1
Response o phy oplank on o enhanced a mosphe ic and i e ine nu ien inpu s 1
in a coas al upwelling embaymen 2
Teixei a, I.G.1,3*; A bones, B.1; F oján, M.1; Nie o-Cid, M.1; Ál a ez-Salgado, X.A.1; 3
Cas o, C.G.1; Fe nández, E.2; Sob ino, C.2; Tei a, E.2; Figuei as, F.G.1 4
1 CSIC, Ins i u o de In es igacións Ma iñas. Edua do Cabello 6, 36208 Vigo. Spain. 5
2 Uni e sidade de Vigo, Campus Lagoas-Ma cosende, 36310 Vigo, Spain. 6
3 CESAM – Cen e o En i onmen al and Ma ine S udies & Depa men o Biology, 7
Uni e si y o A ei o, Campus de San iago, 3810‑193 A ei o, 8
Po ugal. 9
*Co esponding au ho : [email p o ec ed] 10
KEYWORDS: phy oplank on, nu ien s, a mosphe ic deposi ion, i e ine inpu s, 11
upwelling sys em, NW Spain, Ria de Vigo 12
13
ABSTRACT 14
O e he pas decades, as a consequence o human ac i i y, he e was an inc ease in 15
nu ien inpu s o he ocean and hey a e expec ed o enhance e en mo e in he u u e. 16
Coas al a eas, accoun ing o a signi ican p opo ion o ma ine p ima y p oduc i i y, 17
a e he mos ulne able zones o an h opogenic impac s. The esponse o phy oplank on 18
communi ies o an inc ease in o ganic and ino ganic nu ien s le els om na u al 19
alloch honous sou ces was assessed in mic ocosm expe imen s conduc ed in a coas al 20
sys em a ec ed by in e mi en upwelling e en s (Ría de Vigo, NW Ibe ia). Th ee 21
nu ien addi ion expe imen s we e pe o med in sp ing, summe and au umn, when 22
2
su ace wa e was supplemen ed wi h 5 and 10% o a mosphe ic and i e ine ma e . 23
Pico-, nano- and mic ophy oplank on abundances, chlo ophyll a concen a ion (Chl a) 24
and p ima y p oduc ion a es (PP) we e measu ed and compa ed wi h hose in he 25
con ol seawa e sample (wi hou addi ions) a e 48 h o incuba ion. Simul aneous 26
expe imen s wi h con olled addi ions o ino ganic and o ganic nu ien s we e also 27
pe o med in o de o desc ibe he limi ing nu ien o phy oplank on g ow h a each 28
expe imen . The composi ion o he ma e inpu s and he s uc u e o he phy oplank on 29
communi ies de e mined he ype o esponse obse ed. Phy oplank on esponses a ied 30
among seasons, being posi i ely co ela ed wi h dissol ed ino ganic ni ogen (DIN) 31
concen a ions. As expec ed, he phy oplank on esponses o ex e nal nu ien inpu s 32
we e s onge unde low nu ien le els (summe ) han when phy oplank on was al eady 33
g owing in nu ien eple e condi ions (sp ing). Null and nega i e esponses o he 34
na u al inpu s we e obse ed in au umn, which sugges s ha he oceanic phy oplank on 35
ad ec ed o his coas al sys em du ing downwelling e en s could be occasionally 36
inhibi ed by hese nu ien inpu s. In a u u e global change scena io, cha ac e ized by 37
enhanced nu ien inpu s om i e ine and a mosphe ic o igin, he esponse o 38
phy oplank on communi ies will s ongly depend on he concen a ion and chemical 39
composi ion o hese inpu s and on he s uc u e o phy oplank on communi ies able o 40
espond o hem. 41
42
1. In oduc ion 43
Phy oplank on o ganisms a e he main p ima y p oduce s in he sea being 44
esponsible o abou 50% o he wo ld p ima y p oduc ion (Field e al. 1998). Coas al 45
a eas, despi e ep esen ing only 7% o ocean’s su ace, accoun o 15-20% o global 46
3
ma ine p ima y p oduc i i y (Wollas 1993, 1998; La uelle e al. 2009). Enhanced 47
nu ien luxes om con inen al uno , he a mosphe e and he adjacen open ocean, 48
and he e icien nu ien ecycling based on a closed coupling be ween pelagic 49
p oduc ion and ben hic egene a ion a e he easons behind he high p oduc i i y o he 50
coas al zone (Walsh 1991; Wollas 1998). All hese biogeochemical p ocesses a e 51
specially in ensi ied in coas al upwelling egions because o he enhanced en y o 52
nu ien s om he adjacen ocean in esponse o in ense and pe sis en equa o wa d 53
winds (Walsh 1991; Wollas 1998). 54
Due o i s close p oximi y o human popula ions, coas al a eas a e also he mos 55
ulne able zones o an h opogenic o cing (Jickells 1998). Ino ganic and o ganic 56
nu ien s and pollu an s esul ing om human ac i i ies each coas al a eas h ough 57
