Accepted Article View metadata,citationandsimilarpapersatcore.ac.uk du Général LeclercCampus deBeaulieu CS 74205, 35042 Rennes, F This by is protectedarticle reserved. Allrights copyright. doi: 10.1111/1755-0998.12679 lead to differences between this version and the throughbeen the copyediting, pagination typesetting, which process, may and proofreading hasThis article been accepted publicationfor andundergone peerfull but review not has 5 4 2 1 Clio Der Sarkissian past the Ancient analysis DNA identifies marine mollusc shells as new metagenomic archives of Article type :Resource Article PROF. LUDOVIC ORLANDO (Orcid ID 0000-0003-3936-1850): 3 9EZ, United Kingdom;9EZ, United 29280 Plouzané,France; Voldgade 5-7, Copenhagen K 1350,Denmark; Orlando al Butler Paul Institute of Earth Sciences, University of Iceland, Askja, IS-1 Askja, Iceland, of University Sciences, Earth of Institute of University Sciences, Environmental and Life of College CGES, Campus 24-25 Bâtiment 1, Rennes de Université UMR6566, CReAAH, IUEM, Brest, de Université CNRS/UBO/IRD/Ifremer, 6539 UMR Lemar H Natural GeoGenetics, for Centre 1,6 4 Luet Chauvaud Laurent , 1 , Vianney Pichereau Vianney ,

2 Jn Eiríksson Jón , istory Museum of Denmark, Univ Denmark, of Museum istory 2 , Catherine Dupont Catherine , 5 Version of Record. Please Version as this cite ofRecord. article Jms Scourse James , 3 01 Reykjavík, Iceland; , Peter C. Ilsøe C. Peter , Exeter, Penryn, Cornwall, TR10 4 Crsie Paillard Christine , rance; rance; ersity of Copenhagen, Øster Copenhagen, of ersity de Beaulieu, 263 Avenue 263 Beaulieu, de Rue Dumont d'Urville, 1 , Mickael Perrigault Mickael , 2 Ludovic , provided byOpenResearchExeter 2 , brought toyouby CORE Accepted Article eosrcig aaoniomns n takn mluc evolutio mollusc tracking and palaeoenvironments reconstructing po biominerals carbonate shell in Adding composition. biogeochemical and structure (micro-) shell be can pollution, and salinity, availability, food temperature, env (palaeo-) as well as traits history life Their environment. M a r i n e m o ABSTRACT l l u s c s h e l l s e n c l o mollusc shells marine ancient Metagenomics of s e a w e a l t TITLERUNNING h o f i n f o r m a t i o n o [email protected] c o a s t a Copenhagen, Øster Voldgade 5-7, Copenhagen K 1350, Denmark; Fax eaeoi aaye t mrn mluc hls pnig h last the spanning shells mollusc marine to analyses metagenomic high-throughp extraction, DNA applying by potential this assess This by is protectedarticle reserved. Allrights copyright. 6 sequencing KEYWORDS allées JulesGuesde, 3 uoi Olno Cnr fr GeoG for Centre Orlando; Ludovic AUTHOR CORRESPONDING Université de Toulouse, University Paul Sabatier (UPS), Laborat (UPS), Sabatier Paul University Toulouse, de Université

marine 1000 Toulouse, France.

ols sel; nin DA mtgnmc; ihtruhu DNA high-throughput metagenomics; DNA; ancient shells; mollusc etal ofr a oe ad c and novel a offers tentially ntc, aua itr Museum History Natural enetics, traced through the analysis of their of analysis the through traced ironmental conditions, including conditions, ironmental ut shotgunsequencingutDNA and oire AMIS, CNRS UMR 5288, 37 5288,UMRCNRS AMIS, oire omplementary proxy both for to this list the DNA entrapped DNA the list this to ay rjcois Hr, we Here, trajectories. nary ofDenmark, Universityof 700 er. e report We years. ~7,000 +55235 E-mail: +4535322325; : ognss n their and organisms l Accepted Article This by is protectedarticle reserved. Allrights copyright. Butler 2008; Gillikin & (Gröcke dating sclerochronological Trinkler (Chauvaud rates growth including traits, history microstructure their while 2008), Lindberg & b (Ponder instance inference for can morphology Their past. the about information (Marin polys pigments, containing weight) in (0.01-5.00% layer organic consi all species, among variation microstructure despite which (Marin invertebrates of bodies soft Shells are biomineral exoskeletonsINTRODUCTION offering protection from pre management. speciesand aquaculture conser pa include applications future Other and changes. environmental communities, microbial molluscs, potent of the history with evolutionary research, DNA ancient in perspectives new opens gene novel as shells mollusc marine reveal results Our Disease. the o sequences genomic reconstruct we Finally, environment. marine approaches. genome-scal barcodes, We genomes, DNA also mitochondrial of combination find shell biomineralsidentifica taxonomic positive demonstrate We state. disease and to containstructure, biomineral their on dependent highly is arecovery DNA diversi successful DNA extraction from shells,including a variety of a nte suc o ifrain s hi boeceia compositi biogeochemical their is information of source Another (Hiebenthal salinity and temperature seawater in changes Vibrio tapetis et al. et al. 2012). Marine mollusc shells survive in the fossil record and and record fossil the in survive shells mollusc Marine 2012). 2010a). Their internal structure is also commonly examined for atra rm aia lm hls rvosy igoe wt Bro with diagnosed previously shells clam Manila from bacteria et al. 02. olss r poiet rdcr o shells, of producers prominent are Molluscs 2012). et al. et 2012) and diseases (Paillard diseases and 2012) et al. et al. ncient specimens, and find that find and specimens, ncient tion of mollusc species using a using species mollusc of tion accharides, lipids and proteins and lipids accharides, tic archives of the past, which past, the of archives tic y f irba DA rm the from DNA microbial of ty carbonate layer preservation, layer carbonate ain n dscain o the to desiccation and dation vation of endangered mollusc endangered of vation 02 Reynolds 2012; st of both a calcified and an f organisms closely related to related closely organisms f n wt oye ad carbon and oxygen with on, 03 o ucvrn past uncovering or 2013) a rva iprat life important reveal can dt, n metagenomic and data, e ue fr phylogenetic for used e thogens in the face of ial to reconstruct the provide invaluable invaluable provide et al. et al. wn Ring Ring wn 2016). 2004;

Accepted Article 2013; Crosetto 2013; (Bernstein transcriptomics and metagenomics genomics, have that technologies (HTS) sequencing DNA high-throughput in This by is protectedarticle reserved. Allrights copyright. sub-fossilizedof eggshellsavian (Nys 2011). (Beniash biominerals (Smith hydroxyapatite calcium bones of composed and teeth in DNA of preservation long-term the on et al. al. (Lartaud availability (Chauva temperatures past for proxies providing isotopes stable (Pedersen (Pedersen reveal awealth (Orlando yBP) Present, Before years of information Middle including specimens, Plei Genome-scaleancient datafrom aboutmusselsshells offreshwater pearl pastmito single a and microsatellites nuclear nine organisms,of amplification stud single one only knowledge, our of best the To studies. DNA populationhen and shells, mollusc marine in DNA of preservation long-term potenti suchDespite profiling. DNA environmental and community reconstructions demographic and phylogeographic identification, sh during the outside and inside both biomineral from DNA As proxy. complementary formation, offer potentially can shells mollusc marine from extracted DNA shell DNA might be (Penkman reconstruction for stratigraphic used not only f ancient DNA (Oskam (Oskam DNA ancient aie ols sel, lo ae f acu croae T asses To carbonate. calcium of made also shells, mollusc marine 2014) and phytoplankton dynamics (Szymanek 2011); and rates of rates and 2011); (Szymanek dynamics phytoplankton and 2014) 2016) for reviews). This has mainlybeen made possible through et al. 2015; Ermini 2015; et al. et 2015; Orlando 2015; et al. et al. 2010); trace elements for the presence of pollutants (Pérez-Ma pollutants of presence the for elements trace 2010); 2010). This work paves the way for the recovery of DNA from anDNA of recovery the for way the paves work This 2010). et al. Margaritifera margaritifera Margaritifera 2015; Slatkin & Racimo 2016; Leonardi Leonardi 2016; Racimo & Slatkin 2015; et al. et al. et al. 2015). Ancient DNA studies have so far mostly relied relied mostly far so have studies DNA Ancient 2015). 2013; Dabney 2013; 2004) have also been shown to occasionally preserve et al. 2011). 2011). et al. et al. (Geist (Geist 2007; Metzker 2010; SegataMetzker2010; 2007; 2013a; Meyer 2013a; chondrial (mtDNA) barcode from barcode (mtDNA) chondrial stocene remains (120-780,000stocene remains y has reported successful PCR successful reported has y an extremely informative and informative extremely an ce, their potential for ancient for potential their ce, al, no work has evaluated the evaluated has work no al, mrn mluc hl DNA shell mollusc marine s Calcium carbonate matrices carbonate Calcium r aie ols species mollusc marine or et al. ud ad niomns (see environments and s bt lo o bacterial for also but , amino-acid degradation amino-acid constant improvements constant et al. recently revolutionized revolutionized recently ell could be entrapped entrapped be could ell et al. 2008). et al. et 03, hc are which 2003), et al.et 05 ad food and 2005) 2016; MacHugh 2016; 2014, 2016), 2014, yol yol cient cient et al. et

