Specialized mycorrhizal association between a partially mycoheterotrophic orchid Oreorchis indica and a Tomentella taxon

Abstract

The evolution of full mycoheterotrophy in orchids likely occurs through intermediate stages (i.e., partial mycoheterotrophy or mixotrophy), in which adult plants obtain nutrition through both autotrophy and mycoheterotrophy. However, because of its cryptic manifestation, partial mycoheterotrophy has only been confirmed in slightly more than 20 orchid species. Here, we hypothesized that Oreorchis indica is partially mycoheterotrophic, since (i) Oreorchis is closely related to leafless Corallorhiza, and (ii) it possesses clustered, multi-branched rhizomes that are often found in fully mycoheterotrophic orchids. Accordingly, we investigated the nutritional modes of O. indica in a Japanese subboreal forest by measuring the 13C and 15N abundances and by community profiling of its mycorrhizal fungi. We found that O. indica mycorrhizal samples (all 12 samples from four individuals) were predominantly colonized by a single OTU of the obligate ectomycorrhizal Tomentella (Thelephoraceae). In addition, the leaves of O. indica were highly enriched in both 13C and 15N compared with those of co-occurring autotrophic plants. It was estimated that O. indica obtained 44.4 ± 6.2% of its carbon from fungal sources. These results strongly suggest that in the Oreorchis-Corallorhiza clade, full mycoheterotrophy evolved after the establishment of partial mycoheterotrophy, rather than through direct shifts from autotrophy.

Full text

Vol.:(0123456789)
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Mycorrhiza
https://doi.org/10.1007/s00572-020-00999-z
SHORT NOTE
Specialized mycorrhizal association betweenapartially
mycoheterotrophic orchid Oreorchis indica andaTomentella taxon
KenjiSuetsugu1 · TakashiF.Haraguchi2,3· AkifumiS.Tanabe4· IchiroTayasu2
Received: 22 May 2020 / Accepted: 21 October 2020
© Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
The evolution of full mycoheterotrophy in orchids likely occurs through intermediate stages (i.e., partial mycoheterotrophy
or mixotrophy), in which adult plants obtain nutrition through both autotrophy and mycoheterotrophy. However, because of
its cryptic manifestation, partial mycoheterotrophy has only been confirmed in slightly more than 20 orchid species. Here,
we hypothesized that Oreorchis indica is partially mycoheterotrophic, since (i) Oreorchis is closely related to leafless Cor-
allorhiza, and (ii) it possesses clustered, multi-branched rhizomes that are often found in fully mycoheterotrophic orchids.
Accordingly, we investigated the nutritional modes of O. indica in a Japanese subboreal forest by measuring the 13C and
15N abundances and by community profiling of its mycorrhizal fungi. We found that O. indica mycorrhizal samples (all 12
samples from four individuals) were predominantly colonized by a single OTU of the obligate ectomycorrhizal Tomentella
(Thelephoraceae). In addition, the leaves of O. indica were highly enriched in both 13C and 15N compared with those of co-
occurring autotrophic plants. It was estimated that O. indica obtained 44.4±6.2% of its carbon from fungal sources. These
results strongly suggest that in the Oreorchis-Corallorhiza clade, full mycoheterotrophy evolved after the establishment of
partial mycoheterotrophy, rather than through direct shifts from autotrophy.
Keywords Calypsoeae· Corallorhiza· Ectomycorrhizal fungi· 13C natural abundance· 15N natural abundance·
Mycorrhiza· Orchidaceae· Partial mycoheterotrophy· Tomentella
Introduction
Most land plants, from liverworts to angiosperms, form myc-
orrhizal mutualistic relationships wherein fungal partners
receive photosynthetic C from associated plants; in turn,
plants receive N, P, or water gathered by the fungal mycelia
(van der Heijden etal. 2015). However, in some cases, plants
also depend on fungi as essential sources of C by reversing
the polarity of carbon movement (Leake 1994).
Orchids are among the most prevalent of the described
mycoheterotrophic taxa. Indeed, all orchids are initially
mycoheterotrophic during early life stages because their
seeds lack endosperm and possess embryos with only mar-
ginal C reserves (Merckx 2013; Dearnaley etal. 2016). This
characteristic is notable given that Orchidaceae is one of the
most species-rich families in the plant kingdom, with more
than 28,000 known species spanning 763 genera (Christen-
husz and Byng 2016). In fact, orchids comprise nearly half
of the fully mycoheterotrophic plant species reported to date
(Merckx 2013).
