Posted on Authorea 19 Aug 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.162936514.40253306/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. nvriaedsAcrs–Fclyo gainadEvrnetlSine,RaCptoJa ’vl,Sao d’Avila, Joao A¸cores, Portugal. Terceira, Capitao Rua Heroismo, Sciences, do Environmental Angra and 9700-042 Agrarian Pedro, of Faculty A¸cores – dos Universidade 3 . Barcelona, E-08028, 643, Diagonal Av. 2 Barcelona, de Universitat (IRBio), tute 1 patterns biodiversity of Arnedo understanding our Dom`enech on Marc mega-diverse impact of inventories major standardised a into has stages immature groups Incorporating audiences: all For accurate more providing interpretations, our relevant and influence novel inventories. may seasonality, a biological marked provide standardised juveniles with from that areas of question reveal information in not sort also especially do some they findings that, inventories, but our knowledge adult-based quantitative, Although of by extent Species layer provided estimates. certain information informative a the accurate respectively. to of provide relevance especially to are juveniles, the sampling. necessary considered, data and whole be taken metabarcoding of may the were that adults factor in ability stages revealed conversion adults as immature taxonomic results the from when our identified species investigate reduced Moreover, those 331 greatly further to latitudes. were We respect and across communities with between 363 35% communities. composition of obtained in across increase We Differences an inferred diversity represented juveniles local patterns by abundance. to diversity communities only stages on represented oak juvenile the of white inform on contribution from to the effect (1) analyse metabarcoding their assess to to non-adult (2) approach discarding protocols, and of metabarcoding standardised impact with a estimates, the collected use characterising more Peninsula to we representing Iberian devoted informative Here, frequently the been considered in has despite estimates. effort diversity be sorting, little our can during date, on To assessment discarded specimens individuals. often no collected are and the stages biodiversity, of half immature of monitoring than component and inventories largest them. biological the including in are without underrepresented traditionally However, are arthropods programs. , challenging their of Because Abstract 2021 19, August 3 2 1 Arnedo Miquel and Dom`enech patterns Marc biodiversity of understanding major our a on has impact groups mega-diverse into of stages inventories immature standardised Incorporating audiences: all For nvriyo h Azores the of University Troms of University Barcelona of University owga olg fFseySine nvriyo Troms of University Science, Fishery of College Norwegian eateto vltoayBooy clg n niomna cecs&Boiest eerhInsti- Research Biodiversity & Sciences Environmental and Ecology Biology, Evolutionary of Department Ec–Cnr o clg,EouinadEvrnetlCags/AoenBoiest ru and Group Biodiversity Azorean / Changes Environmental and Evolution Ecology, for Centre – cE3c 1 1 wnS Wangensteen S. Owen , 1 wnS Wangensteen S. Owen , 1 ø 2 laEngu´ıdanos Alba , 2 laEngu´ıdanos Alba , 1 ø h rtcUiest fNra,Norway. Norway, of University Arctic The , 1 aoaMalumbres-Olarte Jagoba , 1 aoaMalumbres-Olarte Jagoba , 3,4 iulA. Miquel & 3 , Posted on Authorea 19 Aug 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.162936514.40253306/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. tde ugs htatrpdppltos hc lyafnaetlrl neoytmfntoig are functioning, ecosystem in role fundamental 2018). a (Leather play rate arthropods recent and which alarming hand, many assessments an populations, other of biodiversity at vertebrates the in knowledge arthropod declining On underrepresented than taxonomic that Mart´ın, frequently 2012). Ferr´andez, scales suggest poor & Triantis, are temporal studies the Borges, they However, shorter (Cardoso, and 1997). and programs bioindicators, the Butcher conservation have as spatial & use arthropods finer Yen their times, at 1993, generation limits Doran changes short al and biodiversity and (Taylor et account on rates (Kremen into reproductive inform them high takes to their biodi- it potential no of unless Therefore, credible because biodiversity. considered Moreover, be of 2001). component can abundant program most monitoring and versity (Oliver largest greatest periods the the sampling represent of retrieve Arthropods length biodiversity to and rapid number designed the instance, specifically minimising 1996). For area, strategies Beattie limitation. particular and sampling al a this daun- from efficient et overcome a information yet Barnosky to of is amount fast 2015; devised arthropods are al as been such et protocols have groups (Telfer assessment methods diverse biodiversity and Several to abundant task. highly threats changesting identifying unanticipated ecosystem and monitoring tackle sampling for However, the and methods as 2013). identify detection to early to referred and extinction, essential Bioinventories been to 2011). are has species disturbances al, what numerous et These in driving Barnosky 2016). and described, 2014; al., 2009) al, are et (Hulme, they Barlow species before exotic Cha- 2019; even of Wilcove, (Stuart sometimes rate communities & invasion Valderrama-Sandoval, biological the Socolar, of accelerating abundance 2000; are and al richness et species III the pin altering is activity human Global INTRODUCTION title: Running with areas in standardi- especially Keywords from that, information knowledge accurate of more inventories, inventories. layer providing adult-based relevant biological interpretations, by and sed our provided novel influence information a may the provide seasonality, taxonomic juveniles of marked relevance of that the reveal sort question also some not they estimates. but do informative our quantitative, findings accurate Moreover, extent our provide latitudes. certain