m icrobial ecology - kansas state university micecol.pdfinocybe geophylla [af287835] (agaricales)...

MicrobialEcology

Soil Fungal Communities Underneath Willow Canopies on aPrimary Successional Glacier Forefront: rDNA Sequence ResultsCan 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

Soil fungal communities underneath willow canopiesthat had established on the forefront of a receding glacierwere analyzed by cloning the polymerase chain reaction(PCR)-amplified partial small subunit (18S) of theribosomal (rRNA) genes. Congruence between two setsof fungus-specific primers targeting the same gene regionwas analyzed by comparisons of inferred neighbor-joiningtopologies. The importance of chimeric sequences wasevaluated by Chimera Check (Ribosomal Database Proj-ect) and by data reanalyses after omission of potentiallychimeric regions at the 50- and 30-ends of the clonedamplicons. Diverse communities of fungi representingAscomycota, Basidiomycota, Chytridiomycota, and Zygo-mycota were detected. Ectomycorrhizal fungi comprised amajor component in the early plant communities inprimary successional ecosystems, as both primer setsfrequently detected basidiomycetes (Russulaceae andThelephoraceae) forming mycorrhizal symbioses. Variousascomycetes (Ophiostomatales, Pezizales, and Sordar-iales) of uncertain function dominated the clone librariesamplified from the willow canopy soil with one set ofprimers, whereas the clone libraries of the ampliconsgenerated with the second primer set were dominated bybasidiomycetes. Accordingly, primer bias is an importantfactor in fungal community analyses using DNA extractedfrom environmental samples. A large proportion (930%)of the cloned sequences were concluded to be chimericbased on their changing positions in inferred phylogeniesafter omission of possibly chimeric data. Many chimericsequences were positioned basal to existing classes offungi, suggesting that PCR artifacts may cause frequentdiscovery of new, higher level taxa (order, class) in directPCR analyses. Longer extension times during the PCR

amplification and a smaller number of PCR cycles arenecessary precautions to allow collection of reliableenvironmental sequence data.

Introduction

Fungi perform important ecosystem functions by partic-ipating in the decomposition of dead tissues as well asplant uptake of water and nutrients [6, 34]. Assessmentof fungal community composition is difficult because ofunreliable and ephemeral production of identifiablemacroscopic fruiting bodies [11, 27, 35]. Many fungialso produce microscopic, sexual or asexual fruitingstructures or fruit below ground escaping detection inassessments relying exclusively on the collection ofepigeous fruiting bodies. Pure culture techniques allowfungal community assays of soil and tissue samples in theabsence of identifiable macroscopic fruiting bodies.However, similar to bacteria [38], it is likely that largenumbers of fungi would be missed in such pure cultureassays (see [31, 41]). To overcome these problems infungal community analysis, molecular means specificallytargeting fungi in environmental samples have beendeveloped [3, 9, 14, 25, 28, 32, 33, 40].

Direct molecular assessment of the fungal commu-nities allows analyses without relying on whether or notthe fungi can be grown in pure culture or producefruiting bodies. However, polymerase chain reaction(PCR) artifacts, such as chimeric sequences resultingfrom amplification of more than one template, can causeproblems in environmental samples with unknownsources of diverse initial template DNA [13, 19, 24, 42,43]. Various coextracted substances and low concen-trations of the target template in the presence of highlysimilar competing target and nontarget templates mayfurther influence the fidelity of PCR reactions [42].Correspondence to: Ari Jumpponen; E-mail: [email protected]

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

Table

1.BLAST

andRDPan

alyses

oftheenvironmentalsequencesob

tained

from

underneath

thewillow

canop

iesestablished

ontheforefron

tof

arecedingglacier

Environmentalclone

Chimeraat

RDP

BLA

STmatch

[accession

number]

(Order)

Phylum

Similarity

Frequency

B_C

anop

y_300_01_08[A

Y382401]

Yes

(G20)

Spilocaea

oleaginea[A

F338393]

(Chaethothyriales/Dothidiales)

Ascom

ycota

980.60

B_C

anop

y_300_01_14[A

Y382402]

Yes

(G40)

Spilocaea

oleaginea[A

F338393]


Ascom

ycota

960.20

B_C

anop

y_300_01_16[A

Y382403]

Yes

(G80)

Hym

enoscyphus

ericea

[AY228753](H

elotiales)

Ascom

ycota

95a

0.10

B_C

anop

y_300_01_18b

[AY382404]

Yes

(G40)

Inocybegeophylla

[AF287835]

(Agaricales)

Basidiomycota

970.10

B_C

anop

y_300_02_04b

[AY382405]

Yes

(G20)

DarkseptateendophyteDS16b

[AF168167]

(Unkn

own)

Ascom

ycota

980.22

B_C

anop

y_300_02_05[A

Y382406]

Yes

(G20)

Pezizagriseorosea[A

F133150]

(Pezizales)

Ascom

ycota

990.11

B_C

anop

y_300_02_06[A

Y382407]

Yes

(G20)

Pezizagriseorosea[A

F133150]

(Pezizales)

Ascom

ycota

980.11

B_C

anop

y_300_02_10[A

Y382408]

Yes

(G20)