Interestingly, many fully mycoheterotrophic orchids have
been reported to associate with ectomycorrhizal (ECM)
fungi that are also connected with surrounding trees and
shrubs (Taylor and Bruns 1997, 1999; Selosse etal. 2002;
Roy etal. 2009; Okayama etal. 2012; but also see Ogura-
Tsujita etal, 2009; Suetsugu etal. 2020). Fully mycohetero-
trophic monotropoids and pyroloids (Ericaceae) have also
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s0057 2-020-00999 -z) contains
supplementary material, which is available to authorized users.
* Kenji Suetsugu
kenji.suetsugu@gmail.com
1 Department ofBiology, Graduate School ofScience, Kobe
University, Kobe, Hyogo657-8501, Japan
2 Research Institute forHumanity andNature, Kita-ku,
Kyoto603-8047, Japan
3 Biodiversity Research Center, Research Institute
ofEnvironment, Agriculture andFisheries, Osaka Prefecture,
10-4 Koyamotomachi, Osaka572-0088Neyagawa, Japan
4 Graduate School ofLife Sciences, Tohoku University,
Sendai, Miyagi980-8578, Japan

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been reported to have such ECM associations (Bidartondo
and Bruns 2001; Hynson etal. 2009). These ECM taxa, such
as Sebacina, Russulaceae, and Thelephoraceae, as well as
some ECM-forming Ceratobasidiaceae, are also described
as mycobionts of some green orchids exhibiting a nutritional
mode that combines both autotrophy and mycoheterotrophy
(i.e., partial mycoheterotrophy or mixotrophy; Gebauer and
Meyer 2003; Bidartondo etal. 2004; Selosse etal. 2004).
In contrast, most green orchids associate with rhizocto-
nias, a polyphyletic basidiomycetes group that is typically
saprophytic or endophytic (i.e., Tulasnellaceae, Ceratoba-
sidiaceae, and some Sebacinales; Dearnaley etal. 2012).
These studies suggest that mycorrhizal shifts from non-ECM
rhizoctonias to ECM fungi often precede the evolution of
full mycoheterotrophy (Hynson etal. 2013).
Partial mycoheterotrophy was first observed in Cepha-
lanthera and Epipactis (tribe Neottieae) after identifying
achlorophyllous individuals within otherwise green species
(Selosse etal. 2004; Julou etal. 2005; Selosse and Roy 2009;
Hynson etal. 2013) and the measurement of natural 13C and
15N abundances that differed from those of either autotrophic
or fully mycoheterotrophic plants (Gebauer and Meyer 2003;
Hynson etal. 2013). Partial mycoheterotrophy has been
intensively investigated in the tribe Neottieae (Gebauer and
Meyer 2003; Bidartondo etal. 2004; Selosse etal. 2004;
Selosse and Roy 2009; Suetsugu etal. 2017), and it has
also been reported in the tribes Cymbidieae and Orchideae
(Motomura etal. 2010; Yagame etal. 2012; Gebauer etal.
2016; Schiebold etal. 2018). Partial mycoheterotrophy may
also be implemented by a pale greenish leafless Corallorhiza
trifida (tribe Epidendreae, subtribe Calypsoeae; Chase etal.
2015), whereas all other Corallorhiza species are fully
mycoheterotrophic. Because of its leafless habit, C. trifida
is sometimes considered fully mycoheterotrophic (McKend-
rick etal. 2000; Cameron etal. 2009), but 13C measurements
suggest that the species is likely partially mycoheterotrophic,
obtaining about 25% of its C by photosynthesis (Zimmer
etal. 2008).