Accepted Article This by is protectedarticle reserved. Allrights copyright. bacterium the by caused (BRD) Disease Ring Brown the their in deposits conchiolin dark and lesions growth presented specifically, More S1). and 1 (Tables infection trematode potent damage exhibited shells of number A species. identifying (Reimer (UCIAMS#182492, yBP 6,809-6,962 of range calibrated probability study at radioca the was hav_Mussel sample Kech The records. collection Carbon or 2015)) Cycle AMS Facility,B i r University o c h ceram e dates, a (radiocarbon u context 2 archaeological 0 Califor their 0 on 4based ; A n ddat e were r specimens s The eS1). and n 1 2(Tables 0collections 0 and 8sites ; G i s s i n g e r 2 0We 1 analysed 1 ; C h aclean m p marine a g n e mollusc shells, Shelldescription sample-set i.e., deprivedof any ( quahogs ocean and ( from clams~7 to 5 from dated shells mollusc marine from extracte DNA characterised we ( preservation, long-term its investigate and content MATERIAL races. (past) D (ancient) species, as shells mollusc establishes populations, work ba This molluscs, trematodes. on focus particular and with communities, analyses mapping targeted as ap well We 1). (Table as microstructure host/p biomineral in variability their species, their common occurrence in European archaeological sit Haliotis tuberculata et al.

and METHODS and Ruditapes philippinarum 03) Dansi mcocpc opooia caatr wr u were characters morphological macroscopic Diagnostic 2013)). ), oysters ( oysters ), Arctica islandica Arctica

Crassostrea sp. fr h eooi ad clgcl motne f these of importance ecological and economic the for ) , Ruditapes decussatus Ruditapes ), scallops ( Pecten maximus Pecten R. philippinarum R. Vibrio tapetis , Venerupis corrugata inner surface that are typical of of typical are that surface inner archaeological from tissue, soft et al.et ed between 5and ~7,000 yBP nia Irvine, leading to a 95.4% a to leading Irvine, nia ially indicative of microbial or microbial of indicative ially athogen co-evolutionary arms co-evolutionary athogen es over long time ranges, and ranges, time long over es le bt mtgnmc and metagenomic both plied c, ihc reat (ag & (Large artefacts lithic ics, ,000 yBP.We selected shells Marine13calibration curve A eevis o studying for reservoirs NA , htu-eune and shotgun-sequenced d, bndtd s at f this of part as rbon-dated 02 L Gf & Dupont & Goff Le 2012; tra clrpye and chlorophytes cteria, ), mussels ( mussels ), (Paillard Manila clam shells shells clam Manila et al. ), abalones abalones ), Mytilus sp. Mytilus sed for for sed 1989; ), Accepted Article This by is protectedarticle reserved. Allrights copyright. B (KOR_I treated bleach decontaminationTable 1, Figure (POS_Cla BRD-positive a and (NEG_Clam) healthy S1). a both analysing Based on“m the positive from DNA recovering was of feasibility the assessed first We preliminary investigated results, the or fragment wholethe shell was used. by comparingFor margin. ventral shells’ the of fragment a on performed was shotgunreferre hereafter (and years 60 lastthe from dating shells For HT preparation sample Shell al. Paillard & Maes 1995).Other clam shells pre-datingthe first c under constant mixing. After centrifugation (2,000 RPM; 2 min), 2 RPM; (2,000 centrifugation After mixing. constant under 15 mL digestion (Yang buffer (0.45DNA was mM extracted EDTA, 0.5% fromDNA extraction the N-laurylsarcosyl, shell powder based on the 0. so-called the weregroundto shells powder fine using andmortar a pestle distilled of volumes three in washes three by followed mixing, volu bleach 1% one in incubation through fragments shells/shell both fromFi 1, (Table (KOR_Clam) clam “diseased”Manila BRD-positive a of fragments (KOR_I + Ca) rget o te ae hl. usqet o hs et we test, this to Subsequent shell. same the of fragments _Clam) 1989) also conchiolinweak exhibited deposits. et al. et 1998; Gamba 1998; Gamba - B + _Clam,KOR_I et al. + 2014, 2016).short, 2014, wasIn incubated powder overnight at 37°C + B B - Ca, KOR_I _Clam, + _Clam) and non-treated (KOR_I + B + _Clam)and “healthy” (KOR_I haracterisation of BRD (Paillard d to as “modern”), DNA extraction DNA “modern”), as to d odern” marine mollusc shells by by shells mollusc marine odern” . older shells (“ancient”), either a either (“ancient”), shells older water (Figure S1). After drying, After S1). (Figure water impact of shell pre-extraction - me for 10 min under constant under min 10 for me B the supernatant was filtered filtered was supernatant the - gure S1). DNA was analysed was DNA S1). gure _Clam,KOR_I ) aia lm ~7 yBP; (~27 clam Manila m) Y” ehd ecie in described method “Y1” 25 mg/mL proteinaseK) dtst fr bleach- for datasets S eotmntd the decontaminated - B + - B Ca, KOR_I _Clam, + _Clam)shell in et - Accepted Article End-repair was carriedout at 12 end-repair and ligationEngl (New 454 (0.5 for Set Mix Master Prep Library DNA Quick NEBNext µM Illumina adapter final concentr This by is protectedarticle reserved. Allrights copyright. MgCl mM 4 Buffer, Gold 1X Gold, library DNA diluted 1/20 µL 1 mix: reaction following the of µL libraryclonality, per-samplewasestimatedaon basisqPCR by cyclesrequired amplif were libraries the sequencing, to shotgun Illumina recover to Prior DNAlibrary amounts sufficientof 30 µL and 20µL,respectively. for s ligation and end-repair after purification for kit MinElute the min, fill-in at 37 Seguin-Orlando in described protocol the Illumina for libraries DNA blunt-ended into built were Extracts Library construction and sequencing volume of60µLEB buffer. Purification PCR MinElute the using purified then was DNA min). (Merck unit filter centrifugal 30kDa Ultra-15 Amicon an through min. qPCRswere performed on a LightCycler30 sec, at 30sec,60°Cannealing at72°C for elongation 30 for (5'-AATGATACGGCGACCACCGAGATCTAC- 480 Real-Time PCRcycl40min; cycling10 Syconditions for92°Cactivationwere: at inPE1.0 3’), and 0.8 µL of 1:4:2000 Primer ROX:SybR:DMSO (5’-CAAGCAGAAGACGGCATACGAGATIIIIIIGTGACTGGAGTT-CAGACGTGT DNA dyeprimer mix (ThermoFi 6 Illumina an of µM of 0.2 and ACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) µM 0.2 ° C for 20 min followed by followed min for 20 C ° 2 Cfor 20 min and at 37 (Life Technologies), 1 mg/mL BSA, 0.25 mM of each dNTP, each of mM 0.25 BSA, mg/mL 1 Technologies), (Life

Bst et al. enzymeinactivation at 80 (2013) and Orlando ° C for 15 min, ligation at 20 at ligation min, 15 for C sec; final elongation at72°Cfor 7 final sec; elongation with EB buffer elution volumes volumes elution buffer EB with es of: denaturation at 92°C for for 92°C at denaturation of: es shotgun sequencing based on based sequencing shotgun carried out in duplicates in 20 20 in duplicates in out carried ation) and in 25 µL for fill-in. (inEB buffer), 0.1 units Taq Kit (QIAGEN) in a final elution equencing, while minimizing while equencing, ied by PCR. The number of ilpr) 300 P; 50 RPM; (3,000 Millipore) et al. and BioLabs) in 50 µL for for 50 µL in BioLabs) and sher Scientific). Thermo- Scientific). sher stem instrument (Roche instrument stem ° C for 20 min. We usedWe 20 min. for C (2013). We used the used We (2013). pidxd (‘I’) bp-indexed GCTCTTCCG- ° C for 20 20 for C Accepted Article This by is protectedarticle reserved. Allrights copyright. Centre (Table S1). High-Throu National Danish the at platforms MiSeq and HiSeq2000 sin or paired-end either in sequenced and pooled were libraries laboratory during contamination DNA of levels low with line in detection the below were sequencing) for amplified EBCs (except TapeStationinstrument (High Sensitivity D1000 ScreenTape, Agil EB buffer, the amountlibraries, of 15-29 DNA for in EBC thelibraries; amplifiedand that the number Table of cycleswas derived S1). fromCt After indexed valuesfor ea MinElute m reaction qPCR the of µL 20 to added was extract DNA undiluted librariesp f as conditions same the in performed was amplification Library Eachlibrary was PCR-amplified S1). (Table barcodes, using before indexed one and PCR-amplified being independently identifying were pooled EBCs libraries, corresponding DNA and shell ancient sequenced most for satisfied not with indexed was the this As amplified Il were condition this fulfilling libraries DNA shell Only modern concen (≥128-fold session extraction corresponding the from EBC was inferi value Ct qPCR library’s a successful when extraction were rates success Extraction qPCR. and building, library EBC), extracti during samples as time same the at blanks analysing by reagents/en laboratory the from contamination DNA 4.0. Software the Ct valuee for concentration DNA estimatedof measure relative A Science). Applied using the second derivative maximum meth selected for shotgun sequencing. or tothe Ct value minus 7 ofthe urification and elution in 25 µLelution inurification and ch library (6-21 for shell DNA DNA shell for (6-21 library ch gle-end mode on the Illumina Illumina the on mode gle-end on (Extraction Blank Control, Control, Blank (Extraction on threshold of the instrument, instrument, the of threshold ent). DNA amounts in blanks blanks in amounts DNA ent). or qPCR, exceptthat 5 µL of ach library was provided by by provided was library ach those obtained from their ix without ROX:SybR:DMSO,without ix was quantified on a 2200 tration compared to EBCs). EBCs). to compared tration calculated considering an an considering calculated ancient shell DNA libraries libraries DNA shell ancient lumina primer (Table S1). S1). (Table primer lumina vironment was controlled controlled was vironment ok Te nee DNA indexed The work. od with the LightCycler LightCycler the with od hu DA Sequencing DNA ghput with unique external external unique with Accepted Article FilterUniqueBAM PythonFilterUniqueBAM script (Schubert PALEOMIXreads, the and non-collapsed for (http://broadinstitute.github.io/picard/) withremoved MarkDuplicates Tools in version Picard 1.119 2012), and only reads showing mapping qualities (Li Durbin2009). Seeding & was disabledfor mapping, recommended as in (Schubert mapping against referenceDNA sequences (seebelow Tables BWAand v0.5.9 S2-S6)with This by is protectedarticle reserved. Allrights copyright. markers (“-mpi 100.0”) S7). (Table wlca”) and only keeping (Huson the MEGAN6 top-1% hits (“-top endogen damaged 1.0”) than showingTaxonomic longer be se would classification these that expectation the was then performedcontamina potential of impact the by limit theto order in program disregarded daa- m the o and d parameters default e with 27/07/2016) ( the on F(downloaded i g u (Buchfink v0.8.17 r DIAMOND eprogram shells, S mollusc 1 marine of content ) DNA total . the characterise FTo o r P O S _ C using DIAMOND Metagenomic analyses l a m a n d N E G _ C l a m , n o n - c o l l a p s e d p a i r collapse of overlapping pair-end mates as in described (Orlando et al. Post-sequencing read processing and mapping werecarried out using PALEOMIX (Schubert mapping and read sequence processing DNA 2014), including adapter trimming with AdapterRemoval2 (Schubert et al. 2007, 2011) using the weighted lowest-common algorithm (option et al. et al. 2015) and the BLAST “ 2014) for collapsed reads. ≥ 30 were kept. PCR duplicates were ting modern DNA, building on on building DNA, modern ting quences identical to database to identical quences we used the metagenomic meganizer distributed with distributed meganizer et al. et -sniie alignment “--sensitive” nr ous DNA molecules. molecules. DNA ous ed-end reads were were reads ed-end poen database protein ” 2013), and read et al. 2016), et al. “-

