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(American Journal of Botany. 2008;95:531-541.) doi: 10.3732/ajb.2007171 © 2008 Botanical Society of America, Inc. |
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Bryology and Lichenology |
2 School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London, E1 4NS, UK 3 Meredith College, Department of Biology and Health Sciences, 3800 Hillsborough Street, Raleigh, North Carolina 27601 USA 4 Dipartimento di Scienze ambientali, Seconda Università di Napoli, via A. Vivaldi 43, 81100 Caserta, Italy
Received for publication 30 May 2007. Accepted for publication 15 February 2008.
ABSTRACT
Liverworts form diverse associations with endophytic fungi similar to mycorrhizas in vascular plants. Whereas the widespread occurrence of glomeromycotes in the basal liverwort lineages is well documented, knowledge of the distribution of ascomycetes and basidiomycetes in derived thalloid and leafy clades is more fragmented. Our discovery that the ramified and septate rhizoids of the Schistochilaceae, the sister group to all other ascomycete-containing liverworts, are packed with fungal hyphae prompted this study on the effects of the fungi on rhizoid morphology, host specificity, the cytology of the association, and a molecular analysis of the endophytes. Two species of Pachyschistochila and their fungi were grown axenically. Axenic rhizoids were unbranched and nonseptate. Reinfected with their own fungus and that from the other species, both Pachyschistochila species produced branched and septate rhizoids identical to those in nature. Woronin bodies and simple septa identified the fungus as an ascomycete referable, according to phylogenetic analyses of ITS sequences, to the Rhizoscyphus (Hymenoscyphus) ericae aggregate, also found in other liverwort–ascomycete associations and in mycorrhizas in the Ericales. Healthy hyphae and host cytoplasm suggest that the Schistochila–fungus association reflects a balanced mutualistic relationship. The recent dating of the divergence of the Jungermanniales from the fungus-free Porellales in the Permian and the origins of the Schistochilaceae in the Triassic indicate that these associations in liverworts predate the appearance of the Ericales.
Key Words: ascomycete evolution • coevolution • leafy liverworts • mycorrhiza • Rhizoscyphus ericae • Schistochilaceae • symbiosis
The establishment of endophytic fungal associations is widely accepted as a major step in the evolution and diversification of land plants (Selosse and Le Tacon, 1998
; Read et al., 2000). These associations are widespread in vascular plants (mycorrhizas) and common in hornworts and liverworts (Duckett et al., 1991; Read et al., 2000), although in the latter two it is still debatable whether these are true symbioses (Wang and Qiu, 2006). Recent phylogenetic analyses reveal that associations with glomeromycotean fungi reflect the ancestral type (Wang and Qiu, 2006 and literature therein) (the Glomeromycota are sister to other mycorrhiza-forming fungi [James et al., 2006]), a conclusion underlined by their widespread occurrence in the basal lineages of both vascular plants and liverworts, not to mention palaeozoic fossils (Remy et al., 1994; Taylor et al., 1999; Krings et al., 2007a, 2007b). A more restricted distribution of ascomycetous and basidiomycetous endophytes in more derived clades is thought to reflect secondary acquisition of these fungi following the loss of the primitive glomeromycotean associations (Kottke and Nebel, 2005; Wang and Qiu, 2006; Berbee and Taylor, 2007).
With modern phylogenetic analyses unanimously identifying liverworts as the earliest divergent land plant clade (Qiu et al., 2006; Renzaglia et al., 2007), it may be anticipated that studies on this group will provide important new insights into the evolution of terrestrial plant–fungus associations. While the Glomeromycota–liverwort associations have been explored in detail from both molecular and cellular standpoints (Ligrone et al., 2007), information on fungal associations in more derived liverwort clades remains fragmentary. Recent analyses reveal that members of the Leafy II clade (Davis, 2004) are either nonsymbiotic or form endophytic associations with Basidiomycota or Ascomycota (Sebacina species and members of the Rhizoscyphus ericae aggr., respectively) (Nebel et al., 2004; Kottke and Nebel, 2005; Duckett et al., 2006b). However, 95% of the species in the Leafy II clade have yet to be analyzed for the presence of fungi (Kottke and Nebel, 2005), and in some families (e.g., Schistochilaceae), existing reports are conflicting (Döbbeler, 1978, 1997;Boullard, 1988; Nebel et al., 2004; Kottke and Nebel, 2005).
