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Mycologia, 95(3), 2003. pp. 480--487.
©
200.~by The MycologicalSocietyof America, Lawrence, KS 66044-8897
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q S. Co,fellah.
Taxonomy of the Rhizopogon vinicolor species complex based on analysis of
ITS sequences and microsatellite loci
mushroom genera Suillus, Gomphidius and Chroogomphus and is thought to be derived from an epigeous
(possibly Suillu~qike) ancestor through loss of com-
Annette M. Kretzer ~
S. U.N.Y. Collegeof Environmental Science and
Forestry, Faculty of Environmental and Forest Biology,
350 lllick Hall, Syracuse, Ntno York 13210-2788
plex morphological characters, such as a stipe, vertically oriented tubes and forcible spore discharge
(Bruns et al 1989). Because of its reduced morphology, Rhizopogon is a taxonomically difficult genus and
the current taxonomy has largely been shaped by the
influential work of Smith and Zeller (1966). In addition to describing numerous new species, Smith
and Zeller (1966) also established a detailed subgeneric classification with two subgenera (Rhizopogonella 'and Rhizopogon) that comprise two sections (Rhizopogonella, ~trulatae) and four sections (Pd~izopogon,
Amylopogon, Fulviglebae, Villosuli), respectively. Species in the subgenus Rhizopogonella later have been
transferred to the genus Alpova (Trappe 1975), and
what we recognize as genus llhizopogon today is essentially Smith and Zeller's subgenus 1U~izopogon plus
Alpova olivaceotinclus (from section Rhizopogonella)
which has been transferred back to the genus Rhizopogon by Grubisha et al (2001); it remains to be
determined in future studies, however, if other species from Alpova section Rhizopogonella will have to
be transferred back to genus Rhizopogon as well.
Grubisha et al (2002) recently conducted an ITS
sequence-based phylogenetic study of the genus Rhizopogon. They found that Smith's and Ze[ler's (1966)
section Amylopogon was monophyletic, section Rhizopogon was polyphyletic and section Fulviglebae likely
would have been polyphyletic, if more species had
been included; those species from section Fulviglebae
that were included in the study formed a monophyletic clade nested within a paraphyletic section ViUosuli. Based on these results, Grubisha et al (2002)
have proposed a n u m b e r of changes to the subgeneric classification of Rhizopogon that include reinstitution of an e m e n d e d subgenus Rhizopogon, elevation of sections Amylopogon and Villosuli to subgenus
level and creation of two new subgenera, Roseoli and
Versicolores (for more details see Grubisha et al 2002).
Those species of section Fulviglebae that group with
former section Villosuli were transferred to the new
subgenus ViUosuli. Subgenus Villosuli is a strongly
supported, monophyletic clade that will be the focus
of the study presented below. Members of the subgenus Villosuli share a high level of host specificity
Daniel L. Luoma
.Department of Foresl Science, Oregon State University,
154 Peary Hall, Corvallis, Oregon 97331-7501
Randy MoIina
U.S.D.A. Forest Service, Pat@c Northwest Re.warch
Station, 2300 SWJefferson Way, Corvallis, Oregon
97331
.Joseph W. Spatafora
De4?artment of Botany and Plant Pathology, Oregon
State University, 2082 Cordley Hall, Corvallis, Oregon
97331-2902
Abstract: We are re-addressing species concepts in
the Rhizopogon vinicolor species complex (Boletales,
Basidiomycota) using sequence data from the internal-transcribed spacer (ITS) region of the nuclear ribosomal repeat, as well as genoLypic data from five
microsatellite loci. The R. vinicolor species complex
by our definition includes, but is not limited to, collections referred to as/L vinicolorSmith, R. diabolicus
Smith, R. ochraceisporus Smith, R. parvulus Smith or
tL vesiculosus Smith. Holo- a n d / o r paratype material
for the named species is included. AJmlyses of both
ITS sequences and microsatellite loci separate collections of the/7_ vinicolor species complex into two distinct clades or clusters, suggestive of two biological
species that subsequently are referred to as R, vinicolor sensu Kretzer et al and R. vesiculosus sensu
Kretzer et al. Choice of the latter names, as well as
morphological characters, are discussed.
Key words:
fimgal species concepts, internal
transcribed spacers, microsatellite markers, Rhizopo-
gon
INTRODUC~rlON
Rhiz~pogon is an ectomycorrhizal genus in the Boletales (Basidiomycota) that forms hypogeous, trufflelike fruit bodies. It is closely related to the epigeous
Accepted for publication February 5, 2003.
Corresponding autbor. E-mail: kretzcra@esf.cdu
480
KRETZER ET AI_,:RIIIZOPOGONVINICOLORSPECIES COMI'LEX
a n d are known to associate o n l y with Pseudotsuga spp.
