Culturing and Direct DNA Extraction Find Different Fungi From

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CulturingBlackwell Publishing Ltd. and direct DNA extraction ﬁnd different fungi from the same ericoid mycorrhizal roots

Tamara R. Allen1, Tony Millar1, Shannon M. Berch2 and Mary L. Berbee1 1Department of Botany, The University of British Columbia, Vancouver BC, V6T 1Z4, Canada; 2Ministry of Forestry, Research Branch Laboratory, 4300 North Road, Victoria, BC V8Z 5J3, Canada

Summary

Author for correspondence: • This study compares DNA and culture-based detection of fungi from 15 ericoid Mary L. Berbee mycorrhizal roots of salal (Gaultheria shallon), from Vancouver Island, BC Canada. Tel: (604) 822 2019 •From the 15 roots, we PCR ampliﬁed fungal DNAs and analyzed 156 clones that Fax: (604) 822 6809 Email: [email protected] included the internal transcribed spacer two (ITS2). From 150 different subsections of the same roots, we cultured fungi and analyzed their ITS2 DNAs by RFLP patterns Received: 28 March 2003 or sequencing. We mapped the original position of each root section and recorded Accepted: 3 June 2003 fungi detected in each. doi: 10.1046/j.1469-8137.2003.00885.x • Phylogenetically, most cloned DNAs clustered among Sebacina spp. (Sebaci- naceae, Basidiomycota). Capronia sp. and Hymenoscyphus erica (Ascomycota) predominated among the cultured fungi and formed intracellular hyphal coils in resynthesis experiments with salal. •We illustrate patterns of fungal diversity at the scale of individual roots and compare cloned and cultured fungi from each root. Indicating a systematic culturing detection bias, Sebacina DNAs predominated in 10 of the 15 roots yet Sebacina spp. never grew from cultures from the same roots or from among the > 200 ericoid mycorrhizal fungi previously cultured from different roots from the same site. Key words: fungal ericoid mycorrhizal fungi, fungal diversity, fungal detection, Sebacina, Capronia, Hymenoscyphus, Ericaceae. © New Phytologist (2003) 160: 255–272

primarily fungi in the Ascomycota, this sequence-based Introduction approach found that about half of the fungi were in the Roots of salal (Gaultheria shallon Pursh) a western North Basidiomycota or Zygomycota, rather than the Ascomycota American shrub, are usually heavily colonized by mycorrhizal and several of the sequences could not be classiﬁed into a fungi (Xiao & Berch, 1996). In this study, we compare the familiar fungal order (Vandenkoornhuyse et al., 2002). fungi in an individual mycorrhizal root as detected by direct Like the roots of grasses, ericoid mycorrhizae offer a DNA extraction and analysis, with the fungi as detected by relatively simple biological environment where fungal DNA their growth in pure culture. Failure of bacterial cells to grow sequence diversity can conveniently be assayed. No one had under standard cultural conditions has been reported previously studied the diversity of fungal DNAs from these repeatedly, and as reviewed by Pace (1997), 99% of bacteria roots. However, the DNAs from the roots could be matched observed microscopically are missed in standard culturing against an extensive database of sequences from fungi cultured experiments. Similar losses may take place among fungi from ericoid and epacrid mycorrhizae from around the world cultured from natural environments. Instead of using the (McLean et al., 1999; Bergero et al., 2000; Berch et al., 2002; more traditional isolation in pure culture to detect fungi from Cairney & Ashford, 2002). the roots of a grass, Arrhenatherum elatius (L.) P. Beauv., Our interest in ericoid mycorrhizal fungi has its origin in a Vandenkoornhuyse et al. (2002) extracted total DNA and biological problem, but is also linked to an economic problem. then used fungal speciﬁc PCR primers to amplify and Conifer seedlings used in reforestation of some sites previ- sequence the 18S ribosomal RNA genes of root-associated ously occupied by cedar-hemlock (Thuja plicata Donn./Tsuga fungi. In contrast to most culture-based studies that detect heterophylla (Raf.) Sarg.) forests on Vancouver Island, British

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Columbia, Canada turn yellow and fail to grow while the eri- PCR amplification from segments of highly colonized, mycor- caceous shrub salal thrives. Colonized with fungal mutualists, rhizal salal roots. For comparison with these fungal DNAs, the roots of the salal may be able to access nutrients, especially we would culture fungi from root segments flanking the seg- nitrogen, that may be otherwise unavailable (Read, 1991; ments used for direct DNA extraction, mapping the original Perotto et al., 2002). To contribute to an understanding of the position of each segment from each root. From the mapped biology underlying the conifer growth check problem, Xiao & positions of root segments used for direct DNA extraction Berch (1996, 1999), Monreal et al. (1999), and Berch et al. and for culturing, we could reconstruct the original position (2002) have been investigating the diversity and functioning of each fungal colony or DNA in the original root and track of some of the mycorrhizal fungi collected from salal roots which root segments gave no fungal colonies. To help distinguish from the Salal Cedar Hemlock Integrated Research Project other saprophytes or parasites from likely mycorrhizal part- (SCHIRP) trial site at Port McNeil, Vancouver Island, British ners, we planned to test the fungi in culture for their ability Columbia. Through their series of studies, a succession of dif- to form hyphal coils in the epidermal and cortical cells in ferent techniques have revealed different mycorrhizal fungi. resynthesis experiments with salal. Discrepancies between Using culturing and morphological identification, Xiao & the fungi detected as DNAs and the fungi that grew in pure Berch (1996) recognized four groups of fungi among over 200 culture would suggest a possible systematic detection bias mycorrhizal fungal cultures from salal from the SCHIRP site. against some fungal species. Using a combination of detection Two groups could be identified by their sporulation patterns, methods should optimize the chances of identifying the entire while two remained unidentified and were nicknamed suite of mycorrhizal fungi occupying root systems. ‘Unknown 1’ and ‘Unknown 2’. Monreal et al. (1999) determined ITS2 sequences from all fungi that had been confirmed Materials and Methods as ericoid mycorrhizal symbionts, including ‘Unknown 1’ and ‘Unknown 2.’ Among additional fungi cultured from the A total of 15 mycorrhizal hair roots of salal (G. shallon) were Vancouver Island SCHIRP site, Monreal et al. (1999) found selected for detailed analysis, from samples collected from the isolates with sequences matching the well-characterized eri- SCHIRP study site located between Port McNeil and Port coid mycorrhizal fungus Hymenoscyphus ericae. Lynn Sigler Hardy on northern Vancouver Island, British Columbia (personal communication) later used conidial morphology to (50°60′ N, 127°35′ W). The first sample was collected in confirm Monreal’s isolates as H. ericae. October 1998 and samples two and three were collected Berch et al. (2002) discussed the phylogenetic relationships from plants about 30 m apart in January 1999. Each sample, of sequences from cultured or cloned fungi from about 800 collected with a shovel, consisted of about 500 g of soil, rhizomes salal root tips from the Vancouver Island SCHIRP site and and roots. Because salal reproduces with rhizomes, we noted which of the cultured fungi produced mycorrhizae in could not be sure whether samples were from the same or resynthesis experiments with salal. Berch et al. (2002) discovered different plants. Samples were kept in plastic bags on ice, that isolates similar to most of the ericoid mycorrhizal fungi transported to the University of British Columbia, and held grown in pure culture from salal roots from our Vancouver at 4°C until cleaning. Small roots were removed from the Island SCHIRP site were also found in Europe or Australia. rhizomes, soaked in cool tap water and washed gently to All the ericoid mycorrhizae from pure cultures from salal were remove soil and plant debris. Using light microscopy, roots from ascomycetous fungi. However, Berch et al. (2002) also were screened for healthy appearance (turgid cells and light reported that the predominant fungal clones from PCR color) and for fungal colonization. From these apparently amplifications of DNA extracts from salal mycorrhizae were healthy, colonized roots, sections with a total contiguous from basidiomycetes closest to inconspicuous jelly fungi in length of 4 cm were selected for study. Seven roots (#1011– the genus Sebacina. 1116) were selected for analysis from sample one from Our objective in this study was to analyze the patterns of October. From the January collections, from sample two, two fungal diversity, as detected by culturing and by cloning, at roots (#1302–1322) were selected, and from sample three, six the scale of individual roots. We planned to analyze the fungal roots (#1442–1542) were selected (Fig. 1). Roots 1482 and DNAs that we could detect by direct DNA extraction and 1502 were from the same rhizome as were roots 1522 and

