Microbial Ecology

Soil Fungal Communities Underneath Willow Canopies on a Primary Successional Glacier Forefront: rDNA Sequence Results Can Be Affected by Primer Selection and Chimeric Data Ari Jumpponen

Division of Biology, Kansas State University, 125 Ackert Hall, Manhattan, KS 66506, USA

Received: 7 January 2004 / Accepted: 9 March 2004 / Online publication: 3 November 2006

Abstract amplification and a smaller number of PCR cycles are necessary precautions to allow collection of reliable Soil fungal communities underneath willow canopies environmental sequence data. that had established on the forefront of a receding glacier were analyzed by cloning the polymerase chain reaction (PCR)-amplified partial small subunit (18S) of the ribosomal (rRNA) genes. Congruence between two sets Introduction of -specific primers targeting the same gene region was analyzed by comparisons of inferred neighbor-joining Fungi perform important ecosystem functions by partic- topologies. The importance of chimeric sequences was ipating in the decomposition of dead tissues as well as evaluated by Chimera Check (Ribosomal Database Proj- plant uptake of water and nutrients [6, 34]. Assessment ect) and by data reanalyses after omission of potentially of fungal community composition is difficult because of unreliable and ephemeral production of identifiable chimeric regions at the 50- and 30-ends of the cloned amplicons. Diverse communities of fungi representing macroscopic fruiting bodies [11, 27, 35]. Many fungi Ascomycota, , Chytridiomycota, and Zygo- also produce microscopic, sexual or asexual fruiting mycota were detected. Ectomycorrhizal fungi comprised a structures or fruit below ground escaping detection in major component in the early plant communities in assessments relying exclusively on the collection of primary successional ecosystems, as both primer sets epigeous fruiting bodies. Pure culture techniques allow frequently detected basidiomycetes (Russulaceae and fungal community assays of soil and tissue samples in the Thelephoraceae) forming mycorrhizal symbioses. Various absence of identifiable macroscopic fruiting bodies. ascomycetes (Ophiostomatales, Pezizales, and Sordar- However, similar to bacteria [38], it is likely that large iales) of uncertain function dominated the clone libraries numbers of fungi would be missed in such pure culture amplified from the willow canopy soil with one set of assays (see [31, 41]). To overcome these problems in primers, whereas the clone libraries of the amplicons fungal community analysis, molecular means specifically generated with the second primer set were dominated by targeting fungi in environmental samples have been basidiomycetes. Accordingly, primer bias is an important developed [3, 9, 14, 25, 28, 32, 33, 40]. factor in fungal community analyses using DNA extracted Direct molecular assessment of the fungal commu- from environmental samples. A large proportion (930%) nities allows analyses without relying on whether or not of the cloned sequences were concluded to be chimeric the fungi can be grown in pure culture or produce based on their changing positions in inferred phylogenies fruiting bodies. However, polymerase chain reaction after omission of possibly chimeric data. Many chimeric (PCR) artifacts, such as chimeric sequences resulting sequences were positioned basal to existing classes of from amplification of more than one template, can cause fungi, suggesting that PCR artifacts may cause frequent problems in environmental samples with unknown discovery of new, higher level taxa (order, class) in direct sources of diverse initial template DNA [13, 19, 24, 42, PCR analyses. Longer extension times during the PCR 43]. Various coextracted substances and low concen- trations of the target template in the presence of highly similar competing target and nontarget templates may Correspondence to: Ari Jumpponen; E-mail: [email protected] further influence the fidelity of PCR reactions [42].

DOI: 10.1007/s00248-004-0006-x & Volume 53, 233–246 (2007) & * Springer Science + Business Media, Inc. 2006 233 234

Table 1. BLAST and RDP analyses of the environmental sequences obtained from underneath the willow canopies established on the forefront of a receding glacier Chimera at Environmental clone RDP BLAST match [accession number] (Order) Phylum Similarity Frequency B_Canopy_300_01_08 [AY382401] Yes (G20) Spilocaea oleaginea [AF338393] (Chaethothyriales/Dothidiales) Ascomycota 98 0.60 B_Canopy_300_01_14 [AY382402] Yes (G40) Spilocaea oleaginea [AF338393] (Chaethothyriales/Dothidiales) Ascomycota 96 0.20 B_Canopy_300_01_16 [AY382403] Yes (G80) Hymenoscyphus ericea [AY228753] (Helotiales) Ascomycota 95a 0.10 B_Canopy_300_01_18b [AY382404] Yes (G40) Inocybe geophylla [AF287835] () Basidiomycota 97 0.10 B_Canopy_300_02_04b [AY382405] Yes (G20) Dark septate endophyte DS16b [AF168167] (Unknown) Ascomycota 98 0.22 B_Canopy_300_02_05 [AY382406] Yes (G20) Peziza griseorosea [AF133150] (Pezizales) Ascomycota 99 0.11 B_Canopy_300_02_06 [AY382407] Yes (G20) Peziza griseorosea [AF133150] (Pezizales) Ascomycota 98 0.11 B_Canopy_300_02_10 [AY382408] Yes (G20) Tetracladium marchalianum [AY204613] (Incertae sedis) Ascomycota 99a 0.11 B_Canopy_300_02_12 [AY382419] Yes (G20) Spilocaea oleaginea [AF338393] (Chaethothyriales/Dothidiales) Ascomycota 98 0.33 B_Canopy_300_02_14b [AY382410] Yes (G40) Oidiodendron tenuissimum [AB015787] (Onygenales) Ascomycota 97 0.11 B_Canopy_300_03_06 [AY382411] No Prismatolaimus intermedius [AF036603] (Enoplida; Prismatolaimidae) Contaminant 97 0.08 B_Canopy_300_03_12b [AY382412] Yes (G100) Cladonia sulphurina [AF241544] (Lecanorales) Ascomycota 93 0.15 B_Canopy_300_03_17 [AY382413] Yes (G40) Hypoxylon submonticulosum [AF346544] (Xylariales) Ascomycota 96 0.31 A.

b J B_Canopy_300_03_19 [AY382414] No Neobulgaria premnophila [U45445] (Helotiales) Ascomycota 98 0.46 UMPPONEN B_Canopy_450_01_02 [AY382415] Yes (G80) Pulvinula archeri [U62012] (Pezizales) Ascomycota 94 0.27 B_Canopy_450_01_06 [AY382416] Yes (G40) Hypomyces chrysospermus [AB027339] (Hypocreales) Ascomycota 96 0.20

