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Thébaud Borges This article isprotected Allrights bycopyright. reserved. doi: 10.1111/1755-0998.12617 thisversionandthe between lead todifferences been through the copyediting, typesetting, and proofreading which may pagination process, forpublicati accepted This articlehasbeen Emerson C. Brent forest arthropod biodiversity across spatial scales id molecular surveyand A combinedfield type:Resource Article Article Date:16-Sep-2016Accepted Date:02-Sep-2016Revised Received Date :23-May-2016 8 7 6 5 4 3 2 1 Finnish Museum of Natural History, University of Helsinki, P.O. Helsinki, of University History, Natural of Museum Finnish CNRS-Uni 5174 UMR Biologique, Diversité & Evolution Laboratoire Norwi Anglia, East of University Sciences, Biological of School Island Ecology andEvolution Research Group, Instituto de Produ M PBT Pulmns ééax t i-gesus n iiu Tro Milieu en Bio-agresseurs et Végétaux Peuplements PVBMT, UMR L La de Universidad (Zoología), Animal Biología de Departamento National Muséum marins, Invertébrés collections, des Direction CE3C – Centre for Ecology, Evolution and Environmental Changes

Accepted 7 97 Denis, Cedex 9, Reunion 93002, Island, France. CS Cassin, René avenue 15 Réunion, La de Université Canar Spain. Tenerife, Laguna, La 38206 Sánchez, Francisco Astrofísico France. 05, cedex Paris 75231 Buffon, 61rue CP53, Naturelle, Heroísmo, Açores, Portugal 9700-04 s/n, d’Ávila João Capitão Rua Ambiente, do Engenharia e de –Departamento Açores Universidade dos and Biodiversity Group Helsinki, Finland France 9, Cedex 31062 Toulouse Sabatier-ENFA, Paul UK. 7TJ, NR4 Norwich Park, Spain 38206, Islands, Canary Tenerife, C/Astr Agrobiología, y Naturales Article 5 Néy Mollaret Noémy , 3

1,2 * , Juliane CasquetJuliane 6 Pdo Oromí Pedro , fsc Facso áce 3 La 3, Sánchez Francisco ofísico 3 , Heriberto López Heriberto , on and undergone full peer review buthasnot review undergone fullpeer on and 7 Dmnqe Strasberg Dominique , Version of Record. PleaseVersion cite thisarticle as entification protocol forcomparing protocol entification

1 , Pedro Cardoso Pedro , Box 17, 00014 00014 17, Box h Research ch 8 2, Angra do ad Christophe and , / Azorean / gn, C/ aguna, d'Histoire y Islands, Islands, y Laguna, Ciências 44 Saint Saint 44 4,5 versité versité pical, , Paulo A. V.A.Paulo , ctos This article isprotected Allrights bycopyright. reserved. a in undertaken program sampling comprehensive and recent a by Accepted diversity e complex richness species total a of challenges logistical encompass The function. may they Earth on point given any art many so are there - scale of one simply is obstacle primary ke several for communities higher-order of level the at elusive fundamental(Cardoso biodiversityremains an metrics ter important most in biodiversity arthropod of frontier drivers the Understanding such and challenge as alpha, in ecologicalIntroduction and beta e and gamma o to extended groups andhabitatsbyadapting be can sampling Field studies. independent across of the Linneaninformation taxonomic prior upon shortfallpr assignment species our Because data. distributional and data to yieldsequen diversity mtDNA using species biological presumed with units these demonstrated estimates then and PUs into juveniles and adults classified that can spiders, aoc groupvolcanic the on located lo plots ten with sampled Article We species. limited biological infer to sequencing taxonomicmtDNA c units and parataxonomic arthropods, into distributional of samples sampling mass combine protocols These species. data there may becan a be lack employed of by researchers robustWe provide whose amorphologic setunreliable in result can taxonomicwhich expertise, taxonomic of standardized skills mayfor be ecological arthropods li is oftenbeta alpha, as such metricscomplicated and biodiversity fundamental Obtaining molecular by a lack of prior samplAbstract taxonomi * Contact author: [email protected] etal.

2011). Despite the fact that most terrestrial organisms are ar are organisms terrestrial most that fact the Despite 2011). eanic island of Réunion (Mascar Réunion of island eanic a priori our field sampling protocol acc protocol sampling our field , or taxonomic expertise, it mi it expertise, taxonomic or , Ps, n slcie sampling selective and (PUs), information regarding theregional pool of stimation is exemplified is stimation y reasons. Perhaps the hropod species, and at and species, hropod ordingly. ordingly. e iety compared directly be c information and/or information c and gamma diversity gamma and volutionary research research volutionary ally based estimates. based ally nimises the problem the nimises ene archipelago) for archipelago) ene the reconciliation of reconciliation the for this region. We otocol is not reliant not is otocol etil ecosystems restrial ce data, ecological data, ce n pooos that protocols ing of individuals for for individuals of mited, and where wland rainforest rainforest wland diversity remain remain diversity ther arthropod arthropod ther asfcto of lassification rpcl forest, tropical of form and and form of thropods, okod y olcig pcmn ad ulig eeec collect reference building and specimens collecting by workload Basset m of designation the by process inventory the into incorporated of a meaningfulass biodiversity any of validity, the ultimately and precision, af broaderthat biodiversityminimizethe and impediment to crucial reliable al. gro taxonomicarthropod some of characteristic diversity, species cryptic mo becomes and groups, geogr taxonomic on framework, depending vary will impediment of level The diversity. species “Li arthrop investigate to aim that studies so-called most in itself presents (the impediment taxonomic This ident available. readily suffi when especially regional challenge, species formidable lists and a taxonomy remains are lacking,diversity and when taxon resources. is to seekBorges (e.g. to optimise Snyder 2003; datameth of combination a requiring usually 1996a) Beattie & Oliver return andmuch develope been information reliability have resources and time as minimal possible use that fromprograms an area respon (e.g. glo In Duelli major loss. against of 1997; species era potential Jo an in diversity and inve richness species arthropod re to obstacle access an is area an and within species arthropod standardised inventory of lack A scales. spatial larger across hours, andfinancial backing limit the extent towhich such sam However, resource interms requirements oftaxonomic expertise, tremendous step forwardforest ofPanama,Basset samplin days person 24,354 in of total a and protocols, structured filling the knowledge void of arthro This article isprotected Allrights bycopyright. reserved. km several of area an within plots m 20 x 20 twelve where

AcceptedCicconardi 2013; Article et al. et Exhaustive sampling programs such as thatof Basset h saily xlct quantifica explicit spatially The et al. 2012; Novotny 2012; etal. 2005; Duelli 2005; 2006) that require standardisation for comparison among sites sites among comparison for standardisation require that 2006) etal. et al. 2010). Overcoming the taxonomic impediment can thus be thus can impediment taxonomic the Overcoming 2010). et al. (2012) collected an estimated 6144 arthropod species. species. 6144arthropod estimated an (2012)collected et al. 2002). Parataxonomists can reduce the taxonomic taxonomic the reduce can Parataxonomists 2002). e o hs Rpd Biodiversity Rapid this, to se 1999). The challenge for effective RBA programs programs RBA effective for challenge The 1999). re problematic with apparently apparently with problematic re in f oa atrpd specie arthropod local of tion et al. essment. In the absence the In essment. od species richness and richness species od 2 pling can be expanded expanded be can pling ups (e.g. Cicconardi (e.g. ups were sampled. Using fects the accuracy and and theaccuracy fects nes & Eggleton 2000; 2000; Eggleton & nes of comparisons liable ods (e.g. Scharff(e.g.ods omic expertise is not is expertise omic g in the San Lorenzo tet f ie and time of stment seset (RBA) Assessment field and lab person person lab field and (2012) represent a a represent (2012) be prahs to approaches ible rhseis (e.g. orphospecies os te nt of units the ions, nnean shortfall”) shortfall”) nnean pi rgo and region aphic o biodiversity. pod inl complete ciently a cags and changes bal fcto cn be can ification rcns and richness s t gte as gather to d ih ees of levels high et al. et et

