, Galtier J. 2011. The advantages of thin section preparations over acetate peels in the study of late Paleozoic fungi and other microorganisms. PALAIOS 26:239–244, doi:10.2110/palo.2010. p10-131r Wu WP, Zhuang WY. 2005. Sporidesmium, Endophragmiella and related genera from China. Chiang Mai, Thailand: Fungal Diversity Press. 531 p.