Ectomycorrhizal fungal communities in endangered Pinus amamiana forests
Ectomycorrhizal fungal communities in endangered Pinus amamiana forests
Masao Murata 0 1
Seiichi Kanetani 1
Kazuhide Nara 0 1
0 Graduate School of Frontier Sciences, The University of Tokyo , Kashiwa, Chiba , Japan , 2 Kyushu Research Center, Forestry and Forest Products Research Institute , Chuo-ku, Kumamoto , Japan
1 Editor: Erika Kothe, Friedrich Schiller University , GERMANY
Interactions between trees and ectomycorrhizal (ECM) fungi are critical for the growth and survival of both partners. However, ECM symbiosis in endangered trees has hardly been explored, complicating conservation efforts. Here, we evaluated resident ECM roots and soil spore banks of ECM fungi from endangered Pinus amamiana forests on Yakushima and Tanegashima Islands, Kagoshima Prefecture, Japan. Soil samples were collected from remaining four forests in the two islands. The resident ECM roots in soil samples were subjected to molecular identification. Soil spore banks of ECM fungi were analyzed via bioassays using a range of host seedlings (P. amamiana, P. parviflora, P. densiflora and Castanopsis sieboldii) for 6±8 months. In all remaining P. amamiana forests, we discovered a new Rhizopogon species (Rhizopogon sp.1), the sequence of which has no match amoung numerous Rhizopogon sequences deposited in the international sequence database. Host identification of the resident ECM roots confirmed that Rhizopogon sp.1 was associated only with P. amamiana. Rhizopogon sp.1 was far more dominant in soil spore banks than in resident ECM roots, and its presence was confirmed in nearly all soil samples examined across the major remaining populations. While Rhizopogon sp.1 did not completely lose compatibility to other pine species, its infection rate in the bioassays was highest in the original host, P. amamiana, the performance of which was improved by the infection. These results indicate that Rhizopogon sp.1 is very likely to have a close ecological relationship with endangered P. amamiana, probably due to a long co-evolutionary period on isolated islands, and to play the key role in seedling establishment after disturbance. We may need to identify and utilize such key ECM fungi to conserve endangered trees practically.
Funding: This work received support from The
biodiversity conservation research activity
promotion project from the Yakushima
Environmental and Cultural Foundation (URL:
http://www.yakushima.or.jp/, MM, Describe the
role: data collection and analysis) and a
Grant-inAid for Scientific Research from the Japan Society
for the Promotion of Science (26870163 MM and
Forests have disappeared and deteriorated all over the world due to habitat destruction and
environmental changes caused by human activity [
]. As a result, many tree species are
threatened with extinction [
]. In particular, the dominant trees in a forest play critical roles in
primary production and ecosystem structuring, directly and/or indirectly supporting many other
organisms. Thus, the extinction of a dominant tree species can have a serious impact on
15H02449 KN, URL:
https://www.jsps.go.jp/jgrantsinaid/, Describe the role: the study design,
data collection and analysis, decision to publish, or
preparation of the manuscript).
biodiversity in the entire forest ecosystem, and for this reason, conservation measures are
urgently needed for dominant trees threatened with extinction.
Dominant trees in temperate forests, such as species of the Fagaceae and Pinaceae, are
associated with ectomycorrhizal (ECM) fungi and depend on them for soil nutrients. Without
compatible ECM fungi, these trees cannot grow normally [
]. Moreover, it has become
increasingly clear that the species composition and distribution of ECM fungi in soil affect the
establishment of tree seedlings [
]. Therefore, knowledge of the ECM fungi associated with
an endangered tree species could be key to its conservation.
There are two main infection pathways used by ECM fungi in nature: the mycelium
(mycelial network) extending from the existing ECM roots (e.g. [
]) and spores dispersed from
fruiting bodies [
]. Both types of infection are ubiquitous in soils of developed forests, where host
roots can be easily infected with ECM fungi. Mycelial networks are the predominant infection
pathway in less disturbed forests, while spores that have accumulated in the soil, called soil
spore banks, are the primary source of ECM infection for regenerating seedlings after
disturbances that eliminate existing trees [
]. Therefore, pioneer trees that require disturbance for
regeneration depend on both soil spore banks, at the seedling stage, and mycelial networks, at
the mature stage.
The species composition of ECM fungi often differs between spore banks and resident
ECM roots, with soil spore banks usually composed of fewer ECM fungal species [8, 10±12].
Especially in pine forests, soil spore banks are dominated by pine-specific Rhizopogon [8, 10,
13±15], because their spores have a longer life span and greater environmental resistance than
do those of other ECM fungi [16±19]. All Rhizopogon species produce hypogeous sporocarps
and depend on animal ingestion for spore dispersal [
]. Thus, gene flow of Rhizopogon
species is more restricted than fungi producing epigeous sporocarps and is easily inhibited by
geographical barriers . This limited gene flow leads to genetic differentiation among
isolated Rhizopogon populations [21±23] and eventually to speciation in geographically isolated
]. While most pine species are successful pioneer colonizers after disturbances
throughout the northern hemisphere [
], establishment of their seedlings may be
sustained by unique Rhizopogon species that evolved locally.
The genus Pinus consists of two subgenera, four sections and over 100 extant species, and it
is the largest genus of conifers and the most widespread genus of trees in the Northern
]. While some pine species have wide distribution ranges over a continent, many
have been restricted to small isolated areas [
] as a result of long biogeographic history since
the Early Cretaceous [27±29] and competition with broadleaf trees. Species with small
distribution ranges are prone to extinction caused by environmental and demographic stochasticity
in combination with inbreeding depression [
]. In fact, 19 pine species are classified as
threatened (12 endangered and 7 vulnerable species) [
Pinus amamiana is endemic to Yakushima and Tanegashima Islands, Kagoshima
Prefecture, Japan [
] and is classified as ªEndangered A3ce; B1ab(iii,v)+2ab(iii,v)º on the IUCN Red
List and as ªVulnerableº by the Japanese government [
]. The abundance of P. amamiana has
been drastically reduced by timber harvesting and pine wilt disease, and only 1,500 and 100
individuals persist in Yakushima and Tanegashima Islands, respectively [
]. Although we
know nothing about the ECM fungi colonizing this endangered pine species, P. amamiana may
be associated with unique ECM fungi that coevolved locally and may depend on these fungi for
ECM fungi of endangered trees have long been overlooked, and thus available information
on these fungi is quite limited. However, our recent study in forests of Pseudotsuga japonica
(Pinaceae), another endangered conifer species in Japan, found that soil spore banks are
dominated by Rhizopogon togasawariana that coevolved with the endangered host for more than 30
2 / 19
million years [
]. Notably, this fungus was completely absent in resident tree roots [
Because P. japonica is a light-demanding pioneer tree species, its seedlings are rarely found on
the dark forest floor [
]. These findings strongly suggest that both P. japonica and R.
togasawariana depend on disturbance for regenerating their populations, and that R.
togasawariana plays the key role in the establishment of seedlings during regeneration. The existence of
this ECM fungus specific to the endangered tree species and its potential role in establishment
of endangered tree seedlings have critical implications for conservation; yet, it is difficult to
generalize the results obtained from this single case. Similar studies in Pinus, which includes
far more endangered species than those of Pseudotsuga, would further improve our
understanding of ECM symbiosis on endangered trees.