This article isprotected Allrights bycopyright. reserved. Accepted However plasticity). phenotypic (e.g. entity taxonomic same the PUs may be undiagnosable at the DNA sequenceAlt units. such other from PU that of distinctiveness taxonomic level, suggesting PU a with segregate may variation sequence DNA PUs, of case the (e.g boundaries species of validation the for complement useful biodivers of Emerson (e.g. scales quantification spatial comparative the for further us take s judiciously used m 2012), Francis & Krishnamurthy 2011; gene” DeSalle “barcoding the as such cytochrome discovery, species to approaches c oxidase criticisms valid are there subunitWhile I (COI),(1) Validation or refinement of parataxonomic units as presumed species sensu Hebert offer: hi We regions. documented poorly especial impediment taxonomic the furtheraddress to assessment app an scale propose large with way, integrative we an in deal, to endstep molecular this To advantage. within an that be investment will anything time assessment, their then reduces important, or time workload, Article same taxonomist’s the at but bottleneck, amount to a substantialsignifican taxonomic the revise necessaryto still is expertise impedim taxonomic the alleviate to helps approach parataxonomic task. publis eight If across oneassignment) acceptsthat accuracy accessave an with Arthropoda, the within toidentity taxonomic to aregard g (which is a consequencespecies confirmationby ataskthat ataxonomist, canbechalle consider of without only morphology bothentiti morphological discrete splitting as species of estimations are PUs un parataxonomic as to referred and be best may Basset but 1996b), Beattie (e.g. morphospecies lumping as to referred been have which It has been shown that parataxonomic accuracy can deviate subst etal. 2011; Ji 2011; gis rlac o simplistic on reliance against e cmaios Kel 04. Th 2004). (Krell comparisons hed ing taxonomy. Such PUs then req then PUs Such taxonomy. ing hih fu iprat advantag important four ghlight etal. 2013; Yu 2013; et al. etal. 20) eg Glsen & Goldstein (e.g. (2003) ce of PUs, which may still may which PUs, of ce nging to accomplish. arthropod biodiversity ernatively two ormore et al. et . Riedel. 2012), and provide a provide and 2012), es, based on external on based es, its (PUs, Krell 2004). 2004). Krell (PUs, its o txnms i a is taxonomist ood , consistent with thewith consistent , uch approaches can approaches uch ly in species-rich or species-richin ly , in case of two or they may belong to rage of 76 percent t oe different over ity ige sequence single s ht hs may this that es rcse i PU in processes 2000; Oliver & & Oliver 2000; uire subsequent uire n, taxonomic ent, etal.

biodiversity a oc uig a using roach s wie a while us, antially with antially piie a optimizes itochondrial 2013). In In 2013). This article isprotected Allrights bycopyright. reserved. Accepted spec sampled if represented in adatabase. assessment biodiversity a within identification use be also can databases such this, to Related identification. wo inventory subsequent for 2007) Hebert & (Ratnasingham (BOLD) (Benson GenBank as such databases taxonomic formal p await made be can data species sequence DNA reference their identification, inventoried of subset a if comp Even for available immediately more be to data biodiversity for incr assessment biodiversity in data sequence DNA Incorporating (3) Improvedcomparability inferenc and estimation improving underlying ofbiodiversity components patterns. thus data, sequence DNA their representa with PUs of sorting the complementing by revealed be cryptic Such for. accounted easily be cannot that heterogeneity beta assessments biodiversity inaccura to lead would of it 2009), Ehrlich & Article (Ceballos context considered and the in biodiversity gammacryptic (Cicconardi Collembola class arthropod the diversity b could species r been has as cryptic richness, species described than higher magnitude undescribed of number the cases some in to impossible or difficult becausemorphologically are species Cryptic (Ceba describedcurrently are than species more for account may crypticsequence data. Subsequently(Scheffers it has species beenThe description suggested of cryptic species has grown exponentially that over undescri(2) Detection of cryptic species hypotheses contribute spec cryptic (e.g. PU single involving the sequencing ofmultipleindividuals within aPUwi a within lumped being species more to etal. 02, agl de o nrae pplto lvl apig f D of sampling level population increased to due largely 2012),

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2015) and the Barcode of Life Database Database Life of Barcode the and 2015) etal. 03. lhuh h polm of problem the Although 2013). d to augment taxonomic distinguish among, and among, distinguish ll be required. ll berequired. diversity can however can diversity the past two decades te measures of alpha, of measures te ecently proposed for proposed ecently llos & Ehrlich 2009). 2009). Ehrlich & llos ublicly available via available ublicly bed cryptic species cryptic bed eases the potential the eases rtv purposes. arative s sal not usually is ie apig of sampling tive rk and specimen e ae already are ies e) a strategy a ies), ecito or description a odr of order an e aot the about e unobserved NA This article isprotected Allrights bycopyright. reserved. Cardoso 2009; Accepted cannot usually discard are samples they spider of percent 75 to up meaning species, because forms juvenile ignore to forced are pool and taxonomy(Casquet island this arein investigation prior not wellthe been known.has that group focal challenging a is This Inisland. the general, along spider rainforests lowland within established biodiv plots biodiversity archipelago; (Mascarene Réunion biodiversity our of value the demonstrate We assessment. biodiversity within samplin com molecular to and ecological is assessment aim broad Our species. of pool regional the regarding ofpurpose the of spider sampling,whose taxonomic andemp be skillscan where that faunaprotocols laboratory may and seque field be of limited,set therea provide DNA where maylandscapes a from be may aspec biological incorporating lackpresumed beof inference the for PUs of assessment of 2009), the robust (Cardoso spiders volcanic Mediterranean In o this paper, we extend and develop a semi-quantitative protocArticleGlowska sequen DNA on based species presumed single a as united be will resultingand from splitting thu a sizes samples if particularly identity, species confound also a among gender within or among Differences sequences. DNA their of juvenile or (Garcia-Robledo larvalWongpakam beetles 2014) and forms, spiders 2009), (Raso Hausmann they & can be associated(Ball mayflies including species arthropod in stages with adult used been has data sequence DNA (Krishnamurt variation morphological limited or large either of ma all not where genders or stages life of association the been One of the successes of combining DNA sequence(4) Association of different arthropod life history stages data and genders with tradit etal. 2014;Jusoh etal. 2008b). Our protocol allows us to include both adult and juven and adult both include to us allows protocol Our 2008b). etal. s comprised of different forms different forms of s comprised 2014; Przybylowicz & Tarcz 2015). 2015). Tarcz & 2014;Przybylowicz pooos o drs te taxo the address to protocols g ucsfly o dniy crypti identify to successfully otws Ida Oen, sample Ocean), Indian Southwest etal. et al. 2014). As such, where PUs are comprised comprised are PUs where such, As 2014). 2015) and for which both the species the both which for and 2015) etal. etal. 2013), flies (Pramual & & (Pramual flies 2013), 2005), butterflies (Gossner (Gossner butterflies 2005), within thesamespecies re small. However, PUs subject of only limited y be described becausedescribed be y forms by reference to reference by forms ed (e.g. Cardoso (e.g. ed the eastern coast of loyed by researchers by loyed ies (Mayr 1942). We We 1942). (Mayr ies ol for the analysis of y Facs 2012). Francis & hy a priori ional taxonomy has taxonomy ional

c larval or juvenile juvenile or larval c oi impediment nomic ersity assessments ersity challenging for the for challenging ie standardized bine approach with a ce similarity (e.g. similarity ce e sind to assigned be enc sad of island ceanic dult forms may information d from ten ten from d nce-based etal. ile

This article isprotected Allrights bycopyright. reserved. is habitat and complex is topography if limiting be Cardoso may 2008b) of Accepted those as such areas sampling Larger constraints. compromi a is area sampling of choice Our 2008b). 2009; (2008a; Basset of area sampling ha He area. sampling is sampling study present area the of and 0.25 2008b) hectare 2009; (2008a; (50m x 50m), whichdesig sampling the between isdifference important intermediatAn S2. Appendix met sampling of details Full species. beetle and spider both of s additional of incorporation the Field sampling methods are adapted from Cardoso Sampling procedures of BOLDt theuse would preclude some ma this and region, gene informative taxonomic similarly but alternative, could region barcode the that noted be should It 2007). Hebert groups,(BO Database Life of Barcode the within database identification should (Hebert bi region downstream am for (mtDN DNA mitochondrial the data sequence We S1). Article(Appendix sequence analyses DNA and labelling f with parataxonomic specimens individual of referencing cross the facilitate ent data for template a provide We 1). (Fig. boundaries species sampling parataxonomic field plot-based standardised integrates protocol sampleOur handling and DNA sequencingProtocol overview for the infereMaterialsmethods and relative tobiodiversityassessmen on only based estimates similar compare We diversity. gamma and beta alpha, estimate to spiders et al.