(Massicotte et al 1994, M o l i n a et al 1997). A n o t h e r
d e t a i l e d p h y l o g e n e t i c s t u d y o f N o r t h A m e r i c a n collections o f s u b g e n u s Amylopogon recently has b e e n
p u b l i s h e d by B i d a r t o n d o a n d B r u n s (2002).
In a d d i t i o n to s u b g e n e r i c classification schemcs,
t h e r e has b e e n c o n t r o v e r s y a b o u t species delineations in Rhizopogon. M o s t species c u r r e n t l y recognized in N o r t h Aanerica d a t e b a c k to Zeller a n d
D o d g e (1918), Zeller (1941) a n d Smith a n d Zeller
(1966), with s u b s e q u e n t a d d i t i o n s by S m i t h (1966,
1968), Harrison a n d S m i t h (1968), T r a p p e a n d Guzm~n (1971), H o s f o r d (1975), Cfizares et al (1992)
a n d Allen et al (1999). However, m a n y species morphologically are very similar a n d d e s c r i b e d differences m i g h t r e p r e s e n t m o r p h o l o g i c a l a n d / o r o n t o g e netic variation within b i o l o g i c a l species. A n u m b e r o f
synonymies t h e r e f o r e have b e e n p r o p o s e d (Martin et
al 1998). G r u b i s h a et al (2002) also r e p o r t e d a n n m b e r o f irregularities with s p e c i e s d e l i n e a t i o n s a n d
identifications. O f p a r t i c u l a r i n t e r e s t in t h e c o n t e x t
of this study was the t'act t h a t several collections o f R.
vinicolor were n o t m o n o p h y l e t i c b u t a p p e a r e d to
form a paraphyletic g r a d e . C o l l e c t i o n s of R. diabolicus, R. ochraceisptnn~s a n d R. parvulus were derived
from within the p a r a p h y l c t i c R. vinicolor grade. We
collectively will refer to these taxa as the R. vinicolor
species c o m p l e x . R. vinicolor a n d R. ochraceisporus
can be d i f f e r e n t i a t e d p r i m a r i l y by subtle c o l o r differences in the reaction o f t h e p e r i d i n m to K O H as well
as by an olive-brown versus rusty-brown ( " r u s s e t " )
gleba at maturity, a c c o r d i n g to Smith a n d Zeller
(1966). In R. diabolicus, t h e m a t u r e g l e b a is "russet,"
as in the case o f R. ochraceisporus, b u t m a i n t a i n s a
b r i g h t r u s t y - c i n n a m o n c o l o r afi.er d r y i n g . R. parvulus
is d i s t i n g u i s h e d by s o m e i r r e g u l a r l y s h a p e d spores.
Finally, we also i n c l u d e 1~ vesiculosus in the R. vinicolor species c o m p l e x b e c a u s e S m i t h calls it "scarcely
d i s t i n g u i s h a b l e " f r o m R. vinicolor in the d r i e d stage;
when fresh, R. vesiculosus is d i s t i n g u i s h e d by yellowbrown inflated cells in t h e e p i c u t i s (Smith arid Zellcr
1966).
Despite known difficulties with species d e l i n e a t i o n s
in the R. vinicolor species c o m p l e x , we have c h o s e n
to work with R. vinicolor as a m o d e l taxon to study
the p o p u l a t i o n s t r u c t u r e o f h y p o g e o u s ba~sidiomyceres (false-truffles). Rhizopogon vinicolor is a pred o m i n a n t l y s p r i n g - f r u i t i n g s p e c i e s ( L u o m a et al
1991) that is very a b u n d a n t in the Pacific Northwest.
Most i m p o r t a n t , it f o r m s m o r p h o l o g i c a l l y distinct ect o m y c o r r h i z a e (EM) o n D o u g l a s fir (Pseudotsuga
menziesii) known as t u b e r c u l a t e EM (Zak 1971, Massicotte et al 1992). T u b e r c u l a t e EM consists o f tight
clusters of e c t o m y c o r r h i z a l r o o t s , e n c a s e d in a weft o f
h y p h a e that often is r e f e r r e d to as the " p e r i d i u m " .
481
T h e y l e n d themselves to p o p u l a t i o n g e n e t i c work because they are large (up to several cm across) a n d
are e n c o u n t e r e d m o r e f r e q u e n t l y than frtfit bodies,
m a k i n g t h e m relatively easy to sample in the field.
S p u r r e d by o u r interest in p o p u l a t i o n genetics of
this g r o u p , we arc re-addressing t a x o n o m i c issues a n d
species d e l i n e a t i o n s within the 17~ vinicolor species
c o m p l e x using b o t h ITS s e q u e n c e s a n d microsatellite
markers. C h a r a c t e r i z a t i o n o [ six p o l y m o r p h i c loci
with t r i n u c l e o t i d e r e p e a t motils from 1¢. vinicolorwas
r e p o r t e d e a r l i e r (Kretzer et al 2000). I n c l u d e d in this
study are scveral I°&iztrpogon spp. type collections
f r o m which DNA has n o t b e e n e x t r a c t e d and analyzed b e f o r e .