In the present study, our objective was to characterize the ECM fungal communities of
both soil spore banks and resident ECM roots in remaining P. amamiana forests. Specifically,
we examined the following hypotheses: (1) P. amamiana is associated with some host specific
ECM fungi that have coevolved with this endangered pine, and (2) these fungi are more
frequently detected in soil spore banks than in resident ECM roots as Rhizopogon species in other
ecosystems. We believe that the results of this study can provide key information for in situ
conservation of P. amamiana and has many important implications for the conservation of
other endangered trees.
Materials and methods
This study was conducted in four P. amamiana forests, i.e. Hirauchi (site 1) and Banri (site 2)
on Yakushima Island, and Wasedagawa (site 3) and Injo (site 4) on Tanegashima Island,
Kagoshima Prefecture, Japan (Table 1, S1 Appendix). Sampling permission for the site 1, 2 and 3
was issued by Yakushima forest office, and it for site 4 was issued by the owner of the land.
These forests harbor the majority of remaining P. amamiana populations. At the Yakushima
sites, P. amamiana is usually found on steep slopes or ridges with other ECM trees
(Castanopsis sieboldii, Lithocarpus edulis, Quercus acuta, Q. phillyraeoides, Q. salicina, Tsuga sieboldii). At
the Tanegashima sites, this tree is usually found on hilly land (site 3) or by the seaside (site 4)
along with other ECM trees (C. sieboldii, L. edulis, P. thunbergii, Q. salicina). In addition to
ECM trees, these forests also contain non-ECM sub-trees (i.e. Cleyera japonica, Cryptomeria
japonica, Distylium racemosum, Elaeocarpus japonicas, Ilex pedunculosa, Morella rubra,
Myrsine seguinii, Pieris japonica, Prunus sp., Rhaphiolepis indica var. umbellate, Rhododendron
tashiroi, Syzygium buxifolium, Ternstroemia gymnanthera). The annual mean temperature
ranged from 17.9 to 20.4ÊC, with abundant precipitation (Table 1).
Site area Latitude/longitude
3 / 19
In September 2014, 26, 21 and 32 pairs of soil samples were collected near randomly selected
P. amamiana trees at sites 1, 2 and 3, respectively. In November 2015, an additional 15 and 10
soil samples were collected at sites 1 and 4, respectively. The number of samples collected
depended on the number of surviving trees at each site. Selected trees were >5 m apart. Two
soil samples (5 × 5 × 10 cm depth), one for resident ECM root analysis and the other for soil
spore bank analysis, were collected near each selected tree. Geographical positions were
recorded using a GPS (GPSMAP62SJ, Garmin, Olathe, KS, USA). Samples were placed
separately in plastic bags and kept at 4ÊC until further analyses.
Bioassay experiments were performed to assess the soil spore banks following an established
]. In these experiments, only soil samples collected in 2014 were used. After
removing roots, organic debris and small stones, the soil samples were air-dried for 2±8
months (S2 Appendix). The bioassay containers were 50-mL centrifuge tubes (Ina Optika Inc.,
Osaka, Japan), with two drainage holes near the bottom blocked by a cotton ball to prevent soil
loss. Each tube was filled with approximately 40 mL of the air-dried soil sample.
We used four tree species, P. amamiana, P. parviflora, P. densiflora and Castanopsis sieboldii
(Fagaceae), to evaluate the host specificity of soil propagule banks, particularly the specificity
of the Rhizopogon spp., which are expected in spore banks. While P. parviflora belongs to the
same subgenus (Strobus) as that of P. amamiana, P. densiflora belongs to the subgenus Pinus.
C. sieboldii is a broadleaf tree species that is predominant in warm-temperate forests of the
region. We were able to use only 46 P. amamiana seeds for all bioassay tubes because of the
limited seed availability and low germination rate of this endangered species.
Seeds of all tree species were soaked in tap water for 48 h, surface-sterilized using a 5%
sodium hypochlorite solution for > 10 min, and rinsed with tap water. To induce germination,
surface-sterilized P. amamiana and P. parviflora seeds were placed on sterilized,
well-moistened peat moss in an incubator (25ÊC for 1 month and then 5ÊC for 2 months), while P.
densiflora and C. sieboldii seeds were placed on sterilized Shibanome soil (fine red granular soil of
Pleistocene volcanic origin) in an incubator (25ÊC). For each soil-host combination, one
germinated seed was planted and covered with sterilized Shibanome soil in a bioassay tube, for a
total of 283 seedlings (S2 Appendix). To monitor airborne spore contamination of ECM fungi,
we prepared a control treatment using 10 tubes containing an autoclaved, randomly selected
soil sample (per host). Bioassay seedlings were watered with tap water (2±3 mL per seedling)
every 3±5 days depending on soil conditions and were harvested after 6±9 months of growth
in a growth chamber (MLR-351; SANYO Inc. Tokyo, Japan) set to 25ÊC with 16 h of light (15
fluorescent lamps: 20,000 lx) and then to 20ÊC for 8 h of dark (S2 Appendix).
Identification of ectomycorrhizal fungi
Roots of resident trees collected from field soil samples and bioassay seedlings were gently
washed with tap water. ECM root tips were classified into morphotypes under a dissecting
microscope based on their surface color, texture and emanating hyphae, as described in
previous studies [
]. For molecular identification of ECM fungi, triplicate ECM root tips, if
available, were selected for each morphotype in a soil sample, then placed individually into
2.0-mL tubes. A total of 682 root tips from ECM were used for molecular identification on
resident trees. In addition, 208, 246, 257 and 191 ECM root tips from the bioassays of the P.
amamiana, P. parviflora, P. densiflora and C. sieboldii, respectively, were subjected to DNA
4 / 19
Molecular identification of ECM fungi was performed following Murata et al. [
each ECM tip sample was placed in a 2.0-mL tube containing a zirconia ball and then
pulverized using a bead beater. Total DNA was extracted using a modified cetyl trimethylammonium
bromide method [
]. Internal transcribed spacer regions (ITS1-5.8S-ITS2) of ribosomal
DNA were amplified using the ITS1F or ITS0F-T forward primer and several reverse primers
(ITS4, ITS4Cg, LB-W [37±40] depending on amplification success. Platinum1 Multiplex
PCR Master Mix (Applied Biosystems, Foster City, CA, USA) was used for polymerase chain
The amplified products were purified and subjected to direct sequencing (3730xl DNA
Analyzer; Applied Biosystems) using ITS1 as the sequencing primer. For poorly sequenced
samples, ITS4 was also used as a sequencing primer. The obtained high-quality sequences were
grouped into molecular operational taxonomic units (MOTUs) with 97% ITS sequence
similarity (including 5.8S regions) using ATGC software (ver. 7.0; GENETYX Corp., Tokyo,
Japan). Species identity was assigned based on the results of BLAST searches against known
taxa in international sequence databases (DNA Data Bank of Japan [DDBJ]/EMBL/GenBank).
