Posted on Authorea 7 May 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158888143.31089837 | This a preprint and has not been peer reviewed. Data may be preliminary. akfrsm pce n oreo ueosseismsdnictos hsi seilytu o large for true especially is This many misidentifications. species for numerous found of difficult be that a source still traits only a shells shell can and on plastic shells based species on surveys where turns some based occurrences. 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Venerida. conservation give large the of Results a for species at known. one used performed currently and was if those Unionida Metabarcoding provide with the can basins. compared for major then one all bivalves. over are primers, freshwater spread for of sampling localities methods sets 350 standardized new over with two with France, metabarcoding species using in rare eDNA and mainly using used. species, performed invasive dataset of was perspectives biggest increasingly detection the Sampling promising the early all the being monitoring, present of authors, long-term one here is different for Nevertheless, We protocols, by overview. and and detection. used global methods a are species the for of methods together freshwater standardization different pooled the of easily Because is be plan. surveys cannot eDNA sampling efficient with limited produced allows a data have (eDNA) generally studies DNA most environmental However, of analysis The Abstract 2020 7, May 7 6 5 4 3 2 1 Dejean Vigneron Thibault Jean Pauline Pri´eVincent conservation and of distribution point species eDNA on an view bivalves: freshwater of monitoring Large-scale Spygen Rhˆone du Nationale Compagnie Biodiversit´e la Fran¸cais de Office SPYGEN Fourier Universit´e Joseph Porto of University Naturelle d’Histoire National Museum 7 1 4 aulLopes-Lima Manuel , mleBreugnot Emilie , 5 lrn Lamand Florent , 5 St´ephanie Couprie , 2 ireTaberlet Pierre , 5 lve Gargominy Olivier , 1 3 5 lc Valentini Alice , Ga¨elle Jardin , 1 ahe Rocle Mathieu , 5 ioa Roset Nicolas , 4 ioa Poulet Nicolas , 6 n Tony and , 5 , 5 , Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. rutltefitro h asl a opeeystrtd h oueo lee ae aidacrigto according varied water filtered of volume minutes The 30 saturated. about The for completely watersheds. filtered was was French capsule 0.45 water the main The a of the sample. (B¨urkle GmbH), filter October, each of the Sampler and for until most Vampire tubing May or sterile cover between a disposable to months, using and (Spygen), summer locations performed capsule in under-sampled was mainly and filtration 2019, species water and rare 2015 on between focusing out carried was Sampling results. the of Sampling significance 2.1 the understand properly to necessary are described are methods policies. 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European largest the of and incognita one 2019, from and is known Rhˆone 2018 not River was The in it individuals where live downstream, few range particularly a its mainstream, Rhˆone drainage, data. of River the Loire of observation recent the of quarter the of sectors by south north-eastern several watersheds, presence in the Charente its (ii) and throughout confirmed Dordogne surveys species the field of the upstream where of (i) presence presence collected its Ard`eche Data the revealed Dordogne and River. area Rhˆoneand wetland the confirmed catchment Camargue the from analysis Loire the of collections in the eDNA research downstream museum of intensive by records in south despite known historical updated specimens not been and few not was data) a largely have species records unpublished for have the recent vouchers, (except populations hand, many doubtful coast other generally its Atlantic are with the and the Although there On Saˆone, on 3a. Seine, 2014), range. area Loire, Damme, Figure its catchment Van the in throughout Kebap¸cı, & shown populations of (Lopes-Lima, living as century basins of France last the of the in half over widespread declined north-eastern is the Mussel in River speaking Thick-shelled Thick-shelled the The as such II). species, conservation-concerned for Mussel eDNA with River collected was been data has valuable data Outstanding and expected, were species they Out-of-range where 3.2.1 detected species. and not known introduced were noticeably poorly range, Others about distribution only. 