Posted on Authorea 17 May 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.162126337.74672794/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. o ln pce htciial eedo yoria yboi o edgriainadseln esta- seedling favorable and of germination portion seed influential an for 2007). represent symbiosis may al., mycorrhizal fungi et mycorrhizal Poulsen on appropriate 2007; depend of al., availability critically et the majority blishment, (Clark that the limited species that seed indicating plant extent recruitment, For some habitats seedling to increased addition because are supplemental in recruit that results populations to shown always plant have fail almost of experiments but populations addition dispersed, to and seeds widely introduction al.,2007; of seed Johansson are et (M¨unzbergov´a Most 2005; seeds microsite-limited (Clark 2013). Herben, or limited hand, Eriksson, & habitats, habitat- seed other & suitable considered or the reach are dispersal plants on to considered & unsuitable, If, fail are Nathan are plants seeds 2007). 1992; and When al., al., sites 2014). et (Conditet these al., Poulsen plants at et occur of Getzin will patterns 2002; recruitment distribution microsites Smith, no and favorable & into recruitment Wang seeds shaping 2000; of step Muller-Landau, abundance Dispersal critical and 2007). al., first distribution et the a Clark Ehrl´en, affect is 1992; & that (Eriksson factors populations major plant represent of limitation recruitment and dispersal Both INTRODUCTION bamboo 1. the within randomly distributed are adults. fungi the confusoides provide litter-decaying G. results of Our from not abundance independently plants. and but adult adult and distribution the from litter, forest spatial distance from genus the the distance the to the in the that related mycorrhizal to from abundance evidence significantly related in not fungus first Mycena significantly also switch a was not and germination with was distinct germination consequently abundance associated and a between Fungal plants, adults observed required abundance. while Gymnopus was confusoides fungus, and relationship G. germination show Mycena positive between in results single strong, stage Our a A adult with germination. seed to associated an Gymnopus. of protocorms analyses to patterns which growth (qPCR) spatial in Miseq subsequent PCR affected partners, using quantitative and fungi plants mycorrhizal with germination adult of experiments seed and abundance test that germination seeds To the developing seed random extent forests. with what combined its bamboo associating to within we fungi, investigate fungi fungi litter-decaying Next, mycorrhizal litter-decaying on of main sequencing. parasitic patterns the scattered is identified high-throughput highly first confusoides to we random G. due have hypothesis, Since be seedling to this may of tend forests. distribution patterns often of bamboo confusoides spatial patterns within nonrandom Gastrodia spatial establishment orchid distribution in mycoheterotrophic seedling resulting of fully and plants, patterns the adult germination spatial of from that individuals distance shown contrast, have increasing In works with establishment. decline Previous often conditions. distribution orchids the natural several constrain under may in thus orchids and establishment, of seedling abundance and germination and orchid for essential are associations Mycorrhizal Abstract 2021 17, May 4 3 2 1 Li Yuan-Yuan orchid mycoheterotrophic fully seed a enhances in substrate germination the in abundance fungal Increasing nvriyo Leuven of Science University Natural of Museum National Wetenschappen Faculteit Protection Leuven Plant Universiteit of Katholieke College University Agricultural China 1 agu Boeraeve Margaux , 2 uHi Cho Yu-Hsiu , 1 3 asJacquemyn Hans , 4 n ugILee Yung-I and , 3 Posted on Authorea 17 May 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.162126337.74672794/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. n amtmeaergosi atr sa(rdene l,20;WS,21) l fte r l fully all are them of All forests. 2014). WCSP, sheltered 2005; and moist al., in et occur (Pridgeon that Asia plants Eastern mycoheterotrophic in regions warm-temperate and genus The germination. species seed Study affected 2.1 abundance fungal METHODS in variation AND distances MATERIAL different spatial at how 2. litter assess bamboo to surrounding and the in plants within germination adult abundance seed from fungal nearest promoting in the fungi variation to mycorrhizal spatial communities distance and investigate the fungal the identify the litter, on to compare that twig to adults than hypothesized sequencing confusoides and and litter high-throughput we leaf protocorms the used we forest, with in bamboo hypothesis, abundance associating bamboo this by fungal test the covered To on 2016). within plant. densely dependent al., adult patterns is more et scattered likely the forests (Kinoshita is highly while survival bamboo germination show litter, for of twig may association and surface this fungi leaf ground on litter-decaying bamboo dependent the of not degradation that likely the Given most through nutrients are to fungi genus access litter-decaying the how gain and of to orchid litter mycoheterotrophic fungi species fully bamboo decaying of the in number of fungi germination A mycorrhizal seed of of . distribution patterns spatial suggesting and plants. to abundance adult 2000), relate the they conspecific al., investigated of et we study, distribution (McKendrick this the m In on 1 than of co-associates scale and ectomycorrhizal development spatial their of a rate from at the of or plants growth germination distributed and of that naturally extent adults In of the the between 2002). of presence relationship al., cm the significant et 15 (McKendrick no within plants was buried adult between there packets from hand, observed seed cm non- between 15–25 were In germination at are development seed buried packets that in seedling carbon. difference and species significant fungal no germination are on was seed which depend adult in orchids, solely containing differences plots mycoheterotrophic and significant 2016). fully life example, al., entire for et for Waud orchids their obtained 2016; adult throughout al., been of fungal photosynthetic et rhizosphere have McCormick rhizoctonia the 2008; results their near Read, with Mixed germination & symbiosis Smith seed with mutualistic 2008; for associations a al., microsites maintain et establish favorable orchids (Cameron possibly Jacquemyn adult create 2007; green way may Diez, most this this 2001; Because photosyntheticsymbionts, in al., 2012). in and et al., establishment (Batty fungi et orchids McCormick seedling mycorrhizal adult 2012a,b; and from 2007, of germination distance al., seed availability