The Schistochilaceae (Lepicoleales, Personiellineae; Crandall-Stotler and Stotler, 2000), one of a number of families in the Jungermanniidae (leafy liverworts) with a Gondwanaland distribution (Schuster, 1984), includes some of the largest and most beautiful of all liverworts (Schuster and Engel, 1977, 1985). One of the most distinctive features of the family are their rhizoids (Schuster, 1966; Crum, 2001), which are highly ramified and septate, particularly in the genera where the rhizoids are colorless (Pachyschistochila, Metaschistochila) rather than magenta (Schistochila). Though rhizoid branching is widespread among liverworts and is related to anchorage to the substratum and/or associations with ascomycete fungi (Pocock and Duckett, 1985; Duckett et al., 1991; Read et al., 2000), septation is only known outside the Schistochilaceae in the genera Arachniopsis (Lepidoziaceae) (Schuster, 1965), Xenochila (Plagiochilaceae) (Degenkolbe, 1938) and Vetaforma (Lepioleineae, a suborder allied to the Schistochilaceae) (Fulford and Taylor, 1960). The function of these remarkable rhizoids is unknown, but it is unlikely to be associated with anchorage because many species grow on soft substrata. Schuster (1966) suggested that septation may be associated with the formation of gemmae or may be a mechanism of regeneration. However, as far as we are aware these inferences were based solely upon examination of herbarium specimens collected for taxonomic purposes rather than investigations into the biology of the Schistochilaceae.
In the course of a systematic survey of the distribution of rhizoid-associated, endophytic fungi in leafy liverworts based on freshly collected rather than dried specimens (Duckett et al., 2006b), we made the surprising discovery that the rhizoids throughout the Schistochilaceae are packed with hyphae while the stems lack fungal endophytes. Previous accounts cited an absence of mycobionts in the Schistochilaceae (Nebel et al., 2004; Kottke and Nebel, 2005) nor are any listed by Wang and Qiu (2006). Boullard’s (1988) tabulations included the Schistochilaceae as a liverwort family containing fungi but no details about their histological localization and identity. Döbbeler (1978, 1997) described an Octosporella-like ascomycete that formed appressoria and haustoria on the leaves of Schistochila aligera but made no mention of rhizoid infections.
Our identification, based on electron microscopy, of the rhizoid fungi as ascomycetes led to the experimental and cytological studies described here. Our objectives were (1) to describe the cytology of the rhizoid–ascomycete associations in the Schistochilaceae from observations on a range of freshly collected specimens supplemented by electron microscopy and to compare these with other liverwort–ascomycete infections; (2) to isolate both partners and resynthesize the associations to see whether rhizoid branching and septation is a consequence of fungal infection; (3) to explore host specificity within the Schistochilaceae via cross-infection experiments; and (4) to identify the Schistochila fungus using molecular and phylogenetic techniques.
The results of the current study have major implications in the broad context of the evolution of ascomycete symbioses in land plants.
MATERIALS AND METHODS
A selection of common New Zealand and Chilean Schistochilaceae (Allison and Child, 1975; Schuster and Engel, 1977, 1985; Glenny, 1998) were collected from Nothofagus and mixed podocarp forest at Arthur’s Pass and between Ross and Fox glaciers, South Island, New Zealand and from a Sphagnum magellanicum bog on the shores of Bahia Parry, Provincia Tierra del Fuego, southern Chile. The collections included species with magenta rhizoids [Schistochila alata (Lehm.) Schiffn., S. appendiculata (Hook.) Trevis., S. balfouriana (Hook. f. & Taylor) Steph., S. glaucescens (Hook.) Evans, S. kirkiana Steph., S. lamellata (Hook.) Dum., S. laminigera (Hook.f. & Tayl.) Evans, S. muricata E. A. Hodgs. & Allison, S. nobilis (Hook.) Trevis., S. repleta (Hook. f. & Taylor) Steph., and Paraschistochila pinnatifolia (Hook.) R. M. Schuster] and species with colorless rhizoids [Pachyschistochila childii R. M. Schuster and J. J. Engel, P. succulenta J. J. Engel & R. M. Schuster, P. subimmersa J. J. Engel & R. M. Schuster, and P. splachnophylla J. J. Engel & R. M. Schuster]. Rhizoids were mounted in water and observed using differential interference contrast optics. Although all the species had variously branched and septate rhizoids associated with fungal hyphae, those from Pachyschistochila were the most easily observed. Accordingly, these were selected for electron microscopy and experimental studies.