Although to across a necessary to especially be were are account, may communities data into factor between conversion taken metabarcoding composition were that in Differences revealed stages respect sampling. results with immature adults whole 35% as when further the of species reduced We in increase 331 Iberian local adults an greatly communities. and represented from to the 363 across juveniles identified obtained by stages inferred those in We only to juvenile abundance. patterns represented communities on diversity Species of respectively. inform oak the contribution juveniles, to white and on metabarcoding the of effect from (1) ability their spiders the assess investigate (2) analyse to and protocols, to estimates, standardised estimates. approach diversity diversity with metabarcoding our has collected on effort a Peninsula little specimens use date, non-adult To discarding we individuals. of Here, collected impact the the of characterising half during to discarded than devoted often more are been assessment representing stages no immature frequently and Arthropod biodiversity, despite them. of sorting, including component without informative largest considered the be are inventories can biological arthropods However, in underrepresented programs. traditionally monitoring are and arthropods taxonomy, challenging their of Because [email protected] Email: Spain. Barcelona, ABSTRACT Barcelona, de Universitat IRBio, & Sciences Finland Helsinki, 00014 13), Correspondence Rautatiekatu (Pohjoinen 17 P.O.Box Helsinki, of 4 IR aoaoyfrItgaieBoiest eerh ins uemo aua itr,University History, Natural of Museum Finnish Research, Biodiversity Integrative for Laboratory – LIBRe rna,Dvriy N acdn,IeinPnnua eaacdn,Spiders Metabarcoding, Peninsula, Iberian barcoding, DNA Diversity, Araneae, : noprtn ueie ndvriyestimates diversity in juveniles Incorporating acDmeeh eateto vltoayBooy clg n Environmental and Ecology Biology, Evolutionary of Dom`enech, Department Marc : 2 it asextinction mass sixth Droet (Dirzo Posted on Authorea 19 Aug 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.162936514.40253306/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. ersnsisaudneo ims ntesml a enamte fmc eae eea tde have studies Lamb Several Agust´ı, debate. 2015; & much Gomez-Polo, of Pi˜nol, Mir, matter 2015; a Leese, correctly been and taxon has (Elbrecht certain sample a issue of the this reads in addressed sequence inability biomass of presumed specifically or number its the abundance is extent assessment its specimens what biodiversity represents voucher To for information. the metabarcoding abundance sometimes revise of provide use to are to the impossible or 2019). of it al., back drawback making et potential traced Watts Another sample, sequences be 2020; bulk of cannot found. al., the number were specimens et preparing the sequences individual (Sales of interesting as millions that process if specimens, of is the of order in approach the numbers the lost this proces- allows large even in using and it is for workload of run efficient as the downside cost metabarcoding reducing barcoding, The single more greatly DNA a once, is to from at it Morini`ere respect obtained specimens 2012; addition, with al., of In advantage et number time. Yu clear large sing 2014; a a al., represents of et approach processing Bohmann environmental simultaneous (e.g., This from DNA or 2016). organisms of al., partial remains or et containing entire containing etc.) identifica- sample automated soil, bulk the (water, single in of samples a consisting from amount technique species molecular large multiple developed of recently a tion more such a time- is sequencing very metabarcoding and be DNA amplifying can considerable. juveniles extracting, be of of also each thousands cost would amplifying or samples economic and hundreds However, extracting with the adult-based 2012). manually samplings Moreover, requires Dowton, of large consuming. it & barcodes for example, Wallman, which For containing individually, drawbacks. Meiklejohn, databases some specimen 2010; reference poses al, still to et technique comparison this (Richard individuals through species immature species of identified sequences & to morphologically barcode Ratnasingham, assigned DNA (Hebert, stages. be barcoding immature DNA of can e.g., identification into identification, the species taken ease for 2003), were DeWaard, approaches stages DNA-based be immature of found reliably use these only The could when were that drastically species changed numerous species of abundances that year. few estimates relative out the a account. diversity that pointed of in only and already time effect included juveniles would stages, particular the which as individuals immature that addressed study, adult at partially in This only identified has stages stages. considering morphologically 1999) immature so (Norris, as time, juvenile study Samu, present particular one incorporating are & one only (R´adai, that Kiss, knowledge, at conditions species our area To weather the certain “photograph” the a all they a provide dismiss on although in they completely once, May-June, present depending conducted in species year usually reproducing are the same protocols and of the assessment known March-April biodiversity in are rapid in spiders clutches Since adults wolf 2017). two as some have example, maturing Achitte-Schmutzler, For in also cycles Nadal, and species. can 1984; year life same (Aitchison, per the annual juveniles generations or within have of adults even number to as the 2018), Avalos, present in & are differ they spiders Gonzalez, when of Zanone, year groups different the Sorensen, of of ob- cycles time 2000; life the to Miller, the desirable & that known is Coyle, is (Toti, incorporation It protocols their sampling influence so 2002). biodiversity, significantly short-term even Scharff, may of in or & immatures diversity patterns 1996), Coddington, Disregarding of spatial Silva 1997). estimates and 