Conclusions

Boehm et al. (2009a): Multigene phylogenies indicate that the Hysteriaceae and the Mytilinidiaceae represent two unrelated monophyletic families, situated within the Pleosporomycetidae (Schoch et al. 2007a). We retain the Hysteriales for the Hysteriaceae and establish the order Mytilinidiales for the Mytilinidiaceae. We reserve the genus Glonium for forms closely allied to the type G. stellatum, as circumscribed by von Höhnel (1918) and Petrak (1923a), and place them within the Gloniaceae fam. nov., Pleosporomycetidae incertae sedis. Lastly, we reinstate the genus Psiloglonium Höhn. in the Hysteriaceae, with the type species P. lineare (Fr.) Petrak (Petrak 1923a), to accommodate non-subiculate species previously classified in the genus Glonium, a position supported by Barr (1987). The genus Ostreichnion is transferred to the Hysteriaceae. The apparent lack of appreciable overlap between the Hysteriaceae and the Mytilinidiaceae, and their phylogenetic distance within the subclass, argue for the retention of the character states Barr (1987) initially proposed to delineate the two. These include differences associated with the pseudothecium, peridial wall-thickness, hamathecial type and centrum development, and differences in ascospore symmetry. Although distinct monophyletic core groups have been defined for each family, with high indices of statistical support, genera within the Hysteriaceae display a far higher degree of polyphyly than do those in the Mytilinidiaceae, the genus Ostreichnion notwithstanding (Figs. 2 & 3). We conclude that ascospore septation and pigmentation, features traditionally used to define genera in the Hysteriaceae, are extrememly homoplasious and therefore poor morphological indicators for generic delineation within the family. Similar findings have been reported among other fungal groups (Gueidan et al. 2007; Miller & Huhndorf 2005). Remarkably, the hysterothecium, the classic hallmark of the family Hysteriaceae, long considered a synapomorphic character state, has in fact arisen multiple times among unrelated groups within the Pleosporomycetidae. It is concluded that the hysterothecium defines no fewer than four separate lineages, as represented by the Hysteriaceae, the Gloniaceae, Farlowiella, and by the unusual clade comprising the two geographically disparate isolates of Hg. fraxini. Hysterothecia may offer selective advantages in the form of spore discharge over prolonged periods of time, as a function of relative humidity. These observations suggest that some taxa may be perennial, a view suported by Lohman (1931, 1933a). The ability to perennate, and time spore discharge with environmental conditions suitable for germination, spanning multiple seasons, may be the driving force behind the evolution of the hysterothecium among unrelated groups, since most of these fungi occur on decorticated woody substrates prone to prolonged periods of desiccation. Despite the monophyly of both families, molecular data are clearly at odds with genera delineated by spore septation in the Hysteriaceae, and argue for further, more extensive sampling of taxa for future studies, perhaps involving additional genes and faster evolving characters. Two taxonomic approaches to resolving genera among hysteriaceous fungi are possible. The current genera (e.g., Hysterium, Hysterographium, Gloniopsis, and Ostreichnion) could be split into different genera, or alternatively, the current generic concepts could be considerably widened to include divergent spore septations within a single genus. As for the genus Glonium, there is historical precendent (Petrak 1923a; von Höhnel 1918), for which molecular data are now provided, to split the genus into two unrelated genera, Psiloglonium and Glonium, the former in the Hysteriaceae, the latter in the Gloniaceae. However, until more taxa and/or genes are analyzed, given the cohesiveness of the family as a whole, it is perhaps premature to begin restructuring genera within the Hysteriaceae, the genera Psiloglonium and Glonium notwithstanding. To summarise, our study is the first to provide a large enough range of taxon sampling, combined with a large enough data set, to address evolutionary questions among hysteriaceous fungi. As such, it provides a foundation for future studies. In this study, we have shown that character states previously considered to represent synapomorphies among hysteriaceous fungi, in fact, represent synplesiomorphies and most likely have arisen multiple times through convergent evolutionary processes in response to common selective pressures.

