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Mycologia
ISSN: 0027-5514 (Print) 1557-2536 (Online) Journal homepage: http://www.tandfonline.com/loi/umyc20
Phylogenetic assessment of global Suillus ITS
sequences supports morphologically defined
species and reveals synonymous and undescribed
taxa
Nhu H. Nguyen, Else C. Vellinga, Thomas D. Bruns & Peter G. Kennedy
To cite this article: Nhu H. Nguyen, Else C. Vellinga, Thomas D. Bruns & Peter G. Kennedy
(2016) Phylogenetic assessment of global Suillus ITS sequences supports morphologically defined
species and reveals synonymous and undescribed taxa, Mycologia, 108:6, 1216-1228
To link to this article: http://dx.doi.org/10.3852/16-106
Published online: 30 Jan 2017.
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Phylogenetic assessment of global Suillus ITS sequences supports morphologically
defined species and reveals synonymous and undescribed taxa
Nhu H. Nguyen
1
Department of Tropical Plant and Soil Sciences, University
of Hawai‘iatMa¯noa, Honolulu, Hawai‘i 96822
Else C. Vellinga
Thomas D. Bruns
Department of Plant and Microbial Biology, University of
California, Berkeley, California 94720
Peter G. Kennedy
Departments of Plant Biology and Ecology, Evolution,
and Behavior, University of Minnesota, Saint Paul,
Minnesota 55108
Abstract: The genus Suillus represents one of the most
recognizable groups of mushrooms in conifer forests
throughout the Northern Hemisphere. Although for
decades the genus has been relatively well defined mor-
phologically, previous molecular phylogenetic assess-
ments have provided important yet preliminary insights
into its evolutionary history. We present the first large-
scale phylogenetic study of the boundaries of each
species in the genus Suillus based on the most current
internal transcribed spacer (ITS) barcode sequences
available in public databases, as well as sequencing of
224 vouchered specimens and cultures, 15 of which
were type specimens from North America. We found
that species boundaries delimited by morphological
data are broadly congruent with those based on ITS
sequences. However, some species appear to have
been described several times under different names,
several species groups cannot be resolved by ITS
sequences alone, and undescribed taxa are apparent,
especially in Asia. Therefore, we elevated S. tomentosus
var. discolor to S. discolor; proposed synonymies of S.
neoalbidipes with S. glandulosipes,S. borealis with S. brun-
nescens,Boletus serotinus and B. solidipes with Suillus elben-
sis,S. lactifluus with S. granulatus,S. himalayensis with S.
americanus; and proposed usage of the names S. clinto-
nianus in the place of the North American S. grevillei,
S. weaverae for North American S. granulatus,S. ampli-
porus in the place of the North American S. cavipes,
and S. elbensis in place of the North American S. viscidus.
We showed that the majority of Suillus species have
strong affinities for particular host genera. Although
deep node support was low, geographic differentiation
was apparent, with species from North America,
Eurasia, and Asia often forming their own clades.
Collectively, this comprehensive genus-level phyloge-
netic integration of currently available Suillus ITS
molecular data and metadata will aid future taxonomic
and ecological work on an important group of ectomy-
corrhizal fungi.
Key words: Boletales, bolete, geography, molecular
phylogenetics, Suillaceae, suilloid, systematics, taxonomy
INTRODUCTION
The genus Suillus Gray comprises approximately 100
mushroom-forming species that are widely distributed
throughout the Northern Hemisphere. Suillus species
associate almost exclusively with members of the family
Pinaceae and exhibit strong patterns of host specificity
among different genera (Smith and Thiers 1964a,
Klofac 2013). They can also be found where hosts
have been planted and have subsequently invaded in
the Southern Hemisphere (Chapela et al. 2001, Dickie
et al. 2010, Walbert et al. 2010, Hayward et al. 2015), as
well as Pacific islands (Hynson et al. 2013). Given its
broad distribution and notable host specificity pat-
terns, the genus Suillus has emerged as a model system
for studying linkages among ectomycorrhizal fungal
diversity, ecosystem function, population genetics,
and host-symbiont coevolution (Dahlberg and Finlay
1999, Wu et al. 2000, Branco et al. 2015).
Although many species need taxonomic revisions
or descriptions, the genus Suillus is well circumscribed,
and its species boundaries are seldom disputed (Smith
and Thiers 1964a, 1971; Kretzer et al. 1996; Nguyen
et al. 2012; Klofac 2013). The most comprehensive
morphological work on the genus that focused on
North American taxa was published over 50 y ago
(Smith and Thiers 1964a). This work is still considered
authoritative and has long served as the backbone for
Suillus systematics. Recently, Klofac (2013) published
an overview of the nomenclatural history of the genus,
in which he extensively referenced the existing litera-
ture on Suillus, presented a list of taxa that he accept-
ed, and provided keys to those taxa based mostly
on morphological data. He accepted 94 species in
Suillus. Since then, S. indicus B. Verma & M.S. Reddy,
S. triacicularis B. Verma & M.S. Reddy, S. himalayensis
B. Verma & M.S. Reddy, S. marginielevatus S. Sarwar,
Khalid & Dentinger, S. lariciphilus K. Das, D. Chakr.,
K.P.D. Latha & Cotter, and S. adhikarii K. Das,
D. Chakr. & Cotter have been described from Pakistan
and the Himalayan regions of India and Nepal (Verma
and Reddy 2014a, 2014b, 2015; Adamcˇík et al. 2015;
Submitted 12 May 2016; accepted for publication 31 Aug 2016.
1
Corresponding author. E-mail: xerantheum@gmail.com
Mycologia, 108(6), 2016, pp. 1216–1228. DOI: 10.3852/16-106
#2016 by The Mycological Society of America, Lawrence, KS 66044-8897
1216
Das et al. 2015) along with the very unusual S. foetidus
Y. Li & L.L. Qi from northeastern China (Qi 2016).
The description of many new species in a relatively
short time suggests the possibility of a number of addi-
tional undescribed species in currently understudied
areas.