MA'FERIA[~SAND METHODS
Type collections were obtained from The University of
Michigan Fungus Collection. When collections consisted of
several sporocarps, one well-preserved sporocarp was chosen for DNA extraction and amplification. Tubcrculale EM
amdyzed in this study were collected in the spring of 1998
in two plots, one located at Mary's Peak (44 ° 31.86' N and
123° 32.86' W) in the Oregon Coast Range and the other
at Mill Creek (44° 12.17' N and 122° 13.95' W) in the
Oregon Cascade Range. Plots were within 40-80- and 4050-year-old second-growth forests, respectively, dominated
by Douglas fir (Pseudotsuga menziesii), weslern hemlock
( 7?~uga heterophylla) and western red cedar ( 7"huja plicata).
The plotswere 10 m x 10 m, and an II x I1 point grid
was established with 1 m point intervals. Soil cores (5 cm
wide and approx. 30 cm deep) were taken at each grid
point (121 cores per plot), and soils were sifted through a
W. S. Tyler Company 2 mm sieve. [n total, 30 independcnl
collections of tuberculate EM were ohlatined, 15 fl'om
Mary's Peak and 15 fl'om Mill Creek. Tuberculate EM collections were fi'eeze-dried be[bi'e molecular analysis and will
be identified as MPI-98 (Mary's Peak) and MCI-98 (Mill
Creek) ~mples, respectively. Finally, cultures of R. vinicolor
T20787 and T20874 were kindly provided by Dr. Ari Jumpponen (now at Famsas State University). Voucher collections
of sporocarps from which these cultures were derived arc
housed in the Fungus Collection of the Oregon State University Herbarium (OS()#61147 and OSC#61148).
Genomic DNA was extracted from dried material, as described in Kretzer et al (2000). The ITS region (comprising
ITS-I, the 5.8S rRNA gene, and ITS-If) was l'CR-amplificd
with primers ITSlf and ITS4. When amplification of the
entire region was unsuccessfid, only ITS-I was amplified with
primers ITSlf and ITS2. For primer sequences, see White
et al (1990) and Gardes and Bruns (1993). PCR reactions
contained 50 mM KCI, 10 mM Tris-HCl (pH 9.0 at 25 C),
2.5 mM MgC1._,,0.1% Triton ® X-100, 0.2 mM of each dNTI',
0.5 IxM of each primer, 50 U/m[. Taq DNA polymerase, and
empirical amounts of template DNA. Temperature cycling
conditions were: 2 rain at 94 C, fifllowed by 35--40 cycles of
45 s a t 9 4 C , 30 s a t 5 0 C , 60s + I s/cycle at 72 C a n d a
final extension of 10 rain at 72 C. PCR products were di-
482
MYCOi,O(;IA
gested with AluI and IIinfl to produce restriction fragmentlength polymorphisms (RFI.P's) according to Gardcs and
Bruns (1996). For sequencing purposes, PCR products were
purilied by electrophoresis on 1% agarose gels followed by
extraction with the QIAquick Gel Extraction Kit (Qiagen
Inc.). Nucleotide sequences were determined with a BigDye
Terminator sequencing kit and an ABI 373 automated sequencer. Sequencing Analysis and SeqEd sof~vare was used
to process raw data (PE Applied Biosystems).
PCR conditions for the amplification of microsatellite loci
as well as primer sequences have been reported before
(Kretzer et al 2000). Sizing of PCR product-~ was performed
by acrylamide gel electrophnresis on an ABI 377 auu~mated
sequencer using the "GS500 Tamra" internal size standard.
Band sizes were estimated with GeneScan software (PE Applied Biosystems). Because the mobility of DNA fragments
is inlluenced by base composition as well as the lluorescent
dyes used, size units translate only roughly into numbers of
hasepairs and commonly include fractions of a unit. Nevertheless, they are known to be highly reproducible with
standard deviations for fragments in the 200-300 bp range
commonly -0.15 (e.g., Haberl and Tautz 1999). Alleles
therefore were scored as different whenever a break in the
conli~uous distrihution of allele nletusurements was detectcal. In some cases, that meant thai. allele si~es might differ
hy as little as 0.5 size units. Small differences of this kind
can he explained by single basepair insertions a n d / o r substitutions that are known to occur in the microsatellite
Ilaqking regions (e.g., Orti et al 1997).
Phy]ogenefic analysis of ITS sequences was condt,cted in
PAUP* (Sinauer Associates Inc.). Alignment of ITS sequences was perJbrmed manually with the PAUP* editor
at~d a color fi:mt. Sequences immediately adjacent to the
priming sites of tile ~quencing primers were of poor quality in some of tile sequences, and the respective areas fi'om
the 18S, 5.8S and 28S genes were excluded fi'om the analysis. Fore" additional nucleotide positions were excluded because they appeared to be polymorphic, not only between
taxa hut also within individual collections as indicated by
double peaks in the sequencing chromatograms. Transitions and transversions were weighted equally (=1 step).
