Posted on Authorea 20 Feb 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.161383346.69785032/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. [email protected] author *Corresponding 6845 3 Australia, Western Perth, U1987, Box 6845 Australia, 2 Western Perth, U1987, Box GPP 1 plant N.E. M. associated White, their Heyde, and der invertebrates van of metabarcoding DNA using communities trajectories ambitious the restoration given Evaluating Restoration. salient Ecosystem particularly techniques now on monitoring decade is new UN and the outcomes, incorporating with restoration and associated improving Testing targets to restoration. invertebrate restoration critical of for climate global is providing metabarcoding trajectories harsh metabarcoding locally, DNA likely DNA its foraging how evaluate as show are with to such we invertebrates Pilbara, Thus, used indicate the environment. be results their can in assay and communities trajectory invertebrates Plant between the interactions unclear. while about was communities, data have flooding, climax which most monsoonal was invertebrates, stable recovery unpredictable community airborne relatively Invertebrate and in with conditions. weaker environmental ecosystems were to fidelity patterns in local These evident less communities therefore time. with and over signals, abilities communities com- restoration dispersal plant reference higher clearest and to the invertebrate similar showed identified deter- invertebrates more and about Ground-dwelling to Australia, becoming Western used information metabarcoding. in be provide DNA locations may different chronosequences using also across ecologically communities munities invertebrates can three invertebrate airborne in how and and restoration ground-dwelling evaluate invertebrates mine-site collected we We survey of Here restoration. rapidly many during interacting. to are trajectories are ecosystem there tool invertebrates mine in and a those overlooked expertise, which is taxonomic often with rare are metabarcoding plants require invertebrates DNA often Unfortunately, for- survey, soil to restoration. species. and time-consuming ecological undescribed are herbivory of they decomposition, indicators because pollination, as monitoring identified restoration (e.g. been functions have ecosystem and critical mation), many provide communities Invertebrate Abstract 2021 20, February 1 Majer Heyde der van Mieke communities plant associated of their metabarcoding and invertebrates DNA using trajectories restoration Evaluating niomna rtcinAtoiy 1 abo uy elntn61,NwZealand. New 6011, Wellington Quay, Lambton 215 GPP Authority, University, Protection Curtin Environmental Sciences, Molecular and Life Bentley, of University, School Curtin Laboratory, Sciences, DNA Environmental Life and and Trace Molecular of School Restoration, Site Mine for Centre ARC utnUiest ete Campus Bentley University Curtin 1 rn Wardell-Johnson Grant , 2 eil P. Nevill, , 1,2* uc,M. Bunce, , ,2 1, 1 ihe Bunce Michael , 2,3 1 io,K.W. Dixon, , ioeWhite Nicole , 1 iglyDixon Kingsley , 1 1 enne,K. Fernandes, , 1 n alNevill Paul and , 1 rse Fernandes Kristen , 2 ae,J. Majer, , 1 1 adl-ono,G. Wardell-Johnson, , 1 Jonathan , 1 , Posted on Authorea 20 Feb 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.161383346.69785032/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. ae tpriual icl oietf netbaeseies fe eurn ayepr person-hours expert many requiring communities often invertebrate specimens, within invertebrate diversity identify high to the likely. 1977; difficult change, less particularly restoration ecosystem (Southwood it during of (adversity)-strategists makes changes indicators often directional A excellent are make of being that may Despite ecosystems 1989) selection harsher Majer and in 1985; However, diversity al. 2006). et lower Gross Dunlop the and Suding unpredictable, recovery 2016; naturally of al. is trajectories et expectation restoration evaluate (Mcdonald and to community objective used the be where may convergence, communities environments disturbed or favorable invertebrate in and such, thrive predictable, As which require species, 1989). which r-strategist (Majer species, to generalist K-selected al. from to et shift et responses environments, a (Andersen (Magura unpredictable to restoration in forest and attributed during regenerating variation been composition a have community in 2009) the in millipedes Majer changes ability, 2002; than of dispersal quickly with with more Some mixed, beetles Along recolonize example, 2015). are 2012). to For al., ability. able restoration dispersal Banks, are to to abilities Fr`ere, attributed & responses dispersal be (Cristescu, high may arthropods, classes taxa arthropod of (Gaston, different target groups among diverse restoration incredibly particular the and target capture, Majer, on to to 1998; easy depending tend Folgarait, numerous, Rosenberg, studies being 1997; 1998; mention Because (Folgarait, Sparling, to function 1991). & ecosystem not for 1986), Andersen essential Lehmkuhl, are both 2002; & and in Danks, disturbances al. recovery to ecosystem et sensitive of are indicators (Andersen They as 2009). systems used been terrestrial long have and they 2020; aquatic as Cross interest particular and including of Bateman, are function paid Cross, Invertebrates 2018; been ecosystem (Catterall has many restoration attention ecological greater in facilitate Recently, 2009). and role 2004). Catterall, Majer assess Holland, Poff, 2006; vital that both & to Geber, & a assumption Alarc´on, Andersen, fauna & understanding Bronstein, Ambrose, play (Bronstein, to general and Ness, cycling they (Palmer, the 2013), 2001; nutrient with communities Hunter, because and Banks, Fr´ere,2018; & plant pollination press), important Rhodes, dispersal, in of is (Cristescu, seed case return fauna pedogenesis, al. the the of always et recovery with not Borges the is area Gomes this an 2005; However, recolonize Dixon, Aide 