Smith and Thiers (1964a) divided the genus Suillus
into three sections, Paragyrodon,Boletinus, and Suillus,
although they stated that these sections were recog-
nized “partly for historical reasons and for conve-
nience in dealing with the species in contrasting
groups even if these are based mainly on a single char-
acter”(Smith and Thiers 1964a). Section Paragyrodon
was monotypic and included only Paragyrodon sphaeros-
porus (Peck) Singer, a Quercus-associated species that
Singer (1942) had previously placed in its own genus.
An early mitochondrial mapping study subsequently
confirmed that it did not belong in Suillus (Bruns
and Palmer 1989). Section Boletinus, which was recog-
nized as a distinct genus (Singer 1975), was included
in Suillus and described as having either stipes with a
fibrillose zone or a distinct membranous annulus or a
distinctly fibrillose-squamulose pileus. Section Suillus
was further broken down into series Hirtellini (mis-
spelled as Hirtellinii) and Suilli. Species in series Hirtel-
lini have a glandular-dotted stipe, although the glands
often appear as smears rather than dots. The imma-
ture (button) pileus is covered by a tomentum that in
fully expanded mature specimens aggregates into tufts
or becomes glabrous in some species. For species of
series Suilli, the veil forms a well developed annulus
or it adheres to the pileus margin when it breaks
open, leaving a “cottony roll”on the pileus margin;
young specimens may have a distinct false veil (i.e.
one not attached to the stipe but initially almost cover-
ing the tube cavity). Species with dark brown spores
were excluded by Smith and Thiers (1964a) from
Suillus and treated as members of a separate genus
Fuscoboletinus Pomerl. & A.H. Sm., but mitochondrial
mapping results disagreed with that segregation
(Bruns and Palmer 1989).
The ability to sequence DNA started the modern era
of molecular phylogenetic studies, with the first com-
prehensive molecular sequence-based work on Suillus
conducted by Kretzer et al. (1996). Although that
study was published 20 y ago and included only a limit-
ed sampling of species in the genus, it still represents
the most comprehensive molecular phylogenetic study
to date. It confirmed that Fuscoboletinus was part of
Suillus and was neither a distinct nor a natural genus.
Various other studies have used molecular phyloge-
netics to identify and support morphological taxono-
my of species in their regional mycota, although
species sampling across the genus was limited (Sarwar
et al. 2011; Min et al. 2014; Sarwar and Khalid 2014;
Verma and Reddy 2014a, 2015; Qi et al. 2016).
Sequencing of holotype specimens has been shown to
be essential in assigning correct names to phylogenetic
clades and delimiting species. For example, Nguyen
et al. (2012) showed that by sequencing holotype
specimens S. ponderosus A.H. Sm. & Thiers and S. caer-
ulescens A.H. Sm. & Thiers each contained a variety
of the species S. imitatus A.H. Sm. & Thiers, whereas
Bruns et al. (2009) produced sequences from holo-
types to indicate that S. quiescens T.D. Bruns & Vellinga
indeed had not been described before. The widespread
application of DNA sequencing has also produced
numerous sequences from environmental samples
(i.e. either ectomycorrhizal root tips or soil). These
samples cannot be accurately assigned to species with-
out the use of molecular phylogenetics, so placing
these data in the context of a comprehensive global
specimen-based phylogeny is necessary to assign taxo-
nomic names properly (Bonito et al. 2010; Nguyen
et al. 2012, 2013).
Molecular data has been useful in delineating and
identifying Suillus species when macroscopic charac-
ters are plastic or hard to interpret (Nguyen et al.
2012). Most species distinctions are based on appear-
ance of fresh collections, host associations, and geogra-
phy. Microscopic differences in cystidial position and
clustering, pileipellis structure, or spore color distin-
guish some major groups within the genus. However,
distinctions among closely related species are generally
limited to subtle differences in spore sizes that are not
based on extensive measurements of multiple collec-
tions and rarely hold up to scrutiny. Although most
species are distinct when young, many older specimens
look similar to each other and their appearances
are affected by environmental factors such as heavy
rain, drying winds, or sun exposure that can lead to
misidentifications. Developmental changes in color
are also common. For example, some species always
have white basidiocarps, whereas others start out white
but darken to various colors, while a third group is
made up of white variants of typically colored species.
These variations often make identification based solely
on morphology difficult to impossible. One frequently
used aid to morphological identification is host associ-
ation. In native environments, Suillus species co-occur
with their hosts and are thus restrained by host bioge-
ography. However, a few species have naturally broad
geographic distribution and others have been intro-
duced with novel host species in many regions where
they were not native (Vellinga et al. 2009), further
complicating correct identification.
Along with helping to address morphological and
geographical ambiguities, DNA sequence data of Suil-
lus specimens have also been used to examine patterns
of host-symbiont co-evolution (Kretzer et al. 1996,
NGUYEN ET AL.: GLOBAL SUILLUS ITS PHYLOGENY 1217
Wu et al. 2000). Based on field reports of host associa-
tion, Kretzer et al. (1996) showed that Suillus clades
(although some weakly supported) were associated
with Larix,Pseudotsuga, or with different subgenera of
Pinus, subgenus Strobus (herafter referred to as soft
pines), or subgenus Pinus (hereafter referred to as
hard pines), as well as one species associating with
Quercus. Wu et al. (2000) focused on a small group of
the Pinus-associated species and found evidence of
host specificity for two species groups from North
America and China.
To further facilitate taxonomic, ecological, and evo-
lutionary work on Suillus, we assembled all the available
sequences of the nuclear rDNA internal transcribed
spacers (ITS) for this genus and provided additional
sequences for type material and vouchered specimens.
We analyzed these data phylogenetically and used
those analyses to discuss species boundaries and trends
of host associations across the entire genus, with an
emphasis on North American taxa. Whenever possible,
we used holotype data to inform our assessments of
taxonomic categorization, provided novel taxonomic
treatments for various members of the genus, and
pointed to species and species complexes where more
taxonomic work is still needed.