Fig. 1 Diagrams of 15 mycorrhizal salal roots, showing the cultured or cloned fungi detected from different segments of each root. As sketched at the top, ericoid mycorrhizal root pieces, 4 cm in length were selected for this analysis. To the left are root identification numbers and the total number of different fungal genotypes detected in each root. A different colour indicates each distinct fungal genotype. The middle segments of the diagram show the numbers of clones of Sebacina spp., Capronia sp., Hymenoscyphus ericae, and ‘other’ fungi, obtained by direct DNA extraction and PCR amplification from the middle, 2 cm long segment of each root. The five narrow boxes at each end of each root diagram show the fungi that grew in pure culture from the 2 mm root sections. Empty boxes indicate lack of fungal growth. ‘myc’ designates an isolate that formed hyphal coils in salal root cells (as would be typical of ericoid mycorrhizae) in resynthesis experiments. Absence of a comment on mycorrhizal status usually indicates an isolate that grew too poorly for resynthesis tests. The letter following ‘other’ designates a unique RFLP pattern. A sequence number designates each sequence from a clone or a culture, and the corresponding GenBank accession numbers are provided in Table 1. For the phylogenetic clustering of the ‘other’ sequences, see Fig. 2(a),(b).

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1542. Each of the other roots was from a different rhizome. regions were cloned using InvitrogenTM TOPO TA Cloning® Each root was sterilized in 30% hydrogen peroxide for 1 min kit (Invitrogen Canada Inc. Burlington, ON, Canada) follow- and washed three times in sterile distilled water. The 4-cm ing the manufacturer’s instructions. From the first root, we mycorrhizal root segments were further subdivided into 11 selected approximately 50 colonies with plasmid inserts for pieces (Fig. 1). The largest piece, a 2-cm section from the further analysis. For subsequent roots, we selected 20 colonies middle of the 4 cm root was used for direct DNA extraction with inserts for further analysis when possible. Some roots and PCR amplification. Ten 2 mm pieces, five from each end gave relatively little fungal PCR product, and as a result, fewer of the 2 cm middle section, were set on potato dextrose agar transformants were available for analysis. Plasmid DNAs for culturing. One of the pieces included the root tip. Because extracted using a QIAprep® kit (QIAGEN Inc., Mississauga the hair roots were highly branched, multiple fine lateral root Ontario, Canada) were digested with EcoR I to verify that an tips were present along the main root axis and some of the insert of the correct size was present, and the cloned insert was segments used for cloning or culturing also included a smaller, amplified by PCR. MspI and in some cases, RsaI digests of the lateral root tip. PCR amplified inserts were used to group similar isolates based on their RFLP patterns. To further characterize cultured fungi or cloned DNAs, we Cultures selected fungal DNAs from each RFLP group and used the The original location of each 2 mm piece was recorded to primer ITS3 to generate single-stranded ITS2 sequences allow reconstruction of the original fungal distribution along using Applied Biosystems AmpliTaq DyeDeoxyTM termina- each root. Genomic DNA from each fungus that grew in tor kit following the manufacturer’s instructions (PE Applied culture was extracted and amplified with the fungal specific Biosystems, Foster City, CA, USA). The ITS2 sequences were primer ITS1-F (Gardes & Bruns, 1993) or universal primers subjected to BLAST searches, and aligned with similar ITS4 or TW13 (http://plantbio.berkeley.edu/∼bruns/ sequences from GenBank, using ClustalW PPC (Higgins et al., primers.html#28s). The PCR product of each isolate was 1992) followed by manual adjustment with SeqApp (available digested with MspI. Some of the products were also digested through ftp://iubio.bio.indiana.edu/molbio/seqapp/). with RsaI. The fungal isolates were grouped by RFLP pattern. Given the extensive database of ericoid and epacrid mycor- Fungal isolates were tested for ability to form hyphal coils rhizal fungal ITS sequences, we had expected the ITS2 clone inside of the root cells of salal in Petri-dish growth chambers. sequences to find matches among GenBank’s ITS2 sequences. Fungal isolates that did form coils inside cells of living roots However, many of the cloned sequences failed to find matches. were termed ‘mycorrhizal.’ (Studies to confirm mycorrhizal To provide a more highly conserved sequence region for status would have required additional evaluation of whether phylogenetic identification of the clones, we switched from the fungus benefited the plant.) Seeds of salal were sterilized amplifying and sequencing the ITS2 regions alone to ampli- in 30% hydrogen peroxide for 1 min and placed on water agar fying an approximately 1200 bp region that included about for germination. Seedlings were transplanted to a low nutrient 500 bp of the 5′ end of the LSU gene as well as the ITS2 medium, modified Melin–Norkrans agar MMN (Xiao & region. We then used sequences including the LSU region for Berch, 1992) in plastic Petri dishes and inoculated with a fun- BLAST searches and to cluster clones in a preliminary phyl- gal culture. Roots were extracted from growth chambers and ogeny. For phylogenetic analysis, representatives of groups of stained with aniline blue. Colonization of the root cortical isolates were selected for more extensive sequencing, using cells was confirmed with light microscopy. Fungal isolates that primers ITS3, ITS4, cTB6 and TW13 (http://plantbio.berkeley. formed hyphal coils within root cells were re-tested by cultur- edu/∼bruns/primers.html#28s) to obtain double-stranded ing the fungus from sterilized roots of colonized plants and sequences across the 1200 bp sequence region. The LSU and inoculating the fungus on new plants. ITS plus LSU fragments were also aligned with sequences from BLAST searches from GenBank. To help track and sort sequences, each was assigned a number based on its original Direct DNA extraction, PCR amplification, cloning, and position in a preliminary alignment and phylogeny. GenBank sequence analysis accession numbers for all isolates in our alignments are listed Total genomic DNA was extracted from the middle 2 cm in Table 1. segments of 15, highly colonized, sterilized field collected Some ITS2 sequences were too different to be aligned. Simi- mycorrhizal root pieces using a DNeasy® Plant Mini Kit lar subsets of sequences were aligned in blocks, and gaps were (QIAGEN Inc., Mississauga Ontario, Canada). Genomic inserted to the other, more distant sequences. In this way, for DNA from sterile leaves from salal was extracted and used as example, ITS sequences of ascomycetes in the Leotiomycetes a negative control for each DNA extraction and PCR were aligned with one another, but the basidiomycete sequences amplification. Total fungal DNA was amplified with ITS1-F, were shifted to a different alignment block instead of being in combination with either ITS4 or TW13. PCR products aligned with or compared to the Leotiomycetes sequences. from the ITS and large subunit (LSU) ribosomal RNA gene Some portions of some of the ITS2 basidiomycete sequences

