microbial ecology - rausser college of natural resources · 2007-03-07 · microbial ecology soil...
TRANSCRIPT
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
Tab
le1.
BL
AS
Tan
dR
DP
anal
yses
of
the
envi
ron
men
tal
seq
uen
ces
ob
tain
edfr
om
un
der
nea
thth
ew
illo
wca
no
pie
ses
tab
lish
edo
nth
efo
refr
on
to
fa
rece
din
ggl
acie
r
En
viro
nm
enta
lcl
one
Ch
imer
aat
RD
PB
LA
STm
atch
[acc
essi
onn
um
ber]
(Ord
er)
Ph
ylu
mSi
mil
arit
yF
requ
ency
B_
Can
op
y_30
0_01
_08
[AY
3824
01]
Yes
(G20
)Sp
iloc
aea
olea
gin
ea[A
F33
8393
](C
hae
tho
thyr
iale
s/D
oth
idia
les)
Asc
om
yco
ta98
0.60
B_
Can
op
y_30
0_01
_14
[AY
3824
02]
Yes
(G40
)Sp
iloc
aea
olea
gin
ea[A
F33
8393
](C
hae
tho
thyr
iale
s/D
oth
idia
les)
Asc
om
yco
ta96
0.20
B_
Can
op
y_30
0_01
_16
[AY
3824
03]
Yes
(G80
)H
ymen
oscy
phu
ser
icea
[AY
2287
53]
(Hel
oti
ales
)A
sco
myc
ota
95a
0.10
B_
Can
op
y_30
0_01
_18
b[A
Y38
2404
]Y
es(G
40)
Inoc
ybe
geop
hyl
la[A
F28
7835
](A
gari
cale
s)B
asid
iom
yco
ta97
0.10
B_
Can
op
y_30
0_02
_04
b[A
Y38
2405
]Y
es(G
20)
Dar
kse
ptat
een
dop
hyt
eD
S16b
[AF
1681
67]
(Un
kno
wn
)A
sco
myc
ota
980.
22B
_C
ano
py_
300_
02_
05[A
Y38
2406
]Y
es(G
20)
Pez
iza
gris
eoro
sea
[AF
1331
50]
(Pez
izal
es)
Asc
om
yco
ta99
0.11
B_
Can
op
y_30
0_02
_06
[AY
3824
07]
Yes
(G20
)P
eziz
agr
iseo
rose
a[A
F13
3150
](P
eziz
ales
)A
sco
myc
ota
980.
11B
_C
ano
py_
300_
02_
10[A
Y38
2408
]Y
es(G
20)
Tet
racl
adiu
mm
arch
alia
nu
m[A
Y20
4613
](I
nce
rtae
sed
is)
Asc
om
yco
ta99
a0.
11B
_C
ano
py_
300_
02_
12[A
Y38
2419
]Y
es(G
20)
Spil
ocae
aol
eagi
nea
[AF
3383
93]
(Ch
aeth
oth
yria
les/
Do
thid
iale
s)A
sco
myc
ota
980.
33B
_C
ano
py_
300_
02_
14b
[AY
3824
10]
Yes
(G40
)O
idio
den
dro
nte
nu
issi
mu
m[A
B01
5787
](O
nyg
enal
es)
Asc
om
yco
ta97
0.11
B_
Can
op
y_30
0_03
_06
[AY
3824
11]
No
Pri
smat
olai
mu
sin
term
ediu
s[A
F03
6603
](E
no
pli
da;
Pri
smat
ola
imid
ae)
Co
nta
min
ant
970.
08B
_C
ano
py_
300_
03_
12b
[AY
3824
12]
Yes
(G10
0)C
lad
onia
sulp
hu
rin
a[A
F24
1544
](L
ecan
ora
les)
Asc
om
yco
ta93
0.15
B_
Can
op
y_30
0_03
_17
[AY
3824
13]
Yes
(G40
)H
ypox
ylon
subm
onti
culo
sum
[AF
3465
44]
(Xyl
aria
les)
Asc
om
yco
ta96
0.31
B_
Can
op
y_30
0_03
_19
b[A
Y38
2414
]N
oN
eobu
lgar
iapr
emn
oph
ila
[U45
445]
(Hel
oti
ales
)A
sco
myc
ota
980.