increasing et that however, from with the Results scale, shown sites. decline local by within example, often distributed the limited orchids for regularly At not have, rarely 2014). are Jacquemyn, fungi studies was OrM & previous that widespread orchids (McCormick suggesting introduction scale erratic, more of seed often landscape therefore is distribution using a germination studies are the at Previous fungi that and 2018). generally OrM shown al., orchids suitable is et indeed from it (McCormick orchids, have suggests independently with orchids experiments distributed association of are distribution the the without fungi 2018). than well OrM al., grow & et can that (Rasmussen McCormick fungi seed- assumed orchids 2014; (OrM) in Jacquemyn, of mycorrhizal factor & distribution orchid limiting McCormick Because spatial a 2002; al., and al., be et abundance et to Rasmussen McKendrick the considered 2009; 1998; affects is Rasmussen, Whigham, significantly fungi of & mycorrhizal mycorrhizal that on Rasmussen consist suitable recruitment rely 2008; that of ling protocorms availability Read, of seeds natu- the & development Under dust-like Therefore, early (Smith 2000). have the 2015). supplies Ghani, Jac- and Orchids & nutrient & seeds (Arditti for 2018). orchid McCormick dispersal of associations 2007; al., seed germination of al., et the time et conditions, McCormick the Jacquemyn ral at 2016; 2007; endosperm (Diez, al., without embryos orchids et rudimentary for Waud true 2014; particularly quemyn, is this and microsite, Gastrodia efrhrcmie edgriaineprmnswt uniaiePR(PR nlssto analyses (qPCR) PCR quantitative with experiments germination seed combined further We . .trifida C. ossso bu 6seista r ieydsrbtdars h aetois Oceania, Paleotropics, the across distributed widely are that species 66 about of consists .nidus-avis N. edig eed oeo h blt ftefna ybot otase carbon transfer to symbionts fungal the of ability the on more depends seedlings Gastrodia lnsadposstae tlat5mfo h ers dl,btthere but adult, nearest the from m 5 least at situated plots and plants xlsvl nai abofrssadaeprstco litter- on parasitic are and forests bamboo inhabit exclusively 2 Gastrodia oaoriatrifida Coralorrhiza pce aebe eotdto reported been have species atoi confusoides Gastrodia eti nidus-avis Neottia nteother the on , G. , Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. loehr hr ee8 nqesmls( dl otsmls rtcr ape,2 itrsmlsper samples litter 20 samples, protocorm 4 Taiwan). Taichung, samples, Ltd., root Co. adult described (GMbiolab (4 Plant samples manufacturer samples confusoides the root unique the using G. 88 as by protocorms were or sterilized described fragments there surface as root Altogether, were Kit mycorrhizal rinsed protocorms were and g Purification The protocorms 0.1 fragments DNA The plant. from litter Genomic tissue. extracted individual of and was amount each soil DNA small Afterwards, for excess the above. of remove were sample because which to root pooled fragments, water were mycorrhizal mm colonization. plot with 3-5 each homogeneous mycorrhizal in into for packets a sectioned checked seed followed were create from s, microscopically roots to 30 and colonized for water pooled of solution each distilled then portions hypochlorite from sterile 5-cm sodium roots in distal % three 1 the steps Subsequently, least a rinse stored at with 30-s Additionally, immediately sterilized three CA). were surface by were Carlsbad, samples plant Inc., littler adult Laboratories the orchid BIO (ca. and sampled samples (MO bag, litter Kit sampling point, Isolation sampling sterile each DNA a At using S1). taken -80 (5, (Fig. were distances at adult different weight) orchid five fresh sampled at each g west) around east, 10 cm) south, 500 (north, 200, direction 100, cardinal each 50, in collected were samples of Litter rupture communities the mycorrhizal and of observed 2). were Sampling (Fig. formation 2.3 germination mycorrhiza present variable, a were of be 4 seed embryos under signs can checked enlarging 2016, x clear stages carefully by when October germination (distances) and testa occurred In In seed opened have 5 orchid year. examination. then to As total, until 1 water, considered day germination. was tap about In 1 counting with for for for rinsed towel ground microscope m. gently paper the stereo were 5 in in packages moist left seed of maintained of laboratory, were and radius adult packages the washed orchid seed a gently sampled 80 retrieved, within were each = packages (north, orchids around (plots) 53- direction cm) 4 adult x of 500 cardinal neighboring into (directions) 200, each square visible put 100, in a without 50, directly layer within (5, randomly and litter distances placed 10-m harvesting the different were same into after five October the seeds at buried pooled of west) were 500 were beginning east, packages approximately seeds south, the Seed package, effects at all seed population netting. quality, mature potential Per phytoplankton were seed exclude to seeds in packages. order When differences In seed create populations. plants. studied understory may m¯os¯o the few that the from ( Taiwan by harvested only City, bamboo Taichung dominated were with was capsules District, forest soil 2015, Heping germination bamboo in organic The seed located on level. a Whigham forest ) sea & plant, bamboo above Rasmussen by adult a meter described 1000 in the method at conducted package to seed were distance the Experiments using the plots (1993). along four in varied established germination was experiment seed whether investigate To of experiment capsules germination associations with fungal Seed seeds stalks on 2.2 E). the followed depends elongated After 1D, pollination, roots or and (Fig. October. of flowers tubers to fungi month underground Only September litter-decaying one of from with 1C). development after above-ground reach successful quickly found dispersed, and (Fig. be ripen been rapidly October have can Capsules elongate height orchid in 1B). stalks mycoheterotrophic in dispersal flowering fully vary (Fig. the this seed that cm pollination, and 50 stalks successful dehiscence flowering and After by 20 short 1A). between producing (Fig. height September, cm a 5 in 2015). and flowers al., 2 generally et between species Lee The 2009; al., Taiwan. et (Ogura-Tsujita genera partners fungal confusoides mycorrhizal the Gastrodia major of their fungi several litter-decaying as Interestingly, identified and 2008) been 2015). have (Hsu, al., Taiwan et in 2009; Lee forests al., 2010; bamboo et Bougoure, Ogura-Tsujita & 1911; as Dearnaley (Kusano, such 2009; fungi al., wood-decaying et and/or Martos fungi litter-decaying with associate ° o usqetaayi.GnmcDAfo itrsmlswsetatduigtePowerSoil the using extracted was samples litter from DNA Genomic analysis. subsequent for C atoi