1997). Craig, naturally and Tomlinson, (Cross, will Ruiz-jaen vegetation they of 2019; favor in al. monitoring restoration et in overlooked often are Fauna restoration global ambitious the Introduction Restoration. critical given Ecosystem is salient on for metabarcoding decade particularly UN DNA now trajectories the likely as is with evaluate such and associated to techniques outcomes, targets used monitoring restoration we be new improving Thus, can incorporating are to environment. communities and invertebrates their invertebrate Testing indicate and harsh of results invertebrates restoration. its metabarcoding assay between with DNA Plant interactions Pilbara, how about the show unclear. in data was providing trajectory flooding, locally, and the evident foraging abilities monsoonal while most communities, was dispersal unpredictable recovery climax higher and community stable have climate Invertebrate relatively which conditions. communities with invertebrates, environmental reference ecosystems airborne to in to in fidelity similar weaker local more invertebrates were less becoming patterns Ground-dwelling therefore communities These and with metabarcoding. Australia, time. determine signals, Western DNA over restoration to in using clearest locations used across communities the different invertebrates be in- plant showed ecologically airborne those may and three and which invertebrate communities in ground-dwelling with identified is invertebrate restoration collected plants mine-site metabarcoding how We about of DNA evaluate information restoration. chronosequences we provide during species. also Here survey, trajectories undescribed can to ecosystem many and interacting. time-consuming invertebrates are are are survey Unfortunately, there vertebrates rapidly they and restoration. to because expertise, ecological tool monitoring of taxonomic a restoration indicators rare as in require her- identified overlooked often decomposition, been often pollination, have are and (e.g. invertebrates formation), functions soil ecosystem and critical bivory many provide communities Invertebrate Abstract 2 directional hnei opsto oad reference a towards composition in change Posted on Authorea 20 Feb 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.161383346.69785032/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. igwt h lnsi n rudrsoainsites interac- restoration are around communities and invertebrates invertebrate in how as plants indicating well the by with area. as information ting site recovery functional the of provides outside trajectories Metabarcoding from show iv) sourced not be will may invertebrates less DNA plant with than because associated clearly plants more recovery The of iii) trajectories ecosystems. demonstrate unpredictable communities harsher climax climatically diverse, conditions. stable because environmental invertebrates with local airborne Ecosystems reflect than ii) better better they trajectories abilities recovery dispersal show lower reference will with invertebrates to dwelling (convergence hypotheses: Ground trajectories four i) restoration have we evaluate We whether evaluates to sites. invertebrates, study invertebrates restored airborne This in of and ecosystems. communities) dwelling and metabarcoding climates ground DNA different capture with use that spatially locations two can types multiple use in we trap the Here sites two and sites. monitor study mine mine, reference and to per the per site to invertebrates sites reference closest ground-dwelling were single reference sites a of separated older used metabarcoding as study auto-correlated this DNA spatially however, fidelity were 2019); of results al., high use et have (Fernandes the necessarily restoration explored site not mine has may work they earlier 2020a), for al., Our approaches et DNA Heyde such these plant der conditions. carry of However, (van interaction may local utility 2009). area invertebrates the to the the restoration al., illustrate since the so However, can et outside restoration. context, communities from (Jurado-Rivera during restoration these plants consumed assessing a and that Presumably, invertebrates have in between unknown. suggest they undertaken is studies (Pornon been species visited monitoring previous have not restoration plant arthropods, pollinators hitherto which species of and plant have case and which diversity with studies identify the interacting invertebrate 2016) to In been able only al., 2016). have be not et should they al., detect samples organisms et arthropod to the Pornon from ability identifying DNA 2009; by its al., data is et functional metabarcoding (Jurado-Rivera provide 2016). DNA also restoration al., of to but et advantages responses composition (Beng the arthropod change land-use demonstrate of (Maja- and to One method 2019) used extraction DNA al., been DNA analy- using et has or and (Fernandes estimates 2015) data stored mining abundance Leese, presence/absence readily post Although & be 2018), 2018). (Elbrecht can al., al., bias 2012). data et primer et methods sequencing al., neva (Fernandes by the morphological skewed regulators et benefit, conventional often as Yu added are than such an 2013; party, metabarcoding faster As third al., 2013). and me- a al., et DNA reliable, by et individually, Ji zed accurate, Ji identified 2016; 2016; be be al., al., to to et et shown (Beng has (Beng been specimen each genome has where the tabarcoding invertebrate metabarcoding identification, determine of DNA morphological to regions using to sequencing Compared reduced barcoding 2009). high-throughput be small uses (Majer, can process Lepidoptera) from monitoring This (i.e. diversity invertebrate assignments. value conservation taxonomic with and provide associated to legal (Andersen difficulties of bioindicators the and 2004; good of threatened al., al., be Some et are to et shown (Austin they (Austin previously undescribed or restoration have endemism is to 2002), they of diversity