46B
_C
ano
py_
450_
01_
02[A
Y38
2415
]Y
es(G
80)
Pu
lvin
ula
arch
eri
[U62
012]
(Pez
izal
es)
Asc
om
yco
ta94
0.27
B_
Can
op
y_45
0_01
_06
[AY
3824
16]
Yes
(G40
)H
ypom
yces
chry
sosp
erm
us
[AB
0273
39]
(Hyp
ocr
eale
s)A
sco
myc
ota
960.
20B
_C
ano
py_
450_
01_
13[A
Y38
2417
]Y
es(G
40)
Oid
iod
end
ron
ten
uis
sim
um
[AB
0157
87]
(On
ygen
ales
)A
sco
myc
ota
980.
07B
_C
ano
py_
450_
01_
14[A
Y38
2418
]Y
es(G
40)
Oid
iod
end
ron
ten
uis
sim
um
[AB
0157
87]
(On
ygen
ales
)A
sco
myc
ota
970.
33B
_C
ano
py_
450_
01_
18[A
Y38
2419
]Y
es(G
40)
Oid
iod
end
ron
ten
uis
sim
um
[AB
0157
87]
(On
ygen
ales
)A
sco
myc
ota
980.
13B
_C
ano
py_
450_
02_
02[A
Y38
2420
]Y
es(G
40)
Rh
izoc
ton
iaso
lan
i[D
8564
3](C
erat
ob
asid
iale
s)B
asid
iom
yco
ta95
0.06
B_
Can
op
y_45
0_02
_13
[AY
3824
21]
Yes
(G40
)H
ypom
yces
chry
sosp
erm
us
[AB
0273
39]
(Hyp
ocr
eale
s)A
sco
myc
ota
960.
94B
_C
ano
py_
450_
03_
02[A
Y38
2422
]Y
es(G
40)
Con
ner
sia
rils
ton
ii[A
F09
6174
](E
uro
tial
es)
Asc
om
yco
ta99
/99a
,c0.
14B
_C
ano
py_
450_
03_
05[A
Y38
2423
]Y
es(G
40)
Rac
ibor
skio
myc
eslo
ngi
seto
sum
[AY
0163
51]
(Ch
aeto
thyr
iale
s)A
sco
myc
ota
990.
14B
_C
ano
py_
450_
03_
07[A
Y38
2424
]Y
es(G
40)
Her
potr
ich
iaju
nip
eri
[U42
483]
(Ple
osp
ora
les)
Asc
om
yco
ta97
0.43
B_
Can
op
y_45
0_03
_14
[AY
3824
25]
No
Myc
osph
aere
lla
myc
opap
pi[U
4346
3](C
hae
toth
yria
les)
Asc
om
yco
ta98
0.29
B_
Can
op
y_75
0_01
_01
b[A
Y38
2426
]Y
es(G
20)
Inoc
ybe
geop
hyl
la[A
F28
7835
](C
ort
inar
iace
ae)
Bas
idio
myc
ota
970.
20B
_C
ano
py_
750_
01_
07b
[AY
3824
27]
Yes
(G40
)P
eziz
agr
iseo
rose
a[A
F13
3150
](P
eziz
ales
)A
sco
myc
ota
990.
40B
_C
ano
py_
750_
01_
10b
[AY
3824
28]
Yes
(G40
)A
nam
ylop
sora
pulc
her
rim
a[A
F11
9501
](A
gyri
ales
)A
sco
myc
ota
970.
20B
_C
ano
py_
750_
01_
15b
[AY
3824
29]
Yes
(G80
)P
ulv
inu
laar
cher
i[U
6201
2](P
eziz
ales
)A
sco
myc
ota
970.
20B
_C
ano
py_
750_
02_
13[A
Y38
2430
]Y
es(G
40)
Hyp
omyc
esch
ryso
sper
mu
s[M
8999
3](H
ypo
crea
les)
Asc
om
yco
ta96
0.33
B_
Can
op
y_75
0_02
_15
b[A
Y38
2431
]Y
es(G
40)
Oph
iost
oma
pili
feru
m[A
J243
294]
(Op
hio
sto
mat
ales
)A
sco
myc
ota
97/9
7c0.
44B
_C
ano
py_
750_
02_
19b
[AY
3824
32]
Yes
(G40
)H
ypom
yces
chry
sosp
erm
us
[M89
993]
(Hyp
ocr
eale
s)A
sco
myc
ota
950.
22B
_C
ano
py_
750_
03_
03b
[AY
3824
33]
Yes
(G80
)Sa
rcin
omyc
espe
tric
ola
[Y18
702]
(Ch
aeto
thyr
iale
s)A
sco
myc
ota
950.
