Heterobasidiomycetes form symbiotic associations with hepatics: Jungermanniales have sebacinoid mycobionts while Aneura pinguis (Metzgeriales) is associated with a Tulasnella species

Mycol. Res. 107 (8): 957–968 (August 2003). f The British Mycological Society

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DOI: 10.1017/S0953756203008141 Printed in the United Kingdom.

Heterobasidiomycetes form symbiotic associations with hepatics : Jungermanniales have sebacinoid mycobionts while Aneura pinguis (Metzgeriales) is associated with a Tulasnella species Ingrid KOTTKE1*, Alexander BEITER1, Michael WEISS1, Ingeborg HAUG1, Franz OBERWINKLER1 and Martin NEBEL2 1

Botanisches Institut, Eberhard-Karls-Universita¨t Tu¨bingen, Spezielle Botanik, Mykologie und Botanischer Garten, Auf der Morgenstelle 1, D-72076 Tu¨bingen, Germany. 2 Staatliches Museum fu¨r Naturkunde Stuttgart, Rosenstein 1, D-70191 Stuttgart, Germany. E-mail : [email protected] Received 27 January 2003; accepted 31 May 2003.

In order to evaluate substrate dependence of the symbiotic fungal associations in leafy liverworts (Jungermanniopsida), 28 species out of 12 families were investigated by transmission electron microscopy and molecular methods. Samples were obtained from the diverse substrates : from naked soil, from the forest floor on needle litter, from between peat moss, from rotten bark of standing trees, and from stumps and rotten wood. Associations with ascomycetes were found in most of the specimens independent from the substrate. Seven species sampled from soil were found to contain basidiomycete hyphae. Ultrastructure consistently showed dolipores with imperforate parenthesomes. Molecular phylogenetic studies revealed that three specimens belonging to the Jungermanniales were associated with members of Sebacinaceae, while Aneura pinguis (Metzgeriales) was associated with a Tulasnella species. These taxa are so far the only basidiomycetes known to be symbiotically associated with leafy liverworts. The probability that the associations with Sebacinaceae are evolutionary old, but the Tulasnella associations more derived is discussed. The sebacinoid mycobionts form a similar interaction type with the jungermannialian leafy liverworts as do the associated ascomycetes. The term ‘ jungermannioid mycorrhiza’ is proposed for this distinctive symbiotic interaction type.

INTRODUCTION Distinctive groups of liverworts consistently associate with symbiotic fungi (Read et al. 2000). It was hypothesized that the interactions are of importance to both partners and may be considered as symbiotic, although functional studies have still to be carried out (Read et al. 2000). An argument in favour of a mycorrhiza-like association in the leafy liverworts is that the associated fungi form mycorrhizas with vascular plants. Members of the Glomeromycota (Schu¨ßler, Schwarzott & Walker 2001) are associated with complex thalloid liverworts (Marchantiopsida) and several basic, mostly simple thalloid liverworts in the Jungermanniopsida (Haplomitrium, Pellia, Fossombronia) forming similar structures as in arbuscular mycorrhizas (Stahl 1949, Ligrone & Lopes 1989). Ascomycetes forming ericoid mycorrhizas were found to associate with distinct leafy liverworts (Duckett, Renzaglia & Pell 1991, Duckett & Read 1991, 1995). Basidiomycete hyphae were demonstrated by ultrastructural research in Aneura pinguis, * Corresponding author.

Cryptothallus mirabilis and Southbya tophacea (Pocock & Duckett 1984, Ligrone, Pocock & Duckett 1993, Read et al. 2000). The basidiomycete mycobiont of Cryptothallus mirabilis was identified as a species of Tulasnella by molecular means (Bidartondo et al. 2003) and was shown to form ectomycorrhizas with seedlings of Betula sp. in microcosm experiments suggesting epiparasitism by Cryptothallus (Read et al. 2000, Bidartondo et al. 2003). The jungermannioid liverworts associated with ascomycetes sampled previously were from moist, acidic peat and raw-humus soils where ericaceous plants were mostly present and it was concluded that both groups of plants share the same symbionts in the common habitat belonging to the Hymenoscyphus ericae aggregate (Duckett, Renzaglia & Pell 1991, Duckett & Read 1995, Chambers et al. 1999). By use of the dye 3,3k dihexyloxacarbocyanine iodine (DiOC6(3)), basidiomycetes were suspected in Lophozia excisa, L. ventricosa, Nardia scalaris, Southbya nigrella, Tritomaria quinquedentata, Anastrophyllum hellerianum (syn. Sphenolobus hellerianus), Harpanthus scutatus, and Saccogyna viticulosa (Duckett & Read 1991), but definitely

Symbiotic basidiomycetes in leafy liverworts demonstrated only in Southbya tophacea (Read et al. 2000). The basidiomycetes were not identified and the interactions were not studied further. We questioned whether the habitat or the systematic position of a species in the Jungermanniopsida determined the associations with basidio- or ascomycetes. We therefore sampled specimens from diverse habitats in Europe and investigated them by light and transmission electron microscopy. DNA sequencing and molecular phylogenetic methods were used in order to identify the associated basidiomycetous fungi.