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Research 259 Lineage ... Leotiomycetes; Helotiales; Dermateaceae? 3 UAMH 10106 Accession numbers 2 Y064705 A Source 1 DNA Region (Read) ITS2 & LSU Culture AF081438 Leotiomycetes; UBC M8 UBCtra 1271C UBCtra 241 ITS2 ITS2 & LSU ITS2 GenBank Culture Culture AF081435 AF300750 AF149068 ...... Korf and Kernan UBC M20 UAMH 10130 Helotiales; Helotiaceae UAMH 10073 UAMH 10074 UBC S246UBCtra 1256CUBCtra 179CUBCtra 1311CUBCtra 143CUBCtra 1278CUBCtra 280CUBCtra 305C maius G. L. BarronOidiodendron & Booth) von Arx (Barron Udagawa, Uchiyama & Kamiya ITS2 ITS2 ITS2 ITS2 ITS2 ITS2 ITS2 ITS2 Culture GenBank ITS2 Culture Culture Culture Culture Culture AF081443 AF300737 Culture AF149077 AF300739 AF062798 AF149075 AF300738 Leotiomycetes? ... AF149073 AF149076 ...... Leotiomycetes; Onygenales; Myxotrichaceae ...... Hymenoscyphus ericae H. ericae H. ericae H. ericae Sample (isolate, clone or species) Fungal isolates or clones in phylogenetic analyses able 1 262728 UBCtra 14424 UBCtra 141 ITS2 ITS2 Clone Culture AF300751 AF149067 ...... 29 30 3132.2 Scytalidium vaccinii Dalpé, Sigler & Litten UBCtra 1462.1 ITS2 ITS2 AF081439 Clone ... AY112916 Leotiomycetes? 1 2 3 4 5 6 7 8 9 9.21011 UBCtra 15222 A12 Byssoascus striatosporus1314 Myxotrichum deﬂexum Berk.15 Gymnostellatospora japonica1616.2 Pseudogymnoascus roseus Raillo UBCtra 1157C17 UBCtra Seq67 Seaver18 Pezicula ocellata (Pers. Fr.) Pezicula alba Guthrie19 ITS220 UBCtra 1061 C ITS221 UBCtra 288 ITS2 ITS221.2 ITS222 UBCtra 1182C23 UBCtra 29 ITS223.2 UBCtra Seq68 UBCtra P1322.3B GenBank GenBank24 Clone GenBank25 UBCtra 1025C GenBank UBC S9 UBCtra Seq57 ITS2 ITS2 & LSU UBCtra 1128C GenBank ITS2 AF062817 AF062814 AF062818 and AY064704 AF062819 AY112913 ITS2 Culture AF141199 Clone ... ITS2 ... ITS2 ...... ITS2 ITS2 Culture ITS2 AF300736 Leotiomycetes; Helotiales; Dermateaceae ITS2 Culture AF300743 Culture ITS2 Clone ITS2 ITS2 & LSU Clone Culture AF300742 Leotiomycetes? Culture AF149074 ... GenBank Clone AF300744 Culture AY112914 Leotiomycetes? AF149087 AF300755 AF300741 AF081442 ... AY112915 AF300746 ...... Leotiomycetes? ...... T Seq. Num

260 Research ... Leotiomycetes? Helotiales Mycosphaerellaceae Hypocreales Sordariomycetes Chaetothyriomycetes; Chaetothyriales; Herpotrichiellaceae Lineage Leotiomycetes; Helotiales; Helotiaceae 3 Y112936 ... UAMH 10102 UAMH 10330 A UAMH 10331 UAMH 10105 Accession numbers UAMH 10329 2 Ectomycorrhiza Source 1 DNA Region ITS2 GenBank AJ300332 Dothideomycetes incertae sedis UBCtra 1205C ITS2 GenBank AF300749 Sample (isolate, clone or species) Berkeley & Curtis S.J. Hughes Gams & Jansson UAMH 10103 H. ericae continued able 1 Seq. Num 353636.236.4 UBCtra 27437 UBCtra 1340C UBCtra1436C38 UBCtra1439C394041 UBC M542 UBCtra 32343 UBCtra 1121C44 UBCtra 26445 UBCtra 15346 UBCtra 1254C & Wilcox47 Phialophora ﬁnlandia Wang 48 Phialocephala dimorphospora Kendrick48.2 UBCtra 1314C UBCtra 1253C49 UBCtra 152 ITS2 ITS2 ITS2 & LSU50 UBCtra 180 UBC7-23–5 ITS2 & LSU ITS2 ITS251 Culture52 UBCtra 1016C Culture Cladosporium oxysporium Culture Culture53 ITS2 ITS2 ITS2 UBCtra Seq62 GenBank54 Monodictys castaneae (Wallr.) ITS2 GenBank54.2 ITS2 ITS2 AY219881 coniospora (Drechsler) Drechmeria AY219879 55 AF149069 AF300752 Culture Culture GenBank56 UBCtra 1018C ITS2 UBCtra 1453C AF081441 ITS2 Culture AF08143457 Culture Culture Leotiomycetes? Helotiales 58 UBCtra 1022C ITS259 UBCtra 300 ITS2 ITS2 & LSU ITS2 ... Leotiomycetes? Helotiales? AF081440 ITS2 AF149083 AF300747 Culture60 sp. Microdochium ITS2 Leotiomycetes? Helotiales? Culture61 UBCtra 1170C ... AF149070 Hemlock 61.2 1181C AF149078 AF300745 Culture62 ITS2 Culture62.2 UBCtra Seq66 GenBank ...... Leotiomycetes? GenBank UBCtra Seq46 UBCtra Seq54 AF300754 Culture AF300753 Leotiomycetes? Helotiales? UBCtra Seq53 UBCtra Seq50 Leotiomycetes? ... ITS2 ITS2 & LSU AF149072 Clone AF149071 AJ238678 AF106018 ... ITS2 AF300740 Culture ... ITS2 ITS2 Culture ... Leotiomycetes; Helotiales Dothideomycetes ITS2 AF300730 Hypocreales Clavicipitaceae Sordariomycetes Culture Leotiomycetes? AY219880 ITS2 GenBank Culture ITS2 & LSU ITS2 AF300727 ITS2 ... Culture ITS2 ITS2 Culture AF300728 AJ279481 Clone AF149079 Clone Sordariomycetes? Clone AF300729 Clone Clone AF300735 ... Xylariales Sordariomycetes ... AF300732 AF300731 AY112917 ... AF300733 AY112918 ...... 34 33 UBCtra 1317C ITS2 Culture AF300748 T