1995, Baxter, reliable temporal Stork & tain the & (Kuntner of Russell-Smith cases & inference 2004; extreme Cardoso, the Serrano, in in Malumbres-Olarte, specimens 94% & 2020a, collected to Oliveira, al, the up Silva, et of Malumbres-Olarte Cardoso, 70% last 2020, 2019; and during the al, discarded Crespo, 40% in et are between and stages (Soukainen for visible immature vulva surveys account Coddington, only studies may biodiversity the 1997; diversity are (Dobyns, specimens and and level inventories immature features males species most However, These at in in sorting. identify Thus, females. bulb to 1996). in copulatory Coyle, impossible & or the area) Young, difficult of genital arthropod immatures structure makes exclu- the most which fema- the almost of moult, across is e., degree, modifications identification identification i. lesser (external taxonomic species characters, a Morphological epigyne genitalic for to exception. on structures no and, based main are apparatus sively Spiders the copulatory 1985). are male (Eberhard, systems evolution 3 Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ln ihters ftesmls ecendadseiie l h qimn ihdltdsdu hypo- the using sodium bp diluted 313 with of equipment fragment” the “Leray all COI sterilised the sequenced and and amplified cleaned We spider processed We extractions. batch, cobweb samples. from the sample the nega- in successive the g one included between of of 0.3 were chlorite obtained added rest specimen controls We of We the These a USA). fraction with nitrogen. protocol. control, CA, extraction along a liquid Valencia, positive the extracted of (QIAGEN, in one we 1836), Kit help and Koch, and Isolation the water) DNA sample, with (distilled PowerSoil plot tive juveniles Supporting a homogenised of using collected each replicate S1 the from Table each all (see replicates homogenised 2018) extraction we al. two species et plot, of (Crespo bp) each study (~658 For (COI) previous I a Accessibility). subunit Data from oxidase available and c information were cytochrome adults evaporate. the as to for captured sequences bodies the barcode in DNA alcohol Representative absorbent sequencing remaining plot in the and each specimens allow amplification of the to extraction, individuals placed them immature We DNA weighing balance. the before analytical All minutes an level. 30 using family for family to paper each juveniles stereomicroscope. for 2000 and separately Stemi level ZEISS weighted a species were 48 under to them with identified adults we sweeping identified and We and specimens immature beating plot. and every capture, semi- adult sorted in used direct We of weeks We timed Parks two 1). of for National (Fig. (PO), active man-hours Perdido (PS) six traps 12 Monte Nevada across pitfall combined y Sierra forests that Ordesa and (PA), Caba˜neros oak methods (PC) Monfrag¨ue Maurici (PM), quantitative white (PP), Sant Europa in de de Estany distributed Aig¨uestortes i Picos namely plots Peninsula, 16x1ha Iberian May-June 2020b). Dom`enech, included the in Arnedo, Moya-Lara˜no, Crespo, 2009) design & Malumbres-Olarte, (Cardoso, 2018; sampling COBRA al., The protocol et sampling (Crespo standardised 2014 and the 2013 using specimens the collected We sorting and collection Sample METHODS and AND ap- inventorying MATERIAL metabarcoding biological of standardised use morphology-based are the which traditional, refine schemes. protocols, to to monitoring assessment contribute alternatives biodiversity will efficient rapid and as in changes, proaches insights stages ecosystem important monitor life provide efficiently different of results to all ability Our essential considering the samples. of assess bulk we relevance Finally, from the patterns. into information such estimates. abundance for diversity recover commu- responsible to across factors to patterns ecological contribute metabarcoding biodiversity possible spiders affects the specimens Spe- identify immature immature estimates. and that of diversity nities community inclusion diversity into the additional how spiders investigate of the we juveniles Secondly, quantify including first of impact we the cifically, evaluate 2015). to al., aim (Pi˜nol we et studies Here diet to of seems case variation of between the efficiency the success in the of extraction Aim DNA or different rest 2016) predator 2015), the Dawson, of Leese, reads while & primers G´omez-Daglio, of and Swift, sample, blocking Abboud, number (Elbrecht the (Schiebelhut, the the specificity species in in primer variation or biomass as the tissues such the of different factors in part et to differences certain between due (Soinien a by relationship be only analysed explained data positive found, concentrations a be dietary commonly of While to is range (2019). analyse seems biomass al. the quantitatively et and that Deagle, Deagle to reads be by Thomas, of it may suggested number 2020; conclusions as used al., studies, different even across et these considerably (Kennedy have for the varies taxa explanation or consider One among al., found 2016), et vary 2015). studies others Trites, (Ratcliffe may al., some conditions that 2015), & certain While factors al., under Harsch, inconclusive. correction abundance Pi˜nol et Eveson, taxon’s applying remains 2015; a by answer of Leese, or estimate the and 2020) accurate (Elbrecht an but give limited 2019), to metabarcoding metabarcoding al., of et power Deagle quantitative 2019; al., et 4 iiiinsimile C L. (C. Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ace eefud edsadda otmnns1 OU ihlwnmeso ed htcorresponded that better reads which of for numbers MOTUs low those better, with of were MOTUs identification 16 there the contaminants if updated as checked we discarded manually We and we found. NCBI, the were Ecotag, or to matches by BOLD assigned in MOTUs made matches the assignment recent only taxonomic retaining more the by database After resulting Araneae. the (Bakker order of contaminants rank database. putative assign taxonomic reference out to every local filtered We for tree, the thresholds in taxonomy identity sequences NCBI arbitrary related 2019). the establishing closely al., on require most et Ecotag the not based 2004). the of does protocol, al., ancestor approach from assignment sequence et common This retrieved phylogenetic (Kulikova reference last sequences a repository the local EMBL with to uses the sequences the spiders 2016) built and adult al. We 2007) et of 2016). Hebert, (Boyer sequences al., & our (Ratnasingham analyses et representative downstream combining database (Boyer the in BOLD Ecotag, Ecotag of entities by using assignment single taxonomic required (seeds) as the database MOTU treated performed also each we were of singletons, tetrad) the sequences one removing and After triad juveniles. Antich, one together systems with 2019; the clustered eukaryotic pairs, al., Garc´es-Pastor were for of et (nine 2019; sequences 13 range Swarm al, whose by et wide adults = Siegenthaler as a d present interspecific 2019; in species and of al., clusters, Tur´on, The intra et value Palac´ın, & species-level 2021). discriminating Kemp Wangensteen distance for to 2017; value Turon, a MOTUs optimal & singletons, set approximate (Wangensteen sequences the We as to COI be is, sequences reads. to original that adult shown 50,000 the divergences, been discarding to of has from abundance segment which program initial algorithm, the clustering the their only the prevent kept increased running To we artificially (meta- before fragment. adults, we stages juvenile COI of life and Leray case both the data) the from matching barcode In sequences the algorithm. DNA Operational combined clustering and we Molecular Swarm 2.1.13 (morphology comparable, into Swarm clustering adult sequences data) the in making barcoding cluster For implemented to (MOTUs). algorithm 2015) Units clustering Dunthorn, Taxonomic & aggregation also Vargas, step-by-step we de and the Quince, (Mah´e, abundances, used Rognes, their We of sequences Mah´e, 2016). track primer keeping & the the while using removed sequences) sequences identical and chimeric all removed dataset (grouping aligned reads the the demultiplexed dereplicate we and 40 We with than 2016). command al., bigger et the scores (Boyer using package quality reads metabarcoding paired-end Obitools the the using aligned analyses V3 bioinformatic the a conducted with We UK. MiSeq Salford, Illumina of an University the in Bioinformatics (Perkin-Elmer, at sequenced kit run was DNA-seq partial PCR-free library paired-end NextFlex multiplexed bp the Waltham, 2x250 The using Scientific, library USA). Fisher a Three Thermo Massachusetts, build Kit, USA). Waltham, to Assay CA, used BR Valencia, were dsDNA USA) (Qiagen, purified quantitation Massachusetts, Kit and fluorometric control) Purification Qubit PCR-positive by PCR one test). (determined MinElute and a controls plots a as PCR-negative served for and using two and buffer (except them (including Tris-borate-EDTA processed volume plot in be equal agarose per to by 1% first replicates products in the 4 GelRed electrophoresis were of by they with amplifications as total stained of replicates 10 a quality 3 included giving the obtained profile study, evaluated we We PCR which the The for in PS1, sample. replicate and per PA1 extraction uL each 20 of to up replicates volume 95 of the variability at adjust sequence min to increasing water for DNase-Free end and 5’ the template the in 1 in 10 tag with Ns same included USA), random (the mix CA, tags 2–4 PCR sample of The 8-bp lead twin library. a included the and pair primers) primer reverse Each and 2013). forward al., et the (Leray from primer 5’-GGWACWRGWTGRACWITITAYCCYCC-3’,mlCOIintF modified 2013) mlCOIintF-XT Hawk, primer & forward Parker, the rever- Meyer, and includesthe (Geller, 5’-TAIACYTCIGGRTGICCRAARAAYCA-3’ set jgHCO2198 This primer 2018). Turon, Palac´ın,se (Wangensteen, Guardiola, Leray-XT set primer degenerate ngsfilter ° ,3 ylso 94 of cycles 35 C, eas lee u eune otiigabgosbss ete used then We bases. ambiguous containing sequences out filtered also We . μ fec 5 each of l ® uli cdGlSan(itu,Hyad aiona S) epoe l PCR all pooled We USA). California, Hayward, (Biotium, Stain Gel Acid Nucleic ° i,45 min, 1 C μ owr n ees rmr,0,16 primers, reverse and forward M uchime μ mlTqGl 6 atrmx(ple isses otrCity, Foster Biosystems, (Applied mix Master 360 Gold AmpliTaq l ° i n 72 and min 1 C denovo illuminapairedend 5 loih nVerh(ons lui ihl,Quince Nichols, Flouri, (Rognes, VSearch in algorithm ° i,ad5mna 72 at min 5 and min, 1 C μ eslce eune ihalignment with sequences selected We . fbvn eu lui,2 albumin, serum bovine of l μ ° fteprfidpool purified the of g .W sdtoPCR two used We C. Obiuniq μ fDNA of l to Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ertivdDAbroe f38oto h 7 pce ersne saut ntesmln lt.Atrthe After plots. sampling the in adults as represented species 376 the of out 368 of barcodes DNA retrieved We results Sequencing 2015). Gudmestad, & Hua, Taylor, Pasche, response (p-valueRESULTS other (Yellareddygari, significant for in considered Only R designed were tested model. adjusted residuals rule are each the distributed one-in-ten calculated for models randomly We the fit regression 1. following of and plots Beta goodness 0 in 1995). the global between percentages presence of data Holford, of percentage minimum proportional function the non-independence & the with of a the Feinstein, variables as function as to Concato, 10 a due RRA family as used (Peduzzi, separately the RRA each we family models for the and each to for one sample, to family, one models a models and each regression in the sample, to in applied of the beta applied We taxa in were proportion abundance. apply represents certain models of family the to the Two the a used (RRA), 2010) mass plots. We of of abundance Zeileis, 10 plot. percentage proportion least every & read the at in relative (Cribari-Neto in by family v1.1” or