MATERIALS AND METHODS
We sequenced freshly collected basidiocarp vouchers, exist-
ing collections deposited in herbaria, and cultures for this
study. Loans of types and specimens deemed important
were sent from DBG, MICH, and SFSU. Many of the fresh
specimens from Colorado were contributed by the Pike’s
Peak Mycological Society and the Colorado Mycological
Society on targeted forays to collect Suillus. Other specimens
were contributed by citizen mycologists around the country.
Specimens collected for this project were deposited in the
University of California (UC) Herbarium (SUPPLEMENTARY
TABLE I). Herbarium abbreviations are according to Thiers
(2016 continuously updated).
DNA was extracted from dried or cultured tissue by using a
modified Sigma Extract-N-Amp kit (Sigma Aldrich, St Louis,
Missouri) for fresh or recently collected specimens. For
those that have been stored under herbarium conditions,
especially type specimens, DNA was extracted with a standard
2% CTAB buffer with 2% polyvinylpyrrolidone (PVP, mw
40 000) and chloroform, precipitated with isopropanol,
followed by cleanup with ethanol. Standard PCR conditions
were followed using the primers ITS1F (Gardes and Bruns
1993) and ITS4 (White et al. 1990) initially for all specimens.
For those with degraded DNA (due to storage over decades),
we used a combination of ITS1F and ITS2, ITS3, and ITS4
(Bruns et al. 2009) primers to individually amplify the nuc
rDNA ITS1-5.8S-ITS2 repeat. Amplified products were sequ‐
enced using standard BigDyeHTerminator 3.1 Cycle Sequen‐
cing Kit (Applied Biosystems, Foster City, California).
Each automated sequence was manually interpreted and
corrected for ambiguous base calling. The sequences generated
in this study were deposited in GenBank under acces‐
sion Nos. KX213693–KX213835 and KX230573–KX230654
(SUPPLEMENTARY TABLE I). All ITS sequences of Suillus
from GenBank or those that were released from UNITE
(https://unite.ut.ee) were downloaded as of 1 Nov 2015.
To find environmental sequences not classified under Suillus,
representative sequences of vouchered species were matched
to GenBank sequences with at least a 93% sequence similari-
ty using BLAST. Sequences shorter than 150 bp were dis-
carded, except in the case of a few species where these
were the only sequences available. Locality and host meta-
data for each sequence were downloaded from GenBank
using a custom Perl script.
All sequences were initially aligned using MAFFT and
optimized manually: An initial phylogenetic tree was pro-
duced that allowed the sequences to be reorganized by
clades, and sequences within each clade were realigned man-
ually when necessary. Beginnings and ends of sequences were
coded as missing when not available. Phylogenetic inference
was made using the GTRGAMMA model in RAXML (Stamata-
kis et al. 2005). Maximum likelihood bootstrap support values
were calculated from 1000 replications. Midpoint rooting was
used for the tree, because the ITS sequences of taxa outside
of Suillus (e.g. Truncocollumella,Rhizopogon) could not be
aligned with confidence so no outgroup could be assigned.
In cases where well supported taxa nested inside of a group
of loosely defined taxa (S. weaverae,S. subaureus,S. decipiens,
/suilloides clade), we used a subset of the main align‐
ment for each taxon, realigned as necessary, and re-analyzed
those sequences using Bayesian Inference implemented in
MrBayes (Ronquist et al. 2012). Each subset of data was run
for at least 1 000 000 generations until the trees converged
and average split frequency was ,0.01. The sequence align-
ment for this project is available under TreeBase study No.
S19256. Clade notations are based on Moncalvo et al. (2002),
i.e. a non-capitalized name preceded by a forward slash.
RESULTS
We successfully obtained ITS sequences for 15 type
specimens and 209 sequences from vouchered speci-
mens and cultures. Combined with environmental
sequences, the final alignment contained 1029 sequ‐
ences. The deeper phylogenetic nodes of the tree
could not be reconstructed with confidence, but the
terminal branches of the tree grouped into well
defined and well supported clades (FIG. 1). Due to
the large size of the tree, we have divided it into three
major groups to facilitate interpretation and discus-
sion: the Spectabilis group, the Granulatus group,
and the Tomentosus group (this latter group includes
the morphologically distinct S. bovinus). We readily
acknowledge that these groups are not statistically sup-
ported but fit together using a combination of the ITS
sequences and the morphological data reflecting
Smith and Thiers’s (1964a) sect. Boletinus plus Fuscobo-
letinus (<Spectabilis group), series Suilli (<Granulatus
group), and series Hirtellini (<Tomentosus group).
1218 MYCOLOGIA
The three groups contain multiple clades that typi-
cally reflect morphological species, but each group
also contains clades that require further taxonomic
work. The Spectabilis group contains a suite of species
that associate exclusively with hosts other than Pinus
(i.e. Larix,Pseudotsuga, and possibly Picea). It contains
two clades of Larix-associated species and one clade
of Pseudotsuga-associated species; the /grevillei and
/lakei clades are well supported (.97%), whereas
the /spectabilis clade is not. The species status of
the members of the /lakei and /spectabilis clades is
taxonomically stable, but multiple entities appear that
have been subsumed under the same name for the
/grevillei clade. The Granulatus group represents the
most diverse group of Suillus in this study. Many of
the species have high statistical phylogenetic support,
but some clades contain multiple taxa that could not
be resolved (/placidus and /flavidus clades) due to
the lack of sequences from type specimens to anchor
epithets. Other clades (e.g. /acidus, /glandulosipes,
/albivelatus, and /luteus clades) were well resolved
but each clade contained multiple holotypes. Com-
pared to the other two groups, the Tomentosus group
was held together with moderately high bootstrap
support (89%), even though the data is based on a
single gene region. Many of the recognized species
within the Tomentosus group clustered into clades,
although only a few were well supported.
Species for which multiple names are applied.—We
identified three species that are currently accepted
under multiple names with similar if not identical ITS
sequences. These are species in the /glandulosipes
clade, the /albivelatus clade, and the /luteus clade.
The /glandulosipes clade contains two species, S. glan-
dulosipes Thiers & A.H. Sm. and S. neoalbidipes M.E.