Tetracladium

marchalianu

m[A

Y204613](Incertae

sedis)

Ascom

ycota

99a

0.11

B_C

anop

y_300_02_12[A

Y382419]

Yes

(G20)

Spilocaea

oleaginea[A

F338393]


Ascom

ycota

980.33

B_C

anop

y_300_02_14b

[AY382410]

Yes

(G40)

Oidiodend

rontenu

issimum

[AB015787](O

nygenales)

Ascom

ycota

970.11

B_C

anop

y_300_03_06[A

Y382411]

No

Prism

atolaimus

interm

edius[A

F036603]

(Enop

lida;

Prism

atolaimidae)

Con

taminant

970.08

B_C

anop

y_300_03_12b

[AY382412]

Yes

(G100)

Cladoniasulphu

rina

[AF241544]

(Lecanorales)

Ascom

ycota

930.15

B_C

anop

y_300_03_17[A

Y382413]

Yes

(G40)

Hypoxylon

subm

onticulosum

[AF346544]

(Xylariales)

Ascom

ycota

960.31

B_C

anop

y_300_03_19b

[AY382414]

No

Neobulgaria

prem

nophila

[U45445]

(Helotiales)

Ascom

ycota

980.46

B_C

anop

y_450_01_02[A

Y382415]

Yes

(G80)

Pulvinu

laarcheri[U

62012]

(Pezizales)

Ascom

ycota

940.27

B_C

anop

y_450_01_06[A

Y382416]

Yes

(G40)

Hypom

yces

chrysospermus

[AB027339](H

ypocreales)

Ascom

ycota

960.20

B_C

anop

y_450_01_13[A

Y382417]

Yes

(G40)

Oidiodend

rontenu

issimum

[AB015787](O

nygenales)

Ascom

ycota

980.07

B_C

anop

y_450_01_14[A

Y382418]

Yes

(G40)

Oidiodend

rontenu

issimum

[AB015787](O

nygenales)

Ascom

ycota

970.33

B_C

anop

y_450_01_18[A

Y382419]

Yes

(G40)

Oidiodend

rontenu

issimum

[AB015787](O

nygenales)

Ascom

ycota

980.13

B_C

anop

y_450_02_02[A

Y382420]

Yes

(G40)

Rhizoctonia

solani

[D85643]

(Ceratob

asidiales)

Basidiomycota

950.06

B_C

anop

y_450_02_13[A

Y382421]

Yes

(G40)

Hypom

yces

chrysospermus

[AB027339](H

ypocreales)

Ascom

ycota

960.94

B_C

anop

y_450_03_02[A

Y382422]

Yes

(G40)

Connersia

rilstonii[A

F096174]

(Eurotiales)

Ascom

ycota

99/99a

,c0.14

B_C

anop

y_450_03_05[A

Y382423]

Yes

(G40)

Raciborskiomyces

longisetosum

[AY016351](C

haetothyriales)

Ascom

ycota

990.14

B_C

anop

y_450_03_07[A

Y382424]

Yes

(G40)

Herpotrichiajuniperi[U

42483]

(Pleosporales)

Ascom

ycota

970.43

B_C

anop

y_450_03_14[A

Y382425]

No

Mycosphaerella

mycopappi

[U43463]

(Chaetothyriales)

Ascom

ycota

980.29

B_C

anop

y_750_01_01b

[AY382426]

Yes

(G20)

Inocybegeophylla

[AF287835]

(Cortinariaceae)

Basidiomycota

970.20

B_C

anop

y_750_01_07b

[AY382427]

Yes

(G40)

Pezizagriseorosea[A

F133150]

(Pezizales)

Ascom

ycota

990.40

B_C

anop

y_750_01_10b

[AY382428]

Yes

(G40)

Anamylopsora

pulcherrim

a[A

F119501]

(Agyriales)

Ascom

ycota

970.20

B_C

anop

y_750_01_15b

[AY382429]

Yes

(G80)

Pulvinu

laarcheri[U

62012]

(Pezizales)

Ascom

ycota

970.20

B_C

anop

y_750_02_13[A

Y382430]

Yes

(G40)

Hypom

yces

chrysospermus

[M89993]

(Hypocreales)

Ascom

ycota

960.33

B_C

anop

y_750_02_15b

[AY382431]

Yes

(G40)

Ophiostom

apiliferum

[AJ243294]

(Ophiostom

atales)

Ascom

ycota

97/97c

0.44

B_C

anop

y_750_02_19b

[AY382432]

Yes

(G40)

Hypom

yces

chrysospermus

[M89993]

(Hypocreales)

Ascom

ycota

950.22

B_C

anop

y_750_03_03b

[AY382433]

Yes

(G80)

Sarcinom

yces

petricola[Y18702]

(Chaetothyriales)

Ascom

ycota

950.14

B_C

anop

y_750_03_04[A

Y382434]

Yes

(G20)

Laccaria

pumila

[AF287838]

(Agaricales)

Basidiomycota

980.29

B_C

anop

y_750_03_08b

[AY382435]

Yes

(G20)

Laccaria

pumila

[AF287838]

(Agaricales)

Basidiomycota

960.14

B_C

anop

y_750_03_11[A

Y382436]