responses al. because invertebrate arthropod degree et either at of high taxa looking 75% particular its studies as targeted most with Hebert, much Consequently, have as & Australia 2003). where Austin, Fisher, in & and (Smith, Harvey, especially 2015) Yeates, cryptic identified, al., are et be taxa & Rix to invertebrate Hamilton, 2004; many yet (Pearson, Additionally, worldwide have 2013). dwindling or and al. is costly 2005) et that is expertise Majer process taxonomic This 2011; 1983). on Erwin (Majer dependent taxa and invertebrate consuming, different time in specializing taxonomists multiple from 3 Posted on Authorea 20 Feb 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.161383346.69785032/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. MKni ta. 09.TePlaai infiatmnn einadacut o 9 fgoa rnore iron global of 39% for accounts and region mining significant gum invertebrates a snappy A-strategist is of r-and Pilbara woodlands of The open range 2009). support which al., wide fertility, et a low (McKenzie with for loams select stony to acidic with ( thought are climate are Soils 1989). arid rainfall (Majer, yearly hot, 15 in a of variation minimum Pinder, has large & mean Pilbara Leeuwen, a van have The (McKenzie, Temperatures activity Australia. 30.6 cyclonic flooding. with north-western unpredictable associated causing in and South32). 2009) summer, is from in Pilbara, (Data occurring rainfall sites the most reference location, increased of has percentages third cover cover Total plants South32). The similar species. from recalcitrant Fabaceae to (Data species, and restoration applied native Myrtaceae of is by 100 age superphosphate dominated over with of are with treatment sites seeded one-time restoration South32 stock- ripped, and a Fresh by with Reference then and gravel. supplemented undertaken planted, were and and were was sites area material grasses) restoration restoration The waste (mostly the needed. using All to shaped as areas old. was mined topsoil years newly landscape piled 20 from the transferred and (32.96 mining directly After South32 11, was owners. by topsoil 6, mine run 2, previous now sites the is or restoration which sam- mine and We iron bauxite sites 1975). in the reference (Havel, rich from Myrtaceae sites surfaces and six with Dasypogonaceae, pled gravel Proteaceae, by in Asteraceae, dominated high Fabaceae, Anthericaceae, is and vegetation poor The patens, nutrient E. 1991). are (McArthur, soils mean aluminum lateritic climate; and The Mediterranean Meteorology). hot-summer similar of a Bureau has 8.6 and of 2007) ( temperature al., minimum et Jarrah age (Myers the restoration hotspot 2013). Biodiversity density with in Global Stevens, increased and location & has richness cover Dixon, second species percent (Benigno, The Plant and sites sites, surrounding species. reference reference the plant in than to highest native restoration areas is with direct in and mined included seeding higher and returning and be owners of mine ripping, to aim previous tended topsoil, and the Construction Hanson fresh with by Hanson of done restored was a transfer been restoration at have All sites woodlands. sites Banksia The eight native old. Cyperaceae, sampled years (31.76 Stylidiaceae, we 22 Lexia Anthericaceae, 2018, 11,14, in in October quarry In species sand Materials 1977). non-woody (Trudgen, and Haemodoraceae Proteaceae, and referred Ericaceae, is species, Myrtaceae, ecosystem tree The of dominant 1991). high the dominant McArthur, with recognized less dunes, after 1989; Bassendean include globally Heddle, woodland silicaceous a & Banksia the Australia, (Dodd on as south-western capacity located to is water-holding of mine low region The and broader 2007). minimum acidity al., the mean et of winters; (Myers part wet hotspot biodiversity is cool mild communities. location reference with This criteria: in climate Meteorology). following variation Mediterranean the for warm-summer 12.8 account of in a (2020b), to temperature similarity basis has al. other sites, the Plain each restoration et Coastal on from to Heyde The proximity separate selected der efforts, spatially were least van restoration and at of in sites aspect, and target described (Young), reference and the old previously two slope are years are that locations 9 ecosystems sites than all to less These At similarity sites (Older). two below. of years least briefly Figure sites at 9 see location, and sampled than 1, each we (Figure older At locations, sites sites location. three reference each two all separated within At spatially restora- two sites maps). in with the for along consistency of S1 sampled was aspect were Australia, There site Western age (PB). in and restoration Pilbara apart different climate and km type, (JF); 1000 soil Forest to approaches, Jarrah up tion (SCP); locations Plain three Coastal from Swan sampled namely: were sites reference and Restoration Sites Study Methods and Materials .leucophloia E. ° ,wt 6. mma anal(utainBra fMtoooy.Teufvual odtosand conditions unfavourable The Meteorology). of Bureau (Australian rainfall mean mm 263.8 with C, and .wandoo E. vrhmokgass( grasses hummock over ) ° ,ma aiu 24.7 maximum mean C, uaytstodtiana Eucalyptus h nesoycnit fslrpylu husfo eea aiisincluding families several from shrubs sclerophyllous of consists understory The . ° ,ma aiu f23.7 of maximum mean C, clpu marginata Ecalyptus ° ,115.95 S, roi ien,T aeoi,T lanigera T. basedowii, T. wiseana, Triodia ° and ,adwt 5 mma nulrifl Asrla ueuof Bureau (Australian rainfall annual mean mm 757 with and C, uti floribunda. 