Potential PCR chimeras were removed manually before further analyses, based on inconsistent
BLAST results between segments within each MOTU sequence, e.g., between the initial and
last parts (about 100 bp each) of the sequence. If the ITS similarity with a described species
from herbarium specimens was > 97%, we used that species name for the MOTU. When the
similarity to known species was 90±97% or < 90%, we identified MOTUs at the genus and
family level, respectively. One exception was C. geophilum, which was identified by amplifying
the Cenococcum-specific primer (ITS4Cg) and confirmed by sequencing randomly selected
samples. Although several different MOTUs of C. geophilum were found (< 97% ITS sequence
similarity), they were treated as one MOTU in this study, because no taxonomic agreement
was reached among the many previous studies of ECM fungal communities (e.g., [
Representative sequences of individual MOTUs were deposited in the DDBJ under accession
To confirm the identity of host species in all molecular samples, the plastid trnL or rbcL
region of plant DNA was amplified using primers trnC (50-cgaaatcggtagacgctacg-30)
or rbcLa-F (50- atgtcaccacaaacagagactaaagc- 30) in combination with trnD
(50ggggatagagggacttgaac- 30) or rbcLa-R (50- gtaaaatcaagtccaccrcg- 30). All
the amplicons were purified and subjected to direct sequencing using trnC or rbcLa-F as the
sequencing primer. As with species identification of ECM fungi, the identity of host species
was assigned based on the results of BLAST searches against known taxa in international
The frequency of an ECM fungal species was defined as the number of bioassay seedlings
colonized by each ECM fungal species or, for resident trees, the number of soil samples containing
each ECM fungal species. The frequencies of major fungal species were compared between
different bioassay host species using Fisher's exact test implemented in SPSS (ver. 11.5; SPSS
Japan Inc., Tokyo, Japan). Statistical significance was set at α = 0.05. Estimate S software (ver.
]) was used to calculate the minimum number of total species richness indicators
A fungal community matrix was built for the major host groups (P. amaiana, T. sieboldii
and Fagaceae) at each site. Community compositional similarity was visualized using
nonmetric dimensional scaling (NMDS), implemented in the `vegan' package of R software [
using the Bray-Curtis distance (Manhattan distance for NMDS based on the unit of soil
5 / 19
samples for resident ECM roots) and 999 permutations. Differences in community composition
between resident ECM roots and assayed spore banks were determined using Adonis
(permutation multivariate analysis of variance; [
]) in `vegan', again using the Bray-Curtis distance and
999 permutations [
]. Data dispersions of the communities among groups were analyzed by
the Betadisper test (Permutational analysis of multivariate dispersions; [
]) in `vegan'.
ECM fungal communities of Pinus amamiana forests
Of the 104 soil samples collected from the four study sites, 44 contained P. amamiana ECM
roots. Other dominant host taxa in the belowground ECM tips were T. sieboldii and Fagaceae,
which were found in 9 and 58 soil samples, respectively (Table 2). As a minor host, the
subgenus Pinus was confirmed in one soil sample. No ECM roots were found in 15 soil samples.
Of the 101 putative ECM fungi that were identified in the four sites, 42 species were found
on P. amamiana roots (Table 2, S3 Appendix). Thirteen and sixty-one ECM fungi were found
on T. sieboldii and Fagaceae roots, respectively. The rarefaction curves indicated that neither
the observed ECM fungal richness on P. amamiana nor richness on all hosts approached an
asymptote with the maximum sample size (Fig 1), indicating that additional fungal species
would be found with additional sampling effort. The richness estimator Jackkife2 showed that
at least 229 ECM fungal species should inhabit these forests (Fig 1). The Jackkife2 richness
estimator for ECM fungi on P. amamiana was 102 (Fig 1). The observed ECM fungal richness
Jackkife2 estimators on T. sieboldii and Fagaceae trees were 31 and 141, respectively.
The total ECM fungal community was composed of a few common species and a large
number of rare species. Only eight ECM fungal taxa appeared in five or more soil samples. In
contrast, 73 taxa were found only once (i.e., singletons). Cenococcum geophilum was the most
frequent taxon, found in 39% of the soil samples. The occurrence frequencies of Russulaceae
(48%), Boletaceae (36%), Clavulinaceae (17%), Rhizopogonaceae (14%) and Thelephoraceae
(13%) were also high at the family level. Russulaceae (30 spp.) was the most species-rich ECM
fungal lineage, followed by Boletaceae (19 spp.), Clavulinaceae (eight spp.) and Thelephoraceae
(six spp.), while only two species of Rhizopogonaceae were found (Table 2).
Only Rhizopogon sp.1 was found at all sites (Table 2). In addition, Boletaceae sp.1,
Clavulinaceae sp.3, Phylloporus sp.1 and Thelephoraceae sp.1, among the major ECM fungal species
with >5% relative frequencies, were only found on Yakushima Island (sites 1 and 2). In
contrast, none of the major ECM fungi were exclusive to Tanegashima Island (sites 3 and 4).
Cenococcum geophilum, Clavulinaceae sp.1, Clavulinaceae sp.3 and Phylloporus sp.1 were
found in all of the host groups (two conifer species and Fagaceae, Table 2). In contrast,
Rhizopogon sp.1 and Thelephoraceae sp.1 were only found in P. amamiana, with a >5% relative
frequency, while Boletaceae sp.2, Elaphomyces sp.1, Lactarius sp.1 and Phylloporus sp.2 were only
found in Fagaceae. ECM fungal communities were separated by host groups (Fig 2, S4
Appendix), with statistical significance, as determined by the Adonis test (pseudo-F2,4 = 2.06, R2 =
0.26, P < 0.01) in combination with the Betadisper test (F,2, 7 = 3.241, P = 0.101). The effect of
site was also significant by the Adonis test (pseudo-F3,4 = 2.56, R2 = 0.49, P < 0.01), yet it was
affected by the difference in data dispersion (Betadisper test: F3,6 = 8.484, P = 0.014).