Accepted Article This by is protectedarticle reserved. Allrights copyright. abalone the musselblue oyster Pacific the for published sequences genome sho mapping by examined was DNA nuclear mollusc of presence The sequences reference DNA Mollusc For the clam clam the For Artigaud We next assessed the potential of shell DNA for the molecular i molecular the for DNA shell of potential theassessed next We sequences reference DNA Vibrio ca in <100bp, the corresponding taxa were excluded. taxon; given a for markers the all to mapping length read database the to Hits S1). (Figure (http://www.boldsystems.org/) b a r m second c the of The sequences o identification. species d positive e false s for control a r i sequence reference e each to mapped were reads aDNA sample, each v a ia sequence corresponding the lof end the at added and duplicated a brefer each in bp 30 lfirst the genomes, mtDNA of e circularity the f o r(Figu species related closely or sample-set, our tin represented database h egenom mtDNA M 52 contained database read first The HTS databases. o sequence shotgun l mapping by l EST estimated was u content mtDNA sMollusc c ap NCBI h yet al. l u m the i(https://www.ncbi.nlm.nih.gov/nuc n t h e(http://compgen.bio.unipd.it/ruphibase/), B a r c o d eO RP2-3sequenc genomic to reads HTS shotgun andmapped We shells. positive HH6087, generated as part of this study (Figure S1, T 2012a;b; Ghiselli et al. Mytilus galloprovincialis H. tuberculata R. philippinarum 2014, 2015) and 2015) 2014, cytochrome c oxidase subunit I subunit coxidase cytochrome et al. (Harney 2012; Allam ,transcript sequences were collected from the Ruphibase databa M. galloprovincialis est/) and previous publication previous and est/) (Murgarella et al. et et al. 06, h scallop the 2016), 2014) (Table S3). ee ’eteiy CIP, o wih 88,453 which for (COI5P), 5’-extremity gene et al. Crassostrea gigas (Moreira 2016), as well as to sets of transcripts of sets for to as well as 2016), et al.et P. maximus dentification of of dentification re S1, TableS2). To account for ence sequence were manuallywere sequence ence s in (Vilstrup (Vilstrup in s 2015) (Figure S1, Table S2). Table S1, (Figure 2015) s (Milans ee eotd s h total the as reported were ses where this length was was length this where ses es of two two of es contained database tDNA (Zhang (Zhang es forthe molluscspecies ndependently in order to order in ndependently tgun HTS reads to the able S4) and isolated (Pauletto (Pauletto s to two reference reference two to s et al. et al. Lf Database Life f V. tapetis V. tapetis et al. et 2011; Moreira 2012) et al. 2013). For 2013). in BRD- strains,

and theand 2014; se

Accepted Article to reads wh obtained coverage maximal the depth-of-coverage, average ~7X other every against w e r Th e S4). c (Table o level genome m the p at a characterised r far so especies dt ot h o s e e x p repres one corresponding to sequencesgenomic 63 to alsomapped e c t e dw h e n m of identification positive false potential out rule To a p p i n g r e a d sd e r iMEGAN6 above. analyses described as v i n g f r o (Altschul aligner BLAST2 the using 11/10/2016) Vibrio To verify that shell HTSreads mappingwere for furtherkept analyses. to the correction mismatch for allowing and 77), and 55 33, (21, sizes This by is protectedarticle reserved. Allrights copyright. (Huang each from single-stranded reads (Bankevich v3.7.0 assembler SPAdes the using performed was (Schubert v2.1.5 AdapterRemoval using removed were adapters Sequencing DNA High-Throughput National Danish the at platform sequenc single-end before cycles seven for libraries amplifying h at sec 15 of: cycles (four ultrasonicator Bioruptor diagenode were obtainedby applying the protocoldescribed above onµg1 (Reid2002 in Borrego BRD-positive a from respectively genus, they were aligned to the BLAST “ et al. et V. tapetis et al.et 2012)). The number of reads simulated for each 1996), and from a healthy Atlantic halibut ( halibut Atlantic healthy a from and 1996), et al. reference sequences. 2003). DNA was extracted following (Bidaultfollowing was extracted DNA 2003). Vibrio reference sequence. This was done by simulating 101 bp HiSeq20 bp 101 simulating by done was This sequence. reference Vibrio R. philipinarum R. eoi sqec uig R (eso Mount-Rainier (version ART using sequence genomic V. tapetis clam in France in 1990 (Paillard (Paillard 1990 in France in clam et al. Vibrio nt ncetd dtbs (onodd on (downloaded database nucleotide ” 1990; Camacho 1990; species, shell HTS shotgun reads were reads shotgun HTS shell species, rfrnesqecsblne o the to belonged sequences reference Hippoglossus hippoglossus Vibrio . Only contigs ≥300 bp in length in bp ≥300 contigs Only . et al. n o a Ilmn HiSeq2000 Illumina an on ing of DNA extract sheared using a et al. et al. and off), sec 90 intensity; igh otie mpig patterns mapping obtained e entative sequence per per sequence entative sequence corresponded to a Centre. Post-sequencing, the Post-sequencing, Centre. en mappingshell HTSDNA 2015). Shotgun HTS reads 2016). De novo assembly 02, sn fu k-mer four using 2012), et al. each m 2009), followed byfollowed 2009), Vibrio ) in Scotlandin ) et al. genome 1994; Vibrio 00 Accepted Article complete nuclear reference andmtDNA genomes (assemblyhg19 bui re HTS mapping by estimated was contamination Human S6). (Table (DQ157222), lenticularis This by is protectedarticle reserved. Allrights copyright. ( strands double in deamination of followi the estimated We S8-S10). (Tables obtained was coverage accurate taxonomic assignment, we used as input file the bam al mapped acrossreads samplesand formollusc,investigatedforwas randomly a given marker.(Jónsson v2.0.1 mapDamage using assessed sampling For eachdis size fragment and misincorporation, base fragmentation, markerDNA from and bam e filesPost-mortem DNA damage profiles the minimum number o available): of exception the (with DIAMOND candida the of sequences mtDNA complete the to mapping read HTS tre of presence the investigated also We S5). (Table (HQ914635) AY835431),andAY359242 of genomes chloroplast and Chlorophytagreen algaeWe in shells, attempted by mapping shotgunChlorophyte, to trematode HTS and human DNA reference readssequences confirm the results of the DIAMOND metagenomic emntn i oehns ( overhangsterminating in regions). We then contrasted nuclear and mtDNA damage parameter damage mtDNA and nuclear contrasted then We regions). (LN810504, KT625422),(LN810504,and; Clonorchis sinensis S. japonicum (HM120841), λ (ii) tasomd no 1/ into transformed , (FJ381664), V. tapetis the complete chloroplast genomes of genomes chloroplast complete the Pseudendoclonium akinetum akinetum Pseudendoclonium Opisthorchis viverrini δ , chlorophyte and trematode DNA, considering both all both considering DNA, trematode and chlorophyte , D ad ige tad ( strands single and ) (iii) S. mekongi Schistosoma mansoni et al. the complete plastid genome of genome plastid complete the 2013) and 100,000 MCMC iterations. Damage iterations. MCMC 100,000 and 2013) λ N022) and (NC002529), 1 a rx fr h lnt o overhanging of length the for proxy a –1, for which nocomparative dataare Gnak ceso numbers accession (Genbank ignment for which the maximal the whichfor ignment (HE601612), δ matode DNA in shells through ng damage parameters: rates rates parameters: damage ng S tributions in shell DNA were were DNA shell in tributions , rbblt o ras not reads of probability ), to te trematodes identified by identified trematodes te ld 37.1). 37.1). ld Bryopsis plumosa s for each single ancient ancient single each for s ads to the to ads (i) c sml yedn an yielding sample ach S. spindale the complete mtDNA complete the analyses identifying identifying analyses f sequencing reads reads sequencing f Chlorella variabilis Chlorella S. haematobium Homo sapiens (DQ157223) , Phacotus