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Data may be preliminary. ihrSinic.Sqecn a efre nteIlmn ie ltomuigte30cceV sper as V2 cycle 300 the using platform (Thermo MiSeq Quantitation Illumina Fluorometric the Qubit on QIAquick PippinPrep instructions. using a performed a manufacturer’s quantified using was using cleaned same Sequencing and were (150-450bp) Germany) Scientific). the Libraries that Fisher performed (Qiagen, U.S.A). with primer-dimer was MA, Kit Beverly, done any selection Purification Science, remove were (Sage PCR Size to PCRs cassette size-selected PCR. bromide The was fusion ethidium equimolar prevent that 2% approximately during library to mixes into added. accumulated amplicon pooled the lab was have then PCR stochasticity, DNA were the may a PCR Samples in before produce minimize above. to previously room described to concentrations qPCRs used clean and standard dedicated the duplicate been of as in a had combination conditions tagged done in combinations unique fusion were prepared into tag a PCRs incorporated were primer-MID assigned Fusion were was tags the contamination. 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Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. vrl,teewr ee Ssi h ibr oprdt h osa li rJra,epcal nthe in especially Jarrah, or Plain Coastal the sequences. to we the compared assay, of Pilbara 87.8% plant for the the accounted In sequences/sample. in these 5600 and ASVs count. filtering of fewer read post minimum were the remained a 381 there remaining of with ASVs, The Overall, 23.7% samples plant up 197 511 Arthropoda. initial from phylum made the the sequences only From plant to and minimum filtered belonged fungi, a 13,441,527 2635 or with generated ASVs, unidentified samples initial either 196 5862 were of from ASVs Out sequences invertebrate reads/sample. quality-filtered 3,000 14,780,759 of generated we total, In Caceres restoration Results (De with ‘indicspecies’ package associated is R the taxa communities using the site reference each understand for 2009). to analysis to provides Legendre, multipattern similarity and Finally, a & extra K-strategists ran community we to richness. the sites, in A- or without reference fair. or patterns and and changes, were r- locations with the compositional from the data composition by between whether changing SCP driven trajectory of about the of prediction comparisons tested information the any we on that of additional three, ensure based proportion to all is relationship the years) For This analysis was 11 that This time. relationship and model. over expect years This increase linear we (7 would simple sites Third, sites, calculated. two reference a in location. was using found and sites were tested assay that reference was ASVs each were the is, for site that and separately each al., ASVs, site ‘reference’ at models et Replicates each linear (Oksanen between time. using over group similarity tested communities the the reference and of to similar interval pooled requires more variance confidence trajectory on become of 95% communities restoration analysis based multivariate restored a the permutational (NMDS), using establishing showing ‘vegan’ tested scaling Second, were ellipses the (PERMANOVA). ages multidimensional from draw restoration between function metric to Differences ‘ordiellipse’ Non used 2018). The using dissimilarity. was Bray-Curtis package visualized with R reference was criteria and the table Three This to ASV convergence restoration, 1967). presence/absence or older (Mantel, sites. restoration, recovery younger R Spatial of reference between in different 2018). trajectory and package be al., a should ‘ade4’ showed et composition community the communities Majaneva First, in if 2015; community. determine test Leese, to Mantel & examined (Elbrecht the were We biases transformed using were of tested was counts effects S2). was there Read the verified autocorrelation (Figure continuing. We reduce Copy before abundance. dataset to richness assay. sample ASV presence/absence plant the within and the 0.05% to from depth of for sequencing minimum ‘Plantae’ removed between a and were correlation to assay no filtered removed invertebrate controls were were the sample depth extraction for each sequencing in ‘Arthropoda’ the low numbers phylum with in the Samples in present 2018). ASVs were Team, selected Core that (R 3.5.3 ASVs R and using run were support statistics minimum All a with taxonomy assign to Statistics 2007) al., et Database (Huson Life MEGAN of used 205. Barcode We of the databases. from two extracted the sequences of reference COI arthropod (BOLD: and the against against searched search was ( to also Search table GenBank Centre) Alignment sequence Supercomputing database Local (Pawsey The reference Basic computer the cluster online 2016). equiva- using high-performance (Edgar, a determined are usearch on was ASVs to Taxonomy in (blastn) stage These Tool removed. (ZOTUs) inference (ASV). chimeras units esti- Variants sample and the taxonomic Sequence were the constructed operational Amplicon in using then rates leave used radius R error and then zero in the were errors to performed rates filtered, be lent error was to quality The likely processing were dereplicated. sequences sequence sequences remove were Further where sequences 2018) the 2016) 2018). al., and Team, al., et mated, Core (Wilkinson et package (R (Callahan “insect” package platform the “DADA2” 3.5.3 in R function demultiplex the a on using demultiplexed were Sequences analysis Sequencing https://www.barcodeoflife.org https://www.ncbi.nlm.nih.gov/genbank/ ,bcueteeaerfrnesqecsta r on nqeyo one on uniquely found are that sequences reference are there because ), 6 directional .Ivrert eune were sequences Invertebrate ). hne eepc that expect we change; Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 4wr sindt aiy 6t eu,adol oseislvl hsicue h ant the includes This restoration level. older with species associated were to (12/16) Coleoptera 3 Most Pilbara. only the in and sites reference genus, with associated to , with 16 =0.05) family, (alpha to sanguineus association assigned significant with were ASVs 44 invertebrate ( 82 were restoration there younger locations, three the Across (average Analysis sites with Multipattern reference shared two ASVs the respectively). between of 8 shared number and ASVs the invertebrate (10 3 in the had traps variation to only pitfall Pilbara was compared ‘reference’ the assay there 59.7% in plant 1.2 sites, traps vs the of reference pitfall in (78.6% two The ASVs assay the shared other. plant Between both each of the for proportion a 5). true and higher was had (Figure ASVs) a This traps assay also ‘reference’ vane traps. was the pitfall 22.2% There than Overall, vs samples ASVs). 