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RESULTS before ancient collected 3 as data here considered (i.e. were data data ancient Shell-only between ones. made was distinction a 1844), Mussel INPN Pearl Mussel the Freshwater well Orb Giant on as Nut the available the collection concern, are museum conservation data in of bivalve e.g. species academic), freshwater auricularius focused as 900 validation, For well 32 scientific 2020. as About in (grey (3) programs. database (2) literature various data; France; the through in in the observations provided as situated of data is integration includes observation thus land. the INPN the on after recorded and referenced dated not is 2019) is not species al., species the is et marine e.g. conformity, observation (Gargominy (1) levels register e.g. three consistency, taxonomic on French data various this by the provided of the data in validation and the for programs national habitats ensure national needed natural INPN many and partners. from species, data information comes animal information synthetic and of The (plant 2016). of dissemination Pri´e Cucherat, heritage and restitution natural see the French heritage, the the and geological way to and strategies relatied standardized conservation reports a international is of and in development Heritage) NAtural ensure the the to of expertise, Inventory implemented INPN National by naturel, provided system Patrimoine data du metabarcoding, a national eDNA bivalves (Inventaire freshwater INPN with used. collected were data the compare to order In o h aaooit ti ag,fs-oig ep ubd aiae ie hti eyhard very is that river navigated turbid, deep, fast-flowing, large, a is It malacologist. the for Sege,19) h erse ie Mussel River Depressed the 1793), (Spengler, .crassus U. paru rivicola Sphaerium ctgrzdE yteIC n itdi h uoenhbttdrcieannex directive habitat European the in listed and IUCN the by EN (categorized https://inpn.mnhn.fr/accueil/presentation-inpn?lg=en Lmrk 88 n h ihmObMussel Orb Witham the and 1818) (Lamarck, 4 suaoot complanata Pseudanodonta n agmn,L´eonard, Gargominy, and , .solidum S. (Rossm slr 1835), ¨ assler, (Normand, Pseudunio terra Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. o urnl osdrdo osraincnen h erse ie Mussel are River which species Depressed some The of concern. status conservation conservation worrying of the considered out currently stressed France Meuse, not in the sampling in large-scale data Our new species Rhˆone with River. lower endangered range, the and (Mouthon current along Rare 1989 its all from Ard`eche,3.2.3 Saˆone, and extended date the the has distribution Seine, eDNA its the on the of data of upstream first Analysis the 2012). Ta¨ır although Abacci, 2017), & Forcellini, & (Mouthon recently invasive Peaclam for Ridgedbeak sampling eDNA The of de monitoring. value Atlantic the the Canal and from highlights detection the samples results our early via These of species’ eDNA. None (probably surveys. Mussel Montpellier, watershed traditional Quagga in with already contained Seine observed River has drainages been the (Lez never species into had rivers the it coastal and where first that scattered Bourgogne), Bas-Rhˆone-Languedoc Mediterranean Canal), shows was with available the the Rivers, data it previously into Moselle via eDNA to when and introduced probably 2017). Meuse According also 2010 Fruget, the (Pri´e 2011). being & Rhˆone since only Rivers considerably, Beisel, colonized France and have spread & Rhine in to Vaate the thought known de was in it been (Bij data 3b, basins only Figure Moselle in has shown and It data Meuse negligence. the or in confusion detected species to mussel this due 2019). Zebra that Geist, unnoticed widespread known & the now Huber to is 2017; shape it al., Indeed, Mussel et protection (Donrovich Mussel. Quagga implement apparently species to Pond The native urgent and Chinese be to high, the would threat is it of significant basins, pressure a colonization these absent represents observation the all still For eDNA avoid probably basins. is to the Charente it measures where and that Seine watersheds, show Adour, analyses by Dordogne the 2016 eDNA the methods, and from in hand, traditional out of other Meuse carried with the colonization the surveys On observed bivalves rapid freshwater from River. been the Moselle the during since the detected confirming has and been localities River, scuba-diving, it not few Tarn had where a it by the from add Saˆonewhere collected and River, of River, the Data S`evre field, niortaise downstream 2010). the the (Adam, in known: basin, basin species previously Garonne Rhone identifiable easily not the this and were France of that of expansion south the the confirmed analysis in eDNA spread rapidly It has Italy. which 2007, from Mussel introduced Pond the maybe Chinese in France, The places for few highlighting. species a eDNA species new in an Invasive a detected without 3.2.2 were reliable overlooked is species no been Mussel have the is Swan sure of endemic There Italian for considered sequences 2017). was The would eDNA It al., study, River. 2019). et this al., Loire (Froufe et In upper (Riccardi data Switzerland. field and molecular the Italy in on Mussel it to based identify Swan Italy, to the character Italian in clarify morphological the diagnostic to re-described needed example, recently is an been focus approach not As has barcoding do a status. databases cases, taxonomic of many processes populations’ In validation misidentifications. common, potential and these widespread on are species these Because river. genus databases. the Mussel the of River stretches Swollen of new the Species on and particular) Mussel River in network Thick-shelled 2000 the path (Natura as southward such a Rhˆone affinities provides tumidus Rhˆone the itself northern the its U. While Rhˆone more biome, Hence itself. the bank. Mediterranean of from left the species ecosystems its of