It is also possible that Oreorchis, the sister genus of Cor-
allorhiza, is partially mycoheterotrophic, as partially myco-
heterotrophic orchids are often phylogenetically close to full
mycoheterotrophs (Selosse and Roy 2009). Oreorchis is a
small genus of approximately 16 species that are broadly
distributed from the Himalayas across China to Taiwan,
eastern Siberia, Korea, and Japan (Pearce and Cribb 1997)
and includes Oreorchis indica (syn. Kitigorchis itoana;
Yukawa etal. 2003). Kitigorchis itoana was once considered
endemic to a very narrow range in central Japan and was
regarded as the sole member of the genus (Pearce and Cribb
1997). Pearce and Cribb (1997) suggested that clustered,
multi-branched coralloid rhizomes of K. itoana differed
from subterranean organs (i.e., normal roots) of Oreorchis
and speculated that Kitigorchis represented an intermediate
between Corallorhiza and Oreorchis. However, clustered,
multi-branched rhizomes may represent a plesiomorphic
character in the subtribe Calypsoeae, as some leafy gen-
era (e.g., Cremastra) possess similar rhizomes; hence, the
generic status of Kitigorchisshould not besupported based
on this single characteristic. Indeed, based on herbarium
specimens and species descriptions, Yukawa etal. (2003)
concluded that K. itoana and O. indica were conspecific
(Yukawa etal. 2003).
However, notably, O. indica often possesses coralloid
rhizomes that are a hallmark of mycoheterotrophic plants
(Fig.1; Leake 1994; Imhof etal. 2013). In fact, as suggested
by Pearce and Cribb (1997), the dual subterranean system of
O. indica (i.e., coralloid rhizomes like in Corallorhiza and
normal roots) indicates that the species is at an intermedi-
ate stage in mycoheterotrophic evolution. Therefore, given
that (i) Oreorchis is closely related to leafless Corallorhiza
and (ii) it possesses coralloid rhizomes often found in fully
mycoheterotrophic orchids, we investigated the physiologi-
cal ecology of O. indica to determine whether this species is
partially mycoheterotrophic, based on the molecular identi-
fication of mycorrhizal fungi and the abundance of the 13C
and 15N natural isotopes.
Material andmethods
Study species andsampling locality
The study was conducted in Kitayama, Fujinomiya City,
Shizuoka Prefecture, Japan. The study site is a subboreal
forest (ca. alt. 2200m) dominated by Tsuga diversifolia,
Abies veitchii, and Picea jezoensis var. hondoensis with
sparsely distributed Betula ermanii and Alnus maximowiczii.
We collected leaves, coralloid rhizomes, and roots of four
O. indica individuals and applied molecular and isotopic
analysis to the same individuals. To avoid lethal sampling,
we collected minimum coralloid rhizome and root samples
required for molecular analysis as follows; 2–4 independ-
ent coralloid rhizome and root fragments were harvested by
digging about 20cm away from shoots and then carefully
approaching underground parts of the plant from one side;
after sampling, the hole was refilled with the same soil. The
study site contained about 100 flowering individuals of O.
indica in the investigated year.
Molecular analysis
The excised coralloid rhizomes and roots were investigated
to determine the presence of mycorrhizal colonization under
the light microscope. We picked up mycorrhizal tissues of
both roots and rhizomes (2–3mm in length) whose cells

Mycorrhiza
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were filled with fungal pelotons (Fig.1).Individual colo-
nized root and coralloid rhizome fragments were washed to
remove small particles on the root surface and were surface
sterilized by 1% sodium hypochlorite. In total, the DNA was
extracted from 12 mycorrhizal samples from four individuals
(eight coralloid rhizome samples from four individuals and
four root samples from three individuals), using the cetyltri-
methylammonium bromide (CTAB) method.
We amplified the nuclear internal transcribed spacer (ITS)
region of mycorrhizal fungi using the fungal specific primer
set ITS86F/ITS4 (Gollotte etal. 2004) fused with 3–6-mer
Ns and with the Illumina forward/reverse sequencing primer
by polymerase chain reaction (PCR). For the PCR, Q5 High-
Fidelity DNA Polymerase kits (NEB, Ipswich, USA) were
used, with a temperature profile of 98°C for 3min, fol-
lowed by 35 cycles at 98°C for 10s, 58°C for 20s, 72°C
Fig. 1 Oreorchis indica. a Flowering plants. Bar = 3 cm. b Flow-
ering plant with its underground system. Bar = 3 cm. c Flowers.
Bar=5mm. d Underground system (corm, roots, and multi-branched
coralloid rhizomes). Bar = 1 cm. e Cross section of rhizome with
abundant peloton formation in cortical cells. Bar = 200 m. f Cross
section of root with abundant peloton formation in cortical cells.