5). (49.4% reference (Figure with assay pitfall time shared invertebrate only were over the sequences, that ASVs plant ASVs For ‘reference’ of forest increasing time. proportion Jarrah showed over higher the ASVs Pilbara and ‘reference’ Plain the of Coastal in the proportion traps from the samples in trap increases pitfall significant the showed from communities invertebrate the Only ASVs associated 4). traps, (Figure “reference” traps. pitfall traps of pitfall Pilbara vane the Proportion the the communities in in in plant only communities significant relationship in was reference a change but to not directional Jarrah, similar was The and the more there in communities traps. while became samples pitfall reference communities trap the vane to plant and not the similarity pitfall Similarly, but between the traps, relationship both vane in significant the changes occurred a directional in evident different. observed was were restoration less no communities there was were of plant There the age trajectory Plain, from PB). This results Coastal JF, The the 4). the (in Pilbara. (Figure In traps the forest vane in Jarrah the composition community the in invertebrate absent and in entirely Plain time) or over Coastal SCP) reference the the to from (in similarity traps (increasing pitfall changes the directional in clear showed communities invertebrate The 1). communities (Table reference assay invertebrate to the dissimilarity Similarity community to with compared correlation sequences spatial plant the Similarly, the S2). traps 1). in (Table pitfall Table lower samples from (alpha=0.05, communities was restoration traps another invertebrate (pitfall) vane the one the older in not samples autocorrelation the from but spatial reference or significant the different (vane) showed alpha=0.05). where younger PERMANOVA, tests significantly samples, the Mantel Pilbara 3, The were from the (Figure different in ages community significantly assays plant not restoration and the were of types all exception trap showed the with both analysis (alpha=0.05), restoration for pairwise older locations restoration, the all younger Similarly, in between sites composition community reference analyses. in and subsequent differences the the our taxonomic significant in for in poor were diversity ASVs the while There of Plant to Proteaceae, Because considerations and level. 2). our Dilleniaceae, (Figure family confined Malvaceae Fabaceae, to and we Poaceae Myrtaceae, identified assignments, Fabaceae, by were the be families dominated identified could richest of the was the be ASVs from Pilbara most not forest plant absent could Jarrah up of ASVs largely invertebrate and 99% made were of However, SCP Collembola (67%) also majority level. Araneae. The Hemiptera) order and ASVs. and beyond Collembola Orthoptera with more Coleoptera, Hemiptera, had along Diptera, (Hymenoptera, which traps, Pilbara, Coleoptera, these pitfall Hymenoptera, the of by in Some dominated diversity was traps Lepidoptera. vane and the in diversity Invertebrate S1) (Table ASVs Composition invertebrate Community fewer 17-32% had Pilbara the where traps pitfall ± .AV e ape.Teaon fsae Sswshge ewe h osa li n Jarrah and Plain Coastal the between higher was ASVs shared of amount The sample). per 0.4ASVs hc a soitdwt one etrto ntePlaaadteant the and Pilbara the in restoration younger with associated was which , < er) le ( older years), 9 > er) eeec ie,o obnto TbeS) fthese, Of S3). (Table combination a or sites, reference years), 9 7 oooimrothsteini Monomorium Iridomyrmex Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. -adKsrtgss hstmlt nrdcsatidavriyo -eeto taey hc sselected is which strategy, A-selection or for adversity conditions third the a explaining introduces as well template As system habitats this predictability. of the and variety K-strategists, as favourableness the and condenses their Southwood species which by r- to template’, K-selected further equivalent ‘habitat to developed axes a is shift as two concept (1983) onto a Greenslade This by and 1989). Greenslade followed (Majer, (Pianka and (high community disturbance, species (1977) environments r-selected climax selected the by stable unpredictable dominated of r a are advantage towards in systems from develops take that dispersal) vary K- expected they low is to that as it environments, bodies, restoration, strategies initially unpredictable and large history succession in ecological lifespan, life In dispersal) 1970). long for high fecundity, model opportunistic, (low predictive bodies, selected small a lifespan, is short theory fecundity, selection cases r/K in The pool; invertebrate source taxa using ecosystems of a trajectories unpredictable presence as restoration vs the evaluate act Stable to on could to difficult that dependent leading more vegetation is it, be remnant may this colonize near it However, will were communities. sites, 1997). species sites restoration Dreams’ al., re-established, all isolated of study, of et be ‘Field this (Palmer can the In without habitat function a with populations. recover suitable of of conforms to if attractiveness restoration This ability that and communities. the an posits support plant demonstrating which forage of are hypothesis establishment communities the likely invertebrate the indicate are that following can that indicate intervention invertebrates organisms findings of airborne of Our indicators Because while site. good sites. situ, restoration are & in using invertebrates mobility Herzog, and ground-dwelling reproducing (Knop, more accessing Ollerton, nature, distance & with from sedentary greater