Accepted Article This by is protectedarticle reserved. Allrights copyright. d MetaPhlAn2 the to reads shotgun aligning first by out carried relative and assignment taxon Microbial NEG_Clam. and POS_Clam paired-en non-collapsed analyses, MEGAN in As S7). (Table 2016) u characterised was extracts DNA shell of content microbial The MicrobialmetaBIT profiling using S11). (Table samples modern and sample of overlap between the damage parameter 97.5% confidence interv numbe This altogether. species of set whole the individuallyor i con samples,modern of interval confidence 97.5% the interval overlapped confidence 97.5% parameter’s damage the to belonging sample against modern samples. We compared the proportion of ma irboe rjc Cnotu 21) n 1 si smls (Fierer samples soil 15 and 2012) Consortium Project includi Microbiome metaBIT with analy distributed the profiles in microbial considered comparative were paired-reads, including reads, all (Sunagawa Expedition p all S12), Table N=26; surface, N=14; layer, mesopelagic N=20; samples seawater 60from data purposes,shotgun comparisonFor duplicates werethen removedasdescribed above. Truong only considering taxa showing taxa onlyabundances considering ≥1%in reduce order to (Truong profiles MetaPhlAn2 the of analyses Statistical 2016).

et al. 2015) using the Bowtie2 v2.1.0 aligner (Langmead & Salzberg 20 et al. 2015), were profiled using metaBIT as detailed above, except t except above, detailed as metaBIT using profiled were 2015), et al. 2015)were performedin metaBIT, r was reported as the percentage percentage the as reported was r bihd y h Tr Oceans Tara the by ublished sidering each mollusc species eachmollusc sidering d reads were disregarded for disregarded were reads d noisedue tofalse-positives. (deep chlorophyll maximum, maximum, chlorophyll (deep atabase (Segata (Segata atabase al for the considered ancient sing metaBIT (Louvel (Louvel metaBIT sing abundance calculation were calculation abundance

g 8 hmn (Human human 689 ng et al. ses. We also used the the used also We ses. acet ape that samples ancient n Dmg iterations pDamage 2012; Louvel et al. et 2. PCR 12). 2012; et al. et al. et hat hat

Accepted Article assigned eukaryotic phylum Lokmer (Figure 2015; Wegner 1A), we & identified (Lokmer microbiota theshell expec other that contamination,and from arise Th as assigned 1B). (Figure NEG_Clam to compared POS_Clam in normalization) two the BRD POS_Clam only(2.4%; Figure 1A). HTSreads mapped to th This by is protectedarticle reserved. Allrights copyright. reads m 1.39-3.82% the of of sum 1B)a with (Figure DNA content Thi 1A). (Figure kingdom Bacteria the to assigned being and 1B) mapping tothe human genome(0.03% inbleached redu KOR_Clam of extraction DNA to prior decontamination Bleach 0.12% (POS_Clam) the HTSreads(Figureof 1B) mapped tothe hum fractions minimal contained sam and/or curation, analyses, collection, during contamination datasets The S8). Table 1B, (Figure to transcript mapping (0.4-2.0X ref maximalcoverage) and mtDNA the to assigned was reads bacterial most reads wereassigned BLAST the with to reads the HTS shotgun Bacteria kingdom (Figuremetag samples, both For POS_Clam. and NEG_Clam 1A). clams Manila the A from extracted be could HTS shotgun with compatible amounts DNA decontamination bleach impact of and analyses Preliminary RESULTS was true even after correcting for the amount of shell powder u powder shell of amount the for correcting after even true was shells bleached in genomes mtDNA and transcripts did not result in a loss of reads mapping to the genome of of genome the to mapping reads loss of a in not result did f BRD-positive two the For samples). non-bleached in 0.07-0.13% V. tapetis V. tapetis strains HH6087 and RP2-3 with a 29-369-fold(afterwithenrichmenta RP2-3 and HH6087 strains can be retrieved from BRD-infected marine mollusc shells, that Vibrio nr aaae sn DAOD n MGN rvae that revealed MEGAN6 and DIAMOND using database Vibrio genus forboth shells, and to the species might representmembers of the healthy vs vs 0.05-0.49% non-bleachedin shells; Figure 2.68-2.96% in non-bleached shells. This shells. non-bleached in 2.68-2.96% et al.et V. tapetis 2016). Within Mollusca, the most pling, as 0.17%as (NEG_Clam) andpling, sed (0.05-0.09% in bleached in (0.05-0.09% sed ted s, however,s,preserved mollusc apping to the the to apping erence sequences (3.1-29.0X) RP2-3 (3.22% in bleached fraction (1.3-4.0%) of these of (1.3-4.0%) fraction an reference sequence. ragments, bleach treatment treatment bleach ragments, e genome sequencesgenometheof e R. philippinarum enomic comparison of the of comparison enomic e te rcin f reads of fraction the ced the 27 year-old shells of of shells year-old 27 the f N aiig from arising DNA of is suggests that DNA V. tapetis sequencing effort effort sequencing R. philippinarum speciesfor it does not speciesby vs vs

Accepted Article This by is protectedarticle reserved. Allrights copyright. in length) overhang and deamination of rates (estimated levels treatme bleach Finally, 1B). Figure extraction; for used powder correcti when 0.06% both shells; infected non-bleached in 1.83% aibe hn tepig o dniy bsd n aiu nme o for 20-100% number 2C): (Figure maximum genera mollusc on morphologically-defined based identify, to attempting when variable Within exogenous molluscDNA. contamination from no suggesting vi were Mollusca Importantly, Nematoda. and fungi Basidiomycota abundances two for for except Chlorophyta 2B), (Figure phylum eukaryotic abundant most greenthe algae, Phaeophyceae brown in datasets alga kingdom abundant most was the that Bacteria revealed analyses Metagenomic followed byEukaryota, Archaea content Metagenomic and viruse extraction 2/10for clams, thenone of 2mussels, abalones2/2 5 of rate success lower a yielded samples Ancient (1/3). oyster for shells of number limited a but (3/3), abalones and (11/11) we couldsuccessfully extract DNAfrom 16/21 shells (76%), whic an age sample across variable were rates success extraction DNA rate DNA extractionsuccess de bleach pre-extraction subsequent applied analyses. we content, DNA pathogenic and so DNA exogenous impacted only treatment bleach As S2). (Figure eune. l msdniid pcmn, u n EC, ee assig were EBCs, no but specimens, misidentified All sequenced. sampl of totality the considering when 2) (Table shells ancient and 1/3 ocean quahogs. es extracted, including those not those including extracted, es nt didnot increaseDNA damage scallops (2/5) and one diseased one and (2/5) scallops d species. In modern samples, modern In species. d h included all thosefor clams rtually absent from all EBCs, EBCs, all from absent rtually Mollusca, success rates weresuccessrates Mollusca, was Mollusca 2A). (Figure s mollusc and and mollusc 1 (9) wt successful with (29%), /17 ng for the amount of shell ned to to ned urces and maintained host maintained and urces e, Bacillariophyta diatoms, samples showing highest oen n 010 for 0-100% and modern the majority of HTS otmnto i all in contamination f hits per sample, sample, per hits f Mytilus V. tapetis suggesting , DNA Accepted Article This by is protectedarticle reserved. Allrights copyright. (Louvel database MetaPhlAn the environmen of diversity limited the to due rates negative false molluscs’ mostly coastal environment. metaBIT microbial profili pro S3B), and 3B (Figures diversitymicrobial seawater the from Marine molluscshell microbial profiles showed most affinity wi procedure for hel_Quahog. of limit the exemplifies also it NEG_Clam, for decontamination b explained be can result this Although S4). (Figure 0.62%) and maximal showed also that samples these in contamination derived Clonorchis sinensis high-quali shells, fifteen in found, and parasites, possible as 3,090 (i.e., of over-representation the to due likely misidentification knascae bacteria skin-associated e to due microbiomes, skin/nose human the for affinities showed fe shells mollusc hel_Qua samples, Two S3A). and 3A (Figures diversity microbiome marine the from recovered diversity genu microbial the at distances Bray-Curtis of (PCoA) Analysis Coordinate We further characterisedthe microbial contentMicrobial profiling of shellHTS dat Derbesia ( level genus the at identification BRE5m_Oyst and POS_Clam only Chlorophyta, Within contamination. HTS-based characterisation of the microbiomes in mollusc tissue mollusc in microbiomes the of characterisation HTS-based re ourS5), (Figure identified be couldsediments and seawaters , and , vs Caulerpa 428 for and and ; Opisthorcis viverrini