Acknowledgements: The senior author wishes to acknowledge early encouragement for this study from the late Dr Margaret Barr, to whom this manuscript is dedicated. The authors wish to thank Dr G Kantvilas (Tasmanian Herbarium, Hobart, Tasmania), Dr S Lee (Dept. Plant Pathology, University of Stellenbosch, South Africa), Dr M Gryzenhout (Dept. Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa), and Dr MI Messuti (Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, Quintral, Bariloche, Rio Negro, Argentina), for kindly supplying some of the isolates used in this study. Also Dr Walter Gams (Baarn, the Netherlands) is gratefully acknowledged for the Latin translation and numerous helpful insights into the taxonomic issues raised by this work. Dr. Scott Redhead (National Mycological Herbarium, Agriculture and Agri-Food Canada, Ottawa, Canada) is also acknowledged for his nomenclatural insight into the taxonomic history of the genus Hysterium and the type species. The senior author also wishes to acknowledge support from the President of Kean University, Dr. Dawood Farahi, the Kean University Epsilon Corps., and two Kean University Faculty Foundation Research Awards, awarded in 2007 and 2008. The second and third authors acknowledge funding support from the NSF through a grant (DEB 0717476).

Boehm et al. 2009b: Hysteriaceous fungi are an ancient and ecologically successful group of organisms, as attested by their wide geographic distribution on a multitude of gymnosperm and angiosperm host species. Whereas the Mytilinidiaceae are found almost exclusively on conifers, the Hysteriaceae occur primarily on angiosperms (Zogg 1962). Presumably, the Hysteriaceae underwent rapid speciation in response to the angiosperm radiation of the mid- to late-Cretaceous, 65-100 mya (Palmer et al. 2004). However, this must have occurred prior to the complete loss of continental contiguity, which occurred during the same time period. This is because we see today a remarkable degree of intraspecific stability, in both morphology and sequence data, among geographically disparate accessions (Fig. 1). For example, little morphological or sequence variation was detected in Hysterium angustatum, from North America (CBS 123334), Kenya (GKM 243A), New Zealand (SMH 5211.0), and South Africa (CMW 20409; Lee & Crous 2003). Similarly, little variation was detected in Psiloglonium clavisporum, from Kenya (GKM L172A, GKM 344A) and North America (e.g., CBS 123338), or in Oedohysterium sinense, from South Africa (CBS 123345) and North America (EB 0339). As we are presumably sampling remnants of once contiguous sexual populations, their similarity today must imply that speciation occurred prior to complete genetic isolation. The break-up of Pangea during the Triassic 200 mya, and the formation of the nascent central Atlantic Ocean, separating Gondwana from Laurasia, during the Jurassic, 150 mya, must have effectively disrupted once contiguous populations. Although most flowering plant families were established by the end of the Cretaceous, 65-70 mya, it is now believed that they diversified into their present lineages (e.g., eudicots, Magnoliids and monocots) much earlier, around 140 mya (Davies et al. 2004, Palmer et al. 2004, Moore et al. 2007). This may have allowed for remnants of once contiguous populations to colonise early angiosperm lineages, prior to the complete dissolution of continental integrity during the mid- to late-Cretaceous. Recent studies (Lücking et al. 2009), based on a recalibration of published molecular clock trees, using internally unconstrained, uniform calibration points, have suggested an origin for the fungi between 760 mya to 1.0 bya, with the origin of the Ascomycota set at 500-650 mya. Whatever the timing, hysteriaceous fungi incurred little appreciable intraspecific morphological or genetic (e.g., nuLSU, nuSSU, TEF1 and RPB2) change over significant periods of geologic time, on different continents. Thus, with the exception of Hb. mori, and perhaps, Gp. subrugosa (see below), most members of the Hysteriaceae appear to be stable species.