No

Pezizagriseorosea[A

F133150]

(Pezizales)

Ascom

ycota

990.43

234 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT

B_C

anop

y_900_01_03b

[AY382437]

Yes

(G40)

Russula

compacta[A

F026582]

(Agaricales)

Basidiomycota

960.25

B_C

anop

y_900_01_16[A

Y382438]

Yes

(G40)

Hypom

yces

chrysospermus

[AB027339](H

ypocreales)

Ascom

ycota

970.13

B_C

anop

y_900_01_19[A

Y382439]

No

Russula

compacta[A

F026582]

(Agaricales)

Basidiomycota

980.63

B_C

anop

y_900_02_02[A

Y382440]

No

Oidiodend

rontenu

issimum

[AB015787](O

nygenales)

Ascom

ycota

990.09

B_C

anop

y_900_02_04[A

Y382441]

No

Chaetom

ium

elatum

[M83257]

(Sordariales)

Ascom

ycota

980.18

B_C

anop

y_900_02_06[A

Y382442]

Yes

(G80)

Thelephorasp.[A

F026627]

(Thelephorales)

Basidiomycota

990.55

B_C

anop

y_900_02_10[A

Y382443]

No

DarkseptateendophyteDS16b

[AF168167]

(Unkn

own

Ascom

ycota

980.09

B_C

anop

y_900_02_12b

[AY382444]

Yes

(G80)

Pulvinu

laarcheri[U

62012]

(Pezizales)

Ascom

ycota

970.09

B_C

anop

y_900_03_09[A

Y382445]

Yes

(G40)

Hypom

yces

chrysospermus

[AB027339](H

ypocreales)

Ascom

ycota

940.75

B_C

anop

y_900_03_11b

[AY382446]

Yes

(G80)

Polyporoletus

sublividus

[AF287840]

(Cantharellales)

Basidiomycota

940.13

B_C

anop

y_900_03_17[A

Y382447]

Yes

(G40)

Thelephorasp.[A

F026627]

(Thelephorales)

Basidiomycota

980.13

S_Canop

y_300_01_01[A

Y382448]

Yes

(G60)

Bulgariainquinans[A

J224362]

(Helotiales)

Ascom

ycota

980.11

S_Canop

y_300_01_07[A

Y382449]

No

Inocybegeophylla

[AF287835]

(Agaricales)

Basidiomycota

980.89

S_Canop

y_300_02_01b

[AY382450]

Yes

(G100)

Bulgariainquinans[A

J224362]

(Helotiales)

Ascom

ycota

950.14

S_Canop

y_300_02_11[A

Y382451]

Yes

(G40)

Mortierella

chlamydospora

[AF157143]

(Mucorales)

Zygom

ycota

970.29

S_Canop

y_300_02_13b

[AY382452]

Yes

(G80)

Bulgariainquinans[A

J224362]

(Helotiales)

Ascom

ycota

960.14

S_Canop

y_300_02_14b

[AY382453]

Yes

(G160)

Limnoperdon

incarnatum

[AF426952]

(Aphyllophorales)

Basidiomycota

940.14

S_Canop

y_300_02_19b

[AY382454]

Yes

(G100)

Panellusserotinu

s[A

F026590]

(Agaricales)

Basidiomycota

940.29

S_Canop

y_300_03_04[A

Y382455]

Yes

(G40)

Spizellomyces

acum

inatus

[M59759]

(Spizellomycetales)

Chytridiomycota

970.67

S_Canop

y_300_03_18[A

Y382456]

Yes

(G20)

Laccaria

pumila

[AF287838]

(Agaricales)

Basidiomycota

970.33

S_Canop

y_450_01_02b

[AY382457]

Yes

(G100)

Byssoascusstriatosporus[A

B015776](O

nygenales)

Ascom

ycota

940.25

S_Canop

y_450_01_07[A

Y382458]

Yes

(G80)

Entolom

astrictius[A

F287832]

(Agaricales)

Basidiomycota

930.25

S_Canop

y_450_01_19[A

Y382459]

Yes

(G60)

Bulgariainquinans[A

J224362]

(Helotiales)

Ascom

ycota

980.50

S_Canop

y_450_02_05[A

Y382460]

Yes

(G60)

Ophiostom

astenoceras

[M85054]

(Ophiostom

atales)

Ascom

ycota

951.00

S_Canop

y_750_01_10[A

Y382461]

Yes

(G20)

Thelephorasp.[A

F026627]

(Thelephorales)

Basidiomycota

97/94c

0.40

S_Canop

y_750_01_11[A

Y382462]

Yes

(G20)

Inocybegeophylla

[AF287835]

(Agaricales)

Basidiomycota

960.40

S_Canop

y_750_01_18[A

Y382463]

Yes

(G20)

Inocybegeophylla

[AF287835]

(Agaricales)

Basidiomycota

980.20

S_Canop

y_750_02_09[A

Y382464]

Yes

(G40)

Sporothrix

schenkii[M

85053]

(Ophiostom

atales)

Ascom

ycota

930.92

S_Canop

y_750_02_17b

[AY382465]

Yes

(G160)

Dendrocorticium

roseocarneum

[AF334910]

(Aphyllophorales)

