Vertical stratificaction of bacterial communities driven by multiple environmental factors in the dark waters off the Galician coast (NW Spain)

1 Ve ical s a i ica ion o bac e ial communi ies d i en by mul iple en i onmen al 1 ac o s in he wa e s (0-5000 m) o he Galician coas (NW Ibe ian ma gin) 2 Vladimi Dobal-Amado 1,2, Ma Nie o-Cid3, Elisa Gue e o-Feijoo1, Vic o He nando-3 Mo ales2, E a Tei a2, Ma a M. Va ela-Rozados1* 4 1 Spanish Ins i u e o Oceanog aphy, Oceanog aphic Cen e o A Co uña, 15001, A 5 Co uña, Spain 6 2 Depa men o Ecology and Animal Biology, Uni e si y o Vigo, 36310, Vigo, Spain 7 3 Ins i u e o Ma ine Resea ch o Vigo, CSIC, 36208, Vigo, Spain 8 * Co espondence o: [email p o ec ed] 9 10 ABSTRACT 11 The p ocesses media ed by mic obial plank onic communi ies occu along he en i e 12 wa e column, ye he mic obial ac i i y and composi ion ha e been s udied mainly in 13 su ace wa e s. This esea ch examined he e ical a ia ion in bac e ial abundance, 14 ac i i y and communi y composi ion and s uc u e om su ace down o 5000 m dep h 15 ollowing a longi udinal ansec o he Galician coas (NW Ibe ian ma gin, om 43ºN, 16 9ºW o 43ºN, 15ºW). Communi y ac i i y and composi ion changed wi h dep h. The 17 leucine inco po a ion a es dec eased om he eupho ic laye o he ba hypelagic wa e s 18 by h ee o de s o magni ude, whe eas p oka yo ic abundance dec eased only by one 19 o de o magni ude. The ela i e abundance o SAR11 and Al e omonas, de e mined by 20 ca alyzed epo ed deposi ion luo escence in si u hyb idiza ion (CARD-FISH), 21 dec eased wi h dep h. Meanwhile, he con ibu ion o SAR 202 and SAR324 was 22 signi ican ly highe in he deepe laye s (i.e. NEADW, No h Eas A lan ic Deep Wa e 23 and LDW, Lowe Deep Wa e ) han in he eupho ic zone. Bac e ial communi y 24 s uc u e, assessed by Au oma ed Ribosomal In e genic Space Analysis (ARISA), was 25 dep h-speci ic. A dis ance based linea model (Dis LM) e ealed ha he a iabili y 26 ound in bac e ial communi y s uc u e was mainly explained by empe a u e ni a e, 27 phospha e, dissol ed o ganic ma e (DOM) luo escence, p oka yo ic abundance, 28 leucine inco po a ion and o a lesse ex en salini y, oxygen, CDOM abso bance and 29 dissol ed o ganic ca bon concen a ion. Ou esul s displayed a bac e ial communi y 30 s uc u e shaped no only by dep h- ela ed physicochemical ea u es bu also by DOM 31 quali y, indica ing ha di e en p oka yo ic axa ha e he po en ial o me abolize 32 pa icula DOM sou ces. 33 2 KEY WORDS: Bac e ia, communi y composi ion, luo escen in si u hyb idiza ion 34 (FISH), Au oma ed Ribosomal In e genic Space Analysis (ARISA), DOM, deep 35 wa e s, NW Ibe ian ma gin 36 INTRODUCTION 37 P oka yo es a e an impo an componen o ma ine plank on, accoun ing o a majo 38 ac ion o he o al plank onic biomass in ma ine wa e s (Gio annoni and Rappé, 39 2000). Mo eo e , hey play an impo an ole as media o s o he main biogeochemical 40 cycles (Fu h man e al., 2015). P oka yo ic abundance and ac i i y dec ease one and 41 wo o de s o magni ude, espec i ely, om he eupho ic o he ba hypelagic zone in he 42 ocean (Naga a e al. 2000, A is egui e al., 2009). Such pa e n is de e mined by he 43 e ical a iabili y in he physical and chemical ea u es o he pelagic en i onmen , 44 which also con ibu e o he e ical s a i ica ion o he bac e ial communi ies 45 associa ed o wa e masses (De Long e al., 2006). In addi ion, se e al s udies a 46 di e en oceanic egions a ound he wo ld ha e shown ha whe eas a conside able 47 ac ion o he bac e ial axa is p esen h oughou he wa e column, se e al ope a ional 48 axonomic uni s (OTUs) a e speci ic o he di e en wa e masses, o e all leading o a 49 wa e mass-speci ic clus e ing o bac e ial communi ies in he da k ealm (Díez e al. 50 2004, Tambu ini e al. 2009, Celussi e al. 2010, Galand e al. 2010, Agogué e al. 2011, 51 Lekunbe i e al. 2013, Mapelli e al. 2013,). 