Bar=200m

Mycorrhiza
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for 20s, and a final extension at 72°C for 10min. In addi-
tion, to add Illumina sequencing adapters to the respective
samples, supplemental PCR was performed using forward
fusion primers comprising the P5 Illumina adapter, 8-mer
indices for sample identification, a partial sequence of the
sequencing primer, and reverse fusion primers comprising
the P7 adapter, 8-mer indices, and a partial sequence of the
sequencing primer. The same Q5 kit was used, with a tem-
perature profile of 98°C for 3min, followed by 12 cycles
at 98°C for 10s, 65°C for 20s, 72°C for 20s, and a final
extension at 72°C for 10min. Equal volumes of the PCR
amplicons of the samples were pooled and purified using the
AMPure XP Kit (Beckman Coulter, CA, USA). The ratio of
AMPure reagent to amplicons was set to 0.6 (v/v) in order
to remove primer dimers (i.e., DNA fragments shorter than
200bp).
Bioinformatics
The sequencing libraries of fungal ITS were processed in
an Illumina MiSeq sequencer with MiSeq Reagent Micro
Kit v2 (300cycles, Illumina, USA) at an 8-pM loading con-
centration with 10% PhiX spike-in. The sequence data were
deposited in the Sequence Read Archive of the DNA Data
Bank of Japan (accession number: DRA011027).
After sequencing, non-demultiplexed FASTQ files of
forward (270bp), index1 (8bp), index2 (8bp), and reverse
reads (30bp) were generated using bcl2fastq v1.8.4 pro-
vided by Illumina (last bases of forward and reverse reads
were truncated). Then, non-demultiplexed FASTQ files
were demultiplexed based on index1, index2, and forward
and reverse primer position matching, and the forward
and reverse primer positions were eliminated using the
clsplitseq command of Claident v0.2.2018.05.29 (Tanabe
2018). In this step, no mismatches in index1 and index2
were tolerated; 14 and 15% of mismatches were tolerated in
the forward and reverse primer positions, respectively, and
forward and reverse sequence reads associated with low-
quality (quality score < 30) index reads were discarded.
The 3′ tails of demultiplexed forward reads were truncated
until all quality scores exceeded 29 in 3-mer length sliding
window. Truncated forward reads shorter than 230bp and/
or containing 10% or more low-quality (quality score <30)
positions were discarded, and truncated forward reads longer
than 230bp were trimmed to 230bp. Erroneous reads were
also removed based on the CD-HIT-OTU method (Li etal.
2012) which is re-implemented in the clcleanseqv command
in Claident. The remaining forward reads were clustered
with a sequence identity cutoff of 97% using VSEARCH
v2.8.0 (Rognes etal. 2016) via the clclassseqv command
in Claident, and these clusters were treated as OTUs in this
study. De novo and reference-based chimera removal based
on the UCHIME (Edgar etal. 2011) algorithm implemented
in VSEARCH was also applied to the OTUs. The UNITE
database for UCHIME ver.7.2 was used as reference for
UCHIME (Nilsson etal. 2019). Taxonomic assignment
of OTUs was performed based on the query-centric auto-
k-nearest-neighbor (QCauto) algorithm (Tanabe and Toju
2013) and the lowest common ancestor (LCA) algorithm
(Huson etal. 2007) via the clmakecachedb, clidentseq, and
classigntax commands in Claident. The “overall_genus” ref-
erence database was used; it contains all genus-level identi-
fied sequences from the NCBI nt database. Relaxed LCA,
which tolerates 20% of unmatched neighborhood sequences
and provides the 80% majority-rule consensus of neighbor-
hood sequences, was applied. The OTUs representing only
one sequence in each individual were excluded (Brown etal.
2015). After that, the functional guild of each fungal OTU
was estimated based on the FUNGuild database (Nguyen
etal. 2016).
Stable isotope analysis
The relative abundance of 13C and 15N in plants often var-
ies with their nutritional modes. Mycoheterotrophic plants
generally have enriched 13C and 15N compared with co-
occurring autotrophic plants, and this pattern is mainly
attributed to the relatively high 13C and 15N abundance of
fungal symbionts. Mycoheterotrophic plants exploiting ECM
fungi usually have a higher relative abundance of 15N than
mycoheterotrophs exploiting wood- or litter-decaying sap-
rotrophic fungi (e.g., Ogura-Tsujita etal. 2009) because sap-
rotrophic fungi generally contain less 15N than ECM fungi.