appendiculata Gastrodia 2 lt o N xrcinadsbeun oeua analysis. molecular subsequent and extraction DNA for plot) ltweerosadlte ape eetkn(i.S) h dl rhd eeselected were orchids adult The S1). (Fig. taken were samples litter and roots where plot saflymchtrtohcoci htocr anyi abofrssi central in forests bamboo in mainly occurs that orchid mycoheterotrophic fully a is , atoi confusoides Gastrodia 3 and atoi nantoensis Gastrodia cu rdmnnl in predominantly occur Mycena hlotcy edulis Phyllostachys Gastrodia and Marasmia μ mesh m species, ® Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. mlcn.I hswy rdc dniywscnre oalsmlscniee ob oiieuigthe using positive generated be of to number considered a samples (Fig. of forall specificity sequencing confirmed product was Sanger confirm identity by to product verified analysis way, was each this accuracy after In mol amplification performed 06 addition, amplicons. + was 1.0E In analysis - C). curve 02 S2A, + melting 1.0E A (range, initial curves (OTU5). Standard follows: triplicate. in as amplified run μ curve were standard a Amplifications using quantified S1). (Table OUT 95 target ° at min the 5 were on for Reactions denaturation depending USA). primer, quantify reverse CA, to 1.0 and 25 Carlsbad, USA), utilized of Technologies, CA, were volume Hercules, (Life assays total Laboratories, instrument qPCR a PCR The in based real-time performed validated S1). Flex and (Table developed 6 region was QuantStudio 2 assay (ITS) (qPCR) spacer PCR Mycena transcribed quantitative internal real-time the a i.e. OTUs, taxa, on fungal both For non-rhizoctonia saprotrophic with (OTU5). distinct associating fungi two OrM to abundant OTU analyses. most corresponded an further the that the in revealed in put sequencing used Miseq were Illumina was results which The abundance numbers), fungal retained. read of containing were Assessment cell sample each 2.6 root with orchid table one OTU least x at (sample on table ( found sequences were short which fami- by OTUs mycorrhizal represented entries. orchid-associating OTUs ( Firstly, uncultured/environmental potential similarity for including process. sequence screened (megablast step-wise 2009), using manually a BLAST UPARSE) al., then following by 10 were et lies (selected top BLAST (Benson sequences the by representative database on identified OTU based (nt) OTUs the identities nucleotide of taxonomic GenBank results assigned 1990) the by were al., analyzed thresh- OTUs et was confidence Remaining (Altschul region using algorithm) ITS1 database %. each ITS 70 of fungal taxonomy of UNITE The old the against set. and (http://rdp.cme.msu.edu/) data identified Classifier reference were RDP UCHIME chimeras 97 UNITE and with http://drive5.com/uparse/) clustered the were 7.1 using us- OTUs (version removed (OTUs) UPARSE 2013). units using their (Edgar, taxonomic cutoff 7 two to similarity operational version USEARCH into % according or in clustered assembled implemented were one algorithm were UPARSE run the Miseq with bp discarded. ing Illumina 10 were reads the assembled from than Second, obtained be longer Sequences not overlap were could an bp. characters that had ambiguous 50 Reads containing that sequence. than reads sequences overlap shorter and only score measurements quality Third, were average matching that removed. an primer receiving reads in site mismatches truncated any nucleotide the criteria. at following truncated discarding the with were window, 1.17) reads (version bp QIIME using 300 quality-filtered First, and demultiplexed 250 were files x fastq (2 Raw sequenced paired-end and Bioinformatics concentrations 2.5 equimolar Miseq. in Illumina pooled an then on Corporation, Kit were bp) (Promega Extraction Fluorometer Gel Samples QuantiFluor DNA WI). using AxyPrep quantified the Madison, and using 72 CA) purified City, and of Union cut Biosciences, were extension (Axygen range size final appropriate the 95 a within amplicons at and denaturation s initial 30 0.4 an and of ITS2R China), consisted and Beijing, ITS1F pair 20 Ltd., primer in Co., the out carried using was amplified PCR was 2013). genes μ al., RNA et ribosomal Adams nuclear S1; the (Table of region ITS1 The methods Molecular 2.4 OU n T5,ad3 feogto t72 at elongation of s 30 and OTU5), and (OTU3 C 1 (5 primer each of L -1 eegnrtduigfie1-oddltoso agtDAapie rmpooom OU)o roots or (OTU3) protocorms from amplified DNA target of dilutions 10-fold five using generated were ) OU)and (OTU3) < μ ) 2 M), Gymnopus 0% ihfna pce costersqec eghwr eoe.Scnl,only Secondly, removed. were length sequence their across species fungal with %) 90 μ ° f25m NP,4 dNTPs, mM 2.5 of L ,floe y4 ylso 5so eauaina 94 at denaturation of s 15 of cycles 40 by followed C, OU)DAfo ahpooom oto itrDAsml na ABI an on sample DNA litter or root protocorm, each from DNA (OTU5) ° μ o 5s C rdcswr eaae ygleetohrssand electrophoresis gel by separated were products PCR s. 45 for C ossigo 12.5 of consisting L μ fTransStart of L μ N epae 9.0 template, DNA L ° o i,te 5cce f95 of cycles 35 then min, 3 for C μ ° .Ec apeetatwsapie ndpiaeand duplicate in amplified was extract sample Each C. f5 TransStart 5x of L 4 ® atf oyeae h aaeeso reactions of parameters The Polymerase. FastPfu μ μ fi YRGenPRSprMx(Bio-Rad Mix Super PCR Green SYBR iQ of L ecin otiig1 go eoi N,0.8 DNA, genomic of ng 10 containing reactions l μ H L 2 n 1.25 and O ® atf ue Tase Biotech (TransGen Buffer FastPfu Mycena μ ° 1 M fec forward each of mM) (10 L o 0s n 55 and s, 30 for C < ° < ,3 fanaiga 59 at annealing of s 30 C, OU)and (OTU3) 5 p rhvn low a having or bp) 150 0oe 0b sliding bp 50 a over 20 atoi confusoides Gastrodia Gymnopus ° for C Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iaigsesvre ewe 0%i lt1 0%i lt2 5%i lt3ad6 npo .On 4. plot in % 65 and 3 plot in % of 85 Analysis ( 2, packets. effect seed plot significant individual in no within showed % packets found 90 were seed ger- 1, containing of germination) packets development germination seed plot of for seedling % in percentage data of (0.64 % The stages protocorms 70 2A,B). distinct 3.2 (Fig. between average, and observed varied site were seeds study tubers) minating the root young at and encountered (protocorms regularly was germination Seed ( part Germination minor 3.2 a plots. explained among plot consistent cases, in were observed both results was In concentration fungal ( S3). plant and (Fig. orchid plant the orchid surrounding the litter from of bamboo distance the terms between in relationship significant fungi target mol our 484626.5 of mol quantification 640129.5 accurate being enabling from obtained D), per were S2B, (mol) DNA molecules (Fig. DNA magnitude target of orders five PRsadr uvsbsdo nw ocnrtoso agtDAsoe iercreain(OTU3, correlation linear a showed were DNA study target this under of database in concentrations known SRA