Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. hs hmeswsls rnucd ieecsi oiac ftecmo Tsdieteobserved the drive OTUs common the of Fungal dominance chambers. in between two Differences variation other but the different, pronounced. in significantly abundance less also sequence were was low chambers chambers very ventilation consistently in these and ‘common’ differed as or nursery chambers OTUs rare the fungal waste were were in 13 identified the (28%) that communities We chambers OTUs in locations. waste of community five the OTUs portion all fungal across in large of chambers the ventilation a proportion Notably, and however nursery the total), level. the of the from order terms of domatium. the (28% in chamber the at OTUs chambers into three of unidentified the air abundances The allow dominated sequence that Chaetothyriales and chambers, waste; km. order ‘ventilation’ (13.4%) deposit the 675 and workers from kept; spanning ant are fungi where locations from Overall, brood chambers, five the DNA ‘waste’ where across fungal including: chambers, sequenced type functions ‘nursery’ We ant-associated chamber different ant-plant by ago. serve bipartite epiphytic years differ types the be 100 communities of to over fungal chambers ant-plant thought if domatium epiphytic long distinct an were in three mutualisms discovered in Ant-plant the been noticed have being bipartite. fungi fungi as as despite of such thought microorganisms involving traditionally mutualisms associations multipartite decades, recent In observed types. exists the chamber Abstract fungi among drive involving differs mutualism OTUs fungi multipartite common a of suggests the role This but the of different, types. that dominance chamber significantly and the in also ant-plant abundance of were this Differences sequence each chambers in low for five communities ventilation very pronounced. fungal in and all the less or nursery across in was rare the patterns chambers were chambers in ventilation that Notably, communities these chambers and Fungal level. waste between nursery order the variation chambers. the the in two at ‘common’ from other unidentified as consistently the were OTUs in differed (28%) fungal domatium. OTUs chambers 13 the of identified waste We into portion sequence the workers and large air locations. (13.4%) in a ant allow OTUs however community of that where total), proportion fungal chambers, the chambers, the the of ‘ventilation’ of ‘waste’ terms (28% and in OTUs chambers including: kept; the of are dominated functions abundances Chaetothyriales brood ant-associated order spanning the the different from locations fungi where serve five Overall, an across chambers, types epiphytic in type ‘nursery’ the chamber chamber noticed of waste; by three chambers being differ deposit domatium The communities fungi distinct fungal despite three if bipartite km. the establish tra- from be 675 to DNA associations to Hook.f. fungal in thought sequenced beccarii discovered We long Myrmecodia ant-plant been were ago. years have mutualisms 100 Ant-plant fungi over ant-plant as bipartite. epiphytic such as microorganisms of involving thought mutualisms ditionally multipartite decades, recent In Abstract 2020 11, September 3 2 1 Field mutualism multipartite Greenfield a Melinda suggest strongly range across geographic ant-plants large within a communities fungal of patterns Consistent utainToia ebru,JmsCo University Cook James Herbarium, Tropical Australian Tartu Campus of Cairns University - University Cook James 1 n adaAbell Sandra and , 1 oiLach Lori , 3 1 rdCongdon Brad , 1 1 tnAnslan Sten , ymcdabeccarii Myrmecodia 2 eoTedersoo Leho , okf oestablish to Hook.f. 2 Matt , Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. niomn rtcinadBoiest osrainAt19 Cmowat fAsrla n the and Australia) of (Commonwealth 1999 Act Conservation Biodiversity Australia. and Queensland, Protection ant-plant northern Environment epiphytic to the opportunistic endemic in as ceae), dismissed fungi investigated or first 1974) study were Janzen, Our Fungi shrubs 1978; 2015). Huxley, or 1911). Renner, 1920; trees (Bailey, Miehe, & 1920; are literature (Chomicki (Bailey, ant-plants the epiphytes) in most 1974). (15% rarely whereas (Janzen, Americas mentioned ant-plant ant-plants epiphytic, the epiphytic epiphytic are the and in for ant-plants epiphytes) noticed important ant of (no by particularly 47% deposition Africa be waste region, in reason, to Australasian this believed For the other tree. is like host In domatium their because, from nutrient-limited, the water typically in or are nutrients workers and obtain support not orders do for the they trees Leroy epiphytes, from on 2011). C. yeasts” grow al., “black 2005; usually et are ant-plants (Voglmayr al., Epiphytic far Ascomycota et so phylum Sch (Dejean studied Voglmayr, of systems plant Nepel, Capnodiales ant-plant the 2009; receive from and Voglmayr, to trap, Chaetothyriales isolated & myrmecophyte the nutrients fungi Mayer in host dominant of 2011; role The their transfer al., of structural 2014). et of the C´eline a combination Leroy stems play 2017; facilitate (a fungi al., the and ant-carton The et ants, on 2005). in the secretions) Orivel, build (hairs) & from sugary ants trichomes Corbara, nutrients by Other Ayroles, plant 2009). together Solano, with al., held (Dejean, fungi et tree soil fungal combining (Defossez and their hyphae by on material chewing detritus insects vegetative 2011). depositing and capture al., and fungus, geogra- et to defecating the and Defossez observed traps evolved 2012; of were al., independently ants fragments resident et three transporting the Blatrix in Forpatch, ant-plants, 2013; recently. food these al., of of et relatively one source (Blatrix identified In a associations been (tree) as ant-plant have used distinct are ant-plants phically patches and fungal domatium ants domatium 1995). on fungi, example, Ehleringer, waste (Janzen, involving & 1979; depositing Davidson, herbivores mutualisms Rickson, Treseder, by 1978; as Multipartite plant 1989; Huxley, Toney, such host 1993; & Gay, their enemies Ellgaard, 2011; feed Thien, Blatrix, against workers bodies) & Barber, ant plant some Rico-Gray, Selosse, food McKey, some the In Djieto-Lordon, or and defend (Defossez, 2009), nectar surfaces 2015). al., usually extrafloral et Renner, ants (e.g., Rosumek & resident host 1972; from (Chomicki The their formed leaves from 1985). are or rewards (Beattie, which hypocotyls, food domatia, obtain thorns, called (H ants stems, structures resident as hidden specialised the involve such in ant-plants, and 2011). parts ants complex al., plant to more et Voglmayr and modified space are 2014; plant interactions nesting Blatrix, myrme- the these provide & between as McKey, that Ant-plants Frederickson, - known suggest (Mayer, bipartite plants studies fungi as in recent as such of evolved but Weller,microorganisms thought have - Jeon, long mutualisms ants Cho, were resident complex (Kim, mutualisms its regions, bacteria Ant-plant tropical and protects (“ant-plants”). In fungi that cophytes bacteria pathogenic 2019). streptomyces from and Thomashow, plants 1999); fun- & strawberry Malloch, control the and & that include honeybees Summerbell, bacteria mutualisms Scott, pollinating antibiotic-producing & multipartite Efrony, (Currie, receive and Reshef, parasites of ants organisms Koren, garden attine interacting Examples (Rosenberg, gal fungus-farming viruses the and the a of 2013). an- archaea 2007); two of bacteria, Goodrich-Blair, have Zilber-Rosenberg, involves least consist & can also at mutualisms that (Hussa which that association Multipartite in benefit organisms coral-algae partners positive hosts. other two net their with than a on symbioses more effects of in (positive) association engage prolonged mutualistic bacteria or and (negative) fungi tagonistic as such Microorganisms types. INTRODUCTION chamber among mutualism differs multipartite fungi a of suggests data role This the types. that chamber and KEYWORDS: ant-plant the this of in each exists for fungi communities involving fungal the in patterns ldbe isn 90 n tesoti oedwfo eitrn hyra ntedomatium the in rear they hemipterans from honeydew obtain others and 1990) Wilson, & ¨ olldobler eaacdn,PacBio metabarcoding, , n-ln,mreoht,mtaim yboi,fungi, symbiosis, mutualism, myrmecophyte, Ant-plant, ymcdatuberosa Myrmecodia ymcdabeccarii Myrmecodia 2 ak(uica)(ih,11) u aesnebeen since have but 1911), (Miehe, (Rubiaceae) Jack ymcdabeccarii Myrmecodia ymcdabeccariiMyrmecodia slse svleal ne the under vulnerable as listed is okf Gninls Rubia- (Gentianales: Hook.f. nnegr Mayer, & ¨ onenberger, , hldi cor- Philidris Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. ot (12 north ieo iscinadwsecue.Tesuyae ersnsalrepr ftekondsrbto of distribution known the of at part National domatium large Kutini-Payamu its a in inside represents accessible) decomposing area be was study to Park The canopy National excluded. the River was beccarii in Annan and M. high from dissection too collected of often plant time (and 10 one Park from National rare and Kutini-Payamu communities at were Park, fungal sites plants the two analyse and the at plants), to sites but (9 aimed two Park We residential National from total). plants), River plants collected (10 Annan We (7 Beach at 2017. Cowley site March at