Thus, stable isotope ratios of mycoheterotrophic plants can
be useful tools for estimating their nutritional source.
Here, we conducted stable C and N isotope analysis to
identify the nutritional source of O. indica. First, we set
four quadrats of 1×1m around O. indica. We sampled
the leaves of O. indica and other understory plants that
were found in all the quadrat, as reference plants. We
used this strategy to limit the influence of environmen-
tal factors, such as atmospheric CO2 isotope composi-
tion, the microscale light climate and soil type (Gebauer
and Schulze 1991). The collected leaves were dried at
60°C for 4days, and then ground using scissors and an
agate mortar. The abundances of the stable 13C and 15N
isotopes and C and N concentrations, were measured at
the Research Institute for Humanity and Nature (Kyoto,
Japan) using theDelta V Advantage mass spectrometer
connected to a Flash EA 1112 elemental analyzer via
the ConFlo IV interface (all Thermo Fisher Scientific,
Waltham, MA, USA). The relative abundances of the sta-
ble isotopes were calculated as δ15N or δ13C=(Rsample
/Rstandard−1)×1000 [‰], where Rsample represents

Mycorrhiza
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the 13C/12C or 15N/14N ratio of the sample, respectively,
and Rstandard represents the 13C/12C ratio of Vienna Pee
Dee Belemnite or the 15N/14N ratio of atmospheric
N. The C and N isotope ratios were calibrated using
three laboratory standards: CERKU-01 (dl -alanine,
δ13C=−25.36‰, δ15N=−2.89‰), CERKU-02 (l-ala-
nine, δ13C=−19.04‰, δ15N=22.71‰), and CERKU-
03 (glycine, δ13C=−34.92‰, δ15N=2.18‰), which
are traceable back to the international standards (Tayasu
etal. 2011). The analytical standard deviations (SDs)
were 0.06‰ (δ13C, n=7) and 0.16‰ (δ15N, n=10) for
CERKU-01, 0.025‰ (δ13C, n=5) and 0.33‰ (δ15N,
n=10) for CERKU-02, and 0.025‰ (δ13C, n= 4) and
0.04‰ (δ15N, n=2) for CERKU-03. The total N con-
centrations of the leaf samples were calculated using the
sample weights and the CO2 and N2 gas volumes of the
laboratory standards (Tayasu etal. 2011).
We compared δ13C and δ15N values of O. indica and
autotrophic reference plants using a linear mixed model.
We fitted “plant identity” as a fixed term and “plot” as a
random term in the generalized linear mixed model. We
then used a post hoc pairwise comparison test (Tukey’s
honest significant difference (HSD) to assess whether
the values of O. indica and autotrophic reference plants
differ significantly from each other. In addition, enrich-
ment factors (ε) were calculated from the δ values of each
plant group based on ε=δS−δREF, where δS represents
the δ13C or δ15N value of O. indica and δREF represents
the mean value of all autotrophic reference plants from a
specific sampling plot (Preiss and Gebauer 2008).
Results
Molecular identification ofmycobionts
Community profiling based on the metabarcoding technique
revealed only two fungal OTUs in the mycorrhizal samples.
All mycorrhizal samples (12 samples from four individuals)
were predominantly colonized by one OTU belonging to
the ECM basidiomycete genus Tomentella (Thelephoraceae;
19,293 sequencing reads). The other OTU, belonging to root
endophytic Helotiales, was detected in only one coralloid
rhizome sample (19 sequencing reads). No other fungal
OTUs were detected in the remaining samples.
Stable isotope analysis
The δ13C values of O. indica (−29.0±0.2‰; mean ± SD)
were significantly higher than those of autotrophic refer-
ence plants Parasenecio adenostyloides and Maianthemum
dilatatum (P<0.001; −32.6±1.0‰; Fig.2, TableS1). In
addition, the δ15N values of O. indica (2.6±1.1‰) were
significantly higher than those of autotrophic reference
plants (−6.9±0.9‰; P<0.001). The 13C and 15N enrich-
ment factors of O. indica were 3.6±0.5‰ and 9.5±1.5‰.