By the end of the growth period, three, fifteen, two and seven seedling mortalities occurred
among the P. amamiana, P. parviflora, P. densiflora and C. sieboldii soil samples, respectively
(S5 Appendix). ECM formation was observed in 100%, 81.3%, 75.3% and 80.6% of seedlings in
P. amamiana, P. parviflora, P. densiflora and C. sieboldii, respectively. Five, four, nine and
6 / 19
a F, P, Ps and T indicate Fagaceae, Pinus amamiana, Pinus sp. (subgenus Pinus) and Tsuga sieboldii, respectively.
seven ECM fungi were identified from 208 P. amamiana, 246 P. parviflora, 257 P. densiflora
and 191 C. sieboldii DNA samples, respectively (Table 3 and S3 and S5 Appendices). None of
the control seedlings had ECM roots.
In bioassays of the subgenus Strobus, six ECM fungal species were found, of which three
were shared between P. amamiana and P. parviflora (Table 3, Fig 3). Nine and seven ECM
fungal species were detected in the P. densiflora and C. sieboldii bioassays, respectively. The
estimated species richness (Jackknife2) was 7.9, 7.9, 20.7 and 13.8 in P. amamiana, P. parviflora,
P. densiflora and C. sieboldii, respectively.
Rhizopogon sp.1 was the most dominant ECM fungal species detected in the P. amamiana
bioassay, found in 40 of 43 (93% frequency) soil samples (Fig 3). This fungus was also found in
P. parviflora and P. densiflora samples but at lower frequencies, 67% and 31%, respectively (Fig
3). Rhizopogon sp.1 was not found in any of the C. sieboldii bioassays. Cenococcum geophilum
was the most common fungal species in the P. densiflora and C. sieboldii bioassays (Fig 3).
Among the 16 ECM fungal species found in bioassays, only C. geophilum was shared among
the four host species (Fig 3).
Soil spore bank communities were clearly separated by host and site using NMDS
ordination (Fig 4), with statistical significance determined by the Adonis test (host: pseudo-F3,6 =
12.08, R2 = 0.69, P < 0.01; site: pseudo-F2,6 = 5.12, R2 = 0.2, P < 0.01) in combination with the
Betadisper test (host: F3,8 = 1.351, P = 0.325; site: F2,9 = 1.819, P = 0.206). A significant
difference (S6 Appendix; pseudo-F1,18 = 5.76, R2 = 0.24, P < 0.01) was found between soil propagule
banks and resident ECM fungal communities on mature trees in these same three forests,
however, it was affected by significant difference in data dispersion among the groups (Betadisper,
F1,18 = 44.257, P < 0.001).
We found Rhizopogon sp.1 in all remaining P. amamiana forests. Host identification of the
resident ECM roots confirmed that Rhizopogon sp.1 was associated only with the endangered
9 / 19
Fig 1. Sample-based rarefaction curves for ectomycorrhizal (ECM) fungi found in Pinus amamiana forests. Black circles
and triangles represent observed ECM fungal species richness for all host species and for P. amamiana, respectively.
Jackknife2 minimal species richness estimates of ECM fungi are also shown for all host species (white circles) and for P.
amamiana (white triangles).
species P. amamiana. In soil spore banks, Rhizopgon sp.1 was far more dominant than in
resident ECM roots and was identified in nearly all soil samples examined (93%) among the major
remaining P. amamiana populations. These findings generally support both our hypotheses,
confirming the existence of an ECM fungus specific to the endangered pine species in its
natural settings and the predominance of this fungus in soil spore banks [
]. Rhizopogon sp.1 is
very likely to have a close ecological relationship with the endangered P. amamiana, probably
due to long co-evolutionary periods on isolated islands.
Dominant ECM fungi in soil spore banks generally contribute to establishment of tree
seedlings, especially for pine trees regenerating after a disturbance [46±48]. Thus, the Rhizopogon
sp.1 found in this study likely also contributes to seedling establishment of the endangered P.
amamiana after a disturbance [
]. Although no other native pine species are distributed
in the remaining forests of P. amamiana, some broadleaf trees could compete with P.
amamiana during regeneration. It should be noted here that the dominant broadleaf tree C. sieboldii
was not compatible with Rhizopogon sp.1 in the bioassay experiments. Alternatively, C.
sieboldii bioassay seedlings were dominated by the true generalist C. geophilum, sclerotia of which
10 / 19
Fig 2. Non-metric multidimensional scaling (NMDS) depicting ECM fungal communities of resident trees
in four endangered Pinus amamiana forests. Stress = 0.089. White, gray and black symbols indicate the
communities on P. amamiana, Tsuga sieboldii and Fagaceae, respectively. Circles, diamonds, squares and
triangles represent communities at sites 1, 2, 3 and 4, respectively.
exist in almost all forests investigated (e.g. [
8, 11, 12, 15
]). Murata & Nara [
] suggested that
C. geophilum competes with other ECM fungi, thus affecting the infection rates of other soil
spore bank fungi. While ECM symbiosis is a prerequisite for trees to grow and survive in
], controlling ECM fungal communities may be difficult, especially after forest
development. Eliminating other ECM fungi from soil spore banks in disturbed sites via treatments,
such as heating [12, 52±54] or filtering [
], may allow an increase in the relative frequency
of Rhizopogon sp.1 and eventually increase regeneration of the endangered P. amamiana
We found no sequences that match Rhizopogon sp.1 in an international nucleotide
sequence database (DNA Data Bank of Japan [DDBJ]/EMBL/GenBank), potentially indicating
its endemism to these islands. In fact, based on the morphological characteristics of the
sporocarps and phylogenetic relationships with other described Rhizopogon species, we are now
describing it as a new species, which does not belong to any subgenus proposed by Grubisha
et al. [
] (Sugiyama et al. under revision). The existence of a Rhizopogon fungus specific to an
endangered tree was documented in our recent study of Pseudotsuga species in Japan and
11 / 19
China; i.e., P. japonica is associated with R. togasawariana [
] and P. sinensis with an
undescribed Rhizopogon species . Here, we first confirmed the existence of a Rhizopogon
fungus specifically associated with endangered Pinus, which is a far larger genus than
Pseudotsuga and includes far more endangered species. Given the predominance of the host-specific
fungus in soil spore banks and its potential roles in seedling regeneration, such fungi should be
explored further in other endangered Pinus species that have been geographically isolated for
long periods. Without identifying such key ECM fungi, conservation of endangered pine
species will be difficult.