a mosphe ic deposi ion, con inen al uno and g oundwa e e luen s (Jickells 1998; 58
Doney 2010; S a ham 2012). O e he pas decades, he e was an inc ease in hese 59
nu ien inpu s o he ocean and hey a e expec ed o expand e en mo e in he u u e 60
(Ande son e al. 2002; Galloway and Cowling 2002; Galloway e al. 2004; Duce e al. 61
2008). Al e a ions in he magni ude and composi ion o ma e inpu s in coas al a eas 62
may induce signi ican changes in phy oplank on communi ies inhabi ing hose sys ems 63
(Pae l 1997; Peie ls and Pae l 1997; Sei zinge and Sande s 1999; Spa ha is e al. 2007). 64
Ex eme cases o nu ien en ichmen may in ol e he de elopmen o massi e 65
phy oplank on blooms, including ha m ul algal species (Pae l 1997; Ande son e al. 66
2002). 67
The Ría de Vigo is a p oduc i e embaymen in he coas al upwelling sys em o 68
he No hwes Ibe ian Peninsula (F aga 1981). Seasonal pa e ns in phy oplank on 69
composi ion and p oduc i i y in his sys em a e closely ela ed o he hyd og aphic 70
a iabili y (Figuei as and Ríos 1993; Figuei as e al. 2002). Upwelling e en s, occu ing 71
4
usually be ween Ma ch and Oc obe , in oduce nu ien ich subsu ace wa e s in o he 72
pho ic laye and a e ollowed by high concen a ions o phy oplank on cells and 73
chlo ophyll. On he con a y, he win e pe iod is a o able o downwelling and i is 74
cha ac e ized by lowe biomass accoun ed o small-size phy oplank on cells (Figuei as 75
e al. 2002). Ri e discha ge and a mosphe ic deposi ion may also cons i u e signi ican 76
sou ces o nu ien inpu s o his coas al embaymen , al hough o a lesse ex en as 77
compa ed wi h upwelling episodes (Gago e al. 2005; Rod íguez and Macías 2006; 78
Alonso-Pé ez and Cas o 2014; Fe nández e al. in 2016). The high p oduc i i y 79
cha ac e is ic o his sys em sus ains an in ensi e mussel p oduc ion and signi ican 80
ca ches o se e al ish and shell ish species, which a e ele an o he economy o he 81
egion (Figuei as e al. 2002; F oján e al. 2014). Thus, unde s anding he esponse o 82
he o ganisms in he basis o he ood web o an e en ual inc ease in nu ien s inpu s in 83
he Ría de Vigo may be de e minan o an icipa e i s consequences o he whole 84
ecosys em. 85
The ou comes o an inc ease in nu ien s on coas al mic obial plank on in his 86
sys em was es ed in p e ious expe imen al s udies, which ocused on he e ec o 87
con olled ino ganic and o ganic nu ien addi ions (Ma ínez-Ga cía e al. 2010; Tei a 88
el al. 2011) and na u al addi ions o ainwa e (Tei a e al. 2013; Ma ínez-Ga cía e al. 89
2015). In he p esen s udy, we aimed a examining he esponse o phy oplank on 90
communi ies in he Ría de Vigo o ealis ic inc easing amoun s o dissol ed ma e 91
inpu s om bo h a mosphe ic and i e ine o igin. To calcula e he cu en a e age 92
i e ine and a mosphe ic inpu s o he Ría de Vigo we conside ed he a e age i e low 93
o he Ri e Oi abén-Ve dugo, 17 m3 s–1 (Gago e al. 2005) and he a e age 94
p ecipi a ion o he Ría de Vigo, 7.7 mm d–1. Conside ing he su ace a ea o he ía is 95
(174 km2), he mean su ace mixing laye (2 m) and he a e age lushing ime o his 96
5
laye (5 days), he su ace mixing laye o he ía mus con ain abou 2% o i e and 97
2% o ainwa e . The e o e, he addi ions o 5% and 10% es ed he e would se e o 98
ob ain he esponse o he Ría de Vigo o u u e global change scena ios in which 99
human ac i i ies inc ease he quan i y wi hou al e ing he quali y o i e ine and 100
a mosphe ic inpu s. Fu he mo e, concomi an expe imen s wi h con olled addi ions o 101
ino ganic and o ganic nu ien s we e also pe o med in o de o desc ibe he limi ing 102
nu ien o phy oplank on g ow h a each season. 103
104
2. Ma e ials and Me hods 105
2.1 Sampling 106
Seawa e o he expe imen s was sampled om he middle o he Ría de Vigo 107
(42º14.09’ N, 8º47.18’ W) du ing he p oduc i e season in sp ing (May) and summe 108
(July), and a e he ansi ion o he unp oduc i e pe iod in au umn (Oc obe ) 2013 109
(Fig. 1). Tempe a u e, salini y and in si u luo escence down o 25 m dep h we e 110
ob ained wi h a SBE 9/11 CTD p obe and a Sea ech luo ome e a ached o a ose e 111