McCollin, invertebrates those more (Cranmer, preventing from barriers as their 2018) and other within al., important 2015) or thermophilic et sites al., is Tomlinson of et reference abilities sign 2012; (Magura no to dispersal is quickly similar there in Fortunately, more 2011). is Variation areas Schmid, airborne Beyond taxa PB). recolonize of 1). SCP, to associated recolonization (Table able 5, quicker ‘reference’ samples are suggest (Figure of trap also years number pitfall results few in the our a correlation by traps, compared spatial the traps evidenced vane weaker greater of as the distance Ward, the in invertebrates have Dick, 2004; attraction taxa and & al., to shared in 5), (Jha of et differences (Figure away tend proportion Ness the km greater traps 1980; expected, the 1.8 pitfall (Majer, explain often than dispersal as the also invertebrates, limited more would to and, more This from airborne have 2001). come traps Yen, traps capture may & pitfall pitfall traps New, that by Vane caught organisms as species trap 4). fidelity while can Figure 2010) and local 3, 2018), same (Figure (Hall, recovery pollinators the community show of not indications do traps Vane invertebrate locally. foraging airborne likely vs directional are Ground-dwelling of invertebrates invertebrates. indications that airborne some indicates showed than and also ability composition samples community dispersal invertebrate in with lower changes associated with diversity invertebrates climax plant stable identification, ground-dwelling in Examining taxonomic evident and in more interactions limited were biological 2009; and recovery with complex community even Majer, assess of ecosystems rapidly that trajectories 2002; to These show used al., trajectories. we be restoration study, et establish can this samples (Andersen invertebrate in change of and metabarcoding ecosystem 2007), DNA Moir, of & indicators Brennan, key Majer, are fauna invertebrate with Terrestrial associated were (13/14) ASVs Fabaceae Discussion Most forest. 2). Jarrah (Table and included Plain Pilbara Coastal Species the the level. in 52 in species sites S4), restoration to (Table younger reference 8 association of and significant traps genus, with vane to ASVs 21 younger 59 the family, in were to mainly (Proteaceae) there found assigned were assay, were ASVs plant Apidae which the samples. of trap For vane traps. from were vane those restoration of 13 and sites reference or , on nodrrsoaini h arh and Jarrah, the in restoration older in found 8 uery commixta Duperreya (Convulvulaceae) erpiesquamata Petrophile , on in found Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eore Cak&Dlwt,17;Hrea 98.Ti td ffr rlmnr etn fcnitnyin consistency of those testing use that preliminary invertebrates offers more study in This resulting 1988). flowering, Herrera, and growth 1974; communities (Unpublished). plant invertebrate traps Dallwitz, new pitfall in (April) & from more changes autumn (Clark generated is directional during communities resources there plant detected conducted in spring also study differences the traps no previous In but pitfall A 2019), using 1998). al., the sites al., et between (Fernandes Plain interaction et Coastal the Rico-Gray especially the & 2017; and in Gestel, al., same communities, Van the et plant (Santorufo, whether 2005), (CaraDonna communities confirm Miyashita, two invertebrate cannot & affects we gain Shimazaki Seasonality summer, 2014; to year. spring/early Maisto, with. researchers the the interacting allows throughout in are that exist conducted they was patterns method plants study efficient that the this resource argue and these as We community and Importantly, However, invertebrate costs. time 2019). the from greater cost, of Sigsgaard, DNA and a Alternatively, snapshot & samples is informative 2016). more (Thomsen an samples al., far (PB pollinators arthropod et therefore traps example probable Pornon bulk and vane for identify 2017; using sampling separately, al., in to species-specific et species used sites require (Bell each methods been most loads from also plants. in pollen DNA which has present the with extracting flowers from are interacting and DNA are they invertebrates invertebrates extracting no JF), which isolating by virtually identify and require are cannot family would (PB there we the Unfortunately, that While This traps in 2). evidence 1980). plants pitfall Figure Keighery, also example, JF, the 2012; is For and al., invertebrates. in there by et traps, ASVs visited (Jabaily into otherwise Goodeniaceae pollination falling or insect debris of ingested require from than were richness Goodeniaceae originate that of rather greater cover plants may site, a high are DNA restoration have generating these plant to the characteristics, some observed on site While sites some locally restoration South32). DNA younger plants reflected of in with sequences ASVs limitation (Figure interacting Plant Fabaceae al., acknowledged sites are through. et restoration commonly Lim invertebrates between passing 2018; a composition Heyde signal the only al., community der is that assay et the (van Fernandes plant indicates data that 2017; in area 3) behavior Leese, difference study hypothesized & clear or the Peinert, we the Elbrecht, However, abundance outside as 2016). 