52 P oka yo ic communi y composi ion in he ocean is dep h-s a i ied (DeLong e al., 53 2006; B own e al., 2009), po en ially linked o dep h ela ed changes in he quali y and 54 quan i y o a ailable ene gy sou ces. P e ious s udies in he A lan ic ha e shown ha 55 he abundance o he SAR11 clus e (Alphap o eobac e ia), a slow g owing k-s a egis 56 (Yilmaz e al., 2016), dec eases wi h dep h. By con as he abundance o clus e s like 57 SAR202 (Clo o lexi), which is well adap ed o sca ce ood a ailabili y and o u ilize 58 ecalci an o ganic compounds up ake (Yilmaz e al., 2016), o SAR324 59 (Alphap o eobac e ia) and SAR406 (Del ap o eobac e ia), which possess mul iple genes 60 associa ed wi h a pa icle a ached li es yle (Yilmaz e al., 2016), inc eases wi h dep h 61 (Va ela e al. 2008a; Lekunbe i e al. 2013). O he phylogene ic g oups, such as 62 Al e omonas (Gammap o eobac e ia), show mo e a iable and pa chy dis ibu ions. 63 While Lekunbe i e al. (2013) ound ha he ela i e con ibu ion o Al e omonas 64 inc eased wi h dep h in he opical A lan ic, Scha enho e e al. (2009) showed ha 65 3 Al e omonas was mo e abundan in he eupho ic laye han in mesopelagic wa e s in a 66 la i udinal A lan ic ansec , likely p o i ing om he high abundance phy oplank on 67 s o age molecules (Yilmaz e al., 2016). 68 The e ical a iabili y in he bac e ial communi y composi ion has been shown o 69 co ela e wi h changes in bio ic and abio ic en i onmen al ac o s (Ghiglione e al., 70 2012), indica ing hei con ol on bac e ial popula ions. Recen s udies ha e e ealed 71 ha physical o ces (e.g empe a u e, hyd os a ic p essu e, salini y) a e co ela ed wi h 72 spa ial a ia ion in abundance, ac i i y and di e si y o ma ine mic obial communi ies 73 ( e iewed in Fuh man e al., 2015). Also, p e ious s udies ha e p o ided some 74 e idences ha bac e ial communi y composi ion a ies wi h dissol ed o ganic ma e 75 (DOM) sou ces suppo ing he po en ial s uc u ing e ec o DOM composi ion 76 (Ki chman e al., 2004). Despi e hese ad ances, ou knowledge on he sou ces o DOM 77 in he meso- and ba hypelagic wa e s and he link be ween he composi ion and 78 di e si y o DOM and bac e ial communi ies in he da k ocean is s ill limi ed. 79 Thus, he aim o his s udy was wo old: (i) o cha ac e ize he e ical a iabili y o he 80 abundance, ac i i y, composi ion and s uc u e o he bac e ial communi ies om 81 su ace down o 5000 m dep h along a la i udinal sec ion o NW Spain and (ii) o 82 iden i y he en i onmen al ac o s, including physico-chemical a iables and op ical 83 indices o he DOM, ha bes explain he a ia ion o he bac e ioplank on communi y 84 s uc u e. 85 86 MATERIALS AND METHODS 87 S udy si e and sampling 88 The sampling o he epi- meso- and ba hypelagic wa e s was ca ied ou along a 89 longi udinal ansec o Galicia du ing he oceanog aphic c uise BIOPROF-2 90 (Sep embe 2012) on boa d he R/V Co nide de Saa ed a. In o al, 22 s a ions we e 91 sampled om 43ºN, 9ºW o 43ºN, 15ºW o Cape Finis e e (NW Spain) o he 92 physicochemical cha ac e iza ion, and 6 s a ions we e sampled o he o ganic ma e 93 a iables as well as bac e ial communi y analyses (see Fig. 1). A each s a ion, wa e 94 samples we e collec ed wi h Niskin bo les moun ed on a CTD (conduc i i y-95 empe a u e-dep h) ose e sample om se e al “co e” dep hs, on he basis o di e en 96 empe a u e and salini y cha ac e is ics om each CTD cas : he eupho ic zone (EZ, 0-97 4 100m) he Eas e n No h A lan ic Cen al Wa e (ENACW,250-900m), he laye o he 98 Oxygen Minimum Zone (OMZ); he Medi e anean Wa e (MW, ≈1000m); he 99 Lab ado Sea Wa e (LSW, 1800-2000m); he Eas e n No h A lan ic Deep Wa e 100 (ENADW, 2450-2900m ) and he Lowe Deep Wa e (LDW, ≥4000m). 