2003) to facilitate taxonomic assignment using the specimen specimen the using assignment taxonomic facilitate to 2003)

et al. adult forms to illustrate the b the illustrate to forms adult ampling techniques designed to designed techniques ampling (2012) and the 1 ha sampling area of Cardoso Cardoso of area sampling ha 1 the and (2012) o facilitatetaxonomicassignme t basedonmorphospecies only lfcto b mr successful. more be plification et al. (2008a; 2009; 2008b), with with 2008b), 2009; (2008a; enefits of our approach approach our of enefits hods are presented in LD) (Ratnasingham & (Ratnasingham LD) optimise the capture the optimise disturbed, as is often often is as disturbed, nt. be substituted for an . et al. b peeal for preferable be y ield sampling data, data, sampling ield e between the 0.04 the between e y n crto to curation and ry n of Cardoso of n se among several among se with subsequent subsequent with nce of biological of nce these values to oee, this However, re wedefine a A) COI barcode (2008a; 2009; 2009; (2008a; odiversity et al. et al.

Mastercycler epthermocycler systemin a 25µl total PCR volume (Folmer HCO2198 and LCO1490 primers the with overnight, and then stored at -20 °C prior to DNA amplification ml mg This article isprotected Allrights bycopyright. reserved. Acceptedincubated in PCR plate wells with150 µl of aprotocol 10% Chelex using to classes 96-well available the individuals per PU per plot. with PCR complemented plates was sampling forms, (Casqueteit of composed exclusively were mor that PUs or those for one or classes, within individuals of number sufficient a of absence DN for selected were plot each within PU each for juveniles two male, adult one possible, where this, for account To juveniles. lifeidentificatio species historyfor problems present may spiders case our differences,samples within each PU will also be based uponresources, focal taxon and withof individuals to be sequenced per the PU per plot is a decision to l taxonomic available using but majoritylevel, family or genus to otherwise leve species we to made were recommend identifications taxonomic ArticleProvisional of individuals wascarr shape, copulatory bulb four individualsSorting samp Specimen sorting to parataxonomic units and genetic analyses as to a useful spider 14/01/2013. minimuinvasive PUso terms in on undisturbed relatively be based to deemed usingwas chosen that habitat were locations Plot 2). (Fig. Réunion plant of island morphological species.Strasberg ran altitudinal an within rainforest lowland within established Samplingsampl depleted.Ten easilyfauna get trampled andmay sites the features,Basset wasar sampling Smaller landscapes. mountain rugged within case the conducted including between the e 23/1 -1 DNA extractions were carried out using a fast and cost effectiv et al. et solution of Proteinase K. Plates were then sealed and incubate etal. (2012), are likely to be too small if the sampling effort is r is effort sampling the if small too be to likely are (2012), 2005 for a description of the habitat) along the eastern coast eastern the along habitat) the of description a for 2005 ied out by one of us (JC) using (JC) of us byone out ied et al. et 2012). In brief, single legs were were legs single brief, In 2012). etal. 1994) using an Eppendorf Eppendorf an using 1994) ® solution and 10 µlof a10

be based upon financial a binocular microscope. . PCRs were conductedwere PCRs . ge of 570 - 790 m (see (see m 790 - 570 of ge iterature. The number The iterature. one adult female and ing areas (plots) were (plots) areas ing her juvenile or adult adult or juvenile her containing 16.3 µl of of µl 16.3 containing project objectives. In objectives. project n due to gender and and gender to due n A sequencing. In the In sequencing. A f human impact and impact human f e epce t be to expected led eas such as those of those as such eas cesbe forest accessible wee possible, where l e of these three iyu and pigynum . hie of Choice m. easonable as easonable 0/2012 and and 0/2012 ban four obtain a 5 °C 55 at d chelex e of the This article isprotected Allrights bycopyright. reserved. anotherAcceptedthey if GL a as classified are specimen single a by represented GL. SamplesPUs contained withinas monophyletic within groups a each GL, of(Tamura DNA while sequences GL which minimising were aim tofrom include s al DNA representativethen sequences, the within DNA outlier for, number correction and assessed of each PUsSubsequen reassessed. were samples w misclassified presented that PU were by one by identified subjectmisclassifications of th of origin taxonomic the determine to database toidentification a neighbourwith joining asequencing and amplification the local analysito blast, the wrong PU,misclassif (i) to due orwas sequence DNA (ii)outlier the if determine sample the or laboratory outlierDNAprocedural sequence contam against was s other gen high assessed PUs. exhibit that Inrelative the sequences case outlier for of to a checked positive other against and PU sampleseach match, the within the specime the PU PU.As that a first Outlier step after the sequences DNA sequencing, DNAArticle DNA were sequence sequence alignments w Reconciling parataxonomic units with presumed biological species alignments for each and checked then were sequences PCR products were (http://www.genosc Sanger Genoscope sequenced Centre, with Sequencing both National the forward French and re successfully by checked then was success PCR amplifiedfor 30 s and primerprim extension s, 30 for C 95 at at 72 products (denaturation C forcycles 38–40 40 repeated s); min; and final exte a polymerase Taq U⁄µl were 5 of extract. µl 0.2 then dNTP, mM PCR 10 of was µl outsourced 0.5 carried primer, out under thepurified for following DNA water, conditions: se 5 µl ho of Promegagreen 5x buffer, 0.5µl of a 20 etal. 2013) for the characterisation of genetic lineages (GL). GLs a GLs (GL). lineages genetic of characterisation the for 2013) PU using Geneious Pro, version 5.4 (http//w version 5.4 PU usingGeneiousPro, N sqecn, neune sa unsequenced sequencing, DNA of non-target DNA. If no posit qec ws umte t te B the to submitted was equence running products on a 1% TAE ag miuu bss ald eoe c before called bases ambiguous nsion at 72 C for 7 min. 7 for C 72 at nsion do not cluster within cluster not do e sequence. In light of of light In sequence. e n associated with the the with associated n

ww.geneious.com). ww.geneious.com). ication of the sample the of ication ive match was found found was match ive er annealing at 55 C C 55 at annealing er µM aliquot of each -tr t9 fr 2 for C 95 at t-start first blasted locally ination resulting in resulting ination ere constructed for constructed ere t to the analysis of, analysis the to t qecs sampled equences pe wti PUs within mples l individuals of all all of individuals l verse primers. All primers. verse unig o the to quencing nd 2µl of DNA uig MEGA6 using s tc divergence etic ti a L PUs GL. a ithin L specimen OLD p.n.r. All ope.cns.fr). rs gl and gel, arose sin t, to to, assigns us (PC) for onstructing re defined re This article isprotected Allrights bycopyright. reserved. Acceptedusing R package vegan (Oksanen datasets using BAT and their Spearman correlations with Mantel (βtotal, beta pairwise the calculated βrepl we Finally, above. as correlation and estimato βrich, abundance Chao the seeused (Carvalhoimprove would datasets three the of each from richness species understa To (http://biostat.mc.vanderbilt.edu/wiki/Main/Hmisc). compa three was richness observed their to according plots of percentiles ranking (Cardoso BAT 0.975 package and R using 0.025 replacement without approaches the resamples as calculated limits confidence min the than included smaller were individuals forms i adult only juveniles when with species species biological presumed biological presumed (ii) PUs; with (i) for: richne species observed compare to analysis rarefaction used We SpearmanAlpha and beta diversity across plots rather thanoverestimation, ofbiological species. t correlation to lead should it as conservative, considered be Article can approach and individuals, for likelihood data sampling geographical haplotype and ecological relationshipsbio presumed of estimatedinference the for lineages such of independence using using (Robinson the K2P genera mode19 36 of analysis an from derived species, related sampled closest thebetween divergence corrected (K2P) 2-parameter Kimura 6.77% of Rthe when species biological divergent different to belong may packageGL that a within consider We species. biological presumed single definition, with the simplestBOLD Identification System. case being thata (http://wsc.nmbe.ch), Catalogue the all by provided individuals nomenclature the following assignment taxonomic w We adopt the biological species concept (Mayr 1942) as our work et al. 2009). We evaluate the ecological or geographical geographical or ecological the evaluate We 2009). etal. mm bnac ars pos (30 plots across abundance imum nd representative sequences we sequences representativend