B_Canopy_450_01_13 [AY382417] Yes (G40) Oidiodendron tenuissimum [AB015787] (Onygenales) Ascomycota 98 0.07 : F B_Canopy_450_01_14 [AY382418] Yes (G40) Oidiodendron tenuissimum [AB015787] (Onygenales) Ascomycota 97 0.33 UNGI B_Canopy_450_01_18 [AY382419] Yes (G40) Oidiodendron tenuissimum [AB015787] (Onygenales) Ascomycota 98 0.13 B_Canopy_450_02_02 [AY382420] Yes (G40) Rhizoctonia solani [D85643] (Ceratobasidiales) Basidiomycota 95 0.06 IN THE B_Canopy_450_02_13 [AY382421] Yes (G40) Hypomyces chrysospermus [AB027339] (Hypocreales) Ascomycota 96 0.94 B_Canopy_450_03_02 [AY382422] Yes (G40) Connersia rilstonii [AF096174] (Eurotiales) Ascomycota 99/99a,c 0.14 W B_Canopy_450_03_05 [AY382423] Yes (G40) Raciborskiomyces longisetosum [AY016351] (Chaetothyriales) Ascomycota 99 0.14 ILLOW B_Canopy_450_03_07 [AY382424] Yes (G40) Herpotrichia juniperi [U42483] (Pleosporales) Ascomycota 97 0.43 C

B_Canopy_450_03_14 [AY382425] No Mycosphaerella mycopappi [U43463] (Chaetothyriales) Ascomycota 98 0.29 ANOPY B_Canopy_750_01_01b [AY382426] Yes (G20) Inocybe geophylla [AF287835] (Cortinariaceae) Basidiomycota 97 0.20 b

B_Canopy_750_01_07 [AY382427] Yes (G40) Peziza griseorosea [AF133150] (Pezizales) Ascomycota 99 0.40 S B_Canopy_750_01_10b [AY382428] Yes (G40) Anamylopsora pulcherrima [AF119501] (Agyriales) Ascomycota 97 0.20 OIL B_Canopy_750_01_15b [AY382429] Yes (G80) Pulvinula archeri [U62012] (Pezizales) Ascomycota 97 0.20 ON B_Canopy_750_02_13 [AY382430] Yes (G40) Hypomyces chrysospermus [M89993] (Hypocreales) Ascomycota 96 0.33 A b c G B_Canopy_750_02_15 [AY382431] Yes (G40) Ophiostoma piliferum [AJ243294] (Ophiostomatales) Ascomycota 97/97 0.44 LACIER B_Canopy_750_02_19b [AY382432] Yes (G40) Hypomyces chrysospermus [M89993] (Hypocreales) Ascomycota 95 0.22 b

B_Canopy_750_03_03 [AY382433] Yes (G80) Sarcinomyces petricola [Y18702] (Chaetothyriales) Ascomycota 95 0.14 F B_Canopy_750_03_04 [AY382434] Yes (G20) Laccaria pumila [AF287838] (Agaricales) Basidiomycota 98 0.29 OREFRONT B_Canopy_750_03_08b [AY382435] Yes (G20) Laccaria pumila [AF287838] (Agaricales) Basidiomycota 96 0.14 B_Canopy_750_03_11 [AY382436] No Peziza griseorosea [AF133150] (Pezizales) Ascomycota 99 0.43 A. b G

B_Canopy_900_01_03 [AY382437] Yes ( 40) Russula compacta [AF026582] (Agaricales) Basidiomycota 96 0.25 J B_Canopy_900_01_16 [AY382438] Yes (G40) Hypomyces chrysospermus [AB027339] (Hypocreales) Ascomycota 97 0.13 UMPPONEN B_Canopy_900_01_19 [AY382439] No Russula compacta [AF026582] (Agaricales) Basidiomycota 98 0.63 B_Canopy_900_02_02 [AY382440] No Oidiodendron tenuissimum [AB015787] (Onygenales) Ascomycota 99 0.09 :

B_Canopy_900_02_04 [AY382441] No Chaetomium elatum [M83257] (Sordariales) Ascomycota 98 0.18 F B_Canopy_900_02_06 [AY382442] Yes (G80) Thelephora sp. [AF026627] (Thelephorales) Basidiomycota 99 0.55 UNGI

B_Canopy_900_02_10 [AY382443] No Dark septate endophyte DS16b [AF168167] (Unknown Ascomycota 98 0.09 IN b B_Canopy_900_02_12 [AY382444] Yes (G80) Pulvinula archeri [U62012] (Pezizales) Ascomycota 97 0.09 THE B_Canopy_900_03_09 [AY382445] Yes (G40) Hypomyces chrysospermus [AB027339] (Hypocreales) Ascomycota 94 0.75 b W

B_Canopy_900_03_11 [AY382446] Yes (G80) Polyporoletus sublividus [AF287840] (Cantharellales) Basidiomycota 94 0.13 ILLOW B_Canopy_900_03_17 [AY382447] Yes (G40) Thelephora sp. [AF026627] (Thelephorales) Basidiomycota 98 0.13

S_Canopy_300_01_01 [AY382448] Yes (G60) Bulgaria inquinans [AJ224362] (Helotiales) Ascomycota 98 0.11 C S_Canopy_300_01_07 [AY382449] No Inocybe geophylla [AF287835] (Agaricales) Basidiomycota 98 0.89 ANOPY S_Canopy_300_02_01b [AY382450] Yes (G100) Bulgaria inquinans [AJ224362] (Helotiales) Ascomycota 95 0.14 S