MATERIAL AND METHODS Samples were collected from different substrates such as stumps, the bark of a dead standing tree, rotten wood, from the forest floor covered by needle litter, from naked soil and between peat moss in different habitats and geographical regions of Europe (Table 1). In total 38 samples representing 28 species and 12 families of Jungermanniopsida (sensu Crandall-Stotler & Stotler 2000) were investigated by light and electron microscopy and by molecular phylogenetic methods. Nomenclature follows Grolle & Long (2000) and Missouri Botanical Garden (2002). Systematic arrangements follow the system of Crandall-Stotler & Stotler (2000). Vouchers of the liverworts are stored in the herbarium of the State Museum of Natural History Baden-Wu¨rttemberg, Stuttgart (STU). Vouchers of resin embedded samples were stored in the collection of the Botanical Institute, Systematic Botany and Mycology, Eberhard Karls University Tu¨bingen (TUB). Light microscopy Specimens were cleaned under tap water. Soil particles adhering to rhizoids were removed with a fine paintbrush. Specimens were cleared in KOH (10 %) for 10 min at ca 20 xC, rinsed in tap water three times, exposed to HCl (10 %) for 1 min and stained in aniline blue (0.05 % aniline blue in 90 % lactic acid ; Riedel-de Haen, Taufkirchen) for 30 min at 90 x. Specimens were rinsed in lactic acid twice before transfer to a microscopic slide. Three specimens of each sample were investigated by light microscopy. In case hyphae were detected in the rhizoids, stems or thalli, the material was selected for transmission electron microscopy and DNA sequencing. Transmission electron microscopy Small parts of stems or thalli with rhizoids containing hyphae were fixed in 2 % glutaraldehyde in 0.1 % cacodylate buffer for 1 h under vacuum and post-fixed in osmium tetroxide (1 %) for 1 h. Following dehydration in a graded acetone series, specimens were embedded in Spurr’s resin (acetone :resin 1: 1, 2 :1, for 30 min each, pure resin overnight; low viscosity – longer pot life

958 formulation ; Spurr 1969). Specimens were flat embedded and hardened at 70 x for 3 d. Semithin sections (0.7 mm) were stained with neofuchsin cristal violet. Ultrathin sections were stained by uranyl acetate and lead citrate. Sections were examined in a Zeiss TEM 109 or 902. DNA sequencing Well colonized rhizoids and stems or thalli were selected from three specimens per sample, washed 4 times in sterile water and DNA was isolated using the DNAeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Nuclear rDNA including the D1/D2 domain of the large ribosomal subunit (LSU) was amplified by the polymerase chain reaction (PCR) with the primer pairs NL1 (5k-GCATATCAATAAGCGGAGGAAAAG-3k ; O’Donnell 1993) / NL4 (5k-GGTCCGTGTTTCAAGACGG-3k ; O’Donnell 1993) or LR0R (5k-ACCCGCTGAACTTAAGC-3k ; Moncalvo, Wang & Hseu 1995) / LR5 (5kATCCTGAGGGAAACTTC-3k ; Vilgalys & Hester 1990). PCR reaction volume was 50 ml, with concentrations of 1.5 mM MgCl2, 200 mM of each dNTP (Life Technologies, Eggenstein, Germany), 0.5 mM of each of the primers (MWG Biotech, Ebersberg, Germany), 1 U Taq-polymerase (Life Technologies), using amplification buffer (Life Technologies) and an empirically determined dilution of the DNA extract. Good results were achieved at diluting the DNA extract (200 ml) 1 :10, 1:25 or 1: 100. For PCR we chose a touch-down profile with annealing temperatures of 60–50 x: after initial denaturation at 94 x for 3 min, 10 cycles were run with variable annealing temperatures ranging from 60 x in the first cycle to 51 x, in each cycle decreasing by 1 x, followed by 25 cycles with a constant annealing temperature of 50 x. Each of the cycles consisted of an annealing step of 0.5 min, an elongation step of 72 x for 1 min, and a denaturation step of 94 x for 0.5 min. The PCR was finished with a final elongation phase at 72 x for 7 min, after which the samples were stored at 4 x. Controls without template were run for every PCR experiment. The PCR products obtained were purified using the QIAquick protocol (Qiagen, Hilden, Germany) and sequenced in both directions with sequencing primers NL1 and NL4, or NLMW1 (5kTCAATAAGCGGAGGAAAAGA-3k ; Sampaio et al. 2002), NL4, the reverse complement of NL4, and LR5, respectively, on an automated sequencer (ABI 373A Stretch, Applied Biosystems, Foster City, CA) using the ABI PRISM Dye-Terminator Cycle Sequencing Ready Reaction Kit, version 2 (Applied Biosystems). Sequence editing was done using Sequencher (Gene Codes, Ann Arbor, MI). Molecular phylogenetic analysis We used ClustalX (Thompson et al. 1997) to align the rDNA sequences to D1/D2 sequences published

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Table 1. Species sampled from diverse substrates and locations in Europe, the majority showing symbiotic fungal associations. Species with fungal associations registered for the first time in bold.

Substrate and locationa

Previous investigationsb and results + or x fungal association

Plant group

Species

Fungal association

Blasiales Blasiaceae

Blasia pusilla

x

Wet sandy soil I

6, 7, 11, 15; allx

Fossombroniales Fossombroniaceae

Fossombronia wondraczekii

+

Dense, loamy soil II

6+

Metzgeriales Aneuraceae

Aneura pinguis

+

Soil/Sphagnum II

5, 6, 7, 13, 14; all+

Jungermanniales Pseudolepicoleaceae Geocalycaceae

Blepharostoma trichophyllum Lophocolea heterophylla

+ +

6+, 10x 10x, 15x

Calypogeia azurea C. integristipula C. muelleriana

+ + +

C. neesiana Bazzania flaccida B. trilobata

+ x x

Lepidozia reptans Kurzia pauciflora Cephalozia bicuspidata

+ + +

C. catenulata C. lunulifolia

+ +

Nowellia curvifolia Cephaloziella divaricata Anastrophyllum minutum Barbilophozia barbata Lophozia incisa L. sudetica Mylia anomala M. taylorii Nardia scalaris Marsupella emarginata Diplophyllum albicans D. obtusifolium