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Research 261 Herpotrichiellaceae Herpotrichiellaceae .. Herpotrichiellaceae Herpotrichiellaceae Saccharomycetaceae Lineage 3 UAMH 10104 Accession numbers 2 Source 1 DNA Region ITS2 & LSU GenBank AF050256 ... . et al IMI 182065 ITS2 & LSU GenBank AF284122 Leotiomycetes; Helotiales; Helotiaceae eloschistaceae Sample (isolate, clone or species) T H. ericae continued able 1 Seq. Num 75.276 UBCtra Seq10777879 UBCtra 1046C8081 UBCtra 1041.482 UBCtra 1322.1183 UBCtra 1522.684 UBCtra 1086.1184.2 UBCtra 1522.1285 UBCtra 1.1486 UBCtra 1322.287 UBCtra 1086.12 UBCtra Seq18888.2 UBCtra 1041.9 ITS289 epimyces M. E. Barr Capronia 90 UBCtra 1.0791 sp. Cordyceps ITS2 UBCtra Seq4192 ITS2 & LSU Leotia viscosa Fr. ITS2 & LSU Davidson93 veratri Cash & R.W. Sclerotinia Clone94 ITS2 & LSU Fulgensia schistidii (Anzi) Poelt ITS2 & LSU Clone LSU Culture Clone95 Geosmithia emersonii (Stolk) Pitt96 Pidoplitchkoviella terricola Kiril. Suh & Blackwell ITS2 & LSU Clone Clone LSU97 LSU Ramichloridium anceps (Sacc. & Ellis) de Hoog LSU LSU98 not submitted LSU99 dactylotricha Untereiner Capronia ITS2 GenBank pulcherrima (Munk) E. Müller LSU Capronia Clone ITS2 & LSU AF300734 AF284129100 esculenta (L) Pers. Morchella AF284128 ITS2 & LSU Erysiphe heraclei (DC.) St.-Amans. Clone AF284126 Clone Chaetothyriomycetes; Chaetothyriales; LSU Samuels GenBank villosa G.F. Capronia ITS2 & LSU AF300725 Clone ITS2 & LSU GenBank ITS2 LSU AF050245 Meyen ex Hansen cerevisiae Saccharomyces GenBank Clone GenBank Clone ... GenBank ... GenBank AF300722 ITS2 & LSU ... AF113739 ... Clone ITS2 & LSU AF050284 AF300720 AF300719 Clone LSU AF300718 GenBank ... AF096197 GenBank AF279881 LSU AY112919 AF033387 GenBank AB027378 ... AF300721 ITS2 & LSU Leotiomycetes; Helotiales Sclerotiniaceae Chaetothyriomycetes; Chaetothyriales; ...... AF300723 ... AF050243 GenBank Microascales; Microascaceae Sordariomycetes; GenBank AF113737 Lecanorales; Lecanoromycetes; AY112920 GenBank ... Eurotiales; Trichocomaceae Eurotiomycetes; Hypocreales; Clavicipitaceae Sordariomycetes; Z73326 ...... AB022391 AF050261 Leotiomycetes; Helotiales Unknown U42669 Saccharomycetales; Saccharomycetes; Leotiomycetes; Erysiphales; Erysiphaceae Chaetothyriomycetes; Chaetothyriales; Pezizomycetes; Pezizales; Morchellaceae 64.2656668 UBCtra 1442.26970 UBCtra 1302.1171 UBCtra 1302.5 Gams71.2 W. Chalara microchona 71.5 UBCtra Seq1.1 A72 UBCtra 1011.1373 UBCtra 1522.5 Lojkania enalia75 Cenococcum geophilum Fr. UBCtra 1041.3 UBCtra 1542.5 UBCtra 1.01 LSU ITS2 ITS2 & LSU ITS2 & LSU ITS2 & LSU LSU GenBank Clone ITS2 & LSU Clone Clone ITS2 & LSU AY112935 LSU Clone AF222467 Clone ITS2 & LSU Clone ITS2 & LSU AF284124 Dothideomycetes AY112921 AF284123 ITS2 & LSU Clone Clone Clone AF300726 Leotiomycetes? Clone AF300724 ... AF284133 ...... AY016363 AF284132 ... AF284130 ... Dothideomycetes AF284127 Dothideomycetes Fenestellaceae Hypocreales; Clavicipitaceae Sordariomycetes; Sordariomycetes Chaetothyriomycetes; Chaetothyriales; 6364 Sd2 ITS2 Culture AF269068 ... T

262 Research Lineage ... Hypocreales 3 Accession numbers A202729 2 Source 1 DNA Region LSU GenBank AF291366 ... sensu cup & Talbot r Sample (isolate, clone or species) Wa Oberw. & K. Wells Oberw. K. Wells Saccardo continued able 1 Seq. Num 147148 Neuh. Sebacina epigaea (Berk. & Br.) Tul. Sebacina incrustans (Fr.) LSU LSU GenBank GenBank AF291267 AF291365 ...... 101102104105 globospora DA Reid Tremella 106 Bensingtonia ciliata Ingold107 UBCtra 1086.1108 UBCtra 1086.3109 UBCtra 1011.5110 UBCtra 1542.4113 UBCtra 1011.2114 UBCtra 1522.7115 UBCtra 1522.10116 UBCtra 1482.11 LSU117 UBCtra 1041.6118 UBCtra 1542.2 LSU119 UBCtra 1.17120 UBCtra 1.05121 UBCtra Seq1.1B122 GenBank UBCtra 1542.3123 LSU UBCtra 1.12124 GenBank LSU UBCtra Seq1A125 LSU UBCtra 1482.3126 LSU UBCtra 1011.9127 LSU UBCtra 1322.6 AF189869128 LSU UBCtra 1322.8128.2 LSU Clone Boletus mirabilis Murrill AF189887130 LSU Clone Fr. fragilis (Pers. Fr.) Russula aff. 131 Clone Karst. LSU Pseudohydnum gelatinosum (Fr.) UBCtra Seq48132 Clone ITS2 & LSU Tremellaceae 133 Hymenomycetes; Tremellales; Clone Bref. cerasi (Tul.) Craterocolla Clone LSU Eﬁbulobasidium rolleyi (Olive) K. Wells Clone134 Urediniomycetes ITS2 & LSU LSU Neuh. epigaea (Berk. & Br.) Sebacina aff. AF300779 Clone Clone135 gelatinosa (Murril) LSU Tremelloscypha AF300775 Clone ITS2 & LSU AY112930136 Clone LSU UBCtra Seq1B AF300789 LSU ITS2 & LSU137 LSU Eﬁbulobasidium albescens (Sacc. & Malbr.) AF300782 Clone GenBank LSU LSU AF300794 ITS2 & LSU LSU Hymenomycetes; Auriculariales; Sebacinaceae Clone AF300787 Burt pallidum (Schw.) ITS2 & LSU Tremellodendron ... AF284131 AF300793 Clone Sebacina vermifera Oberw. LSU LSU ... AF300780 LSU ... Clone ITS2 & LSU Clone Clone AF300783 ... Clone AF384861 ... GenBank Clone ITS2 GenBank AF300781 ... GenBank ITS2 & LSU Sordariomycetes AF300777 ... Clone GenBank AF300788 ... GenBank ... AF335443 GenBank AF300778 AF300776 Hymenomycetes; Auriculariales; Hyaloriaceae AF284137 AF291317 AF300792 Clone AF291363 Hymenomycetes; Auriculariales; Sebacinaceae ITS2 & LSU AF335451 AF384860 AF300784 ... ITS2 & LSU AF291308 ... AF300785 AF291376 Hymenomycetes; Russulales; Russulaceae AF384862 ... Clone ...... Hymenomycetes; Boletales; Boletaceae ...... AY112931 Hymenomycetes; Auriculariales; Sebacinaceae ...... AF300774 AF 02728 and Unknown ... 140141142143 Sebacina sp. RoKi 179144 Sebacina dimitica Oberw. subsphaerospora (Litsch.) Liberta Trechispora 145.2 (Murrill) Liberta regularis Trechispora 146 cinctum (Corda) Myrothecium UBCtra 1522.2 B ITS2 & LSU Sebacina vermifera Oberw. GenBank ITS2 & LSU LSU LSU GenBank ITS2 & LSU AF347080 GenBank GenBank GenBank AF347087 LSU Hymenomycetes; Aphyllophorales AJ301997 AF291364 AF291367 ... Clone Sordariomycetes; ...... AF300790 Hymenomycetes; Auriculariales; Sebacinaceae T