S_Canopy_300_02_11 [AY382451] Yes (G40) Mortierella chlamydospora [AF157143] (Mucorales) Zygomycota 97 0.29 OIL S_Canopy_300_02_13b [AY382452] Yes (G80) Bulgaria inquinans [AJ224362] (Helotiales) Ascomycota 96 0.14 ON S_Canopy_300_02_14b [AY382453] Yes (G160) Limnoperdon incarnatum [AF426952] (Aphyllophorales) Basidiomycota 94 0.14 b A S_Canopy_300_02_19 [AY382454] Yes (G100) Panellus serotinus [AF026590] (Agaricales) Basidiomycota 94 0.29 G S_Canopy_300_03_04 [AY382455] Yes (G40) Spizellomyces acuminatus [M59759] (Spizellomycetales) Chytridiomycota 97 0.67 LACIER S_Canopy_300_03_18 [AY382456] Yes (G20) Laccaria pumila [AF287838] (Agaricales) Basidiomycota 97 0.33

b F

S_Canopy_450_01_02 [AY382457] Yes (G100) Byssoascus striatosporus [AB015776] (Onygenales) Ascomycota 94 0.25 OREFRONT S_Canopy_450_01_07 [AY382458] Yes (G80) Entoloma strictius [AF287832] (Agaricales) Basidiomycota 93 0.25 S_Canopy_450_01_19 [AY382459] Yes (G60) Bulgaria inquinans [AJ224362] (Helotiales) Ascomycota 98 0.50 S_Canopy_450_02_05 [AY382460] Yes (G60) Ophiostoma stenoceras [M85054] (Ophiostomatales) Ascomycota 95 1.00 S_Canopy_750_01_10 [AY382461] Yes (G20) Thelephora sp. [AF026627] (Thelephorales) Basidiomycota 97/94c 0.40 S_Canopy_750_01_11 [AY382462] Yes (G20) Inocybe geophylla [AF287835] (Agaricales) Basidiomycota 96 0.40 S_Canopy_750_01_18 [AY382463] Yes (G20) Inocybe geophylla [AF287835] (Agaricales) Basidiomycota 98 0.20 S_Canopy_750_02_09 [AY382464] Yes (G40) Sporothrix schenkii [M85053] (Ophiostomatales) Ascomycota 93 0.92 S_Canopy_750_02_17b [AY382465] Yes (G160) Dendrocorticium roseocarneum [AF334910] (Aphyllophorales) Basidiomycota 92 0.08 S_Canopy_750_03_01b [AY382466] Yes (G100) Bulgaria inquinans [AJ224362] (Helotiales) Ascomycota 95 0.14 S_Canopy_750_03_13 [AY382467] Yes (G20) Termitomyces sp. [AB051891] (Agaricales) Basidiomycota 94 0.43 S_Canopy_750_03_15 [AY382468] Yes (G40) Inocybe geophylla [AF287835] (Agaricales) Basidiomycota 97 0.14 S_Canopy_750_03_18b [AY382469] Yes (G40) Laccaria pumila [AF287838] (Agaricales) Basidiomycota 95 0.14 S_Canopy_750_03_19 [AY382470] Yes (G20) Cyathrus striatus [AF026617] (Nidulariales) Basidiomycota 97 0.14 S_Canopy_900_01_06 [AY382471] Yes (G20) Inocybe geophylla [AF287835] (Agaricales) Basidiomycota 98 0.77 S_Canopy_900_01_11 [AY382472] Yes (G40) Russula compacta [U59093] (Agaricales) Basidiomycota 97 0.23 S_Canopy_900_03_02 [AY382473] Yes (G40) Thelephora sp. [AF026627] (Thelephorales) Basidiomycota 97 1.00 Chimera Check scores in parentheses. Frequency refers to the occurrence of a clone in the library obtained from one sample. RDP = Ribosomal Database Project; BLAST = basic local alignment search tool. a Sequence omitted from the neighbor-joining analyses because of a large insert. b Sequence determined chimeric in analyses after omission of data beyond chimera points. c BLAST matches were partial and did not span over the entire cloned sequence. 235 236 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT

Furthermore, primer sets designed to obtain broad collected in August 2001. Samples were stored on ice specificity to a target group (e.g., fungi) may have biases until processed. In the laboratory, roots were handpicked and preferentially amplify one target group but not from soil, and soil was homogenized manually in plastic another [2, 36]. bags. Approximately 0.25 g of soil was transferred to The overall goal of the presented studies was to the extraction buffer, and DNA was extracted using characterize fungal community composition within UltraClean Soil kit (Molecular Biology Laboratories established willow (Salix spp.) canopies on the forefront Inc., Carlsbad, CA) following manufacturer’s protocol. of a receding glacier. The nuclear small subunit (18S) of Extracted DNA was stored frozen (_20-C) until further the ribosomal RNA gene (rDNA) was amplified with two processing. different sets of fungus-specific primers to estimate the influence of primer selection on the observed community PCR Amplification of the Fungal DNA. A partial structure. To evaluate the influence of chimeric ampli- sequence of the 18S of the fungal rDNA was amplified cons on the obtained 18S phylogenies, data sets were with two different primer sets in 50-2L PCR reaction reanalyzed after omission of the chimeric regions mixtures. First, the reaction to collect data set B identified using Chimera Check software of the Ribo- contained final concentrations or absolute amounts of somal Database Project (RDP, version 2.7 [26]). The reagents as follows: 400 nM of each of the forward and results indicate that diverse fungal communities exist reverse primers (nu-SSU-0817-50 and nu-SSU-1536-30 within the willow canopies, that primer selection strongly [3]), 2 2L of the extracted template DNA, 200 2M of influences the observed fungal community structure, and each deoxynucleotide triphosphate, 2.5 mM MgCl2, 1 U that chimeras are a serious concern in direct PCR of Taq DNA polymerase (Promega, Madison, WI), and applications targeting fungi in environmental samples. 5 2L of manufacturer’s PCR buffer. The PCR cycle parameters consisted of an initial denaturation at 94-C for 3 min, then 40 cycles of denaturation at 94-C for Methods 1 min, annealing at 56-C for 1 min and extension at 72-C for 1 min, followed by a final extension step at 72-C Study Site. - 0 00 - 0 00 Lyman Glacier (48 10 52 N, 120 53 87 W) for 10 min. Second, the reaction to collect data set S is located in the Glacier Peak Wilderness Area in the contained final concentrations or absolute amounts of North Cascade Mountains (Washington, USA). The site reagents as follows: 300 nM of each of the forward and has been utilized in several studies on early plant reverse primers (EF4 and EF3 [32]), 2 2L of the extracted community assembly in recently deglaciated substrate template DNA, 200 2M of each deoxynucleotide (e.g., [20, 23]). Similarly, it has been a focus of studies triphosphate, 1.7 mM MgCl2, 2 U of Taq DNA poly- aiming to examine fungal community assembly in such merase (Promega), and 5 2L of manufacturer’s PCR an environment [19, 21, 22]. The elevation of the present buffer. The PCR cycle parameters consisted of an initial glacier terminus is about 1800 m. The deglaciated fore- denaturation at 94-C for 3 min, then 40 cycles of dena- front is approximately 1000 m long over an elevation drop turation at 94-C for 1 min, annealing at 48-C for 1 min of only 60 m with no distinctive recessional moraines and extension at 72-C for 1 min, followed by a final [4, 20]. The glacier has receded since the 1890s, opening extension step at 72-C for 10 min. All PCR reactions the forefront to colonization by plants and fungi. were performed in a Hybaid OmniCycler (Hybaid Ltd., Periodic photographs and snow survey data have allowed Middlesex, UK). Possible PCR amplification of airborne the reconstruction of the glacier retreat over the last and reagent contaminants was determined using a blank century [20]. sample ran through the extraction protocol simulta- neously with the actual samples and a negative PCR Sampling and DNA Extraction. Shrub willows control in which the template DNA was replaced with (Salix commutata and S. planifolia) comprise the early ddH2O. These remained free of PCR amplicons in all perennial plant communities and are the largest plant trials. individuals during early vegetation development [22]. Twelve shrub canopies–three of approximately equal size Small-Subunit rDNA Clone Library Construction and at distances of 300, 450, 750, and 900 m from the glacier Analysis. Primers specific to fungi and stringent PCR terminus–were selected, and 200-mL soil samples were conditions resulted in amplicons of expected size (about