14B
_C
ano
py_
750_
03_
04[A
Y38
2434
]Y
es(G
20)
Lac
cari
apu
mil
a[A
F28
7838
](A
gari
cale
s)B
asid
iom
yco
ta98
0.29
B_
Can
op
y_75
0_03
_08
b[A
Y38
2435
]Y
es(G
20)
Lac
cari
apu
mil
a[A
F28
7838
](A
gari
cale
s)B
asid
iom
yco
ta96
0.14
B_
Can
op
y_75
0_03
_11
[AY
3824
36]
No
Pez
iza
gris
eoro
sea
[AF
1331
50]
(Pez
izal
es)
Asc
om
yco
ta99
0.43
234 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT
B_
Can
op
y_90
0_01
_03
b[A
Y38
2437
]Y
es(G
40)
Ru
ssu
laco
mpa
cta
[AF
0265
82]
(Aga
rica
les)
Bas
idio
myc
ota
960.
25B
_C
ano
py_
900_
01_
16[A
Y38
2438
]Y
es(G
40)
Hyp
omyc
esch
ryso
sper
mu
s[A
B02
7339
](H
ypo
crea
les)
Asc
om
yco
ta97
0.13
B_
Can
op
y_90
0_01
_19
[AY
3824
39]
No
Ru
ssu
laco
mpa
cta
[AF
0265
82]
(Aga
rica
les)
Bas
idio
myc
ota
980.
63B
_C
ano
py_
900_
02_
02[A
Y38
2440
]N
oO
idio
den
dro
nte
nu
issi
mu
m[A
B01
5787
](O
nyg
enal
es)
Asc
om
yco
ta99
0.09
B_
Can
op
y_90
0_02
_04
[AY
3824
41]
No
Ch
aeto
miu
mel
atu
m[M
8325
7](S
ord
aria
les)
Asc
om
yco
ta98
0.18
B_
Can
op
y_90
0_02
_06
[AY
3824
42]
Yes
(G80
)T
hel
eph
ora
sp.
[AF
0266
27]
(Th
elep
ho
rale
s)B
asid
iom
yco
ta99
0.55
B_
Can
op
y_90
0_02
_10
[AY
3824
43]
No
Dar
kse
ptat
een
dop
hyt
eD
S16b
[AF
1681
67]
(Un
kno
wn
Asc
om
yco
ta98
0.09
B_
Can
op
y_90
0_02
_12
b[A
Y38
2444
]Y
es(G
80)
Pu
lvin
ula
arch
eri
[U62
012]
(Pez
izal
es)
Asc
om
yco
ta97
0.09
B_
Can
op
y_90
0_03
_09
[AY
3824
45]
Yes
(G40
)H
ypom
yces
chry
sosp
erm
us
[AB
0273
39]
(Hyp
ocr
eale
s)A
sco
myc
ota
940.
75B
_C
ano
py_
900_
03_
11b
[AY
3824
46]
Yes
(G80
)P
olyp
orol
etu
ssu
bliv
idu
s[A
F28
7840
](C
anth
arel
lale
s)B
asid
iom
yco
ta94
0.13
B_
Can
op
y_90
0_03
_17
[AY
3824
47]
Yes
(G40
)T
hel
eph
ora
sp.
[AF
0266
27]
(Th
elep
ho
rale
s)B
asid
iom
yco
ta98
0.13
S_C
ano
py_
300_
01_
01[A
Y38
2448
]Y
es(G
60)
Bu
lgar
iain
quin
ans
[AJ2
2436
2](H
elo
tial
es)
Asc
om
yco
ta98
0.11
S_C
ano
py_
300_
01_
07[A
Y38
2449
]N
oIn
ocyb
ege
oph
ylla
[AF
2878
35]
(Aga
rica
les)
Bas
idio
myc
ota
980.
89S_
Can
op
y_30
0_02
_01
b[A
Y38
2450
]Y
es(G
100)
Bu
lgar
iain
quin
ans
[AJ2
2436
2](H
elo
tial
es)
Asc
om
yco
ta95
0.14
S_C
ano
py_
300_
02_
11[A
Y38
2451
]Y
es(G
40)
Mor
tier
ella
chla
myd
ospo
ra[A
F15
7143
](M
uco
rale
s)Z
ygo
myc
ota
970.