sample . Sub-su ace seawa e (3-4 m) was collec ed in 12 L acid-clean Niskin bo les 112
and il e ed h ough a 200 µm po e size mesh o emo e la ge zooplank on in o a la ge 113
acid-clean ca boy o ans e i o he coas al s a ion (Fig. 1) o p epa e he addi ion 114
expe imen s. 115
116
2.2 Ekman anspo and uno 117
The Ekman anspo (Qx; m2 s−1) pe pendicula o he coas , a p oxy o he 118
occu ence and in ensi y o coas al upwelling, was calcula ed om wind di ec ion and 119
eloci y, eco ded a Cabo Sillei o buoy (Fig. 1) acco ding o Bakun (1973): 120

6
Ría de Mu os
Ría de A ousa
Ría de Pon e ed a
A
Cape Finis e e
Ría de Vigo
-10.0 -9.5 -9.0 -8.5 -8.0
Longi ude (E)
42.0
42.5
43.0
43.5
La i ude (N)
Sillei o
Buoy
-8.9 -8.8 -8.7
Longi ude (E)
42.1
42.2
42.3
42.3
42.4
La i ude (N)
75m
20m
40m
B
Ría de Vigo
+
IIM - CSIC
X Ci y Hall
Coas al s a ion
𝑄𝑥= 𝜌𝑎∗𝐶∗ [𝑉]∗𝑉
𝑦
𝑓∗𝜌𝑤 (1) 121
whe e ρa is he ai densi y (1.22 kg m−3), C is an empi ical d ag coe icien (1.3 × 10−3, 122
dimensionless), [V] is he wind speed (m s−1) wi h componen Vy, ƒ is he Co iolis 123
pa ame e a his la i ude (9.95 × 10−5 s−1), and ρw is he densi y o seawa e (~1025 kg 124
m−3). The sign o Qx was changed o associa e posi i e alues wi h o sho e anspo 125
(upwelling) o su ace wa e s. 126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
Fig. 1: (A) Map o he wes Galician coas wi h he loca ion o he Sillei o buoy (◆) and (B) 144
map o he Ría de Vigo wi h he loca ion o he sampling s a ion (●), he i e Oi abén-Ve dugo (▲) and 145
he me eo ological s a ions in he IIM (+) and in he Ci y Hall (X). The coas al s a ion whe e addi ion 146
expe imen s ook place is indica ed by an a ow. 147
148
7
Runo was calcula ed using da a om Ri e Oi abén-Ve dugo (Fig. 1), acco ding o he 149
me hod desc ibed in O e o e al. (2010). 150
151
2.3 P epa a ion o na u al wa e s concen a es o he addi ion expe imen s 152
Ri e and ainwa e samples, and <10 µm a mosphe ic pa icles we e collec ed 153
and p ocessed o ob ain concen a es o he i e ine and a mosphe ic ma e inpu s o he 154
Ría de Vigo. The objec i e was o educe he olume o he o iginal wa e samples 10-155
old main aining hei chemical composi ion, i.e. wi hou addi ion o loss o any 156
componen p esen in he na u al samples. In his way, i was possible o es he e ec 157
o an inc ease in na u al ma e using small wa e olumes and wi hou signi ican 158
changes in salini y. 159
The Ri e Oi abén-Ve dugo (Fig. 1) was sampled a week be o e he addi ion 160
expe imen s in Ap il, July and Oc obe 2013. The wa e samples we e collec ed 161
ups eam o he eshwa e -seawa e in e ace, o gua an ee ha he chemical 162
composi ion o he i e samples ep esen ed ha o he wa e ha mixes wi h he 163
seawa e o he Ría de Vigo. Fi e li e s o each sample we e g a i y il e ed h ough a 164
p e-washed (wi h 10 L o ul apu e wa e ) dual-s age (0.8 and 0.2 µm) il e ca idge 165
(Pall-Ac opak supo Memb ane). The il a e was hen concen a ed 10- old using 166
o a o y e apo a ion wi h a Buchi R215 e apo a o unde mild condi ions (ba h 167
empe a u e: 25 ºC, acuum: 13 mba , condense : ace one/CO2) o a oid b eakage o 168
any o ganic compound p esen in he o iginal wa e samples. Analysis o he 169
concen a ion o ino ganic (ammonium, ni i e, ni a e and phospha e) and o ganic 170
(dissol ed o ganic ca bon and ni ogen) subs a es con i med ha he samples we e 171
concen a ed quan i a i ely bu main aining hei o iginal composi ion. 172
8
A MTX ainwa e sample (model FAS005AB) and a high olume PM10 MCV 173
PM1025 sample (model CAV-A/MS) ins alled a he oo o he Ins i u o de 174
In es igaciones Ma inas (IIM-CSIC) (Fig. 1) collec ed samples o we and d y 175
deposi ion o he Ría de Vigo. The MTX sample was equipped wi h a humidi y senso 176
opening he sys em only when i was aining and allowing sampling jus he we 177
ac ion o he a mosphe ic deposi ion. Rainwa e was collec ed om ou weeks o one 178
week be o e he addi ion expe imen s. Samples we e aken daily and ozen 179
immedia ely a e collec ion. A week be o e he expe imen , he daily samples we e 180