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Research 263 ll others are GenBank Lineage ‘Clone’ indicates a sequence from fungal PCR product directly ‘Clone’ indicates a sequence from fungal PCR product directly 3 2 ing isolation from ericoid mycorrhizal roots. Sequences taken Accession numbers 2 Source 1 DNA Region end of the large subunit ribosomal RNA gene was available for analysis. end of the large ′ Accession numbers beginning with ‘UAMH’ designate cultures in the University of Alberta Microfungus Collection and Herbarium. A 3 Sample (isolate, clone or species) (Sebacinaceae) continued able 1 ‘LSU’ indicates that approximately 500 bp of the 5 Seq. Num accession numbers. ampliﬁed from ericoid mycorrhizal roots of salal. ‘Culture’ indicates a sequence from a fungus that grew in pure culture follow culture indicates a sequence from fungus that grew in pure ericoid mycorrhizal roots of salal. ‘Culture’ ampliﬁed from a database are designated ‘GenBank’. T 150150.2151153 UBCtra1542.10 UBCtra 1011.6155156 UBCtra1041.2157 UBCtra 1.04158 UBCtra Seq61159 UBCtra 1542.6160 UBCtra 1522.15161 Schroeter Clavulina cinerea (Fr.) 162 UBCtra 1522.1 B163 UBCtra 1255C164 UBCtra Seq7165 UBCtra 14421166 UBCtra Seq70167 UBCtra Seq4169 ITS2 & LSU UBCtra Seq69 ITS2 & LSU170 UBCtra Seq2 ITS2 & LSU171 UBCtra 14423 ITS2 & LSU172 Clone UBCtra 15029173 Clone ITS2 & LSU UBCtra 14426 ITS2173.2 GenBank LSU UBCtra Seq51 Clone174 ITS2 & LSU UBCtra Seq52175 UBCtra1092C Clone LSU UBCtra Seq55176177 Clone UBCtra 1456C ITS2 AF284136179 AF335456 Clone AY112932 UBCtra Seq60180 Clone ITS2 UBCtra Seq64 ITS2181 AF284135 UBCtra Seq65 ITS2182 Clone UBCtra P1322.1B AF284134183 ITS2 UBCtra 1462.6 ITS2184 Clone ... UBCtra 1502.1 Hymenomycetes; Cantharellales; Clavulinaceae AY112929 Unknown 185 ITS2 UBCtra 15222 C ITS2 AF300759 Clone186 Clone UBCtra Seq49 AF300791 Hymenomycetes; Auriculariales; Sebacinaceae ITS2187 Clone UBCtra Seq56 ITS2 ... UBCtra Seq9 Clone ITS2 AF300786 Clone UBCtra 1522.1 A1 ITS2 Unknown Mycorrhiza of Neottia nidus-avis AY112933 Clone ITS2 ... ITS2 Clone ... AF300764 Clone ITS2 AF300771 Clone AF300766 ITS2 Hymenomycetes; Auriculariales; Sebacinaceae Clone ITS2 AF300765 Clone ITS2 AF300761 Hymenomycetes? ITS2 Clone Clone AF300768 ITS2 Hymenomycetes; Auriculariales; Sebacinaceae AF300770 ... Clone ITS2 ITS2 AF300769 ITS2 ... Clone AF300772 Clone ... AF300756 ITS2 ... Clone Clone AF300757 ITS2 ... ITS2 AF300758 ... Clone AY112934 ITS2 ... Clone GenBank Clone AF300773 ... AF300762 Hymenomycetes; Aphyllophorales Clone AF300760 ... Clone AY112923 AF300763 ... Clone ... Clone AY052374 AY114156 Hymenomycetes AY112922 AY112924 Hymenomycetes; Auriculariales; Sebacinaceae ... AY112925 ...... AY112926 ... Unknown AY112928 AY112927 Hymenomycetes; Auriculariales; Sebacinaceae ......

showed no obvious sequence similarity to any of the other none of the over 300 previously determined sequences from sequences and were excluded from all analyses. The complete ericoid mycorrhizal fungi matched the Sebacina-like clones. alignment is available from TreeBASE (accession # S905). The Sebacina-like PCR products were produced from 11 The complete alignment, assembled to help cluster similar of 15 mycorrhizal roots (Fig. 1). From the PCR product of sequences, included ITS2 data from most taxa, and about 10 of the 15 roots, most of the DNAs detected were of the 500 bp of sequence the 5′ end of the LSU rDNA for repre- Sebacina type (Fig. 1). The Sebacina-like DNAs prevailed among sentative sequences of most phylogenetic groups (Table 1, mycorrhizal roots collected in October and in January. Of Fig. 2). The alignment included diverse sequences from fungi the 90 sequenced fungal clones, 46 clustered phylogenetically in the Ascomycota and Basidiomycota, as well as sets of with the ITS2 and LSU sequences of Sebacina vermifera numerous, almost identical sequences.  4.0b10 (PPC) (Figs 1, 2a and 3). Of the 66 clones characterized by RFLPs, (Swofford, 1999) was used for all analyses. To show similarity 46 were also of the Sebacina type. among the 191 sequences (ITS2 and/or LSU) in our alignment, The second most common clones from mycorrhizal root we used parsimony bootstrapping without branch swapping DNA clustered in the Ascomycota genus Capronia (Fig. 2b). and showed the resulting trees as phylograms, so that the Eight of the 15 roots, including roots from both collection similarity of isolates could be inferred from horizontal branch dates and from three samples, contained Capronia-like DNA lengths. The complete 191 sequence data set provided little (Fig. 1). Eighteen of the sequenced clones, and nine of the bootstrap support even to clusters of nearly identical clones characterized by RFLP were of the Capronia type. The sequences. Other kinds of analyses were not feasible for the third most common clone matched H. ericae, and two roots complete data set because the sequences were numerous and yielded 14 clones (four sequenced and 10 analyzed by RFLP) diverse, because LSU data were not obtained for most of the of this fungus (Fig. 1). Other fungal clone types were less isolates, and because the highly variable ITS2 regions lacked frequent. One root (1101) gave three clones with sequences sufficient phylogenetic signal to resolve relationships. similar to those of species of wood decay basidiomycetes in the For more critical phylogenetic analysis, sequences with genus Trechispora (Fig. 2a). A clone sequence (UBCtra1302.14) only ITS2 data were excluded from the analysis, and the that appeared to be a chimera of a H. ericae ITS region and a remaining sequences with LSU or with both LSU and ITS2 LSU sequence from the Hypocreales was not included in data were analyzed. We conducted a series of analyses, variously analyses. Clone sequence #9.2 (UBCtra15222A) matched the including only sequences from ascomycetes or only sequences sequence of known ericoid mycorrhizal fungus Oidiodendron from basidiomycetes, or subsets of the ascomycete or basidio- maius (Fig. 2b). Eight clone sequences (not included in the mycete sequences in which numbers of taxa were reduced by fungal total) were from salal or other plants. The remaining excluding all but one to four representative sequences from clone sequences were widely scattered among the Ascomycota groups of similar sequences. We used 500 replicated parsimony and Basidiomycota. Of these remaining sequences, clones searches, without branch swapping, for one estimate of boot- from the same root usually represented genetically different strap support. When analyzing smaller numbers of sequences, fungi and clustered in different clades of the phylogram we used 20 replicated heuristic parsimony searches with (Figs 1 and 2a,b). random addition of taxa to find the most parsimonious trees, and 500 parsimony heuristic searches, with tree-bisection Cultures reconnection branch swapping, for bootstrap analysis. For a neighbor-joining bootstrap analysis, we used distance matri- Even though initial light microscopy indicated that mycorrhizal ces constructed assuming a general-time reversible model of hyphae were abundant in all segments, fungi grew from only sequence evolution, with the values for a proportion of invari- 39 of the 150 segments from the 15 roots (Fig. 1). Only 15 of able sites and for the gamma shape parameter estimated using the 39 cultured fungi entered salal cortical cells to form likelihood and the parsimony bootstrap tree. mycorrhizal coils in resynthesis experiments (Fig. 1). The Sebacina-like fungi that predominated among the directly amplified mycorrhizal DNAs were completely absent among Results the cultured fungi. Among the cultures, the most frequently The 15 mycorrhizal hair roots of salal chosen for analysis were isolated fungi had Capronia-like DNA sequences that matched heavily colonized by fungal mycelia. All the segments chosen the second most common sequence type from the DNAs for analysis contained typical ericoid mycorrhizal hyphal coils. directly amplified from mycorrhizal roots (Figs 1 and 2b). No An average of 3.8 genetically different fungi were detected per sporulation was observed among these Capronia-like isolates. root (Fig. 1). Roots from the three different soil samples A total of eight of these Capronia-like cultures grew from showed similar patterns of diversity. From the DNA extracts segments of three different roots (while direct PCR amplification of mycorrhizal salal roots, clones from the genus Sebacina detected Capronia-like sequences in eight roots). Both direct predominated (Figs 1, 2 and 3). With BLAST searches as PCR amplification and culturing detected the same Capronia- well as phylogenetic analysis (explained below), we found that like fungi from only two roots. In resynthesis experiments,

