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A MORPHOLOGICAL AND MOLECULAR ANALYSIS OF ENDOPHYTIC ASSOCIATIONS OF LIVERWORTS WITH GLOMEROMYCOTEAN FUNGI

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A MORPHOLOGICAL AND MOLECULAR ANALYSIS OF ENDOPHYTIC ASSOCIATIONS OF LIVERWORTS WITH GLOMEROMYCOTEAN FUNGI
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  G LOMEROMYCOTEAN ASSOCIATIONS IN LIVERWORTS : A MOLECULAR ,  CELLULAR ,  AND TAXONOMIC ANALYSIS 1 R OBERTO  L IGRONE , 2,5 A NNA  C ARAFA , 2 E RICA  L UMINI , 3 V ALERIA  B IANCIOTTO , 3 P AOLA  B ONFANTE , 3 AND  J EFFREY  G. D UCKETT 4 2 Dipartimento di Scienze ambientali, Seconda Universita` di Napoli, via A. Vivaldi 43, I-81100 Caserta, Italy; 3 Dipartimento di Biologia vegetale, Universita` degli Studi di Torino, and Consiglio Nazionale delle Ricerche (CNR),Istituto per la Protezione delle Piante, Sezione di Torino, Viale P. A. Mattioli 25, I-10125, Torino, Italy; and 4 School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UKLiverworts form endophytic associations with fungi that mirror mycorrhizal associations in tracheophytes. Here we report a worldwide survey of liverwort associations with glomeromycotean fungi (GAs), together with a comparative molecular andcellular analysis in representative species. Liverwort GAs are circumscribed by a basal assemblage embracing theHaplomitriopsida, the Marchantiopsida (except a few mostly derived clades), and part of the Metzgeriidae. Fungal endophytesfrom  Haplomitrium ,  Conocephalum ,  Fossombronia , and  Pellia  were related to  Glomus  Group A, while the endophyte from  Monoclea  was related to  Acaulospora . An isolate of   G. mosseae  colonized axenic thalli of   Conocephalum , producing anassociation similar to that in the wild. Fungal colonization in marchantialean liverworts suppressed cell wall autofluorescence andelicited the deposition of a new wall layer that specifically bound the monoclonal antibody CCRC-M1 against fucosylated sidegroups associated with xyloglucan and rhamnogalacturonan I. The interfacial material covering the intracellular fungus containedthe same epitopes present in host cell walls. The taxonomic distribution and cytology of liverwort GAs suggest an ancient srcinand multiple more recent losses, but the occurence in widely separated liverwort taxa of fungi related to glomeromycoteanlineages that form arbuscular mycorrhizas in tracheophytes, notably the  Glomus  Group A, is better explained by host shifting fromtracheophytes to liverworts. Key words:  arbuscular mycorrhizas; cell walls; DNA sequencing; Glomeromycota; immunocytochemistry; liverworts;symbiosis; ultrastructure. The establishment of biotrophic associations with fungi isconsidered a major factor involved in the colonization of terrestrial habitats by phototrophic organisms (Selosse and LeTacon, 1998). It is assumed that the common ancestor to theGlomeromycota, Ascomycota, and Basidiomycota srcinatedafter the appearance of land plants (Berbee and Taylor, 2007)and that the association with glomeromycotean fungi, to formthe so-called arbuscular mycorrhizas (AMs), is a plesiomorphy(primitive character) in the tracheophytes. Already present inSiluro-Devonian fossils of protracheophytes and still occurringin the majority of present-day tracheophytes (Selosse and LeTacon, 1998; Wang and Qiu, 