one plants), until site (10 one 2016 total), March plants from (10 1) Cardwell (Fig. Australia Queensland, northern whole 46 collected We high a indicate will ant-plant it the correct, in had both have role different are fungi mutualistic the predictions indicate a in would these have roles it If fungi different mutualism. locations, that ant-plant play five this likelihood fungi the with across of suggest association communities would fungal chambers long fungal If it a distinct sites? domatium chambers, are geographical the across the there of consistent If this each dominate chambers. is in so, fungi distinct if of which are and chambers chambers, communities (a) the domatium among three questions: differ communities the the fungal in answer communities ants, fungal to symbiont the beccarii resident locations its sampled ant-plant, we five this this, of involving across test mutualism functioning To multipartite and fungi. a identity and is the there that and about hypothesized waste questions We the leaving in ant-plants, taxa epiphytic fungal in fungi. two fungi the noted into on (1982) air-flow focussing Huxley allow While studies that chambers 1991). (‘ventilation’) Jebb, of superficial chambers 1982; nursery and 1978, defecate, (Huxley, and system waste the deposit workers ant the the to due where doma- chambers loss The ry’) 2007). habitat Appelman, being & threat Kahler, Bahr, cordata main Lovatt, (Kemp, the trees of with host tium Queensland), its containing of forests (State of destruction 1992 Act Conservation Nature 2. 1. td ie n sampling and sites Study METHODS AND MATERIALS .beccarii M. mt .(yeotr:Friia)(uly 92.Tecabr nld mohwle (‘nurse- smooth-walled include chambers The 1982). (Huxley, Formicidae) (Hymenoptera: F. Smith b r ug nqet aho h he hme ye rsae mn h hmes c do (c) chambers? the among shared or types chamber three the of each to unique fungi are (b) ? ° 11.”,143 41’11.7”S, rmCrwl ntesuh(18 south the in Cardwell from otisantoko utpecabr omnyocpe ytentv ant native the by occupied commonly chambers multiple of network a contains .beccarii M. .cordata P. ymcdabeccariiMyrmecodia ° 00.0E Fg 1). (Fig. 0”E) 20’03. ep t ro eg,lra,adppe,wre ‘at’ hmeswhere chambers (‘waste’) warted pupae), and larvae, (eggs, brood its keeps hywr o netgtdi eal oorkolde hr r odetailed no are there knowledge, our To detail. in investigated not were they , ° 90.” 146 19’09.5”, lnsfrom plants 3 Melaleuca ° 25.”)t uiiPym ainlPr nthe in Park National Kutini-Payamu to 02’58.9”E) .beccarii. M. re tfielctossann 7 min km 675 spanning locations five at trees .beccarii M. Melaleuca lnsfo ahlocation, each from plants re nPr Douglas Port in trees .beccarii M. Philidris M. Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. iscinof Dissection ant week. per Each twice run-off. wool) of cotton where solution point of sucrose ball the 25% small of to beccarii a mL sprayer with 15 1 pressure (plugged approximately with vials garden and FIGURE ant-plants mL week a the 8 per of with plastic once two roots week worms into the meal per sprayed divided two we times with provided time, four was this the to colony During (16 prevent three collection). to Campus after water containers Cairns days tap the University, 21 of to Cook edge 1 James upper Fluon (approximately collected at with the The lined around greenhouse escaping. strip been a from had mm colonies that 25 from ant box a were Tangle-Trapresident plastic in collected and L Australia) ant-plants sides 27 NSW, its All a Murwillumbah, on into accessibility. Qld) immediately Tinbeerwah, and placed Unlimited, and workers, tree ant host of the presence base), the Melaleuca around mm 300 selected We .beccarii M. n-lnswr olce uigtesuy B nw itiuinof distribution Known (B) study. the during collected were ant-plants re tlat2 itn rmayohratpatcletd Each collected. ant-plant other any from distant m 20 least at trees .beccarii M. A a fnrhr uesad utai,soigtefielctoswhere locations five the showing Australia, Queensland, northern of Map (A) ymcdabeccarii Myrmecodia a enfound. been has n-lnsfrcleto ae nsz crufrneo oaimapproximately domatium of (circumference size on based collection for ant-plants n-lnsadcleto fchambers of collection and ant-plants .beccarii M. 4 ® Asrla noooia upisPy Ltd, Pty. Supplies Entomological (Australian n-lnswr eti hi lsi oe in boxes plastic their in kept were ant-plants ° 85.3S 145 48’58.83”S, .beccarii M. .beccarii M. ° 11.3E ni dissection until 41’16.73”E) e osindicate dots red – a eoe from removed was ® Myrmecodia (Livefoods Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. he hme ape oeo ahtp fcabr e n-ln bigacmiaino h chambers 2 the FIGURE of combination a (being study). ant-plant (pilot Beach potential per Cowley dissected only chamber) for The had be above). of which account to as Beach of type slices to Cowley (three each three for dissection ant-plant from of except collected ant-plant per (one dissection ant-plant samples each samples each chamber chamber during during Nine sample three collected open extraction. each were (Fig. left DNA between type) domatium chamber was for ethanol the each 99.5% processing tube of using sample edge control dissections during outer mL during contaminants brown- the 1.5 forceps near being and A structure chambers scalpel type ventilation collected. the honeycomb and nursery flame-sterilised a surfaces, We structures; and (wart-free) 2). surface surfaces smooth wart-like smooth weight with with with sample coloured brown/black coloured (total yellow tubes dark mL being being 1.5 chambers chambers individual For waste into scalpel placed features: domatium. a and using the characteristic collected of domatium were mg middle the ventilation) the 25 from and away from nursery, of chambers (waste, slice collection the type the one sample chamber from and lift each for domatium to removed of selected the chambers were were of 4-5 ant-plants slices MO, side/end slice, Three the Louis, each each with slices). from of cross-sections between St. slice roots mm ethanol one Aldrich, 10 and 99.5% including approximately (Sigma into using leaves, vertically acetate (flame-sterilised stems, sliced knife ethyl was The a domatium total Each colony. mL discarded. ant 10 and domatium the approximately euthanise in whole to soaked each USA) balls placed cotton we five dissection, with of time At sn h ignDes ln iiKt(ignPyLd itra utai)floigmanufacturer’s following samples, Australia) chamber Victoria, different Ltd, the Pty in (Qiagen fungi of Kit identity Mini the Plant determine DNeasy to Qiagen samples the the using from through DNA chambers extracted domatium We the into Greenfield. Sequencing flow M. visible and to pupae Photos: Extraction ant-plant. and air (b) DNA larvae the allow workers; (eggs, of which kept ant surface chambers, are by the ventilation colony on the deposited (c) of pores waste and brood photo); from the this nutrients where in surface absorb smooth that with structures chamber nursery wart-like with chambers waste ± gfrec hme yefrec lc) eietfidtedffrn hmesbsdo their on based chambers different the identified We slice). each for type chamber each for mg 5 (A) ymcdabeccariiMyrmecodia n-ln on ant-plant .beccarii M. Melaleuca 5 .beccarii M. n-ln noa . eldpatccontainer plastic sealed L 8.5 an into ant-plant rea adel uesad (B) Queensland. Cardwell, at tree n-lnswr h rtsto ant-plants of set first the were ant-plants .beccarii M. (a) Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. hmrcsqecsadrmvd.Afrhr1 Tswr eetda hmrcsqecs(reat of (artefacts as sequences detected (394 chimeric singletons being Global as plant). OTUs one detected 33 and were insect another (one OTUs removed in 16 were (resulting OTUs further non-fungal removal Two A or amplification). removed). e-values PCR Fungi and and kingdom sequences kingdom to of e chimeric 2). fungal e-values assignment between = the where E-values cost for to values dataset. extension matches BLASTn the gap reliable 1, from on as OTUs = removed based sequences cost of and OTUs assign opening assignment remaining gap to taxonomic the -1, used for = filtered cluster penalty manually 2013; databases non-singleton 1, further al., reference each = We (K˜oljalg et of reward (v7.2) ecology sequence size=7, UNITE abundant molecular word most and 0.001, fungal Godzik, the = & in GenBank for (e-value (Li set against search (v4.6) commonly BLASTn OTU) CD-HIT used as We (i.e. with similarity, The 2014). 