+ + + + + + + + + + + +

Fir stump V Rotten wood of conifer, needle litter II/V Loamy soil IV/VI Bog, humus V Soil covered by needle litter I/II/V Sphagnum V Dead trunk of beech V Rotten wood of Norway spruce needle litter II/V Rotten wood II/V/VI Sphagnum V Sandy soil, rotten trunk III/VI/V/IV Rotten wood II Rotten wood, stump, wet raw humus, II/V Rotten wood III/V Soil I/II Rotten wood VII Bog, raw humus V Sandy soil VII Wet soil VI Bog, raw humus V Bog, raw humus V Sandy, wet soil IV Wet granite V Loamy soil Ix Loamy soil II

Calypogeiaceae

Lepidoziaceae

Cephaloziaceae

Cephaloziellaceae Jungermanniaceae

Gymnomitriaceae Scapaniaceae

1, 3, 4, 6, 10, 11, 12; all+ 10, 12, 13, 15; all+ 10+ 2+, 10x, 12x 1, 3, 10, 11, 12, 13, 15 ; all+ 6, 10, 11, 12, 13 ; all+ 2, 3, 10, 11, 12, 13, 15 ; all+ 10x, 12+ 10, 12, 15; all+ 10, 12, 13, 15; all+ 17, 10, 11, 12, 13, 15; all+ 10x

6, 8, 9, 10, 12, 13, 15 ; all+ 6+, 9x 3, 9, 13, 15; all+ 3+, 10x 2+, 3+, 6+, 10x, 11+, 15x

a Locations: I, Suebian Forest, Germany; II, Surroundings of Tu¨bingen, Germany; III, Suebian Alb, Germany; IV, German Alps; V, Vosges, France; VI, Pyrenean; and VII, Swedish Lapland. b 1 Nemec (1899); 2 Cavers (1903); 3 Garjeanne (1903); 4 Nemec (1904); 5 Denis (1919); 6 Stahl (1949); 7 Pocock & Duckett (1984); 8 Pocock & Duckett (1985a); 9 Pocock & Duckett (1985b); 10 Pocock & Duckett (1985c); 11 Boullard (1988); 12 Duckett, Renzaglia & Pell (1991); 13 Duckett & Read (1991); 14 Ligrone, Pocock & Duckett (1993); and 15 Duckett & Read (1995).

in GenBank (NCBI; http://www.ncbi.nlm.nih.gov/) that represent major groups of Hymenomycetes, with a focus on heterobasidiomycete taxa (excluding Tremellomycetidae). The DNA alignment was manually corrected with Se-Al, version 1.0 (Rambaut 1996). Ambiguous alignment regions were excluded from phylogenetic analysis ; highly divergent parts of Tulasnella sequences were recoded as ‘missing data ’. We used PAUP* (Swofford et al. 2002) for molecular phylogenetic analysis : neighbour joining (Saitou & Nei 1987) was done using Kimura-2-parameter genetic distances (Kimura 1980), combined with a bootstrap analysis (Felsenstein 1985) from 1000 replicates.

RESULTS Light and transmission electron microscopy Metzgeriales Aneuraceae The rhizoids of Aneura pinguis showed a low degree of fungal colonization, but the basal cells of the thallus contained hyphal coils. Active and collapsed hyphae were found in the living thallus cells (Fig. 1). The hyphae stored glycogen, and contained lipids which occasionally were strongly osmiophilic (Fig. 1). The hyphal walls were thin, electron dense and covered by a fibrillar matrix (Fig. 2). Dolipores with slightly

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Table 2. Basidiomycete-associations in Jungermanniidae : ultrastructural and molecular results.

Species

Systematic position

Basidiomycete with imperforate parenthesome

Aneura pinguis

Subclass Metzgeriidae Metzgeriales Aneuraceae

+

Tulasnella

x

x

Calypogeia azurea C. muelleriana Lophozia incisa L. sudetica Barbilophozia barbata Diplophyllum albicans

Subclass Jungermanniidae Calypogeiaceae Calypogeiaceae Jungermanniaceae Jungermanniaceae Jungermanniaceae Scapaniaceae

+ + + + + +

nda Sebacinoid Sebacinoid Sebacinoid nd nd

+ + x(+) x(+) + x

+ + x x + x

a

Fungus determined by molecular phylogeny (LSU sequences)

Ingrowth peg formation

Ascomycete hyphae

nd, not determined.

Figs 1–2. Fig. 1. Hyphae of Tulasnella sp., alive and collapsed (arrow) in a thallus cell of Aneura pinguis. Asterisk indicates degenerating chloroplast. Hyphae contain osmiophilic bodies, probably lipids. Bar=1 mm. Fig. 2. Hypha with doliporus and dish-shaped, imperforate parenthesome (arrows) in the thallus of Aneura pinguis. Hyphal walls are covered by a mucilage matrix (asterisk). Bar=1 mm.

dish-shaped imperforate parenthesomes were frequently observed (Fig. 2). Clamp connections were not found. The moss cell walls were breached through by the hyphae without formation of a neck of cell wall encasement (data not shown). Jungermanniales Fungal associations of supposed symbiotic state were only missing in three of the species investigated : Blasia pusilla, Bazzania flaccida, and Bazzania trilobata (Table 1). Most of the jungermannialian species were

found to be associated with ascomycetes. In Lophocolea heterophylla, Calypogeia integristipula, Anastrophyllum minutum, and Diplophyllum obtusifolium ascomycetes were registered for the first time. Three species were associated with asco- and basidiomycetes (Calypogeia azurea, C. muelleriana, and Barbilophozia barbata), three species were found associated with basidiomycetes only (Lophozia incisa, L. sudetica, and Diplophyllum albicans ; Table 2). Only specimens collected from the ground, like naked soil, needle layer on forest floor, raw humus or peat were associated with basidiomycete hyphae (Table 1). No basidiomycetes