for different on extension very streams northward lower alpine are a the rather sample, northward allows in valley and to a tributaries bank easier Its right constitutes are 2014). its valley which al., on tributaries, its freshwater streams et investigate Mediterranean Darwall and typical (ex. to area, are subregion performed Mediterranean part Mediterranean northwestern been the the has in of Rhˆone campaign flows extension lower dredging The extensive species. nor bivalves scuba-diving No sample. to hlpsn 78 hs e aacudjsiyteipeetto fcnevto measures conservation of implementation the justify could data new These 1788. Philipsson, risn otiomsbugensis rostriformis Dreissena Anodonta iaoot woodiana Sinanodonta uls compressa Euglesa r oeie icl oietf n r rbbyotnmsdnie in misidentified often probably are and identify to difficult sometimes are risn polymorpha Dreissena Pie 82 a enfral icvrdi rnevery France in discovered formally been has 1852) (Prime, La 84 salreseis nrdcdi rnesince France in introduced species, large a is 1834) (Lea, 5 Aduo,19)i mle pce,smlri shell in similar species, smaller a is 1897) (Andrusov, Pla,17) n hrfr ieyt remain to likely therefore and 1771), (Pallas, ndnaexulcerata Anodonta .complanata P. or,1838 Porro, historically Anodonta Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. rtcl n nlss u eut r hrfr oprbe ohi pc n ie ti o osbeto possible now sampling is it standardized time: with and scale, space large in a both at comparable, surveys therefore eDNA are implemented results have Our we analyses. and here, protocols time scale first large the a For at sampling small eDNA Mussel. a Pea Standardized in Orb 4.1 False once this the analyses for of eDNA habitat status conservation suitable with DISCUSSION worrying Although detected France). the 4 been (north-west confirm Britany data has in eDNA sampling, It Valley once under-sampled, Valley. extensive is Rhone and Rhone Despite River species the the France). Charente in (south-west in the Pea sites Toulouse of eDNA Orb four tributary near False its only record the detect from rare historical is didn’t it fluctuations is it recorded additional we so level although (1987) an and water France, Kuiper France, of report and most and in and covering pollution Mouthon rare range, to everywhere. distribution very intolerant localized wide become a and is has it and gives habitat (2012) waters eDNA Welter-Schultes of Mussel. clean the kind and but This population. clear living 2012). basin, (Welter-Schultes in reported Seine previously areas, a the swampy Mussel to eDNA in stable closed Pea by basin, individuals Orb confirmed Seine False living data the elsewhere Orb The in River, of known once Meuse Nut not records only the is that recent species on it the indicating several collected revealed since single detected are sampling been status a be recently conservation there in have detected could Finally, poor individuals only very eDNA analysis. was Living with sites) eDNA basin. probably work. sampled the this but in 65 of there, the framework of occurs of the downstream still (out in subsist RhˆoneMussels catchment sample to Nevertheless, not the seems areas. did in population catchment we French one site that It large least sector sediments. At the a known. muddy of on currently most more Loire, are alarming. over populations preferring is spread France living available, Mussel, throughout few sampling are Orb very eDNA records concerning Witham situation hundred its the a but than About threatened, less substrate little a the seems of also terms in Mussel demanding Orb be threatened most to Nut a the appears Only Mussel The observation). Orb Mussel its Witham France. Orb after the in makes years Witham which 10 species Seine, the about bivalve lower the results, freshwater occurred probably in sampling observations, eDNA remain, (where shell would to now on population by based According single site mainly reality. last Aisne data, the been the this has But from of it 3d. extirpated preserved picture Figure where Witham be in obsolete places can the shown three which an as but the shell, give banks of thick Mussel), river identifiable one the Orb easily extensively to in its Witham close years given been samples, for available the our is has to in data environment suitable Comprehensive once alive. only of sites observed detected sampling kind been 250 has This Mussel (estimated no substrates. Orb study as sandy our remarkable, Rhˆone on in is lower sampled preferably the water, on well-oxygenated data seems in only Mussel Mussel The Orb River France. Witham Depressed sector. The in this The range for species. its known the of are revealed Rivers. favorable data most Rhˆone analyzed) none few historical from lower 7 drainage, although sites extirpated Similarly, the Loire the favorable be basin. of potentially the studied), in to Garonne out on two sampled Moselle the station studied localities the the one in were the (only substratum) sampled), of of stations Charente limestone any 52 out in the on the detected station and confirm ecosystems of not sampled) (downstream Saˆone data (one was out 65 eDNA stations the eDNA Mussel the France. (9 