Figure 2D).We alsoinvestigated the presence of DNAfrom trema Ruditapes, Ruditapes, Propionibacterium acnes Propionibacterium Pseudendoclonium, Ostreobium Pseudendoclonium, et al. 1 for 21 2016). Although bacterial species typical of marine (Figure 2E). Venerupis 4.% n 5.%, ugsig human- suggesting 54.6%), and (42.8% , and 43 for for 43 and , Mytilus ty hits against against hits ty sults call for a thoroughshotgun a for call sults th those of soils, and segregated tal microbe markers present in present markers microbe tal ng most likely suffers from high from suffers likely most ng y the absence of pre-extraction of absence the y bably not representative of the of representative not bably asets using metaBIT. Principal metaBIT. using asets , s (Desriac (Desriac s Chlorella levelsof human DNA (0.21% markers in the u bec decontamination bleach our hog and NEG_Clam, however NEG_Clam, and hog eae audne i the in abundances levated s level showed that the the that showed level s Arctica l usd te human the outside ll r e t taxonomic to led er Schistosoma mansoni , et al. Bryopsis rte ta to than rather ) 2014; Lokmer 2014; nr , database database Phacotus, todes , Accepted Article ula rfrne eune (Figu sequences reference nuclear for variable more was success identification taxonomic Mollusc for coverageobserved when mapping as against identified mtDNA genomesin absence This by is protectedarticle reserved. Allrights copyright. coverage ≥0.1X for any ancientas MURp_Clam sample, except med_Abalone1/2 scallops (Figure 4C,Table 2).Of note, (0.42 MuRp_Clam, initiall clams, 10/10 for success assignment genus with robust, also was us identification Genus EBCs. in observed also mapping spurious DNA mollusc low show might samples Both (0.003X). data nuclear mis-assig also(0.010X) LEF-B_Scallop was as coverage), maximal to misassigned was MURp_Clam respectiv 2.057-54.142X, and 0.006-2.008X of depths-of-coverage (Figur species, even sometimes and genus, right the to assigned co samples modern most genomes, mtDNA and data nuclear on Based sequence representing the reference genusexpected for agiven c maximal when positive considered was identification taxonomic sequenc comparative a against reads shotgun shell mapping After molluscs of Molecular identification pathogens.and emerging and molluscs between interplay the understand help and sources, (Mustafa Meisterhans 2015; Wegner & R. decussatus et al. R. decussatus R. decussatus 2016). This would improve the resolution power in profiling sh Raayi o te hl mrhlg dd o dsis h identi the dismiss not did morphology shell the of Reanalysis . ae o CIP acds wih s n ie ih h pten of pattern the with line in is which barcodes, COI5P on based . et al. Mytilus trossulus re 4A, Tables 2 and S8), none none S8), and 2 Tables 4A, re 05 Lokmer 2015; based on mtDNA genome mapping (0.057X et al. 06 ad n osa environments coastal in and 2016) y labelledy as sample. es 4A-B, Tables 2and S8)with ned to ned 3/3 abalones,1/1 oysterand climatic changes, pollutants pollutants changes, climatic ing mtDNA COI5P barcodes barcodes COI5P mtDNA ing content, probably leading to leading probably content, ol b caatrsd t a at characterised be could overage was obtained for a for obtained was overage nin sel. Considering shells. ancient l. tnig s exception, as Standing ely. dtbs fr molluscs, for database e of reference sequences reference of H. tuberculata 9X). For ten samples, R. philippinarum l b taxonomically be uld ell microbial microbial ell fication of of fication based on , was was , Accepted Article This by is protectedarticle reserved. Allrights copyright. relatively high coverage (0.145-0.215X) was also recoveredthe to mapping however, for maximal coverage(0.001-0.039X) was obtained for the c mis-assigned shells were the the were shells mis-assigned KM192130-32), KM192124-28, of genome mtDNA complete hav_Mussel, the for For obtained clams. except coverage), (0.223-6.665X samples shotgun ves_Quahog; and reads quahog2_Quahog, (hel_Quahog, available to not mtDNAwas genomes, con mollusc DNA mollusc data nuclear comparative which for and/orlmc2B_Clam;0.001X), endogenous low taxa with samples could in as well be as positivelEBCs, DQ399833), which all form amonophyletic clade ofBaltic Sea ta HH6087) KOR_Clam and (4.195 3 in the shells showing signs of acute BRD: the identifying POS_Clam at aimed next We (7.322 Manila clams infected in of shells Vibrio tapetis of Molecular identification genera (Figure 4C, Tables2and S8). mitochondri considering when samples, ancient all using for successful identification genus positive hav_Mussel, for comparative cont mtDNA mollusc endogenous genome low by explained sequences be could This 0.007X). for thesespecies (Figure 4B edulis of BRD andshowing signs of infection, maximalcoverage was obt Table S9). Maximal coverage across all across coverage Maximal S9). Table of diversity known currently the against alignment read due to spuriousmapping (0.007-0.047X), similar to that observ P. maximus (0.003X) and (0.003X) R. decussatus vs Mytilus edulis Mytilus 1.095X).In Manilaclam shells pre-dating the first descriptio V. tapetis Vibrio H. tuberculata H. and species in BRD-positive Manila clam shells, using species was obtained for the for obtained was species (AY484747) and (AY484747) V. corrugata V. M. galloprovincialis Ruditapes Ruditapes (<0.001X)transcripts was observedin clams identified as Vibrio and M. galloprovincialis vs xa (Zbawicka xa eoe (iue 5 n S6, and 5 (Figures genomes from a closely related species related closely a from ained for the other other the for ained 1.932X for Venerupis y identified for all ancient ancient all for identified y Y922 023) but (0.223X), AY497292 ent and/or by thelack of , Tables 2 andS8). Except l O5 broe was barcodes COI5P al M. trossulus M. .0-.0X. Mapping 0.001-0.004X). aia cvrg was coverage maximal ret eu. Spurious genus. orrect et giBCa and (gripB_Clam tent V. tapetis d n Bs (0.002- EBCs in ed asclosely related M. trossulus M. et al. V. tapetis (DQ198231, (FJ890849, strain RP2- strain 2014). The 2014). V. tapetis strain strain or M. n

Accepted Article 2011) (Figure S6, Table S9). other in 0.177X for obtained was coverage maximal shell, AKKp_Clam abnormal (Figure reconstruction genome robust allow to low too was which BRE2_Abalone, n for did enrichment BRE5p_Oyster) BRE5m_Oyster, BRE3p_Abalone, BRE3m_Abalone, BR in observed those from different deposits conchiolin showing tapetis This (<0.001-0.052X). SELp_Clam other in other for those to similar were genomes This by is protectedarticle reserved. Allrights copyright. In shells showing signsthe of weak BRD infection,recovered from POS_Clam, KOR_Clam, LAN1p_Clam, LAN2p_Clam, and the depths-of-coVibrio (1.910X LAN1p_Clam HH6087: strain presently described at the genome level, as expected when mappi when expected as level, genome the at described presently mapping to theLAN1p_Cl KOR_Clam, POS_Clam, For two a limited inourimpact suggesting dataset (Fi contamination of 145-1,897-f were samples infected in depths-of-coverage effort, (0.672X a a lat 264.-od ihr o the for higher 12.6-45.8-fold least at was BLAST the to alignment V. tapetis reference sequences (Figures S8 and S9). These results are in are results These S9). and S8 (Figures sequences reference DNA content below our analyses’ detection threshold (Figures 5 (Figures threshold detection analyses’ our below DNA content vs Vibrio 0.160X), and LAN3p_Clam (0.510X LAN3p_Clam and 0.160X), species ortoaclosehitherto sequenced. relative not V. tapetis pce) <.0X o MR_lm <.0-.1X, n 0.004-0.0 and (<0.001-0.013X), MURp_Clam for <0.001X species), Vibrio V. tapetis V. tapetis s p e c i e s ) , a k n o w n p a t h o g e n o f m a r i n e o r g a n i s m s ( C h o w d h u r y nt H07 hwd aia cvrg (.6 ad .1, respectively), 0.11X, and (0.06X coverage maximal showed HH6087 database (Figure S7). For these samples, the samples, these For S7). (Figure database (Table S9,Figures 5and S6). Inhealthy med_Abalone and reference sequences were assigned to the the to assigned were sequences reference suggests infection by another p another by infection suggests am, LAN2p_Clam and LAN3p_Clam, and LAN2p_Clam am, vs Vibrio .4X for 0.447X V. tapetis vs species: 0.009X for MATp_Clam (<0.001-0.030X (<0.001-0.030X MATp_Clam for 0.009X species: 0.122X). After normalizing by the sequencing the by normalizing After 0.122X). species than any other other any than species V. tapetis gure 5 and S6, Table S9). S9). Table S6, and 5 gure V. rotiferianus old higher than those in EBCs, EBCs, in those than higher old tan P-) LAN2p_Clam RP2-3), strain ng verage for the two D Manila clams (AKKp_Clam, athogen or a reduction in in reduction a or athogen 5 andS6, Table S9). Inthe V. tapetis LAN3p_Clam to belonging line with the the with line and S6). Likewise,and S6). shells Vibrio 59.5-72.1% of the reads the of 59.5-72.1% Vibrio t hw specific show ot genome coverage (0.263X reads to all other all to reads Vibrio genus after after genus Vibrio V. tapetis vs species 5 for 05X 0.001- DNA et al. V.

Accepted Article This by is protectedarticle reserved. Allrights copyright. C observed we Second, S10). and 6 (Figures a some of DNA nuclear mollusc the in depurination to due starts observed an increase (decrease) of purines (pyrimidines)m shells against ancient from reads shotgun mapping when First, at gen evidencefor provided our authenticity. data recover molecules DNA of preservation the characterised next We Post-mortem DNA damage patterns mapDamage estimates of deamination in double-strands ( double-strands in deamination of estimates mapDamage modern with differences significant showed samples ancient Some where damage parameter distributions for ancient and modern sam modern ancient and for parameterdistributions wheredamage samples (Table S11). Compared to to Compared S11). (Table samples be compared, we obtained overlap values similar to those calcul test median( (median(ancient)=10.1%; shells modern in observed those clams, where10 the in sample 3.0% to NEG_Clam year-old 27 sizes the in 1.4% from allow varied the comparison, 5’-C old thi_Clam to 16.8%va fragments DNA mollusc nuclear ancient of 5’-end the in at rates the ~5,000 year-old (Dabney DNA ancient of typical auzay1B_Clam. In m ah nin sel a 649.% for 6.4-99.4% was shell ancient each 97 the and samples modern all for interval confidence 97.5% the (1/ ends overhanging of length δ increased was overlap this molluscmtDNA, For S13). Figure S11; D , 63.1-98.8% for for 63.1-98.8% , p -value= 4.57 10 δ S -5 , 47.8-99.3% for 1/ for 47.8-99.3% , ) (Figures 6,S11 andS12, Table S10).  ad G and T λ et al. - 1). For nuclearmollusc DNA, therange of overlap between  δ msnoprto pten de o yoie deamination cytosine to due patterns misincorporation A 2013b) (Figures 6 and S11, Table S10). C S10). Table S11, and 6 (Figures 2013b) D , δ δ D λ , 9.7-97.7% for for 9.7-97.7% , S – 1 (TableS11; Figure S14). Forclams and abalones, was 4.1-35.3-fold higher in ancient mollusc nuclear  T rates were significantly higher than higher significantly were rates T δ δ S , D