Rhizopogon sp.1 was associated solely with the endangered P. amamiana under natural
settings. This fungus was also compatible with P. parviflora and P. densiflora in bioassays, but the
colonization frequency was reduced significantly with increasing phylogenetic distance. P.
amamiana is closely related to P. armandii var. armandii and P. armandii var. mastersiana [
], belonging to the same clade of subsection Strobus found in East Asian subtropical areas
], as the divergence of P. amamiana from P. parviflora or P. densiflora dates
approximately 6.4 Mya or 85 Mya, respectively [
]. The partial compatibility with these distantly
related pine species may indicate that the isolation period has not been long enough for
compatibility to be lost completely. In previous studies of R. togasawariana associated with
Pseudotsuga japonica, colonization in P. densiflora was totally absent in a similar bioassay, but it
was compatible with North American Pseudotsuga menziesii [
]. R. togasawariana belongs to
the subgenus Villosuli, which is specific to Pseudotsuga, and its origin precedes the migration
of the host to Asia approximately 34 Mya. Although Rhizopogon is regarded as a host-specific
ECM fungal lineage [
], its compatibility is not strict within the same host genus. Yet, the
compatibility with non-native hosts may not be fully functional in terms of nutrient transfer
and host performance.
12 / 19
Ectomycorrhizal fungal communities in endangered Pinus amamiana forests
Fig 3. Soil propagule banks of ECM fungi in endangered Pinus amamiana forests assayed in four hosts: P.
amamiana (a), P. parviflora (b), P. densiflora (c) and Castanopsis sieboldii (d). The relative frequency
indicates the proportion of soil samples containing each ECM species out of all soil samples examined (S5
Another interesting finding of this study is that P. amamiana was not associated with any
Suillus species, either in ECM roots of resident trees or in soil spore banks. Suillus produces
epigeous sporocarps for spore dispersal by wind but is a phylogenetic sister to Rhizopogon
]. While the life span of Suillus spores is shorter than that of Rhizopogon, it is frequently
recorded in soil spore banks [
], probably because of its strong spore dispersal abilities [
] and good germination rates . Some Suillus species are specific to, or prefer, the
subgenus Strobus within the genus Pinus [63±65], and these fungi have wide intercontinental
distributions. For example, Suillus spraguei is found in Strobus pine forests from North America to
East Asia [
]. In Japan, S. spraguei is associated with P. parviflora, P. pumila and P. koraiensis
], all of which belong to the subgenus Strobus. In addition, S. spraguei and some closely
related species are associated with P. armandii, which is closely related to P. amamiana [63,
Fig 4. Non-metric multidimensional scaling (NMDS) depicting soil propagule bank communities of ECM
fungi in three endangered Pinus amamiana forests. Stress = 0.066. Black, light gray, dark gray and white
symbols indicate the communities assayed with P. amamiana, P. parviflora, P. densiflora and Castanopsis
sieboldii, respectively. Circles, diamonds, and squares represent communities assayed with soils from sites 1, 2,
and 3, respectively.
14 / 19
67]. The nearest pine population belonging to Strobus, P. parviflora, is located in the
Takakumayama mountains on the main island of Kyushu, which is its southern population limit ,
and this population is located >100 km away from the remaining P. amamiana forests by sea.
Therefore, the absence of Suillus species in the remaining P. amamiana forests may indicate
the difficulty of airborne spore dispersal from distant Strobus forests together with local
extinction on Yakushima and Tanegashima Islands.
In the bioassay experiment, it was difficult to characterize the effect of ECM infection on
the initial growth of seedlings, because the experimental period was too short and the bioassay
tubes were too small to evaluate the growth of P. amamiana. Therefore, we did not compare
the growth of seedlings infected with ECM fungi with that of control plants in the bioassays.
Instead, seedlings infected with Rhizopogon sp.1 and uninfected seedlings were raised for 1
year in a separate bioassay experiment, after which growth was compared. Seedlings infected
with Rhizopogon sp.1 exhibited increased growth compared with control seedlings, with
marginal statistical significance (S7 Appendix) and the difference would become much larger with
the growth period. Moreover, P. amamiana seedlings colonized by Rhizopogon sp.1 are much
more tolerant to transplantation than are uncolonized seedlings (S8 Appendix). Thus, even in
a nursery setting, the application of Rhizopogon sp.1 is a promising approach to producing
good seedlings for transplantation to conserved areas.
S1 Appendix. Location of four study sites in Japan.
S2 Appendix. Air-dried periods, growth periods and number of sample of each tree species
in bioassay experiment.
S3 Appendix. Blast results of ectomycorrhizal fungal species identified in this study using
S4 Appendix. Non-metric multidimensional scaling (NMDS) depicting ECM fungal
communities of resident trees in four endangered Pinus amamiana forests based on individual
soil samples. Stress = 0.142. White, gray and black symbols indicate the communities on
Fagaceae, P. amamiana and Tsuga sieboldii, respectively. Circles, squares, diamonds and triangles
represent communities at sites 1, 2, 3 and 4, respectively. The effects of both host and site were
significant (Adonis, P<0.01) after confirming data variance among the groups was not
significant (Betadisper, P>0.05).
S5 Appendix. Summary of bioassays using soil from three endangered Pinus amamiana
S6 Appendix. Comparison of ectomycorrhizal fungal communities between soil propagule
banks and resident trees in endangered Pinus amamiana forests using non-metric
dimensional scaling (NMDS). Each symbol represents a community in each host per site.
Stress = 0.134. White and black symbols indicate the communities assayed with resident trees
and soil propagule banks, respectively.
15 / 19
S7 Appendix. In the bioassay experiment different from this study (unpublished data), the
number of leaves and tree height of seedlings infected with Rhizopogon sp.1 and seedlings
not infected with ECM fungi (control) grown for 1 year. Significant probability between
Rhizopogon sp.1 and Control of leaf number and tree height by T test was p = 0.08 and p = 0.06,
respectively. The bar shows the standard error.
S8 Appendix. From a bioassay experiment separate from this study (unpublished data),
transplantation of seedlings infected with Rhizopogon sp.1 (upper) and uninfected
seedlings (lower) to new pots after 1 year of growth.
We are grateful to the members of the Yakushima forest ecosystem conservation center and to
K. Tetsuka and T. Saito for supporting our field survey. This study was supported by the
biodiversity conservation research activity promotion project from the Yakushima Environmental
and Cultural Foundation.
Conceptualization: Masao Murata, Kazuhide Nara.
Formal analysis: Masao Murata.
Funding acquisition: Masao Murata, Kazuhide Nara.
Investigation: Masao Murata, Seiichi Kanetani, Kazuhide Nara.
Resources: Masao Murata, Seiichi Kanetani, Kazuhide Nara.
Validation: Masao Murata.
Writing ± original draft: Masao Murata, Kazuhide Nara.
Writing ± review & editing: Masao Murata, Seiichi Kanetani, Kazuhide Nara.