Basidiomycota

920.08

S_Canop

y_750_03_01b

[AY382466]

Yes

(G100)

Bulgariainquinans[A

J224362]

(Helotiales)

Ascom

ycota

950.14

S_Canop

y_750_03_13[A

Y382467]

Yes

(G20)

Termitom

yces

sp.[A

B051891](A

garicales)

Basidiomycota

940.43

S_Canop

y_750_03_15[A

Y382468]

Yes

(G40)

Inocybegeophylla

[AF287835]

(Agaricales)

Basidiomycota

970.14

S_Canop

y_750_03_18b

[AY382469]

Yes

(G40)

Laccaria

pumila

[AF287838]

(Agaricales)

Basidiomycota

950.14

S_Canop

y_750_03_19[A

Y382470]

Yes

(G20)

Cyathrusstriatus

[AF026617]

(Nidulariales)

Basidiomycota

970.14

S_Canop

y_900_01_06[A

Y382471]

Yes

(G20)

Inocybegeophylla

[AF287835]

(Agaricales)

Basidiomycota

980.77

S_Canop

y_900_01_11[A

Y382472]

Yes

(G40)

Russula

compacta[U

59093]

(Agaricales)

Basidiomycota

970.23

S_Canop

y_900_03_02[A

Y382473]

Yes

(G40)

Thelephorasp.[A

F026627]

(Thelephorales)

Basidiomycota

971.00

Chim

eraCheckscores

inparentheses.Frequency

refers

totheoccurrence

ofaclon

ein

thelib

rary

obtained

from

onesample.

RDP=Ribosom

alDatabaseProject;BLAST

=basiclocalalignmentsearch

tool.

aSequ

ence

omittedfrom

theneighbo

r-joininganalyses

because

ofalargeinsert.

bSequ

ence

determined

chim

eric

inanalyses

afterom

ission

ofdatabeyondchim

erapoints.

cBLAST

matches

werepartial

anddid

not

span

over

theentire

clon

edsequ

ence.

A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT 235

Furthermore, primer sets designed to obtain broadspecificity to a target group (e.g., fungi) may have biasesand preferentially amplify one target group but notanother [2, 36].

The overall goal of the presented studies was tocharacterize fungal community composition withinestablished willow (Salix spp.) canopies on the forefrontof a receding glacier. The nuclear small subunit (18S) ofthe ribosomal RNA gene (rDNA) was amplified with twodifferent sets of fungus-specific primers to estimate theinfluence of primer selection on the observed communitystructure. To evaluate the influence of chimeric ampli-cons on the obtained 18S phylogenies, data sets werereanalyzed after omission of the chimeric regionsidentified using Chimera Check software of the Ribo-somal Database Project (RDP, version 2.7 [26]). Theresults indicate that diverse fungal communities existwithin the willow canopies, that primer selection stronglyinfluences the observed fungal community structure, andthat chimeras are a serious concern in direct PCRapplications targeting fungi in environmental samples.

Methods

Study Site. Lyman Glacier (48-1005200N, 120-5308700W)is located in the Glacier Peak Wilderness Area in theNorth Cascade Mountains (Washington, USA). The sitehas been utilized in several studies on early plantcommunity assembly in recently deglaciated substrate(e.g., [20, 23]). Similarly, it has been a focus of studiesaiming to examine fungal community assembly in suchan environment [19, 21, 22]. The elevation of the presentglacier terminus is about 1800 m. The deglaciated fore-front is approximately 1000 m long over an elevation dropof only 60 m with no distinctive recessional moraines[4, 20]. The glacier has receded since the 1890s, openingthe forefront to colonization by plants and fungi.Periodic photographs and snow survey data have allowedthe reconstruction of the glacier retreat over the lastcentury [20].

Sampling and DNA Extraction. Shrub willows(Salix commutata and S. planifolia) comprise the earlyperennial plant communities and are the largest plantindividuals during early vegetation development [22].Twelve shrub canopies–three of approximately equal sizeat distances of 300, 450, 750, and 900 m from the glacierterminus–were selected, and 200-mL soil samples were

collected in August 2001. Samples were stored on iceuntil processed. In the laboratory, roots were handpickedfrom soil, and soil was homogenized manually in plasticbags. Approximately 0.25 g of soil was transferred tothe extraction buffer, and DNA was extracted usingUltraClean Soil kit (Molecular Biology LaboratoriesInc., Carlsbad, CA) following manufacturer’s protocol.Extracted DNA was stored frozen (_20-C) until furtherprocessing.