Palm & E.L. Stewart,that have very similar morpholog-
ical characteristics. In particular, these species share
an abundant cottony veil that covers the tubes in
young specimens but differ in that S. glandulosipes is
thought to be a predominantly western species and
to have more conspicuous clusters of caulocystidia
(Palm and Stewart 1984). The holotype specimens of
both species fall within one well defined clade with
100% bootstrap support. Therefore, we consider these
two species as one, with S. neoalbidipes as a synomym of
S. glandulosipes.
The /albivelatus clade includes sequences under
four names: S. albivelatus A.H. Sm., Thiers & O.K.
Mill., S. pseudoalbivelatus B. Ortiz & Lodge, S. wasatchi-
cus Thiers and S. volcanalis Thiers. Suillus albivelatus
was described from the Priest River area in northern
Idaho (Smith et al. 1965); S. wasatchicus was described
from the Wasatch Range of Utah (Thiers 1976), and
S. volcanalis from Lassen County in Northern
California (Thiers 1967). Suillus pseudoalbivelatus is
the only species in this clade not occurring in western
North America; it is reported only from the Dominican
Republic (Ortiz-Santana et al. 2007). All four holotypes
are included in our phylogeny, and because no strong
bootstrap support holds this clade together, we refrain
from synonymizing these names. Should better data
result in a well supported clade, the name S. albivelatus
would supersede the other names.
The /luteus clade, although not statistically sup-
ported, holds all the specimens that have been identi-
fied as S. luteus (L.) Roussel, a common species that
occurs with P. sylvestris in Europe and elsewhere where
P. sylvestris and other Pinus species have been intro-
duced. Two species thought of as North American
endemics, S. borealis A.H. Sm. Thiers & O.K. Mill. and
S. brunnescens A.H. Sm. & Thiers, nested within this
clade. The name S. borealis A.H. Sm., Thiers & O.K.
Mill. is illegitimate because of S. borealis (Peck) Kuntze
described from Labrador, Canada. We are unable to
confirm the identity of S. borealis (Peck) Kuntze, but
the current concept of S. borealis is based on S. borealis
A.H. Sm., Thiers & O.K. Mill. (Smith et al. 1965). This
species has a strong persistent veil, similar to S. luteus,
whereas S. brunnescens has a thin and membranous veil
that disappears with age. The change in color from white
to brown and a weak veil is used to distinguish S. brunnes-
cens from the others, but our collections showed inter‐
mediate characteristics with nearly white basidiocarps
that mature to brown with persistent veils. The related-
ness of S. luteus,S. borealis, and S. brunnescens had not
escaped the attention of Smith et al. (1965), who
suggested that these species belong to stirps Luteus.
However, we showed that the holotype ITS sequences
of S. borealis and S. brunnescens are identical to each
other. Therefore, we consider S. borealis a synonym
of S. brunnescens due to their plastic morphological
characters as well as identical ITS sequences. However,
currently we do not consider this North American
species to be conspecific with S. luteus because of its
occurrence with soft pines (see DISCUSSION). Although
we were not able to obtain holotype sequences for all
the key members from several other clades, we think
it is useful to discuss their potential taxonomic status
below.
The /salmonicolor clade contained specimens that
were identified as S. subluteus (Peck) Snell, S. cothurna-
tus Singer, and S. salmonicolor (Frost) Halling. This
group of species has thin stipes, an annulus that is
persistent, thick, often baggy and slimy, and salmon-
colored flesh in the stipe. Halling (1983) considered
S. subluteus and S. pinorigidus Snell & E.A. Dick syno-
nyms of S. salmonicolor and Smith and Thiers (1964a)
considered S. cothurnatus as a southern USA variant
of S. subluteus. Currently, those who have collected
NGUYEN ET AL.: GLOBAL SUILLUS ITS PHYLOGENY 1219
FIG. 1. ITS maximum likelihood tree of Suillus specimens and environmental sequences. Numbers on branches are
maximum likelihood bootstrap values. Only bootstrap values .80% are shown. Each terminal leaf is annotated as accession
number –species/sequence name –host –locality when they are available. Host abbreviations: Q. 5Quercus,P.5Pinus,
Ps. 5Pseudotusga,L.5Larix. Clade names are preceded by a forward slash.
1220 MYCOLOGIA
NGUYEN ET AL.: GLOBAL SUILLUS ITS PHYLOGENY 1221
and identified Suillus suspect that S. cothurnatus and
S. salmonicolor are synonymous, but there are no se‐
quences available to support this hypothesis and the
type collections for both of these species were made
in the 19th century. If these two species were shown
to be the same taxon, S. salmonicolor would be the
oldest available name.
The /acidus clade contained sequences from the
holotypes of S. lutescens A.H. Sm. & Thiers and S. suboli-
vaceus A.H. Sm. & Thiers. We were unable to obtain a
sequence for the type of S. acidus (Peck) Singer, but
all specimens of what we identified as S. acidus based
on morphology fit into this clade. (Smith and Thiers
1964a) thought that S. lutescens was closely related
to S. acidus, and our sequence data support that rela-
tionship. To confirm the correct name for this
clade, the holotype sequences for S. acidus will need to
be obtained. Given the age of the holotype (before
1905), epitypification may be the best approach.
A similar problem exists within the /placidus and
/flavidus clades, with no holotype sequences (except
S. helenae Thiers & A.H. Sm. in the /flavidus clade)
available to anchor species epiphets. The /placidus
clade was well supported (99%) and contained speci-
mens from both North America and Europe; names
such as S. placidus (Bonord.) Singer, S. subalpinus
M.M. Moser, S. punctatipes (Snell & E.A. Dick) Singer,
and S. anomalus T.J. Baroni, Largent & Thiers have
all been applied to sequences and specimens in this
clade. Suillus placidus has a white pileus that does
not change color as the specimen ages; the species
was described from Germany but is strictly associated
with introduced North American P. strobus (Bonorden
1861). It is thought that this species was introduced
to Europe with its North American P. strobus host. Suil-
lus subalpinus is a high-altitude species that associates
with soft pines. Suillus anomalus has pores that contin-
ue down the stipe, and although it morphologically
appears to be distinct, its ITS sequence was not. The
North American species generally called S. granulatus
also belongs in this clade (see The case of Suillus granu-
latus) and forms a well supported taxon. The /flavidus
clade contained specimens with names such as
S. umbonatus E.A. Dick & Snell, S. megaporinus Snell &
E.A. Dick, S. flavidus (Fr.: Fr.) J. Presl, and S. helenae.