2015; from of Leese, expected, DNA lack & (Elbrecht plant was The metabarcoding carry This 2020a). can associated 4). al., invertebrates diversity et Figure because plant diluted the 3, in be (Figure evident would less samples were invertebrate composition community with in changes directional Generally, samples the complete. invertebrate in Plain from overall with taxa Coastal far associated ‘reference’ is the Plants recovery shared in regard, ecosystem of than that so proportion pressures under 5), low In selection recolonization (Figure particularly by areas Thus, locations. traps a influenced species. the two was pitfall in less as there rich Pilbara and other However, unfavorable less stochastic the forest. also and more Jarrah are at unpredictable be and they may situation as regions, sites two the are Pilbara other habitat that the to of grassland structure with conditions contrast predominantly the compared provide marked original and Here, may restoration; the in sites. areas of reference is structure reference and which the the al. restored resemble 1), et Pilbara to Figure (Gibson between came climate (see shared rapidly ant as were revegetation well that the that compositional as observed driving of landform/hydrogeologic, factors factors (1990), and were main regolith/soil Fletcher the All are Pilbara, composition extent, 2015). the fauna species lesser In the terrestrial sites. results, a in restored our turnover to and to trajectory similar natural similar and but, between a rehabilitation, different show (1985), Pilbara remained not young al. did in climate, recovered et rapidly harsher richness Dunlop and of recovery. unpredictable change proportion more community directional a the a of has shows which in which community. location, 5), reference increases Pilbara (Figure the The significant Plain as of taxonomic Coastal recorded sites that on and toward reference based we Jarrah composition the them in sites community both classify found in in restored to were time older information with they inadequate taxa in whether ‘reference’ was and on expected, there (Greenslade based including since As species taxa environments, strategy, identification. between selection classified Such interaction for we 9 Acacia hus(aafo BHP, from (Data shrubs Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. nesn .N,&Saln,G .(97.At sidctr frsoainsces:Rltosi ihsoil with Relationship : success restoration of tropics. indicators seasonal as Australian Ants the (1997). P. in G. biomass Sparling, microbial & N., A. Andersen, responses. 10.1046/j.1365-2664.2002.00704.x community doi: ant Reposito- 8–17. of M assessment Simplifying D., management: B. land Digital Hoffmann, N., A. Andersen, Dryad the MvH; by done the References was analysis statistical the MvH; in authors. was by involved at all work processed were by bioinformatics and GW-J and edited analyzed and molecular was was NW, MvH; manuscript data available MB, by all PN, processed MvH; by and MvH, performed collected manuscript. were the is Samples wrote design. and experimental study the conducted and MvH data Contributions Author sample and ry: Sequencing Environmental and Trace the Accessibility of bioinformatics. Data members and like workflows the also metabarcoding and would We with collection sampling. support sample for for BHP, with sites Laboratory companies help (TrEnD) the to mining for DNA access from Walters the facilitating funding Sheree thank for thank We with South32 Centre to Australia. Centre and Training Western Supercomputing Material, Transformation of Pawsey Construction Government Industrial Hanson the the Council and and Research (ICI150100041) Government Australian Australian Restoration the Site by Mine supported for was work This and projects Acknowledgements restoration these in meet and them to incorporating metabarcoding potentially required DNA to investment as critical financial outcomes. such is restoration considerable techniques beneficial improving the of monitoring are from new they selection value Testing where the evaluating targets. best on Challenge restoration the Bonn heavily global get The ecosystems ambitious we depends ecosystem. that that reference km restored the remember ensure million fully in must to 350 variation been restore important natural have the to is capture goal sites It to whether sites ecosystems. reference determining different appropriate the consistency and so and how variation, years dynamic, seasonal showing DNA multiple of are using by effects across the the trajectories recovery determine patterns Despite restoration to ecosystem of ecosystems. research Understanding functional community. additional climax require plant indicate will stable the metabarcoding can in with interacting metabarcoding invertebrates reference are dwelling data, towards invertebrates success ground trajectories abundance of that in definition of show the We evident lack enables monitoring. it more restoration as for were important scales is ecosystems time restoration site required establish restored the to a and samples of invertebrate criteria, trajectory of likely sequencing the throughput Defining high trajectories. of use the demonstrated have We years. between Conclusion or within temporally not but space, across patterns restoration https://doi.org/10.5061/dryad.q573n5tgw 2 fdgae ersra cssesb 00(uige l,21)maswe means 2015) al., et (Suding 2030 by ecosystems terrestrial degraded of le,W . rffih,A .(02.Uigat sboniaosin bioindicators as ants Using (2002). D. A. Griffiths, & J., W. uller, ¨ 10 oit o clgclRestoration Ecological for Society ora fApidEcology Applied of Journal , 5 2,109–114. (2), , 39 (1), Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. rs,S . aea,P . rs,A .(00.Rsoaingas h r an tl vrokdin overlooked it? still fauna about are done Why be goals: can Restoration what and (2020). T. ecosystems A. Restoration functioning Cross, and recovering & of W., process P. fauna? the Bateman, restoring L., as same S. the Cross, flora restoring Is rehabilitation. mining (2013). B. and P. 10.1111/1365-2664.12046 koalas Banks, & Cur- from C., learned Australia: Frere, in Lessons J., rehabilitation Rhodes, mine H., in fauna R. of Cristescu, review A directions. (2012). future B. 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Moir, in (Orabi, sites pH restoration soil younger and the structure, in vegetation primarily plants, Found host of presence the to linked is composition species Hemipteran traps. pitfall JF the in restoration Reason younger in bees richness of Higher Family insects sucking of Order - Julida Name Apidae Hemiptera sanguineus Iridomyrmex analysis Melophorus species indicator and interest data of the Taxa of observations general on based interest, of Taxa Table JF-Jarrah Plain, Coastal dissimi- SCP-Swan community shown. and are distances (sites) spatial pooled between and separately, PB-Pilbara. soup: correlation Forest, samples the Biodiversity the showing for test Results (2012). Mantel larity. 