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Figs 3–6. Fig. 3. Calypogeia muelleriana: dense hyphal colonization of the rhizoidal base and invasion into the cortical cells by ingrowth pegs (arrows). Hyphae breach through the rhizoidal wall but become covered by wall material of the cortical cell. Asterisk indicates a dikaryon. Bar=0.5 mm. Fig. 4. Dolipore with straight, imperforate parenthesome of the sebacinoid fungus in C. muelleriana. Bar=0.25 mm. Fig. 5. C. azurea : hyphal colonization of the rhizoidal base and invasion of cortical cells by ingrowth pegs (arrows). Asterisk indicates a doliporus. Bar=1 mm. Fig. 6. Dolipore with imperforate parenthesome of the presumably sebacinoid fungus in Calypogeia azurea. The parenthesome was distorted during the preparation procedure. Bar=0.15 mm. Abbreviations : cc, cortical cell ; hy, hypha ; n, nucleus ; and rb, rhizoidal base.

were found in samples collected from tree bark or rotten wood. No basidiomycetes were found in flagellae, and none of the species associated with basidiomycetes form flagellae. Only species associated with basidiomycetes are considered here in detail as these findings are new.

Calypogeiaceae In Calypogeia azurea and C. muelleriana, the rhizoids and 3–4 layers of the stem cortical cells were well colonized by asco- and basidiomycete hyphae. Occasionally, hyphae of asco- and basidiomycetes were found in

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Figs 7–10. Fig. 7. Dolipore with straight, imperforate parenthesome (arrow) of a sebacinoid fungus in Lophozia sudetica. Bar=0.15 mm. Fig. 8. Dolipore with straight, imperforate parenthesome (arrow) of a sebacinoid fungus in L. incisa. Bar=0.25 mm. Fig. 9. Dolipore with straight, imperforate parenthesome (arrows) of a presumably sebacinoid fungus in Barbilophozia barbata. Bar=0.15 mm. Fig. 10. Dolipore with straight, imperforate parenthesome (arrows) of a presumably sebacinoid fungus in Diplophyllum albicans. Bar=0.25 mm.

the same cell side by side (Fig. 13). Hyphae invaded the moribund rhizoids and formed a dense layer on the rhizoidal base (Fig. 3). When invading the surrounding cortical cells, hyphae were ensheathed by host cell wall material resulting in tube-like ingrowths (Figs 3 and 5). Hyphae digested the cell wall layer of the rhizoid, while the wall layer of the stem cell formed the encasement (Fig. 3). This interaction type was observed not only with ascomycete but also with basidiomycete hyphae. Dikaryons were found and dolipores were visible in the hyphae indicating the basidiomycete nature of the associated fungus (Figs 3 and 5). The dolipores were consistently covered by straight, imperforate parenthesomes (Figs 4 and 6).

Jungermanniaceae Only basidiomycete hyphae were found to be associated with Lophozia incisa and Lophozia sudetica. Dolipores with straight, imperforate parenthesomes were consistently observed (Figs 7–8). The thin-walled hyphae were densely packed in the rhizoid base and formed coils in the cells of the outer three to four layers of the stem (Fig. 11). The hyphae were normally not covered by cell wall material of host origin (Fig. 11). Rarely necks of host cell wall material were formed around invading hyphae and were later broken through by the fungus. Ingrowth pegs were only found in degenerating cells (data not

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Figs 11–14. Fig. 11. Colonization of stem tissue of Lophozia incisa by a sebacinoid fungus. Hyphae containing lipids (arrows) are not covered by cell wall tissue of the liverworts. Bar=1 mm. Fig. 12. Hyphae of an ascomycete colonizing the rhizoidal base of Barbilophozia barbata. Hyphae contain osmiophilic polyphosphate-nitrogen bodies (arrows) and display a more dense and more osmiophilic cytosol than the basidiomycete hyphae (for comparison see Figs 11 and 13). Bar=0.4 mm. Fig. 13. Rhizoidal base of Calypogeia azurea colonized by basidiomycete (asterisk) and ascomycete hyphae (arrow) both invading the cortical cell by hyphal ingrowth pegs. Bar=1 mm. Fig. 14. Stem tissue of Cephalozia lunulifolia colonized by ascomycete hyphae that are covered by cell wall material of the liverwort. The well preserved material shows the typical characters of a symbiotic interaction : enlarged nucleus, active cytosol containing intact mitochondria, well preserved membranes and intimate contact of fungus and plant cell walls in the ingrowth pegs. Bar=0.4 mm. Abbreviations as in Figs 3–6.

shown). Ascomycete hyphae were not detected in the material. In Barbilophozia barbata, moribund rhizoids were well colonized by asco- and basidiomycete hyphae forming dense layers at the rhizoidal base. Hyphae invaded the surrounding cortical cells and three to four cell layers of the stem. Penetrating hyphae became covered by host cell wall material resulting in typical ingrowth pegs. Asco- and basidiomycete hyphae formed the same interaction type. Basidomycete

hyphae displayed a doliporus covered by a straight, imperforate parenthesome (Fig. 9). Scapaniaceae In Diplophyllum albicans, the rhizoids and three to four layers of the stem cells were colonized by basidiomycete hyphae only. The basidiomycete nature was indicated by dolipores with straight, imperforate parenthesomes (Fig. 10). The hyphae in the stem cells were not covered

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Fig. 15. For legend see opposite page.