29S_
Can
op
y_30
0_02
_13
b[A
Y38
2452
]Y
es(G
80)
Bu
lgar
iain
quin
ans
[AJ2
2436
2](H
elo
tial
es)
Asc
om
yco
ta96
0.14
S_C
ano
py_
300_
02_
14b
[AY
3824
53]
Yes
(G16
0)L
imn
oper
don
inca
rnat
um
[AF
4269
52]
(Ap
hyl
lop
ho
rale
s)B
asid
iom
yco
ta94
0.14
S_C
ano
py_
300_
02_
19b
[AY
3824
54]
Yes
(G10
0)P
anel
lus
sero
tin
us
[AF
0265
90]
(Aga
rica
les)
Bas
idio
myc
ota
940.
29S_
Can
op
y_30
0_03
_04
[AY
3824
55]
Yes
(G40
)Sp
izel
lom
yces
acu
min
atu
s[M
5975
9](S
piz
ello
myc
etal
es)
Ch
ytri
dio
myc
ota
970.
67S_
Can
op
y_30
0_03
_18
[AY
3824
56]
Yes
(G20
)L
acca
ria
pum
ila
[AF
2878
38]
(Aga
rica
les)
Bas
idio
myc
ota
970.
33S_
Can
op
y_45
0_01
_02
b[A
Y38
2457
]Y
es(G
100)
Bys
soas
cus
stri
atos
poru
s[A
B01
5776
](O
nyg
enal
es)
Asc
om
yco
ta94
0.25
S_C
ano
py_
450_
01_
07[A
Y38
2458
]Y
es(G
80)
En
tolo
ma
stri
ctiu
s[A
F28
7832
](A
gari
cale
s)B
asid
iom
yco
ta93
0.25
S_C
ano
py_
450_
01_
19[A
Y38
2459
]Y
es(G
60)
Bu
lgar
iain
quin
ans
[AJ2
2436
2](H
elo
tial
es)
Asc
om
yco
ta98
0.50
S_C
ano
py_
450_
02_
05[A
Y38
2460
]Y
es(G
60)
Oph
iost
oma
sten
ocer
as[M
8505
4](O
ph
iost
om
atal
es)
Asc
om
yco
ta95
1.00
S_C
ano
py_
750_
01_
10[A
Y38
2461
]Y
es(G
20)
Th
elep
hor
asp
.[A
F02
6627
](T
hel
eph
ora
les)
Bas
idio
myc
ota
97/9
4c0.
40S_
Can
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_11
[AY
3824
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Yes
(G20
)In
ocyb
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(Aga
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Bas
idio
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Yes
(G20
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[AF
2878
35]
(Aga
rica
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Bas
idio
myc
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980.
20S_
Can
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0_02
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[AY
3824
64]
Yes
(G40
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hen
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[M85
053]
(Op
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mat
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sco
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Yes
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(Hel
oti
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sco
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14S_
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[AY
3824
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Yes
(G20
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Bas
idio
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3824
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Cya
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[AF
0266
17]
(Nid
ula
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Bas
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myc
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970.
14S_
Can
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[AY
3824
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Yes
(G20
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[AF
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(Aga
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Bas
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Can
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3824
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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 and
Analysis. 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.
236 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT
A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT 237
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.
238 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT
A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT 239
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 Composition
Among 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.
240 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT
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
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 the
Glacier 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 Composition
Among 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.
A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT 243
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.