found on NCBI OUT r (xxxxx). based in Numbers OMFs curves Accession deposited standard putative the were qPCR each under reads database for nucleotide sequences sequences GenBank Raw to representative submitted prevailing and S2). one (Table xxxxx and roots protocorms, BioProject in in exclusively solely occurred occurred and in stages dominant adult was and (OTU3) protocorm samples between differed comprised partners root fungal samples that adult litter of the bamboo members and while surrounding to samples, with The sequences), related OTUs were (172552 (excluding S2). OTUs sequences OTUs (Table 1640 with 129 sequences) sequences (168843 comprised 3925341 bamboo OTUs surrounding samples and 427 roots protocorm comprised adult The protocorms, from samples contained litter. set data Miseq quality-filtered The in of associations fungi percentage Mycorrhizal of a the abundance 3.1 as the (i.e., and included success plant was germination adult RESULTS plot related nearest 3. the while we of variable, analysis, distance dependent second the as to a soil. used protocorm) the In a was into packages model. developing seed the seeds the to in in of used protocorm distance effect was the one random ( to Model fungi least related with Mixed key was associating of at Linear protocorm) abundance fungus included into of the Generalized developing was main to A seed or the Plot plant one for plants. adult least factor. nearest (mol/ separately (at adult fixed germination concentration performed with a seed were Fungal associating whether used Analyses see fungus was plant. that factor. the plants adult hypothesis random and adult the a nearest to protocorms test as the distance to model while of used the variables, was distance in dependent Model the Mixed as to Linear used a related Linear was First, Generalized was used Models. abundance we Mixed germination, fungal Linear seed and and verify abundance Models mycorrhizal to Mixed in analyses variation spatial all investigate To with triplicate OTU5) in with analysis respectively. colonized included Data contaminants, 2.7 were roots of or DNA) absence the OTU3 template and with (no amplification colonized reactions target-specific protocorms control from negative extract and (DNA positive value Additional (Ct assays qPCR 2 .9;OTU5, 0.998; = Gymnopus μ 1 -1 N xrc S 22 mol 62923 = (SE extract DNA r μ 1 2 -1 .9)btenlgvle fiptDAadqC hehl ylsoe tleast at over cycles threshold qPCR and DNA input of values log between 0.997) = < N xrc S 38. mol 43787.6 = (SE extract DNA oeOU-908sqecs-5. )i h dl otsmls ti noteworthy is It samples. root adult the in %) 53.3 - sequences 90028 - OTU (one .confusoides G. 9adasnl etn eka h xetdmligtmeaue i.S2). Fig. temperature; melting expected the at peak melting single a and 29 μ N xrc TbeS) h ihs ocnrtoso agtOrM target of concentrations highest The S3). (Table extract DNA L Mycena rtcr n otsmls ihteaeaeOU concentration OTU3 average the with samples, root and protocorm χ ² .0ad6.39, and 4.40 = oeOT-147 eune 55% nteprotocorm the in %) 95.5 - sequences 164772 - OUT (one Mycena 5 μ 1 < -1 ,rsetvl.Frteetotre Ts no OTUs, target two these For respectively. ), μ 0% ftettlvrain niaigta the that indicating variation, total the of %) 10 < 1 and -1 0 oa eune) otoci mycorrhizal orchid Most sequences). total 100 n h vrg T5cnetainbeing concentration OTU5 average the and ) p Gymnopus > χ .confusoides G. ² .5frOU n T5 respectively) OTU5, and OTU3 for 0.05 4.58; = ntelte.Presence/absence litter. the in ) p .3 fdsac oadult to distance of 0.33) = nwihasingle a which in , Gymnopus OU)that (OTU5) Mycena μ L) Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 05 j ta. 05,o ewe noeei tgs(..MCrike l,20;Bdrod Read, & Bidartondo al., 2004; et al., Ercole et McCormick 2006; al., (e.g. et stages McCormick ontogenetic between occur (e.g. or can years 2015), turnover subsequent Temporal al., between availability. temporal et and fungal or Oja Spatial or almost season 2015; show 2021). conditions growing others al., ecological the while et in (Ventre-Lespiaucq partner throughout variation time (Bidartondo same time or reflect the and space may mycorrhizal with 2020) through turnover orchid al., associating partners that et continuously of 2013; (Xing evidence species switching space al., growing some in complete et is with with both highly Long interactions there 2008), substantially vary Furthermore, from De broad Read, can very & 2012c; ranging 2019). but to al., stable, al., with, 2009) not et et are al., Jacquemyn associate Shefferson interactions et 2017; orchids (e.g. (McCormick (Rasmussen, al., simultaneously fungi orchids fungi et fungi of OrM Waud of multiple cycle few number with life a associating the the or orchids in in one role with variation crucial interactions remarkable a specific play is interactions there mycorrhizal that 1995), stages clear developmental is different it the in & Although partner and Yashima fungal forest Japan, 1974; of bamboo (Shidei, in shift ”the types The forests 4.2 of forest thickets. bamboo adaptation other bamboo in to the in occur specific that suggest groups can from results fungal different fungi These among is saprotrophic shared 2010). forests of Nose, be bamboo group could in limited community therefore a fungal and only litter, that bamboo this reported decomposes that suggest it results where These 2015). from al., isolated of sequences spectrum with broader a Mycenaceae, The besides that in showed of found (2019) stage was juvenile al. result the as et similar such the Chen A associations, on fungal forests. depending grow. broadleaved vary species in can the communities found mycorrhizal where orchid type that vegetation suggesting Sebacinaceae, and Russulaceae dominant the also ica were Mycenaceae and Marasmiaceae three nipponica of with members associating Japan, In partners fungal 2015). al., et (Lee Mycenaceae fontinalis Marasmi- trodia and 2010), Bougoure, Mycenaceae with & 2009), from (Dearnaley associating and aceae al., fungi families observations et that fungal these shown (Ogura-Tsujita major have with Mycenaceae two that line the reports are in previous aceae are confirm further results results Our These 2018). S2). with saprotrophic al., (Table associating and Marasmiaceae et fungi and 2016) Ogura-Tsujita dominant al., the 2015; et that ectomy- al., Yagame Tˇeˇsitelov´ashowed with 2015; et 2012; predominantly a al., (Lee al., associate with et fungi et orchids associate Ceratobasidiaceae (Ogura-Tsujita mycoheterotrophic Tulasnellaceae, typically of fungi orchids fully members corrhizal autotrophic and (i.e. (Jac- most partially fungi fungi rhizoctonia Whereas Serendipitaceae), mycorrhizal so-called 2021). and of including al., fungi sets et of different variety Wang with wide 2019; associate Merckx, to & tend quemyn orchids mycoheterotrophic and