every present explained of reads “betareg reads individuals package of of of R proportion proportion number the the the and calculated in biomass we variation of sample, level the the data assess were metabarcoding To parks from southern significance abundance SIMilarity and and species northern Of correlation of between ANalysis the approaches. Assessment composition assess adults+juveniles an species and to applied adults in test also by differences We Mantel recovered approaches. if a equally test applied both We to from 2020). analysis obtained Team, (ANOSIM) matrices Core distance the (R the with R used and between in we alone 2019) adults this, on al, For information et together. the juveniles, with juveniles composition. both community including and data the when presence/absence on adults communities with based analyses among analysis out these (NMDS) patterns find performed To scaling similarity We multidimensional information). the non-metric Supporting a S1, in (Fig. number performed differences we reads the significant of plotting were number curves, increasing rarefaction there an of if against means replicate by replicates per patterns MOTUs sequenced of the community same of on completeness the juveniles the checked of approach. including We match” MOTUs of “best unidentified effect into the unidentified the the translated to (every only of (all respect approaches Evaluation approach with approach those results of ”splitter” “lumper” also either the we using a a in However, purposes, differences using using species). comparative minor same and ways: For the taxon. as species) alternative MOTUs considered same different additional unidentified /family the two database a two as of the match as treated in in best were MOTU the sequence results and If specific MOTU. taxonomic collapsed the the the were the of analysed which they of sequence to same, seed identifier match the best the the to the was is, similar used most that we the level, MOTU, plot. was species least which that the to at of identified assigned required replicates be algorithm we not the assignment could plot, of that two a than MOTUs least in the less clusters at For present with juvenile in as MOTU’s MOTU and counted the the adult be of All (Miller, of to detection up. Gateway Delimitation MOTU and added Science a plot were CIPRES the for plot the in Also, each at reads discarded. of five remotely were replicates run reads the were total All five Analyses 2010). 2018). Schwartz, of Chernomor, means & Vinh, Hoang, by 2013; Pfeiffer, & support Haeseler, branch von Minh assessed positions & we Haeseler, Nguyen, partitioned and specimens (Minh, von We partition, Maximum replicates each adult 2015). by approximation to the Minh, tree bootstrap model of ultrafast the GTR & 1,000 unlinked sequence inferred Haeseler, an We COI We von assigned species. the and Schmidt, pseudogenes. or and codon (Nguyen, to genera by We MOTU v.1.6 families, lab. corresponding every IQ-TREE specific same of MOTUs using to the sequence the MOTUs Likelihood seed in remove unassigned the conducted allocate to using studies help 2017) tree other to al., COI in a et analysed built (Frøslev been also algorithm had that LULU species the non-iberian used of checklist a to 6 metaMDS < ucini h akg vgn (Oksanen “vegan” package the in function .5 oeswt pseudo-R a with models 0.05) 2 ausaddtrie the determined and values betareg ucini the in function 2 > . and 0.5 Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. arcswssgicnl o r=036 .0) hr eesgicn ieecsbtentespecies the between differences distance two significant were the between There homogeneous correlation 0.004). less the = were that p parks revealed 0.336, the test = and Mantel (r distinction, The low north-south adults. significantly clear on individuals was a based show including matrices NDMS not performed the did NMDS in 4b) generally The (Fig. than were com- component. northern stages park first between life same separation the all the clear along of a of communities showed communities ordination southern the This and parks. between munities the distances between the distances the 4a) patterns than (Fig. lower community NMDS on adult-based juveniles the including In parks of of southern number the effect the to the 34) 17, of and to spider 24 Evaluation 12 in sheet-weaving (16, from high parks of northern 11). ranging as number the and parks, twice the from 10 all as While abruptly (7, much across decreased parks. (as similar adults southern as parks very the recovered northern remained in species juveniles the species observed extent) as in of was lesser collected juveniles trend number a MOTUs of opposite the to that the (Linyphiidae) (although than but spiders trend greater PP) Philodromidae sheet-weaving similar was families for adults a hunting Interestingly, as sit-and-wait and the spiders. represented parks, and specific crab the was () PM). some all Thomisidae (Araneidae) spiders in but in orb-weavers cob-weaving and juveniles, higher juvenile adults in and 69.1% of observed of adults and richness also richness in PC the was the richness example, in than For higher species 3). greater identical 45.9% (Fig. much PO, or PS, differences in similar in higher clear a higher (5.4% showed showed park 29.8% families every PA, families in in the adults higher of of that 17.6% Most (44%). than PP, PA2 higher in and PS1 was higher the juveniles (83%), (47%) as 8.2% was adults), PS2 PP4 recovered (92%), PM2 with (54%), species PP1 percentages, of only PO2 (102%), number the (56%), than The PC1 with for PP2 species (112%), (58%), As increase more PP3 PA1 2). (117%), (169% 35% (68%), (Fig. PC4 richness PO1 number a (119%), PP4) of (73%), the PC3 represented (in addition (162%), unsuccessful) 44 juveniles greatest PC2 was to by as the sequencing followed PM2) provided (whose only (in 203 juveniles PM1 and found 24 where Excluding juveniles from adults. species plot total as varied as the a exclusively species found 