River in of Meuse in Depressed also out the the elsewhere station and but Seine, specimens one station) studied), the (only live (one in station of Moselle Seine species one record the the the recent (only stations), of of (two areas no presence catchment Meuse is current the individuals the There the in concern station), species 46 station). (one the these, National (one Somme of of the Rhine presence Out the (Bouchet, of the 3c). stations), recognized to Figure those 15 were (see attest (about France (especially subspecies data in These three collections available recently. where are living museum data), records observed by hundred unpublished a evidenced Paris, About in as 2002). History France, Natural in of distribution Museum wide a had .rivicola S. paru solidum Sphaerium uls pseudosphaerium Euglesa sas pce hrceitco ontemlrervr,poal less probably rivers, large downstream of characteristic species a also is satpcldwsra pce,lvn nlrervr,a depth, at rivers, large in living species, downstream typical a is 6 ar,12 salwadrvr pce htlvson lives that species rivers lowland a is 1927 Favre, Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. oeo h hr rget eapiywt u rmr r altpssae ewe ieetspecies different between shared haplotypes are primers our Mussel with the Pea amplify of Iridescent we most the fragments for (eg. sequences short gene few Mussel the 16S a Orb of the only Oval some have for the We studied or known. species 1832) specimen not this routes. (Jenyns, one is of colonization only species haplotypes species each sometimes invasive different of species, of the diversity understanding map haplotype better to the a Second, possible for it insights makes provide analysis maybe and large-scale complex, eDNA species. this to Clams Asiatic the ( for France leana for documented described largely distinguish species been with three have to phylogeography taxonomy not molecular complex decided of a Corbicula We hazards of the ages. two scars and these protocol. glacial Hybridization the eDNA specimen: the bear our sampled throughout in any just taxa expansions in may two story and DNA these same contractions mt there the now, and species tell for interbreed markers multiple But divergence. independent largely significant two may a these with lineages that lineages evidence distinct namely two no species, reveals is also distinct mtDNA, of male favor in in identified argue would distinct marker two courtillieri female of U. mtDNA favor 1), in Mussel Box eDNA argue River Significance that not Thick-Shelled is do said the experience, be Regarding ancestral field can with from that together least result France, The only in synonyms. may species. be results divergence may sampling mtDNA species scale Their two large shape sympatric. these shell are from and neither retention, lineages discriminable polymorphism not both are where and - anatomy geographically nor overlap largely to seem has markers, database molecular our question From species unsolved. the This remains of 1859) boundaries? status (Hattemann, the the species but within reflect (2014), Puillandre and Pri´e species and by characterize species the to Naiad French within used for addressed markers whereas species been mtDNA clear reflect already not the actually is markers, do It (mtDNA) First, taxonomy. DNA molecular mitochondrial of on uncertainties based the mainly boundaries. by delimitations, biased species potentially our in still perspectives range are eDNA results distribution and Our large taxonomy supposed molecular status. their of conservation despite their Limits and detected on Mussel, 4.2 Mussel light Orb rarely Pearl Nut red were Freshwater the a Mussel Giant Mussel, flashes well-known Orb River which very Depressed France, Witham the the the one for as Only such all samples status. species above eDNA conservation some critical our Furthermore, its False by 2018). supporting The added France, al., species. (Pri´e in be et endangered sites could and two rare only location detect in new to detected valuable was was Mussel sampling Pea eDNA Orb large-scale hand, Clam other the Fingernail On Long the data. example, isolated For one invasive. added colonize not also occurrence are to results were Our species the expected they colonization. transversum introduced documenting revealed where were allow some species could sampling they invasive that regularly eg. the places locations eDNA confirm watersheds, of these main absence in Re-sampling large-scale connecting the expected. also channels (yet) Our confirm not artificial samples and absence. mainly eDNA range Contrarily, e. for 2017). (i. known Fruget, Pri´e routes argue & particularly previously colonization to potential is their the This used in considering occurrence. populations. species’ be species bivalve given invasive a also of detect of evolution to can used the usually it are monitor analyses But to eDNA species: future invasive monitoring the for relevant in sampling our reproduce rm,1867, Prime, p.