Sequence data indicate that Hb. mori occurs in both Clades A and D. However, analysis of Hb. mori specimens originating from each clade (e.g., CBS 123563 / BPI 878731, and others, in Clade A versus GKM 1013 / BPI 879788 in Clade D), failed to find any appreciable difference in either spore morphology (e.g., septation, pigmentation, symmetry, or measurement), substrate-choice, or features associated with the hysterothecium. Likewise, no morphological difference could be detected among genetically unrelated accessions of Gp. subrugosa, from South Africa (CBS 123346 / BPI 878735), in Clade D, versus those from Kenya (GKM 1214 / BPI 879776) and Cuba (SMH 557 / BPI 879777), outside of Clade D. These two examples illustrate a lack of correspondence between the morphospecies concept (Burnett 2003) and the genealogical concordance phylogenetic species recognition concept (Taylor et al. 2000), the latter indicating here the presence of cryptic species within the two morphospecies. Hysterobrevium mori and, to a lesser extent, Gp. subrugosa, may represent examples of convergent evolution, whereby similar ascospores borne in hysterothecia have evolved multiple times within the family. This is supported by the polyphyly inherent in the circumspection of the classical genera of the Hysteriaceae (e.g., Farlowiella, Gloniopsis, Glonium, Hysterium, and Hysterographium), revealed by recent studies (Schoch et al. 2006, Boehm et al. 2009, Mugambi & Huhndorf 2009). Alternatively, Hb. mori and Gp. subrugosa may have retained ancestral character states, and thus may represent evolutionary lineages that did not incur appreciable morphological change, while at the same time accumulating sufficient genetic change to fall, in the case of Hb. mori, into distant clades within the family. If this is the case, then these two taxa may represent examples of speciation in progress, with genetic change preceding morphological change, thus differing from independent convergent character states. Whatever the mechanism, it is difficult to see how Hb. mori, for example, may be classified into different species, in different genera (e.g., Hysterobrevium and Oedohysterium), without a sound morphological basis. We conclude that both Hb. mori and Gp. subrugosa contain genetically unrelated, cryptic, and potentially different biological species, that can not at present be morphologically differentiated.

Although there are examples of interspecific concordance between morphological and molecular data in this study (see below), these are few. For the most part, molecular data support the premise of a large number of convergent evolutionary lineages, sharing similar spore morphologies, but that are not closely related. This resulted in a polyphyletic core set of genera for the Hysteriaceae, and presented us with a complicated picture of past speciation events within the family (Boehm et al. 2009). To achieve a natural phylogeny, that is, one based on the concordance of morphological and molecular data, required that we break-up what were once thought to be stable genera. Thus, two species of Hysterium were transferred to Oedohysterium (Od. insidens and Od. sinense), and two species of Gloniopsis to Hysterobrevium (Hb. smilacis and Hb. constrictum). While Hysterographium, with the type Hg. fraxini, was removed from the Hysteriaceae (Boehm et al. 2009), some of its species remained within the family, transferred here to Oedohysterium (Od. pulchrum), Hysterobrevium (Hb. mori) and Gloniopsis (Gp. subrugosa). New species were described (e.g., Gp. arciformis sp. nov. and Gp. kenyensis sp. nov.) which would previously have been classified in Hysterographium, but are now accommodated in Gloniopsis. Molecular data necessitated that both Gloniopsis and Hysterobrevium include hyaline and pigmented dictyospores, and the genus Oedohysterium, both phragmospores and dictyospores. This, then, de-emphasised spore morphology as a synapomorphic character state. Likewise, the genus Glonium sensu Zogg (1962) was divided into Psiloglonium in the Hysteriaceae and Glonium in the Gloniaceae (Boehm et al. 2009), and, more recently, Anteaglonium in the Pleosporales (Mugambi & Huhndorf 2009). These taxonomic changes were unexpected, as they were not premised on past assumptions of synapomorphy related to spore morphology (Zogg 1962). Although we have included here a total of 59 accessions, representing 22 species in seven genera, for the Hysteriaceae, and another 62 outside of the family (Table 1), taxon sampling may still be insufficient. Clearly, additional species and genera need to be sampled before a complete picture emerges for the family.

The quest for synapomorphic character states that correlate with molecular data was one of the goals of this study. If traditional character states associated with spore septation/pigmentation or the fruitbody (Zogg 1962) can not be relied upon to deduce phylogeny, are there other character states that can be emphasised instead? Two examples are discussed below, the first relating to spore morphology, the second to characters associated with the fruitbody. Although both Oedohysterium and Hysterium possess similar pigmented asymmetric phragmospores, species of Oedohysterium can be morphologically differentiated by the possession of an enlarged supra-median cell. Molecular data also revealed that a species previously classified as Hysterographium, namely Hg. pulchrum, belonged to Oedohysterium, despite the presence of dictyospores. Closer inspection, however, reveals that the dictyospores of Od. pulchrum also possess a swollen supra-median cell. Additionally, a certain number of spores remain as transversely septate phragmospores (Checa et al. 2007), thus reinforcing its placement within Oedohysterium.