2013). (OTUs) 97% al., al., Units of et et Taxonomic Taylor threshold (Bengtsson-Palme Operational (v1.0.9) a genes to ITSx at flanking with clustered (without the 2006) extracted were of sequences were reads middle region sites) ITS the ITS binding Full full in primer module. found 28S; (v1.11.1) were built-in and vsearch sequences 18S PipeCraft primer of with using removed full 1, removed where also = and were reads differences (chimeric read) detected artifacts barcode were Multiprimer (allowed 2010). sequences 2009) al., chimeric al., Potential et (Schloss 2). using (v1.36.1) = Quince, mothur Nichols, differences using Flouri, primer identifiers (Rognes, unique annotation. (v1.11.1) vsearch on taxonomy using based and conducted incorporates clustering were and (fastq platform reads filtering, Mah´e, 3) 2016) analysis CCS & chimera (library the This demultiplexing, 44336 of 2017). filtering, performed filtering 2), Tedersoo, were quality Quality (library & region) for ITS 49839 Hiiesalu, full tools Bahram, 1), (for required data (Anslan, (library sequencing (v1.0) 50461 PacBio the PipeCraft totalling of using reads analyses Bioinformatics CCS 4). yielded (library 52476 cells SMRT 8 The reads. CCS generating for v5.1.0.26411) used GUI Bioinformatics was and the minAccuracy=0.9) Services Analysis (minPasses=3, using Sequencing consensus Link settings Circular SMRT v2.1, cells recommendations. default (v5.1.0.26412, Polymerase SMRT manufacturer’s Link with Sequel SMRT the 8 following on using Pacific min pipeline to instrument 600 (CCS) following Sequel of sequences loaded preparation time PacBio were movie Qubit a library and libraries on using v2.1 SMRTbell chemistry sequenced The quantified to and was protocol. subjected method tube preparation and Diffusion each library libraries in four Amplicon DNA in Biosciences (FavorGen and of arranged Kit PCRs, amount Purification were extraction, PCR The DNA products FavorPrep (for Austria). a procedures using cycles Vienna, all purified 35 throughout Corp., were ( or used amplicons controls 30 were Pooled positive using Australia) and by in sequencing). controls naturally reamplified Negative were occur not band product. does visible PCR no enough yielding obtain 5 samples at to of DNA minutes electrophoresis minutes. gel 15 20 running initial approximately by 72 an in estimated at was conditions: amplified quantity minutes were thermo-cycling relative 10 following samples of the All activity. in cycle proofreading enzyme per out This 3’-5’ carried 0.011% reduction). and were rate 95 rate error activity PCRs error x exonuclease and 5 units, 5’-3’ estimated duplicate 99.5% units, both (0.5% polymerase, has polymerase Taq 2 proofreading mixture (modified modified of a polymerase consisted and DNA base) cocktail base FIREPol PCR 10-11 17 HOT with The and contains tagged Estonia) were for S1). Tartu, primers barcode Table The Biodyne, formal (Supplementary 2012). 0.5 the multiplexing al., extract, (ITS1-5.8S-ITS2), for et DNA region (Schoch identifiers al., (ITS) studies unique spacer et molecular pair (Tedersoo transcribed (TTCCTSCGCTTATTGATATGC) in (GGTCATTTAGAGGAAGTAA) internal fungi ITS4ngs full ITS1Fngs of primer identification the primer reverse target forward and to 2015) the 2014) al., using et (PCR) 50 (Tedersoo reaction eluted we chain where step polymerase final the at except instructions, ° ,floe y2 ylso 95 of cycles 25 by followed C, enovo de n eeec aaaebsdfitrn aantUIEUHM ees 72 Aaekvet (Abarenkov v7.2) release UCHIME UNITE (against filtering database-based reference and μ aho h rmr (20 primers the of each L ae fastq , 1 maxee ° .PRpout o aho h ulct ape eecmie n their and combined were samples duplicate the of each for products PCR C. ° μ o 0scns 55 seconds, 30 for C fdul-itle ae.TeHTFRPlBlend FIREPol HOT The water. double-distilled of L iln=5,fastq 50, = minlen μ ) 5 M), -20 6 μ n e and f5xHTFIREPol HOT x 5 of L μ ° fprfidDAisedo 100 of instead DNA purified of L o 0scns 72 seconds, 30 for C an ) h lee aawsdemultiplexed was data filtered The 0). = maxns -50 eemnal hce gis h e best ten the against checked manually were μ fDAsml n1 grs e for gel agarose 1% on sample DNA of L Cantharellus > e -20 ° ® o iueadafinal a and minute 1 for C eecniee unknown considered were ln atrMx(Solis Mix Master Blend p rmArc that Africa from sp. ® μ .W performed We L. ® uie PCR Purified . atrMix Master ® < e -50 Biotech were μ L Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. eue h akgs‘hlsq v.80 MMri oms 03,‘ea’(...)(kae tal., et (Oksanen (v.2.5.6) ‘vegan’ 2013), Holmes, & chamber (McMurdie each (v1.28.0) for the ‘phyloseq’ locations of packages five each the the for used across models We communities separate fungal (adjusted ran p-values in comparisons. (all also differences their pairwise We for and types using procedure). test type statistics chamber across to resampling test three types and step-down univariate chamber chambers a the calculate three the using to among an in testing argument abundant comparisons compute multiple “p.uni” significantly to pairwise for the were for mvabund with that used “LR” term in OTUs we = model interaction function fungal locations, test the an identify anova binomial”, To for and the combined). “negative table location, used locations = We deviance type, family ”pit.trap”. chamber of including = included differences analysis arguments function which resamp significant manyglm default The and model with for locations. a (likelihood-ratio-test), location, test five fit and the to across to type 2012) chambers chamber used Warton, three 2018). was the & Chen, mvabund in Wright, (H. communities Eddelbuettel, in OTU Naumann, (v1.6.20) Naumann, fungal (Wang, Wang, ‘VennDiagram’ the were (v4.0.1) package 2020; among OTUs ‘mvabund’ containing Warton, R package fungal dataset shared the analysis & the and abundance using Wilshire, Unique using multivariate diagram samples. the 2019) Venn 135 Team. used from a We Core OTUs creating (R, fungal by 164 3.6.1 the investigated version for R data in abundance sequence conducted were analyses for controls Statistical negative and positive removed. The analyses also controls. Statistical were because extraction total) samples or chamber in of dissection plant (8 dissection other other PCRs any the any the from during contaminated removed lab not not was had the ( OTU1029 it in samples. OTU1029 chamber of (open samples ant-plants AN03 occurrence tube 135 46 the single contained control from of a the controls dissection contained dissection/extraction total from AN03) One 28 a chambers plant removed. the the waste left were of Twenty-seven This 45 and 2020). contamination independent. and mean al., no chambers, there not ant-plant et ventilation in are this Greenfield 44 plant ([dataset] resulting in collected chambers, chambers – per each nursery the plant type) for of 46 chamber per sample interconnectedness chamber comprising each samples waste the (for 3 one because samples to and chamber necessary sample multiple plant 332 was chamber remaining per This ventilation the chambers one collected. collapsed sample, plant We of chamber from dataset. number nursery the 11 multiple one samp- from River, chamber being the excluded Annan Three were reduce Douglas). from they Port to 17 so (27 from Beach, fungi, samples chambers 30 Cowley amplify and waste Kutini-Payamu, from to 119 from failed 9 21 and les Cardwell, Cardwell, Douglas) from from from 30 Port 15 30 and Beach, River, from Park, Cowley Annan 30 National Kutini-Payamu from and from (26 Kutini-Payamu, from 20 chambers from (27 Beach, ventilation chambers Cowley 97 nursery from Douglas), 116 13 Port of Cardwell, comprised from were 26 cham- the River, samples domatium Annan chamber the and 335 from 90-97% The collected controls). samples at dissection/extraction amplicon matched DNA fungal of (32.9%) 335 bers OTUs including at samples 54 371 taxa further sequenced closest the We to A Of matched 2019). UNITE). (24.4%) al., OTUs and et 40 of (GenBank (Nilsson identity remaining the respectively databases taxonomic inside levels, the existing matched phylum taxa (42.7%) in and fungal OTUs class, dominant 70 order, OTUs, the family, 164 genus, on species, is of to thresholds study roughly similarity this sequence of remove used focus We to the filtered further because we of performed which chambers was domatium 2020) step al., across abundances et final OTUs occurrences read Greenfield 374 This 2 containing total that ([dataset] (only dataset with singletons) 2 a doubletons OTUs left of (all OTU any This abundances similarity. OTUs two erroneous sequence read removed five low potentially minimum also and with avoid coverage We with to along sequence controls. low removed removed positive with were was dataset) the dataset) the OTU in the control only in PCR found occurrence were positive one single only The with sequences. each total, in OTUs .beccarii M. n 6lbrtr otos( oiiecnrl n eaiePRcnrl,ad28 and controls, PCR negative 4 and controls positive (4 controls laboratory 36 and .beccarii M. . < 0 evn aae ihattlo 6 uglOU TbeS2). (Table OTUs fungal 164 of total a with dataset a leaving 10, > 97%, > 90%, 7 < > 0 eunesimilarity. sequence 90% 85%, Tremellomycetes > > 7 opeeitn uglISsequences ITS fungal pre-existing to 97% 80%, > 5,and 75%, p)s hsOUwsremoved was OTU this so sp.) > 0 omthOTUs match to 70% Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. h oa 6 Ts(i.3) fte14OU,4 eeuietfibea h re ee n 5o these of 15 and level sequence order of terms the 3B). in at (Fig. order unidentifiable total dominant were the the of 46 was 28% Chaetothyriales OTUs, of Fungi. up 164 57 kingdom highest making the beyond for abundance the Of assigned accounted using with 3A). collectively be level orders which not (Fig. Ascomycota, species could Eurotiales, The OTUs phylum to and Mortierellomycota. 164 from of Capnodiales, classified phylum were total 33.5% Chaetothyriales, were orders the from level, were 15.9% 25 one order OTUs and the and of the genus, of number Basidiomycota, Below to Sixteen phylum orders. 