2015). 2015). (ho 94 i BT n ue S used and BAT in 1984) (Chao r etal. 2012)) among all plots with the three three the with plots all among 2012)) ti a L eog o a to belong GL a ithin diversity components components diversity 1 spider species from species spider 1 y exceed the average the exceed y tests for significance for tests logical species using species logical e underestimation, he their correlation we correlation their re submitted to the the to submitted re cue, n; (iii) and; ncluded, l with MEGA6. Our Our MEGA6. with l etal. s cos l plots all across ss d f estimating if nd e bten the between red and used was 0) species and their and species tN lineages mtDNA their maximum maximum their A ubr of number A . among 1000 1000 among World Spider 2015). The The 2015). ing species ing pearman Hmisc This article isprotected Allrights bycopyright. reserved. 12 representing extraction, DNA for chosen individuals 1588 the Acceptedf obtained were sequences DNA Overall, sequence. no yielded 233 well only for the forward primer, indivi 66 sequenced 1171 pri reverse and forward the both for sequences DNA goodyielded which well of only sequencing, for th DNA for submitted were (97,8%) in 30 of amplification successful third (following re-exPCR round of firstattempts. A PCR three after a with individuals 126 another performed t and a second in attempt, extraction resulted PCR on the 66 individuals second PCR a that of did no round first (3 av 596 further A a with (50,4%), individuals 800 an of amplification plot. and per juveniles, PU 984 per and individuals individuals adult 604 representing and extraction DNA for chosen were individuals 1588 of total A DNA sequencing Sy Tetrablemmidae. Oonopidae, Nesticidae, the Mysmenidae, to Lycosidae, Cyatholipidae, assigned U PUs of Theridiidae, placement Pholcidae, Linyphiidae, reliable No Theridiosomatidae). Gnaphosidae, Article(Araneidae, liai we Réunion, on taxonomists diversity species for information published f families to assigned PUs For possible. when species taxonomic descriptions, species and genus to PUs compared we Thomisidae), who Hahniid were(Clubionidae, Réunion on information published able is there which for families to belonging comprised to taxonomicallyinto 148 only PUs, of which77 were of comprisedS 1). (Table 935 and 372 between ranged plot per individuals of adults, ofboth adult and juve andwere co adult were spiders male, individual 33 5558 of 824 total were were a plots adult ten all Across female place comprised and3954 (71%) were juveSpecimen sorting and assignment to parataxonomic units of PUs onlyResults juvenil from seven

dividuals. Thus a total of 155 of total a Thus dividuals. information was available for t for available was information ae, Miturgidae, Nephilidae, Sal Nephilidae, Miturgidae, ae, olwn fmle: Anapidae, families: following

4 out of 1588 individuals individuals 1588 of out 4 traction) resulted in the resultedin traction) about species diversityspecies about 7,5%) amplifying with amplifying 7,5%) pecimens weresortedpecimens e reverse primer, and primer, reverse e lce, f hc 780 which of llected, t successfully amplify amplify successfully t he further taxonomic or which there is no ticidae, Sparassidae, ticidae, mers, 84 sequenced sequenced 84 mers, 3 (83%) of the 148 the of (83%) 3 nile forms, 38 were were 38 forms, nile C amplification, PCR or 1350 (85%) of (85%) 1350 or assigning PUs to PUs assigning nile. The number The nile. s Fr samples For es. mphognathidae, mphognathidae, e wt local with sed duals (75,3%) (75,3%) duals he successful successful he rg o 2.6 of erage tep. We attempt. Corinnidae, loboridae, loboridae, families This article isprotected Allrights bycopyright. reserved. the is GL-12 species. biological Acceptedt lineages both infer therefore we and GL-47b), for 38% only to GL-47 the of (80% ratios adult to juvenile different present do ecological sampling. Whilesegregation they do not exhibit obviouslyis GL-47, differ withexc divergences with GL comprisedsecond The species. biological probable the of6 3 1-3(Fig. plots from sampled GL-01c of 14individuals all and PUs two divergent samp GL-01b s that of individuals 61 of all with discrete, terms geographically comprise in lineages differing not While (GL-47aGL-47, andniche than GL-01b GL-47 and and GL-01c, and we consider no itdifferenc toThis be amethods. probab differenand GL-01c weresampling methods almost (FBN, AAS, FSN, BLM,fro obtained were GL-01a TWS, of individuals However GL-01. comprise LIT exclusively and PIT), while three comprises GL-01, lineages these, obtained of (GL-01a, first The GL-01b species. congeneric with and GL-01c) only that show diverg theRobinson no genetic segregation presented three LIT GLs, 54 the a Across w GL-44. in 100 25 PUs with Articletogether grouped specimens four remaining the while specime nine which from specimens, sequenced 13 comprises PU-93 a were (PU-93) one but all PUs, 115 the Of 2). (Table PUs eight compri each PUs, 115 the from delineated were GLs 54 of total A assignment Reconciling parataxonomic units with presumed biological species, and taxonomic 115 PUs. DNA with individuals 1289 of total a yielding PUs), alternative to reassigned contam were individuals to resulting 121 attributed further sequences A in the error. to removal laboratory due of excluded 8 PUs (i.e. were individuals all se individuals either with these withinreconciling wi sequences outlier with specimens all re-examining After PUs. et al.

’s (2009) 6.77% K2P corrected average divergencebetween closes e suggests GL-01a represents a lineage o lineage a represents GL-01a suggests e third GL exceeding the thresho the exceeding GL third unig ro o icret PU incorrect or error quencing mln tcnqe, L0b and GL-01b techniques, ampling these PUs reassigned to reassigned PUs these ), and we), and treat each asa ld, with three divergentthree with ld, sequences, assigned to assigned sequences, a are adults, compared adults, are a ent distributions, they distributions, ent ssigned to a single GL. single a to ssigned o represent probable represent o eeding the threshold the eeding led from plots 4-10, 4-10, plots from led le biological species. biological le f broader ecological ecological broader f m a broad range of range broad a m , 27, 48, 70, 94 and and 94 70, 48, 27, , sed of from one to one from of sed ne i ecs of excess in ences samples of GL-01b of samples hn ah U and PU, each thin ith the 7 PUs that that PUs 7 the ith genetic divergent nd PIT sampling b) that show no sinet 61 assignment, dfeet PU, different a e n em of terms in ce ns form GL-46, form ns GL01c are nto or ination t This article isprotected Allrights bycopyright. reserved. correlated,Accepted indicating that the rankingdifferent of plots is similar (0. for all ten plotsthis was done for all individual (Fig unsequ plot. the withincryptic species individualsthat sequenced for of proportion the as plot i species cryptic presumed given a of number the estimated thus spec biological cryptic presumed was it level, individual the At Alpha and beta diversity across plots recordedfor inRéunion. thefirst time Theridiosoma ( genera seven b Trachelidae), Europe, to introduced fa Three been Réunion. have from unrecorded to known is that origin Asian be to GL-27 reveals match final inaurata Nephila including ArticleArgyrodes sequences previously consisten were obtained by matches one of us (JC) seven in a separatethe s thati studies limited numberof 99% or higher.database identification Seque specimen BOLD 2). the to (Table submitted species level The family or lack genus to assigned of 36 remaining matching forbiological many species species, is not sur (doi:10.5061/dryad.8g5d2). Dryad on available with a furthalignmen full and (KX762052-KX762107), GenBank on available are presumed 57 the of each from sequences Representative species. differentiation among theselineages. lineages, we treat GL-12 as a singl However, in the absence of any clear ecologicalor ge The richness valuesobtained by the three approaches are signif species to assignment Taxonomic sp., Arachnura scorpionoides , A Trachela . borbonicus from several Indian Ocean islands (Kuntner & Agnarsson 2011). T 2011). Agnarsson & (Kuntner islands Ocean Indian several from , n a lat 1 pce (l bt n uietfe) are unidentified) one but (all species 11 least at and ), and Nesticella s, and for adult forms only. only. forms andforadult s, 4. eetees vle are values Nevertheless, 4). . not possible to assign unsequen assign to possible not nclude spider species fromIndi species spider nclude r w tnaie pce level species tentative two er A. minax ies GL-01a, GL-01b, GL-01c and and GL-01c GL-01b, GL-01a, ies Nesticella mogera from Madagascar (Cheng & Kuntner 2014) and