2006), the AMs have beenreplaced by associations with basidio- or ascomycetes inseveral derived lineages of higher plants (Wang and Qiu, 2006;Berbee and Taylor, 2007). Endophytic fungal associations not only occur in tracheophytes but also in the gametophytes of liverworts and hornworts, while they appear to be absent inmosses (Read et al., 2000; Renzaglia et al., 2007).The fungal associations in members of the Marchantiopsida (complex thalloid liverworts) and Metzgeriidae (simple thalloidliverworts) are cytologically similar to AMs (Strullu et al.,1981; Pocock and Duckett, 1984; Ligrone and Lopes, 1989;Ligrone and Duckett, 1994). Similar associations have alsobeen described in  Haplomitrium  and  Treubia  (Carafa et al.,2003; Duckett et al., 2006a), two taxa recently placed in a cladethat is sister to all other liverworts (Forrest and Crandall-Stotler, 2004, 2005; Heinrichs et al., 2005; Forrest et al., 2006).With the application of molecular techniques, the fungalsymbiont in  Marchantia foliacea  has been identified asbelonging to the glomeromycotean genus  Glomus , group A(Russell and Bulman, 2005). An assemblage of simple thalloidliverworts and the leafy liverworts (Jungermanniidae) form a diversity of endophytic associations with asco- or basidiomy-cetes or are fungus-free (Kottke et al., 2003; Nebel et al., 2004;Duckett et al., 2006b).With reference to the topology of liverwort phylogeny asrevealed by recent molecular work (Davis, 2004; Forrest andCrandall-Stotler, 2004, 2005; Heinrichs et al., 2005), it hasbeen suggested that the association with glomeromycoteanfungi is a plesiomorphy in the liverworts (Nebel et al., 2004;Kottke and Nebel, 2005). Moreover, considering that theliverworts are almost unanimously recognized as the earliest- 1 Manuscript received 24 February 2007; revision accepted 16 August 2007.This work was funded by grants from the Seconda Universita` di Napoliand Regione Campania, Italy (LR 5, 2003). The research in Torino wasfunded by the Biodiversity Project of CNR, Italy. The authors thank M.Hahn (Complex Carbohydrate Research Center, University of Georgia,USA) and J. P. Knox (Centre for Plant Sciences, University of Leeds, UK)for the generous gift of the antibodies used in this study, and V.Gianinazzi-Pearson (INRA, Dijon, France) for supplying the spores of   G.mosseae  and  G. clarum.  The authors also thank K. Renzaglia, the staff at the IMAGE Center (Southern Illinois University), and the staff at theCISME (University of Naples  ‘‘ Federico I, ’’  Italy) for laboratory andelectron microscopy facilities; K. Pell (QMUL) for technical assistance;the Department of Plant and Microbial Sciences, University of Canterbury,Christchurch, New Zealand, for laboratory facilities; the New ZealandDepartment of Conservation for granting collecting permits; and B.Butterfield (University of Canterbury) and D. Glennie (Landcare, Lincoln,New Zealand) for their help in the collection of the specimens used in thisstudy. J.G.D. was supported by an overseas travel grant from the RoyalSociety (UK) in New Zealand and by a DEFRA Darwin Initiative grant inChile. R.L. was supported by a grant from CNR (Italy) in New Zealand. 5 Author for correspondence (e-mail: roberto.ligrone@unina2.it)American Journal of Botany 94(11): 1756–1777. 2007.  