Rhizoids from wild-collected P. succulenta were fixed and embedded for transmission electron microscopy (TEM) as described previously (Carafa et al., 2003). Scanning electron microscopy (SEM) was carried out on critical-point-dried axenic and reinfected rhizoids of P. subimmersa and P. splachnophylla.
For examining the effects of fungi on their hosts, axenic cultures of P. subimmersa and P. splachnophylla were established from spores on 1/10 Parker nutrient medium solidified with 1% Phytagel (Sigma, St. Louis, Missouri, USA) following surface sterilization of mature undehisced capsules in 1.5% sodium hypochlorite solution (Duckett et al., 2004). The cultures were maintained at 15°C under constant illumination from fluorescent tubes with an irradiance of 50 W•m-2. After ~3 months in axenic culture, the liverworts had formed colonies 5–10 mm in diameter.
Rhizoidal fungi from the two Pachyschistochila species were isolated by surface-sterilizing 20 individual rhizoids from 10 different stems of wild-collected plants in 1.5% hypochlorite for 2–3 min. Following thorough washing in sterile, distilled water, the rhizoids were transferred to 1% Phytagel plates containing no nutrients. After ~3 months, the fungi growing from the isolated rhizoids were added to well-established liverwort colonies, and their ability to reproduce rhizoidal associations like those found in nature was monitored for 6 months. Typical examples from the resyntheses were fixed and embedded for electron microscopy and their cytology compared to wild specimens.
DNA extraction and molecular methods
Pure cultures of fungi isolated from Pachyschistochila subimmersa and P. splachnophylla (two from each species) that had been found to produce typical associations by in-vitro resynthesis were used for DNA extraction and sequencing. Mycelia were ground in 2% sodium dodecyl sulfate (SDS) extraction buffer (0.15 M NaCl, 50 mM Tris [pH 8.0], 10 mM Na2EDTA, and 2% SDS [v/v]) and incubated 10–18 h at room temperature. Proteins were removed and separated with phenol:chloroform: isoamyl alcohol (1:1:24) followed by centrifugation, and DNA was precipitated using 100% isopropanol. Pelleted DNA was washed in 70% ethanol and eluted in Tris (10 mM) EDTA (1 mM). The ITS1, 5.8S, and ITS2 regions of nrDNA were amplified by PCR (polymerase chain reaction) using the primers ITS1 and ITS4 (White et al., 1990) or, if this failed, ITS1F (Gardes and Bruns, 1993) and ITS4, using a ThermoHybaid PCR Express thermocycler with 35 cycles of 1 min at 94° (denaturing), 30 s at 50° (annealing), and 1 min at 72° (extension). PCR amplicons were purified using Qiaquick spin-columns (Qiagen, Valencia, California, USA) according to manufacturer protocols or using a modified shrimp alkaline phosphatase protocol (Werle et al., 1994). Sequencing PCR used Big Dyes v.2.0 (Applied Biosystems, Foster City, California, USA) and an ABI Prism 3700 or 3730 (Applied Biosystems) and the same primers used for amplification. Sequences were compiled and edited using the program Sequencher version 4.2 (Gene Codes Corp., Ann Arbor, Michigan, USA). ITS sequences were compared to the GenBank database using the BLAST algorithm (Altschul et al., 1997) for preliminary identification. Sequences were also compared to those obtained from endophytes isolated from Schistochila appendiculata (Hook.) collected in New Zealand as part of a nontargeted survey of photosynthetic liverwort tissues (Davis, 2005). The ITS sequences obtained in this study have been submitted to GenBank (EU221876 bankit1024273 1 of a total of 2, fungus from P. subimmersa; EU221877 bankit1024274 2 of a total of 2, fungus from P. splachnophylla).