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Fig. 2 Phylogram comparing clone sequences detected from DNA extracts from salal mycorrhizae with sequences from cultures from salal mycorrhizae, with the Basidiomycota in 2a and Ascomycota in 2b. All sequences with ‘UBC’ are from salal mycorrhizae and the clone sequences are in gray boxes. Arrows indicate cultures that formed endomycorrhizae with salal in in vitro tests. Each sequence begins with an identiﬁcation number. The last four digits in the names of UBC sequences from this study designate the 4 cm salal root segment that was the source of the sequence. Other reference sequences from GenBank are also included in this analysis. The phylogram divides the fungal sequences between the Basidiomycota (2a) and the Ascomycota (2b). The clone sequences are concentrated in Sebacina (Basidiomycota) and in Capronia (Ascomycota) while the sequences from cultures are predominantly in clades in the Ascomycota. This phylogram is the product of 500 parsimony bootstrap replicates without branch swapping. Bootstrap numbers are given when > 50%. Horizontal branch lengths are proportional to number of substitutions.

Fig. 2 continued.

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Fig. 3 Phylogram showing the relationships among the Basidiomycota from salal mycorrhizae. The clone sequences from DNA directly extracted from mycorrhizal salal roots all include ‘UBCtra’ and the last four digits in their names designate the particular salal root that yielded the clone. Most of the sequences from the mycorrhizae formed a monophyletic group united at Node A with 87% bootstrap support. These sequences formed the sister group to Sebacina vermifera and were nested in the Sebacinaceae. The phylogram is based on sequences of approx. 500 bp of the 5′ end of the ribosomal LSU, and on the ITS2 regions where available. The phylogram is the product 500 parsimony bootstrap replicates without branch swapping, and ascomycetes Morchella esculenta and Saccharomyces cervisiae served as the outgroups. Horizontal branch lengths are proportional to number of substitutions.

five of the Capronia-like isolates formed hyphal coils inside However, mycorrhizal isolates close to ‘Unknown 1’ (#2, salal root cells. When the Capronia-like isolates failed to UBCtra1256C) and ‘Unknown 2’ (#24, UBCtra1128C) were colonize salal root cells, it was usually because the fungus did isolated from roots that were not used for cloning, although not grow well enough to contact the plant. Four isolates they were from the same soil samples as the 15 roots used for from three roots were identified as H. ericae (Fig. 1). Both detailed analysis. direct PCR amplification and culturing detected H. ericae in one of the roots. All isolates of H. ericae formed hyphal coils Phylogenetic analysis in salal root cells in in vitro synthesis experiments. Only two of the 39 cultures from the 15 root segments were from the The simple clustering of sequences based on fast parsimony Basidiomycota and ITS2 sequence of one of these (#173, bootstrapping divided 191 sequences in our alignment between UBCtra1092C) matched clone sequences from the same root the Ascomycota and Basidiomycota (Fig. 2a,b). The alignment (root 1101) and clustered with Trechispora (Fig. 2a). included sequences from our cultures and clones of ericoid Some mycorrhizal fungi that were frequently detected in mycorrhizae, along with other sequences from GenBank. previous studies from the same Vancouver Island SCHIRP While only two of the sequences from fungi cultured from the site were absent or infrequent on the 15 roots emphasized in 15 ericoid mycorrhizal roots were from the Basidiomycota, this study. Although we came across the common mycorrhizal most of the clones of PCR amplified DNAs were from the species Oidiodendron maius once among the cloned DNAs, Basidiomycota (Fig. 2a,b). Most of the basidiomycete clone we did not find the species among any of the fungal cultures sequences were similar to one another and to sequences from the 15 roots. We did not detect the sterile and uniden- from the orchid mycorrhizal fungus Sebacina vermifera tified taxa ‘Unknown 1’ or ‘Unknown 2’ among our 15 roots. (Fig. 2a). The nodes clustering the Sebacina-like fungi in the