PCR Amplification of the Fungal DNA. A partialsequence of the 18S of the fungal rDNA was amplifiedwith two different primer sets in 50-2L PCR reactionmixtures. First, the reaction to collect data set Bcontained final concentrations or absolute amounts ofreagents as follows: 400 nM of each of the forward andreverse primers (nu-SSU-0817-50 and nu-SSU-1536-30

[3]), 2 2L of the extracted template DNA, 200 2M ofeach deoxynucleotide triphosphate, 2.5 mM MgCl2, 1 Uof Taq DNA polymerase (Promega, Madison, WI), and5 2L of manufacturer’s PCR buffer. The PCR cycleparameters consisted of an initial denaturation at 94-Cfor 3 min, then 40 cycles of denaturation at 94-C for1 min, annealing at 56-C for 1 min and extension at72-C for 1 min, followed by a final extension step at 72-Cfor 10 min. Second, the reaction to collect data set Scontained final concentrations or absolute amounts ofreagents as follows: 300 nM of each of the forward andreverse primers (EF4 and EF3 [32]), 2 2L of the extractedtemplate DNA, 200 2M of each deoxynucleotidetriphosphate, 1.7 mM MgCl2, 2 U of Taq DNA poly-merase (Promega), and 5 2L of manufacturer’s PCRbuffer. The PCR cycle parameters consisted of an initialdenaturation at 94-C for 3 min, then 40 cycles of dena-turation at 94-C for 1 min, annealing at 48-C for 1 minand extension at 72-C for 1 min, followed by a finalextension step at 72-C for 10 min. All PCR reactionswere performed in a Hybaid OmniCycler (Hybaid Ltd.,Middlesex, UK). Possible PCR amplification of airborneand reagent contaminants was determined using a blanksample ran through the extraction protocol simulta-neously with the actual samples and a negative PCRcontrol in which the template DNA was replaced withddH2O. These remained free of PCR amplicons in alltrials.

Small-Subunit rDNA Clone Library Construction andAnalysis. Primers specific to fungi and stringent PCRconditions 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-0817-5

0and nu-SSU-1536-3

0[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 andneighbor-joining analyses performed in PAUP* [37]. Numbers above the nodes refer to the occurrence of that node in 1000 bootstrapreplicates. Values 950% are shown.


780 bp in set B and about 1400 bp in set S) when thePCR products were visualized on 1.5% agarose gels. Themixed populations of PCR products were ligated into alinearized pGEM-T vector (Promega). The circularizedplasmids were transformed into competent JM109 cells(Promega) by heat shock, and the putative positivetransformants were identified by !-complementation[30].

Twenty putatively positive transformants from eachclone library were randomly sampled, and the presenceof the target insert was confirmed by PCR amplificationin 15-2L reaction volumes under the same reactionconditions as described above. To select different plas-mids for sequencing, these PCR products were digestedwith endonucleases (HinfI, AluI; New England BioLabs,Beverly, MA) and were resolved on 3% agarose gels [15].The PCR screening of clone libraries combined withrestriction fragment length polymorphisms (RFLP) en-abled the selection of different RFLP phenotypes forsequencing. Sequences from each different RFLP pheno-type in all clone libraries were obtained by use offluorescent dideoxy-terminators (ABI Prism\ BigDyeiApplied Biosystems, Foster City, CA) and an automatedABI Prism\ 3700 DNA Analyzer (Applied Biosystems)at the DNA Sequencing and Genotyping Facility atKansas State University (GenBank accession numbersAY382401–AY382473). Vector contamination was re-moved with the automated vector trimming function inSequencher (Version 4.1, GeneCodes, Ann Arbor, MI).The similarities to existing rDNA sequences in theGenBank database were determined at the NationalCenter for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/ [1]) by standard nucleotidebasic local alignment search tool (BLAST, version 2.2.1)without limiting queries and Sequence Match (version2.7) at the RDP (http://rdp.cme.msu.edu/html/ [26]).

The environmental sequences and sequences fromGenBank were aligned in 830 positions (data set B) andin 1623 positions (data set S) using Sequencher and weremanually adjusted to maximize conservation. Regionsadjacent to the priming sites were omitted because ofhigh frequency of ambiguous sites. Data set B containedone nontarget contaminant (B_Canopy_300_03_06 mostsimilar to Prismatolaimus intermedius, Enoplida, inBLAST searches; Table 1) and three clones that containedlarge insertions and were unalignable with other fungalsequences (B_Canopy_300_01_16, B_Canopy_300_02_10, and B_Canopy_450_03_02; Table 1). Although

large insertions have been observed in the rDNA ofHelotiales, Lecanorales, and Onygenales (see [3, 16, 17,29]), the unalignable sequences were omitted becausetrue insertions and chimeric PCR products could not beidentified reliably. The taxonomic relationships amongthe fungal sequences were inferred by neighbor-joining(NJ) analyses in phylogenetic analysis using parsimony(PAUP*) [37]. A chytridiomycetous fungus (Monoble-pharis hypogyna) was selected for the outgroup. Datamatrices were left uncorrected, rates for variable siteswere assumed equal, and no sites were assumed invari-able. Sites with missing data, ambiguous nucleotides, orgaps, were randomly distributed among taxa. Therobustness of the inferred NJ topologies was tested by1000 bootstrap replicates. The most parsimonious treeswere obtained using random addition sequence and abranch-swapping algorithm with tree bisection recon-nection. The number of equiparsimonious trees wasexpected to be high attributable to several closely relatedsequences in the clone libraries. As a result, the maxi-mum number of retained trees was restricted to 1000.The consensus (50% majority rule) and NJ topologiesplaced the environmental sequences similarly (data notshown).