divergent clade in the phyletic tree of land plants (Nickrent et al., 2000; Dombrovska and Qiu, 2004; Groth-Malonek et al.,2005; Qiu et al., 2006) and that the Glomeromycota are basal tothe other mycorrhiza-forming fungi (Schu¨ßler et al., 2001;James et al., 2006), it has been suggested that glomeromyco-tean associations (GAs) in liverworts predated the arbuscular mycorrhizas in vascular plants (Nebel et al., 2004; Kottke andNebel, 2005; Duckett et al., 2006a; Wang and Qiu, 2006). Analternative scenario, i.e., secondary host shift of glomeromy-cotean symbionts from tracheophytes to liverworts, has beenconsidered by Selosse (2005), mainly on the basis of Russelland Bulman’s (2005) identification of the fungal endophyte of   Marchantia paleacea  as a member of the  Glomus  Group A,i.e., a derived group in the phyletic tree of Glomeromycota (Schu¨ßler et al., 2001).In spite of the growing interest in fungus–liverwort associations in recent years, current information on their cytology and physiology is remarkably sparse. In particular, asconcerns putative GAs, the information available for most of the taxa reported by Nebel et al. (2004) is from light microscopy and generally does not go beyond the notion of the presence/absence of fungal endophytes tentatively referredto as glomero, basidio- or ascomycetes. Owing to the smallnumber of taxa investigated in detail to date, it is impossible toreach any general conclusions about the cytology of putativeGAs in liverworts.The general aim of this long-standing investigation was toprovide an exhaustive survey of the biology of GAs inliverworts and specifically to (1) identify the fungal endophytesthrough molecular analysis in selected liverwort taxa; (2)determine the taxonomic and geographical distribution of GAsin liverworts through a morphological (light and electronmicroscopy) analysis of taxa collected worldwide; (3) inves-tigate the level of cellular compatibility between liverworts andfungi through a detailed immunocytochemical analysis of their contact surfaces; and (4) confirm Koch’s postulates through invitro synthesis of GAs from axenic liverwort cultures andspores of known glomeromycotean fungi. The data presentedare discussed in the context of the srcins of GAs in liverwortsand their evolutionary relationships with AMs.MATERIALS AND METHODS The liverwort species examined, their taxonomic position, fungal status, andgeographical srcin are listed in Table 1. Liverwort taxonomy follows Crandall-Stotler and Stotler (2000) and Heinrichs et al. (2005). With the exception of fewexceedingly rare species, the diagnosis for fungal status was based on the studyof samples from at least two separate collection sites and from freshly-collectedspecimens. At least 20 plants were examined for each sample. For voucher information of the taxa examined in this study, see the Appendix.  Molecular analysis  —  Molecular analysis of fungal endophytes was carriedout for the following liverwort species:  Haplomitrium chilensis ,  Conocephalumconicum, Monoclea gottschei, Fossombronia echinata ,  Pellia endiviifolia. Healthy thalli or, in the case of   H. chilensis , subterranean mycotrophic axes(Carafa et al., 2003) were carefully rinsed with distilled water, and colonizedparts were isolated with a razor blade under a dissecting microscope. Thesamples, each about 50–100 mg, were surface-sterilized with cloramine T (3%)and streptomycin (0.3%) followed by two rounds of sonication. A mininum of two samples for each liverwort species were processed separately.DNA was extracted using the Dneasy Plant Mini kit (Qiagen, Valencia,California, USA) according to manufacturer protocols. Partial small-ribosomal-subunit (SSU) DNA fragments (550 bp) were amplified using the universaleukaryotic primer NS31 (Simon et al., 1993) and the Glomeromycota-specificprimer AM1 (Helgason et al., 1998). DNA extracts from  Glomus mosseae (BEG12) and  Gigaspora rosea  (BEG9) isolates were used as positive controls,while DNA extracts from fungus-free apical parts of the thalli were used asnegative controls.The PCR reaction was performed in a total volume of 25  l L containing 2  l Lof template solution, 0.2 mM of each dNTP, 10 pmols of each primer, 1 U of REDTaq DNA polymerase (Sigma, St. Louis, Missouri, USA) and 1 3 REDTaqReaction buffer (SIGMA). Amplification was performed in a GeneAmp PCRsystem 9700 (PerkinElmer, Waltham, Massachussets, USA) programmed asfollows: 1 3 3 min at 95 8 C; 35 3 1 min at 95 8 C, 1 min at 58 8 C, 2 min at 75 8 C;1  3  7 min at 72 8 C. Electrophoretical analysis of the PCR products revealed a single band of 550 bp. This fragment was purified from gel using the QIAquickpurification kit (QIAGEN), cloned into a pGEM-T Easy Vector (Promega,Madison, Wisconsin, USA), and then transformed into  Escherichia  coli JM109High Efficiency Competent Cells (Promega).Thirty putatively positive transformant clones (white colonies) from eachliverwort sample were selected manually, and the DNA extracted from eachclone was amplified using the PCR mix and program detailed previously. For RFLP (restriction fragment length polymorphism) analysis, aliquots of 4  l L of each PCR amplicon were mixed with 16  l L of digestion mix containing 2.0  l Lbuffer 10 3 , 0.2  l L bovine serum albumin, 13.3  l L H 2 O, and 0.5  l L of therestriction enzyme  Hinf  I or   Hsp 92II (Promega) for 3 h at 37 8 C. Fragment patterns were analyzed on agarose gel containing 0.84 % agarose (Sigma) and1.5% high-resolution agarose (Sigma). One to four PCR amplicons weresequenced for each restriction pattern and species, using the vector-specificprimers T7 and SP6, at the DNA Sequences Naples Facilities. The sequenceshave been deposited in GenBank under the accession numbers reported inTable 3.Forward and reverse sequences were analyzed using the program BioLign4.0.6 (http://en.bio-soft.net/dna/BioLign.html). DNA sequences were comparedto GenBank database using the BLAST algorithm (Altschul et al., 1997) for identification. Data bank sequences with high homology to our sequences wereincluded in the data set, using the profile alignment function CLUSTAL W(Thompson et al., 1994) for multiple alignment. The nearest relatives of eachsequence were inferred with the neighbor-joining algorithm (Saitou and Nei,1987) and the Kimura two-parameter model (Kimura, 1980), using the PHYLIPpackage (Felsenstein, 1989). The confidence of branching was assessed using1000-bootstrap resampling (Felsenstein, 1985).  