Alignmem gaps were treated as missing data, but parsimony-informative alignment gaps were recoded by changing
one character per alignment gap to a new state ' T ' (lbr
" i n d e l ' ) . The new state ' T ' was introduced in the same
position, when alignment gaps were identical in size and
positiml and in dil'l~rent positions otherwise. Change fl'om
any nt,clcotide to an "I" was weighted as one step. Parsi,nony analysis was perf~)rmed using the henristic search option with 10 random sequence additions. Bootstrap analysis
was based on 10 000 replicates using the fast stepwise addition option. Tile dataset minus the exclt, ded positions was
d(,posited at TreeBase.
Microsatellite data were anal~ed by neighbor-joining
(NJ) analysis of genotypic distances. Allele frequency-based
analyses were not possible because of the relatively small
munber of type collections available fbr many of the species
analyzed here and because the group is taxonomically so
difficul! that existiqg species concepts call be tested reliably
only cm type material. We used a simple allele sharing dis-
tance, which is [1 - (no. of shared alleles/2 X no. of loci
compared)] (Bowcock et al 1994). Distances based on stepwise mutation models were not suitable for our dataset, because at some loci alleles sizes were not strictly spaced in
multiples of three nucleotides as would be expected for loci
with trinucleotide repeats under a stepwise mutation model.
As already was discussed, irreg-ular spacing can be caused
by small insertions/deletions a n d / o r substitutions in the
microsatellite flanking regions, and was particularly pronounced when collections belonging to different species
were analyzed togethel:
RESULTS
A total of 14 new ITS s e q u e n c e s were d e t e r m i n e d in
this study a n d are listed in TABLE I with their GenBank accession n u m b e r s . Most new sequences were
derived from holo- a n d paratype material designated
by A l e x a n d e r Smith (Smith a n d Zeller 1966) a n d
made available to u s by T h e University of Michigan
F u n g u s Collection. Collection AHS68673, the holotype for R. parvulus, was n o t available to us tor DNA
extraction b e c a u s e of small collection size, a n d DNA
extraction and amplification from collection
AHS65267, the holotype for R. ochraceisporus, was unsuccessful. All t u b e r c u l a t e EM c o l l e c t i o n s were
screened before s e q u e n c i n g by ITS-RFLP's, using the
PCR primers I T S l f a n d ITS4 a n d the restriction enzymes Alul a n d HinfI. Two different fungal ITSRFLP p a t t e r n s were observed with the restriction enzyme Alul, a n d o n e e x e m p l a r y collection of each pattern was c h o s e n for s e q u e n c i n g (for details o n b a n d
sizes see discussion).
ITS s e q u e n c e s d e t e r m i n e d in this study were
a l i g n e d a n d a n a l y z e d t o g e t h e r with 18 s e q u e n c e s
from Rhizopogon s u b g e n u s Villosuli that were determ i n e d in a p r e v i o u s study ( G r u b i s h a et al 2002).
T h e c o m p l e t e ITS dataset i n c l u d e d 32 taxa a n d 542
characters, o f which 47 c h a r a c t e r s were p a r s i m o n y
i n f o r m a t i v e . P a r s i m o n y a n a l y s i s r e s u l t e d in 100
m o s t - p a r s i m o n i o u s trees that were 81 steps long.
O n e of the m o s t - p a r s i m o n i o u s trees is shown in FIG.
1A, a n d b r a n c h e s s n p p o r t e d by all 100 trees are
h i g h l i g h t e d in b o l d lines. N u m b e r s above b r a n c h e s
indicate s u p p o r t f r o m b o o t s t r a p analysis. Collections
of the R. vinicolorspecies c o m p l e x fall into two wellresolved a n d s t r o n g l y s u p p o r t e d clades that f u r t h e r
on we will refer to as R. vinicolor sensu Kretzer et al
a n d R. vesiculosus s e n s u Kretzer et al (for choice o f
clade names, see d i s c u s s i o n ) ; t h e r e was n o resolution b e t w e e n c o l l e c t i o n s within e i t h e r clade d u e to
little or n o s e q u e n c e variation. Both R. vinicolorsensn Kretzer et al a n d R. vesiculosus sensu Kretzer et
al clades i n c l u d e e x e m p l a r y s e q u e n c e s from tuberculate EM, i n d i c a t i n g that taxa in b o t h clades f o r m
KRETZER ET AL: RItIZOPOGON V1NICOLOR SPECIES COMPLEX
483
TAm.~;I. Collections from the R_ vinicolorspecies complex studied by either ITS sequence or microsatellite analysis or both.
Additional ITS sequences from Rhizopogon subgenus ViUosuli were taken from Grubisha et al. (2002)
Collection
No.