Members of this clade exhibit the largest diameter of
pores in the genus (up to 1 cm). The name S. flavidus
has been used for European specimens, whereas
the name S. umbonatus is applied to the western North
American specimens, and S. megaporinus is applied
only to specimens collected in the Sierra Nevada of
California. The holotype sequence of S. helenae (named
for Helen Smith, Alexander Smith’s wife) also fits
within the group and was described from a single col-
lection in Oregon. If holotypes of the aforementioned
species fit in this clade, S. flavidus is the oldest name
and should be used for this species. Finally, Suillus
subaureus, the lone angiosperm-associated species in
this genus, was well supported (99%) and although
nested, is separate from the rest of the sequences in
the /flavidus clade.
Suillus americanus is the most naturally widespread
Suillus species in the Northern Hemisphere. It is the
only species found in North America, Eastern Europe
through Pakistan, India, Russia, and Japan. Previously
it was known under two names, S. americanus and
S. sibiricus. Klofac (2013) subsumed these species under
S. americanus. The lack of discernable phylogenetic
structure for the /americanus clade agrees with this
taxonomic change. The species currently has two vari-
eties (var. americanus, var. reticulipes) and three formae
(f. americanus,f.helveticus, and f. sibiricus). These with-
in-species morphological variants are expected for
such a widespread distribution. These forms are not
easily distinguishable with the ITS, but inclusion of
data from other genes may allow their separation. Suil-
lus himalayensis B. Verma & M.S. Reddy was described
and accepted as a species even though the two repre-
sentative sequences did not form a well supported
clade, and they were nested within S. americanus
(Verma and Reddy 2014b). Our results from a much
larger sampling of the species (FIG. 1B) show that the
two sequences of S. himalayensis fit within S. americanus,
and we consider the morphological differences as var-
iations within S. americanus. We therefore consider S.
himalayensis a synonym of S. americanus. Unfortunately,
the epithet americanus takes precedence because
the epithet sibiricus more clearly speaks to its distribu-
tion in higher latitudes or altitudes in the Northern
Hemisphere.
Undescribed taxa and new combinations.—We think that
the set of species discussed below has strong enough
phylogenetic signal in our analysis to be considered
new species. However, we note that in some cases,
these species may have already been described under
Boletus, and studies must be conducted to transfer
those species to Suillus.
The /viscidus clade contains the currently accepted
name Suillus viscidus (L.) Roussel, which has been
applied broadly to three distinct clades of mushrooms
occurring in North America, Europe, and Asia. The
name S. viscidus, described as Boletus viscidus L. from
Europe is best applied to the European clade, whereas
the name Suillus elbensis (Peck) Kuntze best fits the
North American taxon. Other North American names
that would fit this taxon are Boletus serotinus Frost and
Boletus solidipes Peck, but both names are younger
than Boletus elbensis Peck.The clade from eastern Asia
does not appear to have been critically compared to
1222 MYCOLOGIA
related European and North American taxa and may
require a new name. The consortium of sequences
that we call the /bresadolae “clade”is loosely held
together with specimens from Europe and India. Fur-
ther studies and collection of Indian specimens will
help determine whether they should be segregated
from the European S. bresadolae.
Suillus grevillei (Klotzsch) Singer was monophyletic
(with an 87% bootstrap support) although the clade
was split between specimens from North America/
Eurasia and those strictly from Europe. Korhonen et al.
(1993) suggested that this group contained two spe-
cies, where S. grevillei occurring mostly in Europe with
lemon-yellow pileus, hyaline hyphae in the pileipellis,
and smaller spores compared to S. clintonianus (Peck)
Kuntze occurring mostly from Siberia to eastern Asia
and in North America with dark reddish-brown
pileus, mainly encrusted hyphae in the pileipellis and
larger spores. Phylogenetic distance between these
two groups, combined with morphological distinction,
provide convincing evidence to recognize them as
separate species. Therefore, we suggest that the name
S. grevillei be reserved for the European taxon and
S. clintonianus be applied to the North American/
Asian taxon. The occurrence of S. clintonianus in
North America and Asia observed by Korhonen et al.
(1993) based on morphology and confirmed here
with molecular data hints at a biogeographic pattern
that may be linked to the distribution of their Larix
hosts with which they fruit abundantly.
The /cavipes clade contained species that are mostly
well defined morphologically and well supported by
phylogeny (FIG. 1A). Suillus cavipes (Opat.) A.H. Sm.
& Thiers itself may be separated into two species, one
from Eurasia and the other from North America. It
was originally described as Boletus cavipes Opat. from
subalpine habitats in Styria, Austria (Opatowski 1836).
Therefore, the name is best applied to the European/
Asian clade. The name Suillus ampliporus (Peck) Kuntze
is available for the North American taxon. Index
Fungorum (www.indexfungorum.org) erroneously lists
Boletinus porosus (Berk.) Peck as a synonym of S. cavipes,
but that species is considered a synonym of Boletinellus
merulioides (Schwein.) Murrill (Both 1993). The Chinese
species S. cavipoides (Z.S. Bi & G.Y. Zheng) Q.B. Wang &
Y.J. Yao might be synonymous with the Eurasian
S. cavipes. Unfortunately, no sequences of S. cavipoides
were available for this study (Wang and Yao 2004).
The /subluteus (Japan) clade was sister to S. subalu-
taceus (A.H. Sm. & Thiers) A.H. Sm. & Thiers. It was a
moderately supported clade of mostly environmental
samples, with the majority of sequences from Japan
and one from Russia. The clade contained three
specimens that were identified as S. subluteus (Peck)
Snell. These sequences from Japan clearly do not
represent S. subluteus, a species in the /salmonicolor
clade. Instead, this represents an unnamed species
native to East Asia.
The /spraguei clade was well supported (100%).