hawed a ambien empe a u e, mixed in one olume (6 L), and quan i a i ely 181
concen a ed ollowing he same p ocedu e as o he i e ine samples. Rainwa e was 182
collec ed only o he expe imen s in sp ing (May) and au umn (Oc obe ) 2013 because 183
we deposi ion was e y sca ce he weeks be o e he summe expe imen in July (36 184
mm accumula ed om 11 June o 10 July; me eo ological s a ion o he Vigo ci y hall – 185
Fig. 1). The high olume sample was used o collec a mosphe ic pa icles (1−10 µm) 186
on p ecombus ed (450 ºC, 4 h) 140 mm GF/F il e s (48 h sampling). The pa icles we e 187
collec ed he week be o e each o he h ee addi ion expe imen s, ope a ing du ing 48 h 188
a a a e o 30 m3 h–1. Then, he wa e -soluble ac ion (WSF) o one eigh h o he il e 189
was ex ac ed in 400 mL o he co esponding ainwa e concen a e by mechanical 190
s i ing du ing 40 min. In he summe expe imen , he WSF was ex ac ed in milli-Q 191
wa e . These p opo ions (1/8 o he il e in 400 mL o wa e ) we e decided o ob ain 192
10- old he expec ed concen a ions based on p e ious in o ma ion abou he 193
composi ion o we and d y deposi ion o he Ría de Vigo (Tei a e al. 2013; Ma ínez-194
Ga cía e al. 2015). Final mixed ex ac s we e il e ed h ough p ecombus ed (450 ºC, 4 195
h) 47 mm diame e Wha man GF/F il e s in an acid-cleaned glass il a ion sys em, 196
unde low N2 low p essu e, o be chemically cha ac e ized and used in he expe imen s 197
9
NATURAL
Ri e ine A mosphe ic
0%
5%
10%
Con ol
Ino ganic
O ganic
Mixed
CONTROLLED
as a mosphe ic concen a e. Simila ly o i e ine concen a es, quan i a i e 198
concen a ion was main ained excep o he silica e because he educ ion o he 199
ainwa e olume was ca ied ou in a glass o a y e apo a o and he a mosphe ic 200
pa icles we e collec ed on o a glass ibe il e . 201
202
2.4 Na u al and con olled addi ion expe imen s 203
Fo bo h na u al and con olled addi ion expe imen s, 4 L UV- anspa en Whi l-204
pak® bags we e gen ly illed unde dim ligh condi ions un il 2 L o capaci y. Fo he 205
na u al addi ion expe imen s, i e ine and a mosphe ic concen a es we e added o 206
subsu ace seawa e , collec ed as desc ibed in Sec ion 2.1, in p opo ions o 0% (1% 207
ul apu e wa e : 99% seawa e ), 5% (0.5% concen a e: 0.5% ul apu e wa e : 99% 208
seawa e ) and 10% (1% concen a e: 99% seawa e ) o he o iginal (p e ious o 209
concen a ion) i e ine and a mosphe ic ma e ials (Fig. 2). These p opo ions also 210
ensu ed ha he inal salini y o he samples was kep cons an independen ly o he 211
amoun o ex ac added. 212
213
214
215
216
217
218
219
220
Fig. 2: Schema ic ep esen a ion o he na u al and con olled addi ion ea men s pe o med a each 221
season (see de ails in sec ion 2.4). 222
223
16
The le els o signi icance o - es s, pe o med be ween each ea men and he con ol, a e: *p<0.05, 354
**p<0.01, ***p<0.001. 355
In summe , Chl a and PP a es esponded posi i ely in all ea men s, including 356
na u al and con olled addi ions (Fig. 4c, d). The e we e only wo excep ions: PP a es 357
did no espond o o ganic inpu s and he esponse o Chl a o he 10% i e ine inpu 358
was ma ginally signi ican (P=0.07). In gene al, he esponse a io in his expe imen 359
was highe han in sp ing, wi h he esponse a ios o he na u al inpu s being highe 360
han 1.5 and o he con olled ino ganic inpu s eaching 4.5 o Chl a and 7 o PP (Fig. 361
4a-d). In au umn, he e was a nega i e esponse in Chl a o he na u al inpu s (~0.85) 362
and o he o ganic con olled addi ions (~0.40) (Fig. 4e). On he con a y, ino ganic and 363
mixed con olled addi ions led o a sligh inc ease in Chl a concen a ions. PP a es 364
inc eased in esponse o he 10% i e ine inpu s and o he mixed ea men , and 365
dec eased in esponse o he o ganic addi ions (Fig. 4 ). 366
Table 3: Ou pu o mul i a ia e Gene al Linea Model analysis o Chl a, PP and he se e al 367
phy oplank on g oups’ esponse a ios as dependen a iables and Expe imen and T ea men as ixed 368
ac o s. Synecho: Synechococcus- ype cyanobac e ia, APF: au o ophic pico lagella es, ANF: au o ophic 369
nano lagella es. 370
Tes s o Be ween-Subjec s E ec s
Sou ce
F
Sig.