Type
IL diabolic~
IL diabolictL,
IL diabolicTL~
tL diabolicus
IL ochraceisporus
R. ochraceisporus
IL ochraceisporu.s
R. ochraceisporus
IL ochraceisporus
R. parvulus
IL v~ic~zlosus
IL vesiculosus
IL vinicolor
1L vinicolor
1~ vinicolor
R. vinicolor
FL vinicolor
tL vinicolor
R. vinicolor
R. vinicolor
R. vinicolor
R. vinicolor
R. vinicolor
IZ vinicolor
R. vinicolor
R. vinicolor
R. vinicolor
R. vinicolor
1.L vinicolor
Tuberculate EM
Tuberculate EM
Tuberculate EM
Tuberculate EM
AHS68404
AHS68424
AHS68489
AHS70305
AHS58928
AHS65963
AHS68395
T17916
T17944
AHS68364
AHS68040
AHS68041
Tru2195
AHS68071
AHS68092
AHS68177
AHS68179
AHS68189
AHS68227
AHS68576
AHS68579
AHS68595
AHS68602
AHS68690
AHS68705
T17899
T19383
T20787
T20874
MC1-98-H9 = type I
M C 1 - 9 8 - B=
0 type II
MC1-98-XX
MP1-98-XX
paratype
paratype
paratype
holotype
paratype
paratype
paratype
--paratype
holotype
paratype
holotype
paratype
paratype
paratype
paratype
paratype
paratype
paratype
paratype
paratype
paratype
paratype
paratype
-~
~
~
~
-~
--
Origin
Pend Oreille Co.,
Pend Oreille Co.,
Bonner Co., ID
Idaho Co., ID
Idaho Co., ID
Adams Co., ID
Pend Oreille Co.,
OR
OR
Bonner Co., ID
Bonnet Co., ID
Bonner Co., ID
Boise Co., ID
Bonner Co., ID
Pend Oreille Co.,
Bonner Co., ID
Bonner Co., ID
Bonner Co., ID
Pend Oreille Co.,
Bonner Co., ID
Bonner Co., ID
Bonner Co., ID
Bonner Co., ID
Bonner Co., 1D
Bonner Co., ID
Benton Co., OR
Coos Co., OR
Adams Co., ID
Bonner Co., ID
Lane Co., OR
I,ane Co., OR
Lane Co., OR
Benton Co., OR
ITS accession
WA
WA
WA
WA
WA
AF366386
Grubisha et
AF366387
AF366388"
AF366389
Grubisha et
AF366390"
Grubisha et
Grubisha et
Grubisha et
AF366391
AF366392
AF263929
AF418268
AF263930
-AF263931
---
ai., 2002
al., 2002
al., 2002
al., 2002
al., 2002
--AF263932
--Grubisha et al., 2002
Grubisha et al., 2002
Grubisha et al., 2002
-AF263938
AF263939
---
* Only the ITSI region was successfully amplified and sequenced; see methods.
ec'tomycorrhizae with t u b e r c u l a t e m o r p h o l o g y o n
Douglas fir.
We subsequently have scored microsatellite alleles
at five loci for 22 s p o r o c a r p v o u c h e r collections from
the R. vinicolor species c o m p l e x (again mostly type
material, see Fie,. 1B a n d TAm.E I) a n d 30 collections
of tuberculate EM from two plots described in the
methods. Characterization of six p o l y m o r p h i c microsatellite loci from R. vinicolor T20787 was r e p o r t e d
previously (Kretzer et al 2000). PCR p r i m e r s develo p e d for five of the six loci also were f o u n d to amplify
fragments of expected size from collections of R. vesiculosus sensu Kretzer et al; those were the primers
for loci Rv02, Rv15, Rv25, Rv46 a n d Rv53, which consequently were used in this study. Microsatellite markers revealed that the 30 collections of tubet:culate EM
m a d e in two 10 m by 10 m plots r e p r e s e n t e d a total
of 11 different multilocus g e n o t y p e s ( = genets); six
were genets of R. vinicolor sensu Kretzer et al, a n d
five were genets of R. vesiculosus sensu Kretzer et al
as i n d i c a t e d by ITS-RFLP analysis c o n d u c t e d earlier
(see first p a r a g r a p h of the results section). Details o n
the size a n d d i s t r i b u t i o n of genets across both plots
will be p u b l i s h e d elsewhere. To avoid redundancy,
only o n e representative tuberculate EM sample of
each g e n e t was i n c l u d e d in the cluster analysis pres e n t e d below; in addition, o n e g e n e t was omitted
from the analysis because of missing data at o n e locus.