Within it were several subclades separated by geogra-
phy, with an American clade and a Japanese clade.
The separation of these two clades reflects an earlier
study by Burchhardt et al. (2011). Based on relative
branch lengths of each of these clades compared to
the rest of the genus, we propose that the Japanese
clade does not belong to the same species as the North
American S. spraguei (Berk. & M.A. Curtis) Kuntze.
The North American species is better known as
S. pictus (Peck) Kuntze, but that name is based on
the illegitimate name Boletus pictus Peck. Both the
American and Japanese species associate with soft
pines. Suillus decipiens (Peck) Kuntze, an American spe-
cies that associates with hard pines, nests within the
Japanese clade (although Bayesian analysis showed
that it was not nested, SUPPLEMENTARY FIG. 1). Related
to the /spraguei clade are two collections that were
identified as S. sibiricus (Singer) Singer. However, these
are not closely related to true S. americanus (Peck)
Snell (syn. S. sibiricus) and instead appear to be an
unnamed species found at high elevation in the
southern Rocky Mountains of Colorado, USA. This
species occurs with soft pines, most likely Pinus flexilis.
We plan to describe this species once more specimens
become available.
Suillus tomentosus var. discolor A.H. Sm., Thiers &
O.K. Mill. nested well within the Tomentosus group
(100% clade support). Specimens identified as this
variety grouped well together (97% clade support)
and were separate from S. tomentosus (Kauffman) Singer.
Furthermore, var. discolor associates with soft pines,
whereas S. tomentosus associates with hard pines. There-
fore, we elevate this variety to species as Suillus discolor
(see TAXONOMY).
The case of Suillus granulatus.—Within the /placidus
clade was a well supported clade (100%) of a well
known North American species that has long been
called Suillus granulatus (L.) Roussel. Whether this spe-
cies is the same as the European species under that
same name was fervently debated between Singer
(1945) and Smith and Thiers (1964a, 1966). Singer
(1945) described the North American specimens as
S. granulatus ssp. snellii based on spore size, spore
color (isabella), and an association with soft pines
(P. strobus), which distinguishes the subspecies from
the European taxon that associates with hard pines.
As a result of this debate, no firm name had been
accepted for general use, and the name S. granulatus
has been in continual use in North America to the
present. Our molecular evidence indicates that the
NGUYEN ET AL.: GLOBAL SUILLUS ITS PHYLOGENY 1223
species in North America that associates with soft pines
is different from the European S. granulatus. Surpris-
ingly, the holotype sequence of S. weaverae described
by Smith and Shaffer (1965) fits firmly within this
clade. Suillus weaverae was described based on its
“avellaneous to cacao brown”spore color, which does
match specimens of S. granulatus ssp. snellii (cinna-
mon) as described by Singer. The photo of original
material of S. weaverae shows a dark variant of S. granu-
latus sensu American authors with an infection of
aHypomyces (http://iucn.ekoo.se/iucn/species_view/
415953). Unfortunately, we have not been able to
obtain a holotype sequence for ssp. snellii, but even if
the holotype of ssp. snellii fits firmly in this clade, the
species level status of S. weaverae supersedes the subspe-
cies level of S. granulatus ssp. snellii as the only available
name for this clade. With morphological, photograph-
ic, and molecular evidence, we propose S. weaverae as
the correct name for this species.
The /granulatus clade is the best fit for the true
S. granulatus. Our phylogeny showed an Asian sub-
clade, and although different, the branch length of
this unsupported clade was not long enough to consid-
er it a separate species without other evidence. North
American specimens identified as S. lactifluus (With.)
A.H. Sm. & Thiers also nested within this clade. It
has long been suspected that this is an eastern
North American introduction of the European S. gran-
ulatus (Murrill 1910, Singer 1965), but Smith and
Thiers (1964b) thought that this species has “improp-
erly been regarded as a synonym of S. granulatus”.
Our data show that this species is indeed the same as
the European S. granulatus or at least not the same
species as the American S. granulatus. Although there
is no type collection for S. granulatus (L.) Roussel,
the concept of this species in Europe is clear.
The /granulatus (Eurasian) clade contains speci-
mens called S. granulatus,S. variegatus (Sw.) Richon &
Roze, S. collinitus (Fr.) Kuntze, and S. fluryi Huijsman.
The names S. variegatus and S. collinitus were misap-
plied to the respective specimens because S. variegatus
belongs in the Tomentosus group (see DISCUSSION)
and S. collinitus is sister to the /granulatus (Eurasian)
clade in consideration. Further studies will be neces-
sary to determine the exact taxonomy of this clade.
Recently diverged species and species complexes.—The /pun-
gens clade contained two species that appear recently
diverged even though their morphologies are distinct
and they can be separated easily. Suillus pungens
Thiers & A.H. Sm. is a common species occurring
with P. muricata,P. radiata, and introduced P. pinea,
P. pinaster, and P. halapensis along the California coast
(USA), whereas S. kaibabensis Thiers is a common
species occurring with P. ponderosa in the southwestern
USA. One ectomycorrhizal root tip sequence from
Mexico nested within this group, although it is unclear
whether it best fits within S. kaibabensis or S. pungens.
More material from Mexico may reveal that this
complex has species that recently diverged through
geographic and climatic separation between coastal
California, Mexico, and the mountains of the south-
western USA.
The /pseudobrevipes clade contained four sub-
clades or species that are similar morphologically and
are difficult to distinguish due to evanescent veils often
absent at maturity or in adverse weather. It included
the widespread S. albivelatus (discussed above), S. pseu-
dobrevipes that occurs with P. ponderosa in western North
America, and S. occidentalis Thiers that occurs in the
southwestern USA. The /sp. (Florida) clade might be
the same taxon as S. pseudogranulatus (Murill) A.H.
Sm. & Thiers, but unfortunately we were not able to
examine the fresh specimens from Florida to confirm
the evanescent veil and color characteristics that would
identify them as that species. Suillus floridanus Murrill
is more closely related to S. decipiens and S. spraguei.
Furthermore, one sequence from an ectomycorrhizal
root tip found in Mexico may represent a distinct sub-
clade of this complex. In this clade, morphological
similarity appears to be reflected by ITS similarity.