Light and electron microscopy  —  Both fresh and fixed samples wereexamined by light microscopy. The visibility of fungal hyphae in rhizoids andhand-cut sections of the thallus was improved by staining with 0.05% trypanblue in lactophenol (Ligrone and Lopes, 1989) or 0.05% aniline blue in lacticacid. Autofluorescence of liverwort cell walls was observed on fresh hand-cut sections using an excitation filter at 365 nm and a barrier filter with a transmission cutoff at 397 nm.Colonized areas of the thallus of fungus-containing specimens were cut intosmall pieces under a dissecting microscope and fixed with a mixture of 3%glutaraldehyde, 1% freshly prepared formaldehyde, and 0.75% tannic acid in0.04 M piperazine-N,N 0  –bis(2-ethanesulfonic acid) (PIPES) buffer, pH 7.0, for 2 h at room temperature under gentle vacuum. The samples were then rinsed in0.08 M PIPES buffer and twice in 0.08 M Na-cacodylate buffer, and postfixedin 1% OsO 4  in 0.08 M Na-cacodylate buffer, pH 6.7, overnight at 4 8 C.Following dehydration in a step gradient of ethanol and one step in propyleneoxide at 4 8 C, the samples were slowly infiltrated with Spurr’s resin(Polysciences, Warrington, Pennsylvania, USA) at 4 8 C, transferred topolypropylene dishes, and cured at 68 8 C for 24 h. For light microscopy, 0.5- l m-thick sections of resin-embedded samples were cut with a diamondhistoknife, stained with 0.5% toluidine blue O in 1% Na-tetraborate, andphotographed with a Zeiss Axioskop (Zeiss, Jena, Germany) light microscopeequipped with a Sensicam QE (Applied Scientific Instrumentations, Eugene,Oregon, USA) digital photocamera. For transmission electron microscopy(TEM), ultrathin sections were cut with a diamond knife, collected on 300-mesh uncoated nickel grids, stained with 3% uranyl acetate in 50% methanolfor 15 min and in Reynold’s lead citrate for 10 min, and observed with a Jeol1200 EX2 (Jeol, Tokyo, Japan) electron microscope.For scanning electron microscopy (SEM), the samples were cut with a razor blade and taken through a 1 : 1 ethanol:acetone series to remove the cytoplasm,osmicated for 48 h in aqueous 2% OsO 4 , and stored in 70% ethanol. Thesamples were then dehydrated in anhydrous ethanol and critical point driedusing CO 2  as the transfusion fluid, mounted on stubs, and sputter-coated with390 nm palladium-gold. The samples were viewed using a Hitachi (Hitachi,Tokyo, Japan) S570 scanning electron microscope. November 2007] L IGRONE ET AL .—G LOMEROMYCOTEAN ASSOCIATIONS IN LIVERWORTS  T ABLE  1. Fungal associations in liverworts. Liverwort taxa Fungal status Geographic origin Haplomitriopsida:Haplomitriales  Haplomitrium blumei  (Nees) R.M. Schust. a  G Malaysia (2)  H. gibbsiae  (Steph.) R.M. Schust. a  G New Zealand (7), Uganda (1)  H. hookeri  (Smith) Nees a  G UK (5)  H. intermedium  Berrie ac G Australia (1)  H. ovalifolium  R.M. Schust  a  G New Zealand (2)  H. chilensis  R.M. Schust. ac G Chile (3)Treubiales Treubia lacunosa  (Colenso) Prosk. a  G New Zealand (1) T. lacunosoides  Pfeiffer, Frey & Stecha  ac G New Zealand (6) T. pygmaea  R.M. Schust. ac G New Zealand (6)Marchantiopsida (complex thalloid liverworts):Blasiales  Blasia pusilla  L. a   — UK (4), USA (1)Sphaerocarpales Sphaerocarpos michelii  Bellardi c  — Italy (1), UK (1) S. texanus  Austin c  — UK (1) Geothallus tuberosus  Campb. bc  — USA (1)  Riella americana  M.Howe & Underwood c  — USA (1)  Riella helicophylla  (Boryet Mont.) Mont. c  — Greece (1)Monocleales  Monoclea forsteri  Hook . a  G New Zealand (7)  M. gottschei  Lindb . a  G Chile (2), Mexico (1), Venezuela (1)MarchantialesAytoniaceae  Asterella bachmanii  (Steph.) S.W.Arnell