Detection and Analysis of Chimeric Sequences.Chimeric sequences may be frequent in environmentalsamples with diverse, mixed populations of competingtemplates [19, 24, 42]. To identify the most likelychimera breakpoints, all sequenced clones were analyzedby the Chimera Check program of the RDP (version 2.7[26]). To test the effects of the chimeric sequences on theplacement of the environmental clones in the obtainedNJ topologies, the data were reanalyzed after exclusion ofdata upstream and downstream of the most commonlyencountered chimera breakpoints (positions 1–391 and502–830 in data set B alignment and positions 1–730 and902–1623 in data set S). The obtained topologies werecompared to detect clones that clearly changed positionsin different analyses.

Results

Fungal Community Analyses. A total of 480 rDNAclones in 24 libraries were screened, and unique RFLPphenotypes were identified and sequenced to assay fungalcommunity composition within established Salix spp.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 numbersof the GenBank-obtained sequences are shown in parentheses. Sequence data were aligned in Sequencher and neighbor-joininganalyses performed in PAUP* [37]. Numbers above the nodes refer to the occurrence of that node in 1000 bootstrap replicates.Values 950% are shown.


http://www.ncbi.nlm.nih.gov/BLAST/http://www.ncbi.nlm.nih.gov/BLAST/http://rdp.cme.msu.edu/html/

exclusion of likely chimeric sequences, data set Bcontained 24 and data set S 18 unique clones. BLAST(Table 1) and NJ analyses (Figs. 1 and 2) placed thecloned environmental sequences into the kingdom Fungi.The target sequences broadly represented fungi includingAscomycota, Basidiomycota, Chytridiomycota, andZygomycota. Overall, the cloned sequences indicatedthe presence of various groups of fungi in the soilunderneath the willow canopies at the receding glacierforefront. Nontarget contaminants were rare; one clonewas determined to be a nematode (P. intermedius). Threeadditional sequences were omitted because they containedlarge unalignable inserts whose origin could not beconfirmed to be fungal.

The majority of clones obtained with both primerpairs were placed among hymenomycetes and filamen-tous ascomycetes. The clones included taxa with likelyaffinities within the ascomycetous Sordariomycetes andbasidiomycetous Russulales and Thelephorales (Figs. 1and 2; Table 1). Two general points are noteworthy.First, various basidiomycete clones likely representectomycorrhizal fungi. Clones in data sets B and S hadwell-supported affinities within Russulaceae (B_Cano-py_900_01_19 in data set B and S_Canopy_900_01_11 indata set S) and Thelephoraceae (B_Canopy_900_02_06and B_Canopy_900_03_17 in data set B and S_Cano-py_750_01_10 and S_Canopy_900_03_02 in data set S).Second, some ascomycete clones, similarly, are likely toform associations with willow roots. Both data setscontained clones with well-supported affinities to Sor-dariales (B_Canopy_900_02_04 in data set B andS_Canopy_450_02_05 and S_Canopy_750_02_09 in dataset S). These sordarialean fungi are likely similar to thoseforming ectomycorrhizas with willows as reported earlierby Trowbridge and Jumpponen [39].

Most clone libraries were dominated by a singlesequence type (Table 1). In two cases (samples S_Canopy_450_2 and S_Canopy_900_03), the libraries containedonly one sequence type. These libraries were unlikelyto be representative because data set B contained morethan one sequence type in those samples. The dominant,nonchimeric sequence types in data set B were notidentical with those in data set S suggesting primer bias(see below).

Congruence in Fungal Community CompositionAmong the Two Data Sets. Analysis of the 18SrDNA with two different primer sets designed to bespecific to fungi congruently identified several groups.

These included well-supported groups with affinitieswithin Sordariales, Russulaceae, and Thelephoraceae.However, after exclusion of all suspected chimeric data,several incongruences were also evident (Figs. 3 and 4).Data set B (20 ascomycete clones of the 24 total clones)contained a larger number of ascomycete sequences thandid data set S (4 ascomycete clones of the 18 totalclones). Many of the groupings were not supported inbootstrap analyses, but three ascomycete groups exem-plify the more abundant detection of ascomycetes in dataset B. First, two clones (B_Canopy_450_03_14 andB_Canopy_450_03_17) were placed among Dothideomy-cetes with reasonably high bootstrap support in NJanalyses (Fig. 1). Second, three clones (B_Canopy_300_02_05, B_Canopy_300_02_06, and B_Canopy_750_03_11) were grouped with Peziza griseorosea with 100%bootstrap support, strongly indicating an affinity withinPezizaceae. Third, five clones (B_Canopy_300_03_17,B_Canopy_450_01_06, B_Canopy_450_02_13, B_Cano-py_750_02_13, and B_Canopy_900_03_09) from fivedifferent samples were placed on a sister clade toOphiostomatales. None of these well-supported groupsoccurred in data set S.

Data set S contained well-supported groups withinChytridiomycota (S_Canopy_300_03_04; Fig. 2) andZygomycota (S_Canopy_300_02_11; Fig. 2). In contrast,data set B contained no clones representing lower fungi.This result was not attributable to mere exclusion ofchimeric data, as no lower fungi were detected in data setB in BLAST analyses. Data set S also included a largegroup of basidiomycetes with likely affinities withinCortinariaceae representing at least two distinct taxa(Cortinarius sp. and Inocybe sp.). No clones had well-supported affinities to Cortinariaceae in data set B,although at least three sequences were determined mostsimilar to Inocybe geophylla in BLAST analyses.