n 1.-79 fr 1/ for 12.4-97.9% and ) and single-strands ( single-strands and ) ried from 2.2% in the 106 year- 106 the in 2.2% from ried omic positions just before read before just positions omic ollusc reference sequences,we ollusc reference with ranges of 62.9-97.7% for for 62.9-97.7% of ranges with ated considering all modern .5% confidence interval for ples of the same species canspecies same the plesof samples when considering erod R5_ytr In BRE5m_Oyster. year-old rates these samples, odern ed from shells, which also also which shells, from ed cet n mdr shells modern and ncient oen=.% Wilcoxon modern)=1.8%;  T substitution substitution T δ S λ ), and of the – 1 (Table Accepted Article This by is protectedarticle reserved. Allrights copyright. shells, Clam Manila BRD-positive year-old 12-32 from recovered pre also was DNA ancient of typical misincorporation Nucleotide shells. from recovered molecules vari the reflect and DNA ancient of characteristic are patterns (Table mtDNA mollusc ancient in higher 10.9-107.3-fold and DNA y apn sel htu HS ed aant opee tN seque mtDNA complete against reads HTS shotgun shell mapping by ide metagenomic DIAMOND-based the confirm robustly not could We oftrematodesMolecular detection histones. with associated LAN1p_Clam, showed coverage above 0.10X (0.14X to the to (0.14X 0.10X above coverage showed LAN1p_Clam, In varying from 0.8% in POS_Clam to 1.7% in LAN1p_Clam and LAN2p_C being absent from mollusc mtDNA and and mtDNA mollusc from absent being (Pedersen protection and MATp_Clam nuclea mollusc SELp_Clam, LAN3p_Clam, LAN2p_Clam, Interestingly, LAN1p_Clam, in distributions S18). and showed 6 (Figures BRE5p_Oyster a~10 bp periodicityB observed _ was S fragmentation low c shells, modern a from l DNA nuclear l previously o p ,(Fi (gue_Abalone) Bsizes fragment long and Rthi_Clam) ves_Quahog, E 1 proposed _ lmc3B_Clam A (lmc1B_Clam, b fragmentation intermediate a ves_Quahog), lt o n auzay4B_ e auzay3B_Clam, auzay2B_Clam, , auzay1B_Clam, (hav_Mussel, B R E 3bp m43-50 average with fragmentation _high showed shells Ancient A b fragment DNA a damage-driven l of levels o various observed n we Third, e , K O R _ C l S16). Figure a m , B R E 2 _ A b a l o n e , V. tapetis DNA, δ S estimates were 9.8-42.2 fold higher than higherfold 9.8-42.2 were estimates et al. 2014) (Figures 6 and S18). This is supported by this periodici V. tapetis DNA (Figures 6, S15 and S19), which arenot S. mekongi δ blt i peevto o DNA of preservation in ability D , as expected (Table S10 and gures 6 and S17). In mollusc In S17). and 6 gures mollusc DNA fragment sizes fragment DNA mollusc S10). These damage-drivenTheseS10). ih C with sent in the the in sent BRE3p_Clam, AKKp_Clam reference sequence), but but sequence), reference ation across the dataset. dataset. the across ation med_Abalone, nis_Clam, nis_Clam, med_Abalone, ntification of trematodes of ntification lam (Figures 6 and S15). lm quahog2_Quahog, Clam, cs Ol oe sample, one Only nces. n E-_clo, ROS- LEF-B_Scallop, in DA rget size fragment DNA r BRE5m_Oyster, and rfet nucleosome reflect o  rts t 5’-ends at rates T V. tapetis DNA ty Accepted Article for was DIAMOND by indicated chlorophytes of presence putative The chlorophytes of Molecular detection referencetrematode sequence. no attack, trematode with compatible signs showing BRE5p_Oyster netgtd i nt hw h sm DA xrcin ucs rates success extraction DNA same the show not did investigated ~7,000 last the covering specimens ancient including molluscs, extrac be can DNA endogenous authentic that demonstrate we Here Long-term DNA preservation in marine mollusc shell biominerals DISCUSSION ofdamage authentication withidentification robust compatible r chlorophyte these to mapping reads DNA high-quality unique of (<0.0 EBCs in than depths-of-coverage higher showed enrichment) h and enrichment), 10.8-fold (0.427X; BRE5m_Oyster enrichment), For enrichment). 2.0-fold (0.090X; AKKp_Clam 2. (0.124X; hel_Quahog enrichment), 5 2.0-fold (0.270X; (0.170X; MATp_Clam BRE5m_Oyster enrichment), 4.6-fold (0.707X; POS_Clam sequencin for normalizing (after For respectively. sequences, genome reference chloroplast and that and trematodes, sequence,despiteshellthe thatexhibitedfactthe perforation This by is protectedarticle reserved. Allrights copyright. n o D N A d a m a g e s i g n a l .N o r e a d sf r o mn i s _ C l a m m a p p e d a g a i n s tt h e C. variabilis variabilis C. and B. plumosa B. S. mansoni when mapping shell shotgun HTS reads to their complete plastid complete their to reads HTS shotgun shell mapping when was detected in metagenomic analyses. For BRE5m_Oysterand For analyses. metagenomic in detected was g effort) was observed compared observed was effort) g B. plumosa lesions proposed to be causedby signatures in mapDamage. C. variabilis C. PSCa (.8X 4.2-fold (0.486X; POS_Clam , to EBCs (<0.001-0.039X) for (<0.001-0.039X) EBCs to 01-0.027X). The low number eference sequences was not was sequences eference years. Themollusc species tedfrom the shellof marine lQao (.9; 2.1-fold (0.09X; el_Quahog supported only marginally only supported oig o hi different their to owing , -od nihet, and enrichment), 1-fold reads mapped toany

S. mansoni .1-fold enrichment), enrichment), .1-fold , a slight enrichment enrichment slight a , reference reference

Accepted Article This by is protectedarticle reserved. Allrights copyright. incl These, DNA. ancient of characteristic patterns degradation biominera carbonate calcium shell within survival DNA Long-term recovery. especially taphonomy biominerals, and proteins in DNA, calcium between interactions carbonates, and toinvestigatianalyses.Further DNA ancient for samplesselecting assess laye inner the temporal of integrity the to paid be ranges attention particular depositi during aragonite of dissolution the to due layer inner 5,000 yBP and yielding low amounts examination of DNA, Macroscopic revealed layer. inner aloss the of of t preservation the on We observed that DNAextraction success1996), have shell. increased this may content in particular DNA ratesfrom ancient mari and/or the recruitmentthat of the hemocytes molluscp defenceproviding oyster only the inwas mechanisms, state, the diseased a of hemolymph indicative which dep involve brown important and increased an extra- showed hydroly which mi BRE5_Oyster, post-mortem Interestingly, against protection better a DNA shell offer microstructures nacreous/prismatic matric foliated of structure denser theAdditionally, crossed-lamellar than weight) in (~1% co microstructures andprism content, nacre as in shell organic cont DNA in differences date, to documented been not has shells pre the Although microstructure. shell inner calcite foliated a sc and oysters than aragonite, nacreous columnar or homogeneous DN successful state. disease and preservation, microstructures, biomineral for clams, abalonesand ocean quahogs, witha shell and G and  A mutations at read termini and ~10-bp periodicity in the size the in periodicity ~10-bp and termini read at mutations A on. We therefore recommend that recommend thereforeWe on. sence of nucleic acidsin mollusc ntain alarger ntain componentorganic

on is required to understand theunderstand to required is on ositive results. We hypothesize hypothesize We results. ositive (rfrby rgntc when aragonitic) (preferably r he “porcelaneous” aspect of the the of aspect “porcelaneous” he ent could be due to differences to due be could ent dn icesd ae o C of rates increased uding es compared to calcite might f lm hls ae ~200- dated shells clam of allops, both characterised by characterised both allops, neralisation and cell density density cell and neralisation ne mollusc shells depended depended shells mollusc ne optbe ih hl DNA shell with compatible ala fud (Paillard fluid pallial ne lyr opsd of composed layer inner etato ws more was extraction A sis or microbial attack. attack. microbial or sis eadn biomolecular regarding osit on the inner layer layer inner the on osit ls was supported by distribution of nuclear of distribution (Marin et al. 2012). et al.  T