While no strong bootstrap support suggests that these
four species could be collapsed into one, these names
are well known, the species are widespread, and mor-
phological characteristics can be used to differentiate
them when observed carefully. We choose to retain
the current nomenclature as a case of recent speciation.
TAXONOMY
Summarizing our results, we consider: (i) S. neoalbidipes
M.E. Palm & E.L. Stewart a synonym of S. glandulosipes
Thiers & A.H. Sm.; (ii) S. borealis A.H. Sm. Thiers &
O.K. Miller is a synonym of S. brunnescens A.H. Sm. &
Thiers; (iii) Boletus serotinus Frost and Boletus solidipes
Peck synonyms of Suillus elbensis (Peck) Kuntze; (iv)
S. lactifluus (With.) A.H. Sm. & Thiers a synonym of
S. granulatus (L.) Roussel; and (v) S. himalayensis
B. Verma & M.S. Reddy a synonym of S. americanus
(Peck) Snell. Further, we propose usage of the names:
(xi) S. clintonianus in the place of the North American
S. grevillei; (xii) S. ampliporus in the place of the
North American S. cavipes; (xiii) S. elbensis in place of
the North American S. viscidus; and (xiv) S. weaverae
in place of the North American S. granulatus.
Suillus discolor (A.H. Smith, Thiers & O.K. Miller)
N.H. Nguyen, comb. & stat. nov. FIG.2
MycoBank MB816996
1224 MYCOLOGIA
Basionym: Suillus tomentosus var. discolor A.H. Smith,
Thiers & O.K. Miller, Lloydia 28:134. 1965.
Typification: USA: IDAHO. Reeder Bay, Priest Lake,
Kaniksu National Forest between Priest Lake and
Upper Priest Lake, 3 Oct 1964, A.H. Smith Sm-71001
(holotype MICH341813). GenBank ITS KX213790.
This taxon was separated from var. tomentosus based
on the color of the dried specimens, having a vina-
ceous brown cap with no yellow and reddish tones
present in and over the base of the stipe. However,
S. tomentosus associates with hard pines (subgenus
Pinus), whereas S. discolor associates with soft pines
(subgenus Strobus, in particular P. monticola and P. albi-
caulis in western North America). We have observed
these host patterns in the field, which has also been
reported by Mohatt et al. (2008). Molecular data
showed that S. discolor is closely related to S. tomentosus
but is not nested within S. tomentosus, therefore further
supporting its elevated species status. The species is
reported from a broad range throughout western
North America from Idaho, Montana, Colorado, New
Mexico, Washington, Oregon, and California.
DISCUSSION
By combining all the sequences available from Gen-
Bank, our own specimens, and host and geographic
metadata attached to each sequence, we assembled
the newest species concepts for the genus Suillus.In
particular, the availability of multiple sequences per
species allowed us to infer genetic variations within
and among species. In conjunction with sequencing
of type specimens, this approach is a good start toward
solving current taxonomic difficulties and connecting
morphologically identified species with phylogenetic
species. However, the weakness of a single gene study
is concerning. Apparently, many of the species-level
clades remain unresolved and recently diverged taxa
could not be distinguished readily from each other.
Combining the ITS region with other genes such as
EF1-afrom vouchered specimens represents a better
approach to delimit species and detect biogeographic
signals as shown by the global study of Pluteus
(Justo et al. 2014). Unfortunately, sequencing only
vouchered specimens would exclude most, if not all,
of the environmental sequences that provided much
useful associated host and geographic data seen in
this study.
Despite those concerns, we think ITS still serves as a
good barcoding gene region that mostly reflects spe-
cies boundaries set by careful studies of morphological
species in Suillus. Overall, we observed strong congru-
ence between morphological species and ITS-defined
phylogenetic species (FIG. 1). As expected, when multi-
ple specimens or sequences were available for
comparison, they grouped well together and often
apart from sister clades. In some cases, we observed
that genetic distance of some sequences within a clade
was larger than distance between clades (e.g. long
branches). This may be due to the broad geographic
nature of some species or poor quality sequences
from environmental samples. Because those divergent
sequences were submitted without their chromato-
grams, it was not possible for us to determine the qual-
ity of the nucleotide base calls. Long branches also
occurred in some clades where it was necessary to
include partial sequences of either the ITS1 or ITS2
regions. Despite these anomalies in the dataset, clades
of one species tended to fall well apart from clades of
other species.
In a few cases, well supported taxa nested inside of a
group of loosely defined taxa (S. weaverae,S. subaureus,
S. decipiens, /suilloides clade). Separating the align-
ments of those individual groups and re-analyzing
them with maximum likelihood did not resolve their
position, but Bayesian inference showed that S. subaur-
eus and S. decipiens formed well supported un-nested
clades, whereas S. weaverae and the S. suilloides clades
remained nested within larger clades (SUPPLEMENTARY
FIG. 1). Therefore, we attribute this inconsistency to
the methodology and the minimal character informa-
tion that the ITS gene region could provide. Addition
of more loci will likely improve clade topology.
From sequences obtained from ectomycorrhizal
root tips and field basidiocarp records, we observed
that Suillus is associated with at least two of the four
subfamilies of the Pinaceae: Pinoideae (Pinus), Lari-
coideae (Larix,Pseudotsuga), and possibly Piceoideae
(Picea,Cathaya) but not Abietoideae (Abies,Cedrus,
Keteleeria,Tsuga). Note that associations with Picea and
Cathaya remain unconfirmed. Field records indicate
that Suillus glandulosus (Peck) Singer occurred in
mixed woods with Picea mariana and Abies balsamea
and that S. sinuspaulianus (Pomerl. & A.H. Sm.) E.A.
Dick & Snell occurred in areas with P. glauca,
P. mariana, and L. laricina, but the occurrence of these
two species on ectomycorrhizal root tips of Picea has
not been confirmed. These host species overlap in
habitat with L. laricina. Based on our phylogeny, we
think that the primary host is L. laricina with Picea
spp.or A. balsamea likely acting as secondary hosts.