References
1. Altschul, SF, Madden, TL, Schaffer, AA, Zhang, J, Zhang, Z, Miller,DJ, Lipman, DJ (1997) Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs. Nucleic Acids Res25: 3389–3402
244 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT
2. Anderson, IC, Campbell, CD, Prosser, JI (2003) Potential bias offungal 18S rDNA and internal transcribed polymerase chainreaction primers for estimating fungal biodiversity in soil. EnvironMicrobiol 5: 36–47
3. Borneman, J, Hartin, RJ (2000) PCR primers that amplify fungalrRNA genes from environmental samples. Appl Environ Microbiol66: 4356–4360
4. Cazares, E (1992) Mycorrhizal fungi and their relationship to plantsuccession in subalpine habitats. PhD Thesis, Oregon StateUniversity
5. Chandler, DP, Fredrickson, JK, Brockman, FJ (1997) Effect of PCRtemplate concentration on the composition and distribution oftotal community rDNA clone libraries. Mol Ecol 6: 475–482
6. Cornelissen, JHC, Aerts, R, Cerabolini, MJA, Werger, MJA, vanderHeijden, MGA (2001) Carbon cycling traits of plant species arelinked with mycorrhizal strategy. Oecologia 129: 611–619
7. Dahlstrom, JL, Smith, JE, Weber, NS (2000) Mycorrhiza-likeinteraction by Morchella with species of the Pinaceae in pureculture synthesis. Mycorrhiza 9: 279–285
8. Danielson, RM (1984) Ectomycorrhiza formation by the opercu-late discomycete Sphaerosporella brunnea (Pezizales). Mycologia76: 454–461
9. Dickie, IA, Xu, B, Koide, RT (2002) Vertical niche differentiationof ectomycorrhizal hyphae in soil as shown by T-RFLP analysis.New Phytol 156: 527–535
10. Egger, KN, Paden, JW (1986) Biotrophic associations betweenlodgepole pine seedlings and post-fire ascomycetes (Pezizales) inmonoxenic culture. Can J Bot 64: 2719–2725
11. Egli, S, Ayer, F, Chatelain, F (1997) Die Beschreibung derDiversitat von Macromyceten. Erfahrungen aus pilzolologischenLangenzeitstudien im Pilzreservat La Chaneaz, FR. Mycol Helv 9:19–32
12. Farrelly, V, Rainey, FA, Stackebrandt, E (1995) Effect of genomesize and rrn gene copy number on PCR amplification of 16S rRNAgenes from a mixture of bacterial species. Appl Environ Microbiol91: 2798–2801
13. Ford, JE, McHeyzer-Williams, MG, Lieber, MR (1994) Chimericmolecules created by gene amplification interfere with the analysesof somatic hypermutation of murine immunoglobulin genes. Gene142: 279–283
14. Gardes, M, Bruns, TD (1993) ITS primers with enhancedspecificity for higher fungi and basidiomycetes: application toidentification of mycorrhizae and rusts. Mol Ecol 2: 113–118
15. Gardes, M, Bruns, TD (1996) ITS-RFLP matching for theidentification of fungi. In: Clapp, JP (Ed.) Methods in MolecularBiology, Vol. 50: Species Diagnostics Protocols: PCR and OtherNucleic Acid Methods. Humana Press Inc., Totowa, NJ, pp 177–186
16. Gargas, A, DePriest, PT, Taylor, JW (1995) Positions of multipleinsertion in SSU rDNA of lichen-forming fungi. Mol Biol Evol 12:208–218
17. Holst-Jensen, A, Vaage, M, Schumacher, T, Johansen, S (1999)Structural characteristics and possible horizontal transfer of groupI introns between closely related plant pathogenic fungi. Mol BiolEvol 16: 114–126
18. Hugenholtz, P, Goebel, BM, Pace, NR (1998) Impact of culture-independent studies on the emerging phylogenetic view ofbacterial diversity. J Bacteriol 180: 4765–4774
19. Jumpponen, A (2003) Soil fungal community assembly in aprimary successional glacier forefront ecosystem as inferred fromrDNA sequence analyses. New Phytol 158: 569–578
20. Jumpponen, A, Mattson, K, Trappe, JM, Ohtonen, R (1998)Effects of established willows on primary succession on LymanGlacier forefront: evidence for simultaneous canopy inhibition andsoil facilitation. Arct Alp Res 30: 31–39
21. Jumpponen, A, Trappe, JM, Cazares, E (1999) Ectomycorrhizal
fungi in Lyman Lake Basin: a comparison between primary andsecondary successional sites. Mycologia 91: 575–582
22. Jumpponen, A, Trappe, JM, Cazares, E (2002) Occurrence ofectomycorrhizal fungi on a receding glacier forefront. Mycorrhiza12: 43–49
23. Jumpponen, A, Vare, H, Mattson, KG, Ohtonen, R, Trappe, JM(1999) Characterization of Fsafe sites_ for pioneers in primarysuccession on recently deglaciated terrain. J Ecol 87: 98–105