101 En i onmen al pa ame e s 102 Dissol ed oxygen samples we e aken in py ex “iodine i a ion” lasks wi h la ed 103 necks and g ound glass s oppe s, wi h a nominal olume o abou 115 mL, and 104 measu ed, on boa d a e sampling collec ion, ollowing he Winkle po en iome ic 105 me hod a e Langdon (2010). Aliquo s o ino ganic nu ien s de e mina ion (ni a e, 106 ni i e, ammonium, phospha e and silica e) we e collec ed in insed polye hylene bo les 107 and ozen a -20ºC un il u he analysis by s anda d colo ime ic me hods wi h a B an-108 Luebbe segmen ed low analyse . 109 Wa e samples o he analysis o dissol ed o ganic ca bon (DOC) we e collec ed in 110 glass ampoules, and acidi ied wi h H3PO4 o pH < 2 be o e hea sealing. Samples we e 111 measu ed in a Shimadzu TOC-CSV analyse by high- empe a u e P -ca aly ic oxida ion. 112 Fluo escence in ensi y was measu ed a wo ixed exci a ion/emission wa eleng hs o 113 320 nm/410 nm (FDOM-M), cha ac e is ic o ma ine humic-like subs ances, and o 280 114 nm/350 nm (FDOM-T), cha ac e is ic o p o ein-like ma e ials using a Pe kin Elme 115 LS55 and ollowing Nie o-Cid e al. (2006). The abso p ion spec a o he ch omopho ic 116 DOM we e acqui ed on a Beckman Coul e DU800 spec opho ome e equipped wi h 117 10 cm qua z cells. Spec al scans we e collec ed om 250 o 700 nm, p o iding he 118 ollowing indexes (G een and Blough, 1994): aCDOM 254 (abso p ion coe icien a 119 254 nm), aCDOM 340 (abso p ion coe icien a 340 nm), aCDOM 365 (abso p ion 120 coe icien a 365 nm), and sCDOM 275/295 (slope o he abso p ion spec um be ween 121 275 and 295 nm). 122 123 P oka yo ic abundance 124 The p oka yo ic abundance (PA) was quan i ied by low cy ome y ollowing Gasol e 125 al. (1999). B ie ly, wa e samples (1.8 mL) we e ixed wi h pa a o maldehyde and 126 glu a aldehyde (2-4% inal concen a ion), lash- ozen in liquid N2 o 10 min and 127 s o ed a -80°C un il u he analysis. P io o analysis, he samples we e hawed o 128 oom empe a u e and he p oka yo ic cells s ained wi h Sy o13 in he da k o 10 min. 129 Subsequen ly, luo escen la ex beads (app oxima ely 1 x105 mL-1) (Molecula P obes, 130 5 In i ogen, Ca lsbad, CA) we e added o all he samples as in e nal s anda d. The 131 p oka yo es we e enume a ed using a FACSCalibu low cy ome e (Bec on Dickinson, 132 F anklin Lakes, NJ) acco ding o hei signa u e in igh angle ligh sca e and g een 133 luo escence. 134 135 P oka yo ic leucine inco po a ion a e 136 3H-Leucine inco po a ion a e (Leu inco p.) was de e mined as a p oxy o p oka yo ic 137 p oduc ion (Ki chman, 2001). Subsamples om he eupho ic and lowe meso-pelagic 138 (down o 500 m) we e amended wi h 20 nmol L-1 [3H]-leucine ( inal concen a ion, 139 speci ic ac i i y 160 Ci mmol L-1, GE Heal hca e, Ame sham, Bucks, UK). T iplica e 140 1.2 mL samples and duplica e TCA ( ichlo oace ic acid)-killed blanks (50% inal 141 concen a ion) (Simon and Azam, 1989) we e incuba ed in he da k a in si u 142 empe a u e-con olled chambe s o 2-6 h depending on he expec ed ac i i y. 143 Incuba ions we e e mina ed by adding TCA (50% inal concen a ion) o he samples. 144 Bac e ial p o eins we e p ecipi a ed by wo successi e cen i uga ions (12000 pm, 10 145 min), including a washing s ep wi h 1 mL o 5% TCA acco ding o he me hod o 146 Ki chman (1985) wi h sligh modi ica ions by Smi h and Azam (1992). 