Figure 1. Neighbor-joining analysis of environmental partial 18S sequences (see Table 1 for accession numbers; AY382401–AY382473) obtained with primer set B (nu-SSU-0817-50 and nu-SSU-1536-30 [3]) from willow canopy soil on the forefront of a receding glacier. Accession numbers of the GenBank-obtained sequences are shown in parentheses. Sequence data were aligned in Sequencher and neighbor-joining analyses performed in PAUP* [37]. Numbers above the nodes refer to the occurrence of that node in 1000 bootstrap replicates. Values 950% are shown. A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT 237 238 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT

780 bp in set B and about 1400 bp in set S) when the large insertions have been observed in the rDNA of PCR products were visualized on 1.5% agarose gels. The Helotiales, Lecanorales, and Onygenales (see [3, 16, 17, mixed populations of PCR products were ligated into a 29]), the unalignable sequences were omitted because linearized pGEM-T vector (Promega). The circularized true insertions and chimeric PCR products could not be plasmids were transformed into competent JM109 cells identified reliably. The taxonomic relationships among (Promega) by heat shock, and the putative positive the fungal sequences were inferred by neighbor-joining transformants were identified by !-complementation (NJ) analyses in phylogenetic analysis using parsimony [30]. (PAUP*) [37]. A chytridiomycetous fungus (Monoble- Twenty putatively positive transformants from each pharis hypogyna) was selected for the outgroup. Data clone library were randomly sampled, and the presence matrices were left uncorrected, rates for variable sites of the target insert was confirmed by PCR amplification were assumed equal, and no sites were assumed invari- in 15-2L reaction volumes under the same reaction able. Sites with missing data, ambiguous nucleotides, or conditions as described above. To select different plas- gaps, were randomly distributed among taxa. The mids for sequencing, these PCR products were digested robustness of the inferred NJ topologies was tested by with endonucleases (HinfI, AluI; New England BioLabs, 1000 bootstrap replicates. The most parsimonious trees Beverly, MA) and were resolved on 3% agarose gels [15]. were obtained using random addition sequence and a The PCR screening of clone libraries combined with branch-swapping algorithm with tree bisection recon- restriction fragment length polymorphisms (RFLP) en- nection. The number of equiparsimonious trees was abled the selection of different RFLP phenotypes for expected to be high attributable to several closely related sequencing. Sequences from each different RFLP pheno- sequences in the clone libraries. As a result, the maxi- type in all clone libraries were obtained by use of mum number of retained trees was restricted to 1000. fluorescent dideoxy-terminators (ABI Prism\ BigDyei The consensus (50% majority rule) and NJ topologies Applied Biosystems, Foster City, CA) and an automated placed the environmental sequences similarly (data not ABI Prism\ 3700 DNA Analyzer (Applied Biosystems) shown). at the DNA Sequencing and Genotyping Facility at Kansas State University (GenBank accession numbers Detection and Analysis of Chimeric Sequences. AY382401–AY382473). Vector contamination was re- Chimeric sequences may be frequent in environmental moved with the automated vector trimming function in samples with diverse, mixed populations of competing Sequencher (Version 4.1, GeneCodes, Ann Arbor, MI). templates [19, 24, 42]. To identify the most likely The similarities to existing rDNA sequences in the chimera breakpoints, all sequenced clones were analyzed GenBank database were determined at the National by the Chimera Check program of the RDP (version 2.7 Center for Biotechnology Information (http://www. [26]). To test the effects of the chimeric sequences on the ncbi.nlm.nih.gov/BLAST/ [1]) by standard nucleotide placement of the environmental clones in the obtained basic local alignment search tool (BLAST, version 2.2.1) NJ topologies, the data were reanalyzed after exclusion of without limiting queries and Sequence Match (version data upstream and downstream of the most commonly 2.7) at the RDP (http://rdp.cme.msu.edu/html/ [26]). encountered chimera breakpoints (positions 1–391 and The environmental sequences and sequences from 502–830 in data set B alignment and positions 1–730 and GenBank were aligned in 830 positions (data set B) and 902–1623 in data set S). The obtained topologies were in 1623 positions (data set S) using Sequencher and were compared to detect clones that clearly changed positions manually adjusted to maximize conservation. Regions in different analyses. adjacent to the priming sites were omitted because of high frequency of ambiguous sites. Data set B contained Results one nontarget contaminant (B_Canopy_300_03_06 most similar to Prismatolaimus intermedius, Enoplida, in Fungal Community Analyses. A total of 480 rDNA BLAST searches; Table 1) and three clones that contained clones in 24 libraries were screened, and unique RFLP large insertions and were unalignable with other fungal phenotypes were identified and sequenced to assay fungal sequences (B_Canopy_300_01_16, B_Canopy_300_ community composition within established Salix spp. 02_10, and B_Canopy_450_03_02; Table 1). Although canopies in a primary successional ecosystem. After