128 yielded matching of species adults, of lifestages addition the as both all The exclusively of richness to found stages. combination species respect were life of The 160 values respectively. those, both final 331, Of as the and species. fragment, different 363 COI 491 were the of juveniles by indistingible and were adults that for species replicates the the collapsing of clusters After two juvenile and reads. and PM1 of of numbers adult low replicate of all very remaining in Delimitation yielded only at plateau which the a in were of reached exceptions presence all plot The PS2, every and species. replicates. almost of plot different its that 350 showed a 331 of to information) some left in corresponded Supporting or plot replicates S1, that assignment, 5 less (Fig. in taxonomic curves with least present Rarefaction their as MOTUs at by replicates out with its collapsed Filtering MOTUs of when species. only 2 different that, least considering 524 MOTUS Finally, to 411 species. match MOTUs left different best were COI 888 their reads the that the to and 5 assigned MOTUs species databases unassigned than library) the genetic remaining reference (merging online the subsequent checking assigning the MOTUs from fragment, and in these assignments, COI samples taxonomic LERAY of improve these the assignment to by omitted tree taxonomic indistinguishable we the be to so refining of found in sequencing unsuccessful involved The was steps information). PM1 The Supporting plot S2, of Table of MOTUs in replicates consisted 455 dataset four juveniles analyses. eliminated (final for the pseudogenes non- MOTUs removing dataset of juvenile removing of final three final process Araneae, the 888 The only sequences), MOTUs. left 1,343 (retaining adult and contained in added 140 process distributed the which artificially curation reads of of removing 9,956,432 the MOTUs, sequences and non-singleton the After taxa, 4,668 adding sequences. in iberian total manually artificial sequences a After all generated adult sequences. grouped samples exclusively unique algorithm pool 3,839,513 Swarm juvenile and the of adults, reads sequencing sequence the process, 15,805,993 removal of chimaera and filtering, cleaning, 7 Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ihesi h otenprs(smc stiea ihi P,btlwri h otenprs Linyphiids parks. southern the in lower but PP), in juvenile high than as higher twice was as richness much species (as adult parks whose northern sheet-weavers, the linyphiids in of large richness case are the juveniles is as interesting exclusively Also found obser- were This that (araneids) 1973). orb-weavers araneids (Levi, Larger the earlier as Araneus family. of mature such this most to orb-weavers, tend of as of species findings, phenology species smaller our found greater the while diversity matches immatures to almost zones, additional fully as related was The temperate vation captured be stage. juveniles in species this autumn also captured of for in may of number species mature number spiders the additional juvenile the made sampling have as that of may chances only fact adults the import- the of increased had araneids, number some it of the stages, because juvenile case as in high the and as In adult twice 3). in richness (Fig. species adults. differences Although similar ant on a ones. related recovered exclusively be northern families may most latitudes. in performed difference Although the both than are suspect between we parks study, delay that further southern phenological bioinventories require a in would spider pattern to large higher this by a species behind was that reasons of ignored juveniles the indicate number knowing of be results total These contribution the may adults. samplings, the diversity however, with individual Interestingly, diversity, obtained the the to richness of contributed of the half considering stages doubled almost part obtained immature than In we which more plots. richness to juveniles our the degree including all than across higher The constant 35% sampling. of spiders not species, combination whole immature was The different the techniques. as 491 sampling in methods, of direct adults total sampling by detect a only the to adult yielded to difficult the stages more related in life than be artifact stage may both an juvenile and the adults be in than well This species smaller fewer adults. inventories could are were of finding there spider that samplings, this than in the However, lower of stages was stage. time metabarcoding juvenile the by at of juveniles that, from relevance suggest estimated could the species on of which number insight 1996), the Silva Overall, first them. 1995, a from Stork offers et derived & Malumbres-Olarte and conclusions Russell-Smith 2020, the 70%, 2004; al, and al et to 76% (Soukainen et 40% studies - Cardoso other from 39% 2019; in between ranged al, obtained ranging et those average, juveniles Malumbres-Olarte to on by 2020a, similar specimens al, represented is captured result mostly the This is all plot. of time per 59% given represented a specimens at Immature biodiversity of component important An below. Gnaphosidae orb-weavers, mostly DISCUSSION Araneidae were Theridiidae funnel-weavers, spiders and In Agelenidae wolf spiders below. while Lycosidae crab mostly line, and Philodromidae were 1:1 ground-dwellers ground-dwellers, the (Clubionidae) Clubionidae above above ground-dwellers mass, were were and and cob-weavers (Theridiidae) (Anyphaenidae) RRA RRA spiders linking spiders linking cob-weaving plots plots ghost the the and while In (Clubionidae). (Agelenidae) line. line, third 1:1 funnel-web 1:1 the varied the in below individuals, the slope or different of above its rather consistently abundance but were models, observations Salticidae) and all the and families in p (Lycosidae some a positive families in had Also, the was models of both abundance where two families or in three overlapping weight the positive of in a and Even still proportion