16 / 19
17 / 19
18 / 19
1. FAO. Global Forest Resouces Assessment 2010 . FAO, Italy, Rome. FAO forestry paper 163 . 2010 .
2. IUCN. IUCN Red List of Threatened Species 2016 [ cited 2016 July] . Available from: www.iucnredlist. org.
3. Smith SE , Read DJ . Mycorrhizal symbiosis . Third Edition ed. London: Academic Press; 2008 .
4. Nara K. Ectomycorrhizal networks and seedling establishment during early primary succession . New Phytol . 2006 ; 169 : 169 ± 78 . doi: 10 .1111/j.1469- 8137 . 2005 . 01545 .x. ISI:000233530400017. PMID: 16390428
5. Nara K. Pioneer dwarf willow may facilitate tree succession by providing late colonizers with compatible ectomycorrhizal fungi in a primary successional volcanic desert New Phytol . 2006 ; 171 : 187 ± 98 . https:// doi.org/10.1111/j.1469- 8137 . 2006 . 01744 . x PMID : 16771994
6. Simard SW , Beiler KJ , Bingham MA , Deslippe JR , Philip LJ , Teste FP . Mycorrhizal networks: mechanisms, ecology and modelling . Fungal Biol Rev . 2012 ; 26 : 39 ± 60 .
7. Ishida TA , Nara K , Tanaka M , Kinoshita A , Hogetsu T. Germination and infectivity of ectomycorrhizal fungal spores in relation to their ecological traits during primary succession . New Phytol . 2008 ; 180 : 491 ± 500 . doi: 10 .1111/j.1469- 8137 . 2008 . 02572 .x. ISI:000259526300022. PMID: 18657211
8. Taylor DL , Bruns TD . Community structure of ectomycorrhizal fungi in a Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communities . Mol Ecol . 1999 ; 8:1837±50 . ISI:000084444300008. PMID: 10620228
9. Frank JL , Anglin S , Carrington EM , Taylor DS , Viratos B , Southworth D. Rodent dispersal of fungal spores promotes seedling establishment away from mycorrhizal networks on Quercus garryana . Botany-Botanique . 2009 ; 87 : 821 ±9. doi: 10 .1139/B09-044. ISI: 000270733200002 .
10. Glassman SI , Peay KG , Talbot JM , Smith DP , Chung JA , Taylor JW , et al. A continental view of pineassociated ectomycorrhizal fungal spore banks: a quiescent functional guild with a strong biogeographic pattern . New Phytol . 2015 ; 205 : 1619 ± 31 . doi: 10 .1111/nph.13240. ISI:000349386300031. PMID: 25557275
11. Miyamoto Y , Nara K. Soil propagule banks of ectomycorrhizal fungi share many common species along an elevation gradient . Mycorrhiza . 2016 ; 26 : 189 ± 97 . doi: 10 .1007/s00572-015-0658-z. ISI:000372908100002. PMID: 26231215
12. Murata M , Nagata Y , Nara K. Soil spore banks of ectomycorrhizal fungi in endangered Japanese Douglas-fir forests . Ecol Res . 2017 ; 32 : 469 ± 79 .
13. Visser S. Ectomycorrhizal Fungal Succession in Jack Pine Stands Following Wildfire . New Phytol. 1995 ; 129 : 389 ± 401 . doi: 10 .1111/j.1469- 8137 . 1995 .tb04309.x. ISI:A1995QR30600002.
14. Baar J , Horton TR , Kretzer AM , Bruns TD . Mycorrhizal colonization of Pinus muricata from resistant propagules after a stand-replacing wildfire . New Phytol . 1999 ; 143 : 409 ± 18 . doi: 10 .1046/j.1469- 8137 . 1999 . 00452 .x. ISI:000082206200018.
15. Huang J , Nara K , Zong K , Lian CL . Soil Propagule Banks of Ectomycorrhizal Fungi Along Forest Development Stages After Mining . Microb Ecol . 2015 ; 69 : 768 ± 77 . doi: 10 .1007/s00248-014 -0484-4. ISI:000353295800005. PMID: 25213652
16. Kjoller R , Bruns TD . Rhizopogon spore bank communities within and among California pine forests . Mycologia . 2003 ; 95 : 603 ± 13 . doi: 10 .2307/3761936. ISI:000185913200006. PMID: 21148969
17. Rusca TA , Kennedy PG , Bruns TD . The effect of different pine hosts on the sampling of Rhizopogon spore banks in five Eastern Sierra Nevada forests . New Phytol . 2006 ; 170 : 551 ± 60 . doi: 10 .1111/j.1469- 8137 . 2006 . 01689 .x. ISI:000236738600014. PMID: 16626476
18. Bruns TD , Peay KG , Boynton PJ , Grubisha LC , Hynson NA , Nguyen NH , et al. Inoculum potential of Rhizopogon spores increases with time over the first 4 yr of a 99-yr spore burial experiment . New Phytol . 2009 ; 181 : 463 ± 70 . doi: 10 .1111/j.1469- 8137 . 2008 . 02652 .x. ISI:000261792900020. PMID: 19121040
19. Nguyen NH , Hynson NA , Bruns TD . Stayin' alive: survival of mycorrhizal fungal propagules from 6-yrold forest soil . Fungal Ecol . 2012 ; 5 : 741 ±6. doi: 10 .1016/j.funeco. 2012 . 05 .006. ISI: 000310717800012 .
20. Ashkannejhad S , Horton TR . Ectomycorrhizal ecology under primary succession on coastal sand dunes: interactions involving Pinus contorta, suilloid fungi and deer . New Phytol. 2006 ; 169 : 345 ± 54 . doi: 10 .1111/j.1469- 8137 . 2005 . 01593 .x. ISI:000234482900013. PMID: 16411937
21. Grubisha LC , Bergemann SE , Bruns TD . Host islands within the California Northern Channel Islands create fine-scale genetic structure in two sympatric species of the symbiotic ectomycorrhizal fungus Rhizopogon . Mol Ecol . 2007 ; 16 ( 9 ): 1811 ±22. doi: 10 .1111/j. 1365 - 294X . 2007 . 03264 .x. ISI:000245697000004. PMID: 17444894
22. Dunham SM , Mujic AB , Spatafora JW , Kretzer AM . Within-population genetic structure differs between two sympatric sister-species of ectomycorrhizal fungi, Rhizopogon vinicolor and R. vesiculosus. Mycologia . 2013 ; 105 ( 4 ): 814 ± 26 . doi: 10 .3852/ 12 - 265 . ISI:000322849500003. PMID: 23709483
23. Abe H , Tabuchi A , Okuda Y , Matsumoto T , Nara K. Population genetics and fine-scale genetic structure of Rhizopogon roseolus in the Tottori sand dune . Mycoscience . 2017 ; 58 ( 1 ): 14 ± 22 . doi: 10 .1016/j.myc. 2016 . 07 .009. ISI: 000390435900002 .