32.3% UNITE. from taxonomic family, and eight fungal a GenBank 25 to databases into classified public available classified were were OTUs across of (OTUs) OTUs 164 chambers units the 164 taxonomic domatium the operational the fungal of from distinct percent collected 164 from samples sequences 135 42,747 the of total a detected We an included types. also chamber We the type. of chamber each (Lenth, (v1.4.5) for and package locations emmeans location included between the used for effects comparisons We pairwise Fixed overdispersion. term for for optimization. OTU interaction account 2020) bobyqa to fungal an and effect compare and random (Bates, poisson level to (v1.1.21) type observation = package 2) chamber lme4 family of the with and in abundances 2015) function) location Walker, read (glmer & model (minimum effects Bolker, OTUs mixed Maechler, the 374 linear modes generalized the trophic with a assign containing using created richness to dataset were 2016) the plots al., used bar et We and OTUs. (Nguyen The fungal ”local” (v1.0) OTUs. abundant then to FUNGuild significantly abundant were used set the differentially type We to fitType chamber identify ‘ggplot2’. ”Wald”, on and to to ‘phyloseq’ contrasts DESeq2 set package Pairwise with test “Benjamini-Hochberg”. significance out to with carried set first location correction was and inference phyloseq data type multiple the further OTU using chamber to Abundance DESEq2 used both chambers. into was different included imported package the and ‘phyloseq’ Phyloseq in and into OTUs 2014) loaded Anders, fungal locations. & abundant across Huber, differentially differences (Love, investigate examine (v1.24.0) to depth) package separately sequencing the ‘DESeq2’ type standardized on chamber The and we each measure sampling then distance First, differences and Bray-Curtis varying locations. chambers), visualise with for (all (NMDS) five to account dataset scaling the whole multidimensional order (to across non-metric Hellinger-transformation in chambers performed a then plots different and with three ordination matrix the create OTU in to our communities 2016) fungal (Wickham, the (v.3.3.0) in ‘ggplot2’ and 2019) 2. 1. oiatfnii h oaimchambers domatium the in fungi Dominant RESULTS 8 .beccarii M. to ee2fnto.TeDSq model DESeq2 The function. deseq2 SplmnayTbeS) Seventy S2). Table (Supplementary Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. n h etlto n at hmessae h eetOU 9i oa)mkn pol .%o the of sequences 0.7% of 1.1% only for up the accounted making collectively of total) types 3.7% chamber in up the (9 of made sequences OTUs one the up which fewest to of made OTUs the unique 5.6% (Fig.4). OTUs OTUs shared 28 comprising shared 17 chambers OTUs, shared The 94 waste 16 chambers were nursery these abundances. shared and ventilation OTUs) the of chambers ventilation waste 164 and in abundances the and the nursery read OTUs and nursery The of 142 The The (57.3% chambers, 4). abundances. OTUs total. waste sequence (Fig. Ninety-four the the types in of chambers. chamber OTUs 88.8% ventilation three 125 the the were in among there shared OTUs detected, 138 OTUs and chambers 164 chambers, (B) domatium total the the OTUs). in Of 164 OTUs (total fungal of order shared chambers order. and each the abundant The Unique in to most found assigned the dominant. OTUs is OTUs are fungal Chaetothyriales fungal showing Eurotiales 164 of orders, the and number of of Capnodiales, chambers the abundances Chaetothyriales, the Relative indicate in showing brackets found orders, in (OTUs) fungal numbers units to taxonomic operational assigned fungal and of proportion Relative (A) 3 FIGURE oiatfna aooi resi h hmeso h ant-plant the of chambers the in orders taxonomic fungal Dominant 9 .beccarii M. n sindt fungal to assigned and ymcdabeccarii. Myrmecodia .beccarii M. Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. l oain Tbe1.Fna T iheswssgicnl ihri h etlto hmesa Port at chambers ventilation the in the higher chambers, fungal significantly nursery The across was differed richness the 1). composition OTU (Table for community Fungal locations and OTU of 1). fungal comparisons, comparisons (Table showed pairwise pairwise chambers locations ventilation the the all the of of in three one communities for OTU but differed communities all OTU for p fungal with locations (LRT=887, varied across location p composition and differed community 939.9, p type OTU 1165, = chamber fungal = (LRT between The (LRT chambers chambers chambers. ventilation waste the and the of for each for location locations among of chambers differed three the of of each distribution 609, analysed We geographic = the (LRT variation across pronounced were differences less communities community with Fungal fungal but different chambers, Significantly ventilation 5A). and (Fig. nursery p chambers the p two between 1300, other found = the (LRT also nursery from (LRT the distinct types p both was chamber in 1872, which communities = the fungal (LRT among the chambers ventilation from communities different fungal was the chambers the waste of in p each differences 2546, with significant = associated indicated OTUs. OTUs analysis 138 of chambers Our ventilation number chambers and total domatium OTUs; 142 The among chambers communities nursery Fungal OTUs). OTUs; overlapping) fungal 125 (not chambers 164 unique waste (total and is: areas) type chambers shaded chamber (overlapping chambers each among to and between shared ant-plant OTUs fungal the of chambers different 4 FIGURE < .0)(i.5A). (Fig. 0.001) < .0) ariecmaioso h hme ye hwdta h uglcmuiyi the in community fungal the that showed types chamber the of comparisons Pairwise 0.001). endarmo nqeadsae ugloeainltxnmcuis(Ts mn h three the among (OTUs) units taxonomic operational fungal shared and unique of diagram Venn ymcdabeccarii. Myrmecodia < .0) uglcmuiisi h at hmesfre cluster a formed chambers waste the in communities Fungal 0.001). .beccarii M. < .0)(i.5-) u nlssas niae ninteraction an indicated also analysis Our 5B-D). (Fig. 0.001) < < 10 .0) ariecmaiosfrtewsechambers waste the for comparisons Pairwise 0.001). eaaeyt eemn ftefna T communities OTU fungal the if determine to separately .0) usr hmes(R 9.,p 990.5, = (LRT chambers nursery 0.001), h ubr ipae ersn h ubrof number the represent displayed numbers The ymcdabeccarii Myrmecodia < .0)and 0.001) < 0.002), Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. usr n etlto.TeLTi h ieiodrtots.pvlei h dutdpvlecalculated p-value adjusted the testing). is in p-value correlation for test. account (to ratio resampling likelihood PIT-trap the via is iterations LRT resampling 999 The using ventilation. and Curtis nursery chambers Bray three depth. the beccarii sequencing Myrmecodia of and one sampling 1 from varying TABLE collected dimensions. for community k=3 account fungal with to used a used was of distance was sample chambers transformation a waste (dark Hellinger (purple), is locations and Douglas A five Park plot Port ventilation, the and National a nursery, of (red), one River on the Park from (blue) Annan point for National ventilation Kutini-Payamu Each plots surveyed: waste blue), and NMDS the locations (light grey). (orange) Beach in separate five nursery Cowley communities are the (yellow), the fungal D Cardwell across in the and differences communities showing showing C combined, fungal respectively, B, locations the Plots all from for distinct chambers. types are chamber (grey) three chambers the for of is chambers ventilation A and nursery waste, the in compositions 5 FIGURE types chamber three the of each for locations five S4). the (Table between significant richness not OTU were fungal 24.0 of (mean comparisons Beach pairwise Cowley and p=0.0018) 40.0 (mean Douglas ariecmaioso uglOUcmuiycmoiini h oaimcabr of chambers domatium the in composition community OTU fungal of comparisons Pairwise o-ercmliiesoa cln ND)odntosdslyn uglOUcommunity OTU fungal displaying ordinations (NMDS) scaling multidimensional Non-metric cosfielctos(h brvainN ainlPr) hmesicuewaste, include Chambers Park). National = NP abbreviation (the locations five across ± .7S)cmae otevniaincabr tCrwl ma 22.3 (mean Cardwell at chambers ventilation the to compared SE) 3.27 . hsodnto lticue 6 ugloeainltxnmcuis(OTUs). units taxonomic operational fungal 164 includes plot ordination This ± . E p SE, 2.6 11 < .08 Tbe 3adS,Fgr 2.Alother All S2). Figure S4, and S3 (Tables 0.0078) ymcdabeccariiMyrmecodia cosfielctos Plot locations. five across ± .3SE, 2.13 Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. adelv otDuls218003*206006*382006** * 0.006 0.023 ** 0.003 * 0.023 * * 0.023 0.023 * 0.023 328.2 280.6 386.1 * 0.023 164.7 * 0.015 204.1 252.6 * 0.023 * ** 258.6 0.046 0.002 0.177 258.1 0.112 293.2 0.177 0.061 258.5 0.177 250.6 360.3 191.9 0.177 * 0.046 216.5 165.3 237.2 0.177 ** 0.006 * 0.023 * 198.6 0.049 * 195.3 0.023 * 251.5 0.023 0.075 186.7 280.7 * 0.023 231.8 191.4 * 0.034 * 0.049 241.9 237.5 Douglas * Port 0.043 142.1 vs NP Douglas Kutini-Payamu Port vs Beach Cowley NP Kutini-Payamu 235.2 vs Beach Cowley Douglas Port vs Cardwell 207.6 NP Kutini-Payamu vs 195.3 Cardwell Beach Cowley Douglas 199.7 vs Port Cardwell vs NP River NP Kutini-Payamu Annan vs NP Beach River Cowley Annan vs NP River Cardwell Annan vs NP River Annan T07+Trichomeriaceae OTU0170+ OTUID 999 “ns” using abbreviation it calculated The but were testing). interaction, p-values in mvabund an correlation The for was non-significance. account there location. to (to which where resampling refers or PIT-trap the and OTUs via type from location fungal iterations chamber abundant resampling which and/or which differentially identified identify type significantly change analysis not chamber be fold the mvabund does to log2 for control The found the abundant (to were for significantly testing results). estimate 34 were multiple (mvabund error OTUs, for standard analysis 41 adjustment the abundance these value is Benjamini-Hochberg multivariate Of the SE size the and LFC rate). effect types) is discovery 2. the chamber p-value false base of is adjusted best to contrast Change The the scale pairwise log2Fold logarithmic the is estimate. samples, the a in all taxon of on changed average over Fungal abundance reported the taken OTU is is contrasts factors, the Mean OTUs. Pairwise much size Base study. fungal (how by results). this estimate abundant divided (DESeq2 in abundant most values, collected differentially count 10 OTUs significantly normalized be Top fungal to the the OTUs to 41 in databases found included GenBank/UNITE in is found of OTU chambers match chamber the the in for indicates (OTUs) significant units + OTUs taxonomic operational 2 fungal and abundant only, location 2). 