Figure 2. Neighbor-joining analysis of environmental partial 18S sequences (see Table 1 for accession numbers; AY382401–AY382473) obtained with primer set S (EF4 and EF3 [32]) from willow canopy soil on the forefront of a receding glacier. Accession numbers of the GenBank-obtained sequences are shown in parentheses. Sequence data were aligned in Sequencher and neighbor-joining analyses performed in PAUP* [37]. Numbers above the nodes refer to the occurrence of that node in 1000 bootstrap replicates. Values 950% are shown. A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT 239 240 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT exclusion of likely chimeric sequences, data set B These included well-supported groups with affinities contained 24 and data set S 18 unique clones. BLAST within Sordariales, Russulaceae, and Thelephoraceae. (Table 1) and NJ analyses (Figs. 1 and 2) placed the However, after exclusion of all suspected chimeric data, cloned environmental sequences into the kingdom Fungi. several incongruences were also evident (Figs. 3 and 4). The target sequences broadly represented fungi including Data set B (20 ascomycete clones of the 24 total clones) Ascomycota, Basidiomycota, Chytridiomycota, and contained a larger number of ascomycete sequences than Zygomycota. Overall, the cloned sequences indicated did data set S (4 ascomycete clones of the 18 total the presence of various groups of fungi in the soil clones). Many of the groupings were not supported in underneath the willow canopies at the receding glacier bootstrap analyses, but three ascomycete groups exem- forefront. Nontarget contaminants were rare; one clone plify the more abundant detection of ascomycetes in data was determined to be a nematode (P. intermedius). Three set B. First, two clones (B_Canopy_450_03_14 and additional sequences were omitted because they contained B_Canopy_450_03_17) were placed among Dothideomy- large unalignable inserts whose origin could not be cetes with reasonably high bootstrap support in NJ confirmed to be fungal. analyses (Fig. 1). Second, three clones (B_Canopy_300_ The majority of clones obtained with both primer 02_05, B_Canopy_300_02_06, and B_Canopy_750_ pairs were placed among hymenomycetes and filamen- 03_11) were grouped with Peziza griseorosea with 100% tous ascomycetes. The clones included taxa with likely bootstrap support, strongly indicating an affinity within affinities within the ascomycetous Sordariomycetes and Pezizaceae. Third, five clones (B_Canopy_300_03_17, basidiomycetous Russulales and Thelephorales (Figs. 1 B_Canopy_450_01_06, B_Canopy_450_02_13, B_Cano- and 2; Table 1). Two general points are noteworthy. py_750_02_13, and B_Canopy_900_03_09) from five First, various basidiomycete clones likely represent different samples were placed on a sister clade to ectomycorrhizal fungi. Clones in data sets B and S had Ophiostomatales. None of these well-supported groups well-supported affinities within Russulaceae (B_Cano- occurred in data set S. py_900_01_19 in data set B and S_Canopy_900_01_11 in Data set S contained well-supported groups within data set S) and Thelephoraceae (B_Canopy_900_02_06 Chytridiomycota (S_Canopy_300_03_04; Fig. 2) and and B_Canopy_900_03_17 in data set B and S_Cano- Zygomycota (S_Canopy_300_02_11; Fig. 2). In contrast, py_750_01_10 and S_Canopy_900_03_02 in data set S). data set B contained no clones representing lower fungi. Second, some ascomycete clones, similarly, are likely to This result was not attributable to mere exclusion of form associations with willow roots. Both data sets chimeric data, as no lower fungi were detected in data set contained clones with well-supported affinities to Sor- B in BLAST analyses. Data set S also included a large dariales (B_Canopy_900_02_04 in data set B and group of basidiomycetes with likely affinities within S_Canopy_450_02_05 and S_Canopy_750_02_09 in data Cortinariaceae representing at least two distinct taxa set S). These sordarialean fungi are likely similar to those (Cortinarius sp. and Inocybe sp.). No clones had well- forming ectomycorrhizas with willows as reported earlier supported affinities to Cortinariaceae in data set B, by Trowbridge and Jumpponen [39]. although at least three sequences were determined most Most clone libraries were dominated by a single similar to Inocybe geophylla in BLAST analyses. sequence type (Table 1). In two cases (samples S_Canopy_ 450_2 and S_Canopy_900_03), the libraries contained Detection and Importance of Chimeric Sequences. only one sequence type. These libraries were unlikely A majority of the environmental sequences were deter- to be representative because data set B contained more mined to be likely chimeric by Chimera Check of the than one sequence type in those samples. The dominant, RDP. Further testing by reanalyses identified 17 chimeras nonchimeric sequence types in data set B were not in data set B and 8 in data set S (Figs. 3 and 4). identical with those in data set S suggesting primer bias Exceptionally high scores (980) in Chimera Check were (see below). always confirmed chimeric in the reanalyses. Lower scores did not indicate nonchimeric origin of a Congruence in Fungal Community Composition sequence, but many sequences could be confirmed Among the Two Data Sets. Analysis of the 18S chimeric in the NJ analyses (Table 1). Many of the rDNA with two different primer sets designed to be chimeric sequences were likely a result of combined PCR specific to fungi congruently identified several groups. products of templates representing fungi from different