Phylogenetic analyses
Based on preliminary identification using the BLAST search, the ITS sequence obtained from the fungus inhabiting P. splachnophylla was determined to belong to the Rhizoscyphus ericae species aggregate. To test this preliminary identification further, published ITS sequences were downloaded from GenBank and used for phylogenetic analyses. Twenty-nine sequences from R. ericae, 14 outgroups sequences from the genus Hymenoscyphus, and two outgroup sequences from the genus Cudoniella were aligned with the ITS sequence from the fungus inhabiting P. splachnophylla using Sequencher (Appendix 1). Regions of nonoverlap were deleted from the matrix, for a total aligned length of 532 bases, including gaps. Phylogenetic analyses were conducted using a heuristic search and maximum parsimony optimality criteria in the program PAUP* version 4.0b10 (Swofford, 2002) with 100 random addition sequences. The steepest descent option was on, and no more than 100 trees were saved per addition sequence. Support for branching relationships was assessed using the maximum parsimony bootstrap, with 100 replicates and the same search strategy employed in the heuristic search. Hymenoscyphus and Cudoniella (along with R. ericae) are in the Helotiaceae and were chosen to root the phylogeny because they are presumably close relatives of R. ericae.
RESULTS
Observations
Figure 1A, B show infected wild rhizoids of Pachyschistochila splachnophylla and P. subimmersa with branching and septation typical of the genus. The septate apices of the wild-infected rhizoids were packed with hyphae, which often formed a more or less continuous layer over the outer surface (Fig. 1A). The penetration of presumably noncompatible fungi (Martínez-Abigair et al., 2005) was prevented by the formation of ingrowths (pegs) of host wall material (Fig. 1B).
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When cultured with their own fungus and that from the other species, 18 of 20 cultures of P. subimmersa and 19 of 20 of P. splachnophylla produced typical associations, characterized by branched and septate rhizoids (Fig. 2). As in their wild counterparts (Fig. 1A, B), these apices were surrounded by and packed with fungal hyphae.
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Transmission electron microscopy confirmed the parenchymatous nature of the cells at the tips of infected rhizoids (Fig. 3A). While the internal walls of the parenchyma cells forming these structures were extremely thin, the external walls were thick, multilayered, and invested with abundant mucilage (Fig. 3B, C). The uninfected parenchyma cells had a large, central vacuole, several oil bodies, inconspicuous plastids with a rudimentary thylakoid system and little or no starch, and a spheroidal nucleus with prevalently noncondensed chromatin. Fungal hyphae infected the cells by crossing the external walls from the outside, with a dense collar of host wall material around the hyphae marking the entry sites (Fig. 3B). Within the cells, the fungus proliferated to form coils of hyphae about 0.6–0.8 µm in diameter (Fig. 3C). The presence of simple septa associated with Woronin bodies indicated that the endophyte was an ascomycete (Fig. 3F). The intracellular fungus was separated from the host cytoplasm by a perifungal membrane continuous with the host plasmalemma. In young intracellular hyphae, the narrow space between the perifungal membrane and fungal wall had no visible contents (Fig. 3C), while in more mature hyphae, this space contained an abundant interfacial matrix of dense fibrillar material forming prominent outgrowths (Fig. 3D, E). In senescent stages, the hyphae collapsed and aggregated into membrane-bound masses, while the host cells survived fungal degeneration (Fig. 3B). One of the most evident effects of fungal colonization on host cells was a general increase in the electron-opacity of the cytoplasm and the nucleus (Fig. 3E), which obscured structural details. No evidence was found for repeated cycles of infection nor for cell-to-cell hyphal spread.
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Phylogenetic analyses
Parsimony searches returned 9500 trees, one of which is shown in Fig. 4. Bootstrap support values 
70 are shown in Fig. 4. Support for monophyly of the R. ericae clade, including the sequence from the fungus that inhabits P. splachnophylla, is 100%.