Expe imen
Chl a
23.860
0.001
PP
34.076
0.000
Synecho
9.405
0.010
APF
20.515
0.001
ANF
24.452
0.001
Dia oms
1.333
0.323
T ea men
Chl a
15.742
0.001
PP
24.922
0.000
Synecho
3.788
0.052
APF
57.782
0.000
ANF
10.840
0.003
Dia oms
0.946
0.519
Expe imen *
T ea men
Chl a
3.385
0.057
PP
10.331
0.002
Synecho
1.638
0.262
APF
11.154
0.002
ANF
3.579
0.050
Dia oms
1.073
0.484

17
Table 4: Bon e oni pos hoc mul iple compa isons om Gene al Linea Models analysis wi h he 371
esponse a ios o Chl a, PP and he se e al phy oplank on g oups as dependen a iables and he 372
expe imen and ea men as ixed ac o s. Synecho: Synechococcus- ype cyanobac e ia, APF: au o ophic 373
pico lagella es, ANF: au o ophic nano lagella es. Fo ea men , only signi ican esul s a e shown. 374
Expe imen
Sig.
Chl a
Sp ing
Summe
0.024
Au umn
0.011
Summe
Au umn
0.001
PP
Sp ing
Summe
0.001
Au umn
0.052
Summe
Au umn
0.000
Synecho
Sp ing
Summe
0.012
Au umn
0.136
Summe
Au umn
0.510
APF
Sp ing
Summe
0.545
Au umn
0.001
Summe
Au umn
0.012
ANF
Sp ing
Summe
0.182
Au umn
0.001
Summe
Au umn
0.013
Dia oms
Sp ing
Summe
1.000
Au umn
0.682
Summe
Au umn
0.499
T ea men
Sig.
Chl a
Ino ganic
Na u al
<0.01
O ganic
0.003
PP
Ino ganic
All
<0.01
Synecho
A m 10%
Mixed
0.037
APF
Mixed
All
<0.01
o ganic
All
<0.01
ANF
Ino ganic
Na u al
<0.05
O ganic
0.002
Mixed
O ganic
0.017
375
376
377
378
18
S a is ical analyses (Table 3) demons a ed ha bo h expe imen and ea men 379
had a signi ican e ec in he esponse a ios o Chl a and PP. The in e ac ion be ween 380
hese wo ac o s was also signi ican o PP esponse a ios. Thus, Chl a and PP 381
esponse a ios we e signi ican ly di e en among he h ee expe imen s (p < 0.01), 382
excep o he PP esponse a io be ween sp ing and au umn (Table 4). Among 383
ea men s, signi ican di e ences we e ound o Chl a and PP esponse a ios be ween 384
na u al inpu s and he ino ganic ea men in he con olled addi ions. The esponse o 385
he ino ganic ea men was also signi ican ly di e en om he o ganic ea men o 386
Chl a and om he mixed and o ganic ea men s o PP. No signi ican di e ences 387
we e ound in Chl a and PP esponse a ios among he samples ea ed wi h bo h ypes 388
and le els o na u al inpu s: i e ine and a mosphe ic. 389
The di e en phy oplank on g oups also esponded di e en ly o he 390
expe imen s and ea men s (Fig. 5, Table 3 & 4). While Synechococcus showed a 391
signi ican inc ease in he o ganic and mixed ea men in sp ing (Fig. 5a), a signi ican 392
dec ease was obse ed in au umn in esponse o a mosphe ic and o ganic addi ions (Fig. 393
5g). In summe , he low abundances o e en absence o Synechococcus in some samples 394
did no allow in e ing eliable esponses o he ea men s. Among APF, posi i e 395
esponses we e obse ed o 10% a mosphe ic, ino ganic and mixed addi ions in sp ing 396
and summe , and nega i e in all expe imen s o o ganic addi ions (Fig. 5b, e, h). ANF 397
showed posi i e esponses in he con olled addi ions and in he 5% addi ion o i e ine 398
inpu s in sp ing (Fig. 5c), and in he ino ganic and mixed ea men s in summe (Fig. 399
5 ). No esponse was obse ed in he au umn expe imen o ANF, excep a dec ease in 400
he esponse o he o ganic addi ions (Fig. 5i). Dia oms showed a high posi i e esponse 401
o he ino ganic ea men in he summe expe imen (Fig. 5d) while a sligh posi i e 402
esponse was also obse ed o he mixed addi ion. In sp ing and au umn, no esponses 403
19
o nega i e e ec s we e obse ed on dia oms, especially o o ganic and mixed 404
addi ions (no shown). 405
406
407
408
409
410
411
412
413
414
415
Fig. 5: Response a ios ( ea men /con ol) o phy oplank on g oups showing signi ican esponses o 416
ea men s in sp ing (a-c), summe (d- ) and au umn (g-i). Synecho: Synechococcus- ype cyanobac e ia, 417
APF: au o ophic pico lagella es, ANF: au o ophic nano lagella es. Ho izon al line deno es he esponse 418