147 Leu inco p. o he samples om he ba hy- and mesopelagic wa e s (below 1000m) was 148 measu ed by adding 5 nmol L-1[3H]-leucine ( inal concen a ion, speci ic ac i i y 160 Ci 149 mmol L-1, GE Heal hca e, Ame sham, Bucks, UK) o duplica e 40 mL samples and 150 duplica e o maldehyde-killed blanks (2% inal concen a ion) (Simon and Azam, 151 1989). Samples and blanks we e incuba ed in he da k a in si u empe a u e in 152 empe a u e- con olled chambe s o 10-24 h depending on he expec ed ac i i y. 153 Incuba ions we e e mina ed by adding o maldehyde (2% inal concen a ion) o he 154 samples. A e 10 min, he samples and he blanks we e il e ed on o 0.2 µm 155 polyca bona e il e s (25 mm il e diame e , Millipo e). Subsequen ly, he il e s we e 156 insed h ee imes wi h 10 mL o 5% TCA. The ea e , he il e s we e ans e ed in o 157 scin illa ion ials and d ied a oom empe a u e. 158 Finally, liquid scin illa ion cock ail was added o all samples and a e 18 h, he 159 adioac i i y was de e mined in a scin illa ion coun e (LKB Wallac). The mean 160 disin eg a ions pe minu e (DPM) o he blanks we e sub ac ed om he mean DPM o 161 he espec i e samples and he esul ing DPM con e ed in o leucine inco po a ion 162 a es. 163 Enume a ion o speci ic g oups o Bac e ia by CARD-FISH and FISH 164 6 CARD-FISH was used o de e mine he abundance o speci ic g oups o bac e ia (Table 165 1) ollowing he me hod desc ibed by Pe n hale e al. (2002). Immedia ely a e 166 collec ing he samples om he Niskin bo les, 20-80 mL o wa e we e ixed wi h 167 pa a o maldehyde (2% inal concen a ion) and s o ed a 4°C in he da k. A e 12-18 h, 168 he samples we e il e ed h ough 0.2 µm polyca bona e il e s (Millipo e GTTP, 25-169 mm il e diame e ) suppo ed by ni ocellulose il e s (Millipo e, HAWP, 0.45 µm), 170 washed wice wi h 10 mL Milli-Q wa e , d ied and s o ed in a mic o uge ial a -20°C 171 un il u he p ocessing in he labo a o y. Fil e s we e cu in sec ions and hyb idized 172 wi h speci ic ho se adish pe oxidise (HRP)-labelled oligonucleo ide p obes o he 173 ollowing g oups: Eubac e ia, SAR11 (Alphap o eobac e ia), SAR324 174 (Del ap o eobac e ia), SAR406, and Al e omonas (Gammap o eobac e ia). Signal was 175 ampli ied by adding y amide-Alexa488. FISH wi h Cy3-labeled p obes was used o 176 enume a e he membe s o he SAR202 (Chlo o lexi) clus e ollowing he p o ocol o 177 Mo is e al. (2004). Fil e sec ions we e ca e ully moun ed in slides and coun e s ained 178 wi h wi h a DAPI-mix [5.5 pa s o Ci i luo (Ci i luo ), 1 pa o Vec ashield (Vec o 179 Labo a o ies) and 0.5 pa s o phospha e-bu e ed saline (PBS) wi h 4′,6-diamidino-2-180 phenylindole (DAPI) ( inal concen a ion 2 µg mL-1)]. The p obes and hyb idiza ion 181 condi ions o he indi idual g oups a e gi en in Table 1. Enume a ion o DAPI-s ained 182 cells and cells s ained wi h he speci ic p obes was pe o med unde a Nikon Eclipse 80i 183 epi luo escence mic oscope equipped wi h a Hg lamp and app op ia e il e se s o 184 DAPI, Cy3 and Alexa448. A minimum o 500 DAPI-s ained cells was coun ed pe 185 sample. 186 187 Bac e ioplank on communi y s uc u e by ARISA inge p in ing 188 A olume o 10-15 L o wa e was il e ed h ough s e ile S e i ex 0.22 µm po e size 189 il e s (Millipo e, USA). Subsequen ly, 1.8 mL o lysis bu e (40 mM EDTA, 50 190 mMT is-HCl, 0.75 M suc ose) was immedia ely added o he il e s be o e s o ing hem 191 a -80ºC un il ex ac ion. The DNA ex ac ion began by adding lysozyme (1 mg mL-1, 192 inal concen a ion (FC); SIGMA-ALDRICH) o he S e i ex, ollowed by 30 min 193 incuba ion a 37ºC. Then, p o einase K (0.5 mg mL-1, FC; SIGMA-ALDRICH) and 194 sodium dodecyl sulpha e (SDS) (1%, FC) we e