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This study reveals that the branched and septate rhizoids in the Schistochilaceae are intimately and obligately associated with ascomycete fungi. Our demonstration that the fungus from Pachy schistochila subimmersa is able to colonize and induce branching and septation of the rhizoids in P. splachnophylla and vice versa is closely in line with earlier cross-infection experiments showing a wide host range for liverwort rhizoidal ascomycetes, even extending to the Ericales (Duckett and Read, 1995). Glomeromycotean endophytes in thalloid liverworts also appear to have wide host ranges (Ligrone et al., 2007), whereas basidiomycete infections in leafy liverworts appear to be more host specific (Kottke et al., 2003; Duckett et al., 2006b).
Molecular findings
The fungal ITS sequences from the fungi isolated from both species of Pachyschistochila confirmed these as ascomycetes. In the case of P. splachnophylla, the BLAST search and phylogenetic analyses strongly suggest that the fungus rests within the Rhizoscyphus ericae aggregate. ITS sequences with 98.4% and 97.9% similarity to this fungus were obtained previously from isolates from the Antarctic liverwort Cephaloziella exiliflora (Chambers et al., 1999). No fewer than 18 of the ITS sequences obtained from much more critical and detailed sampling of the same liverwort are also extremely close to the Pachyschistochila fungus (Upson et al., 2007).
Although Rhizoscyphus ericae was shown in resynthesis experiments to form both rhizoid infections and ericoid mycorrhizas (Duckett and Read, 1995), all these and molecular findings to date are based solely on fungi that can be cultured. Direct sequencing of endophytes in situ, thus removing any bias in favor of culturable fungi, would provide further support for the identity of the fungus involved in the rhizoid infection. Previous nontargeted molecular surveys (designed to provide an estimate of the total number of fungal endophyte communities throughout the plants) revealed that Schistochila appendiculata stems and leaves contained a basidiomycete and both pleosporalean and xylarialean ascomycetes (Davis, 2005) and that a Xylaria species was present in other ascomycetes-associated liverworts (Davis et al., 2003). On the basis of our results, the fungus that infects P. splachnophylla may belong to the Rhizoscyphus ericae group. However, because R. ericae is now known to contain a number of closely related taxa with wide ecological attributes (Vralstad et al., 2000, 2002; Hambleton and Sigler, 2005), for the future, wild-collected rhizoids should be used directly both for sequence analyses and cross-infection studies to establish precisely which strains of fungus produce rhizoid infections and the kinds of mycorrhizal expression these might produce in ectomycorrhizal and ericoid plant hosts.
Developmental considerations
The axenic culture and reinfection experiments clearly demonstrate that rhizoid branching and septation in Pachyschistochila are promoted by the presence of the fungus, but occasional septation in axenic cultures indicates that the potential for cell divisions at the tips of the rhizoids is inherent in the genus. However, we found no evidence, even after many months in culture, that the septate rhizoids in Pachyschistochila are capable of regenerating into new plants. Thus they do not function as asexual propagules as suggested previously (Schuster, 1966). Why, unlike the rhizoids in most other liverworts, nuclear and cell divisions are able to take place in the rhizoid apices of the Schistochilaceae is not immediately apparent, because their cytological organization, with the nucleus occupying a peripheral position behind the apical dome (Fig. 1D), is initially the same as in other unicellular rhizoids (Alfano et al., 1993). However, preceding the first nuclear division the rhizoid nucleus migrates to a central location at the base of the apical dome (Fig. 1E, F), exactly matching the position of the regularly dividing nuclei in the tip cells of moss caulonemata (Duckett at al., 2004). Infected rhizoid apices of P. succulenta and P. splachnophylla are mostly branched (Fig. 1A), whereas those of P. subimmersa are swollen (Fig. 2A) and septation extends well down their shafts (Fig. 2A).