a io = 1. A m.: A mosphe ic inpu s; I: con olled ino ganic addi ions; O: con olled o ganic addi ions; M: 419
con olled mixed addi ions. The le els o signi icance o - es s, pe o med be ween each ea men and 420
he con ol, a e: *p<0.05, **p<0.01, ***p<0.001. In he expe imen s o July and Oc obe , only one bag o 421
each ea men was analyzed o nano and mic ophy oplank on a 48h, so we don’ ha e s a is ical 422
signi icance le els o ANF and dia oms a hese expe imen s. 423
424
I is no iceable ha APF abundances we e be e s imula ed by he combina ion 425
o ino ganic and o ganic compounds ( he mixed ea men ) when compa ed wi h he 426
addi ion o only ino ganic o only o ganic nu ien s (Fig. 5b, e, h). In ac , he o ganic 427
20
ea men seems o be in some way de imen al o APF, showing lowe abundances 428
han in he con ol. By con as , ANF and dia oms ypically g ew mo e wi h he addi ion 429
o only ino ganic nu ien s (Fig. 5c, d, ) han in he mixed ea men . Synechococcus 430
showed a simila pa e n o APF o ganisms, wi h highes g ow h unde he combina ion 431
o o ganic and ino ganic compounds (Fig. 5a, g). Howe e , in his case, he o ganic 432
ea men seemed o be ha m ul o hese o ganisms only in au umn (Fig. 5g). 433
Also o he di e en phy oplank on g oups’ esponse a ios, he expe imen and 434
he ea men we e s a is ically signi ican ac o s (Table 3). Fo Synechococcus, 435
signi ican di e ences we e ound be ween he sp ing and summe expe imen s, while 436
o he small lagella es (APF and ANF) he signi ican di e ences occu be ween he 437
au umn expe imen and he o he wo expe imen s (Table 4). No di e ences we e ound 438
be ween he esponses o he di e en na u al addi ion ea men s. Mos signi ican 439
di e ences in esponse a ios be ween ea men s we e ound be ween na u al and 440
con olled addi ion ea men s. 441
442
Table 5: Pea son co ela ion coe icien s o analyses pe o med be ween Chl a concen a ions, PP a es 443
and abundances o he se e al phy oplank on g oups a he end o he incuba ion, and ini ial ino ganic o 444
o ganic nu ien s le els (including in si u and added) a each expe imen . Synecho: Synechococcus- ype 445
cyanobac e ia, APF: au o ophic pico lagella es, ANF: au o ophic nano lagella es. 446
Chla
PP
Synecho
APF
ANF
Dia oms
SPRING
DIN
0.975***
0.930***
0.563
0.729*
0.920***
-0.229
DOC
0.422
0.236
0.565
0.341
0.216
-0.497
DON
0.421
0.232
0.573
0.349
0.210
-0.520
SUMMER
DIN
0.902***
0.687*
0.506
0.271
0.922***
0.710*
DOC
0.117
-0.221
0.038
-0.216
0.099
-0.118
DON
0.120
-0.214
0.088
-0.254
0.117
-0.093
AUTUMN
DIN
0.657*
0,413
0.642*
0.070
0.104
0.120
DOC
-0.208
-0.290
0.281
-0.563
-0.686*
-0.775**
DON
-0.151
-0.289
0.340
-0.621
-0.697*
-0.738*
447
21
In o de o explain he di e en phy oplank on esponses a each expe imen , we 448
analyzed hem sepa a ely sea ching o co ela ions wi hin he se e al a iables. Chl a 449
concen a ion was posi i ely co ela ed wi h DIN in he h ee expe imen s (Table 5). 450
Simila ly, also se e al phy oplank on g oups showed posi i e co ela ions wi h DIN, 451
bu no o all expe imen s. DIN was posi i ely co ela ed wi h APF and ANF in sp ing, 452
wi h ANF and dia oms in summe and wi h Synechococcus in au umn. No signi ican 453
co ela ions we e ound be ween he se e al phy oplank on g oups and o ganic 454
compounds (bo h DOC and DON) in any o he h ee expe imen s, excep o ANF and 455
dia oms in au umn which showed a nega i e signi ican co ela ion wi h o ganic 456
compounds. 457
Some di e ences in phy oplank on communi y composi ion we e obse ed in 458
esponse o nu ien amendmen s (Fig. 6). In sp ing, he p opo ion o dia oms inc eased 459
wi h i e ine and a mosphe ic inpu s whe eas wi h con olled addi ions he ela i e 460
dia oms abundance was dep essed and ANF a o ed (Fig. 6a). In au umn an inc ease in 461
he p opo ion o Synechococcus abundance was obse ed in esponse o he o ganic 462