Detection and Importance of Chimeric Sequences.A majority of the environmental sequences were deter-mined to be likely chimeric by Chimera Check of theRDP. Further testing by reanalyses identified 17 chimerasin data set B and 8 in data set S (Figs. 3 and 4).Exceptionally high scores (980) in Chimera Check werealways confirmed chimeric in the reanalyses. Lowerscores did not indicate nonchimeric origin of asequence, but many sequences could be confirmedchimeric in the NJ analyses (Table 1). Many of thechimeric sequences were likely a result of combined PCRproducts 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 upstreamdata (positions 502–830) as identified by Chimera Check. Arrows on the right show the new placement of environmental sequencesafter the omission of potentially chimeric downstream data (positions 1–391). The environmental sequences with unstable placementsin these reanalyses were concluded to be chimeric and were excluded from analyses shown in Fig. 1.


divisions as indicated by placement among Ascomycotain analyses utilizing only 50-end of the sequences andamong Basidiomycota in analyses utilizing only 30-endof the sequences (e.g., B_Canopy_900_02_12 andB_Canopy_750_03_03 in data set B–Fig. 3; andS_Canopy_750_02_17 in data set S–Fig. 4). Data set Swas expected to have a greater proportion of chimeras, astheir likelihood was anticipated to increase withincreasing amplicon length. Surprisingly, data set Bcontained 40% (17/43) chimeric sequences, whereasdata set S contained only 31% (8/26) chimeras.

Discussion

Fungal Communities Within Willow Canopies in theGlacier Forefront Soil. Fungal PCR amplicons weresuccessfully obtained from environmental soil samplescollected at the forefront of a receding glacier. A largeproportion of the sequences was determined to bechimeric by the Chimera Check software of the RDP.Analyses conducted after exclusion of the sequence datapotentially obtained from another target organismconfirmed many chimeras, but the placement of mostcloned sequences was insensitive to the exclusion of thepotentially chimeric data. In other words, the placementof a majority of the cloned sequences was similar whetheror not the data identified as possibly chimeric byChimera Check were included in the analyses.

After exclusion of chimeric data, 24 and 18 environ-mental sequences were analyzed in the two data sets.Most of the basidiomycetes detected in these analyseslikely represented ectomycorrhizal associates of thewillow plants. Earlier studies on sporocarp occurrencehave indicated that Cortinariaceae (Inocybe spp. andCortinarius spp.) and Tricholomataceae (Laccaria spp.)are common throughout the primary successional glacierforefront [21, 22]. Neither primer set produced clonedsequences that would find strongly supported affinity toLaccaria spp. in the NJ analyses, although both data setscontained nonchimeric sequences that were deemedsimilar to Laccaria pumila in BLAST analyses. Theabsence of support in NJ analyses is likely because ofthe poor resolution within the Agaricales that the 18SrDNA data provide. Several sequences similar to Corti-nariaceae were detected in both data sets, although onlydata set S had well-supported affinities to Cortinariusiodes and I. geophylla. Additional infrequently fruitingectomycorrhizal fungi exclusive to areas adjacent to theterminal moraine (Russulales representing genera Lactar-

ius and Russula [21, 22]) were detected in the soilsamples collected 900 m from the glacier terminus byboth primer sets. Finally, ectomycorrhizal fungi withinconspicuous fruiting bodies (Thelephoraceae) weredetected within the willow canopies furthest from theglacier terminus by both primers.

Although functional roles of the ectomycorrhizalbasidiomycetes are often simple to decipher from theiraffinities to taxa available in sequence databases, thefunction of a majority of ascomycetes detected in theseanalyses remain unclear. Data set B contained cloneswith affinities to Pezizales (P. griseorosea), and both datasets contained clones with well-supported affinities toSordariales. Several taxa within Pezizales have variousassociations ranging from pathogenicity to mycorrhizalsymbiosis with ectomycorrhizal hosts [7, 8, 10]. Recentstudies at the Lyman glacier site have suggested that taxawith affinities to Sordariales may, unexpectedly, becommon mycorrhizal associates of the shrub willows[39]. Although it is very likely that many cloned ascomy-cetes represent these (facultative) biotrophic associations,various groups of the detected ascomycetes (e.g., taxawith affinities to Dothideales) are soil-inhabiting saprobes.