added, ollowed by 2 h incuba ion a 195 55ºC. Lysa e was hen ex ac ed wice wi h phenol-chlo o o m-isoamyl alcohol 196 (25:24:1; sa u a ed wi h 10 mM T is, pH 8.0, 1 mM EDTA; SIGMA-ALDRICH) and 197 once wi h chlo o o m-isoamyl alcohol (24:1; 99% pu i y o molecula biology; 198 7 SIGMA-ALDRICH) a 4500 pm 10 min. The aqueous phase was concen a ed in 199 mic oconcen a o (Amicon wi h Ul acel-100 memb ane; Millipo e), washed wice 200 wi h s e ile wa e , and educed o app oxima ely 200 µL. The eco e ed DNA was 201 quan i ied in a Nand op spec opho ome e . All DNA ex ac ions we e dilu ed o 202 wo king concen a ions o 10 ng μL-1 o empla e DNA, and s o ed a -20ºC. ARISA-203 PCR was conduc ed on a s anda d amoun o DNA on each sample by using he p ime 204 se ITSF, 5´-GTC GTA ACA AGG TAGGCC GTA-3´and ITSReub, 5´-GCC AAG 205 GCA TCC ACC 3´, as p e iously desc ibed (Ca dinale e al., 2004). PCR eac ions 206 (40L) con ained inal concen a ions o 1x PCR bu e (Genec a ), 2.5 mM MgCl2 207 (Genec a ), 250 M o each dNTP (Genec a ), 250 nM o each p ime , 40 ng L-1 208 bo ine se um albumin, 3.5 U o BioThe mD-TM Taq DNA Polyme ase (GeneC a ) 209 and 0.13 ng L-1 o empla e DNA. The eac ion mix u e was held a 94ºC o 2 min, 210 ollowed by 32 cycles o ampli ica ion a 94ºC o 15s, 55ºC o 30 s and 72ºC o 3 211 min, wi h a inal ex ension o 72ºC o 10 min. ARISA agmen s we e sepa a ed using 212 he ABI P ism 3730XL (Applied Biosys ems) gene ic analyze applying he in e nal 213 s anda d LIZ 1200 (20-1200 pb, Applied Biosys ems). Peaks showing a heigh alue 214 <20 luo escence uni s we e emo ed om he ou pu peak ma ix be o e binning. 215 P o ile peaks we e binned and ea anged by ope a ional axonomic uni s (OTUs), by 216 using R au oma ic and in e ac i e binning sc ip s as ollows. The o al peak a ea pe 217 sample was no malized o one in o de o accoun o un- o- un a ia ions in signal 218 in ensi y (Yanna ell and T iple , 2005) and each ARISA peak was de ined as a di e en 219 ope a ional axonomic uni (OTU). 220 S a is ical analysis o bac e ial communi ies 221 The mic obial communi y da ase was co ela ed wi h he physico-chemical and he 222 DOM- ela ed da ase (see below o a iables included in each da a se ) by a 223 compa a i e Man el ype es , using he RELATE unc ion o he so wa e PRIMER 6. 224 Hie a chical Clus e Analysis was ca ied ou o explo e he bac e ioplank on 225 communi y s uc u e (simila i ies be ween samples), based on he esemblance ma ix 226 gene a ed using B ay Cu is simila i y on he p esence/absence o he OTUs wi hin each 227 sample. Signi ican di e ences in mic obial communi y s uc u e among wa e masses 228 we e in es iga ed by pe mu a ional analysis o a iance (PERMANOVA, Ande son 229 2001). Fu he mo e, pa simonious models we e buil o iden i y he bes ac o s 230 explaining a ia ion in he communi y s uc u e (Dis LM, Ande son e al., 2008). Th ee 231 8 se s o a iables we e conside ed o s udy he bac e ioplank on communi y s uc u e: (i) 232 physico-chemical a iables: empe a u e, salini y, oxygen, ni a e, phospha e, silica e; 233 (ii) DOM- ela ed a iables: DOC, FDOM-T, FDOM-M, aCDOM 254, aCDOM 340, 234 aCDOM 365, sCDOM 275/295; and (iii) mic obial communi y a iables: PA, Leu 235 inco p. P e iously, all a iables we e analyzed o es he colinea i y be ween hem 236 using a Pea son co ela ion ma ix, elimina ing he a iables wi h 2 > 0.95. In o de o 237 assign he con ibu ion o each a iable and each se o a iables aken alone (physico-238 chemical, o ganic ma e - ela ed and mic obiological), an “all speci ied” selec ion 239 p ocedu e was ca ied ou using he “ 2” as selec ion c i e ion. The con ibu ion o each 240 a iable