Autotrophic with associating communities Mycorrhizal 4.1 DISCUSSION ( 4. relationship significant of strong, of abundance A abundance and shown). and germination not germination seed (data between litter observed the was in 0.001) buried were packages seed 10-m the within plants naiigbodevdfrsswr hw osmlaeul soit ihetmcrhzlfnifrom fungi ectomycorrhizal with associate simultaneously to shown were forests broadleaved inhabiting Mycena rwn ndffrn eeaintps(ioht ta. 06.Hwvr niiul of individuals However, 2016). al., et (Kinoshita types vegetation different in growing ) OU)sqecsioae from isolated sequences (OTU3) - Gymnopus .elata G. 2 lt Fg ) o a hr n eainhpbtentefu ietosi which in directions four the between relationship any there was nor 3), (Fig. plots Resinicium .nantoensis G. . fMrsica Lee l,21)and 2015) al., et (Lee Marasmiaceae of Gymnopus atoi appendiculata Gastrodia Gastrodia , Hyphodontia atoi sesamoides Gastrodia ( Mycena χ and ² 0.04, = atoi confusoides Gastrodia .confusoides G. pce ( species .appendiculata G. .confusoides G. tani atclryaatdt rwi aboforest, bamboo in grow to adapted particularly is strain 6 , p Sistotrema .4 Fg 4). (Fig. 0.84) = .confusa G. Gastrodia - Mycena rtcrssae ihsqec similarity sequence high shared protocorms – eesporpi ug fteMycenaceae the of fungi saprotrophic were Mycena Marasmius , rwn nohrbmo oet(e et (Lee forest bamboo other in growing Tricholoma species: , atoi pubilabiata Gastrodia fMcnca Lee l,2015), al., et (Lee Mycenaceae of atoi nantoensis Gastrodia atoi elata Gastrodia ntelte,btntbtenseed between not but litter, the in Gastrodia Gastrodia atoi confusa Gastrodia and and Campanella Russula pce”t particular to species” pce.I a been has It species. hc smainly is which , and , χ a eetdin detected was ² - 51.91; = fMarasmi- of – .nippon- G. Mycena Mycena Gastrodia Gas- p of of < Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. itdwt itr rwo-eaigfni u o ntmeaemchtrtohcocis ntemper- In orchids. mycoheterotrophic temperate in asso- not as orchids but such mycoheterotrophic orchids, Asian fungi, mycoheterotrophic subtropical wood-decaying forests, in or ate primarily litter- occur with to ciated seems turnover partner Complete case, with this associates In stage in of fungi. adult found wood-decaying stages than been with adult groups has associated with fungal and replacement orchid diverse association total juvenile more of the both with pattern required of associated similar stage sequencing tuber juvenile high-throughput the across the However, partner of that fungal development 2000). in further Guo, shift the & similar and A germination in (Marasmiaceae). seed observed stage been adult has the stages ontogenetic to most (Mycenaceae) others, that in stage replaces demonstrated and protocorm that data partners and 2021). sequencing fungal orchids al., high-throughput its et Our in of (Ventre-Lespiaucq meta-analysis subset frequently scenario recent a frequent occurs A retains most partners individual the 2004). mycorrhizal an was al., whereby of et replacement, turnover (McCormick partial lower temporal cases much that was shown protocorms has in detected example, diversity For fungal 2011). al., et Jacquemyn 2008; oei h usqetgot fselnsit dl lns ncnrs,pooom soitdmainly associated play protocorms to contrast, starts In only but plants. germination, adult seed into in seedlings involved of of that not abundance growth fact is the subsequent the fungus and with the this by germination in higher that seed explained role supporting between suggesting be a correlation 4), locations can no necessarily (Fig. found This not (OTU5) also We are germination. plants protocorms. seed and with adult for germination associate to exclusively and required almost plant 2007, closer plants fungi adults mother al., locations mycorrhizal et the Jacquemyn that from of 2007; distance seed indicating abundances Diez, the determining 2001; 2014). 3), plants between al., adult relationship factors Jacquemyn, et of significant (Fig. Batty & critical presence/absence no 2002; (McCormick the found are 2000, between we sites al., soil relationships 2012a,b), et within strong (McKendrick the orchids shown germination have in seed of studies and fungi distribution of number OrM spatial a the suitable Whereas hence of and germination abundance germination seed abilities and and acquisition fungi distribution of resource mycorrhizal plants The in of adult differences distribution and Similar spatial protocorms and in 2021). Abundance partners al., 4.3 mycorrhizal of et in growth switch (Ogura-Tsujita rapid observed forest the the the For explain in may 2017). wood al., more the et access (Kyaschenko i.e. to productivity abiliity In plant the elata sustaining trees 2015). G. for individual (Bever, on provide allocation it constraints pools composition when nutrient and severe community occur diverse acquisition pose fungal to resource may ectomycorrhizal expected of and in be terms 2021) differentshifts can in across partners al., benefits partners switching et fungal orchids, serious (Ventre-Lespiaucq in mycoheterotrophic provides costs shift of complete severe dynamics a population with Although the come 2002). may 2000, stages al., life et McKendrick 1997; Bruns, & nidus-avis n bnac fteocisi o ag atrltdt h itiuinadaudneof abundance and In distribution distribution 2015). the the to al., hence related et and part that Liebel large trees, fact 2008; a by the for al., supported is by et orchids are explained (Cameron the fungi be of fungi the abundance can the fungi, and 2016) support ectomycorrhizal al., hence with et and associating relationship (Waud to orchids strong plants carbon associated adult contribute the associating of orchids and orchids location plants In the adults the with. around and associate fungi be germination they of may fungi between accumulation OrM orchids the of mycoheterotrophic fungi, groups and rhizoctonia different with of autotrophic needs between nutritional patterns the by germination explained seed germination. in seed differences of These patterns spatial driving factor determining of the abundance the and germination Mycena ue,cagn uglprnr to partners fungal changing tuber, sal soit ihnro ldso coyoria ug uigteretr iecce(Taylor cycle life entire their during fungi ectomycorrhizal of clades narrow with associate usually OU)adi hscs hr a ihysgicn,pstv eainhpbtenseed between relationship positive significant, highly a was there case this in and (OTU3) Armillaria Mycena Uaae l,2013). al., et (Umata .elata G. Fg ) niaigta h oa itiuino hsfnu is fungus this of distribution local the that indicating 4), (Fig. iuai discolor Tipularia Armillaria Gymnopus ehlnhr austiniae Cephalanthera sn an using 7 ytsaseptentrionalis Cyrtosia a nbeteuiiaino agrcro pool, carbon larger a of utilization the enable may hc a opeeyasn ngriaigseeds