2 (Table for TABLE effect significant significant chambers interaction OTUs OTUs more 10 an different or only, 6 and type one three location, chamber location in the for type, and abundant significant of significantly OTUs type more be 22 chamber identified to or analysis for OTUs mvabund one the 41 we in these, found locations, of abundant analysis across and significantly DESeq2 compositions Our were community fungal that types. the chamber individually in OTUs differences fungal were identified there why understand better To Type Chamber Type Chamber Type Chamber Type Chamber Type Chamber Type Chamber contrasts Pairwise ariecnrssadmliait bnac nlsst dniysgicnl differentially significantly identify to analysis abundance multivariate and contrasts Pairwise sp taxon Fungal 585 .240.7261 4.5244 15.8755 Mean Base sults re- DESeq2 .250.7564 6.7255 Change Fold log2 sults re- DESeq2 R -au R -au R p-value LRT Ventilation Ventilation p-value Nursery LRT Nursery p-value Waste LRT Waste 12 F Eadjusted SE LFC sults re- DESeq2 p-value sults re- DESeq2 < < .01waste 0.0001 waste 0.0001 ymcdabeccariiMyrmecodia ventilation vs. nursery vs. types chamber between contrasts sults re- DESeq2 h symbol The . < type chamber sults re- mvabund .0 sns ns 0.001 oaininter- location sults re- mvabund action sults re- mvabund Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. T06 Talaromyces OTU0469 Mycosphaerellales OTU0313 Mycosphaerellales OTU0302 Eurotiomycetes OTU0283+ Debaryomycetaceae OTU0263 Trichomeriaceae OTU0214 Chaetothyriales OTU0202+ Candida OTU0171 OTUID sp sp sp sp sp sp sp fluviatilis taxon Fungal .79424 0.7708 4.2540 5.3789 0.9049 2.7155 0.6314 0.9331 5.4152 7.3770 0.5795 6.1078 28.2797 0.8178 3.6296 1.4426 0.7087 4.2244 5.9390 0.7441 5.3425 10.9030 0.7408 3.4016 3.4609 sults re- DESeq2 .570.7927 5.8557 0.9169 3.1878 0.9461 5.9907 0.5974 7.1054 0.8289 4.1561 0.7237 5.2024 0.7509 5.5793 0.7660 5.2003 sults re- DESeq2 13 sults re- DESeq2 sults re- DESeq2 < < < < < < < < < < < < < < < < .01waste 0.0001 waste 0.0001 waste 0.001 waste 0.001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 ventilation vs. nursery vs. ventilation vs. nursery vs. ventilation vs. nursery vs. ventilation vs. nursery vs. ventilation vs. nursery vs. ventilation vs. nursery vs. ventilation vs. nursery vs. ventilation vs. nursery vs. sults re- DESeq2 < < < < < < < < sults re- mvabund .0 sns 0.001 ns 0.001 0.001 0.001 0.001 0.001 ns 0.001 ns 0.001 < < < < < < sults re- mvabund .5ns 0.05 ns 0.05 0.05 ns 0.001 ns 0.01 ns 0.01

< sults re- mvabund 0.001 Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. T04+Fusarium OTU0544+ Eurotiomycetes OTU0438 Chaetothyriales OTU0347+ Talaromyces OTU0300 Talaromyces OTU0281 Tremellomycetes OTU1029+ Tremellomycetes OTU1028 Tremellomycetes OTU1026+ Chaetothyriales OTU0518+ OTUID sp sp sp sp sp sp sp sp sp taxon Fungal 172 .450.5006 3.2445 11.7027 0.7077 3.0895 5.0590 0.6169 5.3899 41.1045 0.7015 3.0174 0.8275 7.3471 3.8785 waste 2.7017 0.0008 0.6642 0.8772 6.5169 19.2890 3.1547 0.5729 0.9057 6.4035 10.6870 0.9531 6.1675 7.9028 sults re- DESeq2 .150.7094 3.9125 ventilation 0.6077 0.0002 2.8327 0.6309 2.5572 0.6925 4.5797 0.6723 6.9120 0.9152 6.2846 0.9638 6.5854 sults re- DESeq2 14 sults re- DESeq2 sults re- DESeq2 < < < < < < < < < < < < < < .01nursery 0.0001 ventilation 0.0001 nursery 0.0001 nursery 0.0001 nursery 0.0001 nursery 0.0001 waste 0.0001 nursery 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste 0.0001 waste vs. waste vs. waste vs. ventilation vs. waste vs. waste vs. ventilation vs. ventilation vs. ventilation vs. ventilation vs. nursery vs. ventilation vs. nursery vs. ventilation vs. nursery vs. sults re- DESeq2 ventilation vs. < < < < < < < < sults re- mvabund < .0 sns ns ns 0.001 ns 0.001 ns 0.001 ns ns 0.001 ns 0.01 0.001 ns ns ns 0.001 ns 0.001 .0 sns ns 0.001 < < sults re- mvabund .0 ns 0.001 0.001

< sults re- mvabund 0.05 Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. T08 Capnodiales OTU0783 Capnodiales OTU0782 Capnodiales OTU0780 Exobasidiales OTU0677 Exobasidiales OTU0670 Exobasidiales OTU0648+ Hysteriales OTU0563+ OTUID sp sp sp sp sp sp sp taxon Fungal .81276 .39000 ventilation 0.0006 0.7309 2.7067 1.0831 0.9293 nursery 4.1842 0.0016 1.2627 0.7840 2.7730 2.0527 0.6091 3.2921 0.7402 3.6334 3.5540 1.0327 0.6214 3.9134 7.1766 0.7920 4.4893 13.3790 sults re- DESeq2 .19094 .05ventilation 0.0025 0.9241 3.3189 ventilation 0.0007 0.5443 2.1719 0.5907 5.4640 ventilation 0.0011 0.5580 2.1552 0.6151 ventilation 6.0686 0.0074 0.8049 2.3656 sults re- DESeq2 .850.7823 3.3875 ventilation 0.0027 0.4550 1.6127 0.4999 4.8572 15 sults re- DESeq2 sults re- DESeq2 < < < < < < < < < .01ventilation 0.0001 ventilation 0.0001 nursery 0.0001 ventilation 0.0001 ventilation 0.0001 nursery 0.0001 nursery 0.0001 .01ventilation 0.0001 ventilation 0.0001 nursery vs. waste vs. nursery vs. waste vs. waste vs. waste vs. nursery vs. waste vs. waste vs. waste vs. waste vs. sults re- DESeq2 waste vs. waste vs. waste vs. nursery vs. waste vs. < < < < < sults re- mvabund < < .0 sns ns 0.001 ns ns ns 0.001 ns 0.001 ns ns ns 0.001 ns 0.05 .1n ns ns 0.01 ns ns 0.05 sults re- mvabund sults re- mvabund Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. T06 Hysteriales OTU0567 Hysteriales OTU0561 Talaromyces OTU0457 Chaetothyriales OTU0373 Chaetothyriales OTU0372 Ustilaginomycotina OTU0291 Sporothrix OTU0221 Fellomyces OTU0938 Cuniculitremaceae OTU0898 OTUID sp sp sp sp sp sp eucalyptigena sp sp taxon Fungal .82353 .90001 nursery 0.9187 0.0014 3.9853 0.9930 5.3501 3.5635 6.2862 0.7004 3.0144 3.8679 0.7829 nursery 4.5343 0.0040 5.8739 1.14302 nursery 3.7334 0.0089 3.0937 0.8336 waste 2.5056 0.0075 4.3914 0.9430 2.7588 2.3369 0.5802 2.9681 1.6562 0.7263 3.4739 4.2562 sults re- DESeq2 .150.7912 4.7105 0.5676 3.8377 0.7291 3.6656 sults re- DESeq2 .300.7073 3.1380 nursery 0.0018 1.15231 4.2716 0.8282 nursery 4.1387 0.0092 0.9409 2.9882 16 sults re- DESeq2 sults re- DESeq2 < < < < < < < < < < .01nursery 0.0001 nursery 0.0001 nursery 0.0001 nursery 0.0001 ventilation 0.0001 nursery 0.0001 ventilation 0.0001 nursery 0.0001 .01nursery 0.0001 ventilation 0.0001 waste vs. waste vs. ventilation vs. waste vs. waste vs. waste vs. waste vs. waste vs. sults re- DESeq2 waste vs. waste vs. waste vs. ventilation vs. ventilation vs. ventilation vs. waste vs. ventilation vs. ns ns < < < sults re- mvabund ns ns ns ns < < .0 sns ns 0.001 ns ns 0.001 0.01 0.001 0.01 < < < sults re- mvabund < < < .1ns 0.01 .1ns 0.01 ns 0.01 .0 ns 0.001 ns 0.01 .1ns 0.01 sults re- mvabund Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. fte1 omnfna Tsi h at hmeswr sindt rpi oe yFNul with FUNGuild by Five modes 6). trophic were Figure to OTUs 3, assigned common (Table were 13 chambers chambers ventilation waste The the and of 3). in nursery 90% (Table OTUs the than chambers fungal in more all common absent were across 13 or OTUs the OTUs chamber abundance of waste these in common of low 13 very each Tremellomycetes these either for and of abundances (1) each Eurotiomycetes total of class (1), the abundances Eurotiales the percentage (4), Chaetothyriales from The orders and the (3). from (2), OTUs Mycosphaerellales fungal most common (2), contained 13 Saccharomycetales 3). found chambers nursery we (Table waste chambers, of OTU waste the and 34.8% the this abundance, 2 In samples, for of Tables chamber abundance terms OTUs 6, waste total in 27 all OTUs (Fig. the but the in chambers of abundances 27 of ventilation found (96.4%) different Each These of was significantly S1). 9.1% values). sp) with 2) and (Fig. (Eurotiomycetes shaded but (Table chambers abundances OTU0283 grey type abundant sequence example, 3, chamber differentially highest occurred For Table one significantly the that 3). 