ea men (Fig. 6c). In his same ea men , i was obse ed a dec ease in he p opo ion 463
o APF abundance (Fig. 6c). 464

22
465
Fig. 6: Rela i e abundance o majo phy oplank on g oups a he end o he expe imen in sp ing (a), 466
summe (b) and au umn (c). Synecho: Synechococcus- ype cyanobac e ia, APF: au o ophic 467
pico lagella es, ANF: au o ophic nano lagella es. C: Con ol (no addi ions); I: con olled ino ganic 468
addi ions; O: con olled o ganic addi ions; M: con olled mixed addi ions. 469
470
471
%
0
20
40
60
80
100
%
0
20
40
60
80
100
%
0
20
40
60
80
100
Synecho
APF
ANF
Dia oms
Pigmen ed dino lagella es
Sp ingSumme Au umn
a
b
c
Ri e ine A mosphe ic
C5%10% C I O MC 5%10% Con olled
23
4. Discussion 472
Pe o ming he expe imen s in sp ing (May), summe (July) and au umn 473
(Oc obe ) allowed in es iga ing he di e en hyd og aphic condi ions and biological 474
communi ies ypical o his coas al embaymen (Figuei as and Ríos 1993; Figuei as e 475
al. 2002; A bones e al. 2008). Jus a e he i s in ense upwelling pe iod o he yea in 476
sp ing, high Chl a and nu ien concen a ions we e obse ed in su ace wa e s, wi h a 477
ypical communi y domina ed in biomass by chain o ming dia oms (Chae oce os spp.). 478
On he con a y, in summe , despi e he p e ious upwelling e en s, we ound ha he 479
su ace laye was poo in nu ien s and domina ed by small plank on o ganisms. This 480
ac was due o he sinking o he su ace phy oplank on communi ies (domina ed by 481
la ge dia oms de eloped in he p e ious weeks - da a no shown), esul ing om he 482
elaxa ion o upwelling a o able winds. In au umn, ypical oceanic communi ies we e 483
ound a he su ace as a esul o a p e ious s ong downwelling e en which pushed 484
con inen al shel wa e s in o he Ría. High abundance o Synechococcus deno es he 485
p esence o his o sho e wa e inside he Ría de Vigo (Rod íguez e al. 2006). 486
The s a is ically di e en esponse a ios o phy oplank on among he 487
expe imen s pe o med in he h ee di e en seasons indica e ha he ini ial condi ions 488
a each expe imen we e impo an o de e mine he magni ude and he ype o esponse 489
o phy oplank on communi ies o nu ien s inpu s. Di e ences in ini ial condi ions 490
among he expe imen s which can in luence hese phy oplank on esponses may include 491
dissimila i ies in en i onmen al a iables (i.e. nu ien s a ailabili y), in he composi ion 492
o he na u al ma e inpu s o in he s uc u e o phy oplank on communi ies (Ma ínez-493
Ga cía e al. 2015). The high posi i e co ela ions ound be ween Chl a (and se e al 494
phy oplank on g oups) and DIN concen a ions in he incuba ion bags sugges ha DIN 495
a ailabili y is an impo an ac o de e mining he esponse o phy oplank on 496
24
communi ies in his in es iga ion. In addi ion, he highes phy oplank on esponses o 497
he se e al ea men s we e obse ed in summe , when in si u DIN le els we e he 498
lowes . In his case, phy oplank on g ow h seemed o be se e ely limi ed by nu ien s 499
and bo h ypes o inpu s (na u al and con olled) p omo ed enhanced phy oplank on 500
g ow h. A simila si ua ion, al hough wi h a lowe magni ude, occu ed in sp ing. In 501
au umn, howe e , when in si u ino ganic nu ien le els we e he highes , phy oplank on 502
did no espond o e en dec eased in abundance. In his case, in e ac ions be ween 503
se e al ac o s besides DIN mus ha e occu ed as discussed la e on. Ni ogen has been 504
widely ecognized as he main limi ing nu ien in ma ine sys ems when ligh in ensi y 505
is su icien (Wollas 1998). In ac , se e al wo ks ha e also obse ed s imula ion in 506
phy oplank on communi ies due o inc eases in ino ganic ni ogen om a mosphe ic 507
deposi ion (Pae l e al. 1990; Cui e al. 2016) o in con olled addi ions (Piehle e al. 508
2004; Xu e al. 2014). 509