Figure 3. Reanalyses of data set B. Phylogram obtained by neighbor-joining analysis after the omission of potentially chimeric upstream data (positions 502–830) as identified by Chimera Check. Arrows on the right show the new placement of environmental sequences after the omission of potentially chimeric downstream data (positions 1–391). The environmental sequences with unstable placements in these reanalyses were concluded to be chimeric and were excluded from analyses shown in Fig. 1. A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT 241 242 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT 243 divisions as indicated by placement among Ascomycota ius and Russula [21, 22]) were detected in the soil in analyses utilizing only 50-end of the sequences and samples collected 900 m from the glacier terminus by among Basidiomycota in analyses utilizing only 30-end both primer sets. Finally, ectomycorrhizal fungi with of the sequences (e.g., B_Canopy_900_02_12 and inconspicuous fruiting bodies (Thelephoraceae) were B_Canopy_750_03_03 in data set B–Fig. 3; and detected within the willow canopies furthest from the S_Canopy_750_02_17 in data set S–Fig. 4). Data set S glacier terminus by both primers. was expected to have a greater proportion of chimeras, as Although functional roles of the ectomycorrhizal their likelihood was anticipated to increase with basidiomycetes are often simple to decipher from their increasing amplicon length. Surprisingly, data set B affinities to taxa available in sequence databases, the contained 40% (17/43) chimeric sequences, whereas function of a majority of ascomycetes detected in these data set S contained only 31% (8/26) chimeras. analyses remain unclear. Data set B contained clones with affinities to Pezizales (P. griseorosea), and both data sets contained clones with well-supported affinities to Discussion Sordariales. Several taxa within Pezizales have various Fungal Communities Within Willow Canopies in the associations ranging from pathogenicity to mycorrhizal Glacier Forefront Soil. Fungal PCR amplicons were symbiosis with ectomycorrhizal hosts [7, 8, 10]. Recent successfully obtained from environmental soil samples studies at the Lyman glacier site have suggested that taxa collected at the forefront of a receding glacier. A large with affinities to Sordariales may, unexpectedly, be proportion of the sequences was determined to be common mycorrhizal associates of the shrub willows chimeric by the Chimera Check software of the RDP. [39]. Although it is very likely that many cloned ascomy- Analyses conducted after exclusion of the sequence data cetes represent these (facultative) biotrophic associations, potentially obtained from another target organism various groups of the detected ascomycetes (e.g., taxa confirmed many chimeras, but the placement of most with affinities to Dothideales) are soil-inhabiting saprobes. cloned sequences was insensitive to the exclusion of the potentially chimeric data. In other words, the placement Congruence in Fungal Community Composition of a majority of the cloned sequences was similar whether Among the Two Data Sets. Differential PCR ampli- or not the data identified as possibly chimeric by fication may be a result of various factors including Chimera Check were included in the analyses. template concentration, numbers of template molecules, After exclusion of chimeric data, 24 and 18 environ- GC content of the template molecules, efficiency of mental sequences were analyzed in the two data sets. primer-template hybridization, polymerase extension Most of the basidiomycetes detected in these analyses efficiency for different templates, relative substrate ex- likely represented ectomycorrhizal associates of the haustion for different templates, and primer specificity willow plants. Earlier studies on occurrence [5, 12, 36, 42, 44]. The presented results of rDNA have indicated that Cortinariaceae (Inocybe spp. and analyses using two sets of primers confirmed predicted Cortinarius spp.) and (Laccaria spp.) EF4–EF3 primer bias toward basidiomycetes and lower are common throughout the primary successional glacier fungi [2, 32]. Only 4 of the 18 nonchimeric clones in forefront [21, 22]. Neither primer set produced cloned data set S were ascomycetous, whereas ascomycetes sequences that would find strongly supported affinity to comprised a majority of nonchimeric clones in data set Laccaria spp. in the NJ analyses, although both data sets B (20 ascomycetes of the total of 24 nonchimeric contained nonchimeric sequences that were deemed sequences). Although not observed in the present study, similar to Laccaria pumila in BLAST analyses. The primers for data set B do amplify chytridiomycetes absence of support in NJ analyses is likely because of and zygomycetes from environmental samples [3, 19]. the poor resolution within the Agaricales that the 18S The observed incongruences are therefore likely to rDNA data provide. Several sequences similar to Corti- have resulted either from true primer bias or from nariaceae were detected in both data sets, although only stochastic variation within an environmental DNA ex- data set S had well-supported affinities to Cortinarius tract. However, the two different fungus-specific primers iodes and I. geophylla. Additional infrequently fruiting congruently identified several groups. These included ectomycorrhizal fungi exclusive to areas adjacent to the well-supported groups with affinities within Sordariales, terminal moraine (Russulales representing genera Lactar- Russulaceae, and Thelephoraceae. The congruence among