families. RRA was spider and there across between reads fitted, relation the be of the not for proportion regression could Although models relating model beta appropriate adequate models variables. built an two provided the which we the families for for between communities, the families so correlation the 10 of did In Seven families least 5). families. (Fig. six at individuals 14 and in weight, remaining present of the were proportion for that data families with spider models the data selecting metabarcoding After from p=0.078). abundance (R=0.136, species (R=0.944, juveniles of species including adult Assessment dataset only the containing dataset with the not with but communities p=0.0005), southern and northern of composition species. rip lobata Argiope als 1772, Pallas, 8 aiiie patagiatus > .5 h w uvswr almost were curves two the 0.05, Cec,15)o several or 1757) (Clerck, Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. diinlsuiswt pdr n te aawudhl eemn fti sacnitn rn n,i so, if and, trend consistent a spiders. is analyses. from this metabarcoding apart if in groups determine sample other the help to would in 1:1 applicable taxa proportion underrepresented the is weight other or below it over real and or if respectively their spiders above be to clearly with might respect not sizes studies juvenile were with Additional large counts mg) or 3 reads small and mass to their with 5.5 (2.5 by taxa in (between mass that individuals while juvenile suggests proportion juvenile mass), intermediate This large in to line. an with the mg those above with 2.4 RRA to Families mostly and corresponded mg). relating families 0.9 line (between 18.7 spider identity plots individuals the the juvenile inspection, the below small mostly detailed with in families Upon families line. to families 1:1 corresponded the the line identity below of or some above consistently across of were 2016). mass factor observations al., and the et RRA the in or (Thomas Interestingly, 2020) differences groups al., taxa animal these and et all other (Kennedy found weight for families in also counts both spider taxa have across read different in abundance studies from individual the proportion information similar and dissipates abundance Indeed, its RRA which 2019; derive linking analyses. families, to al., to spider metabarcoding factor related et across in correction Lamb constant positively unique obtained not 2018; is a is al., using taxon relation of et this a possibility of Deagle of strength 2016; the (RRA) al., However, abundance abundance. et Kleinb read (Thomas in Geisen, relative metabarcoding taxa Schenck, of the other 2017; power or al., quantitative the 2020) et investigating Krehenwinkel al., studies et other (Kennedy with accordance spiders in and expected, information As might abundance comparing individuals provide taxa. all taxa different may or animal across stage data here constant life other Metabarcoding certain is describe on a trend we of Studies this individuals that organisms. if only reveal using phenomenon other community help with the Crivelli, a of & performed that estimates Seitz, inventories diversity Veith, suspect alpha to Poizat, we Jakob, apply year 1987; 1997), the also Epstein, of Sgardelis, might & times & (Scott different groups at be Lazaridou-Dimitriadou present animal might stages 2003; many we life across different stages common with very adult community phenologies certain are exclusively marked a account and in seasonality into that adult that taking as compositions. Given suggests present community by stages be among so might community, life differences species another the all a accentuating in sampling, of than inadvertently juvenile the different considering specimens of as more when time on as still the recovered based compositions and In community not are. NMDS higher see really was pronounced to the at they lead only less communities in what may much adults communities between structure among was with lower distinction differences marked found This pattern phenological the individuals. latitudes this lower addition, the parks, at In all different two juveniles. communities from communities includes and the other communities that to between latitudes to similar NMDS similarities more than the were community at communities park in in most communities same adults differences between only the revealed with composition within taking NMDS also species that the showed plots in in communities ANOSIM While NMDS differences the approaches. of and the The compositions stages, reduced latitudes. species life account the all different of into in individuals differences specimens or substantial immature specimens are adult there only considering that when additional communities revealed among test important coming similarities Mantel provide in improve.The of could increase will interpretation studies will species our analyses. metabarcoding databases changes nominal our in juveniles reference to assignments any Identifying of caveats taxonomic to of some completeness assigned accuracy still that the are be expected and there is years not level), different it could (order of Nevertheless, that scope existence information. taxonomic MOTUs the large the indicate a All may had differences however, more data The our south, a latitudes. climate. Although have and both warmer which north at and parks, linyphiids between drier within northern richness significantly phenologies in a juvenile predominant Pek´ar, with species and (Cardoso, parks, tropics total adult and southern more subtropics in in the found than in indeed than climate, regions We continental temperate 2011). in diverse Coddington, more Jocqu´e & much be to known are lig rusugr 09,orrslsidct that indicate results our 2019), Traunspurger, & ¨ olting, 9 Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. nih . aa´n . agnte,O . uo,X 22)T eos rt lse?Ta sntthe not is That cluster? to or denoise To metaphylogeography. and (2021) 22-177. metabarcoding X. , COI