ac G South Africa (1)  A. muscicola  (Steph.) S.W.Arnell c G Lesotho (1)  A. wilmsii  (Steph.) S.W.Arnell ac G South Africa (1)  A. tenera  (Mitt.) R.M. Schust  . ac G New Zealand (4)  A. australis  (Hook.f. & Taylor) Verd . ac G New Zealand (4) Cryptomitrium oreoides  Perold c  — Lesotho (2)  Mannia angrogyna  (L.) A. Evans a   — Italy (1)  M. fragrans  (Balb.) Frye & L. Clark — China (1), Germany (1)  Plagiochasma exigua  (Schiffn.) Steph. c G South Africa (2), Lesotho (2)  P. rupestre  (J.R.Forst. & G.Forst.) Steph. a  G (1) South Africa (3), Lesotho (2)  Reboulia hemispherica  (L.) Raddi a  G Italy (2), UK (3), Chile (2)Wiesnerellaceae Wiesnerella denudata  Schiffn. b  — (2) Japan (1), Java (1), Nepal (1), Sikkim (1)Conocephalaceae Conocephalum conicum  (L.) Dumort. a  G France (1), Italy (2), UK (6), USA (2) C. salebrosum  Szweykowski, Buczkowska & Odrzykoski c G UK (2), USA (3)Lunulariaceae  Lunularia cruciata  (L.) Dumort. ex Lindb. a  G France (1), Italy (2), UK (4)Marchantiaceae  Bucegia romanica  Raddi bc  — Rumania (2)  Dumortiera hirsuta  (Sw.) Nees a  G Chile (1), France (1), Venezuela (1), UK (1)  Marchantia berteroana  Lehm. & Lindb. c G Chile (1), Venezuela (1)  M. foliacea  Mitt. c G Chile (1), New Zealand (2)  M. pappeana  Lehm. a  G Lesotho (2)  M. polymorpha  subsp.  polymorpha  Gottsche, Lindb. & Nees .  — UK (4)  M. polymorpha  subsp.  ruderalis  Bischl. & Boisselier — (1) UK (3)  M. polymorpha  subsp.  montivagans  Bischl. & Boisselier  a  G UK (2)  Neohodgsonia mirabilis  (H. Perss.) H. Perss. a  G (3) New Zealand (2)  Preissia quadrata  (Scop.) Nees a  G Italy (1), UK (4)Monosoleniaceae  Monosolenium tenerum  Griffith c  — Germany (from aquarium) (1), Japan (1)  Peltolepis grandis  Lindb. b  — Norway (1), Russia (Siberia) (1), Switzerland (1)Cleveaceae  Athalamia hyalina  (Sommerf.) S. Hatt. c G Italy (1), USA (1)  A. pinguis  W. Falc. bc G India (1) Sauteria alpina  (Nees) Nees b  — Switzerland (2)Exormothecaceae  Aitchisoniella himalayensis  Kash. bc  — India (1)  Exormotheca holstii  Steph. — Lesotho (1)  E. pustulosa  Mitt. c  — Lesotho (1) Stephensoniella brevipedunculata  Kash. bc  — India (1)Cyathodiaceae —  Cyathodium cavernarum  Kunze c  — Uganda (1) A MERICAN  J OURNAL OF  B OTANY  [Vol. 94  T ABLE  1. Continued. Liverwort taxa Fungal status Geographic origin C. foetidissimum  Schiffn. a   — (2) Italy (1)Corsiniaceae Corsinia coriandra  (Spreng.) Lindb. a  G (1) Italy (2) Cronisia fimbriata  (Nees) Whittem. & Bischl. bc  — Brazil (1)Monocarpaceae  Monocarpus sphaerocarpus  Carr  c  — Australia (1)Targionaceae Targionia hypophylla  L . a  G France (2), Italy (2), New Zealand (3), UK (2)Oxymitraceae Oxymitra incrassata  (Broth.) Sergio & Sim-Sim a   — Italy (1) O. cristata  Garside ex Perold c  — Lesotho (1)Ricciaceae  Riccia  subgenus  Ricciella R. canaliculata  Hoffm. c  — UK (2)  R. cavernosa  Hoffm. — Lesotho (2), UK (2)  R. crystallina  L. c  — Lesotho (3), UK (1)  R. fluitans  L. — UK (4)  R. huebeneriana  Lindb. — UK (1)  R. stricta  (Lindb.) Perold c  — Lesotho (2), Botswana (1)  Riccia  subgenus  Riccia R. albolimbata  S.W.Arnell c  — Botswana (1)  R. beyrichiana  Hampe ex Lehm. — UK (1)  R. crozalsii  Levier  c  — Italy (1), UK (2)  R. glauca  L. — UK (4)  R. montana  Perold c  — Lesotho (1)  R. nigrella  DC . c  — Italy (1), Lesotho (2), New Zealand (1), UK (2)  R. okahandjana  S.W.Arnell c  — Botswana (1)  R. sorocarpa  Bisch. — UK (2)  R. subbifurca  Croz. c  — UK (3)  Ricciocarpus natans  (L.) Corda — UK (2)Jungermanniopsida, Metzgeriidae (simple thalloid liverworts):Phyllothalliaceae  Phyllothallia nivicola  A. E. Hodgs. c  — Chile (1), New Zealand (1)Fossombroniaceae  Austrofossombronia australis  (Mitt.) R.M. Schust. c G New Zealand (1)  Fossombronia angulosa  (Dicks.) Raddi G France (1), Italy (1), UK (3)  F. caespitiformis  De Not. ex Rabenh. c G Italy (1)  F. echinata  MacVicar  ac G Italy (2)  F. pusilla  (L.) Nees G UK (3)  F. maritima  (Paton) Paton G UK (!)  F. wondraczeckii  (Corda) Dum. ex Lindb. G UK (3)  Petalophyllum ralfsii  (Wils.) Nees & Gottsche a  G Italy (1), UK (3)Allisoniaceae  Allisonia cockaynii  (Steph.) R.M. Schust. ac G New Zealand (4)Pelliaceae  Noteroclada confluens  Tayl. ex Hook. & Wilson a  G Chile (3), Venezuela (1)  Pellia endiviifolia  (Dicks.) Dum. a  G Italy (1), UK (4)  P. epiphylla  ( L.) Corda  a  G UK (5), USA (2)  P. neesiana  (Gottsche) Limpr. G UK (4)Pallaviciniaceae Greeneothallus gemmiparus  Hassel ac G Chile (1)  Jensenia connivens  (Colenso) Grolle ac G Venezuela (1)  J. wallichii  Colenso ac G Venezuela (1)  Moerckia hibernica  (Hook.) Gottsche a   — (3) UK (2)  M. blyttii  (Moerch) Brockm. G Switzerland (1), UK (4)  Pallavicinia connivens  (Colenso) Steph. ac G New Zealand (2)  P. xiphoides  (Hook.f. & Taylor) Trevis. a   — (3) New Zealand (2)  P. tenuinervis  (Hook.f. & Taylor) Trevis. c  — New Zealand (2)  P. indica  Schiffn. c  — Malaysia (1)  P. lyellii  (Hook.) Gray a   — (2) UK (1), USA (1)  Podomitrium phyllanthus  (Hook.) Mitt. c G New Zealand (1) Symphyogyna brasiliensis  Nees & Mont. a  G South Africa (1), Venezuela (1) S. brogniartii  Mont. a  G Venezuela (1) S. hymenophyton  (Hook.) Mont. & Nees c G New Zealand (4) S. subsimplex   Mitt. c G New Zealand (2) S. undulata  Colenso c G New Zealand (2)  Xenothallus vulcanicolus  R.M. Schust. ac G New Zealand (1)Hymenophytaceae  Hymenophyton flabellatum  (Labill.) Dum. a  G (1) New Zealand (4) November 2007] L IGRONE ET AL .—G LOMEROMYCOTEAN ASSOCIATIONS IN LIVERWORTS   Immunocytochemistry  —  Epitopes associated with cell wall polysaccha-rides and proteins were localized immunocytochemically in  Marchantia polymorpha  subsp.  montivagans  and  Conocephalum conicum . Colonized partsof the thalli were cut into 0.5-mm-thick slices and fixed with 3% glutataldehydein 0.05 M PIPES buffer, pH 7.4 for 2 h at room temperature. After carefulrinsing in buffer, the samples were dehydrated in a step gradient of ethanol,slowly infiltrated with LR White resin (Polysciences, Warrington, Pennsylva-nia, USA), and cured at 60 8 C for 24 h. The protocols followed for immunohistochemistry and immunogold electron microscopy have beendescribed in detail in Ligrone et al. (2002). The antibodies tested, their specificity, and source are listed in Table 4. For both light and electronmicroscopy, controls were routinely made by omitting the incubation step withthe primary antibody and were always completely negative.  