germinating in absent completely was which , nvitro in Physisporinus .confusoides G. dlsascaewt ait ffni while fungi, of variety a with associate adults utr ytm nwhich in system, culture Mycena nue edgriain hl the while germination, seed induces , wthdfna atesfo the from partners fungal switched Corallorhiza nte mycoheterotrophic another , ny(hne l,21) A 2019). al., et (Chen only pce and species Mycena iu sylvestris Pinus Tomentella .elata G. .confusoides G Armillaria Gymnopus promoted Neottia showed fungi (Xu . , Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. odt . ubl,S . otr .B 19) erimn ercnpcfi dlsadtemaintenance the and forest. adults limited? neotropical conspecific seed near a populations Recruitment in plant diversity (1992). Are B. shrub R. and Foster, (2007). tree & W. P., of C. S. Hubbell, Osenberg, experiments. R., & addition during Condit, seed J., communities of D. fungal Levey, meta-analysis the of and R., Dynamics measuring critique J. A Poulsen, (2019). receiving: J., and al. C. Giving et Clark, Y. (2008). L. R. Qin, C., J. growth. Y. Leake, and Wang, & orchid, L., germination green J., Chen, orchid the D. in during Read, mycorrhizas bottlenecks of I., cost specificity Johnson, carbon Fungal D., D. (2008). Cameron, J. D. Read, environ- and & development. stability symbionts. I., root the M. their and and Bidartondo, feedbacks, GenBank. plants physio-evolutionary between (2009). allocation, mutualism W. of Preferential patterns E. symbiotic mental (2015). Sayers, to & D. Constraints J., J. Ostell, Bever, J., (2001). issue), D. (Database K. Lipman, 37 Sivasithamparam, Research, I., Acids Karsch-Mizrachi, & bushland. A., M., Mediterranean D. a Brundrett, Benson, in seed W., orchid orchid of K. terrestrial properties of physical Dixon, germination and L., Numerical search 110. A. alignment No. are Batty, local Review air implications. Tansley Basic biological indoor (2000). (1990). their A. in J. and K. D. fungi A. seeds Ghani, Lipman, & & microbes: J., W., in Arditti, E. Dispersal Myers, W., (2013). Miller, distances. W., short tool. al. Gish, at F., et limitation S. dispersal W., Altschul, show J. and Taylor, air M., outdoor by Miletto, dominated I., the R. wrote Y.Y.L. H.J. Adams, Y.I.L., and Y.I.L. experiments. analyses. molecular statistic REFERENCES to conducted contributed H.J. Y.H.C. analyses. and bioinformatic manuscript. Y.Y.L. performed M.B. research. and the designed Y.I.L. CONTRIBUTIONS AUTHOR blooming of photograph the providing ACCESSIBILITY for DATA Yang K. C. Dr. are thank We symbionts between partner. different patterns a transport to resource assess switch and ACKNOWLEDGEMENTS about plants fungi details adult both more why with particular, interaction understand the In to of needed switching. basis physiological of and distributed cost functional are from the the results fungi independently detail litter-decaying Our and more of partners. in forest abundance investigate mycorrhizal and bamboo in distribution the switch spatial orchid within prompt the mycoheterotrophic randomly a that the by evidence accompanied in first is plant the provide that adult process an two-stage to a growth represents subsequent and germination Seed bamboo the within distribution CONCLUSION random a 5. have covered to usually expected is are ground they recycling, the forest. nutrient forests, for Since bamboo trunks litter. In and bamboo sticks 2009). of layer al., thick et a (McCormick with trees ectomycorrhizal their and ora fMlclrBooy 215, Biology, Molecular of Journal M irbooy19, Microbiology BMC oeua clg,17, Ecology, Molecular 158. 3707-3716. D26-31. e htlgs,145, Phytologist, New 403-410. Mycena odearepens Goodyera and mrcnNtrls,140, Naturalist, American 8 Gymnopus mrcnNtrls,170, Naturalist, American .confusoides G. 367-421. r ffiin eopsr fbmo leaves, bamboo of decomposers efficient are . e htlgs,180, Phytologist, New e htlgs,152, Phytologist, New atoi confusoides Gastrodia e Phytologist New SEJunl 7, Journal, ISME dls uuersac should research Future adults. 261-286. 128-142. 176-184. 0,1503-1514. 205, , 511-520. plants. atoi elata Gastrodia .confusoides G. 1262-1273. Nucleic Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. fArclueIpra nvriyo oy,4, associated Tokyo, and of communities University fungal Imperial Agriculture in of Shift (1911). managed (2017). S. of Kusano, gradient al. age et an A. along Hagenbo, activities enzyme K., in Clemmensen, study fungal J., case do A Kyaschenko, How plants? (2016). mycoheterotrophic T. Yukawa, of & preferences T., habitat . Hashimoto, and early evolution H., and the Sato, seeds, affect H., dust partners Umata, of Y., Ogura-Tsujita, germination species. A., limitation, Pyroleae Kinoshita, Recruitment six in (2013). in seedlings O. modes underground Eriksson, trophic of Ove of development & evolution the A. and V. symbioses Johansson, Mycorrhizal (2019). T. F. S. plants. V. mycorrhizal Merckx, in & Variation five H., of (2012c). seed Jacquemyn, B. populations ground Lievens, among B.,& below fungi Bailarote, Tulasnelloid in Cachapa with K., variation associations Dehert, Spatial A., co-occurring Deja, three H., of (2012b). Jacquemyn, segregation T. spatial Wiegand, with correlate & species. associations Non-random orchid B., mycorrhizal Lievens, divergent (2012a). and T. R., germination three Wiegand, Brys, of & zone H., B., hybrid Jacquemyn, Lievens, a I., in (2011). Roldan-Ruiz, orchids B. of O., Lievens, Honnay, structuring & spatial R., O., Brys, Honnay, H., A., Jacquemyn, P. genus B. the in Cammue, specificity mycorrhizal D., on Tyteca, constraints (Orchidaceae). phylogenetic R., reveals architecture network Brys, of Analysis V., spatially Merckx, A H., (2007). orchid Jacquemyn, T. terrestrial Wiegand, the & in I., recruitment Roldan-Ruiz, seedling 448-459. O., Honnay, of K., analysis Vandepitte, explicit R., Brys, H., Jacquemyn, of Taxonomy tree (2008). Taipei. of University, C. associations T. adult–recruit driven Hsu, Stochastically (2014). P. Island. S. Colorado populations. 20140922. plant Hubbell, Barro in & on recruitment T., species of Wiegand, limitation S., meadow microsite Getzin, and wintergreen Seed the variation (1992). in Temporal J. 91, abundance cologia, Ehrlen, (2015). isotope & S. O., Perotto stable Eriksson, & nitrogen M., and Girlanda, carbon G., and Gebauer, reads. orchid diversity M., amplicon mycorrhizal Rodda, microbial M., in from Adamo, sequences E., OTU and Ercole, accurate proximity highly adult UPARSE: to linked 10, (2013). Methods, germination C. orchid R. symbiotic Edgar, of preference patterns Mycorrhizal Hierarchical (2013). gradients. M. (2007). environmental L. M. Egerton-Warburton, J. & orchid: W., Australian Diez, K. native a Dixon, Marasmi- by D., saprotrophic invasion N. for habitat Swarts, evidence promotes R., molecular J. and Long, Isotopic De (2010). of J. rhizomes J. in Bougoure, mycobionts & aceae W., D. J. Dearnaley, mrcnJunlo oay 103, Botany, of Journal American ncmtsmorio Anacamptis ora fEcology,107, of Journal 360-364. 