Although the fungus remains confined to the rhizoids in all ascomycete–liverwort rhizoid associations described to date, host rhizoid responses to the fungus differ considerably between taxa. Many members of the Lepidoziaceae produce swollen and often almost spherical rhizoid apices even in the absence of the fungus, whereas swelling and irregular branching of rhizoid tips occur only in the presence of a compatible fungus in the Cephaloziaceae (Duckett et al., 1991; Duckett and Read, 1995). In members of the Calypogeiaceae (N
mec, 1899, 1904; Duckett et al., 1991; Kottke et al., 2003), the fungal hyphae penetrate the basal walls of rhizoids and form peg-like structures projecting into the adjoining parenchyma cells of the stem, but further development in the stem tissue is stopped, possibly due to the deposition of abundant interfacial material of host origin (Duckett et al., 1991). Pachyschistochila is the only documented instance of a liverwort where ascomycetous endophytes colonize a parenchyma tissue, although this tissue is derived from the rhizoids. In the taxa where the rhizoids remain unicellular, the nucleus becomes highly pleomorphic and has a dispersed chromatin arrangement after colonization by a compatible fungus (J. Duckett and R. Ligrone, unpublished data), whereas the nuclei in infected cells of septate rhizoids in Pachyschistochila retain a compact, spheroidal shape as in noninfected cells. The absence of cell-to-cell hyphal spread in Pachyschistochila rhizoidal parenchyma and the multiple penetration sites (Fig. 2G) suggest that, as in single-celled rhizoids, fungal colonization of the parenchyma cells entirely depends on external hyphae.
Functional considerations
The almost labyrinthine deposits of interfacial material, recalling the wall labyrinth of transfer cells, in mature hyphae in Pachyschistochila (Fig. 3E), mirror a similar feature in monotropoid mycorrhizas in angiosperms (Read and Smith, 1997). These wall deposits may reflect an amplification of the host cell membrane, facilitating exchange of nutrients, or alternatively, may be a physical barrier produced by the host to restrict fungal growth. The general increase in the electron-opacity of the cytoplasm and the nucleus in infected cells of Pachyschistochila suggests that accumulation of osmiophilic phenolic compounds may further control the growth of the fungus. The presence of masses of collapsed hyphae in apparently healthy host cells in Pachyschistochila supports the conclusion that the host cells survive fungal colonization while the life-span of the intracellular hyphae is relatively short. In other associations of leafy liverworts with compatible ascomycetes, as well as with basidiomycetes (Duckett et al., 2006b), there is no evidence of hyphal digestion by the hosts, and deterioration of the host cytoplasm precedes that of the intracellular fungus. Absence of fungal digestion is also a characteristic of ericoid mycorrhizas (Read and Smith, 1997). Overgrowths of host wall material at hyphal entry points into the rhizoids are also seen in other liverwort fungal associations, but the extreme electron opacity of this material and the constricted diameter of the hyphae within the original host walls appear to be particular to ascomycete associations. The healthy hyphae and host cytoplasm, including plastids, microbodies, and mitochondria, are features indicative of balanced mutualistic relationships, unlike the necrotic rhizoid cytoplasm associated with sites of failed fungal entry seen in wild rhizoids (Fig. 1B) and in mosses and other liverworts in response to parasitic fungi (Martínez-Abigair et al., 2005; Duckett et al., 2006a). These failed entry sites, marked by prominent pegs of host wall, were never observed in reinfection experiments (Fig. 2A–C).
Although it is reasonable to assume that the fungi obtain carbohydrates from their phototrophic hosts, at present we have no clues as to the possible benefits to the liverworts because the growth and vigor of resynthesized associations was not notably different from that of the liverworts grown axenically. This similarity is possibly because our media contained nutrients and, for the future, growth of the associations under conditions limiting growth of the liverworts may reveal nutritional benefits from the presence of the fungus.
In the context of nutrient transfer from a structural standpoint, it is paradoxical to find the host–mycobiont interface confined to the tips of the rhizoids in the Schistochilaceae and Lepidoziaceae; in both families few or no hyphae grew down the shafts of the rhizoids. In contrast, the peg-like structures developing at the interface between the rhizoid bases and underlying stem cells in Calypogeia and Odontoschisma (Duckett et al., 1991) and similarly in other leafy liverworts infected by basidiomycetes (Duckett et al., 2006b) are suggestive of intense nutrient transfer between the partners. Similarly perplexing in the Schistochilaceae, particularly considering the large size and erect habit of many species with rhizoids confined to their basal regions, was our failure to find any evidence for the occurrence of food conducting cells, like those seen throughout mosses and in some complex thalloid liverworts (Ligrone et al., 2000), that might translocate nutrients to and from the fungus.