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I. Kottke and others by a cell wall layer of host origin. Only occasionally, hyphae colonizing cells close to the rhizoid base were covered by wall ingrowths. The hyphal walls were thin and not ensheathed by a fibrillar matrix. Hyphae contained lipids and sometimes osmiophilic bodies, probably lipids or pigments. No ascomycete hyphae were found in D. albicans. General aspects of fungal colonization While only basidiomycete hyphae were found to form coils inside stem cells (Lophozia spp. and Diplophyllum albicans, Fig. 11), asco- and basidiomycetes formed ingrowth pegs when invading the stem cells of the jungermannialian hepatics (Fig. 13). Proof of the fungal group was given by the pore type of the fungal septum, which is simple and accompanied by Woronin bodies in ascomycetes, and a dolipore in basidiomycetes. Ascomycete hyphae could also be discriminated from basidiomycete hyphae in the liverworts tissues by several ultrastructural criteria. The cytosol of ascomycete hyphae appeared more electron dense, containing smaller vacuoles than the basidiomycete hyphae (Figs 11–14). While the basidiomycete hyphae stored lipid droplets (Fig. 11), the ascomycete hyphae frequently contained typical polyphosphate-nitrogen bodies (Fig. 12). The nitrogen content was proved by Electron-Energy-Loss Spectroscopy (data not shown). In well-preserved material the host plasma membrane surrounded the intracellular hyphae and the ingrowth pegs (Fig. 14). The cytosol contained mitochondria and rER indicating an active state of the tissue (Fig. 14). The membranes in the hepatics are, however, very sensitive and frequently the cellular state was not well-preserved. Phylogenetic position of the associated basidiomycetes Fungal DNA sequences were obtained from only eleven liverwort species; the fungi mostly belonged to ascomycetes and are not considered here further. Sequencing was hampered by multi-fungal invasions which gave multiple bands of PCR products in agarose gel electrophoresis. The molecular phylogenetic analysis of the basidiomycete sequences (Fig. 15) allows us to assign with high bootstrap support mycobionts of the following liverworts to distinct lineages of Hymenomycetes : (1) Aneura pinguis has a mycobiont belonging to Tulasnella ; and (2) Calypogeia muelleriana, Lophozia incisa, and Lophozia sudetica have mycobionts that belong to the Sebacinaceae. In case of samples of Calypogeia azurea,

965 Barbilophozia barbata, and Diplophyllum albicans, sequencing was unsuccessful. DISCUSSION The general aspect of fungal colonization of the 27 members of Jungermanniidae was found to be similar to the one described previously (Duckett, Renzaglia & Pell 1991, Read et al. 2000). Typical hyphal ingrowth pegs were shown for ascomycete associations by these authors, but are demonstrated here for the first time in basidiomycete associations. The ingrowth pegs resemble transfer cells which occur frequently in plants where a short-distance exchange of solutes takes place (Gunning & Pate 1969). According to the ultrastructural observations there is no indication of a defence reaction of the plant cells against the invading fungus. Our findings therefore support the suggestion of Read et al. (2000) that the pegs are the major site of nutrient exchange and are indicative of a symbiotic interaction. As this type of interaction is restricted to leafy liverworts of the subclass Jungermanniidae (sensu Crandall-Stotler & Stotler 2000), we suggest the term ‘jungermannioid mycorrhiza ’. The liverworts do not form roots and therefore the term ‘mycorrhiza ’ appears not to be correct if considered from an etymological point of view. However, the symbiotic interaction most likely improves the nutrient uptake and is thus functionally similar to the mycorrhizas of vascular plants. The term ‘mycorrhiza ’ in a functional sense is scientifically well-established, as in case of orchid protocorms as well (Rasmussen 2002). The term ‘mycothallus ’ was suggested by Boullard (1988) in case where gametophytes are associated with symbiotic fungi. However, leafy liverworts do not form thalli and this term would thus be misleading in case of Jungermanniidae. The colonization of basidiomycetes differed between the associated liverworts. In Diplophyllum albicans, Lophozia incisa, and L. sudetica, no or very few ingrowth pegs were found. Instead, the hyphae formed hyphal coils in the stem cells. These specimens were not associated with ascomycetes in our material. In Barbilophozia barbata, Calypogeia azurea, and C. muelleriana, hyphal ingrowth pegs were formed by basidio- and ascomycetes. These specimens were regularly associated with fungi from both fungal groups (Table 2). It is unclear if these differences are due to the liverworts, to the different basidiomycete taxa (Fig. 15) or to cellular interactions between ascoand basidiomycetes.

Fig. 15. Phylogenetic placement of the liverwort mycobionts. Neighbour-joining analysis of an alignment of nuclear rDNA coding for the D1/D2 region of the ribosomal large subunit using Kimura-2-parameter genetic distances. The topology was rooted with the group of Sebacinaceae. Branch lengths are scaled in terms of expected numbers of nucleotide substitutions per site; marked branches of the tulasnelloid clade were reduced to half of their length. Numbers on branches are bootstrap values inferred from 1000 replicates, numbers below 50 % are not shown. Liverwort mycobionts detected in this study are printed in bold.