996. ora fEooy 100, Ecology, of Journal e htlgs,192, Phytologist, New atoi elata Gastrodia ora fEooy 95, Ecology, of Journal . e Phytologist New 1567-1581. atoi sesamoides Gastrodia rceig fteRylSceyo odnB ilgclSine,281, Sciences, Biological B: London of Society Royal the of Proceedings n t yboi soito with association symbiotic its and Gastrodia 207-220. 518-528. 1328-1337. 0,1308-1319. 205, , Ociaee nTia.Mse hss ainlTaiwan National thesis, Master Taiwan. in (Orchidaceae) 1-66. 159-170. iu sylvestris Pinus 9 . Orchis uglEooy 3, Ecology, Fungal irtsmedia Microtis species. Dactylorhiza oay 91, Botany, rhspurpurea Orchis rilai mellea Armilllaria stands. e htlgs,193, Phytologist, New . naso oay 111, Botany, of Annals 288-294. 17-24. species. SEJunl 11, Journal, ISME . LSOE 7, ONE, PLoS e htlgs,176, Phytologist, New . ora fCollege of Journal 454-464. 863-874. Gastrodia 409-418. e42212. Nature Orchis Oe- Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. gr-sjt,Y,Ykw,T,&Knsia .(01.Eouinr itre n yoria associations mycorrhizal fungi. and saprotrophic histories on Evolutionary dependent (2021). plants A. mycoheterotrophic Kinoshita, of & T., Yukawa, Y., mycoheterotrophicOgura-Tsujita, giant J. (2018).The Schweiger, T. Yukawa, M., & Kubota, 27, S., Ecology, K., Isshiki, Tetsuka, M., H., Maki, Umata, N., orchid Maekawa, Y., Fukasawa, S., Yamashita, H., I., Xu, 276, M. and G., Sciences, novel Gebauer, B-Biological for Y., Series Evidence Ogura-Tsujita, London of (2009). and Society T. determinants Yukawa, Royal & orchid their the H., the of Proceedings dispersal, Umata, parasitism: seed T., mycorrhizal Hashimoto, of specialized G., patterns Gebauer, Spatial Y., limitation: Ogura-Tsujita, recruitment (2000). and C. habitat H. recruitment. for microsite, Muller-Landau, consequences & dispersal, limitations. of R., Seed, studies Nathan, in concepts (2005). and T. terms of Herben, identification & Z., Munzbergova, development and germination Symbiotic (2002). devel- J. orchid D. and Sebacina Read, mycoheterotrophic & germination L., Symbiotic the D. Taylor, of R., (2000). J. Leake, J. L., S. D. McKendrick, Read, of & ontogeny L., nature: D. fungi. in Taylor, mycorrhizal plants R., myco-heterotrophic distri- J. of orchid Leake, opment affect fungi L., Mycorrhizal S. in (2018). McKendrick, patterns A. Germination Canchani-Viruet, dynamics. & (2016). population F., and Jr. D. bution K. Whigham, fungi. R. K., mycorrhizal Burnett, M. of & McCormick, abundance F., to D. relate Whigham, orchids L., terrestrial populations? D. three Taylor, orchid (2012). K., of J. distribution M. the McCormick, O’Neill, constrains & What F., (2014). D. H. 202, Whigham, Jacquemyn, Phytologist, & K., K., picture. R. M. simple Burnett, McCormick, a K., not Juhaszova, recruitment: L., orchid on D. fungal Taylor, Limitations of and Abundance K., tips M. root (2009). McCormick, ectomycorrhizal Orchid-fungus al. of et abundance (2006). P. reflects B. J. composition. population community O’Neill, Hodkinson, established F., an & D. within photosynthetic K. (Orchidaceae) Whigham, O’Malley, in K., diversity D., M. McCormick, Mycorrhizal last? Sloan, to F., meant (2004). marriage D. a P. Whigham, fidelity: J. K., (2009). O’Neill, M. A. & McCormick, M. F. Selosse, D. & Whigham, orchids. J., terrestrial K., Fournel, orchids. M. achlorophyllous A., tropical McCormick, Faccio, by P., partners mycorrhizal Bonfante, fungi 184, as between Phytologist, T., fungi exchange New saprotrophic Pailler, nitrogen of and M., recruitment carbon Independent Dulormne, Are F., (2015). non- G. Martos, saprotrophic Gebauer, with & associations I., orchid of the M. and Bidartondo, importance T., The from H. evidence Liebel, novel (2015). under-estimated: G. currently Gebauer, is orchids Asia. & mycoheterotrophic sub-tropical K., fully among C. fungi Yang, Rhizoctonia I., Y. Lee, rtrrhsaltissima Erythrorchis spp. 1324-1337. e htlgs,154, Phytologist, New odearepens Goodyera 392-400. naso oay 116, Botany, of Annals e htlgs,145, Phytologist, New e htlgs,163, Phytologist, New 668-681. clgclMngah,79, Monographs, Ecological rnsi clg vlto,15, Evolution, & Ecology in Trends sascae anywt iegn e fwo-eaigfungi. wood-decaying of set divergent a with mainly associated is ffce yirradiance? by affected eti nidus-avis Neottia e htlgs,219, Phytologist, New clg,87, Ecology, 233-247. 523-537. 425-438. 423-435. 903-911. atoi confusa Gastrodia 619-635. 10 nntr n t eurmn o oal distributed locally for requirement its and nature in naso oay 115, Botany, of Annals 1207-1215. oeua clg,21, Ecology, Molecular oalriatrifida Corallorhiza eooi,145, Oecologia, ora fPatRsac,134, Research, Plant of Journal 278-285. an abnfo saprotrophic from carbon gains ora fEooy 104, Ecology, of Journal 1-8. 761-767. 251-261. 1511-1523. n hrceiaino its of characterization and oalriaodontorhiza Corallorhiza 744-754. 19-41. Molecular Mycena New . Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ad . rs . a ady,W,Lees . aqey,H 21) yoria pcfiiydoes specificity Mycorrhizal (2017). species. H. orchid Jacquemyn, endangered & an B., of Lievens, distribution W., orchids. the Landuyt, limit terrestrial Van not two R., Brys, in M., abundance Waud, establishment seedling mycorrhizal switching Nonrandom Symbiont (2016). in (2021). H. decline 211, T. Jacquemyn, Phytologist, & F. distance-dependent B., S. Lievens, with V. R., Merckx, corresponds Brys, & T., Wiegand, A., M., R. Waud, Orchidaceae. Vos, in F., shifts I. loop. mode S. trophic dispersal Gomes, and seed H., the Jacquemyn, D., Closing Wang, (2002). B. T. Smith, my-379-386. & in C., turnover orchids. B. Temporal with Wang, (2021). concept M´endez, M. of & proof N., a H. interactions: Rasmussen, corrhizal H., Jacquemyn, orchid A., myco-heterotrophic Ventre-Lespiaucq, fully the of Germination 54, (2013). Mycoscience, al et M. Yamada, septentrionalis Y., stages. Ota, ontogenetic H., and Umata, habitats various in Sebacinales green 24, widespread for Two by preference (2015). mutualism al. mycorrhizal ectomycorrhizal et show J. of Jersakova, invasions M., Kotilinek, specialized T., Tesitelova, Independent, (1997). D. T. orchids. nonphotosynthetic Bruns, two & L., D. Taylor, (2008). J. D. Press. Read, & & E., S. J., Smith, forest. Downing, bamboo in T., Yukawa, flora Japanese). fungal R., (in of 251-257 T. Report Kartzinel, (1974). interactions? T. I., ecological Shidei, broad Y. in Lee, specificity determine C., history 107, C. evolutionary Ecology, Cowden, Does (2019). W., H. Bunch, Jiang, P., R. requirements. establis- Shefferson, seedling of Tˇeˇsitelov´a, and complex & Germination a J., Jersakova, (2015). orchids: W., T. in style. K. hment