The second example relates to character states associated with the fruitbody. Fruitbody morphology clearly supports the transfer of the genus Glonium out of the Hysteriaceae to its own family, the Gloniaceae, closely allied with the Mytilinidiales. The Gloniaceae possess a modified hysterothecium, one in which the frutibodies frequently bifurcate to a greater (e.g., G. stellatum and G. circumserpens) or lesser (e.g., G. graphicum) degree, the former two species with radiating stellate composites, usually seated on subicula. This is in contrast to hysterothecia found in the Hysteriaceae which may be gregarious, but are never laterally anastomosed to form radiating composites. Additionally, the morphology of the dehiscence slit found in the Gloniaceae is unlike that found in the Hysteriaceae. In the Gloniaceae, the aperture is in most cases evaginated, forming a miniscule crest, similar to the more extended version found in some species in the Mytilinidiaceae; whereas, in the Hysteriaceae, hysterothecia have deeply invaginated slits. Also, hysterothecia found in the Gloniaceae, like those in the Mytilinidiaceae, are considerably more fragile, as compared to those found within the Hysteriaceae. These character states were either not noted before (e.g., swollen supra-median cell in Oedohysterium and evaginated slit in Glonium), or were noticed, but given less taxonomic weight (e.g., modified hysterothecium in Glonium; Zogg 1962). These examples illustrate that morphological features can be found that correlate with molecular data, despite the anomalies presented by Hb. mori and Gp. subrugosa, mentioned earlier.    

The hysterothecium, thick-walled, navicular, and with a prominent longitudinal slit, has long been considered synapomorphic, defining the Hysteriales. However, this type of fruitbody has evolved convergently no less than five times within the Pleosporomycetidae (e.g., Farlowiella, Glonium, Anteaglonium, Hysterographium and the Hysteriaceae). Similarly, thin-walled mytilinidioid (e.g., Ostreichnion) and patellarioid (e.g., Rhytidhysteron) ascomata have also evolved at least twice within the subclass, the genera having been transferred from the Mytilinidiaceae and Patellariaceae, respectively, to the Hysteriaceae. As such, character states relating not only to the external features of the ascoma, but to the centrum as well (e.g., cellular pseudoparaphyses versus trabeculae, etc.), previously considered to represent synapomorphies among these fungi, in fact, represent symplesiomorphies, and most likely have arisen multiple times through convergent evolutionary processes in response to common selective pressures. Similar findings have emerged for a number of other ascomycete lineages within the Pezizomycotina (e.g., Schoch et al. 2009). One selective advantage of the hysterothecium may be spore discharge over prolonged periods of time, since some, if not most, species may be perennial (Lohman 1931, 1933a). The thick-walled peridium further contributes to xerotolerance, as many of these fungi persist on decorticated, weathered woody substrates prone to prolonged periods of desiccation. Thus, the ability to perennate, and time spore discharge with environmental conditions suitable for germination, spanning multiple seasons, may be the driving force behind the repeated evolution of the hysterothecium.

Acknowledgements: The authors wish to thank Alain Gardiennet (Veronnes, France), Gintaras Kantvilas (Tasmanian Herbarium, Hobart, Tasmania), Marieka Gryzenhout (Dept. Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa), Maria Inéz Messuti and Laura Emma Lorenzo (Departamento de Botanica, Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, Quintral, Bariloche, Rio Negro, Argentina), Eunice Nkansah (Kean University, Union, NJ, U.S.A.), and Meredith Blackwell (Dept. Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, U.S.A.) for kindly supplying some of the isolates used in this study (Table 1). The authors wish to thank Walter Gams (Baarn, The Netherlands) for the Latin translations, and for his numerous helpful insights into the taxonomic and nomenclatural issues raised by this work. We also wish to acknowledge Scott Redhead (National Mycological Herbarium, Agriculture and Agri-Food Canada, Ottawa, Canada) who provided helpful suggestions on the manuscript prior to submission. E.W.A. Boehm wishes to acknowledge support from the National Science Foundation (NSF) Major Research Instrumentation Grant DBI 0922603. A.N. Miller acknowledges funding from the NSF through a BS&I award (DEB0515558) and from Discover Life in America (DLIA 2005-15) Work performed by C.L. Schoch after 2008 was supported in part by the Intramural Research Program of the NIH, National Library of Medicine. Part of this work was also funded by a grant from NSF (DEB-0717476) to J.W. Spatafora (and C.L. Schoch until 2008).