Using the mean value of the published enrichment factors
in fully mycoheterotrophic orchids (ε13C= 8.0‰ and
ε15N=11.5‰; Hynson etal. 2016), the fungal-derived C
and N proportions in O. indica was estimated as 44.4±6.2%
and 82.6±12.6%, respectively (Hynson etal. 2016). The
total N concentrations of O. indica leaves (3.0±0.3%) were
Fig. 2 Mean (± standard devia-
tion) of δ13C and δ15N values in
the leaves of Oreorchis indica
and its neighboring autotrophic
plants Parasenecio adeno-
styloides and Maianthemum
dilatatum

Mycorrhiza
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significantly higher than those of the leaves from autotrophic
reference plants (1.7±0.7%; P<0.01).
Discussion
Community profiling revealed that the analyzed O. indica
rhizomes and roots were almost exclusively colonized by
a single Tomentella OTU (Thelephoraceae) belonging to
the obligate ECM basidiomycete genus. Isotopic analysis
showed that the orchid was partially mycoheterotrophic,
obtaining almost 50% of its C from its fungal partner.
The abundance of 13C and 15N in O. indica provided
insight into its nutritional mode. Oreorchis indica dis-
played significantly higher δ13C (3.6±0.5‰) and δ15N
(9.4±1.5‰) values than those of co-occurring autotrophic
plants, indicating that the orchid was partially mycohetero-
trophic, obtaining nearly half of its C from Tomentella spe-
cies. It is also known that both fully and partially mycohet-
erotrophic plants that exploit ECM fungi are significantly
enriched in 15N, possibly because the fungi themselves
typically contain more 15N than 13C (Gebauer and Meyer
2003; Ogura-Tsujita etal. 2009; Hynson etal. 2016). In
fact, the enrichment factor of O. indica is in accordance
with the mean enrichment factors reported for other par-
tially mycoheterotrophic orchids exploiting ECM fungi
(3.2± 0.2‰ in ε13C, n=189 and 9.6 ±0.3‰ in ε15N,
n=197; Hynson etal. 2016). Using the mean value of the
published enrichment factors in fully mycoheterotrophic
orchids (ε13C=8.0‰ and ε15N=11.5‰; (Hynson etal.
2016), it was estimated that O. indica obtained 44.4±6.2%
and 82.5± 12.6% of its C and N, respectively, from its
fungal associations. These values agree with the mean val-
ues reported for other partially mycoheterotrophic orchids
exploiting ECM fungi (39.6% and 82.6%; Hynson etal.
2016). In contrast, rhizoctonia-associated orchids, such
as Ophrys insectifera and Platanthera bifolia, have been
reported to obtain less C (ca. 20%) from mycorrhizal fungi
(Schweiger etal. 2018). Hence, O. indica can achieve higher
fungal exploitation by switching its mycorrhizal association
from saprotrophic rhizoctonias to ECM fungi.
Interestingly, in Corallorhiza, the sister genus of Ore-
orchis, the association with members of Thelephoraceae
represents the ancestral condition (Taylor and Bruns 1997,
1999; Zimmer etal. 2008; Barrett etal. 2010; Freudenstein
and Barrett 2014). For example, C. trifida forms specialized
interactions with Thelephora–Tomentella complex from seed
germination to maturity (McKendrick etal. 2000; Zimmer
etal. 2008). These findings might indicate that the establish-
ment of symbioses by Oreorchis with ECM Thelephoraceae
taxa serves as a pre-adaptation for the evolution of full myco-
heterotrophy in Corallorhiza. Given that approximately 15%
of the net C fixed by ECM trees is allocated to their fungal
partners (Finlay and Söderström 1992), a stable and abun-
dant supply of C from ectomycorrhizae may be a principal
prerequisite for increasing heterotrophy (McKendrick etal.
2000; Zimmer etal. 2008). In fact, similar mycorrhizal shifts
have also occurred in several other orchid genera, including
Cephalanthera, Cymbidium, Neottia, and Platanthera (e.g.,
Gebauer and Meyer 2003; Bidartondo etal. 2004; Motomura
etal. 2010; Yagame etal. 2012).
It is noteworthy that O. indica is a specialist to the Tomen-
tella taxon, as many leafy partially mycoheterotrophic
orchids are generally associated with multiple ECM fungi
and also occasionally with rhizoctonias (Dearnaley etal.