6, than as recorded OTUs identified more OTUs (Fig. those fungal previously in type 27 OTUs selecting occurred chamber these fungal by of a 41 OTUs) 10 for the 164 and samples of of the subset dataset a of were the 50% least (from at OTUs in common 27 identified We taxa fungal Dominant Kockovaella OTU0981 Candida OTU0821 Capnodiales OTU0815 Capnodiales OTU0746 Capnodiales OTU0674 Pezicula OTU0667 Cryptodiscus OTU0623 OTUID sp sp sp sp sp radicicola sp taxon Fungal .13214 .46007 ventilation 0.0078 waste 0.7486 0.0073 ventilation 2.1745 1.3734 0.0029 ventilation 2.1113 4.0572 0.7953 0.0008 1.0624 2.5703 0.9349 nursery 2.1288 3.3808 0.0066 1.3859 0.7384 2.2966 1.2467 1.2609 nursery 5.3410 0.0019 6.2013 0.9948 3.4673 3.7826 sults re- DESeq2 sults re- DESeq2 .74076 .08ventilation 0.0008 0.7361 ventilation 2.6744 0.0004 1.2750 4.8684 0.9956 4.5019 17 sults re- DESeq2 sults re- DESeq2 < < .01nursery 0.0001 .01ventilation 0.0001 waste vs. sults re- DESeq2 waste vs. ventilation vs. waste vs. waste vs. waste vs. waste vs. waste vs. waste vs. waste vs. ns sults re- mvabund ns < ns < < < .0 sns ns 0.001 ns 0.001 ns 0.01 ns ns 0.001 < sults re- mvabund < < < .1ns 0.01 .1ns 0.01 .0 ns 0.001 ns 0.01 sults re- mvabund Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. 2 uglOU nttl costefielctossree.Fna aaaetebs ac on in found match OTU best the OTUs, the 27 are common Taxa most Fungal the of surveyed. one as locations selected five be To the of UNITE). chambers across and different total) (GenBank three databases in the available in OTUs (OTUs) fungal units (27 taxonomic operational fungal common 6 FIGURE 2, (Table OTU0898 type and chamber OTU0347 with including effect common location OTUs interaction 12 for and an significant five chambers had nursery also 6). and the were and Fig. in two type), OTUs location location, common chambers, chamber for six for ventilation the significant (for abundant to Of were significantly abundant OTU1029. OTUs and also significantly OTU0283 two were were including Also, OTU0469) chambers and waste 2). OTU0302 (Table fungal the OTU0263, eight in 3). OTU0214, These (Table OTUs (OTU0202, chambers (2). common ventilation Tremellales the 13 and in fungal (3), The abundances common Capnodiales percentage from other (3), highest eight both Exobasidiales the contained OTUs, had orders chambers OTUs common the ventilation two to The contained belonging (OTU0300). also OTUs 77.2% nursery chambers in and found nursery both (OTU0281) were The of 84.5% that ( 50% Eurotiales, 3). with OTU0544 least (Table order ( but the abundances) Hypocrealean at chambers chambers nursery total the in ventilation the the the and occurring and in of Likewise, abundances OTU0438 Despite nursery low abundance. Eurotiomycetes the in total (72.8%). the both occurred the chambers of chambers, of nursery ventilation half 83.3% chambers the over was and ventilation in chambers just of nursery in abundance 56.8% orders the found and highest the in chambers was from OTU nursery OTU0563 (Table were this of Eurotiomycetes Hysteriales OTUs of 71.7% class chambers four abundance to in ventilation These classified the found and OTU was but chambers. one nursery OTU0347 these and The Chaetothyreales both (1) to Hypocreales 3). 3). common (1), (Table Hysteriales were symbiotrophs (1), that Chaetothyriales as OTUs two fungal and four saprotrophs had as assigned three A a sqec)audne A n eaie(eune bnacs()o h most the of (B) abundances (sequence) Relative and (A) abundances (sequence) Raw (A) > 0 ftenreycabr ihrltvl ihaudne of abundances high relatively with chambers nursery the of 50% 18 < 7)cmae otevniaincabr ( chambers ventilation the to compared 27%) ymcdabeccarii Myrmecodia Fusarium > 70% sp.) Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. T08 Talaromyces OTU0281 Tremellomycetes OTU1029 Tremellomycetes OTU1028 Tremellomycetes OTU1026 Chaetothyriales OTU0518 Talaromyces OTU0469 Mycosphaerellales OTU0313 Mycosphaerellales OTU0302 Eurotiomycetes OTU0283 Debaryomycetaceae OTU0263 Trichomeriaceae OTU0214 OTU0202 Candida OTU0171 Trichomeriaceae OTU0170 OTUID for modes: samples trophic of ranking: FUNGuild 50% confidence waste. least = represent at WC symbols (in ventilation, ++possible. common and = and PATH=pathotroph was VC +probable nursery, percentage OTU and = the and SAP=saprotroph NC indicates samples, SYM=symbiotroph, Chambers: shading chamber type). Grey in chamber OTU. abundance that the sequence sequence with number, percentage chambers hypothesis abundance, of species UNITE sequence number, (%), or accession similarity GenBank one, UNITE), in the ant-plant and the occur GenBank in of also databases types OTUs chamber may the 13 type of common’, and one chamber ‘most least a chambers, of for ventilation definition OTUs the 3 this TABLE common Under types. in most chamber OTUs types. the other chamber 12 the of the chambers, of each of nursery both, that one Note the least in at chambers. of OTUs waste 50% 6 least were at there in occur to had a sp sp sp sp sp sp sp sp sp sp sp sp Chaetothyriales fluviatilis sp taxon Fungal uglOeainlTxnmcUis(Ts htaesgicnl ieetal bnati at in abundant differentially significantly are that (OTUs) Units Taxonomic Operational Fungal U434H9180F1 0358. . 235. 362. SAP+ - - 26.7 97.8 60.0 13.6 - 11.4 0.0 52.2 71.1 - 13.0 SAP+ 12.3 57.8 4.3 4.5 - 90.7 64.4 95.1 3.2 0.0 6.5 60.0 - 3.4 9.1 84.5 0.0 96.1 2.2 57.8 0.0 - 5.9 19.6 SAP++ 375 99.9 KU141384SH194198.07FU100 4.9 2.6 100.0 2.3 2650 2.2 92.0 62.2 SH477174.07FU87 SYM+ JX999048 0.0 1.4 81 9.1 99.0 4.3 SH477174.07FU86 0.8 JX999048 68.9 - 0.0 1397 0.1 34.8 99.2 SH477174.07FU87 SAP++ JX999048 0.0 80.0 7.1 9.1 2.2 1283 96.4 HM239979SH212163.07FU93 66.7 SYM+ 0.3 1.0 21.7 602 9.1 99.2 KP143766SH209380.07FU99 2.3 68.9 4.5 0.5 93.5 17.4 97 0.0 GU117898SH1541673.08FU83 19.6 1.4 9.1 731 94.9 GU117898SH1541673.08FU84 2.2 0.8 3977 92.4 32.6 DQ914677SH206547.07FU83 3.2 4.3 255 93.9 KP109748SH192552.07FU94 0.4 1.8 829 KU195499SH491217.07FU94 1.4 7.2 1678 HQ634649SH196444.07FU99 4.7 472 HQ652068SH200664.07FU98 2308 KU195499SH491217.07FU95 ber num- sion Acces- GenBank ber num- SH UNITE (%) Simi Seq ymcdabeccariiMyrmecodia abund Seq 19 CV CN CWC VC NC WC VC NC ples sam- ber cham- in dance abun- quence se- % ihfna ao coetmthi online in match (closest taxon fungal with ples sam- ber cham- in dance abun- quence se- % ples sam- ber cham- in dance abun- quence se- % OTU with ples sam- ber cham- of %

OTU with ples sam- ber cham- of %

OTU with ples sam- ber cham- of % FUNGuild trophic mode Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. T06 Hysteriales OTU0561 Talaromyces OTU0457 OTU0373 OTU0372 Ustilaginomycotina OTU0291 Sporothrix OTU0221 Fellomyces OTU0938 Cuniculitremaceae OTU0898 Capnodiales OTU0783 Capnodiales OTU0782 Capnodiales OTU0780 Exobasidiales OTU0677 Exobasidiales OTU0670 Exobasidiales OTU0648 Hysteriales OTU0563 Fusarium OTU0544 Eurotiomycetes OTU0438 OTU0347 Talaromyces OTU0300 OTUID d c b sp sp sp Chaetothyriales sp Chaetothyriales sp eucalyptigena sp sp sp sp sp sp sp sp sp sp sp sp Chaetothyriales sp taxon Fungal F771H2240F9 2 783. . 482. 78- 17.8 SAP+ - 25.0 40.0 PATH- 26.7 - 34.8 34.1 44.4 - 22.7 15.6 3.4 47.8 2.3 6.7 45.7 11.4 38.7 15.2 23.9 43.2 30.4 3.4 PATH- 57.8 14.3 38.2 34.8 5.7 2.6 - 70.5 728 8.9 0.3 KF675741SH021234.07FU98 0.9 94.0 - - 4.5 6.7 75.0 512 - KF366489SH209380.07FU100 61.5 78.3 - 13.3 89.9 767 2.2 59.1 43.5 HQ634653SH025817.07FU98 2.2 20.9 309 - - 56.8 2.2 424 88.6 43.5 HQ634648SH212029.07FU100 1.5 52.3 13.3 580 21.7 99 63.6 8.9 37.0 AB180368SH025674.07FU91 3.1 64.8 13.0 61.4 19.6 84.1 4.5 0.2 56.5 33.7 KU865592- 28.3 1.0 - 41.3 69.3 0.6 73.1 - 40.4 270 SH204460.07FU100 AJ608646 94.1 3.4 15.6 26.1 83.2 0.5 26.8 20.0 579 KY103846SH1523569.08FU91 PATH- 52.1 4.9 59.1 16.2 176 73.4 52.3 605 KC222753SH025821.07FU96 22.2 44.5 54.3 205 26.1 167 56.5 KC222753SH025821.07FU99 - 100.0 88 355 SAP+ 1190 3.2 KC222753SH025821.07FU95 37.8 87 5.3 73.9 44.4 70.1 KP730059- 88 56.8 21.9 3.2 KP730059- 25.0 26.7 71.7 72.8 72.6 KP730059- 69.6 679 1637 2.4 24.2 KF675741SH205606.07FU97 20.0 86 14.3 1606 SH025137.07FU98 JQ905732 2.8 83.3 KX908623- 77.2 4070 KC951221SH212029.07FU97 859 SH194198.07FU99 KJ608116 ber num- sion Acces- GenBank ber num- SH UNITE (%) Simi Seq abund Seq 20 ples sam- ber cham- in dance abun- quence se- % ples sam- ber cham- in dance abun- quence se- % ples sam- ber cham- in dance abun- quence se- % OTU with ples sam- ber cham- of %

OTU with ples sam- ber cham- of %

OTU with ples sam- ber cham- of %

FUNGuild trophic mode