Accepted Article (Bos (Bos enrichment target in-solution whole-genome or sequencing deeper candidat bac possible and trematode for chlorophyte, of screen presence the for to confirmation useful proved MEGAN and DIAMOND metage the here, used methods computational different the Among mollusc marine shells Metabarcoding of This by is protectedarticle reserved. Allrights copyright. available. are databases appropriate comparative e. available, publicly being data of amount the by limited also iden morphologicalrobust for fragmentary too are samples shell always not is which target, to genera/species mollusc regarding re however approach This sequences. reference target to mapping ac and specificity more purpose, that For identification. taxon estimates abundance taxon relative the biases sizes, marker for subje and representativenessin comparativedatabases heavy which, in addition computationally however is approach metagenomic Orlando 2014; Willerslev & (Pedersen depths-of-coverage and p misincorporation from reconstructed be s now can epigenetics maps future nucleosome and for methylation potential suggests also fragments, DNA based metabarcoding applicable to marine mollusc shell DNA (Gei DNA shell mollusc marine to applicable metabarcoding based showhere that molluscmtDNA barcodebe will datasets mollusc comparative more that sequences confident be can canbe recovered decussatus et al. 2011; Schroeder 2011; Nvrhls, ih h icesn apiaino HS o non-m to HTS of application increasing the with Nevertheless, . et al. et al. et 2015; Hanghøj 2015; Ávila-Arcos et al. 2016). et al.et et al. et 2015; Cruz-Dávalos 2014; Gokhman Gokhman 2014; to the absence of normalization . hr, for here, g., , possibly leading to erroneous to leading possibly , tification. Targeted mapping is mapping Targeted tification. available, e.g.,in cases where made availablealsomadeWesoon. uay ee civd when achieved were curacy st for specific DNA markersterial DNA would require nomic approach based on on based approach nomic et al. from shells, making PCR- making shells, from quires prior knowledge uis n olss as molluscs, in tudies ten o nucleotide of atterns 2008), provided that ct to uneven taxon taxon uneven to ct et al. dl raim, we organisms, odel A. islandica tx. Further taxa. e et al. 2014; Orlando 2014; 2016). The 2016). and R. Accepted Article This by is protectedarticle reserved. Allrights copyright. Our results,in particular therecovery of DNA likely belonging pathogenic of and the studies environmental Potential bacteria for environmentschanging (Sanger 1993). popul human ancient in usage resource on light shedding thereby reconstruction s mollusc marine of application interesting Another management. in to of order in alleles past adaptive candidate specific identify and humanass time, diets through diversity genetic in changes track help could from shell Sarkissian (Der species endangered mammalian middendone previously assemblageAs species. mollusc endangered of conservation (Parker hin can atmosphere, the in excess dioxide carbon of dissolution acid ocean as such changes, environmental future and current to 2016). This information could inturnhelpmollusc demo predict Penkman2011;Szymanek (Chauvaudpalaeoenvironmental proxies of the from tracked be can outbreaks)that pollutants,pathogenic change environmental past of impact the investigate to possible (Leonardi molluscs of trajectories use be could sequences mtDNA and mapp targeted from resulting alignments sequence future, the In studies evolutionary for Potential of BRD, also indicate the potential of ancient shell DNA to rec to DNA shell ancient of potential the indicate also BRD, of diseases. BRD first appeared in Brittany in 1987 and has since since has (Paillard clams Manila of aquacultures and European 1987 in Brittany in appeared first BRD diseases. et al. 2013; Gazeau 2013; et al. et al. 2011; Butler2011; 2013). Additionally, ancient shell DNA could be applied to the to applied be could DNA shell ancient Additionally, 2013). et al. dt or e c o n s t r u c t t h e e v o l u t i o n a 06. apig hls n iesre wud ae it make would time-series in shells Sampling 2016). et al. et al. 2005; Thébault2005; et al. 2013; Pérez-Mayol2013; et al. 1989, 1994; Borrego1994; 1989, 05, eprly ape DA data DNA sampled temporally 2015), onstruct the historyof pathogenic to same specimens through a range form and improve conservation ess population genetic viability genetic population ess graphic and adaptive responses der shell formation in molluscs molluscs in formation shell der et al. ification, which, caused by the the by caused which, ification, V. tapetis ations and their adaptation to adaptation their and ations ing against reference nuclear reference against ing (.. tmeaue salinity, temperature, (e.g., s with museum material from material museum with hell DNA time-series is the asd ih otlt in mortality high caused 2009; Lartaud2009; et al. s at archaeological sites, sites, archaeological at s y n demographic and ry , the etiological agent etiological the , 2014; Reynolds 2014; et al. et 1996; Allam1996; et al. 2010; et al. et

Accepted Article prevalence of severe human diseases caused by by caused diseases human severe of prevalence Baker-Austin 2016; su sea of rise the following years, 50 last the within Atlantic increasingl become has which 1996), (Colwell environment marine various from DNA detected we Finally, aquaculture. to tools new for potential the hold material shell of analyses (Trinkler deposition of a mineral layer covering brown depositsbetween and, henc generally also helpof the whole identify BRD time series, are however required the to refine th genomicvi of characterization strains, additional of sequencing genome changes underlyingbefor Brittany in present were epizooty BRD the for responsible the sho strains that suggest to tend results These Brittany. in BRD This by is protectedarticle reserved. Allrights copyright. (Vezzulli the for affinity while beforethe colle first and BRD BRD of outbreak characteristic deposits conchiolin showed brown small highest genetic affinity f al. warming. as emergence (re-) disease human future predict to information help detect similar trends inwider temporal and geographical r

2000; Paillard et al. V. tapetis et al. 2016; Baker-Austin 2016; 00; , 01; ; Jeffroy b; 2011a; b, 2010a; et al. n Mnl cas wih ae eeoe rcvr poess throu processes recovery developed have which clams, Manila and et al. V. tapetis 2008). Interestingly, we found that DNA extracted from shells from extracted DNA that found we Interestingly, 2008). 2016). This spread has also been associated with an increase i increase an with associated been also has spread This 2016). strain RP2-3 was observed in shells post-dating the emergence the post-dating shells in RP2-3observed was strain et al. Vibrio 2016). Screening for for Screening 2016). et al. speciesin shells. V. cholerae 03. u rsls lo ugs ta DNA that suggest also results Our 2013). , V. parahaemolyticus rface temperatures (Vezzulli temperatures rface wing lower virulence than Vibrio rulence genes, and DNA analyses DNA and genes, rulence anges, thus providing invaluable invaluable providing thus anges, e history of BRD. This will more will This BRD. of history e or the or detect and monitor diseases in e 1987. Further1987.includingwork, e Vibrio e, a resistance capacity to BRD to capacity resistance a e, ehnss f co-evolution of mechanisms td n rtay or years four Brittany in cted species in shell DNA could y abundant in the North North the in abundant y cneune f global of consequence a is a diverse genusof the V. tapetis strain HH6087, strain and V. vulnificus vulnificus V. exhibiting exhibiting

strains h the gh n the the n et al. of

Accepted Article ITN604802); “Chairesthe d'Attractivité 2014” IDEX, University Net Training Initial ARAMACC Curie Marie EU the (NE/H023356/1); En Natural UK the d’Avenir”; “Investissements program the under ( LabexMER excellence of cluster the Scientifique; Recherche la PaleoC initiative APEGE the (2010-2012); Bretagne de Européenne National (FNU, Sciences Natural Research, Independent for Council Danish Thisupport. Centre for Sequencing DNA National High-Throughput Researchand Gabriel Renaud for for Bidault fruitful Foundation discussions,for the providing PALEOMIXZoolog the at Sørensen Vinther Martin and Schiøtte Tom thank groupWe historical ACKNOWLEDGMENTS (DNRF94); shellsamples pathogen of detection outbreaks. surveillan biological the as from well as species, mollusc the important mon genetic the the Invertebrate species, threatened for programmes conservation EPT Co shells PROXACHEOBIO re to information chan climate invaluable and ecology community marine couldbiology, evolutionary provide to potential the also hold thus be appliedsediment cores, shell lags fro on the marine seabed, of record shell archaeological to middens and palaeontological and important mus study living communities, inclu This by is protectedarticle reserved. Allrights copyright. (m to opens This reservoir. DNA ancient new a as shells mollusc as old as shells mollusc marine ancient from DNA recovering By Conclusion Vibrio N etato, rsin agø fr ehia assac, Mik assistance, technical for Hanghøj Kristian extraction, DNA of of Toulouse, France. ANR-10-LABX-19; METHOMOL)ANR-10-LABX-19; eum collections. Future studies Future collections. eum ce of aquaculture and early early and aquaculture of ce ols sel, on i e.g., in found shells, mollusc s work was supported by the OO of the Centre National de National Centre the of OO vironment Research Council Council Research vironment and the staff of the Danish ge. DNA analyses of mollusc of analyses DNA ge. ical Museum of Copenhagen of Museum ical 4002-00152B); the Danish Danish the 4002-00152B); lcin W tak Adeline thank We llection. ~7,000 years, we validated validated we years, ~7,000 ig h dvlpet of development the ding work (FP7-PEOPLE-2013- work eta)genomic trn o economically of itoring search in archaeology, archaeology, in search Université m