(fnigr enad,&Sri,20;Pgere l,21) ee eddntdsigihthe distinguish not did we Here, 2011). al., et Pigneur 2002; Streit, & Reinhardt, (Pfenninger, spp. “pictorum” Sy 89 a enrcre nytiei rnewt rdtoa ehd,adeN only eDNA and methods, traditional with France in twice only recorded been has 1829) (Say, idcosa azacy,ZjcadZjc(06 hwdta nidpnetmarker, independent an that showed (2016) Zajac and Zajac Kaczmarczyk, Mioduchowska, . “crassus” sensus ru,ie o France for i.e. group, ru,ie o France for i.e. group, inu ta.21) lhuhoehpoyeof haplotype one although 2011), al. et Pigneur .fluminea C. .ovale S. .crassus U. .mancus U. OF M (O.F. A ´rsa,10) e uprigifrain.Adyet, And information). Supporting see F´erussac, 1807), (A. .casscrassus crassus U. 7 le,1774), uller, ¨ aseishvn obyUiaetlInheritance, Uniparental Doubly having species (a aac,11 and 1819 Lamarck, .mancus U. .fluminalis C. and hlpsn 78and 1788 Philipsson, .pictorum U. .fluminalis C. .pictorum U. OF M (O.F. sdfie ymtDNA by defined as , em ob proper be to seems le,17)and 1774) uller, ¨ Lnau,1758), (Linnaeus, .c courtillieri c. U. .crassus U. E Sphaerium . pulchella and C. Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. Aduo,19)i etr uoe rtosrainfo France. from observation first mussel quagga 10.3391/ai.2011.6.S1.016 Europe: the doi: Western of expansion in Range (2011). 1897) J.-N. (Andrusov, Beisel & A., Vaate, de Rhˆone-M´editerran´ee.Bij bassin le colonise qui esp`ece introduite chinoise une L’Anodonte (2010). B. Adam, (FCT) Technology and Science for References Foundation Portuguese by and funds. ERDF, national the through through Freshwater of Union Conservation European the for the approach particular missing N (OFB). also the in Charneau Project ConBiomics: samples, would Bivalves Mathieu under We the developed (Aquascop), of studies. also Saget analysis was the Grand research Mathieu laboratory of This and DREAL (Biotope), part sampling Philippe The the financed Laurent studies. in also (OFB), participated Tarn main Irz who du Pascal the those DDT all for the thank Loire-Anjou to and like CPIE Occitanie Vilaine, Aquitaine, Gaz, d’Espaces GRT Nouvelle EPTB SpyGen, Conservatoires program, Est, France, NGO, the Mussel de Freshwater Limousin, Caracol Giant Navigables of data consultancies, + Voies Park Aquascop The LIFE Natural and the financing. Regional Midi-Pyr´en´ees Aquitaine, Biotope Rhˆone, the its Naturels Nouvelle by du and to Nationale out R´egionale de Compagnie contributed carried Direction the work d’Occitanie the Spygen includes Biodiversit´e Logement and NGO, here Caracol la du presented by Fran¸cais de jointly l’Am´enagement et Office out de carried The was consultancy. l’Environnement, bivalves for Biotope method and analysis company eDNA view. the of of development point The eDNA may studies an further just here as is present species This we of taxonomy. what Acknowledgements freshwater distribution molecular that actual of monitoring mind the state-of-the-art in and reflect current kept not preserving the be may long-term jeopardize should They for and distribution. it pro- Large-scale tool haplotypes also species, the species. 16S valuable known but both is endangered poorly a ”presence”, document of the represent regarding contractions reliably only However, clearly range to bivalves. not surveys as possible providing well metabarcoding now of as becomes eDNA potential species, it invasive the methods of have gression standardized protocol, Using optimal data. access an ”absence” of with difficulty the analyses, species, to downstream eDNA due alike for managers policies and declining conservation environments. naturalists and these consistent important by erosion to implement considered biodiversity very insufficiently of to highlight currently context essential results are a seems which In areas eDNA rivers. it human-impacted Our major most threa- sizes, of ). the most . populations downstream . in effluents. the species located also for industrial and issues are plains, conservation upstream, They agricultural from species-rich. threats areas, most cumulated the (populated and are diffuse ecosystems facing downstream tened, data, eDNA to According Mussel Mussel River Pea Conclusion Moroccan Caserta 4.3 the The of of haplotypes mtDNA unknown samples male the previously respect, characterizing many that allowed In field. Italy the and in foucauldianus diversity Morocco genetic investigate in to tool performed interesting an remains distribution. eDNA haplotypes But is as here present referenced of we haplotype What haplotype the biased. rare If a fragment). or also 16S Mussel distribution was short Pea species Short-ended our reflect the of of detected haplotypes haplotypes haplotypes the various to example, the related For closely if species. is sure unknown not between is shared it are Therefore, 2020). al., (Pri´e et alr,13 n h tla ie Mussel River Italian the and 1936 Pallary, º OT-104-EE-326 ofiacdb OPT 00 otgl22 and 2020 Portugal 2020, COMPETE by co-financed NORTE-01-0145-FEDER-030286, .subtruncata E. iaoot woodiana Sinanodonta h itiuingvnhr for here given distribution the , .pulchella E. 8 .elongatulus U. La 84 Mluc,Bvli,Uinde : Unionidae) Bivalvia, (Mollusca, 1834) (Lea, skonfo nyoe1Ssqec,which sequence, 16S one only from known is .subtruncata E. MalaCo .casertana E. qai Invasions, Aquatic .pulchella E. .Peffr 85 n nMorocco, in and 1825, Pfeiffer, C. ,278-287. 