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. Resul s o Dis LM model ob ained wi h he bes explana o y en i onmen al 566
a iables on he basis o AIC as selec ion c i e ion, showing he sequen ial es s, wi h 567
he signi icance (p- alue), he p opo ion o a iabili y explained o each a iable 568
(P op) and he cumula i e pe cen age a iance (Cumul). 569
570
Sequen ial es
Va iable
Pseudo-F
p- alue
P op.
Cumul.
NO3
33.833
0.001
0.3196
0.3196
SiO3
12.423
0.001
0.1013
0.4209
Temp
5.0704
0.001
0.0802
0.5011
PA
3.3167
0.001
0.0513
0.5524
Sal
2.6607
0.008
0.0502
0.6026
FDOM-T
2.2483
0.047
0.0489
0.6515
Leu inco p.
2.1611
0.012
0.0209
0.6724
aCDOM 365
2.0241
0.020
0.0208
0.6932
sCDOM275/295
1.3583
0.024
0.0102
0.7034
23
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