Symbiotic basidiomycetes in leafy liverworts Few of the members of Jungermanniales studied lacked fungal associations (Blasia pusilla, Bazzania flaccida, and B. trilobata) supporting earlier findings on the widespread association between liverworts and endophytic fungi (Stahl 1949, Pocock & Duckett 1985a, b, c, Duckett et al. 1991, Duckett & Read 1991, Read et al. 2000). The majority of our specimens were associated with ascomycetes. Species that were found solely associated with ascomycetes were members of the Lepidoziaceae, Cephaloziaceae, and Cephaloziellaceae confirming previous results on the fungal associations in these families (Duckett et al. 1991, Duckett & Read 1991, 1995). Associated basidiomycetes were suspected previously within the Jungermanniidae in Jungermanniaceae, Geocalycaceae, Gymnomitraceae, and Ptilidiaceae (Pocock & Duckett 1985b, Duckett & Read 1991), but were unambiguously demonstrated only in Southbya tophaceae (Arnelliaceae) (Read et al. 2000). The six jungermannioid species shown here to be associated with basidiomycetes are members of the families Calypogeiaceae, Jungermanniaceae, and Scapaniaceae (Table 2). The scattered occurrence of basidiomycete associations in the Jungermanniidae, one taxon in Arnelliaceae and in Scapaniaceae, two taxa in Geocalycaceae, and nine in Jungermanniaceae, is difficult to understand, but is most probably just due to insufficient data. The basidiomycetes associated with jungermannioid liverworts are characterized by dolipores with straight, imperforate parenthesomes (Table 2). Dolipores with imperforate parenthesomes are characteristic of heterobasidiomycetous Hymenomycetes and basal Homobasidiomycetes such as Auriculariales, Dacrymycetales, Tulasnellales, and Hymenochaetales (Oberwinkler 1985, Currah & Sherburne 1992, Wells 1994). According to our molecular phylogenetic results, the basidiomycetes in Lophozia incisa, Lophozia sudetica, and Calypogeia muelleriana are members of the Sebacinaceae (Fig. 15), a group that has been included in the Auriculariales by Bandoni (1984), but was recently shown to represent an independent lineage of Hymenomycetes (Weiß & Oberwinkler 2001). We suspect that molecular techniques missed the sebacinoid mycobionts in the other three species (Calypogeia azurea, Barbilophozia barbata, and Diplophyllum albicans) as sequencing was hampered by the multi-fungal invasions and insufficiently preserved material. We rely on the ultrastructural data to suggest that all six species are associated with members of Sebacinaceae. Sebacinoid mycobionts were only found by us in samples collected from the ground (Table 1). Recent findings support the view that Sebacinaceae are root-related fungi : Sebacinaceae form typical ectomycorrhizas with temperate deciduous trees (Corylus avellana, Carpinus betulus, Fagus sylvatica, Tilia cordata, Picea abies ; Selosse, Bauer & Moyersoen 2002, Urban, Weiß & Bauer 2003) and with Melaleuca uncinata and Eucalyptus marginata in Australia (Warcup

966 1988, Glen et al. 2002). Sebacinaceae also associate with myco-heterotrophic orchids (Warcup & Talbot 1967, McKendrick et al. 2002, Selosse et al. 2002) and with Ericaceae (Berch, Allen & Berbee 2002). We suggest that liverworts were the first that became symbiotically associated with Sebacinaceae and acted as vectors for fungal transfer to the other more recently evolved plant families. Read et al. (2000) suggested a loss of specificity in these widespread liverwortassociated basidiomycetes. However, our alternative hypothesis is that members of the Sebacinaceae preserved the ability to associate with a broad host range during the coevolutionary processes. According to molecular data, liverworts are considered now as the earliest land plants (Kenrick & Crane 1997, Lewis et al. 1997, Qiu et al. 1998). This view is supported by DNA sequences (Lewis et al. 1997), the lack of three mitochondrial introns (Qiu et al. 1998), fossil evidence (Edwards et al. 1995) and morphology (Crandall-Stotler & Stotler 2000, Willis & McElwain 2002). Although there is still disputation on the phylogenetic relationship within the liverworts (Lewis et al. 1997), the phylogenetic trees presented by these authors are supported by the mycorrhizal associations that without doubt evolved in coevolution between plants and fungi (Brundrett 2002). The arbuscular mycorrhiza, the evolutionary oldest association, which involves members of the Glomeromycota (Schu¨ßler et al. 2001), is restricted to the basal groups of the liverworts : the Haplomitriales (Calobryales), the Marchantiidae, and one clade of the Jungermanniopsida, the Metzgeriidae, including Pellia, Pallavicina, Petalophyllum, and Fossombronia but not the Aneuraceae. The second clade of the leafy liverworts, the Jungermanniidae, are either associated with ascoand basidiomycetes forming the jungermannioid type of mycorrhiza or lack mycorrhization (Pocock & Duckett 1985c). We suggest that a basal member of the Jungermanniidae became associated with a sebacinoid species, replacing the ancient glomeralean associations. If this hypothesis holds true, it would suggest that the jungermannioid mycorrhiza with members of the Sebacinaceae is a model for the most ancient basidiomycete mycorrhiza so far demonstrated. The association with ascomycetes may have occurred in parallel, earlier or later. In this context it is interesting to note that sebacinoid fungal associations with plant roots occur frequently together with ascomyceteassociations of the Hymenoscyphus ericae aggregate (Warcup 1988, Selosse et al. 2002, Berch et al. 2002, Urban et al. 2003). Dating of the origin of these two fungal groups could help to clarify if the co-occurrence is due to coevolution, to similar ecological requirements, or both. The loss of symbiotic associations in the jungermannioid liverworts is considered as a more recent event connected to epiphytism and colonization of pioneer habitats (Kottke 2002). Aneura pinguis and Cryptothallus mirabilis, its mycoheterotrophic relative, were found to be associated