Basidiomycota received no bootstrap support from the data analysis, we excluded the basidiomycetes and all sequences set of all 191 sequences. To further investigate the relationship lacking LSU data. With the smaller number of sequences of our isolates to Sebacina and to other fungi in the and 500 fast parsimony bootstrap replicates, node E, uniting Sebacinaceae, we excluded 20 Sebacina-like sequences for the H. ericae isolates and clones, received 86% bootstrap which we had only ITS2 data. We re-analyzed the remaining support (Fig. 4). The monophyly of the Capronia-like sam- 26 Sebacina-like sequences along with two ascomycete ples from salal (node H) received 98% support and the node outgroups, and all other basidiomycete LSU sequences from uniting Capronia villosa with the clones and isolate from salal our alignment. Our salal isolates clustered together (united at (Node G) received 93% support (Fig. 4). Neighbor-joining Node A, Fig. 3) with 87% fast parsimony bootstrap support. bootstrapping, with a GTR model of substitution, and an Node B united salal clones with Sebacina vermifera sensu estimated 35% of sites invariable with a gamma shape parameter Warcup & Talbot, with 86% bootstrap support, and Node C of 0.81 provided 100% support to the H. ericae node E and united all the above isolates with Sebacina vermifera Oberw. 96% support to Capronia Node G. When the LSU fragments (Fig. 3). All sequenced members of the Sebacinaceae are were analyzed alone (excluding the ITS2 region), bootstrap united at Node D, with 59% bootstrap support (Fig. 3). Only support for nodes E-H ranged between 53% and 100%, nodes C and D appeared in neighbor-joining bootstrap trees, depending on the node and the kind of analysis (not illustrated). and the reason seemed to be that some of the Sebacina-like sequences were short fragments with data missing from the Variation among Sebacina-like and Capronia-like 5′ end of the LSU gene. These short sequences tended to clones cluster at the base of Node C, rather than with the other salal Sebacina-like salal sequences. As a further experiment, four of Several of the Sebacina sequences differed from one another the Sebacina-like sequences having full length ITS2 regions (Figs 2a and 3). To minimize sequencing errors, each variable and 550 bp LSU fragments (#124 UBCtra1322.6; #150 position in the DNA sequences was double-checked by com- UBCtra1011.6; #151 UBCtra1041.2; #153 UBCtra1.04) were paring the electropherograms to the alignment. For all of these included in an analysis along with the other basidiomycetes sequences, the quality of the electropherograms was good and from the analysis for Fig. 3. The ascomycete outgroups and all sequencing ambiguities were not the source of the variation. other Sebacina-like isolates were excluded. These Sebacina- For sequences #124, 150, 151 and 153, most of variable sites like sequences that remained in the analysis represented the were confirmed from sequence in both directions. range of different sequence types within the Sebacina-like If the variation was caused by enzymatic nucleotide misin- cluster (Fig. 3). We analyzed the reduced number of taxa by corporation during the PCR amplification process, the errors parsimony heuristic searches with tree-bisection-reconnection should be randomly distributed and not repeated in different (TBR) branch swapping, and 20 replicated searches with PCR reactions. Sequences from different roots originated random addition of taxa and found two most parsimonious from different PCR reactions and so PCR error would not be trees with a length of 1611. Nodes A, B, C and D were present expected to lead to shared variation in different roots. To find in both, most parsimonious trees. We used 500 replicated out whether substitutions were shared among roots, we began parsimony bootstrap replicates, with both ITS and LSU data with the 25 Sebacina-like ITS2 sequences. By eliminating and with TBR branch swapping, and found the following identical sequences, we reduced the data set to 15 unique support for nodes: A, 95%; B 93%; C 70%; and D 91% (not sequences and found all of the 53 possible, equally parsimo- illustrated). Excluding the ITS data, support for nodes was: nious trees, also of length 119, using a branch and bound A 91%; B 84%; C 76% and D 51%. With neighbor-joining search. We then used the ‘describe trees’ option in  to bootstrap analysis of the ITS and LSU data, assuming a GTR map all nucleotide changes to the branches where they most substitution model, an estimate that 20% of sites were parsimoniously would have occurred, for one of the 53 trees. invariable, and a gamma shape parameter estimate of .577, Some nucleotide substitutions were shared across roots. For support for nodes was: A 86%; B 95%; C 84% and D 74%. example, fungal sequences #150, 169 and 181, from roots Continuing with neighbor joining, excluding the ITS data, 1502 and 1542, shared 10 nucleotide substitution characters 22% of sites were estimated to be invariable, the gamma shape that distinguished them from sequences of the other roots. parameter was .504, and support for the four nodes of interest Sequences from roots 1011, 1116, 1056 and 1041, and was: A 36%; B 57%; C 63% and D 44% (not illustrated). sequence #179 from root 1322 shared at least three characters Among the ascomycetes, the fast parsimony bootstrap that distinguished them from other sequences, including a including all 191 sequences revealed a cluster of sequences second sequence, #124, from root 1322. similar to H. ericae, united by node E (Fig. 2). A second group Sequences from a root were usually more similar to other of sequences appeared to be nested in the genus Capronia sequences from the same root than would be expected if (Nodes F, G, and H in Fig. 2). Again, none of these nodes sequence variation had been randomly distributed within and received any bootstrap support when the entire range of among roots. The most parsimonious trees, constrained so sequences was included in the analysis. For a more rigorous that sequences from each root formed a monophyletic group,

Fig. 4 Phylogram showing the relationships among the Ascomycota from salal mycorrhizae. The sequence names from ericoid mycorrhizal salal roots all include ‘UBCtra.’ Gray shading indicates clone sequences. The last four digits in sequence names designate the particular salal root that yielded the clone or culture. The phylogram is based on sequences of approx. 500 bp of the 5′ end of the ribosomal LSU, and on the ITS2 regions where available. The phylogram is the product 500 parsimony bootstrap replicates without branch swapping. Horizontal branch lengths are proportional to number of substitutions. Morchella esculenta was chosen as the outgroup. required 121 substitutions, only two more than the most par- random trees. Instead, many of the random trees (55 of 1000) simonious trees. The most parsimonious, unconstrained trees, were the same length as the most parsimonious, 13-step trees. and the most parsimonious, constrained trees, were all con- siderably shorter than the lengths of 100 000 random trees Discussion that varied in length from 153 to 226 steps. Unlike the Sebacina-like sequences, the 18 Capronia-like Sebacina-like sequences dominated among the directly sequences, including 11 sequences with ITS2 regions, were ampliﬁed fungi almost identical to one another (Figs 2b and 4). The most divergent sequence, #79 from root 1522, differed from the Finding that Sebacina-like DNA was present in 11 of the 15 others at four positions in the ITS2 region. Explaining all the mycorrhizal roots and that 92 out of the 156 cloned DNAs substitutions among the 18 sequences required 13 steps in were of the Sebacina type suggests that Sebacina spp. regularly parsimony analysis. Unlike the substitutions among the associate with salal roots at our Vancouver Island SCHIRP Sebacina-like isolates, substitutions among the Capronia-like research site. If Sebacina spp. produced the most abundant sequences did not show any obvious phylogenetic structure. fungal DNA in the 15 roots, they were probably also present If the substitutions had a phylogenetic structure, then the most in at least some of the 150 segments of the same roots that parsimonious trees would be much shorter than almost all were used for culturing. However, no Sebacina-type fungus