We did not find records of Suillus associating with the
monotypic Cathaya agyrophylla from southern China.
Horton et al. (2005) studied roots of Tsuga and Pseudo-
tusga in the Pacific Northwest and found S. punctatipes
(based on a 3-enzyme digest RFLP pattern) associating
with Pseudotsuga but not Tsuga. Results from seedling
inoculations of several Suillus species also confirmed
that S. tomentosus,S. clintonianus (North American
S. grevillei), and S. caerulescens colonize their respective
NGUYEN ET AL.: GLOBAL SUILLUS ITS PHYLOGENY 1225
hosts but not Tsuga (SUPPLEMENTARY FIG. 2). These two
lines of evidence suggest at the least that Tsuga is not a
host for Suillus, and the lack of ectomycorrhizal root
tips associated with Abies,Cedrus, and Keteleeria suggests
that the subfamily Abietoideae is not associated with
Suillus.Suillus subaureus from the midwestern USA
and Ontario, Quebec, and Nova Scotia in Canada
represents an exceptional case of host specificity, as it
appears to have jumped to associating with angio-
sperm hosts in the genera Quercus and Populus (Smith
and Thiers 1964a, 1971) and is able to produce basi-
diocarps with these hosts when Pinus individuals are
locally absent.
Although deep-branch support was not obtainable
based on data from a single gene region, our results
do suggest some patterning of host association. Species
that associate with Pseudotsuga,Larix, and perhaps Picea
appear to cluster in the Spectablis group. Whether
these groups evolved as basal to the genus as stated
in Kretzer et al. (1996) remains to be demonstrated
with a multilocus phylogeny. Pinus is the most common
host for Suillus, and there have been multiple switches
between Pinus subgenus Pinus and subgenus Strobus.
However, one large clade (contains species such as
S. mediterraneensis,S. brevipes, and S. luteus) in the Gran-
ulatus group associates exclusively with hard pines,
with the exception of S. brunnescens (FIG. 1C).
Suillus luteus is the most widespread species in the
world, occurring in the Northern and Southern Hemi-
sphere. It is native to Europe, where it associates with
P. sylvestris and P. nigra, but it has also been introduced
into North and South America, Asia, and Australasia
with P. sylvestris. Vellinga et al. (2009) observed that it
is able to grow with other Pinus species and has been
repeatedly reported growing in New Zealand growing
with P. radiata (Hedger 1986, Dunstan et al. 1998,
Dickie et al. 2010). In addition, we collected specimens
in two distant forests of pure P. resinosa in northern
Minnesota. Our phylogeny showed that S. luteus was
also found on ectomycorrhizal root tips of P. tabulifor-
mis in Shaanxi, China as well as on P. densiflora in South
Korea (FIG. 1C). This ability to associate with many dif-
ferent hosts is rare in the genus, where host jumps
for Suillus species are often to a different host species
within the same host subgenus. Specifically, the case
of S. brunnescens in the /luteus clade is notable, as that
species associates with soft pines within a large clade
that associates exclusively with hard pines. Given this
contrast in host associations, we think that the /luteus
clade makes an excellent model to study biogeography
(through recent introductions to new geographic
areas) and the co-evolution of hosts and symbionts (i.e.
determining the genomic mechanisms that allow host
jumps across broad host phylogenetic distance).
The number of Suillus species in North America
far exceeds that in Europe (Smith and Thiers 1964a,
Klofac 2013), but exact numbers for Asia are not avail-
able, and it clearly has been undersampled. The num-
ber of unresolved taxonomic issues is also higher in
North America than in Europe, but this may correlate
with the amount of taxonomic efforts put into the
genus. As seen repeatedly in fungal systematics, Euro-
pean names applied to other parts of the world mask
regional diversity that could only be revealed through
DNA sequencing and careful study of numerous
specimens. Our combined analysis of the available
sequences in public databases with sequencing of spec-
imens and type material provide an enticing glimpse
into the taxonomic problems and novelties present
even within a well known group of ectomycorrhizal
fungi. We have solved a few of those problems in the
current study, but many more await the availability of
holotype ITS sequences and genomic data from mod-
ern high-throughput sequencing. Ongoing and future
work will contribute to Suillus as a well defined genus
to the North American Mycoflora (http://www.northa
mericanmycoflora.org/).
ACKNOWLEDGMENTS
This work is a culmination of that which the first author
(NHN) started 10 y ago. He thanks Tom Bruns for putting
the genus on his radar when he first joined his lab as a grad-
uate student, for the continuing support and excitement for
anything in the genus, and for having never tossed a
maggot-ridden Suillus at him. He thanks Else Vellinga for
the many years of educating him about mushroom taxonomy
and stimulating times annotating the working Suillus tree,
and Peter Kennedy for the unyielding support and excitement
about Suillus. The work could not have been accomplished
without the type and important specimens that were loaned
by MICH, DBG, and SFSU. We thank Todd Osmundson and
Valerie Wong for the Perl script that gathers metadata from
GenBank sequences. Special and continuing thanks to the
Colorado mycology group: Vera Evenson (DBG) and the
Colorado Mycological Society, along with Brian Barzee and
the Pike’s Peak Mycological Society, contributed significant
numbers of specimens from Colorado to this project. Brian
Barzee hosted and field-guided two successful collecting
forays to study Suillus in Colorado, and sequences of speci-
mens from those forays can be found in this study. Sara
Branco, Noah Siegel, Beatrice Senn-Irlet, Matthew Smith,
Roy Halling, Debbie Viess, David Rust, Ernst Both, Walt
Sturgeon, Michael Kuo, and Donna Mitchell, in addition to
the many others who have contributed specimens to this
study. Some specimens were sequenced by undergraduates
Lisa Rosenthal and Blake Boeing. Beatriz Ortiz-Santana
provided sequences from the Dominican Republic and
Belize, among other locations; Sara Branco, Rytas Vigalys,
and Lotus Lofgren provided some sequences of respective
regional specimens. This study was partially funded by
1226 MYCOLOGIA
NSF GRFP to NHN and DEB 1554181 to Rytas Vigalys
and PGK.
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