I. Kottke and others with basidiomycetes containing dolipores with imperforate parenthesomes (Ligrone et al. 1993). These authors observed slight differences in the structure of the dolipores and the parenthesomes of the basidiomycetes associated with A. pinguis sampled from different habitats, probably indicating diverse fungal species. According to the descriptions given, the parenthesomes in our material were similar to those in the fungi from specimens collected in sand dunes (type A1), that means slightly dish-shaped without curved ends and without a light layer inside. By use of molecular phylogenetic analysis we suggest that the basidiomycete in A. pinguis is a species of Tulasnella. Tulasnella species closely related to each other were recently also identified in Cryptothallus mirabilis (Bidartondo et al. 2003). One of the corresponding D1/D2 rDNA sequences (Cryptothallus isolate AY192482) is included in our molecular phylogenetic analysis (Fig. 15). It shows that our isolate is likely to represent a different species. Our ultrastructural and molecular phylogenetic data suggest in parallel that basal Hymenomycetes are associated with the liverworts. According to molecular phylogenetic results, the family Sebacinaceae is a basal lineage in Hymenomycetes (Weiß & Oberwinkler 2001). Tulasnellaceae represent a more derived clade which according to recent molecular phylogenetic analyses is closely related to Multiclavula and Cantharellus (Fig. 15; Hibbett, Gilbert & Donoghue 2000, Bidartondo et al. 2003). These findings permit us to suggest that the jungermannioid mycorrhiza is an ancient event, but the aneuracean mycorrhiza may be of more recent origin. The association involving Tulasnella and Aneuraceae may have evolved after loss of mycorrhization with Glomeromycota in the Metzgeriidae. No symbiotic fungal associations were found in Metzgeria up to now and the state of Riccardia is uncertain. In spite of the limited molecular sequence data that are still available in Tulasnella (Bidartondo et al. 2003), our phylogenetic tree may indicate that the Tulasnella’s associated with Aneura and Cryptothallus belong to a derived group of this genus. This may support the speculation that the association with Aneuraceae was a more recent event (Read et al. 2000).

REFERENCES Bandoni, R. J. (1984) The Tremellales and Auriculariales: an alternative classification. Transactions of the Mycological Society of Japan 25 : 489–530. Berch, S. M., Allen, T. R. & Berbee, M. L. (2002) Molecular detection, community structure and phylogeny of ericoid mycorrhizal fungi. Plant and Soil 244: 55–66. Bidartondo, M. I., Bruns, T. D., Weiß, M., Se´rgio, C. & Read, D. J. (2003) Specialized cheating of the ectomycorrhizal symbiosis by an epiparasitic liverwort. Proceedings of The Royal Society of London, B, Biological Sciences 270 : 835–842. Boullard, B. (1988) Observations on the coevolution of fungi with hepatics. In Coevolution of Fungi with Plants and Animals (K. A. Pirozynski & D. L. Hawksworth, eds): 107–124. Academic Press, London.

967 Brundrett, M. C. (2002) Coevolution of roots and mycorrhizas of land plants. New Phytologist 154: 275–304. Cavers, F. (1903) On saprophytism and mycorrhiza in hepaticae. New Phytologist 2: 30–36. Chambers, S. M., Williams, P. G., Seppelt, R. D. & Cairney, J. W. G. (1999) Molecular identification of Hymenoscyphus sp. from rhizoids of the leafy liverwort Cephaloziella exiliformis in Australia and Antartica. Mycological Research 103: 286–288. Crandall-Stotler, B. & Stotler, R. E. (2000) Morphology and classification of the Marchantiophyta. In Bryophyte Biology (A. J. Shaw & B. Goffinet, eds): 21–70. University Press, Cambridge, UK. Currah, R. S. & Sherburne, R. (1992) Septal ultrastructure of some fungal endophytes from boreal orchid mycorrhizas. Mycological Research 96: 583–587. Denis, M. (1919) Sur quelques thalles d’Aneura de´pourvus de chlorophylle. Comptes rendues de Sciences, Paris 148: 64–66. Duckett, J. G. & Read, D. J. (1991) The use of the fluorescent dye, 3,3k-dihexylcarbocyanine iodide, for selective staining of ascomycete fungi associated with liverwort rhizoids and ericoid roots. New Phytologist 118 : 259–272. Duckett, J. G. & Read, D. J. (1995) Ericoid mycorrhizas and rhizoidascomycete associations in liverworts share the same mycobiont : isolation of the partners and resynthesis of the associations in vitro. New Phytologist 129 : 439– 447. Duckett, J. G., Renzaglia, K. S. & Pell, K. (1991) A light and electron microscope study of rhizoid-ascomycete associations and flagelliform axes in British hepatics with observations on the effects of the fungi on host morphology. New Phytologist 118 : 233–257. Edwards, D., Duckett, J. G. & Richardson, J. B. (1995) Hepatic characters in the earliest land plants. Nature 374 : 635– 636. Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791. Garjeanne, A. J. M. (1903) U¨ber die Mykorrhiza der Lebermoose. Beihefte zum Botanischen Zentralblatt 15 : 471– 482. Glen, M., Tommerup, I., Bougher, N. L. & O’Brien, P. A. (2002) Are Sebacinaceae common and widespread ectomycorrhizal associates of Eucalyptus species in Australian forests? Mycorrhiza 12: 243–247. Grolle, R. & Long, D. G. (2000) An annotated check-list of the Hepaticae and Anthocerotae of Europe and Macronesia. Journal of Bryology 22: 103–140. Gunning, B. E. & Pate, J. S. (1969) ‘Transfer cells ’ – plant cells with wall ingrowths specialized in relation to short distance transport of solutes – their occurence, structure and development. Protoplasma 68: 107–133. Hibbett, D. S., Gilbert, L.-B. & Donoghue, M. J. (2000) Evolutionary instability of ectomycorrhizal symbioses in basidiomycetes. Nature 407: 506–508. Kenrick, P. & Crane, P. R. (1997) The origin and early evolution of plants on land. Nature 389 : 33–39. Kimura, M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111–120. Kottke, I. (2002) Mycorrhizae – Rhizosphere determinants of plant communities. In Plant Roots: the hidden half (Y. Waisel, A. Eshel & U. Kafkafi, eds): 919–932. 3rd edn. Marcel Dekker, New York. Lewis, L. A., Mishler, B. D. & Vilgalys, R. (1997) Phylogenetic relationship of the liverworts (Hepaticae), a basal embryophyte lineage, inferred from nucleotide sequence data of the chloroplast gene rbcL. Molecular Phylogenetics and Evolution 7: 377–393. Ligrone, R. & Lopes, C. (1989) Cytology and development of a mycorrhiza-like infection in the gametophyte of Conocephalum conicum (L.) Dum. (Marchantiales, Hepatophyta). New Phytologist 111: 423– 433. Ligrone, R., Pocock, K. & Duckett, J. G. (1993) A comparative ultrastructural study of endophytic basidiomycetes in the parasitic achlorophyllous hepatic Cryptothallus mirabilis and the closely related allied photosynthetic species Aneura pinguis (Metzgeriales). Canadian Journal of Botany 71: 666–679.