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(Kunth, 1865) from easternPoland. , 396 , 106 , 555–567. , , 407–417. 104 to BrownRing Disease in the Diseases of Aquatic of OrganismsDiseases Vibrio vian) Interglacialvian) recordedin DNA extraction from ancient from extraction DNA , 8–16. and associated human ntic. ntic. nant mitochondrial mitochondrial nant Proceedings of the al environments? environments? al ty n WDS and etry , 289 1275– , , 105 , , Accepted Article This by is protectedarticle reserved. Allrights copyright. “I fragment; non-bleached non-infected ”, BLAST of number analyses: Metagenomic (A) shells. Figure 1. Preliminary detectiontaxon tests: from bleached and non-bleached modern FIGURES CDereagents. and LO the wrote article. analyses, with inputm performed CDe from study. the coordinated and conceived LO and CDe VP,MP and LO.VP, CDu, TS, PCI, CP CONTRIBUTIONS: AUTHOR a (project PRJEB20113). from available are study this in generated data sequencing All ACCESSIBILITY DATA re-adjusti EBCsabsent from only taxa and after (B) Considering pro microbial genus-level between distances Bray-Curtis of PCoA and human-associated diversity Figure inshells, 3. seawater,samples. soil Microbial for totaxa assigned eachtaxonomic level. The colour gradientrepresents the proportionFigure 2. Taxon detection bymetagenomic analyses. of the totalnumb h ttl ubr f ed rtie atr ult fleig (ret filtering Quality, quality MQ≥30) reads, after normalized,of number total retained the considering forcalculated is each coverage reads mapping: sample, of by the tota number total the infected bleachedinfected “I fragment; + B + ”, infected bleached fragment. fragment. bleached ”, infected + B - nr , netd o-lahd rget “I fragment; non-bleached infected ”, database hits, normalized, for each sample, by sample, each for normalized, hits, database the European Nucleotide ArchiveNucleotide European the ng relative abundances. l number of retained reads. “I reads. retained of number l unique high-quality (Mapping (Mapping high-quality unique er of assigned reads that were that reads assigned of er files.(A) Considering all taxa. ndLO provided samples and ained reads). (B) Targeted Targeted (B) reads). ained olecular and computational and olecular - B + , non- ”, - B - Accepted Article (100,0000alignment iterations). mapDamage siz and considered fragment were reads (E), (MQ≥30) and high-quality unique (D) in positions read ten first the along HTS reads abovewere mapped are indicated plot.each Nucleotide distrib size fragment fi LAN For trimming). quality (post- the within (A) shown fragmentation describe DNA (C) and to misincorporation, considered were sequences This by is protectedarticle reserved. Allrights copyright. the to mapping reads hav_Mussel, For Figure 6. Post-mortem DNAdamage patterns. retai against the full reads of (se shownare species per genome representative one results for number speci For obtained. total was depth-of-coverage maximal the the which for by sample, dot black the sample, BRD each For filtering. trimming/quality each high-qual for unique normalized, considering mappi sequence, when genome obtained reference coverage species the represents gradient colour The 5. Figure <0.1 depths-of-coverage the panels. signifies corresponding “*” of value the obtained, was depth-of-coverage maximal the which For each comparative database and sample, theunique yellow dot high-quality indica refer barcode mtDNA (C) and (MQ≥30)genome, mtDNA (B) nuclear, (A) each reads, and normalized, forThe colour each gradient sam represents the coverage, scaled by sample, Figure 4. Mollusc taxon identification. Vibrio Vibrio species identification. panel). panel). 1p_Clam and ROS-B_Scallop, the ROS-B_Scallop, and 1p_Clam Mytilus galloprovincialis . indicates the reference sequence reference the indicates obtained when mapping against mapping when obtained e Figure S7 for mapping results mapping for S7 Figure e dsrbtos n F-J. Only (F)-(J). in distributions e which is specified at the top of top the at specified is which tes the reference sequence for sequence reference the tes ula tasrp reference transcript nuclear reference sequences to which sequences to reference misincorporations areshown ple, by the sequencing effort. effort. sequencing the by ple, es other than other es ence sequence, considering sequence, ence s 1 b fo ra ends read from bp 10 rst t (Q3) ed, and reads, (MQ≥30) ity v.2 was run on the full ng against each ution, (B) nucleotide e atr adapter after ned Vibrio tapetis Vibrio ,

AcceptedThis by is protectedreserved.article Allrights copyright. ROS-B_Scallop LEF-B_Scallop BRE3p_Abalone BRE3m_Abalone BRE2_Abalone BRE1_Abalone NEG_Clam MURp_Clam LAN3p_Clam LAN2p_Clam LAN1p_Clam SELp_Clam MATp_Clam AKKp_Clam ArticleKOR_Clam POS_Clam MODERN name Sample 1. information Table Sample and sequencing statistics.

name name Species philippinarum tuberculata Ruditapes maximus Haliotis Pecten

Roscanvel, France Brest, France Le Faou, France Brest, France Brest, France Landéda, France Murano, Italy Landéda, France Landéda, France Landéda, France Seloy, Norway Matsusaka, Japan Akkeshi, Japan Cheju, Korea location Date/Age Geographical Brest, France Landéda, France 2003 AD 1997 AD 2003 AD 2003 AD 1960-1970 AD 2005 AD 1990 AD 2012 AD 2012 AD 2003 AD 2003 AD 1988 AD 1983 AD 1983 AD 1983 AD 1988 AD microstructure Inner shell A Healthy NA A Healthy NA Weak BRD HA A Healthy NA Weak BRD HA Weak BRD Abnormal brown deposit HA Acute BRD HA HA A ct rw eoi n oig ,8,2 9,123,253 9,184,721 Acute brown deposit and boring NA Healthy HA Brown deposit Brown deposit HA Brown deposit HA HA A eoee fe ct R 3,7,2 33,530,237 36,371,525 Recovered after acute BRD HA C Healthy Healthy FC FC diagnostic #Sequences Shell 05515 20,104,424 20,565,155 07526 10,739,804 10,795,266 13,667,939 13,812,143 10,402,596 10,499,178 18,001,838 18,091,072 34,501,909 35,083,934 03381 50,694,338 50,373,801 ,0,8 7,447,143 7,506,486 ,8,6 7,040,427 7,080,764 9,675,009 9,749,078 8,373,378 8,455,559 ,3,2 9,847,558 9,932,924 7,925,697 7,981,394 9,640,133 9,709,393 #Retained

AcceptedThis by is protectedreserved.article Allrights copyright. auzay1-4B_Clam, gripB_Clam, lmc1-3B_Clam EBC7_Control Extraction blank controls for samples: ves_Quahog quahog2_Quahog hel_Quahog gue_Abalone med_Abalone thi_Clam nis_Clam lmc3B_Clam lmc2B_Clam lmc1B_Clam gripB_Clam Articleauzay4B_Clam auzay3B_Clam auzay2B_Clam auzay1B_Clam hav_Mussel ANCIENT BRE5p_Oyster BRE5m_Oyster

Crassostrea sp. decussatus decussatus tuberculata Mytilus sp. Venerupis Venerupis Ruditapes corrugata islandica Haliotis Arctica

Mediterranean Sea Vestmanna, Faroe Islands Hellebæk, Denmark Thisted Bredning, Denmark Nissum Bredning, Denmark 16-17 La Maison Champlain, Brouage, France 16-17 La Maison Champlain, Brouage, France 16-17 La Maison Champlain, Brouage, France 7 France Gripperie-Saint-Symphorien, La Middle Neolithic 6 Les Châtelliers du Vieil-Auzay, France Havnø, Denmark Brest, France Brest, France sine loc. Guernesey Island, UK Middle Neolithic 6 Les Châtelliers du Vieil-Auzay, France Middle Neolithic 6 Les Châtelliers du Vieil-Auzay, France Middle Neolithic 6 Les Châtelliers du Vieil-Auzay, France 1886AD 1901 AD 1832 AD Before 1804 AD 1909 AD 1886 AD 2005-2007 AD 2005-2007 AD Before 1804 AD Late Mesolithic ~7,000 yBP t h cent. AD t t t h h h cent. AD cent. AD cent. AD 3 , 4 NA 5 5 5 NA NA t t t t h h h h mill. BP

mill. BP mill. BP mill. BP mill. 2 2 2 2 1 NA Healthy NA Healthy NA Healthy NA Healthy NA Healthy A Healthy HA Healthy NA A Healthy Healthy HA HA Healthy NA Healthy Perforations NA NA A Healthy NA C ct rw eoi n oig ,2,2 7,686,285 7,724,821 Acute brown deposit and boring Healthy FC FC

elh 17,964,100 Healthy 12,983,981 Healthy elh 10,555,462 Healthy 8,914,833 8,653,416 30936 12,674,074 13,089,376 11,183,672 10,515,059 12,191,311 13,565,581 12,008,593 11,095,140 13,414,246 10,913,477 88176 18,494,781 18,871,776 13,868,126 13,947,789 11,430,195 11,484,012 15,240,748 15,030,940 09185 10,811,007 10,911,865 ,2,9 9,699,275 9,823,899 8,301,630 8,386,634 ,6,9 8,153,984 8,264,494 8,8 475,317 481,087 17,845,554 12,888,532 10,484,321

foliated foliated calcite; "BRD", RingDisease. Brown "AD", Anno Domini; "yBP",years Before Present; "mill.",millen

This by is protectedreserved.article Allrights copyright. 1 The indicates the "p" alone in thethat shel suffix sample name EBC12_Control MATp_Clam EBC11_Control nis_Clam, and hav_Mussel, gue_Abalone, med_Abalone, ves_Quahog EBC9_Control quahog2_Quahog, thi_Clam MURp_Clam EBC8_Control hel_Quahog Accepted (Andersen 2008); Article 2 (Large & Birocheau 2004); 3 (Gissinger 2011); l fragment showedl fragment of infection; signs "m" no of signs infection ium; "cent.",century; “HA”, homogeneous aragonite; “NA”, nacre 4 (Le Goff & Dupont 2015); 5 (Champagne (Champagne et al. 2012) . . ous aragonite;ous“FC”, 106,077 74,324 9,597,406 8,798,083 9,974,348 3,146,625 7,557,184 3,570,529 Accepted Article This by is protectedarticle reserved. Allrights copyright. "mtDNA",mitochondrial DNA Table 2. Mollusc taxon identification success rates.

ols aa rus eaeois ula N mDAgnms mtDNAbarcodes mtDNAgenomes DNA Nuclear Metagenomics Groups taxaMollusc Abalones Abalones Quahogs Scallops Mussels Ancient Oysters Clams Modern Clams Clams Clams

2 10 0 100 100 100 20 10 10 100 100 0 100 (0) 0 (0) (100) (100) (0) (100) (100) (100) (100) (10)

10 8) 0 (100) (0) (100) (80) (0) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) 5)(0 (0 (50) (50) (50) (50) (100) (90) (33) (100) 0 0 10 0 100 100 0 0 10 100 100 100 0 0 100 0 100 80 02 2 20 20 20 33 3 33 33 33 310 0 100 90 33 100 Success rates across all samples % (sequenced only) (sequenced Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.