24. Mujic AB , Hosaka K , Spatafora JW . Rhizopogon togasawariana sp nov., the first report of Rhizopogon associated with an Asian species of Pseudotsuga . Mycologia. 2014 ; 106 : 105 ± 12 . doi: 10 .3852/ 13 - 055 . ISI:000332429400011. PMID: 24396108
25. Koizumi T , Nara K. Two new species of Rhizopogon associated with Pinus pumila from Japan . Mycoscience . 2016 ; 57 ( 4 ): 287 ± 94 . doi: 10 .1016/j.myc. 2016 . 04 .002. ISI: 000378057300008 .
26. Huang J , Nara K , Zong K , Wang J , Xue SG , Peng KJ , et al. Ectomycorrhizal fungal communities associated with Masson pine (Pinus massoniana) and white oak (Quercus fabri) in a manganese mining region in Hunan Province, China . Fungal Ecol . 2014 : 1 ± 10 . doi: 10 .1016/j.funeco. 2014 . 01 .001. ISI: 000336951300001 .
27. Farjon A. Pines : drawings and descriptions of the genus Pinus . 2nd ed. Leiden; Boston: Brill; 2005 . 235 p. p.
28. Ryberg PE , Rothwell GW , Stockey RA , Hilton J , Mapes G , Riding JB . Reconsidering Relationships among Stem and Crown Group Pinaceae: Oldest Record of the Genus Pinus from the Early Cretaceous of Yorkshire, United Kingdom . Int J Plant Sci . 2012 ; 173 ( 8 ): 917 ± 32 . doi: 10 .1086/667228. ISI: 000308908800006 .
29. Hao ZZ , Liu YY , Nazaire M , Wei XX , Wang XQ . Molecular phylogenetics and evolutionary history of sect. Quinquefoliae (Pinus): Implications for Northern Hemisphere biogeography . Mol Phylogen Evol . 2015 ; 87 : 65 ± 79 . doi: 10 .1016/j.ympev. 2015 . 03 .013. ISI:000353368700006. PMID: 25800283
30. Frankham R , Ballou JD , Briscoe DA . Introduction to conservation genetics . 2nd ed. Cambridge, UK; New York: Cambridge University Press; 2010 . xxiii, 618 p. p.
31. Ministry of Environment. Red list of Japanese threatened species 2015 [ cited 2016 July] . Available from: http://ikilog.biodic.go.jp/Rdb/.
32. Murata M , Kinoshita A , Nara K. Revisiting the host effect on ectomycorrhizal fungal communities: implications from host-fungal associations in relict Pseudotsuga japonica forests . Mycorrhiza . 2013 ; 23 : 641 ± 53 . doi: 10 .1007/s00572-013 -0504-0. ISI:000325969000003. PMID: 23702643
33. Yatoh K. Materials for the botanical study on the forest flora of the Kii Peninsula. Analysis and classification of the forest communities (in Japanese) . Bull Fac Agr Mie Univ . 1958 ; 18 : 105 ± 67 .
34. Yamanaka T. Ecology of Pseudotsuga japonica and other coniferous forests in eastern Shikoku (in Japanese with English summary) . Memoirs of the National Science Museum . 1975 ; 8 : 119 ± 36 .
35. Nara K , Nakaya H , Wu BY , Zhou ZH , Hogetsu T. Underground primary succession of ectomycorrhizal fungi in a volcanic desert on Mount Fuji . New Phytol. 2003 ; 159 : 743 ± 56 . ISI: 000184616400021 .
36. Ishida TA , Nara K , Hogetsu T. Host effects on ectomycorrhizal fungal communities: insight from eight host species in mixed conifer-broadleaf forests . New Phytol . 2007 ; 174 : 430 ± 40 . Epub 2007/03/29. https://doi.org/10.1111/j.1469- 8137 . 2007 . 02016 .x PMID: 17388905 .
White TJ , Bruns T , Lee S , Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics . Innis MAG D. H.; Snisky J. J.; White T. J ., editor. Berkeley, CA, USA: Academic Press; 1990 .
38. Gardes M , Bruns TD . ITS primers with enhanced specificity for basidiomycetesÐapplication to the identification of mycorrhizae and rusts . Mol Ecol . 1993 ; 2 : 113 ± 8 . Epub 1993/04/01. PMID: 8180733 .
39. Tedersoo L , Jairus T , Horton BM , Abarenkov K , Suvi T , Saar I , et al. Strong host preference of ectomycorrhizal fungi in a Tasmanian wet sclerophyll forest as revealed by DNA barcoding and taxon-specific primers . New Phytol . 2008 ; 180 : 479 ± 90 . Epub 2008/07/18. https://doi.org/10.1111/j.1469- 8137 . 2008 . 02561 . x PMID : 18631297 .
40. Bahram M , Polme S , Koljalg U , Tedersoo L . A single European aspen (Populus tremula) tree individual may potentially harbour dozens of Cenococcum geophilum ITS genotypes and hundreds of species of ectomycorrhizal fungi . FEMS Microbiol Ecol . 2011 ; 75 ( 2 ): 313 ± 20 . doi: 10 .1111/j.1574- 6941 . 2010 . 01000 .x. ISI:000285877100012. PMID: 21114502
41. Douhan GW , Huryn KL , Douhan LI . Significant diversity and potential problems associated with inferring population structure within the Cenococcum geophilum species complex . Mycologia . 2007 ; 99 : 812 ± 9 . ISI:000253080600003. PMID: 18333505
42. Colwell RK , Chao A , Gotelli NJ , Lin SY , Mao CX , Chazdon RL , et al. Models and estimators linking individual-based and sample-based rarefaction, extrapolation and comparison of assemblages . Journal of Plant Ecology . 2012 ; 5 :3± 21 . doi: 10 .1093/Jpe/Rtr044. ISI: 000299103600002 .
43. Borcard D , Gillet F , Legendre P. Numerical Ecology with R. Numerical Ecology with R. 2011 : 1 ± 300 . doi: 10 .1007/978-1- 4419 -797 6 . ISI: 000286903500001 .
44. Anderson MJ . A new method for non-parametric multivariate analysis of variance . Austral Ecol . 2001 ; 26 : 32 ± 46 . doi: 10 .1111/j.1442- 9993 . 2001 . 01070 .pp. x. ISI:000167002000004.
45. Oksanen JBG , Kindt R , Legendre P , O' Hara B , Simpson GL , Solymos P , et al. Package 'vegan' community ecology package . R package version 1.17±2 . 2010 .
46. Buscardo E , Rodriguez-Echeverria S , Martin MP , De Angelis P , Pereira JS , Freitas H . Impact of wildfire return interval on the ectomycorrhizal resistant propagules communities of a Mediterranean open forest . Fungal Biology . 2010 ; 114 : 628 ± 36 . doi: 10 .1016/j.funbio. 2010 . 05 .004. ISI:000280928100004. PMID: 20943174
47. Kipfer T , Moser B , Egli S , Wohlgemuth T , Ghazoul J . Ectomycorrhiza succession patterns in Pinus sylvestris forests after stand-replacing fire in the Central Alps . Oecologia . 2011 ; 167 : 219 ± 28 . Epub 2011/ 04/07. https://doi.org/10.1007/s00442-011-1981-5 PMID: 21468664 .