6, , risn otiomsbugensis rostriformis Dreissena .pulchella E. Pl,19)wr amplified. were 1791) (Poli, Ml,15)(ee known (seven 1855) (Malm, norrfrnelibrary reference our in (up.1,71–74. 1), 6(Suppl. ol eheavily be could Unio Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. agmn,O,Lenr,L,Pie . uhrt .(06.D ’tltedu netienational. inventaire l’utilit´e d’un De 67-87. (2016). : X. 12 Cucherat, , & Pri´e, diversity V., S., L´eonard, mussel L., coast. Varandas, O., freshwater Gargominy, the Adriatic A., on Teixeira, Croatian curtain J., the and Lifting Lajtner, Peninsula (2017). I., 10.1007/s10531-017-1403-z E. Italian Vanetti, A. environmental the S., Bogan, using & Zaccara, of R., detection N., Sousa Species A., Riccardi, Zieritz, (2008). Pri´e, M., V., P. Lopes-Lima, Lamberti, Taberlet, E., E., & Froufe, M. eDNA F., Pfrender, via Pompanon, samples. R., diversity Sta- water C., species A. from Invento- from amphibian Miaud, Mahon, DNA and F., L., Retrieved Special fish C. G. Preliminary mesocosm Ore- Jerde, Ficetola, 6. of Y., Interagency Quantification Management, Li, (2015). Region Species: R., M. Land D. C. OR. Service, metabarcoding. Turner, Lodge, Mollusk of P., & Forest B. A., Portland, Bureau G. Olds, Agriculture, Aquatic T., Interior, N. 3.1. of Evans, of for Department Version Department U.S. Protocol https://www.blm.gov/or/plans/surveyandmanage/files/10-mollusks U.S. Sampling, and Program. Survey gon/Washington Presence/Absence Species (2008). tus/Sensitive and N. ry Duncan, fish. host of mussel crossresistance Ecosystems pond quagga inducing Reichard, Chinese by Invasive L., and reproduction (2017). Huan-zhang, mussel R. Slav´ık, zebra ve O., Sousa, P., invasive & Horky, Rylkov´a, M., of K., V., Lopes-Lima, Plechingerova, surveillance M., K., and Douda, S., detection Donrovich, early markers: eDNA using mussels. system (2011). Sepp river (2014). C. K., Rhine Miaud, con- Kopp, & K. L., Ventura, P., species De Taberlet, ecosystems. doi: Smith, F., freshwater ecosystems Informing Pompanon, in & S., DNA Pellier-Cuit, nal.pone.0023398 Hotspot: freshwater environmental A., J., of Duparc, Basin Persistence A., Freyhof, in Valentini, Mediterranean https://portals.iucn.org/library/sites/library/files/documents/SSC-OP-052.pdf. T., V., Dejean, planning the Barrios, in development C., 10.2305/IUCN.CH.2014.SSC-OP.52.en. Areas Numa, and website: Biodiversity S., servation Key Carrizo, methods DNA-based hwater Adopting W., management: of to Darwall, environments. molecules system From aquatic unusual (2011). in R. invasions The 10.1016/j.envres.2011.02.001 A. biological (2007). enough? Mahon, monitoring P. & for one A., Blier, J. Isn’t & Darling, mtDNA: W., of Hoeh inheritance D., 10.1016/j.tig.2007.05.011. Stewart, uniparental unix-inspired a H., Doucet-Beaupr´e, doubly obitools: (2016). S., E. Coissac Breton, & P., Taberlet, Y., metabarcoding. Bras, 0998.12428 DNA Le for A., package Bonin, software C., Mercier, F., Boyer, (Eds.), Falkner M. r´ef´erence & de bibliographie Ripken, annot´ee liste J. et r´ef´erentiel52. E. ; nouveau un France Th. un taxonomique, Falkner, de France: G. continentaux de In aquatiques perspectives. et nouvelles d´epart, terrestres de nouveau Mollusques (2002). P. Bouchet, aaeeto ilgclInvasions, Biological of Management -.di 10.1002/aqc.2759. doi: 1-9. , oeua clg Resources Ecology Molecular ilgclLetters, Biological ¨ al ,K,&Jkl,J 21) rcn h ugamse nainaogthe along invasion mussel quagga the Tracing (2017). J. Jokela, & K., ¨ a, oeua clg Resources, Ecology Molecular ,4345 o:10.1098/rsbl.2008.0118 doi: 423–425. 4, ,1112 o:10.3391/mbi.2017.8.1.10 doi: 101–112. 8, 6 94.di 10.1111/17550998.12433 doi: 29-41. 16, , 9 idvriyadConservation, and Biodiversity qai osrainMrn n Freshwater and Marine Conservation Aquatic niomna Research, Environmental LSONE, PLoS p.52) ai,PtionsNaturels Patrimoines Paris, 5-20). (pp. rnsi genetics, in Trends v3-1.pdf iaoot woodiana Sinanodonta 6 7–8.di 10.1111/1755- doi: 176–182. 16, eree rmIUCN from Retrieved . ,e39.di 10.1371/jour- doi: e23398. 6, 6 2537.doi: 3255-3274. 26, 1,9898 doi: 978–988. 111, 3 6-4 doi: 465-74. 23, hetn nati- threatens Mollusques MalaCo Fres- Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. aac,11 Bvli:Uinde n t cetdsubspecies. accepted of reevaluation its H., molecular and kill: Zornig, Ecosystems, rivers can Unionidae) taxonomy M., large (Bivalvia: Bad (2012). Schabuss, in 1819 P. Bouchet, biodiversity Lamarck, N., & fish N., Roset, Puillandre, of A., V., patterns Prie, Maire, quantitative P., reveals transportation. Jean, DNA downstream R., Environmental its Civade, despite (2018). A., T. Valentini, Dejean, M., & Rocle, D., Phylogeny Pont, (2011). K. Doninck, Van & J.-P., Biology, Descy, invasivenary E., the Etoundi, in different K., androgenesis between Roland, and J., hybridization Marescaux, cryptic L.