enced individuals equal to the the to equal individuals enced t with our taxonomic assignment iis Smhgahde Tetra (Symphognathidae, milies , Parasteatoda ), and a further two from published studies th of 18 for possible was level Zag L 21) a pce of species a 2013), Li & (Zhang , Patu , 74 < r < 0.75; 0.013 < p < p < 0.013 0.75; < r < 74 Prasonicella For each cryptic specieseachcryptic For ihy n significantly and highly an Oceanislands. Six of tudy ( tudy e presumed biological presumed e ced individuals to the the to individuals ced sinet, n the and assignments, GL-47a, GL47b. We We GL47b. GL-47a, n a given sampling given a n yielded matches of matches yielded rsn, ie the given prising, ilgcl species biological ts for each GL are cs rm seven from nces Tetragnatha blemmidae and and blemmidae rprin of proportion ut until now e 57 inferred 57 e , fu being four , Tetrablema ographical icantly sp., sp., he , This article isprotected Allrights bycopyright. reserved. i while Additionally, 4). (Fig. plots within estimates richness Accepted3 of 57 were comprisedpresum of number onlythe in increase of juveniles), By not only restricting this it While individuals. 5558 to resulted 1604 from size sample our in our subst sampling to only adult forms we were ab defining PUsisadvocated. contain as PUs, individual of definition the to insensitive individuals is approach p worth is It PUs. from species biological presumed extrapolate from more thanrelatedness onesequence DNA a of use species.Our much. how by identify to is challenge Thus ofbiological a PUso conservaoverestimate an are PUs our species, overcomes that know we Réunion from spiders but in taxonomi the that absence out pointing 2004), of this a robust a prioripreviousl challenge,has morphospecies to PUs from extrapolating of danger tax variatio morphological and species, within sam variation gender and the in providing together grouped represent PUs our part, most were the for Thus, species. biological above), details see (PU-93, anPU objecindiv species, biological one than more Articlecomprise to inferred is to the same biological species. Withpart they the do represent exception collections of individuals of that are GL-01 ulti and clear that biological our presumed PUs 57 represent represent to inferred a poor lineages, approximationgenetic of biologicalth were which 116, for obtained was data sequence DNA which for par 151 to assigned were that spiders 5558 yielded sampling Our Discussion 3). species using adults only,usually but not appro between each correlated for PUs cases). all in richness 0.200 < < p 0.033 0.67; < and< r (0.44 correlations species species Estimating cases). usin all between in 0.014 PUs and species using all individual

ed biological species sampled a sampled species biological ed acrc i cmrmsd In compromised. is accuracy c Betadiversity values were mately inferred to belong to inferred mately t is perhaps unsurprising perhaps is t partitions of life history life of partitions onomic framework, the framework, onomic iduals from all but one but all from iduals antially higher species higher antially resulted in a marginal marginal a in resulted GL-53, each of which long as they do not species, for the most the for species, ointing out that this that out ointing g adults only (Table (Table only adults g wti tee The these. within n step to identify the identify to step cross all plots (only plots all cross pce. hl i is it While species. y been noted (Krell noted been y s or the latter and en collapsed to 54 to collapsed en ach worsened all all worsened ach ataxonomic units, ataxonomic tive approach to ie rtro to criterion tive f the number of number the f h cs o the of case the le to increase to le presumed e This article isprotected Allrights bycopyright. reserved. am structuring geographic exhibits also GL-01b within evolutionary variation of Accepted period substantial a over Fournaise la de Piton from haplotypes mtDNA of movement of absence an indicating 3), divergence coalescent deep by marked is GL-01b within variation the values with consistent is This 3). between (Fig. dispersal to barrier a be plots 3 andFou 4la de Piton from extend that fields (Fig. lava open extensive the 4). Infurthe plots three additionthe to restricted is GL-01c while Fournaise, to thisGL-01b vicariant. vicageographically includes subcanopy habitat. consi techniques, sampling other individua GL-01a sequenced 59 the of 61% litter, leaf and traps 92% While were GL-01c or divergences. GL-01b either to mtDNA assigned individuals sequenced with consistent are differ GL-01, data of sampling case the In threshold. a as species congeneric Robinson based species, cryptic for evidence revealed plots biodiversity enable protocol sampling our and lineages mtDNA divergent with co-segregate Articlevariation of sources however, infer of support merit the among about debated been for has Much the species. speciescryptic existence variat genetic of structuring geographic the as of such variation, based reveal cryptic also to potential the has it that species is on species biological mtDNA isdelim the improv into step DNA a incorporating of consequence useful A divergence speciesand Cryptic values (e.g.that canshapearthropod communitystructure. Taylorelucidating for critical prove may and estimates diversity beta wil juveniles include to & sampling expanding H that indicating 3), estimates samples adults only from derived from estimates diversity adult beta that and juvenile samples, the correlation is g hl nt ifrnitd y clgcl apig G-1 ad GL- and GL-01b sampling, ecological by differentiated not While et al. ’s (2009) average of 6.77% K2P corrected divergence between clo between divergence corrected K2P 6.77% of average (2009) ’s cryptic diversity was sampled in the seven plots stent with a broader ecological broader a with stent

s this to be explored. Sampling tews undetectable otherwise r south, suggesting that that suggesting south, r ecological mechanisms ecological rnaise to the coast may coast the to rnaise high βtotal and βrich o wti seis or species, within ion on the application of application the on ls were captured with captured were ls niche for GL-01a that in pten mtDNA pattern, riant enerally weak (Table (Table weak enerally yed oe realistic more yield l north of Piton de la s of up to 4.8% (Fig. (Kress presumed of itation ed when additional additional when ed r creae with correlated are ne i ecological in ences time. The mtDNA captured in pitfall in captured north to south of of south to north ig boundaries ring cos h ten the across n te seven the ong etal. arris 2012), 2012), arris of the 61 01c are are 01c 2015), 2015), sest This article isprotected Allrights bycopyright. reserved. Accepted beofint that may GL-17), GL-12, add provides protocol our divergenceTablegenetic(e.g. valueshigh withsignalling GLs from data sequence linea mtDNA diversified The of species. identification the for interest management be to unlikely is biodiversity diversity cryptic undetected Such diversity. to protocol number our of ability analyses The th noted be should it However, mayabove. cases the by demonstrated still be at an underestimate, the community with thetwolineages. phen divergent indicating GL-53b), for 38% vs GL-53a for adults to due adult different very their to by provided scale, is further species biological undetected butHowever, support for the hypothesis that these divergent lineag may on based differentiation ecological discernable any present not be wit of lineages two Article con The exceedingproposedthe 6.77% K2P species. cryptic of inferences for support that was stages life juvenile j including or from adult outcome unexpected only of solely comprised PUs associate to us enabled with stages life immature associate to routine becoming is It (Kress this of confirmation comprehensive population genetic analysis including nuclear mar but limitation, dispersal and species two (Croucher species di sex-biased for evidence is there as species, single a within GL-01b and GL-01c couldwithin be argued 3) this to (Fig. be the plots taxon.product sampling northern The of female geographi d etal.