was assessed using “ma ginal es s” o assess he s a is ical signi icance and 241 pe cen age con ibu ion o each a iable and each se o a iables aken alone. Finally, 242 all a iables we e in oduced in he model using he “s ep wise” selec ion p ocedu e o 243 he Dis LM and he “Akaike” in o ma ion c i e ion (AIC). Such p ocedu e allowed us o 244 ind he bes combina ion o en i onmen al a iables ha explained he highes 245 p opo ion o a iabili y ound in he bac e ioplank on communi y s uc u e 246 esemblance ma ix. A “sequen ial es ” was employed o e alua e he cumula i e e ec 247 o each a iable once he p e ious a iable (s) had been accoun ed o . A dis ance-based 248 edundancy analysis (dbRDA) was used o g aphical isualiza ion o he Dis LM 249 esul s. All analyses we e pe o med, using he so wa e packages PRIMER6 & 250 pe mano a+ (Ande son e al., 2008). 251 252 RESULTS 253 En i onmen al cha ac e is ics 254 Physical and chemical cha ac e is ics o he main wa e masses a e summa ized in Table 255 2. The LDW was ound below 4000 m dep h, cha ac e ized by low salini y (34.9), low 256 empe a u e (2.5ºC) and high silica e concen a ions (32.8-44.9 µmol kg-1). The 257 ENADW was clea ly iden i iable be ween 2450 and 2900 m dep h. ENADW di e ed 258 om LDW in showing sligh ly highe empe a u e (2.5-3.5ºC) and highe oxygen 259 con en (Table 2). Two ypes o in e media e wa e s we e ound; he LSW (1800-2000 260 m) ha showed a minimum o salini y (35.0-35.4) and ela i ely high oxygen 261 concen a ion (197.5-262.8 µmol kg-1), and MW (1000 m) ha was clea ly iden i iable 262 by high salini y (35.0-36.2). OMZ was loca ed a abou 900 m displaying oxygen 263 concen a ions a ound 180-241 µmol kg-1. Rega ding DOC concen a ions, he a e age 264 9 alues dec eased p og essi ely om he shallowe dep hs (68.8 and 55.1 µmol L-1, o 265 he EZ and ENACW-OMZ, espec i ely) o he deepe wa e masses (50.8, 48.5, 46.1, 266 42.7 µmol L-1 o MW, LSW, ENADW and LDW, espec i ely) (Fig. 2) 267 Dep h dis ibu ion pa e n o DOM- ela ed a iables 268 Dissol ed o ganic ca bon anged om ~70 µmol C L-1 a he su ace wa e s o 40-45 269 µmol C L-1 in he deepe wa e masses (Fig. 2A). In he same way, FDOM-T dec eased 270 om ~1.0 o 0.4 QSU (Fig. 2C). On he con a y, FDOM-M inc eased wi h dep h, 271 displayed lowe alues a su ace (~0.7 QSU) han a he deep laye s (1.0 QSU, Fig. 272 2B). Rega ding he op ical indexes o he colou ed DOM, in gene al hey exhibi ed a 273 dec ease wi h dep h, mo e signi ican ly o he aCDOM 254 (Fig. 2). 274 275 Dep h dis ibu ion pa e n o p oka yo ic a iables 276 P oka yo ic abundance (PA) anged om 1.1 x 105 o 4.7 x 105 cell mL-1 in he eupho ic 277 zone, and dec eased exponen ially wi h dep h a all s a ions. The minimum alues we e 278 ound in he ENADW ( anging om 1.5 x 104 o 2.7 x 104 cell mL-1; Fig. 3A). The 279 leucine inco po a ion a e (Leu inco p.) showed a simila e ical end, dec easing h ee 280 o de s o magni ude om he EZ (14.2 ± 8.3 pmol Leu L-1 h-1) o he LDW (6.7 ± 3.8 x 281 10 -3 pmol Leu L-1 h-1; Fig. 3B). Cell-speci ic ac i i y (p oka yo ic Leu inco p. di ided 282 by PA) dec eased also wi h dep h. Maximum cell-speci ic ac i i y was ound in he 283 eupho ic laye (1.9 ± 1.3 x 10-3 mol cell-1 d-1) (Fig 3). In he meso- and ba hypelagic he 284 minimum cell-speci ic ac i i y was measu ed a he LDW (4.7 x 10-6 mol cell d-1) and 285 he maximum a he MW (1.7 ± 1.0 x 10-3 mol cell d-1). 