Calculation of multilocus pairwise distances (see
m e t h o d s ) a n d n e i g h b o r - j o i n i n g analysis resulted in
the p h e n o g r a m shown in FIG. lB. It separates collections into two distinct clusters, which c o r r e s p o n d to
R. v i n i c o l o r s e n s u Kretzer et al and R. vesiculosus sensu Kretzer et al clades in Fie,. 1A. W i t h i n the two
clusters, however, again there is n o clear evidence for
484
MYCOLOGIA
A
R. vinicolor Tru2195 H
R. vinicolor AHS68092 P
R. vinicolor T17899
R . _ ~ .(~hvinicolor T20787
R. ochraceisporus AHS65963 P
raceisporus AHS58928 P
clade h
tuberculate
EM type I
R. vinicolor
89
R. diabolicus AHS68424 P
sensu Kretzer et al.
R. diabolicus AHS70305 H
R. diabolic'us AHS68404 P
R, parvulus AHS68364 P
R. ochraceisporus T17916
R. ochraceisporus T17944
R. ochraceisporus AHS68395 P
R. vinicolor AHS68071 P
R. vinicolor AHS68602 P
R. vinicolor AHS68179 P
clade I1:
100
R. vinicolor T19383
R. vesiculosus
tuberculate EM type II
sensu Kretzer et al.
R. vesjculosus AHS68040 H
R. vesiculosus AHS68041 P
R diabolicus AHS68489 P
R. parksii T17679
parksii T19446
Villosulus AHS59143
R. hawkerae AHS68417 P
ersii T17228
Ilescens T17681
R. colossus AHS49480 H
76
R, villosulus T19466
ITS nucleotide sequences:
one out of 100 mp trees.
1 step
B8
. ~ R
~
R
• sp. nov. T17466
B
Genotypic data from 5 microsatellite loci:
NJ phenogram.
o.
-
-
%.
o.o5
"
,~
~L
~
""
"
'°
A¢ f
Rvi T20787
'a~8o8,o
"--n:= =0 %~e.o
o
FIr,. I. (A) O n e of 100 most-parsimonious (mp) trees obtained from nucleotide sequences of the internal-transcribed
spacer region. Branches that are supported by all 100 m p trees are highlighted in bold; numbers above branches indicate
support from bootstrap analysis. The tree is unrooted. (B) Neighbor-joining phenogram based on genotypic data from five
m i c r o s a t e l l i t e loci. A b b r e v i a t i o n s are: R d = R_ diabolicus, Ro = R. ochraceisporus, R p = R. p a r v u l u s , Rve = R. vesiculosus,
Rvi = R. vinicolor, H = h o l o t y p e , P = p a r a t y p e . MC1 a n d M P I s a m p l e s a r e f r o m t u b e r c u l a t e m y c o r r h i z a e ; see Me t hods .
A r r o w i n d i c a t e s b r a n c h s e p a r a t i n g R. vinicolor s e n s u K r e t z e r e t al a n d H. vesicula~us s e n s u K r e t z e r e t al.
any sort o f sub-clustering that would correlate with
c u r r e n t taxonomy or otherwise indicate the presence
o f m u l t i p l e b i o l o g i c a l species. I n t e r n a l b r a n c h
lengths are longer in the R. vinicolor sensu Ki-etzer
et al cluster than in the R. vesiculosus sensu Kretzer
et al cluster. This simply reflects the fact that the microsatellite markers originally were developed for R_
vinicolor, and it is n o t u n c o m m o n for microsatellite
markers to be less p o l y m o r p h i c in nontarget species
than in target species (e.g., FitzSimmons et al 1995).
KRETZER ET AL: RHIZOPOGON VINICOLOR SPECIES COMPLEX
DISCUSSION
Both the ITS sequences and the microsatellite loci
analyzed in this study separated collections of t h e / L
vinicolor species complex into two distinct clades or
clusters indicative of two biological species. These
findings are in taxonomic conflict with the five or
more species names applied to this group. All species
in question were described by Alexander Smith
(Smith and Zeller 1966), who intended to document
all observed morphological diversity and to establish
narrow species concepts, potentially including ontogenetic or environmental variants as discrete species
(Smith and Thiers 1971). To address this possibility,
a re-evaluation of the main morphological characters
used by Smith to separate the species is warranted. A
key character in differentiating R. vinicolor sensu
Smith and R_ ochraceisporus is the color reaction of
the peridium to KOH. The differences, however, not
only are subtle but also are influenced by the developmental stage. Rhizopogon vinicolorsensu Smith and
R_ ochraceisporus also differ in the color of the gleba
at maturity, which is "dark olive-brown" in the former and "rusty cinnamon-brown" (="russet") in the
latter. Color characteristics of immature fruit bodies
are given in much detail for R. vinicolor but are absent from the description of R. ochraceisporus. We
think that R. ochraceisporus constitutes a mature color
variant of R. vinicolor. Similarly, R. diabolicus is characterized by a "russet" gleba at maturity that retains
a "bright rusty cinnamon" color when dried. We exa m i n e d type material for b o t h R. diabolicus
(AHS70305 = holo, AHS68404 = para) and R. ochraceisporus (AHS65267 = holo, AHS68395 = para) and
found the color of the dried glebas comparable if not
identical. R. diabolicus AHS68489 (=paratype) did
not have a "rusty c i n n a m o n " gleba, an observation"
that is consistent with its placement within the R. vesiculosus group by both ITS sequence and microsatellite data (FIG. 1). Mature R. diabolicus sporocarps
strongly resemble R. ochraceisporus ("ochraceous" to
"rusty brown" peridium staining "rusty brown" in
KOH and dark "olive" in FeSOt, "russet" gleba), according to Smith's description, while immature sporocarps bear much resemblance to R. vinicolar, including the white peridium with "vinaceous" flushes,
the "lilaceous" or "vinaceous" reaction to KOH and
the weak reaction to FeSO4. Finally, spores of all
three species (described by predominant size and extremes) are comparable in size (approx. 6.5-9.0 ×
3.0--4.5 i~m) (Smith and Zeller 1966). In conclusion,
we believe that R. vinicolor, IL ochraceisporus and R.