Resynthesis experiments  —  The apical parts of wild thalli of   C. conicum ,about 2 mm long, were isolated and surface-sterilized with hypochlorite for 3min, washed thoroughly in sterile distilled water, and placed on 0.25% phytagel(Sigma) plates either lacking nutrients or containing one-fourth MS nutrient solution (Murashige and Skoog, 1962). The plates were kept in a Sanyo MLR-350 H growth chamber (Sanyo, Msrcuchi City, Osaka, Japan)under a 12 h/12h day/night photoperiod with a light irradiance of 50 W  m  2 and a 12/10 8 C day/  T ABLE  1. Continued. Liverwort taxa Fungal status Geographic origin Makinoaceae Verdoornia succulenta  R.M. Schust. ac B New Zealand (2)Aneuraceae  Aneura lobata  subsp.  australis  R.M. Schust. ac B New Zealand (4)  A. maxima  Schiffn. (Steph.) a  B (3) USA (1)  A. novaeguineensis  Hewson ac B New Zealand (1)  A. pinguis  (L.) Dum. a  B UK (4)  A. pseudopinguis  (Herzog) Pocs ac B Lesotho (2) Cryptothallus mirabilis  Malmb a  B UK (4)  Riccardia intercellula  E.A.Brown c B New Zealand (1)  R. pennata  E.A.Brown c B New Zealand (1)  R. chamedryfolia  (With.) Grolle — (3) UK (4)  R. cochleata  (Hook.f. & Taylor) Kuntze c  — New Zealand (1)  R. eriocaula  (Hook.) Besch. & C.Massal. c  — New Zealand (2)  R. incurvata  Lindb. — (4) UK (5)  R. latifrons  (Lindb.) Lindb. — (4) UK (3)  R. multifida  (L.) Gray — (3) UK (3)  R. palmata  (Hedw.) Carruth. — (3) UK (3)Metzgeriaceae  Apometzgeria pubescens  (Schrank) Kuwah. — UK (2)  Metzgeria conjugata  Lindb. — UK (3)  M. decipiens  (Massal.) Schiffn. & Gotts. — Chile (3)  M. temperata  Kuwah. a   — UK ( 2)  M. furcata  (L.) Dum a   — UK (4)  M. fruticulosa  (Dicks.) A. Evans — UK (2)Pleuroziaceae  Pleurozia purpurea  Lindb. — UK (3)  P. gigantea  (Web.) Lindb. c  — Malaysia (1)  Notes:  G, glomeromycotean endophytes; B, basidiomycotean endophytes; —, fungal endophytes absent. Numbers in parentheses after each country of srcin refer to the number of voucher specimens examined (see Appendix). a  Liverwort taxa examined by electron microscopy in this and our previous studies. b Herbarium specimens only. c Liverwort taxa not included in the previous survey by Nebel et al. (2004); under column  ‘‘ Fungus status ’’ : (1) taxon reported by Nebel et al. (2004) asnonmycorrhizal or as associated with (2) glomeromycotean fungus, (3) unidentified fungus, or (4) basidiomycetous fungus. Unless indicated otherwise, thefungal status agrees with that reported by Nebel et al. (2004) for the same species. No glomeromycotean associations were found in the Jungermanniidae(leafy liverworts).T ABLE  2. Restriction profiles of fungal small-subunit rDNA 550-bpamplicons from fungus-associated liverworts. Restriction enzyme Restriction profiles (bp)  Hinf  I H1 (383, 120, 5)H2 (244, 188, 90, 25)H3 (383, 141, 25)H4 (334, 190, 25)H5 (334, 141, 49, 25)H6 (278, 244, 25)  Hsp 92II S1 (249, 148, 90, 23)S2 (291, 163, 93)S3 (291, 258)S4 (291, 142, 116) T ABLE  3. Combinations of restriction profiles with  Hinf  I (H1-H6) and  Hsp 92II (S1-S4), and GenBank sequence codes (in parentheses) of fungal small-subunit rDNA 550-bp amplicons from fungus-associatedliverworts.  Haplomitrium chilensis  H2S2 (AM412526, AM412528) Conocephalum conicum  H4S2 (AM412536, AM412537, AM412538)H4S3 (AM412539)H3S4 (AM412540, AM412541)  Monoclea gottschei  H1S1 (AM412542, AM412543, AM412544,AM412545)  Fossombronia echinata  H5S3 (AM 412532, AM412535)H2S3 (AM 412534)H2S2 (AM 412533)  Pellia endiviifolia  H3S3 (AM 412529)H6S2 (AM 412530) A MERICAN  J OURNAL OF  B OTANY  [Vol. 94
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