SAP- SAP- SAP- SYM+ SYM++ SYM++ Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. ftenme ffna Tsadteaudne fOU.Tedmnneo heohrae fungi Chaetothyriales of dominance The OTUs. of abundances of the chambers and domatium OTUs the fungal in of dominant number were the Chaetothyriales order of the chambers from domatium Fungi the in orders fungal of Dominant chambers the the of in terms each OTUs in beccarii. for fungal dominated communities M. of fungi fungal however Chaetothyrialean abundances type the to-date, and chamber in studied each numbers systems found to the The ant-plant we unique other of patterns not variation. with the generally As pronounced were driving types. less chambers km. of are chamber the 675 with abundances chambers of spanned OTU but domatium each waste which in in differences, the surveyed differences found the exhibited in locations OTUs also in five fungal communities of chambers all community fungal type ventilation across overlooked fungal chambers and long two The nursery other the The the compare ant-plant. from and distinct epiphytic identify consistently an to of first the chambers is study This DISCUSSION species: (ant Malaysia in salpinioideae) 2011) al., et plant afer momyrmex ant-plant of domatia match: species: a Kockovaella OTU0981 Candida OTU0821 Capnodiales OTU0815 Capnodiales OTU0746 Capnodiales OTU0674 Pezicula OTU0667 Cryptodiscus OTU0623 Hysteriales OTU0567 OTUID uglOU22–coetmth oai fant-plant of domatia match: closest – OTU0202 Fungal etahispida Keetia Crematogaster sp sp sp sp sp radicicola sp sp taxon Fungal d uglOU33–coetmth oai fant-plant of domatia match: closest – OTU0373 Fungal Friia) h1(lti ta. 2013) al., et (Blatrix Kh-1 (Formicinae)) Y088H7390F9 8 884. . 484. 11PATH- SAP++ 11.1 37.8 - 45.5 - 0.0 11.1 34.8 2.2 - 2.2 38.6 5.3 47.7 PATH- 15.6 99.4 39.1 SAP+ 46.0 8.7 45.5 8.9 0.0 4.6 - 48.8 2.2 37.0 20.5 0.7 48.7 0.6 8.9 47.7 285 19.6 KY103848SH176359.07FU95 96.8 3.5 46.8 157 27.3 26.1 SH203686.07FU96 JQ683772 59.1 1.3 2.5 263 47.8 SH025821.07FU95 0.2 JQ760724 37.4 48.5 279 KC222753SH025821.07FU98 3.0 51.5 50.2 198 KC222753SH025821.07FU94 37.0 48.3 619 HQ889715SH201622.07FU100 60.0 536 SH210980.07FU99 AJ877182 600 KF675741SH021234.07FU98 ber num- sion Acces- GenBank Rbaee nCmro atspecies: (ant Cameroon in (Rubiaceae) p Mriia) hk- Vgmy ta. 2011) al., et (Voglmayr KhNk3-2 (Myrmicinae)) sp. enroaarcn letouzeyi africana Leonardoxa ber num- SH UNITE ldmrapetalae Cladomyrma (%) Simi Seq abund Seq 21 c uglOU32–coetmth oai fant- of domatia match: closest – OTU0372 Fungal rmtgse margaritae Crematogaster Fbca:Ceapnoda)(n species: (ant Caesalpinioideae) (Fabaceae: etahispida Keetia Friia) AP Vgmy ta. 2011) al., et (Voglmayr MACP1 (Formicinae)) ples sam- ber cham- in dance abun- quence se- % aaathaipingensis Saraca ples sam- ber cham- in dance abun- quence se- % Rbaee nCmro (ant Cameroon in (Rubiaceae) b uglOU37–closest – OTU0347 Fungal ples sam- ber cham- in dance abun- quence se- % hk-b(Voglmayr KhNk2-2b ) .beccarii M. .beccarii M. Fbca:Cae- (Fabaceae: OTU with ples sam- ber cham- of %

nterms in

Apho- OTU with ples sam- ber cham- of % was

OTU with ples sam- ber cham- of %

FUNGuild trophic mode

SAP- SAP+ SYM++ Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. n ossetyfudars h v oain uvyd he omnOU eeietfidt the to identified were OTUs common Three of surveyed. chambers locations waste are five the in fungi the abundant Chaetothyriales across and specificity. these found ant common and/or if consistently also plant host determine and were any fungi to show they (non-Chaetothyriales) chambers region whether the Other or Australasian investigate systems, could ant-plant the allowed research epiphytic Future in that other according in Chaetothyrieales. ant-plants similarities symbiotrophs widespread order putative sequence epiphytic the as had other to (both Trichomeriaceae Chaetothyriales locations other of family three five the the other and the to The FUNGuild) across OTUs 2017). to these chambers al., of waste et two (Vasse the of origin identification according in geographic cluster found or not OTUs ant-plant, do in host fungal fungi found species, Chaetothyriales ant ant-associated OTU the some fungal to that reporting Chaetothyriales study A phylogenetic recent continents. different by on separately ( sub-families evolved Azteca different have from ant-plant are systems species the ant plant of the domatium Rubiaceae, Chaetothyriales family the order the in cordata from the found are from sequence species ant-plant fungi a two to four similarity included 99% pathogens. chambers hispida at against waste antimicrobial matched compounds as the in (OTU0202) defence such in fungi these as metabolites Alternatively, colony OTUs secondary ant their waste. fungal the some for from Common by least used nutrients used or at of be that food releasing could sources as expect and that cultivated surfaces represent to breakdown compounds be chamber reasonable and the may waste is deposits chambers in the it waste waste involved of Therefore, the colony’s are most trimming the fungi 1982). were on contain 1978, these workers mats (Huxley, chambers of ant thick acquisition waste the nutrient black The that plant to Miehe was for ant-plants. brown by explanation dissected dense noticed possible observed all first only ant-plant also environmentof was the epiphytic We ant-plant an and the neatly, an creating of hyphae. cut chambers in and/or fungal waste workers been waste), the ant had other and/or in by (and that or maintaining mats Java) fungi OTUs fungal are faeces of chamber observed workers in Maintenance who waste ant (1911) that fungi. common (e.g. suggest chambers specific 13 chambers) waste for these other suitable the of the to abundances from, fungi high absence transporting The or in, some surveyed. abundances chambers, low locations across five the found another. fungal across or being chamber of spreads OTUs chambers one potentially waste many in chambers The often interconnectedness despite among more the However, workers significantly given (88.8%) ant occurred unexpected most types. (94 OTUs of not up chamber OTUs fungal movement is made other fungal OTUs the collectively of shared across also and number of that or chambers particles high number chambers identity domatium the high three the the from The the in of clear among abundances. differences is shared total unique were This the by that variation chambers. of than OTUs) different by 164 rather the primarily the OTUs, in driven of specific OTUs were of fungal communities of abundances fungal number sequence somewhat in relative varied differences that the chambers, communities in fungal Between different harboured locations. chambers across ventilation and before nursery, chambers sequenced waste, at domatium been The unclassifiable never the UNITE). were have in and that OTUs (GenBank communities ant-plant Forty-six comparison fungal this for 2015). The in available fungi al., databases of et online Eurotiales species Chen the of are to (K. 2016; there presence according general al., suggesting The associated level the et 2008). order animal in Nepel the al., as Capnodiales well et 2009; order (Schlick-Steiner as the al., nests species from ant et OTUs and Defossez fungal in 2011) fungi of 2012; al., numbers et al., high (Voglmayr et also ant-carton Blatrix were There of 2013; chambers 2011). al., al., et et (Blatrix Voglmayr Guiana French and in .beccarii M. rmtgse margaritae Crematogaster Dlcoeia)in (Dolichoderinae) .beccarii M. p et on nests sp. Rbaee nCmro acsinnme Q369 Vgmy ta. 01.Atog these Although 2011). al., et (Voglmayr HQ634649) number (accession Cameroon in (Rubiaceae) .beccarri M. scnitn ihohrsuiso o-ppyi n-ln oai nCmro,Malaysia Cameroon, in domatia ant-plant non-epiphytic of studies other with consistent is sntsrrsn ie hsodro ug r bqiosi aueadicuesaprotrophic include and nature in ubiquitous are fungi of order this given surprising not is Cecropia .beccarii M. hc,tgte ihCattyilsfni aeas enfudascae with associated found been also have fungi, Chaetothyriales with together which, .beccarii M. nCmro Nple l,21;Vsee l,21) u eerhspot the supports research Our 2017). al., et Vasse 2016; al., et (Nepel Cameroon in re nCsaRc a loioae rmdmtaof domatia from isolated also was Rica Costa in trees otie 3fna Tsta eesgicnl bnatadcommon and abundant significantly were that OTUs fungal 13 contained and Crematogaster 22 p Mriia)in (Myrmicinae) sp. .hispida K. ymcdatuberosa Myrmecodia .hispida K. n hs ant- these and ) .beccarii M. occupied Philidris . n of One Keetia (in Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. eedffrn cosallctosadwr h nycabrtp ohv infiatdffrne nfungal in differences significant have to chambers ventilation type the chamber in only of communities the amounts) fungal were (and The and types locations different locations. all transporting/depositing different workers across some at ant different at chambers abundant the were waste more to significantly the due were chambers into OTUs this be domatium fungal waste the may and chamber in this waste fungi bacteria common of and numbers abundances 13 Different as locations the the alter such domatia. of to Seven outside likely microorganisms occupy. also micro-climates are they different other of ant-plants example, different possibly chambers in for workers to, ant domatium and of due the fungi locations surveyed. in across other locations vary fungus five with could the any interactions across of abundances by in occurrences influenced variation chamber large and between indicates interaction abundances OTUs and these OTUs The fungal of two the of for 18 location of locations and abundances in five variation the The geographic significant across The chambers. harbouring communities of ventilation OTU likelihood the fungal dur- the in in chambers increases Consistency fungi ventilation environment Capnodiales the outside as the Cameroon of in such to in ant-carton occurrence fungi chambers ant-carton observed the opportunistic ventilation in often explain of found we may exposure been and greater which have study, order 2011) fungi the this al., Capnodiales to mor- identified ing et their before. be and (Voglmayr only on recorded monocots Malaysia could based Lauraceae, been study and groups Ericanae, this never in from four have found plants into and sequences including Fungi divided Exobasidiales level parasitize, The are they Capnodiales. 