286 Rela i e abundance o speci ic g oups o Bac e ia 287 Speci ic phylogene ic g oups o Bac e ia showed di e en pa e ns o dis ibu ion 288 acco ding o he wa e mass (Table 3). While SAR 11 and Al e omonas con ibu ion o 289 o al bac e ial abundance was signi ican ly highe in he EZ (ANOVA-Tukey, p < 290 0.0001) han in deepe wa e s, SAR202 ela i e abundance was signi ican ly highe in 291 he LDW, LSW and ENADW (Tukey, ANOVA, p < 0.0001) han in he EZ. The 292 abundance o SAR324 inc eased sligh ly wi h dep h (ANOVA-Tukey, p > 0.05). 293 Con e sely, he abundance o SAR406 a ied be ween 12.6-17.0% and we did no ind 294 signi ican di e ences among wa e masses (ANOVA-Tukey, p < 0.05). 295 Bac e ial communi y s uc u e de e mined by ARISA inge p in ing 296 16 he sampling and analysis was suppo ed by he Local Resea ch P og am o Xun a de 492 Galicia g an “Biodi e sidade Funcional do Mic oplanc on nas p o undidades ma iñas 493 de Galicia” (BIO-PROF, Re . 10MMA604024PR, 2010-2012) and “Relación en e la 494 di e sidad uncional del bac e ioplanc on y la ci culación de ca bono en el océano 495 cos e o en el con ex o del cambio global” (DIFUNCAR, Re . CTM2008-03790). V. D-496 A. was unded by a Campus do Ma Mas e Schola ship. M.N.-C. was unded by he 497 CSIC P og am “Jun a pa a la Ampliación de Es udios” co- inanced by he ESF. 498 He nando-Mo ales V. was suppo ed by he MICINN p og am “Fo mación de Pe sonal 499 In es igado ” (FPI), Re . g an BES-2009-028186. 500 501 502 503 17 FIGURE LEGENDS 504 Figu e 1. Ba hyme ic map o NW o Ibe ian Peninsula and loca ion o sampling 505 s a ions o Cape Finis e e du ing he oceanog aphic c uise BIOPROF-2. Numbe s 506 show he s a ions whe e he abundance, leucine inco po a ion and bac e ial communi y 507 composi ion and s uc u e we e de e mined (mic obiological s a ions). 508 Figu e 2. A e age e ical p o iles o dissol ed o ganic ca bon (A), humic-like 509 luo escence (B), p o ein-like luo escence (C), abso p ion coe icien a 254 nm (D), 510 abso p ion coe icien a 365 nm (E), and op ical slope be ween 275 and 295 nm (F) 511 ound in mic obiological s a ions along he Finis e e sec ion. Samples we e g ouped by 512 wa e masses. 513 Figu e 3. A e age e ical p o iles o p oka yo ic abundance (A), leucine inco po a ion 514 a e (B) and cell-speci ic ac i i y (C) ound in mic obiological s a ions along he 515 Finis e e sec ion. Samples we e g ouped by wa e masses. E o ba s ep esen SE o 2-516 14 measu emen s. 517 Figu e 4. Clus e ing o indi idual samples based on B ay Cu is simila i y ma ix 518 ob ained by ARISA inge p in ing o bac e ial communi ies. Di e en symbols deno e 519 wa e masses; illed squa es co esponding o EZ samples, iangles co esponding o 520 ENACM-OMZ samples, ci cles co esponding o MW samples, c osses co esponding 521 o LSW samples, plusses signs co esponding o ENADW samples and as e isks 522 co esponding o LDW samples. Fo wa e mass abb e ia ions, see Table 2. Di e en 523 line-boxes delinea e di e en clus e s. Di e en lines and le e s deno e g oups. 524 Figu e 5. (A) P incipal componen analysis pe o med on he bio ic and abio ic 525 en i onmen al da a. (B) Dis ance-based edundancy analysis (dbRDA) o dina ion o 526 he i ed model o bac e ial communi y s uc u e based on B ay-Cu is simila i y o 527 ARISA p esence/absence da ase o e laid wi h he pa ial co ela ions o he es ed 528 bio ic and abio ic en i onmen al a iables explaining he clus e o samples. Fo wa e 529 mass abb e ia ions see Table 2. 