has ever been detected in a fungal culture from these or any conditions have already been optimized for recovery of the other ericoid root segments, including the hundreds of other mycorrhizal fungi, the rates of recovery of fungi in pure culture ericoid mycorrhizal fungal cultures from the SCHIRP site were still 10 times the 1% recovery rate for bacteria. If the (Xiao & Berch, 1996; Monreal et al., 1999; Berch et al., 2002). frequency of fungal individuals were proportional to the Most of the ascomycetous mycorrhizal fungi reported from proportion of clones, then about half of the mycorrhizal root the SCHIRP research site have also been reported from other segments may have contained Sebacina species and our inabil- continents (Berch et al., 2002). Even if the Sebacina-like fungal ity to culture these Sebacina species could have accounted for involvement in ericoid mycorrhizae is similarly geographically a substantial fraction of the missing fungal cultures. widespread, it may be systematically overlooked in surveys of root fungi that detect fungi only when they grow in pure Capronia species as possible mycorrhizal symbionts culture under standard conditions. Other species in the genus Sebacina are probably mycor- In both direct PCR amplification and isolation in culture, rhizal and the genus is geographically widespread (Warcup, Capronia-like fungi were common. Although Capronia-like 1988; Glen et al., 2002; Selosse et al., 2002a; Selosse et al., fungi are not usually considered to be mycorrhizal, Bergero 2002b; Urban et al., 2003). Lacking a culture of the Sebacina et al. (2000) repeatedly isolated a sterile fungus, ‘Sd2’ in Italy, species, we cannot test our fungi for the ability to form mycor- from Erica arborea L. (Ericaceae) and from Quercus ilex L. (an rhizae. Most species of Sebacina do not grow readily on arti- oak) that falls phylogenetically among the Capronia isolates ficial media (R. J. Bandoni, pers. comm.). However, our clone and Ramichloridium anceps, a related asexual state (Fig. 2b). sequences cluster phylogenetically with > 87% bootstrap sup- Isolate ‘Sd2’ formed typical ericoid mycorrhizae in resynthesis port with Sebacina vermifera, one of the few Sebacina species experiments. In resynthesis experiments, our Capronia-like that has been cultured and shown to be mycorrhizal with or- isolate clearly colonize the cortical cells of salal roots but whether chids and deciduous trees (Warcup, 1988). The LSU and ITS the species are truly mycorrhizal requires physiological testing sequences of at least two other Sebacina-like fungi were to determine whether the salal benefits from the fungal detected as common ectomycorrhizal associates of eucalyptus colonization. (Glen et al., 2002). Selosse et al. (2002a) showed using DNA Suggesting that the viable part of each fungal individual sequence data and electron microscopic evidence that a Sebacina was small (at least after washing and sterilization), Capronia sp. species appeared to be a mycorrhizal partner of an achloro- never grew from adjacent root segments (Fig. 1). Capronia phyllose orchid, Neottia nidus-avis L. Rich. and of deciduous sp. DNA in a root did not reliably predict that a Capronia trees in France. Similarly, Urban et al. used DNA sequence culture would grow from a neighboring segment of the same data and electron microscopy to identify Sebacina spp. as ecto- root. Instead, Capronia sp. cultures grew from only two of the mycorrhizal partners of deciduous trees from Austria. eight roots that contained Capronia DNAs. Similarly, finding As with the Sebacina-like species from mycorrhizae from a Capronia sp. in a culture did not necessarily indicate that the eucalyptus (Glen et al., 2002) and from achlorphyllous orchids genus could be detected in DNA extracts from a mycorrhizal (Selosse et al., 2002b), DNAs of Sebacina-like fungi from salal root. Capronia sp. cultures were isolated from both the left show patterns of variation consistent with underlying natural and right flanks of root 1056 and yet Capronia sp. was not variation. The differences in sequences from fungi from a sin- detected in the DNA extracts from middle segment of the gle root could be due to heterogeneity in the sequences of the same root (Fig. 1). ribosomal repeats of a single nucleus; to allelic differences in Little is known about the natural habitat of the vegetative dikaryotic nuclei of a hypha, or to the presence of different mycelial stage of Capronia species. Capronia species attract the genets or different species in different individual mycelia. most attention when causing opportunistic infections of Enzymatic misincorporations of nucleotides during PCR humans, but these infections are uncommon and they are amplification were not likely to have caused the substitutions probably an evolutionary dead end for the fungus. Capronia shared among independently amplified fungal DNAs from species’ sexual fruiting bodies are usually found on decorticated different roots. or well-rotted wood and they may be hyperparasites on other As in studies of bacterial cell viability in cultures, lack of fungi (Untereiner & Malloch, 1999). Long-term accumula- fungal growth was the most frequent outcome of attempts tion of coarse woody debris and logging on our Vancouver to grow cultures from salal mycorrhizae. Of our 150 ericoid Island research site had left abundant cedar and hemlock mycorrhizal root segments, 74% gave no slow-growing fungal wood, and perhaps the Capronia isolates were relatively com- cultures. This is consistent with results from Xiao and Berch mon because the salal roots were growing in and around a (1996), who obtained cultures of ericoid mycorrhizal fungi high concentration of well-rotted wood. Untereiner & Malloch from 16% of the 1120 salal mycorrhizal root tips they exam- (1999) found that Capronia species generally had no ability ined, even though 90% of the host roots’ cortical cells were to use cellulose or starch and suggested that some may be colonized. Possibly because the original population of fungal mycoparasites. Our isolates penetrated the roots cells of salal cells in a root segment was high, or possibly because cultural suggesting that they have cellulases.

the hundreds of fungi from salal roots indicates a systematic Evidence for the presence of mixed ascomycetes and detection bias. We plan to explore alternative culturing basidiomycetes in ericoid mycorrhizae techniques to recover Sebacina sp. like isolates in culture and Most ericoid mycorrhizal root contained mixtures of DNA to subject them to mycorrhizal testing. types. Some of the fungi that contributed to the DNAs, the Trechispora spp. for example, were probably saprobic. Some of Acknowledgements the fungi may have been endorhizal, able to penetrate living root cells, without conferring the physiological benefit to the We thank Dr Keith Egger for assistance with phylogenetic salal that would make the fungi truly mycorrhizal. However, analysis, Dr Sara Landvik for critical advice on manuscript because of the way that ericoid mycorrhizae develop, some and Dr Lee Taylor for access to unpublished sequences of roots may have had mixed mycorrhizal fungi. In ericoid Sebacina vermifera. Sea Ra Lim contributed to the sequencing. mycorrhizae, a hypha usually penetrates an individual cell and Lynn Sigler (University of Alberta Microfungus Collection then forms a hyphal coil within one cell. The hypha within and Herbarium) verified the identity of H. ericae cultures the cell does not usually grow into adjacent cells (Cairney & deposited in their collection. Funding for this research was Ashford, 2002; Perotto et al., 2002). As a result, unlike most provided by Forest Renewal B.C and Forestry Innovation ectomycorrhizae where one fungal species predominates in Investment BC grants for the Salal Cedar Hemlock Integrated one root tip, adjacent cells in an ericoid root can be mycorrhizal Research Program and by a grant to M. Berbee from the with different fungal species (Pearson & Read, 1973; Canadian National Science and Engineering Research Council. Gianinazzi-Pearson et al., 1995; Nafar, 1998; Monreal et al., 1999; Perotto et al., 2002). Finding mixed ascomycete and basidiomycete DNAs asso- References ciated with salal roots is consistent with ultrastructural studies Allen WK, Allaway WG, Cox GC, Valder PG. 1989. Ultrastructure of of mycorrhizal roots from other plants in the Ericaceae. Asco- mycorrhizas of Dracophyllum secundum R. Br. Ericales: Epacridaceae). mycetes can be recognized by their simple septal pores that are Australian Journal of Plant Physiology 16: 147–153. often associated with spherical, electron opaque, Woronin Berch SM, Allen TR, Berbee ML. 2002. Molecular detection, community bodies. In the Sebacinaceae, as in other ‘jelly fungi’ in the structure and phylogeny of ericoid mycorrhizal fungi. Plant and Soil 244: 55–66. Auriculariales, septal walls have a central pore, surrounded by Bergero R, Perotto S, Girlanda M, Vidano G, Luppi AM. 2000. Ericoid a torus-like swelling. 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