An evolutionary perspective
The discovery of ascomycete associations in the Schistochilaceae, when considered in relation to the position of the family in liverwort phylogenies (Davis, 2004; Heinrichs et al., 2005, 2007; Forrest et al., 2006; Hentschel et al., 2006), has implications for the understanding of the evolution of fungal symbioses across both liverworts and vascular plants. Whatever the finer configurations of the leafy liverwort trees, the Schistochilaceae are always resolved as sister to all the other fungus-containing liverwort lineages clustering together in a clade designated as either Leafy II (Davis, 2004) or the Jungermanniales (Heinrichs et al., 2005), while the rest of the leafy liverworts cluster together in the clade reported as Leafy I (Davis, 2004) or the Porellales (Heinrichs et al., 2005) and are not known to establish mycorrhiza-like associations (Nebel et al., 2004; Kottke and Nebel, 2005; Duckett and Ligrone, in press). Most clades within the Leafy II group include members with rhizoidal ascomycetes, whereas the distribution of basidiomycetous endophytes is much more restricted and associations with glomeromycotean fungi are unknown (Duckett et al., 2006b; Ligrone et al., 2007).
These patterns suggest that ascomycete associations predated those with basidiomycetes. Taking account of the predominantly southern hemisphere/Gondwanaland distributions of most of these ascomycete-containing liverworts along with recent dating of the divergence of the Jungermanniales from the fungus-free Porellales in the early to middle Permian and the origins of the Schistochilaceae in the Triassic (Heinrichs et al., 2007), the origins of ascomycete associations in liverworts perhaps date back to in excess of 250 million years ago (Ma). Thus land plant–ascomycete associations might have evolved in liverworts long before their mycorrhizal counterpart in the Ericales, an angiosperm group calculated to have appeared around 106–114 Ma (Wikström et al., 2001). However, as concerns the Schistochilaceae, the highly specialized nature of their ascomycete association and in particular its restriction to the rhizoid apices, suggests a derived origin relative to other liverwort–ascomycete associations. In contrast to these uncertainties concerning the origin of ascomycetes land plant symbioses, the close similarities between glomeromycotean associations in Devonian fossils (Berbee and Taylor, 2007; Krings et al., 2007a, 2007b), thalloid liverworts (Ligrone et al., 2007), and pteridophytes (Duckett and Ligrone, 2005) underlines their undoubted antiquity and suggest a substantial homology in terms of cellular interactions (Ligrone et al., 2007). The possible early origins of the liverwort–ascomycete associations may also have major implications for interpreting the evolution of the ascomycetes per se. A very recent and highly robust fungus phylogeny places the leotiomycetes, the clade containing Rhizoscyphus, as highly derived (James et al., 2006). Although this phylogeny may suggest a relatively recent origin for the liverwort symbioses, dating studies on fungal phylogeny are now needed to test this hypothesis. Instead, such a dating study may reveal that major radiations within the ascomycetes are very old, in line with descriptions of ascomycetes from the Lower Devonian (Taylor et al., 1999) and that liverworts may have played a key role in the evolution of ancient mycorrhizas.
Appendix 1. GenBank accession numbers, taxon names, and references for publicly available ITS sequences used for phylogenetic analyses.
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FOOTNOTES
1 S.P. thanks the National Environmental Research Council (UK) for support from a CASE studentship with the Royal Botanical Gardens, Kew, and a DEFRA Darwin Initiative grant in Chile. J.G.D. was supported by an overseas travel grant from the Royal Society (UK) in New Zealand and by a DEFRA Darwin Initiative grant in Chile. R.L. was supported in New Zealand by a grant from CNR (Italy). This work was in part supported by a grant from Regione Campania, Italy (LR 5, 2003). The ultrastructural observations were in part done at the CISME (University of Naples "Federico II", Italy). E.C.D. acknowledges support from Assembling the Liverwort Tree of Life: A Window into the Evolution and Diversification of Early Land Plants, NSF grant 0531730 to J. Shaw at Duke University in Durham, NC, collaborative project. The authors thank the Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand, and K. S. Renzaglia, Southern Illinois University, Carbondale, Illinois for providing laboratory facilities and the New Zealand Department of Conservation for granting collecting permits. 
5 Author for correspondence (e-mail: s.pressel{at}qmul.ac.uk)
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