05, n te noprto o mDA eunig no u prot our into sequencing mtDNA of incorporation the and 2015), etal. 2011; Gillespie & Oxford 1998). Thus our data is indicative of indicative is data our Thus 1998). Oxford & Gillespie 2011; erest for subsequent more deta cal patterns of mtDNA structure mtDNA of patterns cal spotn a yohss f di of hypothesis a supporting , divergenceRobins of threshold eel rsmd rpi species cryptic presumed reveal 2: GL-01b, GL-03, GL-11, GL-03, GL-01b, 2: spersal in some spider some in spersal iled investigation. ueie ais (80% ratios juvenile sampling techniques. sampling es represent different kers. kers. among and within and among on at inferred species inferred at vnl frs An forms. uvenile they can provide ispersal limitation ispersal lge associated ologies ges and endemic endemic and ges osqeta for consequential pra limitation spersal i G-3 while GL-53, hin eurs more a requires their adult forms rpi species cryptic et al. et evto and servation d au by value ed iest is diversity (2009), do (2009), ocol ocol

This article isprotected Allrights bycopyright. reserved. sequenc with assistance researchAccepted for Ravagni Sara thank also We wasHoareau. supportedDanfl Samuel Fournel, Jacques Sadeyen, Joëlle sorting: specimen by the ERA-Net Acknowledgements. Net-Biome researchhabitats by adapting thefield sa other framewto extended be can protocol identification molecular and co our that expect we Thus, assessments. ultimately biodiversity arthropod and accuracy, the impedes otherwise that impediment wi reduces sequencing sorting mtDNA with boundaries sample species of parataxonomicestimation and sampling field standardised i expertise taxonomic and pools species regional on information (Linneanreducethe possibleto is it that demonstrate to able been have shortfall)By applying the protocols developed in thisConclusions study of Réunion sp to quantify samples. for comparing can history evolutionary which through biodive step first the providing produce that arthropod processes the investigate to Articleopportunities complexityspecies presentdiver Réunio biological the accurately describe to withinimportant obviously to endemic a region. species obs.). pers. undescribed Casquet, More (J. be importantly richnessMascarenes to likely are perhaps, which of i on previously recorded been andnot had that families to belonging Réunion) of area protocols In this study, using a combination of standardizedas spiders, hold ecologicalgreat potential to a disclosetaxonomi strong new a species with taxa recordtarget on focused when inventories, is not towe one the like inventories, plot-based of purpose primary The simply dive New speciesrecords,andthe improve taxonom

applied tosamples collected overa total of 2.5 ha(i.e. 1/100

, e ee be to able were we We thank for following for assistance with field sampling and mpling protocol accordingly. mpling protocolaccordingly. rsity of Réunion spiderfauna ofRéunion rsity

ic knowledge within a region. H dniy 1 e seis eod, ie f which of five records, species new 11 identify Improving coverage of taxonomic diversity is is diversity taxonomic of coverage Improving

taxonomic impediment taxonomic s and undescribed taxa. taxa. undescribed and s nd molecular sampling sity and the ecological be taken into account into taken be arthropod groups and groups arthropod describe in this study, ider communities we communities ider the island, and many and island, the s lacking. Combining lacking. s ous, and Dominique and ous, mbined field survey field mbined t also provides new provides also t c impediment, such impediment, c st patterns rsity h biodiversity the diversity when when diversity ,000 of the total total the of ,000 h vldt, of validity, the eiig This editing. e wvr such owever, r, financed ork, o t the to or n h objective th , by Cardoso P, Scharff N, Gaspar C Gaspar N, Scharff P, Cardoso This article isprotected Allrights bycopyright. reserved. AcceptedCardoso P, Rigal C F, Gaspar Carvalho S, Henriques P, Cardoso JC(2015) LC Pereira C, GasparBAT P, Cardoso - BiodiversityAsses Cardoso Standardization (2009) P Cardoso P, Erwin TL,J Amaral C, Aguiar PAV, Borges Borges PAV,Benson DA, Clark C, New Karsch-Mizrachi biodi Quantifying (2000) R TR Pyle I SE, Miller V, (2011) Novotny Y, Basset TheP Cuenoud L, Cizek Y, Basset sevenArticle impediBall SL, Hebert PDN, Burian SK, Webb JM (2005) Biological ident References The NationalParkofRéunionpro Reunion 2.02) Orga andOSU Réunion the provided logistical from support for S20141203_002597 s Autonomo grant MAGRAMA Spanish co- and CGL2013-42589-P, FEDER, grant Parques MINECO Spanish by supported also was Nacionales. DGADD/PE/201 contract Réunion La de Université (DS), activities The n°11-EBIM-001-01grant (CT); ANR-NETBIOME field FCT- Portuguese FEDER; research by cofinanced a (PO) SE-12/02 grants ACIISI Government Island stationCanary through: of Mare forest. forest. M a in study case a sampling: semi-quantitative using (Araneae) 236. akg fr h maueet n etmto o apa n bt ta beta and alpha of estimation and measurement diversity.functionalphylogenetic and the for package scrubland. Mediterranean a in Conservation spiders of assessment composition inMediterranean cork oak forests. 144 them. overcome to how and conservation invertebrate case ofIberian spiders. arthropods. epigean soil Conservation of sampling standardized using 43 Guine New Papua in photography Guyana. digital and parataxonomists with Science barcodes. Society Benthological DNA using (Ephemeroptera) , D30-D35. D30-D35. , , 2647-2655. 2647-2655. , Insect Conservation and Diversity

338 Bioscience , 1481-1484. 1481-1484. ,

14 13 , 2029-2060. 2029-2060. , , 45-55. 45-55. ,

24 , 899-908. 899-908. , , etal. , et al. Biodiversity and Conservation , 508-524. 508-524. , , etal. , etal. vided thenecessaryauthorisati n otmzto o arthropod of optimization and , etal. (2012) Arthropod Diversity in aTropical Forest. (2008b) Rapid biodiversity assessment of spiders of assessment biodiversity Rapid (2008b) (2005) Ranking protected areas in the Azores the in areas protected Ranking (2005) (2008a) Assessing spider species richness and and richness species spider Assessing (2008a) , et al. (2009) Species richness and composition composition and richness Species (2009) NETBIOME grant 0003/2011 (PB); French French (PB); grant 0003/2011 NETBIOME (2015) GenBank. GenBank. (2015) Methods inEcology and Evolution

1 , 71-84. 71-84. , Région Réunion council for research for council Réunion Région Journal of the North American Acta Oecologica Acta

18 Biological Conservation , 3949-3962. 3949-3962. , Nucleic AcidsResearch Nucleic ons forsampling. ifications of mayflies of ifications ampling on Réunion. on ampling est: experience versity: mn Tos a R an Tools, sment nd SE-12/04 (BE), (BE), SE-12/04 nd Journal of Insect 08 (S. BCE (DS). 20585 netre - the - inventories Biodiversity and Longue (P.O.E. (P.O.E. Longue

33 editerranean , 114-127. 114-127. , iacd by financed et in ments

6 nismo and a , 232- , xon,

Glowska E, Dragun-Damian A, Brida L, Dabert J, Dabert M (2014) M Dabert J, Dabert L, Brida A, Dragun-Damian E, Glowska polymorph color the on Selection (1998) GS Oxford RG, Gillespie TL Erwin WJ, Kress CL, Staines EK, prim Kuprewicz C, DNA Garcia-Robledo (1994) R Vrijenhoek R, Lutz W, Hoeh M, Black O, Folmer This article isprotected Allrights bycopyright. reserved. AcceptedEmerson BC, Cicconardi F, Fanciulli PP, Shaw PJA (2011) Phyloge Duelli P,Duelli P (1997) Biodiversityevaluation inagricultural landsca Obrist MK, SchmatzCroucher PJP, OxfordGS, DR Gillespie (1999) RG (2011)Population structu BiodiversityCicconardi F,Article Nardi F, Emerson BC, b Frati the F, Fanciulli Collembola, PP (2010) (2013) BC Emerson PP, Fanciulli F, Cicconardi evaluation an nondirectional suggests Phylogeny (2014) M Kuntner RC, Cheng species Chao mammal A new (1984) of Discoveries Nonparametric (2009) PR Ehrlich G, estimationCeballos of the number boilin without Chelex (2012) RG of Gillespie C, classes Thebaud J, Casquet Casquet J, Bourgeois relative the Determining (2012) P Gomes P, YXC, Cardoso JC, Carvalho Cruaud C eae iopim n yigpii mts Atntihd: Prost (Actinotrichida: mites syringophilid in dimorphism female Evolution spiders:fromhappy-faceevidence C Biological Journal of the (Coleoptera: Linnean Society beetle rolled-leaf Cephaloleia a in associations larva and egg novel detect to library barcode DNA comprehensive 299. invertebrates. metazoan c mitochondrial of amplification Philosophical Transactions of theRoyal Society B phylobetadiversity 33-64. insects. above-ground landscapes: different scales. and theLinnean Society molecularspider area reveal markers mitochondrial and nuclear landscape: patchy (He invertebrates analysis forest 386-400. of persistence basin. Mediterranean North-Western the in Collembola) long-term and divisions of biological22 concept, and the underestimation of global species richness. com 2872. spiders. argiopine in dimorphism size sexual of evolution Scandinavian Journal of Statistics, Theory and Applications services. ecosystem National Academy of Sciences of theUnited States of America and conservation for implications stored spiders. ampli stable obtain to technique spiders. Mascarene and Hawaiian of Biogeography comparison a islands: beta generating in differences estimates. richness species and replacement , 5382-5396. 5382-5396. , Theridion californicum Theridion