Figure 4. Reanalyses of data set S. Phylogram obtained by neighbor-joining analysis after the omission of potentially chimeric upstream data (positions 902–1623) as identified by Chimera Check. Arrows on the right show the new placement of environmental sequences after the omission of potentially chimeric downstream data (positions 1–730). The environmental sequences with unstable placements in these reanalyses were concluded to be chimeric and were excluded from analyses shown in Fig. 2. 244 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT the data sets could possibly have been improved by after the omission of potentially chimeric regions increasing the number of clones sampled from each included such groups. Both B and S data sets included library. However, there is often a compromise between cloned sequences that were basal to ascomycetous the number of clones sampled from each library and the Saccharomycetales (e.g., B_Canopy_750_02_19 in Fig. 3 number of samples to be processed. Clearly, choice of and S_Canopy_300_02_01 in Fig. 4) and basidiomyce- primers and the number of sampled transformants within tous hymenomycetes (e.g., B_Canopy_300_01_18 and the clone libraries have a pivotal importance on the observed S_Canopy_750_03_18). Data set S included a sequence community structure. Comparisons among multiple (S_Canopy_300_02_19) that was positioned basal to extracts of the same sample, two or more primer sets, as higher fungi (i.e., Ascomycota and Basidiomycota). None well as multiple replicate samples may be necessary to obtain of the sequences placed in these basal positions were a more comprehensive view of the fungal communities. consistent in the reanalyses of the partial data sets and were therefore concluded to be PCR artifacts. Detection and Importance of Chimeric Sequences. Chimera Check overestimated the number of chimeric sequences as determined in confirmatory NJ analyses. Conclusions However, sequences with high scores (980) in the The results indicate that ascomycetous and basidiomy- Chimera Check were always confirmed chimeric. Lower cetous ectomycorrhizal fungi comprise a substantial scores included sequences that were determined chimeric component in the fungal communities associated with and many that appeared stable in their position in the established willow canopies in primary successional confirmatory NJ analyses. ecosystems on the forefront of a receding glacier. Use of The NJ analyses presented here aimed to identify and different primers yielded different results and supported detect sequences whose positions in the obtained topo- different conclusions. It seems therefore necessary to logies were inconsistent when only 50-ends or only 30-ends view the results of direct molecular assessments with of the sequences were utilized. Reanalyses of partial data some caution. Finally, chimeras seem to comprise a large sets identified 17 chimeras in data set B and 8 in data set S, proportion of the environmental sequence data as more than 30% of all analyzed sequences. Similar chimera determined by the Chimera Check of RDP and data frequencies have been observed in bacterial community reanalyses. Many of the chimeric reads appeared to analyses [43] and analyses of somatic mutations [13]. comprise novel taxa at least on the level of an order. Chimeric sequences are particularly frequent if sequence However, because it is possible that these sequences may similarity among the competing templates and the be but PCR artifacts, the discovery of novel taxa without number of PCR cycles are high [13, 43]. Accordingly, microscopic or culture-based confirmation may be simple precautionary measures, such as longer extension premature. times and fewer PCR cycles [42, 43], to minimize the generation of chimeras seem necessary. It was hypothesized that longer target amplicons Acknowledgments would be more susceptible for chimera formation. This work was supported by Kansas State University Unexpectedly, the data set with shorter target amplicon BRIEF program, National Science Foundation EPSCoR had greater number of identified chimeras. This obser- Grant No. 9874732 with matching support from the vation may be a result of the larger number of competing State of Kansas, and National Science Foundation Grant templates with fairly high similarity when primers with No. OPP-0221489. I am grateful to Dr. Francesco T. lesser bias were used (data set B; see [13, 43]). Overall, Gentili, Nicolo Gentili, Anna Jumpponen, and Dr. James more data (longer amplicons) are usually beneficial, as M. Trappe for their assistance during sample collection, they often allow better resolution in inferred topologies transport, and preparation in August 2001 and to Emily L. [18]. This is especially important when using conserved King and Justin Trowbridge for their assistance in clone gene regions such as the 18S of the rDNA. It appears that library screening and plasmid preparation. Dr. Charles L. the generation of chimeras is stochastic, and that Kramer, Nicholas B. Simpson, and Dr. James M. Trappe targeting shorter amplicons may be unnecessary in fear provided helpful comments on early drafts of this man- of poor-quality environmental sequence data if steps to uscript. Nicholas B. Simpson edited the manuscript. minimize chimera formation have been taken. Recent studies that utilize direct PCR from environ- mental samples have suggested frequent occurrences of References novel fungal phyla, which find positions basal to 1. Altschul, SF, Madden, TL, Scha¨ffer, AA, Zhang, J, Zhang, Z, Miller, filamentous ascomycetes or hymenomycetes [31, 41]. DJ, Lipman, DJ (1997) Gapped BLAST and PSI-BLAST: a new The preliminary analyses conducted prior to exclusion of generation of protein database search programs. Nucleic Acids Res chimeras as well as the analyses using partial sequences 25: 3389–3402 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT 245