-M., Pigneur, for genus Evidence clam (2002). invasive B. the Biology Streit, of & lineages F., evolutionary Reinhardt, d’ M., fossile Pfenninger, et actuelle American esp`ece formes rare. North une des the Sphaeriidae), France R´epartition(Bivalvia, of en France (2018). in J. presence Mouthon, the of evidence Genetic 10.3391/bir.2017.6.3.07 (2017). M. 1940. Forcellini, species before & Europe Western J., in Mouthon, introduced (2018). species T. ’hidden’ Haaren, a van America, & North M., Forcellini, taxon. J., new surrounding a Mouthon, confusion of taxonomic birth The possible (2012). the K. idae): Abbaci, Tair Inventaire & (Collection J., MNHN Paris, Mouthon, France. Flore). de Sphaeriidae de des et Inventaire Faune (1987). de J. Kuiper, & J., Mouthon, studies. 10.1002/jez.2055 phylogeographical doi: and identification 610–625. mussel mitochondrial sex freshwater Gender-associated endangered for (2016). the J. of implications Sell, tissues & somatic T., in heteroplasmy Zajac DNA K., Zajac, A., Kaczmarczyk, M., Mioduchowska, 2020. April 28 exceeds on (2014). far D. differentiation Damme, Van Species DNA & mitochondrial Kebap¸cı, U., M., Paternal Lopes-Lima, (1996). metabarcod- mussel, S.-K. freshwater (eDNA) the Wu, in . differentiation DNA & Lakes. allozyme Environmental and B., DNA Great mitochondrial J. maternal the (2017). Mitton, in H.-P., A. species Liu, C. invertebrate Stepien, invasive & detect T., doi:10.1371/journal.pone.0177643 to N. assays invasive Marshall, ing the E., of K. success Klymus, Reproduction species. (2019). mussel native J. to Geist, relation & new V., a ( as Huber, DNA mudsnails Environmental 10.1899/13-046.1 Zealand (2013). doi: New P. L. of 792–800. Waits, & 32, detection J., early Baumgardt for A., Ray, (2019). method A., Sepulveda, L. S., Poncet, https://inpn.mnhn.fr/docs-web/docs/download/302170 C. Paris & Goldberg, History, P., Natural Daszkiewicz, fop diffusion P., Museum et National Dupont, œuvre from m´ethodologie, en T., mise : Ramage, France r´ef´erentiel la R´egnier, v13, C., pour TAXREF S., taxonomique Tercerie, O., Gargominy, Evolution uls compressa Euglesa 04 .2764452.di 020/UNU.041RT.2764452.n Downloaded 10.2305/IUCN.UK.2014-1.RLTS.T22736A42465628.en. doi: e.T22736A42465628. 2014: 55,8889 o:10.1046/j.1420-9101.2002.00440.x. doi: 818-829. 15(5), , 0 5-5.di 10.1111/j.1558-5646.1996.tb03907.x doi: 952-957. 50, , 0() o:10.1051/kmae/2012014 doi: 405(8). 1 4.di 10.1186/1471-2148-11-147 doi: 147. 11, rm,15,(iava Sphaeriidae). (Bivalvia, 1852, Prime, Corbicula ilgclInvasions Biological cetfi Reports, Scientific oi conchyliologica, Folia Basteria lm Bvli,Criuia)i etr Europe. western in Corbiculidae) (Bivalvia, clams Corbicula 6(-) 126-130 (4-6): 76 10 1 4136.di 10.1007/s10530-019-02060-3. doi: 3451-3465. 21, , uls compressa Euglesa oaoygsantipodarum Potamopyrgus nocrassus Unio ,131 doi:10.1038/s41598-018-28424-8 10361. 8, Vnria Bivalvia). (Veneroida, 5 3–8. 45, iIvsosRecords, BioInvasions ora fEprmna Zoology, Experimental of Journal nweg n aaeeto Aquatic of Management and Knowledge iaoot woodiana Sinanodonta . nocrassus Unio h UNRdLs fThreatened of List Red IUCN The Bvli,Sheide,ntv of native Sphaeriidae), (Bivalvia, uls ucel Jenyns pulchella Euglesa LSONE PLoS Basteria, Pisidium ndnagadsgrandis grandis Anodonta ora fEvolutionary of Journal ). bvli:unionidae): (bivalvia: rswtrScience, Freshwater 213,50-54. 82(1-3), () 2-3.di: doi 225-231. 6(3), 25,e0177643. 12(5), , Bvli,Sphaeri- (Bivalvia, M Evolutio- BMC La13)in 1834) (Lea nomancus Unio Retrieved . 1832 , 325A, Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. ii enadDja.Otie udn:Pue,Pi,RceadDja.Fedsmln:Pi,Breugnot, Dejean. Prie, and sampling: Taberlet Field Lopes-Lima, content: Prie, Dejean. Figures and intellectual supervision: and Rocle Tables Study important Prie, Valen- Vigneron. for Gargominy, Poulet, Roset, manuscript Prie, Lamand, funding: the analyses: Jardin, Obtained data Couprie, of Dejean. and revision and bioinformatics Jean Critical elaboration, tini, Protocol Prie. Lopes-Lima. and manuscript: the Taberlet data) the OFB of the mnhn.fr. for Drafting future, inpn. near History, a Natural in of CONTRIBUTIONS integrate Museum to AUTHOR National intended the is of (or base integrated data is here national presented data the All inheritance. AVAILABILITY DNA DATA Mitochondrial (1994). R. K. Freeman, & 10.1038/368818a0 Gottingen: C., doi: Saavedra, identification. 368(818). A.O., species Ball, for E., guide Zouros, a molluscs, moni- for non-marine Editions. conservation Poster European in Planet tool (2012). emerging F. an Welter-Schultes, – DNA biodiversity. Environmental present (2015). and E. past Willerslev, toring & F., P. Thomsen, DNA. Environmental 10.1111/j.1365-294X.2012.05542.x (2012). H. doi: L. Rieseberg, 1789–1793. & 21, M., Hajibabaei, E., Coissac, sequence-to-sample P., reducing Taberlet, – illuminated jumps Tag (2015). T. 0998.12402 M. studies. Gilbert, metabarcoding & in K., misidentifications endangered Bohmann, the B., for I. Schnell, tool ecosystems, monitoring Freshwater a and as Marine DNA Environmental Conservation (2016). Aquatic ( a J. mussel Geist, with pearl & R., freshwater Unionidae) Huehn, (Bivalvia: Na- C., S., B. Anodontini Stoeckle, Zaccara, European S., Varandas, of I., Phylogeny Vanetti, (2019). A., linnean/zlz136. Teixeira, M. A., of Lopes-Lima, Zieritz, redescription E., & A. K.