52 Global Ecology and Biogeography , 775-783. 775-783. ,

42 Molecular Ecology Resources

104 Agriculture, Ecosystems &Environment , 39-50. 39-50. , , 600-620. 600-620. , Molecular Marine Biology and Biotechnology (Araneae: Theridiidae). (Araneae: , et al. tcrm c xds sbnt f I subunit oxidase c ytochrome ibeN fo sal mut of amounts small from fiableDNA genetic structure and tempora Agriculture, Ecosystems& Environment (2015) Community assembly on remote remote on assembly Community (2015)

110

21 , 189-198. 189-198. , 12 , 760-771. 760-771. , , 136-141. 136-141. ,

336 , 2391-2404. 2391-2404. ,

Biological Journal of the pes: an approach at two 62 Deep phylogeographic Deep Molecular Ecology Molecular

11 DNA barcodesDNA reveal , 81-91. 81-91. , re and dispersal in a ny, phylogeography,ny, Evolution g, a rapid and easy Proceedings of the

106 , 265-270. 265-270. , Molecular Ecology Molecular oe o species of roles n population. a in ooia species iological s i Haiaiian in ism (2013) Using a a Using (2013) hrysomelidae). n agricultural in , 3841-3846. 3841-3846. , l fluctuations. fluctuations. l effectsin the rom diverse diverse rom isometric d l host plant host l and their Journal of

68 -diversity ethanol- munities.

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This article isprotected Allrights bycopyright. reserved. AcceptedRaso L, Sint D, Rief A, Kaufmannunravel dimorphism sexual Strong (2015) S Tarcz L, R, Przybylowicz Traugott (Diptera fly black of Association M (2014) K (2014) Wongpakam P, Pramual Molecularsurr as morphospecies Invertebrate (1996b) AJ Beattie I, Oliver inverte cost-effective a Designing (1996a) AJ Beattie I, Oliver R Kindt FG, Blanchet J, Oksanen SE Miller Y, Basset V, Novotny (1942) E Mayr ut Kuntner the on M, review Agnarsson critical A (2012) I (2011) RA Francis Phylogeography PK, Krishnamurthy of a successful ae Kress WJ,Article García-Robledo C, Uriarte M, Erikson stud biodiversity DL in (2015) taxonomy vs. DNA Parataxonomy ba (2004) F-T Krell Jusoh WFA,tro in assemblages termite Sampling (2000) P Eggleton DT, Jones HashimJi NR, Y, Ashton SL, Ball L, Pedley Saaksjarvi A, Cywinska S PDN, Hebert identif the enables barcoding DNA (2009) A Hausmann IE, MM, Gossner Adam NA,Goldstein PZ, DeSalle R (2011) Integrating DNA Wahlberg barcode data and N (2014) communities. communities. classificati Pseudothyretes adult of and understanding juvenile better 173 a Arctiinae). linyphiid Erebidae: (Lepidoptera: towards - and theridiid stages usingDNAbarcode. spiders in alpine case study. gl 607. assessm rapid for methods package version 2.0. insects inatropical forest. C Harvard UniversityPress, 11 golden orb spider 1901-1919. conservation. biodiversity in barcoding evolution, and conservation. 812. sorting. ‘morphospecies’ of applicability Lampyr DNA barcodes. (Coleoptera: fireflies mangrove Malaysian of delineation 203. a biodiversity rapid a biodiversity via metabarcoding. barcodes. DNA through geome (Lepidoptera: oak alien and Mitteilungen Muenchener Entomologischen Gesellschaf onnative feeding Ciconichenophilus caterpillars and Determination, discovery, and description. phoeniconaias Stibarokris phoeniconaias areconspecific. Cheyletoidea): , 119. 119. , , 22-54. 22-54. , Systematics and the origin of species from theviewpoint of azoologist. Conservation Biology PLoS ONE Coleopterists Bulletin Coleopterists , et al. Nephila Nephila ssessment protocol. protocol. ssessment (2013) Reliable, verifiable, and efficient monitoring of of monitoring efficient and verifiable, Reliable, (2013)

Proceedings of the Royal Society of London B 9(7) , etal. , et al. Nature Journal of Asia-Pacific Entomology on Indian Ocean islands. Ocean Indian on ambridge, Massachusetts. ent of biodiversity. of ent Trends in Ecology & Evolution , e101755. e101755. , Folia Parasitologica ear J (03 Booia i Biological (2003) JR DeWaard (2015) Vegan: Community Ecology Package. R R Package. Ecology Community Vegan: (2015)

Ecology Letters (2002) Low host specificity of herbivorous herbivorous of specificity host Low (2002) 10

416 , 99-109. 99-109. ,

68 Zoological Journal of theLinnean Society , 841-844. 841-844. , , 703-711. 703-711. , BiodiversityConservation and Journal ofApplied Ecology Bioessays Biodiversity and Conservation

16 Ecological Applications Ecological , 1245-1257. 1245-1257. , 61

33 BMC Evolutionary Biology

, 272-276. 99 , 135-147. 135-147. , rt sre: ts of test a survey: brate , 135-140. 135-140. ,

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This article isprotected Allrights bycopyright. reserved. Acceptedresults and commented on the manuscript. B.C.E analyse ofGLs. assignment conducted alpha andbeta diversi led J.C. sorting. and sample th collecting, sample sorting and classification of samples to PU. Author contributions: (doi:10.5061/dryad.8g5d2). accessibilitystatement: Data recent troglobites: your lineage, Ancient (2013) S Li Y, Zhang Yu DW, Ji YQ,Taylor Emerson MEG (2013) S HR, Kumar A, Filipski Harris D, Peterson BC G, Article Stecher WE K, Tamura (2012) An emergent DM Richardson science M, Rouget D, Strasberg on optim the an of brink Development (2006) P of Sierwald ML, Draney BA, Snyder Scheffers BR, Joppa LN, PimmSL, Laurance WF (2012) Whatwe kno Scharff N, Coddington JA, GriswoldRobinson EA, BlagoevCe, GA, Hebert PDN, Hormiga Adamowicz SJ (2009) Prospe G, De Place Bjor Riedel A, Sagata K,Suhardjono Y Ratnasingham S, Hebert PDN (2007) BOLD: The Barcodeof Life Dat Nesticella Ecology and Evolution biomonitoring. and assessment biodiversity rapid for arthropods 377-388. barcoding. DNA of years 8 past the of review 30 prio 6.0. conservation Version Analysis Genetics Evolutionary broad-scale identifying for basis a Biodiversity and Conservation as Ocean) Is (Mascarene Island Réunion La on transformation and diversity 288. (Diplopoda). millipedes for protocol about Earth’smissi European northern forest. a in richness species spider Estimating quit? barcoding to identify spiders inspeciesrich genera. in Zoology the fast track - towards more sustainabilityMolecular Ecology Notes in biodiversity re , 2725-2729. 2725-2729. , Journal of Arachnology spiders.