In addi ion o ino ganic nu ien s, i has been obse ed ha o ganic compounds 510
may also s imula e phy oplank on g ow h in coas al a eas (Peie ls and Pae l 1997; 511
Sei zinge and Sande s 1999). In ac , p io esul s in he Ría de Vigo showed ha 512
o ganic ni ogen concen a ion also con ibu ed o he a iabili y in he esponse o 513
p ima y p oduc ion and Chl a o ainwa e addi ions (Ma ínez-Ga cía e al. 2015). 514
Howe e , in ou wo k, no signi ican co ela ions we e ound be ween phy oplank on 515
abundance and o ganic compounds, excep a nega i e co ela ion wi h ANF and 516
dia oms in au umn. The ew signi ican posi i e (al hough low) inc eases in 517
phy oplank on abundance in esponse o he o ganic ea men (Synechococcus and ANF 518
in sp ing and Chl a in summe ) may be ela ed o he occu ence o mixo ophy 519
(including di ec consump ion o o ganic compounds) in bo h seasons (Flynn and Bu le 520
1986; Eile 2006) o o an inc ease in nu ien s emine aliza ion media ed by enhanced 521
25
bac e ial ac i i y in he o ganic ea men in summe (Tei a e al. 2016). Nu ien 522
emine aliza ion in sp ing is unlikely o be impo an , because he e we e high in si u 523
nu ien le els and bac e ia did no espond o he o ganic inpu s in his expe imen 524
(Tei a e al 2016). As he chemical composi ion o he ainwa e addi ions he e was e y 525
di e en om ha in Ma ínez-Ga cía e al. (2015), i should be expec ed ha di e en 526
phy oplank on esponses may occu depending on he composi ion o na u al ma e 527
inpu s in he Ría de Vigo. 528
Mo eo e , some phy oplank on g oups appea ed o be a o ed by he 529
combina ion o ino ganic and o ganic o ms. This is he case o APF in sp ing and 530
summe , which p esen ed a highe esponse o he mixed ea men han o he ino ganic 531
o o ganic addi ions alone (Fig. 5b, e). This esponse can be ela ed o he concomi an 532
use o o ganic o ms, namely amino acids, by hese small phy oplank on cells (Flynn 533
and Bu le 1986; He nández-Ruiz e al., 2018). The same end was obse ed o 534
Synechococcus in sp ing (Fig. 5a) likely ela ed o hei mixo ophic capabili ies (Eile 535
2006). O he explana ions may also include he need o ex e nal seconda y me aboli es 536
o phy oplank on g ow h (eg. B12 i amin) p oduced by he e o ophic bac e ia (C o e 537
al. 2005; P ie o e al. 2016) o he occu ence o bac e i o y by he small euka yo es 538
(Sande s and Gas 2012; Ha mann e al. 2013). In bo h cases, phy oplank on would be 539
bene i ing om he s imula ion o bac e ial g ow h by he combina ion o he o ganic 540
and ino ganic compounds in he mixed ea men (Tei a e al. 2016). On he o he hand, 541
he lowe esponse o ANF and dia oms o he mixed compa ed o he ino ganic 542
ea men could be explained by compe i ion o ino ganic nu ien s wi h he o ganisms 543
s imula ed by he mixed ea men , including APF and bac e ia (Join e al. 2002; 544
Ma ínez-Ga cía e al. 2010). 545
32
Join , I., Hen iksen, P., Fonnes, G.A., Bou ne, D., Things ad, T.F., Riemann, B., 2002. 684
Compe i ion o ino ganic nu ien s be ween phy oplank on and bac e ioplank on in 685
nu ien manipula ed mesocosms. Aqua . Mic ob. Ecol. 29, 145-159. 686
Ké ouel, K., Amino , A.1997. Fluo ime ic de e mina ion o ammonia in sea and 687
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La uelle, G.G., Roubeix, V., S e a o e,A., B odhe , B., Ciu a,D., Conley, D. J., Du , 689
H. H., Ga nie , J., Lancelo , C., Le Thi Phuong, Q., Meunie , J.-D., Meybeck, M., 690
Michalopoulos, P., Mo iceau, B., Nı´ Longphui , S., Loucaides, S., Papush, L., P es i, 691
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



Fig.S1:Tempo ale olu iono  heupwellingindexand uno  alues h oughou 2013.
Expe imen da esa ema kedwi hdiscon inuouslines.




Upwelling index (m3 s-1 km-1)
-4000
-2000
0
2000
4000
Runo (m3 s-1)
0
50
100
150
200
250
300
350
Jan Feb Ma Ap May Jun Jul Aug Sep Oc No Dec
Sp ing Summe Au umn