48. Glassman SI , Levine CR , DiRocco AM , Battles JJ , Bruns TD . Ectomycorrhizal fungal spore bank recovery after a severe forest fire: some like it hot . Isme J . 2016 ; 10 ( 5 ): 1228 ± 39 . doi: 10 .1038/ismej. 2015 . 182. ISI:000374377200019. PMID: 26473720
49. Kanetani S , Gyokusen K , Ito S , Saito A . The distribution pattern of Pinus armandii Franch. var. amamiana Hatusima around Mt. Hasha-dake in Yaku-shima Island (in Japanese) . J Jpn For Soc . 1997 ; 79 : 160 ± 3 .
50. Kanetani S , Gyokusen K , Ito S , Saito A . The floristic composition of Pinus armandii var. amamiana forests on Yaku-shima Island, southwestern Japan (in Japanese) . Res Bull Kagoshima Univ For . 2010 ; 37 : 49 ± 61 .
51. Murata M , Nara K. Ectomycorrhizal fungal communities at different soil depths in a forest dominated by endangered Pseudotsuga japonica (in Japanese) . J Jpn For Soc . 2017 .
52. Izzo A , Canright M , Bruns TD . The effects of heat treatments on ectomycorrhizal resistant propagules and their ability to colonize bioassay seedlings . Mycol Res . 2006 ; 110 : 196 ± 202 . doi: 10 .1016/j.mycres. 2005 . 08 .010. ISI:000235919200008. PMID: 16387485
53. Peay KG , Garbelotto M , Bruns TD . Spore heat resistance plays an important role in disturbance-mediated assemblage shift of ectomycorrhizal fungi colonizing Pinus muricata seedlings . J Ecol . 2009 ; 97 : 537 ± 47 . doi: 10 .1111/j.1365- 2745 . 2009 . 01489 .x. ISI:000265035400016.
54. Kipfer T , Egli S , Ghazoul J , Moser B , Wohlgemuth T. Susceptibility of ectomycorrhizal fungi to soil heating . Fungal Biology . 2010 ; 114 : 467 ± 72 . doi: 10 .1016/j.funbio. 2010 . 03 .008. ISI:000279389000009. PMID: 20943157
55. Grubisha LC , Trappe JM , Molina R , Spatafora JW . Biology of the ectomycorrhizal genus Rhizopogon . VI. Re-examination of infrageneric relationships inferred from phylogenetic analyses of ITS sequences . Mycologia . 2002 ; 94 : 607 ± 19 . ISI:000177089800006. PMID: 21156534
56. Wen ZG , Murata MS , Xu ZY , Chen YH , Nara K. Ectomycorrhizal fungal communities on the endangered Chinese Douglas-fir (Pseudotsuga sinensis) indicating regional fungal sharing overrides host conservatism across geographical regions . Plant Soil . 2015 ; 387 : 189 ± 99 . doi: 10 .1007/s11104-014- 2278- 3 . ISI: 000348318100014 .
57. Watanabe A , Shiraishi S. Classification of Haploxylon distributed in Asia using DNA sequences (in Japanese) . Transactions of Kyushu Branch of the Jpn For Soc . 1996 ; 49 : 57 ± 8 .
58. Kanetani S , Kawahara T , Kanazashi A , Yoshimaru H. Diversity and conservation of genetic resources of an endangered Japanese five-needle pine species, Pinus armandii Franch . var. amamiana (Koidz.) Hatusima. USDA Forest Service Proceedings . 2004 ;RMRS-P- 32 : 188 ± 91 .
59. Molina R , Trappe JM , Grubisha LC , Spatafora JW . Rhizopogon. In: J.W.G. C, S.M. C, editors. Ectomycorrhizal Fungi: Key Genera in Profile . Berlin: Springer Verlag; 1999 . p. 129 ± 61 .
60. Peay KG , Schubert MG , Nguyen NH , Bruns TD . Measuring ectomycorrhizal fungal dispersal: macroecological patterns driven by microscopic propagules . Mol Ecol . 2012 ; 21 : 4122 ± 36 . doi: 10 .1111/j. 1365 - 294X . 2012 . 05666 .x. ISI:000306897500019. PMID: 22703050
61. Hynson NA , Merckx VSFT , Perry BA , Treseder KK . Identities and distributions of the co-invading ectomycorrhizal fungal symbionts of exotic pines in the Hawaiian Islands . Biol Invasions . 2013 ; 15 ( 11 ): 2373 ± 85 . doi: 10 .1007/s10530-013-0458- 3 . ISI: 000325555300003 .
62. Nara K. Spores of ectomycorrhizal fungi: ecological strategies for germination and dormancy . New Phytol. 2009 ; 181 : 245 ±8. doi: 10 .1111/j.1469- 8137 . 2008 . 02691 .x. ISI:000261792900002. PMID: 19121026
63. Wu QX , Mueller GM , Lutzoni FM , Huang YQ , Guo SY . Phylogenetic and biogeographic relationships of eastern Asian and eastern north American disjunct Suillus species (Fungi) as inferred from nuclear ribosomal RNA ITS sequences . Mol Phylogen Evol . 2000 ; 17 ( 1 ): 37 ± 47 . doi: 10 .1006/mpev. 2000 .0812. ISI:000089952700005. PMID: 11020303
64. Hirose D , Shirouzu T , Tokumasu S. Host range and potential distribution of ectomycorrhizal basidiomycete Suillus pictus in Japan . Fungal Ecol . 2010 ; 3 ( 3 ): 255 ± 60 .
65. Liao HL , Chen Y , Vilgalys R. Metatranscriptomic Study of Common and Host-Specific Patterns of Gene Expression between Pines and Their Symbiotic Ectomycorrhizal Fungi in the Genus Suillus . PLoS Genet . 2016 ; 12 ( 10 ). ARTN e1006348 doi: 10.1371/journal.pgen.1006348. ISI:000386683300017. PMID: 27736883
66. Kikuchi J , Futai K. Spatial distribution of sporocarps and the biomass of ectomycorrhizas of Suillus pictus in a Korean pine (Pinus koraiensis) stand . J For Res . 2003 ; 8 ( 1 ): 17 ± 25 .
67. Zhang R , Mueller MM , Shi X , Liu P . Two new species in the Suillus spraguei complex from China . Mycologia . 2017 ; 109 ( 2 ): 296 ± 307 . https://doi.org/10.1080/00275514. 2017 .1305942 PMID: 28463625
68. Hayashi Y. Taxonomical and phytogeographical study of Japanese conifers . Tokyo: Norin; 1960 .