Symbiotic basidiomycetes in leafy liverworts McKendrick, S. L., Leake, D. J., Taylor, D. L. & Read, D. J. (2002) Symbiotic germination and development of myco-heterotrophic plants in nature and its requirement for locally distributed Sebacina spp. New Phytologist 154: 233–247. Missouri Botanical Garden (ed.) (2002) TROPICOS W3MOST data base; http://mobot.org/Pick/Search/most.html. Moncalvo, J. M., Wang, H.-H. & Hseu, R.-S. (1995) Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87: 223–238. Nemec, B. (1899) Die Mykorrhiza einiger Lebermoose. Berichte der Deutschen Botanischen Gesellschaft 17 : 311–317. Nemec, B. (1904) U¨ber die Mykorrhiza von Calypogeia trichomanes. Beihefte zum Botanischen Zentralblatt 16 : 253–268. Oberwinkler, F. (1985) Anmerkungen zur Evolution und Systematik der Basidiomyceten. Botanische Jahrbu¨cher fu¨r Systematik, Pflanzengeschichte und Pflanzengeographie 107: 541–580. O’Donnell, K. (1993) Fusarium and its near relatives. In The Fungal Holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematics (D. R. Reynolds & J. W. Taylor, eds): 225–233. CAB International, Wallingford. Pocock, K. & Duckett, J. G. (1984) A comparative ultrastructural analysis of the fungal endophytes in Cryptothallus mirabilis Malm. and other British thalloid hepatics. Journal of Bryology 13: 227–233. Pocock, K. & Duckett, J. G. (1985a) The alternative mycorrhizas: fungi and hepatics. Bulletin of the British Bryological Society 45: 10–11. Pocock, K. & Duckett, J. G. (1985b) Fungi in hepatics. Bryological Times 31: 2–3. Pocock, K. & Duckett, J. G. (1985c) On the occurrence of branched and swollen rhizoids in British hepatics: their relationships with the substratum and associations with fungi. New Phytologist 99: 281–304. Qiu, Y.-L., Cho, Y., Cox, C. & Palmer, J. D. (1998) The gain of three mitochondrial introns identified liverworts as the earliest land plants. Nature 395: 671–674. Rambaut, A. (1996) Se-Al. Sequence Alignment Editor. Version 1.0. Department of Zoology, University of Oxford; http://evolve. zoo.ox.ac.uk/software/. Rasmussen, H. N. (2002) Recent developments in the study of orchid mycorrhizas. Plant and Soil 244 : 149–163. Read, D. J., Duckett, J. G., Francis, R., Ligrone, R. & Russell, A. (2000) Symbiotic fungal associations in ‘lower’ land plants. Philosophical Transactions of the Royal Society of London, B, Biological Sciences 355: 815–831. Saitou, N. & Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406 – 425.

968 Sampaio, J. P., Weiß, M., Gadanho, M. & Bauer, R. (2002) New taxa in the Tremellales: Bulleribasidium oberjochense gen. et sp. nov., Papiliotrema bandonii gen. et sp. nov. and Fibulobasidium murrhardtense sp. nov. Mycologia 94 : 873–887. Schu¨ßler, A., Schwarzott, D. & Walker, C. (2001) A new fungal phylum, the Glomeromycota: phylogeny and evolution. Mycological Research 105: 1413–1421. Selosse, M.-A., Bauer, R. & Moyersoen, B. (2002) Basal Hymenomycetes belonging to the Sebacinaceae are ectomycorrhizal on temperate deciduous trees in silva: microscopical and molecular evidence. New Phytologist 155: 183–195. Selosse, M.-A., Weiß, M., Jany, J.-L. & Tillier, A. (2002) Communities and populations of sebacinoid basidiomycetes associated with the achlorophyllous orchid Neottia nidus-avis (L.) L. C. M. Rich. and neighbouring tree ectomycorrhizae. Molecular Ecology 11: 1831–1844. Spurr, A. R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructural Research 26: 31– 43. Stahl, M. (1949) Die Mykorrhiza der Lebermoose mit besonderer Beru¨cksichtigung der thallosen Formen. Planta 37: 103–148. Swofford, D. L. (2002) PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4b10. Sinauer Associates, Sunderland, MA. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997) The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24: 4876– 4882. Urban, A., Weiß, M. & Bauer, R. (2003) Ectomycorrhizae involving sebacinoid mycobionts. Mycological Research 107: 3–14. Vilgalys, R. & Hester, M. (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238– 4246. Warcup, J. H. (1988) Mycorrhizal associations of isolates of Sebacina vermifera. New Phytologist 110: 227–231. Warcup, J. H. & Talbot, P. H. (1967) Perfect states of Rhizoctonia’s associated with orchids. New Phytologist 66: 631–641. Wells, K. (1994) Jelly fungi, then and now! Mycologia 86: 18– 48. Weiß, M. & Oberwinkler, F. (2001) Phylogenetic relationships in Auriculariales and related groups: hypotheses derived from nuclear ribosomal DNA sequences. Mycological Research 105: 403– 415. Willis, K. J. & McElwain, J. C. (2002) The Evolution of Plants. Oxford University Press, Oxford.

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