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(4), ifl netbaeJ .6 .0 .8 0.205 0.189 0.001 0.161 JF Invertebrate Pitfall Trap aeIvrert F0110010270.104 0.237 0.011 0.131 JF Invertebrate Vane sa Location Assay ln F006 .4 0340.916 -0.314 0.147 0.0466 0.706 -0.1858 JF 0.064 0.069 Plant JF Plant utaingnso n soitdwt one etrto ie nbt h C n B pce nti eu r nw s’u-oig Adre ta.20)adaeotnfudi etrto ie ihlreaeso aegon Adre,Hffan n oe 2003). Somes and Hoffmann, (Andersen, ground bare of areas large with sites restoration in found often are and 2002) al. et (Andersen ’sun-loving’ as known are PB. genus in this sites in restoration Species younger PB. with and Associated SCP the both in sites restoration younger with Associated ant meat Northern ant of genus Australian otgs ilpd naiedtiioeseisfudi ra bnac nteSP atclryi le etrto n eeec ie.Feso itr hc smr vial ntoests h otgeemlieei nw ob iepedtruhu otenAsrla(ae ta.2013). al. et (Baker Australia southern throughout widespread be to known is millipede Portuguese The sites. those in available more is which litter, on Feeds sites. reference and restoration older in particularly SCP, the in abundance great in found species detritivore Invasive millipede Portugese 0) –4 o:10.1111/1755-0998.13148 doi: 1–14. (00), , C .1 .2 0390.874 0.319 -0.359 0.079 0.555 0.125 0.548 0.007 -0.074 -0.084 0.115 0.233 0.789 0.851 0.082 0.838 -0.2365 SCP -0.117 -0.203 0.116 0.114 0.018 PB 0.785 0.349 0.508 0.566 SCP -0.077 -0.0118 0.001 PB 0.002 SCP 0.375 0.308 PB SCP PB er Preprints PeerJ cec fteTtlEnvironment Total the of Science 16 ora fIsc Conservation Insect of Journal p r p Sites Sites r Samples Samples o:10.7287/peerj.preprints.26812 doi: . , 7 231–241. , ehd nEooyand Ecology in Methods , , 749 5 1,4–3 doi: 47–53. (1), 422 doi: 142262. , Iridomyrmex pce r mn h rtt ooiervgttdsts(nesn19) uhlike Much 1993). (Andersen sites revegetated colonize to first the among are species Melophorus , Iridomyrmex pce r trce oaeso aegon nnwyrvgttdsts(nesn omn n oe 2003). Somes and Hoffman, (Andersen, sites revegetated newly in ground bare of areas to attracted are species Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. hw ihtenme fyasrsoainfo o2 er.Rfrnestssonblw SCP-Swan below. shown sites Reference years. 22 to 1 sites from PB-Pilbara. Restoration restoration Forest, years JF-Jarrah collected. Plain, were of Coastal samples number invertebrate the where with restoration shown mining of Chronosequences Figure South32). from Data BHP, from (Data locations these in sites restored at rapidly establish to tend shrubs Acacia PB. and JF in restoration younger with associated strongly are family this in ASVs Reason family Legume Name Anigozanthos microptera Goodenia Fabaceae interest of Taxa agropwAscae ihyugrrsoaini C ifl rp.Teego ucl wti er nSPrestoration. SCP in year) a (within quickly grow 2015). These Barrett, traps. and pitfall (Barrett February-October SCP between in flower restoration to younger known with species, Associated plant arid Pilbara Common sites. reference PB of traps vane the in predominantly found species pollinated insect An paw Kangaroo Goodenia Narrow-winged 17 Anigozanthos r nw ob rdmnnl olntdb etrfeigbrs(yee l 00 n ol eecuaigo anlrclnzto frsoe sites. restored of recolonization faunal of encouraging be could and 2020) al. et (Ayre birds nectar-feeding by pollinated predominantly be to known are Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. rp.Sostenme fAV nec re ivrerts rfml pat)a l etrto and restoration all at (plants) family or (invertebrates) order each vane in and pitfall ASVs from of detected sites. number communities reference the (below) plant Shows and (above) traps. invertebrates of Composition Figure 18 Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ie niaelna oeswt 5 ofiec nevlsonwt hdn.Pvle o h iermodels linear the for P-values sites. shading. reference with shown in interval communities confidence to 95% (years) with ages models different linear of indicate sites Lines restoration of (Bray-Curtis) Similarity Figure group. the of interval confidence 95% indicate and package (alpha=0.05). Ellipses facets R sites. all vegan reference for the and significant in restoration were PERMANOVAs in ‘Ordiellipse’ communities using plant drawn and Invertebrate were or ordinations NMDS Figure 19 Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. infiac fterltosi ewe h rprino eeec Ssadaeo etrto (years). restoration of age the and indicate ASVs P-values reference communities. those of plant and b) proportion sites and the reference invertebrate between in a) relationship present into were the Separated that of ASVs sites. significance into reference separated in sites, found different not the in richness ASV Figure (from traps vane from communities relationships invertebrate the change of not exception did the years) (7,11 with SCP models, the p=0.053) the in to sites of p=0.036 extra significance two the the Removing or plot. each for shown 20 Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. REF REF B REF A 22 yrs 14 yrs 7 yrs 3 yrs 1 yr SCP REF REF B REF REF A 2 yrs 20 yrs 11 yrs 6 yrs 21 JF 4 yrs 7 yrs 9 yrs 15 years REF REF B REF A PB Communities Reference YOUNGER Restoration OLDER Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary.

NMDS2 Plan Invertebrate t NMDS1 22 Restoration type Trap Restoration type Trap Pitfall Vane Reference Older Younger Pitfall Vane Reference Older Younger Posted on Authorea 20 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161383346.69785032/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. b a) ) ASV Richness ASV Richness b) a)

Community Similarity to Reference Plant Invertebrate Restoration Restoration 23 Trap type Trap Vane Pitfall