2012). For example, Ogura-Tsujita etal. (2012) reported
that putatively initially mycoheterotrophic Cymbidium daya-
num is dependent on saprotrophic Tulasnellaceae, whereas
partially mycoheterotrophic C. goeringii and C. lancifolium
associate with both saprotrophic Tulasnellaceae and several
ECM families. Furthermore, nearly mycoheterotrophic C.
macrorhizon and C. aberrans exhibit specialized interactions
with ECM Sebacina (Ogura-Tsujita etal. 2012; Suetsugu
etal. 2018). Therefore, Ogura-Tsujita etal. (2012) suggested
that gradual shifts in fungal partners occur through phases
in which ECM fungi and saprotrophic rhizoctonias coexist
and that such phases play a crucial role in the evolution of
mycoheterotrophy. Despite a partially mycoheterotrophic
nutritional mode, a leafless mixotrophic species including
not only C. macrorhizon and C. aberrans but also C. trifida
associate predominantly with narrow clades of ECM fungi
(Zimmer etal. 2008; Ogura-Tsujita etal. 2012). The strict
specialization towards certain ECM fungi likely reflects
evolution toward full mycoheterotrophy in these leafless
species more than in leafy partially mycoheterotrophic spe-
cies (Zimmer etal. 2008; Ogura-Tsujita etal. 2012). In con-
trast, the specialization to narrow ECM fungi in leafy O.
indica is exceptional (but also see Yagame etal. 2012). The
inferred high mycorrhizal specificity of O. indica might be
exaggerated if the single OTU contained multiple biologi-
cal species and/or some additional fungi were not amplified
due to primer bias. However, we consider that a large error
due to these reasons is unlikely, since DNA metabarcoding
with the selected primer pair (ITS86F/ITS4) is known to
be highly suitable for studying fungal diversity (e.g., Waud
etal. 2014, Beeck etal. 2014). Therefore, it is worth investi-
gating whether the dual association of saprotrophic rhizocto-
nias and ECM fungi or diverse ECM associations arise first
in other Oreorchis species, based on a credible phylogenetic
framework.
Notably, although O. indica is widely distributed from
the western Himalayas to Japan, it is extremely rare on a
local scale (Yukawa etal. 2003). In Japan, O. indica popu-
lations are typically small (tens of individuals), isolated,
and prone to local extinction; as such, the species has been
classified as critically endangered at the national level

Mycorrhiza
1 3
(Ministry of Environment of Japan 2015). The dependence
of O. indica on a single Tomentella taxon may contribute
to its rarity. McCormick etal. (2009) reported that fewer
ECM trees and root tips hosted Tomentella species in areas
with few or no C. odontorhiza, whereas one of the domi-
nant Tomentella taxa in C. odontorhiza rhizomes was only
found immediately adjacent to C. odontorhiza. Therefore,
the abundance of specific mycobionts may be a critical
factor in determining the distribution of C. odontorhiza
(McCormick etal. 2009). However, high mycorrhizal
specificity does not lead to rarity or restricted geographic
ranges if the associated fungus is widely and abundantly
distributed (Ogura-Tsujita and Yukawa 2008; McCor-
mick and Jacquemyn 2014). Further studies are needed
to investigate the distribution patterns and abundance of
associated Tomentella species and determine the effect of
mycorrhizal symbiosis on the rarity of O. indica.
Overall, our results support that O. indica is a partially
mycoheterotrophic orchid. Given that Oreorchis is sister
to the leafless genus Corallorhiza, it is likely that a fungal
shift served as a pre-adaptation for the evolution of full
mycoheterotrophy in the Oreorchis-Corallorhiza clade.
Future studies are needed to investigate both the degree of
mycoheterotrophy and properties of fungal associations in
other Oreorchis and Corallorhiza species, based on plau-
sible phylogenetic relationships.
Acknowledgments We thank Masayuki Sato for his help with the field
study. We also thank Takako Shizuka and Hidehito Okada for valuable
support in fungal DNA analysis.
Funding This work was financially supported by the JSPS KAKENHI
[Grant Numbers 17H05016 (KS) and 16H02524 (IT)], Joint Research
Grant for the Environmental Isotope Study of Research Institute for
Humanity and Nature, and Joint Usage/Research Grant from the Center
for Ecological Research, Kyoto University.
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