2002). and range and (Begerow, pathogens host palms Exobasidiales plant plant order the be the and to from phology known also OTUs are we ant-plants. by Exobasidiales However, other dominated from in findings. were al., recorded these chambers et been support ventilation (Nepel not study The other have this each that from to fungi OTUs related Chaetothyriales fungal closely other Chaetothyriales found are four previously the sequenced and and 2014), isolated fungi were symbiont two OTUs domatium contained These and iso- also frequency. low sequence Cameroon chambers chambers. but nursery a in abundances nursery high The to with the match 373) with 2013). and 97% matched in al., 372 a (OTUs et abundances fungi had (Blatrix Chaetothyriales high and other KC951221) number locations ant-plant in (accession our the found Cameroon of of were four domatia from at some Chaetothyriales lated as common order types was chamber the OTU0347 two Chaetothyriales from these the between fungi and overlap larvae. ant-plant Three was the this with there in and why associated changes nursery be explain temperature/humidity the the may further between to in fungi may brood response rarely moving brood but in workers chambers, of be ant the the ventilation movement observed may why and in we This nursery absent) explain and the (or chambers. pupae) might both ventilation uncommon only This in relatively then brood (and are stem. found chambers the that often the waste taxa in we beneath fungal Also, chambers domatium some chambers. nursery shared waste the the chambers of ventilation into portion differed and lead OTUs nursery upper/middle chambers of fungal the ventilation common domatium towards The the the more of of types. most chamber part of pronounced two lower/middle abundances so former the not however were the chambers chambers, between ventilation waste occurrence and little the nursery very between with communities with compared role. fungal but functional the abundance) in yet-to-be-established high differences a with The suggesting (some It types, fungal chambers non-symbiotic non-Chaetothyriales chamber waste or these the other species. found of the opportunistic, Pleosporales, we Chaetothyriales 50% in However, Hypocreales, 2017), than of 2013). Eurotiales, al., more mycelium al., as in et et determined, the such OTUs (Vasse (Blatrix be orders domatia on contaminants to ant-plant from mycoparasites likely in yet fungi competitors most is as of chambers are act occurrence waste Saccharomycetales (Weiss, yeasts the the and pathogens that these in suggested animal any) that been (if or possible has role mycoparasites is Their are it 2014). that but Oberwinkler, yeasts & mostly Sampaio, contains Bauer, which Tremellomycetes class > 8 dniysmlrt osqecsioae rmteant-plant the from isolated sequences to similarity identity 98% aaathaipingensis Saraca Fusarium enroaarcn letouzeyi africana Leonardoxa T04 hc a bnati l etlto chambers. ventilation all in abundant was which OTU0544 .beccarii M. Fbca)i aasarsetvl Vgmy ta. 01.All 2011). al., et (Voglmayr respectively Malaysia in (Fabaceae) hra h at hmestn ob concentrated be to tend chambers waste the whereas , 23 atspecies: (ant etahispida Keetia .beccarri M. pooymxafter Aphomomyrmex slkl obe to likely is (Rubiaceae) in ) Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. ...dsge h td,cnutdtefil n a ok olce h aa nlsdtedt,cetdthe created data, the analysed data, the collected work, lab and field the conducted study, the designed M.J.G. and Kading, Greta CONTRIBUTION Underwood, Pauline AUTHOR Avril advice Coldman, Nuske, technical Ian Susan their included Bransgrove, for project Kaylene University Connor. this Muir, Cook Brad during Jenn James at and helped Hucks Barry who multiplexing Louise Volunteers Lenancker, the and regarding techniques. Dillon thank training molecular Natalie We and regarding respectively. advice Dr. technical Park, and for National process, collecting Estonia) River and PCR Tartu, finding Annan of in and Yuku-Baja-Muliku (University support Ya’u field Park Puusepp the daily Kuuku Rasmus National and and Kutini-Payamu Northern guidance River the their the for the from Lockhart Cooktown of ant-plants and in in Program owners Norway Trust Ranger traditional of Sea Land and the Council Land RNTBC supported Research thank Corporation and We the Aboriginal Oslo of Kanthanampu Authorities. of programs University Health ”Infrastructure” Regional the and by Southeastern Genomics” Centre hosted Sequencing ”Functional Norwegian platform the the by technology by provided was national service a sequencing The (www.sequencing.uio.no), Douglas. Port in streets residential mutualisms. ant-plant The epiphytic of stability unravel and ultimately maintenance, could trees evolution, ACKNOWLEDGEMENTS questions host fungi the these not) the Whether in Answering (or and important generally. common colony. workers, environment are of and ant ant the fungi widespread Sampling resident in the how whether research. and their explain for ant-plants future help as these of could food well in live, focus of as ant-plants are the epiphytic ant-plants, source which be epiphytic a in should safe other habitat ant-plants as chambers of and in nursery used chambers functions the the be these in in may of fungi brood other all, fungi various the or or any, keep include so pathogens, perform may in that involved fungi ant-plant fungal compounds are mutualism this antibiotic or fungi this in of However, bacterial in role production from chambers. fungi their the waste of and as the community role organisms, such in The fungal other functions waste ant-plant. specific with organic this interactions of a of whether breakdown different distribution contain determining many the the chambers include in of waste to step portion unequivocally likely the first large yet is a the that not over achieved showing have constant have waste by we is we mutu- the that While exists ant-plant, this in this mutualism in distinct chambers. in players multipartite is waste play potential community a the three fungal fungi from the the role/s nutrients chambers, where what absorbs waste chambers determined waste plant the the in the waste in purposes and is deposit different chambers, ants It serve ant-plant the plant. epiphytic of intersect: the this alism and specimens of colony of chambers ant frag- different inclusion the their The the for given populations. and extraordinary suburban range is and broad chambers parks ant-plant a among across communities distribution fungal mented in patterns consistent domatium The the likely in is found it were and here patterns chambers discussed Conclusions locations, OTUs these across fungal in differences common workers these the ant Despite of types. of by significant, abundances chamber chambers tended fungi. and three unwanted were are occurrences the of chambers colony the free of the nursery in chambers stable of the these most ant brood keep for the workers the possible are locations the because chambers is of across nursery be it differ the comparisons ant could and could in observed This pairwise that communities domatium also the chambers fungal We on ventilation of the present the suggesting fungi. three of into sometimes different exposed environment Only are of outside most range the that the locations. from a are It pores fungi to they large Cardwell. transport given therefore workers via and variation, and Beach domatia most environment Cowley the entering outside to had workers the compared chambers to ventilation Douglas chambers the Port all that at surprising higher not being is locations, across richness OTU emt(o IK6005 rne oM refil yteDprmn fEvrnetadHeritage and Environment of collect to Department Greenfield the M. by to permission Greenfield granted M. Council Shire to Douglas granted Protection. WITK16204015) (No. Permit ymcdabeccarii Myrmecodia .beccarii M. . n-lnscletddrn hspoetwr oes ne cetfi Purposes Scientific a under so done were project this during collected ant-plants 24 .beccarii M. .beccarii M. rmbt national both from n-lnsfrom ant-plants Posted on Authorea 11 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159986299.94896964 — This a preprint and has not been peer reviewed. Data may be preliminary. lti,R,Dbu,S,SlsLpz . on . eot . ce,D . j´t-odn .(2013). Dji´eto-Lordon, C. . Symbioses. Ant-Plant-Fungus . Evolved . Independently B., D. in 8 McKey, Specificity L., One, Cultivar Benoit, Plos H. Fungal C., of R. Born, Evolution A., Nilsson, Salas-Lopez, Repeated S., . Debaud, R., data. . fungi Blatrix, sequencing of . sequences environmental ITS A., of doi:10.1111/2041-210x.12073 ribosomal Godhe, from analysis 914-919. ITS2 Z., for (10), and Wang, eukaryotes ITS1 S., of other extraction Branco, and and M., detection software Hartmann, Improved M., (2013). Ryberg, J., hypothesis. Bengtsson-Palme, evolutionary An Exobasidiales: The doi:10.1007/s11557-006-0018-7 (2002). D. using Begerow, Models Mixed-Effects Linear Fitting (1985). Press. (2015). J. S. A. Walker, Beattie, & M., B. Bolker, M., lme4. Fungi. Maechler, and for Ants D., between toolkit Bates, Relations open-source Some Flexible (1920). W. PipeCraft: I. Bailey, (2017). L. data. sequencing Tedersoo, amplicon & U. high-throughput 17 Koljalg, I., custom of . Hiiesalu, perspectives. analysis future M., . bioinformatics and Bahram, . updates S., S., recent Erland, - Anslan, U., fungi Eberhardt, of J., identification 186 I. molecular (2020). Phytologist, Alexander, for New S. H., database UNITE K. Abell, locations The Larsson, & five H., (2010). 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