24. Kopczynski, ED, Bateson, MM, Ward, DM (1994) Recognition ofchimeric small-subunit ribosomal DNAs composed from genesfrom uncultivated microorganisms. Appl Environ Microbiol 63:3614–3621
25. Kowalchuk, GA, Gerards, S, Woldendorp, JW (1997) Detectionand characterization of fungal infections of Ammophila arenaria(marram grass) roots by denaturing gradient gel electrophoresis.Appl Environ Microbiol 63: 3858–3865
26. Maidak, BL, Cole, JR, Jr, Parker, CT, Garrity, GM, Larsen, N, Li, B,Lilburn, TG, McCaughey, MJ, Olsen, GJ, Overbeek, R, Pramanik,TM, Schmidt, TM, Tiedje, JM, Woese, CR (1999) A new version ofthe RDP (Ribosomal Database Project). Nucleic Acids Res 27: 171–173
27. O’Dell, TE, Smith, JE, Castellano, M, Luoma, D (1996) Diversityand conservation of forest fungi. In: Pilz, D, Molina, R (Eds.)Managing Forest Ecosystems to Conserve Fungus Diversity andSustain Wild Mushroom Harvests. U.S. Forest Service GeneralTechnical Reports, PNW-GTR-317. US Department of Agricul-ture, Forest Service, Pacific Northwest Research Station. Portland,OR, pp 5–18
28. Pennanen, T, Paavolainen, L, Hantula, J (2001) Rapid PCR-basedmethod for the direct analysis of fungal communities in complexenvironmental samples. Soil Biol Biochem 33: 697–699
29. Perotto, S, Nepote-Fus, P, Saletta, L, Bandi, C, Young, JPW (2000)A diverse population of introns in the nuclear ribosomal genes ofericoid mycorrhizal fungi includes elements with sequence simi-larity to endonuclease-coding genes. Mol Biol Evol 17: 44–59
30. Sambrook, J (1989) Molecular Cloning—A Laboratory Manual,2nd ed. In: Fritsch, EF, Maniatis, T (Eds.) Cold Spring LaboratoryPress, New York
31. Schadt, CW, Martin, AW, Lipson, DA, Schmidt, SK (2003)Seasonal dynamics of previously unknown fungal lineages intundra soils. Science 301: 1359–1361
32. Smit, E, Leeflang, P, Glandorf, B, vanElsas, JD, Wernars, K (1999)Analysis of fungal diversity in the wheat rhizosphere by sequencingof cloned PCR-amplified genes encoding 18S rRNA and temper-ature gradient gel electrophoresis. Appl Environ Microbiol 65:2614–2621
33. Smit, E, Veenman, C, Baar, J (2003) Molecular analysis ofectomycorrhizal basidiomycete communities in Pinus sylvestris L.stand reveals long-term increased diversity after removal of litterand humus layers. FEMS Microbiol Ecol 45: 49–57
34. Smith, SE, Read, DJ (1997) Mycorrhizal Symbiosis. AcademicPress. London
35. Straatsma, G, Krisai-Greilhuber, I (2003) Assemblage structure,species richness, abundance, and distribution of fungal fruitbodies ina seven year plot-based survey near Vienna. Mycol Res 107: 632–640
36. Suzuki, MT, Giovannoni, SJ (1996) Bias caused by templateannealing in the amplification mixtures of 16S rRNA genes byPCR. Appl Environ Microbiol 62: 625–630
37. Swofford, DL (2001) PAUP, Phylogenetic Analysis Using Parsi-mony (and Other Methods), Version 4. Sinauer Associates.Sunderland, MA
38. Torsvik, V, Goksøyr, J, Daae, FL (1990) High diversity on DNA ofsoil bacteria. Appl Environ Microbiol 56: 782–787
39. Trowbridge, J, Jumpponen, A (2004) Fungal colonization of shrubwillow roots at the forefront of a receding glacier. Mycorrhiza 14:283–293
A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT 245
40. Vainio, EJ, Hantula, J (2000) Direct analysis of wood-inhabitingfungi using denaturing gradient gel electrophoresis of amplifiedribosomal DNA. Mycol Res 104: 927–936
41. Vandenkoornhuyse, P, Baldauf, SL, Leyval, C, Straczek, J, Young, JPW(2002) Extensive fungal diversity in plant roots. Science 295: 2051
42. vonWintzingerode, F, Gobel, UB, Stackebrandt, E (1997) Deter-mination of microbial diversity in environmental samples: pitfallsof PCR-based rRNA analysis. FEMS Microbiol Rev 21: 213–229
43. Wang, GCY, Wang, Y (1996) The frequency of chimericmolecules as a consequence of PCR co-amplification of 16SrRNA genes from different bacterial species. Microbiology 142:1107–1114
44. Zheng, D, Alm, EW, Stahl, DA, Raskin, L (1996) Characterizationof universal small-subunit rRNA hybridization probes for quanti-tative molecular microbial ecology studies. Appl Environ Micro-biol 62: 4504–4513
246 A. JUMPPONEN: FUNGI IN THE WILLOW CANOPY SOIL ON A GLACIER FOREFRONT