530 531 18 532 TABLES 533 534 535 Table 1. P obe and he hyb idiza ion condi ions used in he (CARD-) FISH 536 537 P obe O ganism Sequence (5’ – 3’) % FA Re e ence Eub 338 Bac e ia GCT GCC TCC CGT AGG AGT 55 Amann e al., 1990 Eub 338 II Bac e ia GCA GCC ACC CGT AGG TGT 55 Daims e al., 1999 Eub 338 III Bac e ia GCT GCC ACC CGT AGG TGT 55 Daims e al., 1999 NON 338 Nega i e con ol ACT CCT ACG GGA GGC AGC 55 Wallne e al., 1993 SAR 11 -152 SAR 11 ATT AGC ACA AGT TTC CYC GTGT 45 Mo is e al., 2002 SAR 11 -441 SAR 11 TAC AGT CAT TTT CTT CCC CGAC 45 Mo is e al., 2002 SAR 11 -542 SAR 11 TCC GAA CTA CGC TAG GTC 45 Mo is e al., 2002 SAR 11 -732 SAR 11 GTC AGT AAT GAT CCA GAA AGYTG 45 Mo is e al., 2002 SAR 202 -104 SAR 202 GTT ACT CAG CCG TCT GCC 35 Mo is e al., 2004 SAR 202 -312 SAR 202 TGT CTC AGT CCC CCT CTG 35 Mo is e al., 2004 SAR 324 -1412 SAR 324 GCC CCT GTC AAC CTC CAT 35 Scha enho e e al., 2009 SAR 406 -97 SAR 406 CAC CCG TTC GCC AGT TTA 65 Fuchs e al., 2005 Al 1413 Al e omonas, Colwellia TTT GCA TCC CAC TCC CAT 55 Eile s e al., 2000 % FA, pe cen o mamide 538 539 540 541 19 Table 2. Physical and chemical cha ac e is ics o he wa e masses sampled along he Finis e e 542 sec ion (43 º N 9.3 º W o 43 º N 14.9 W). The maximum and minimum alues a e showed. LDW, 543 Lowe Deep Wa e ; ENADW, Eas e n No h A lan ic Deep Wa e ; LSW, Lab ado Sea Wa e ; MW, 544 Medi e anean Wa e ; ENACW-OMZ, Eas e n No h A lan ic Cen al Wa e - Oxygen Minimum Zone; 545 Temp, empe a u e; Sal, salini y; Oxy, oxygen; NO3, ni a e; PO4, phospha e; SiO3, silica e. 546 Wa e mass Dep h (m) Temp. (ºC) Sal. Oxy (µmol kg-1) NO3 (µmol kg-1) PO4 (µmol kg-1) SiO3 (µmol kg-1) EZ ≤ 100 12.8-19.9 35.7-35.9 189.8-269.2 0.1-8.7 0.1-0.7 0.2-4.4 ENACW-OMZ 250-900 10.1-12.8 35.6-36.1 180.5-241.8 7.3-18.2 0.5-1.0 2.1-7.9 MW 1000 3.6-11.4 35.0-36.2 180.6-261.7 13.0-19.2 0.7-1.3 6.6-12.7 LSW 1800-2000 3.6-6.3 35.0-35.4 197.5-262.8 15.5-19.5 1.0-1.3 10.8-16.6 ENADW 2450-2900 2.5-3.5 34.9-35.0 235.1-253.4 14.6-23.0 1.0-1.5 10.6-34.1 LDW ≥ 4000 2.5 34.9 232.0-236.5 18.3-23.2 1.4-1.6 32.8-44.9 547 548 549 550 20 Table 3. A e age ± SD (mean ± s anda d de ia ion) o he con ibu ion o Bac e ia o he o al 551 p oka yo ic communi y (% o DAPI coun s) and speci ic Bac e ial g oups o he o al bac e ial 552 abundance (% o Eubac e ia coun s) de e mined by (CARD)-FISH in he di e en wa e masses o 553 he Finis e e sec ion. (EZ, n = 8; ENACW-OMZ, n=8 MW, LSW and ENADW,n=4; LDW, n = 554 2). 555 Wa e mass Eubac e ia SAR 11 SAR324 SAR406 Al e omonas SAR 202 (% DAPI coun s) (% Eubac e ia coun s) EZ 65.0 ± 10.2 31.8 ± 5.6 12.3 ± 4.4 12.6 ± 2.9 31.4 ± 3.1 7.4 ± 4.0 ENACW-OMZ 54.6 ± 4.3 27.2 ± 9.2 14.7 ± 4.1 13.5 ± 3.2 28.7 ± 5.0 13.3 ± 5.6 MW 50.1 ± 2.1 20.5 ± 5.6 16.1 ± 2.0 14.3 ± 3.4 21.5 ± 6.8 26.0 ± 5.5 LSW 45.6 ± 1.8 12.3 ± 2.1 22.3 ± 7.2 16.1 ± 2.7 13.3 ± 2.2 33.7 ± 7.6 ENADW 42.1 ± 5.8 12.0 ± 5.2 19.2 ± 8.0 17.0 ± 4.4 13.8 ± 1.7 36.5 ± 4.1 LDW 49.1 ± 8.0 7.7 ± 1.4 23.4 ± 0.3 15.0 ± 5.6 12.8 ± 4.5 40.5 ± 12.3 * Fo wa e mass abb e ia ions see Table 2 556 557 21 Table 4. Resul s o Dis LM model ob ained wi h ‘‘all speci ied” a iables as 558 selec ion p ocedu e and R2 as selec ion c i e ion, showing he ma ginal es s on each 559 a iable, wi h he signi icance (p- alue), he p opo ion o a iabili y explained o each 560 a iable (P op) alone, and he o al a ia ion explained o each se o a iables alone 561 (% Va ). 562 563 Ma ginal es Se Va iable p - alo P op % Va Physico-chemical Temp 0.001 0.2934 50.4 Physico-chemical Sal 0.001 0.1560 Physico-chemical Oxy 0.001 0.1257 Physico-chemical NO3 0.001 0.3196 Physico-chemical SiO3 0.001 0.1868 Physico-chemical PO4 0.001 0.3020 O ganic ma e FDOM-M 0.001 0.2781 38.1 O ganic ma e FDOM-T 0.001 0.1701 O ganic ma e aCDOM 254 0.001 0.0889 O ganic ma e aCDOM 340 0.007 0.0416 O ganic ma e aCDOM 365 0.020 0.0364 O ganic ma e sCDOM 275/295 0.001 0.0718 O ganic ma e DOC 0.001 0.0230 Mic obiological PA 0.001 0.2635 28.3 Mic obiological Leu inco p. 0.001 0.1933 564 565 22 Table 5. 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