-O., Bogan, gel, E., Froufe, N., for Riccardi, (2020). D´ejean, results T. and & sub-region). P. methods (European Taberlet, assessment: Palearctic N., biodiversity Western surveys bivalves Poulet, the freshwater M., years’ for Rocle, metabarcoding ten Jug´e, P., E., DNA Froufe, Environmental waters: B., M., troubled Lopes-Lima, Adam, A., of N., Valentini, Pri´e, V., Patry, exploration N., Challenging mussel Legrand, (2018). pearl L., freshwater doi:10.1007/s10750-017-3456-0 M. mus- Philippe, giant K. the X., quagga Wantzen, of Cucherat, freshwater & R., invasive N., Araujo, the Richard, J., of Soler, records Pri´e, V., new Ecosystems, south: Aquatic of Heading Management (2017). J.-F. sel Fruget, & V., Pri´e, Biodiversit´e). et Inventaires ´editions, Mus´eum Publications Biotope (collection M`eze, : du France. Paris scientifiques de douce French d’eau bivalves of Na¨ıades autres Pri´e, (2017). et distribution V. and taxonomy phylogeny, Molecular Unionidae). (2014). (Bivalvia, N. Puillandre, & Pri´e, V., risn otiomsbugensis rostriformis Dreissena ndnaexulcerata Anodonta Hydrobiologia, agrtfr margaritifera Margaritifera Aduo,19)i rneadfrhrperspectives. further and France in 1897) (Andrusov, 3() 510 o 10.1007/s10750-013-1571-0 : doi 95–110. 735(1), ilgclConservation, Biological 1,3.di 10.1051/kmae/2017023 doi: 37. 418, . agrtfr auricularia Margaritifera olgclJunlo h ina Society Linnean the of Journal Zoological oeua clg Resources, Ecology Molecular Hydrobiologia 11 .:asbttt o lsia oioigapproaches? monitoring classical for substitute a L.): 66,12–19 o:10.1002/aqc.2611 doi: 1120–1129. 26(6), o:10.1007/s10750-020-04260-8 doi: . 8,41.di 10.1016/j.biocon.2014.11.019 doi: 4-18. 183, nEurope. in 5 2910.doi:10.1111/1755- 1289–1303. 15, Hydrobiologia, l16 o:10.1093/zoo- doi: zlz136. , oeua Ecology, Molecular 1() 157–175. 818(1), nweg and Knowledge Unio Nature, species Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. IUE1 FIGURE conservation monitoring-of-freshwater-bivalves-an-edna-point-of-view-on-species-distribution-and- image1.emf Our (1996). male file Wu both and Hosted collect Ball, Mitton often we (Zouros, Liu, Therefore, samples. mussels by fragment. our marine mtDNA mussels in male for freshwater 16S mtDNA DUI in the coined amplify detected tissues, originally also firstly gonadal primers been and eDNA their has 1994) in This Freeman, mitochondria egg offspring. & father’s the their Saavedra, their immedia- to to retain are them spermatozoid specimens mitochondria transmit the males paternal bivalves, and from where DUI Doubly transferred organisms, in are having uniparental eliminated, males mtDNA tely freshwater) the to from (including Contrary mitochondria fecundation. groups DUI, during bivalve with species some bivalve In of particularity a (DUI). Inheritance mtDNA: Uniparental female and Male Significance 1 Box Significant 1 TABLE oe n eune ftepiesue o mlfigevrnetlDAo bivalves. of DNA environmental amplifying for used primers the of sequences and Codes . itiuino h pce ihesaogsamples among richness species the of Distribution : vial at available oeSqec ’3 Reference Vene01 5’-3’ Unio01 Sequence Code Vene01 Unio01 AAGAAACCCPiee l 2020 al. Pri´e et 2020 al. Pri´e et AAGACGAAAAGACCCCGC GCTGTTATCCCCGGGGTAR R F STTACCRGT r´ ta.2020 al. Pri´e et 2020 al. Pri´e et TTDTAAAAGACGAGAAGACCC CSCTGTTATCCCYRCGGTA R F https://authorea.com/users/317031/articles/448890-large-scale- 12 and female Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. ag ies oto h orrsmlsaefo od rusra csses ossz codn othe to according size Dots ecosystems. upstream or ponds from are samples species. poorer of the number of Most rivers. large 2 FIGURE pce’rcns itiuini rne h ihs ie oe 5seisprsml)aein are sample) per species 15 (over sites richest The France. in distribution richness Species’ : 13 Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. Fig.2_SpeciesRichness.eps file Hosted Information. Supplemental from see inferred maps range precise distribution hatched more for data; actual here, eDNA shown area: stars: not shaded red data sampling; absence data; available eDNA eDNA samples. dots: before eDNA Black distribution samples. supposed eDNA area: our from inferred distributions bugensis 3 FIGURE n otatn c erse ie Mussel River Depressed (c: contracting and ) xmlso xadn a hc-hle ie Mussel River Thick-Shelled (a: expanding of Examples : vial at available https://authorea.com/users/317031/articles/448890- 14 .complanata, P. .crassus U. :Wta r Mussel Orb Witham d: :Qag Mussel Quagga b: , .solidum S. .r. D. ) Posted on Authorea 7 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158888143.31089837 — This a preprint and has not been peer reviewed. Data may be preliminary. and-conservation scale-monitoring-of-freshwater-bivalves-an-edna-point-of-view-on-species-distribution- 1_Primers.docx Table file Hosted and-conservation scale-monitoring-of-freshwater-bivalves-an-edna-point-of-view-on-species-distribution- Fig.3_Distribution.eps file Hosted distribution-and-conservation large-scale-monitoring-of-freshwater-bivalves-an-edna-point-of-view-on-species- vial at available vial at available https://authorea.com/users/317031/articles/448890-large- https://authorea.com/users/317031/articles/448890-large- 15