diabolicus are synonyms, an interpretation that also is
supported by neighbor-joining analysis of multilocus
genotypic distances (FIG. 1B). None of the three spe-
485
cies names has priority over the others, and we therefore chose R. vinicolor (sensu Kretzer et al) tor this
group, because it currendy is the most widely used
name and therefore provides the greatest taxonomic
continuity.
The taxonomic status of R. parvulus is more difficult to interpret, largely because only a single collection (AHS68364 = paratype) was available to us for
DNA extraction. This collection clusters tightly with
R. vinicolor sensu Kretzer et al in the ITS tree, a finding that is consistent with most morphological characters (Smith and Zeller 1966). In the microsatellitebased phenogram, however, it falls just slightly outside the R. vinicolor sensu Kretzer et al cluster. It is
different from R. vinicolor sensu Smith primarily in
that it has slightly larger (8-10 × 4-5 p.m) and often
irregularly shaped spores. Even if R. parvulus should
constitute an independent species or hybrid, iris unlikely to confound future population genetic work
because itis very rare.
In both the ITS tree and the microsatellite phenogram, a large n u m b e r of apparently misidentified
I-L vinicolor collections group strongly and distinctly
with R. diabolic'us AHS68489 (discussed above) and
with two collections of R. vesiculosus, including the
holotype (AHS68040). Because again there is no indication for sub-clustering within this group from either dataset, we shall refer to it collectively as R. vesiculosus sensu Kre~er et al (Fw,. 1A). As noted by
Snfith and Zeller (1966), R. vesiculosus is "scarcely
distinguishable" from R. vinicolor in the dried stage.
This high degree of similarity might explain the large
n u m b e r of misidentified collections, not only among
Smith's own "vinicolo¢' collections but also among
OSC herbarium material (data not shown). When
flesh, the color of the mature gleba distinguishes
both species, an observation that is consistent with
our own (see below). Unfortunately, we have not
been able to observe another diagnostic character for
R. vesiculosus that Smith and Zeller (1966) describe
as "yellow-brown (fresh) inflated ceils" found in the
epicutis that are "similar in size and shape to those
found in many species of section Villosuli". Although
these cells readily can be observed in dried material
from section Villosuli, Smith reports that they collapse in R_ vesiculosus upon drying and then are "not
so readily demonstrable" thus making use of this
character difficult to test. We have not undertaken
microscopic studies of this character in fresh material
but have been unable to observe the inflated cells
either in Smith's collections or in our own dried collections. In addition, R. vesiculosus sensu Kretzer et
al differs from Smith's description of R. vesiculosus
in having a wider range of spore lengths (approx. (5)
486
MYCOLOGIA
6--9 (10) ~m versus 6--6.5 I~m) and in growing under
Douglas fir rather than lodgepole pine.
Based on our extensive sampling of spring-fruiting
Rhizopogon species under Douglas fir around Mary's
Peak in the Oregon Coast Range, we find that the
most useful characters for differentiating sporocarps
of R. vinicolor sensu Kretzer et al and R. vesiculosus
sensu Kretzer et al are the colors of the fresh peridium and gleba. However, not all stages of developm e n t are equally distinctive and m o r e than one developmental stage often is needed to unambiguously
assign material to either species: Both species begin
with a white p e r i d i u m that b r u i s e s pinkish-red
(Smith's "vinaceous"), but only R. vinicoltrr sensu
Kretzer et al develops vivid yellow patches during early maturity. At maturity, both species are light yellowish brown (Smith's "ochraceous") and turn various
shades of brown from handling, the shades typically
reflecting the color of the mature gleba underneath
(see below). In R. vinicolor sensu Kretzer et al, the
gleba develops from white when immature through
pale yellow and pale greenish yellow-brown, to dark
greenish brown (Smith's "olive-brown") or brown or
more rarely reddish brown (Smith's "rusty cinnamon-brown" or "russet"). On the other hand, the
gleba of R. ve~siculosus sensu Kretzer et al appears to
develop from white to greenish brown (a stage that
apparently is short and was not noted in the type
d e s c r i p t i o n ) to d a r k b l a c k i s h - b r o w n ( d o m i n a n t
stage). Finally, we find that tuberculate EM of both
species also differ somewhat in morphology. The weft
of darkly pigmented hyphae that encases the clustered ectomycorrhizae is fluffy in R. vinicolor and attached to the ectomycorrhizae in such a way that it
molds to the ectomycorrhizae and cannot be peeled
back readily. In R. vesicmlosus, it is appressed but detached from the ectomycorrhizae such that it can be
peeled back in large patches. Although a more detailed and formalized description of morphological
differences at the EM level would be desirable, it was
not the goal of this study.