life of mycophagous Dixon, studies a N., of in H. implications Rasmussen, challenge mycorrhiza: special Orchid a (2009). N. 118, phase: F. Oikos, Rasmussen, underground & The N., H. Rasmussen, (1998). and technique F. study D. new populations. a Whigham, orchid situ: & terrestrial in N., seeds dust H. of Rasmussen, ecology ecology. Seed terrestrial (1993). in F. application D. its Whigham, Press, & University Cambridge N., 4: H. plant. Rasmussen, vol. mycotrophic Orchidacearum, to fish: seed Genera USA. from reef NY, (2005). orchids: as York, N. Terrestrial New Plants F. (1995). Rasmussen, N. UK. (2007). H. & Oxford, M. Rasmussen, Press, W., B. University M. Oxford Bolker, Chase, & 1). J., (part P. J., Epidendroideae Cribb, D. M., Levey, A. J., recruitment. Pridgeon, C. mycorrhizal seedling orchid of Clark, form of W., functional patterns C. the Temporal Osenberg, fitting pyrosequencing. (2015). R., 454 U. by J. Koljalg revealed Poulsen, & as forests T., and Kull, meadows L., in Tedersoo, fungi P., Kohout, J., Oja, 1122–1134. 334-345. 1582-1593. scaatrzdb o uglseict n osntrqiedrc edmcbotcontact. seed-mycobiont direct require not does and specificity fungal low by characterized is 255-264. 343-352. oaia ora fteLnenScey 126, Society, Linnean the of Journal Botanical rceig fteNtoa cdm fSine,USA, Sciences, of Academy National the of Proceedings yoria symbiosis. Mycorrhizal mrcnJunlo oay 80, Botany, of Journal American e Phytologist New naso oay 116, Botany, of Annals mrcnNtrls,170, Naturalist, American 11 e htlgs,230, Phytologist, New npes https://doi.org/10.1111/nph.17414. press. in , rnatoso yooia oit fJpn 15, Japan, of Society Mycological of Transactions r d e ok Y odn K Academic UK: London, & NY, York, New ed. 3rd oeua clg,26, Ecology, Molecular e Phytologist New 391-402. 1374-1378. rnsi clg vlto,17, Evolution, & Ecology in Trends 49-64. 1690-1699.. Neottia 167-183. 1687-1701. 0,1608-1618. 205, , pce (Orchidaceae) species 4 4510-4515. 94, oeua Ecology, Molecular ora of Journal Cyrtosia New Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. dl lns(m esrdi orposi abofrs nTaiwan. in forest bamboo in plots four in measured 50 (cm) = plants bar basal adult Scale the 3. (arrows). colonized hyphae have Fig. Fungal (arrow) region. hyphae packet. basal the fungal the from germination, at tubers of colonization root 50 stage young = early sowing. and bar the after protocorms Scale μ At of months stages (C) 12 embryo. different at of mm. of packet part selection 2 single A = a (B) bar from mm. Scale protocorms E, 2 = Developing = roots. (A) bar bar of Scale Scale fungi. cells litter. mycorrhizal cortical 10 of = bamboo in (arrows). presence bar decaying fungi seeds Scale 2. mycorrhizal in released hyphae. of (arrows) fungal and Fig. presence roots H, opened the cells; mycorrhizal have showing cortical with fruits C, Scale section tube mature cells; microscopy pollinated. The epidermal pollination, Light successfully (D) been (E) of have cm. cm. weeks flowers 1 two 1 after After = elongation (C) bar Stem (B) cm. Scale cm. 10 1 = = bar bar Scale litter. bamboo 1. Fig. the surrounding litter Figures bamboo of in List observed was qPCR by ( determined plant abundance orchid fungal and plant orchid the S3 Fig. confusoides S2 Fig. (5, distances radial surrounding five litter at S1 and directions Fig. cardinal cm). 500 four and in 200 of obtained 100, analysis samples 50, qPCR litter using bamboo quantified surrounding mean and the of deviation standard S3 Table platform. Miseq Illumina the using S2 Table S1 Table Moso-bamboo of mixture information of Supporting/Supplemental degree the along flora of macrofungal evolution Japanese). forest. of the Changes broad-leaved during and (2010). shifts I. thicket partner Nose, Fungal & T., (2016). Yashima, al. et A. Kinoshita, in mycoheterotrophy Y., Ogura-Tsujita, T., of cultivation Yagame, the of research the architecture on The Retrospect medicine. (2020). (2000). Chinese S. S. Guo, Guo, & & J., Z., Xu, co-occurring Zhao, nine H., in Jacquemyn, 8, interactions L., Evolution, orchid–fungus Guo, and of S., network Shao, Kew. Y., the Gardens, Gao, of Botanic Q., Royal Liu, the X., by Xing, Facilitated http://apps.kew.org/wcsp/. families. Internet: plant the selected on of Published checklist World (2014). WCSP. E h eeoigpooomhseogtdwt h eitmtcclsa h prgo n h fungal the and region up the at cells meristematic the with elongated has protocorm developing The (E) m atoi confusoides Gastrodia For eainhpbtentepretg edgriainof germination seed percentage the between Relationship ailsmln lnapidfrtese emnto xeietadtecleto fbamboo of collection the and experiment germination seed the for applied plan sampling Radial ieetsae ntegriainpoesof process germination the in stages Different uniaiePRrslsfrOU ( OTU3 for results PCR Quantitative umr ffna prtoa aooi nt OU)dtce noci rtcrsadroots and protocorms orchid in detected (OTUs) units taxonomic operational fungal of Summary ito rmr sdi hsstudy. this in used primers of List encnetaino T3( OTU3 of concentration Mean rtcrso ot,icuigmligcreadsadr uv analyses. curve standard and curve melting including roots, or protocorms Mycena χ ² .0ad6.39, and 4.40 = atoi confusoides Gastrodia hns eia ora,113, Journal, Medical Chinese 130. Neottia OU)and (OTU3) . nisntrlhbtt A dl lnswt loigflwr rwn between growing flowers blooming with plants Adult (A) habitat. natural its in mrcnJunlo oay 103, Botany, of Journal American nulRpr fteIhkw rfcua oetRsac,42, Research, Forest Prefectural Ishikawa the of Report Annual μ D h nagdeby a utrdtese ot cl a 50 = bar Scale coat. seed the ruptured has embryo enlarged The (D) m p Gymnopus > .5frOU n T5 respectively). OTU5, and OTU3 for 0.05 individuals. Mycena OU) osgicn eainhpbtentedsac from distance the between relationship significant no (OTU5), Mycena 686-692. n T5( OTU5 and ) 12 atoi confusoides Gastrodia n T5( OTU5 and ) 1630-1641. Dendrobium atoi confusoides Gastrodia μ atoi confusoides Gastrodia Gymnopus m. atoi elata Gastrodia Gymnopus μ m. ed n niain fthe of indications and seeds N (mol DNA ) species. eetdin detected ) rnir nEcology in Frontiers l aetraditional rare a Bl, n itnefrom distance and rtcrs roots protocorms, μ l -1 including ) 43.(in 34-37. Gastrodia Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. i.4. Fig. noaprotocorm. a into of germination eainhpbtenteaudneof abundance the between Relationship atoi confusoides Gastrodia edgriainwsepesda h ecnaeo ed developing seeds of percentage the as expressed was germination Seed . Mycena 13 and Gymnopus (mol μ L -1 n ecnaeseed percentage and ) Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 14 Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 15 Posted on Authorea 17 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162126337.74672794/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 16