Congruence in Fungal Community CompositionAmong the Two Data Sets. Differential PCR ampli-fication may be a result of various factors includingtemplate concentration, numbers of template molecules,GC content of the template molecules, efficiency ofprimer-template hybridization, polymerase extensionefficiency for different templates, relative substrate ex-haustion for different templates, and primer specificity[5, 12, 36, 42, 44]. The presented results of rDNAanalyses using two sets of primers confirmed predictedEF4–EF3 primer bias toward basidiomycetes and lowerfungi [2, 32]. Only 4 of the 18 nonchimeric clones indata set S were ascomycetous, whereas ascomycetescomprised a majority of nonchimeric clones in data setB (20 ascomycetes of the total of 24 nonchimericsequences). Although not observed in the present study,primers for data set B do amplify chytridiomycetesand zygomycetes from environmental samples [3, 19].The observed incongruences are therefore likely tohave resulted either from true primer bias or fromstochastic variation within an environmental DNA ex-tract. However, the two different fungus-specific primerscongruently identified several groups. These includedwell-supported groups with affinities within Sordariales,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 upstreamdata (positions 902–1623) as identified by Chimera Check. Arrows on the right show the new placement of environmental sequencesafter the omission of potentially chimeric downstream data (positions 1–730). The environmental sequences with unstable placementsin these reanalyses were concluded to be chimeric and were excluded from analyses shown in Fig. 2.


the data sets could possibly have been improved byincreasing the number of clones sampled from eachlibrary. However, there is often a compromise betweenthe number of clones sampled from each library and thenumber of samples to be processed. Clearly, choice ofprimers and the number of sampled transformants withinthe clone libraries have a pivotal importance on the observedcommunity structure. Comparisons among multipleextracts of the same sample, two or more primer sets, aswell as multiple replicate samples may be necessary to obtaina more comprehensive view of the fungal communities.

Detection and Importance of Chimeric Sequences.Chimera Check overestimated the number of chimericsequences as determined in confirmatory NJ analyses.However, sequences with high scores (980) in theChimera Check were always confirmed chimeric. Lowerscores included sequences that were determined chimericand many that appeared stable in their position inconfirmatory NJ analyses.

The NJ analyses presented here aimed to identify anddetect sequences whose positions in the obtained topo-logies were inconsistent when only 50-ends or only 30-endsof the sequences were utilized. Reanalyses of partial datasets identified 17 chimeras in data set B and 8 in data set S,more than 30% of all analyzed sequences. Similar chimerafrequencies have been observed in bacterial communityanalyses [43] and analyses of somatic mutations [13].Chimeric sequences are particularly frequent if sequencesimilarity among the competing templates and thenumber of PCR cycles are high [13, 43]. Accordingly,simple precautionary measures, such as longer extensiontimes and fewer PCR cycles [42, 43], to minimize thegeneration of chimeras seem necessary.

It was hypothesized that longer target ampliconswould be more susceptible for chimera formation.Unexpectedly, the data set with shorter target ampliconhad greater number of identified chimeras. This obser-vation may be a result of the larger number of competingtemplates with fairly high similarity when primers withlesser bias were used (data set B; see [13, 43]). Overall,more data (longer amplicons) are usually beneficial, asthey often allow better resolution in inferred topologies[18]. This is especially important when using conservedgene regions such as the 18S of the rDNA. It appears thatthe generation of chimeras is stochastic, and thattargeting shorter amplicons may be unnecessary in fearof poor-quality environmental sequence data if steps tominimize chimera formation have been taken.

Recent studies that utilize direct PCR from environ-mental samples have suggested frequent occurrences ofnovel fungal phyla, which find positions basal tofilamentous ascomycetes or hymenomycetes [31, 41].The preliminary analyses conducted prior to exclusion ofchimeras as well as the analyses using partial sequences

after the omission of potentially chimeric regionsincluded such groups. Both B and S data sets includedcloned sequences that were basal to ascomycetousSaccharomycetales (e.g., B_Canopy_750_02_19 in Fig. 3and S_Canopy_300_02_01 in Fig. 4) and basidiomyce-tous hymenomycetes (e.g., B_Canopy_300_01_18 andS_Canopy_750_03_18). Data set S included a sequence(S_Canopy_300_02_19) that was positioned basal tohigher fungi (i.e., Ascomycota and Basidiomycota). Noneof the sequences placed in these basal positions wereconsistent in the reanalyses of the partial data sets andwere therefore concluded to be PCR artifacts.

Conclusions

The results indicate that ascomycetous and basidiomy-cetous ectomycorrhizal fungi comprise a substantialcomponent in the fungal communities associated withthe established willow canopies in primary successionalecosystems on the forefront of a receding glacier. Use ofdifferent primers yielded different results and supporteddifferent conclusions. It seems therefore necessary toview the results of direct molecular assessments withsome caution. Finally, chimeras seem to comprise a largeproportion of the environmental sequence data asdetermined by the Chimera Check of RDP and datareanalyses. Many of the chimeric reads appeared tocomprise novel taxa at least on the level of an order.However, because it is possible that these sequences maybe but PCR artifacts, the discovery of novel taxa withoutmicroscopic or culture-based confirmation may bepremature.

Acknowledgments

This work was supported by Kansas State UniversityBRIEF program, National Science Foundation EPSCoRGrant No. 9874732 with matching support from theState of Kansas, and National Science Foundation GrantNo. OPP-0221489. I am grateful to Dr. Francesco T.Gentili, Nicolo Gentili, Anna Jumpponen, and Dr. JamesM. Trappe for their assistance during sample collection,transport, and preparation in August 2001 and to Emily L.King and Justin Trowbridge for their assistance in clonelibrary screening and plasmid preparation. Dr. Charles L.Kramer, Nicholas B. Simpson, and Dr. James M. Trappeprovided helpful comments on early drafts of this man-uscript. Nicholas B. Simpson edited the manuscript.

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