Ultimately, sporocarps and tuberculate EM of both
species most readily and reliably can be difterentiated
by ITS-RFLP's with the restriction enzyme AluI.
When PCR primers I T S l f and ITS4 are used, a single
undigested band of size 743 bp characterizes R. vesiculosus sensu Kretzer et al while three bands of sizes
419 bp, 224 bp and 97 bp are most typical of R. vinicolor sensu Kretzer et al (a C to T transition is occasionally observed in one of the restriction sites of
1~ vinicolor and results in only two bands of sizes 516
bp and 224 bp). Exact band sizes have been deduced
from nucleotide sequences.
Our data support the conclusion that, within our
sampling range (Oregon, Washington and Idaho),
the R. vinicolor species complex is composed of two
sympatrically distributed, phylogenetic species (indicated by ITS sequence analyses), which correlate with
biological species (indicated by microsatellite g e n o typic distances). In future population genetic work,
both species readily can be differentiated from either
r e p r o d u c t i v e or vegetative structures using ITSRFLP's as described.
From a taxonomic point of view, we have shown
that, in Rhizopogon subgenus ViUosuli, paratype material in many cases cannot be relied on to represent
the holotype. That puts us in a difficult position for
making taxonomic changes with respect to R. ochraceisporus because DNA from the holotype was not successfully amplified. Two lines of evidence, however,
lead us to believe that R. vinicolor and R. ochraceisporus should be regarded as synonyms: (i) Morphological characters as discussed above do not provide
strong evidence against synonymy. In particular, we
believe that morphology strongly supports synonymy
o f / L ochraceisporus and R. diabolicus (see above); the
latter in turn is supported by our molecular data to
be synonymous with R. vinicolor. (ii) The three paratype collections of R. ochraceisporus analyzed here actually did cluster within the same clade, suggesting
that paratype material for this particular species
might be more consistent than other species. In the
next section, we therefore formally propose synonymization of R. vinicolor, R. ochraceisporus a n d / L diabolicus. Although we believe that these changes best
reflect the current state of knowledge, this study does
not claim to be an exhaustive treatment of the R.
vinicolor species complex. The study was guided primarily by our desire to clarify species delineations in
the Pacific Northwest, which is the geographic center
of o u r o n g o i n g p o p u l a t i o n genetic work, and,
through incorporation of type material, to provide
valid names for the two species identified. The selection of taxa to be included in this study was based
largely on pre-existing molecular evidence that suggested particularly close affiliations of these t a x a with
I~ vinicolor (Grubisha et al 2002). A filture, more
comprehensive study should include vinicolor-like
"collections from a wider geographic range, as well as
type material from other morphologically similar species, such as R. cinnamoraeus Harrison and Smith, R.
subcinnamorneus Smith, R. olivaceoJitscus Smith, R.
pachyspora Hosford and others.
TAXONOMY
Rhizt~pogon vinicolor A. H. Smith.
Basionym: Rhizopogon vinicolorA. H. Smith. In Smith and
Zeller, 1966: Mem. N. Y. Bot. Gard. 14 (2): 67-69.
Synonyms: Rhizopogon ochraceisporus A. H. Smith. In
KRFTZER ET AI.: PuHIZOPO(;ONVINICOLOR SPE(:IES COMPI.EX
Smith and Zeller, 1966: Mere. N. Y. Bot. Gard. 14 (2): 62.
Rhizopogon diabolicu~$ A. H. Smith. In Smith and Zeller,
1966: Mere. N. E Bot. Gard. 14 (2): 64-65.
ACKNOWLEDGMENTS
The authors thank Caprice Rosato fi~r running countless
GeneScan gels, and Nancy Adair and Gi-Ho Sung tbr help
with sequencing. Susie Dunham, Jim Trappe and two anonymous reviewers have made valuable comments at different
stages of this mannscript's development. This work would
not have been possible without the type material housed at
the University of Michigan Fungus Collection and provided
to us by Robert Fogel and Patricia Rogers. The work was
financed byjoint venture agreement No. PNW-98-5113-1JVA
and was supported partially by co-op agreement No. 01-CA11261993-09~PNW, both from the U.S.D.A. Fores! Service
Pacific Northwest Research Station. Christopher Baycura
h~s helped with design of the cover image.
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•