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7 R, Tänzler R, Balke M (2013) In M(2013) Balke TänzlerR, R, , 355-364. 355-364. ,

14 31 21) idvriy op Mtbroig of Metabarcoding soup: Biodiversity (2012) , 3015-3032. 3015-3032. , , 246-273. 246-273. , Trends inEcology &Evolution , etal. Journal of Insect Conservation .O. designed the J.C.study. le 20) n seset f habitat of assessment An (2005)

13 Molecular Biology and Evolution Molecular Ecology Resources Ecology Molecular , 183. 183. , N.M. assisted J.C. withfieldwork editing and alignment. P.C. andalignment. P.C. editing J.C. undertook taxonomic the and all authors discussed the ZooKeys colonization of caves by caves of colonization tegrative taxonomy on n P (2003) When to to When (2003) P n

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within each GL. within that GL.K2Pindicates the of comprise each maximum number PUs assigned toeach number of the individuals is Indiv assignment. and (PU) andmtDNA sequencing, unit parataxonomic to assignment This article isprotected Allrights bycopyright. reserved. Table 2. Réunion. Table 1. GL b X X X X X X X X X X 1b 1a X X X X X X X X X 1cX X X X X X X X LcsdeGn p 20 6.0 2 200 Lycosidae sp. Gen. X X X X X X X X 4 X X X X3 X X2 X X X Lycosidae X X Gen. sp. 2 1 NA Accepted5 X Article lt dl ae dl eae ueie Total Juvenile Adultfemale Adult male Plot 0 1 16 3 765 539 106 110 10 8 6 32 492 425 517 342 372 280 448 402 283 63 308 64 55 42 82 77 72 51 5 42 4 61 3 2 1 16 4 68 935 609 517 648 524 392 372 388 140 110 89 78 146 104 55 9 57 8 7 6 2 4 6 8 10 9 8 7 6 5 4 3 2 1 Spider samples obtained across Genetic lineages (GL) inferred fo lineages inferred (GL) Genetic Plot Plot ten lowland rainforest plots on r adultand spider juvenile sa Ledouxia allauadi allauadi Ledouxia Prepotelus curtis Prepotelus curtis Trachelas Trachelas Trachelas aooi D ni. U K2P PUs Indiv. ID Taxonomic ()s. 0 7 4.8 7 109 (?)sp. ()s. 5 7 2.0 7 155 (?)sp. ()s. 2 0.2 7 32 (?)sp. GL and PU GLandPU theindicates K2P genetic divergence their taxonomic their mples after sample the island of the island 2 3.7 1 12 1 1 NA NA 1 1

This article isprotected Allrights bycopyright. reserved. 8 X X X X iyhia e.s. 2 4.1 0.9 3 2 X 62 X 3 X X sp. Gen. Linyphiidae X sp. Gen. Linyphiidae X X X X X X X X X X 30 X 29 X X X X 28 X X X X 27 X X X 26 X X X X 25 X X X X X X X X 24 X X X X X X X 23 X X 22 0 X X X X X X X X X X X X X X X X X X 21 X 20 19 18 X X X 1 X X X hrdia e.s. 9 5.6 1 69 sp. Gen. Theridiidae X X X X X X X X X X X X X X 12 11 4 X hlia e.s. 1 0.0 1 3 X sp. Gen. Pholcidae X X X X X X X X X X X X X X X X X X X X X 17 X X X 16 X 15 14 13 Theridiidae X Gen. sp. 1 1 NA 10 X X X X X X X X X X X X X X X X X X X X X X X X X 9 X X 8 7 X Accepted Article X X X X X X X X X X 6 Cheiracanthium insulare Lophostica mauritiana Lophostica Prepotelus lanceolatus Prepotelus lanceolatus Pseudemathis trifida Alistra mendenai Afroneta longipalpis Clubiona nemorum nemorum Clubiona Miagrammopes Xeropheaus Nesticella mogera Nesticella Theridiosoma Helsdingenia Helsdingenia Lophostica nova nova Lophostica Tetragnatha Zosis geniculata Xerophaeus Xerophaeus Tetrablema Ulwembua Tylorida Hahnia (?) sp. 2 1 NA NA 1 2 sp. (?) p 6 0.9 2 6 sp. s. 7 3 4.3 3 374 sp. s. 8 8.0 2 38 sp. sp. 8 1 NA NA 1 8 sp. p 4 1.1 2 4 sp. s. 0 2 5.3 2 204 sp. s. 9 2.6 1 29 sp. p 3 0.2 1 3 sp. p 1 1 0.3 1 17 sp. (?) 2 1 2.7 2.7 1 2 (?) 7 0.7 3 27 5 0.5 1 65 1042 2 0.8 0.8 2 1042 6 2 1.1 2 1.1 263 8 387 2 1 1.3 1 1.6 228 4 471 9 0.2 2 19 2 0.2 2 6 This article isprotected Allrights bycopyright. reserved. 7 X X X X X X X X X X 47b 7 X X X X X X X X X X 47a 4 X X X X X X X X 0.0 X X X X 1 X X X X 10 X X X X 54 X X sp. Gen. Theridiidae X X X X 53 X X X X X X X X X 52 X X X X X X X X X X X51 X X X X X X X X50 X X X X X X X X 49 X X 48 46 45 X X X X 4 X X X X X X X X X X X X X X 44 X X X X X X X X X X X X X X X X X 33 X X X X X 32 X X 31 Accepted 0.2 1 9 X sp. Gen. Clubionidae X X X X X X X 43 X X X 42 X X X X Article 41 X X X X 40 X X 39 X 38 X X X X X 37 X X X X X X X 36 X X X X X X 35 X X 34 Arachnura scorpionoides Nephilengys borbonica borbonica Nephilengys Argyrodes borbonicus Cyrtophora citricola Neoscona triangula triangula Neoscona Thwaitesia Thwaitesia Theridiosoma Nephila inaurata Argyrodes minax Parasteatoda Theridion Theridion Theridion Theridion Platnicka Platnicka Cyrtarachne Prasonicella Prasonicella Rhomphaea Poltys kochi kochi Poltys Argyrodes Leucauge Leucauge Patu Chrysso Chrysso (?) sp. 4 1 0.3 0.3 1 4 sp. (?) ? p 9 0.2 1 9 (?) sp. ? p 1 NA 1 1 (?) sp. ? p 1 2 3.6 2 16 (?) sp. ? p 11 2.7 7 121 (?) sp. s. 2 6 1.1 0.3 6 123 6 54 sp. sp. ? p 6 4 1.0 4 65 sp. (?) sp. p 18 0.9 4 128 sp. s. 5 0.2 3 35 sp. ? 1 2 0.5 2 18 (?) p 8 3 1.7 3 84 sp. p 6 1.3 2 6 sp. s. 1 3 1.4 3 210 sp. s. 4 1 2.7 1 343 sp.

3 1 0.8 1 239 0 1.1 1 10 3 0.9 1 33 5 2 0.3 2 1.1 6 72 8 0.6 1 0.2 28 1 26 1 0.3 1 7 differences in species richness. richness. in species differences βrepl=beta diversit diversity, parataxonomic units parataxonomic (PUs)units andsp Table 3. This article isprotected Allrights bycopyright. reserved. sample ID. T groupings. re coloured as differently range, its within variation sequence mtDNA of structuring sampled fr sampled were GL-01b from of locations individuals the All to change. the three mutational northof of Piton locations de D la Fournaise, of supportvalBootstrap evolution. model 2-parameter Kimura the while to using tree the all likelihood indimaximum south. GL-01bFigure 3. also exhi eastern coastofRéunion. Figure 2. species from largecollect field and classification parataxonomic Figure 1. Figure legends (from *p<0.05, Mantel **p<0.01,*** tests): Significance Accepted Article (adults) (all) xSpecies Species PUs x Species (adults) PUs x Species (all) Spearman correlations between beta diversity values calculated Locations of 50m x 50m sampling plots within lowland rainfores MtDNA sequence variation within GL-01b and GL-01c, presented a oprsn ttl βrepl βtotal Comparison Flow chart summarising themain steps for the application of

y due to species replacement, β y duetospecies

ions ofarthropod samples.See ecies (including juveniles a or ues above 80 are shown. The sca The shown.are80 above ues tN sqecn fr h infer the for sequencing mtDNA 0.490** 0.093 0.487** xn aes niae sampling indicate labels axon

0.582*** 0.243 0.528** p <0.001

rich =beta duerich to diversity

dults only). βtotal =totalbeta main text for details. 0.444** 0.370* 0.370 vidualsof GL-01c were le bar represents 1% βrich using lt ubr and number plot biological of ence rsne b the by presented is geographical bits

om the seven A sequence NA t along the along t a s This article isprotected Allrights bycopyright. reserved. Accepted Articlejuveniles included (D). plot sampling neighbouring between Pa text). (see resamples 1000 among percentiles 0.975 and 0.025 limits Confidence included. were forms adult only when species ( and; included,juveniles with species biological presumed (B) Figure 4. Species richness estimated using rarefaction across all plots f plots all across rarefaction using estimated richness Species s estimated for presumed biol presumed for estimated s C) presumed biological presumed C) irwise beta diversityirwisebeta ogical species with species ogical acltd s the as calculated r () PUs; (A) or:

This article isprotected Allrights bycopyright. reserved. Accepted Article