Turon, for & pipelines S., Optimizing spiders. question. O. active Wangensteen, Palac´ın, winter C., A., of Antich, phenology The (1984). W. C. Aitchison, https://doi.org/10.5061/dryad.z08kprrcv. Dryad, specimens: immature REFERENCES of sequences https://doi.org/10.5061/dryad.g79cnp5q0. Dryad, with file specimens: Fasta adult - of sequences with JMO file authors. Fasta and all - MD of OW, contributions with work. manuscript. manuscript final laboratory the ACCESSIBILITY the the DATA drafted approved performed MA and MD and revised JMO and have MD, authors AE All analyses. study. data the was the designed support performed MD Additional and JMO Barcelona. MA, of University MA). Government. (to the Catalan CONTRIBUTIONS #485/2012) the from AUTHOR (OAPN from fellowship Nacionales MA) PhD (to Parques 2017SGR73 APIF de by Autonomo by field. provided samplings Organismo the supported the in the was to us by contributed MD supported funded that that Aniz was people rangers Merced work the park This all Maria the to (Ordesa), all grateful and to Villagrasa are (Cabaneros), as Elena We well Manzaneque Nevada). Europa), individuals as Gomez (Sierra de following Angel Losada (Monfrague), (Picos the Ramos Martin Hoz by Blanca Rodriguez provided la Angel kindly de (Aiguestortes), permits M. Montes accurate Miguel corresponding more institutions: the provide and under still conducted could were sample Collections the in taxon data. presence/absence a simple of ACKNOWLEDGEMENTS than a occurrence composition to taxon-specific the community with are of the transformed data related proxy about appropriately counts metabarcoding information a positively read already that as of as was use families, suggesting factors the across families correction sample, constant Nonetheless, not studies. spider the was however, former in certain relation, in this reported abundance of for strength and The metabarcoding weight quantitative. extent from in certain proportion obtained effective their more reads enables to of sample a proportion decisions. in The conservation present comparisons better-informed stages regarding ultimately all data and the of accurate strategies, analysis question more monitoring Metabarcoding provides not of composition. juvenile protocols previously do Adding community level knowledge assessment inventories. of findings biological of biodiversity the from layer These rapid derived alters relevant interpretations to yet other. also the novel information of each it a some influence to as add may but richnesses, that compare inventories, overlooked species they adult-based by modifying how found provided not simply and information is juveniles beyond communities as goes present among species impact similarity relevant the a of This has proportion adults. initiatives considerable bioinventorying a among in because spiders estimates, of diversity stages on immature developed effect incorporating that be suggests to study Our need factors correction during controls these quantitative CONCLUSIONS as However, included the 2019). 2020). communities in al., mock al., presence/absence taxon et using using (Lamb a et example uncertainty, than metabarcoding of for Kennedy of taxa, composition occurrence 2019; different community degree the for of the al., certain individually surrogate of a et a information with (Lamb as precise and data factors more caution correction provides with corresponding still albeit the sample that, with RRA stating the studies using previous with agree We 10 ora fArachnology of Journal M Bioinformatics BMC , 12 249–271. , , 2021 Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. 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Data may be preliminary. iue2 ubro pce MTs on nya dls nya ueie n sbt iesae nthe in stages life both as and juveniles as only adults, as only found (MOTUs) species of Number 2. Figure Aig – PA communities. studied 16 the of Monfrag – Location Caba˜neros; PM 1. Figure FIGURES reads. of S1 Figure article: S2 this Table of version online the S1 in Table found be may Information Supporting Additional INFORMATION SUPPORTING Evolution ia aae otiigalteMTso mauesae fsiesotie ymetabarcoding. by obtained spiders of stages immature of MOTUs the all containing dataset Final , nomto nteautsqecdseiesue nti study. this in used specimens sequenced adult the on Information 3 aeato uvssoigtenme fMTsprrpiaeaanta nraignumber increasing an against replicate per MOTUs of number the showing curves Rarefaction 613–623. , e O–Ods ot edd;P io eErp;P iraNevada. Sierra – PS Europa; de Picos – PP Perdido; Monte y Ordesa – PO ue; ¨ 16 etre sayd atMuii C– PC Maurici; Sant de Estany i uestortes ¨ Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 6smldplots. sampled 16 17 Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 18 Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iue5 lt fterlto ewe eaiera bnac RA n rprino as(e)and (red) mass of proportion and (RRA) p abundance with read models relative Only between (blue). relation individuals the of of proportion Plots 5. Figure all using NMDS b) (stress=0.074). individuals adult communities Bray- only southern the using (stress=0.065). and using NMDS juveniles) colors data a) and cold presence/absence (adults in colors. individuals on represented warm based are in composition communities represented community Northern are the index. of dissimilarity ordinations Curtis NMDS 4. Fig for (blue) juveniles as found MOTUs of number and (yellow) Park. adults National as every found in species family of each Number 3. Figure 19 > .5ada dqaegons ffi r shown. are fit of goodness adequate an and 0.05 Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. P M P P P 20 P S C P O P A Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 21 Posted on Authorea 19 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162936514.40253306/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. a A d u l t s 22 b A d u l t s

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