2. Anderson, IC, Campbell, CD, Prosser, JI (2003) Potential bias of fungi in Lyman Lake Basin: a comparison between primary and fungal 18S rDNA and internal transcribed polymerase chain secondary successional sites. Mycologia 91: 575–582 reaction primers for estimating fungal biodiversity in soil. Environ 22. Jumpponen, A, Trappe, JM, Ca´zares, E (2002) Occurrence of Microbiol 5: 36–47 ectomycorrhizal fungi on a receding glacier forefront. Mycorrhiza 3. Borneman, J, Hartin, RJ (2000) PCR primers that amplify fungal 12: 43–49 rRNA genes from environmental samples. Appl Environ Microbiol 23. Jumpponen, A, Va¨re, H, Mattson, KG, Ohtonen, R, Trappe, JM 66: 4356–4360 (1999) Characterization of Fsafe sites_ for pioneers in primary 4. Ca´zares, E (1992) Mycorrhizal fungi and their relationship to plant succession on recently deglaciated terrain. J Ecol 87: 98–105 succession in subalpine habitats. PhD Thesis, Oregon State 24. Kopczynski, ED, Bateson, MM, Ward, DM (1994) Recognition of University chimeric small-subunit ribosomal DNAs composed from genes 5. Chandler, DP, Fredrickson, JK, Brockman, FJ (1997) Effect of PCR from uncultivated microorganisms. Appl Environ Microbiol 63: template concentration on the composition and distribution of 3614–3621 total community rDNA clone libraries. Mol Ecol 6: 475–482 25. Kowalchuk, GA, Gerards, S, Woldendorp, JW (1997) Detection 6. Cornelissen, JHC, Aerts, R, Cerabolini, MJA, Werger, MJA, van and characterization of fungal infections of Ammophila arenaria derHeijden, MGA (2001) Carbon cycling traits of plant species are (marram grass) roots by denaturing gradient gel electrophoresis. linked with mycorrhizal strategy. Oecologia 129: 611–619 Appl Environ Microbiol 63: 3858–3865 7. Dahlstrom, JL, Smith, JE, Weber, NS (2000) Mycorrhiza-like 26. Maidak, BL, Cole, JR, Jr, Parker, CT, Garrity, GM, Larsen, N, Li, B, interaction by Morchella with species of the Pinaceae in pure Lilburn, TG, McCaughey, MJ, Olsen, GJ, Overbeek, R, Pramanik, culture synthesis. Mycorrhiza 9: 279–285 TM, Schmidt, TM, Tiedje, JM, Woese, CR (1999) A new version of 8. Danielson, RM (1984) Ectomycorrhiza formation by the opercu- the RDP (Ribosomal Database Project). Nucleic Acids Res 27: 171– late discomycete Sphaerosporella brunnea (Pezizales). Mycologia 173 76: 454–461 27. O’Dell, TE, Smith, JE, Castellano, M, Luoma, D (1996) Diversity 9. Dickie, IA, Xu, B, Koide, RT (2002) Vertical niche differentiation and conservation of forest fungi. In: Pilz, D, Molina, R (Eds.) of ectomycorrhizal hyphae in soil as shown by T-RFLP analysis. Managing Forest Ecosystems to Conserve Fungus Diversity and New Phytol 156: 527–535 Sustain Wild Harvests. U.S. Forest Service General 10. Egger, KN, Paden, JW (1986) Biotrophic associations between Technical Reports, PNW-GTR-317. US Department of Agricul- lodgepole pine seedlings and post-fire ascomycetes (Pezizales) in ture, Forest Service, Pacific Northwest Research Station. Portland, monoxenic culture. Can J Bot 64: 2719–2725 OR, pp 5–18 11. Egli, S, Ayer, F, Chatelain, F (1997) Die Beschreibung der 28. Pennanen, T, Paavolainen, L, Hantula, J (2001) Rapid PCR-based Diversita¨t von Macromyceten. Erfahrungen aus pilzo¨lologischen method for the direct analysis of fungal communities in complex Langenzeitstudien im Pilzreservat La Chane´az, FR. Mycol Helv 9: environmental samples. Soil Biol Biochem 33: 697–699 19–32 29. Perotto, S, Nepote-Fus, P, Saletta, L, Bandi, C, Young, JPW (2000) 12. Farrelly, V, Rainey, FA, Stackebrandt, E (1995) Effect of genome A diverse population of introns in the nuclear ribosomal genes of size and rrn gene copy number on PCR amplification of 16S rRNA ericoid mycorrhizal fungi includes elements with sequence simi- genes from a mixture of bacterial species. Appl Environ Microbiol larity to endonuclease-coding genes. Mol Biol Evol 17: 44–59 91: 2798–2801 30. Sambrook, J (1989) Molecular Cloning—A Laboratory Manual, 13. Ford, JE, McHeyzer-Williams, MG, Lieber, MR (1994) Chimeric 2nd ed. In: Fritsch, EF, Maniatis, T (Eds.) Cold Spring Laboratory molecules created by gene amplification interfere with the analyses Press, New York of somatic hypermutation of murine immunoglobulin genes. Gene 31. Schadt, CW, Martin, AW, Lipson, DA, Schmidt, SK (2003) 142: 279–283 Seasonal dynamics of previously unknown fungal lineages in 14. Gardes, M, Bruns, TD (1993) ITS primers with enhanced tundra soils. Science 301: 1359–1361 specificity for higher fungi and basidiomycetes: application to 32. Smit, E, Leeflang, P, Glandorf, B, vanElsas, JD, Wernars, K (1999) identification of mycorrhizae and rusts. Mol Ecol 2: 113–118 Analysis of fungal diversity in the wheat rhizosphere by sequencing 15. Gardes, M, Bruns, TD (1996) ITS-RFLP matching for the of cloned PCR-amplified genes encoding 18S rRNA and temper- identification of fungi. In: Clapp, JP (Ed.) Methods in Molecular ature gradient gel electrophoresis. Appl Environ Microbiol 65: Biology, Vol. 50: Species Diagnostics Protocols: PCR and Other 2614–2621 Nucleic Acid Methods. Humana Press Inc., Totowa, NJ, pp 177– 33. Smit, E, Veenman, C, Baar, J (2003) Molecular analysis of 186 ectomycorrhizal basidiomycete communities in Pinus sylvestris L. 16. Gargas, A, DePriest, PT, Taylor, JW (1995) Positions of multiple stand reveals long-term increased diversity after removal of litter insertion in SSU rDNA of lichen-forming fungi. Mol Biol Evol 12: and humus layers. FEMS Microbiol Ecol 45: 49–57 208–218 34. Smith, SE, Read, DJ (1997) Mycorrhizal Symbiosis. Academic 17. Holst-Jensen, A, Vaage, M, Schumacher, T, Johansen, S (1999) Press. London Structural characteristics and possible horizontal transfer of group 35. Straatsma, G, Krisai-Greilhuber, I (2003) Assemblage structure, I introns between closely related plant pathogenic fungi. Mol Biol species richness, abundance, and distribution of fungal fruitbodies in Evol 16: 114–126 a seven year plot-based survey near Vienna. Mycol Res 107: 632–640 18. Hugenholtz, P, Goebel, BM, Pace, NR (1998) Impact of culture- 36. Suzuki, MT, Giovannoni, SJ (1996) Bias caused by template independent studies on the emerging phylogenetic view of annealing in the amplification mixtures of 16S rRNA genes by bacterial diversity. J Bacteriol 180: 4765–4774 PCR. Appl Environ Microbiol 62: 625–630 19. Jumpponen, A (2003) Soil fungal community assembly in a 37. Swofford, DL (2001) PAUP, Phylogenetic Analysis Using Parsi- primary successional glacier forefront ecosystem as inferred from mony (and Other Methods), Version 4. Sinauer Associates. rDNA sequence analyses. New Phytol 158: 569–578 Sunderland, MA 20. Jumpponen, A, Mattson, K, Trappe, JM, Ohtonen, R (1998) 38. Torsvik, V, Goksøyr, J, Daae, FL (1990) High diversity on DNA of Effects of established willows on primary succession on Lyman soil bacteria. Appl Environ Microbiol 56: 782–787 Glacier forefront: evidence for simultaneous canopy inhibition and 39. Trowbridge, J, Jumpponen, A (2004) Fungal colonization of shrub soil facilitation. Arct Alp Res 30: 31–39 willow roots at the forefront of a receding glacier. Mycorrhiza 14: 21. Jumpponen, A, Trappe, JM, Ca´zares, E (1999) Ectomycorrhizal 283–293 246 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT

40. Vainio, EJ, Hantula, J (2000) Direct analysis of wood-inhabiting 43. Wang, GCY, Wang, Y (1996) The frequency of chimeric fungi using denaturing gradient gel electrophoresis of amplified molecules as a consequence of PCR co-amplification of 16S ribosomal DNA. Mycol Res 104: 927–936 rRNA genes from different bacterial species. Microbiology 142: 41. Vandenkoornhuyse, P, Baldauf, SL, Leyval, C, Straczek, J, Young, JPW 1107–1114 (2002) Extensive fungal diversity in plant roots. Science 295: 2051 44. Zheng, D, Alm, EW, Stahl, DA, Raskin, L (1996) Characterization 42. vonWintzingerode, F, Go¨bel, UB, Stackebrandt, E (1997) Deter- of universal small-subunit rRNA hybridization probes for quanti- mination of microbial diversity in environmental samples: pitfalls tative molecular microbial ecology studies. Appl Environ Micro- of PCR-based rRNA analysis. FEMS Microbiol Rev 21: 213–229 biol 62: 4504–4513